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Tesis Laura Higueras - Roderic

1. e di E P ALL eur p R D Am H m ur eS 2 n TCU BL ya LAT os D 9 CE d t a oon E s f gt TESIS DOCTORAL Quitosano como matriz biopolim rica para el desarrollo de envases activos antimicrobianos de alimentos Laura Higueras Contreras Valencia Octubre de 2015 Directores Dra Pilar Hern ndez Mu oz Dra Gracia L pez Carballo Dr Rafael Gavara Clemente cr Instituto de Agroqu mica y Tecnolog a de Alimentos Consejo Superior de Investigaciones Cient ficas Departamento de Conservaci n y Calidad de Alimentos Laboratorio de Envases Tutora Dra M Jestis Lagarda Blanch Universitat de Valencia Facultat de Farmacia Departament de Medicina Preventiva i Salud Publica Ci ncies de l Alimentaci Toxicologia i Medicina Legal Ci ncia dels Aliments Quitosano como matriz biopolim rica para el desarrollo de envases activos antimicrobianos de alimentos VNIVERSITAT B VALENCIA Departament de Medicina Preventiva i Salud P blica Ci ncies de l Alimentaci Toxicologia i Medicina Legal Ci ncia dels Aliments UP VNIVERSITAT J IDGVALENCIA TESIS DOCTORAL Quitosano como matriz biopolim rica para el desarrollo de envases activos antimicrobianos de alimentos Laura Higueras Contreras Directores Dra Pilar Hern ndez Mu oz Dra Gracia L pez Carballo Dr Rafael Gavara Clemente Tutora Dra M Jes s Lagarda Blanch Valencia
2. Li X Shao T Shi Q Hu M 2013 A diaryl Schiff base as a photo and pH responsive bifunctional molecule Rsc Advances 3 22877 22881 Lovett J Francis D W Hunt J M 1987 Listeria monocytogenes in raw milk detection incidence and pathogenicity Journal of Food Protection 50 188 192 Mohamad A 2013 Reactivity of base catalysed hydrolysis of 2 pyridinylmethylene 8 quinolinyl Schiff base iron II iodide complexes solvent effects Chemick Zvesti 67 464 476 Muhamad S G 2011 First photolysis of benzidine Schiff base in non aqueous solvents International Journal of Chemistry 1 3 142 145 Muriel Galet V L pez Carballo G Gavara R Hern ndez Mu oz P 2012 Antimicrobial food packaging film based on the release of LAE from EVOH nternational Journal of Food Microbiology 157 239 244 Nazzaro F Fratianni F De Martino L Coppola R De Feo V 2013 Effect of essential oils on pathogenic bacteria Pharmaceuticals 6 1451 1474 Renault F Sancey B Crini G 2009 Chitosan for coagulation flocculation processes An eco friendly approach European Polymer Journal 45 1337 1348 Sashiwa H Aiba SL 2004 Chemically modified chitin and chitosan as biomaterials Progress in Polymer Science 29 887 908 Shahidi F Arachchi J K V Jeon Y J 1999 Food applications of chitin and chitosans Trends in Food Science amp Technology 10 37 51 Vallapa N Wiarachai O Thongchul N
3. McCormick K E Han LY Acton J C Sheldon B W Dawson P L 2005 In package pasteurization combined with biocide impregnated films to inhibit Listeria monocytogenes and Salmonella Typhimurium in turkey bologna Journal of Food Science 70 M52 M57 McGuire R G Hagenmaier R D 2001 Shellac formulations to reduce epiphytic survival of coliform bacteria on citrus fruit postharvest Journal of Food Protection 64 1756 1760 Mecitoglu C Yemenicioglu A Arslano lu A 2007 Antimicrobial and antioxidant activity of edible zein films incorporated with lysozyme albumin proteins and disodium EDTA Food Research International 40 80 91 Mecitoglu C Yemenicioglu A Arslanoglu A Elmaci Z S Korel F Cetin A E 2006 Incorporation of partially purified hen egg white lysozyme into zein films for antimicrobial food packaging Food Research International 39 12 21 Mehyar G F Al Qadiri H M Abu Blan H A Swanson B G 2011 Antifungal effectiveness of potassium sorbate incorporated in edible coatings against spoilage molds of apples cucumbers and tomatoes during refrigerated storage Journal of Food Science 76 M210 M217 321 Anexos Mild R M Joens L A Friedman M Olsen C W McHugh T H Law B Ravishankar S 2011 Antimicrobial edible apple films inactivate antibiotic resistant and susceptible Campylobacter jejuni strains on chicken breast Journal of Food Science 76 M163 M168 Miltz J
4. Trinetta V Floros J D Cutter C N 2010 Sakacin A containing pullulan film an active packaging system to control epidemic clones of Listeria monocytogenes in ready to eat foods Journal of Food Safety 30 366 381 Tire H Ero lu E zen B Soyer F 2011 Effect of biopolymers containing natamycin against Aspergillus niger and Penicillium roqueforti on fresh kashar cheese International Journal of Food Science and Technology 46 154 160 Tire H Ero lu E Soyer F zen B 2008 Antifungal activity of biopolymers containing natamycin and rosemary extract against Aspergillus niger and Penicillium roqueforti International Journal of Food Science and Technology 43 2026 2032 Unalan LU Korel F Yemenicioglu A 2011 Active packaging of ground beef patties by edible zein films incorporated with partially purified lysozyme and NazEDTA International Journal of Food Science and Technology 46 1289 1295 Valencia Chamorro S A Palou L Del Rio M A P rez Gago M B 2008 Inhibition of Penicillium digitatum and Penicillium italicum by hydroxypropylmethylcellulose lipid edible composite films containing food additives with antifungal properties Journal of Agricultural and Food Chemistry 56 11270 11278 Valencia Chamorro S A Palou L Del Rio M A P rez Gago M B 2011 Performance of hydroxypropyl methylcellulose HPMC lipid edible coatings with antifungal food additives during cold storage of Clemenules
5. Agente antimicrobiano alcoholes Biopolimero Alimento Microorganismo Referencia etanol etanol etanol HPMC quitosano shellac tomate uvas sin semilla Thompson Autumn naranja Valencia S Montevideo Botrytis cinerea E coli Enterobacter aerogenes Zhuang et al 1996 Romanazzi et al 2007 McGuire y Hagenmaier 2001 EDTA nisina lisozima EDTA ED semilla de pomelo EDTA EDTA sorbato de potasio EDTA ovotransferrina EDTA nisina lisozima EDTA EDTA nisina EDTA nisina EDTA lisozima EDTA ED semilla de uva EDTA ED semilla de uva nisina EDTA nisina EDTA c m lico ED semilla de uva nisina EDTA lisozima lactoferrina lisozima EDTA EDTA c l urico EDTA nisina EDTA lisozima EDTA lisozima carbonato de potasio carbonato de amonio fosfato de amonio hidrogenocarbonato de potasio hidrogenocarbonato de sodio alginato de sodio K carragenato K carragenato gelatina HPMC PLA prote na de soja ze na prote na de soja prote na de suero de leche quitosano ze na ze na ze na alb mina de garbanzos alb mina de suero bovino HPMC HPMC medio de cultivo medio de cultivo pechuga de pollo jam n cocido mortadela medio de cultivo medio de cultivo medio de cultivo medio de cultivo salchichas de pavo medio de cultivo medio de cultivo medio de cu
6. OMS WHO PA PE PG PLA PP PS PVOH RH SCENIHR SEM TEM TGA UE UNEP USDA cido desoxirribonucleico aceite esencial cido ribonucleico cido ribonucleico mensajero espectroscop a de infrarrojos por transformada de Fourier con reflexi n total atenuada ciclodextrina Comunidad Europea Comunidad Econ mica Europea quitosano chitosan dalton US Energy Information Administration European food safety authority US Environmental Protection Agency copol mero de etileno y alcohol vin lico United Nations Food and Agriculture Organization US Food and Drug Administration glicerol absorci n at mica de horno de grafito Government Superannuation Fund Authority Generally Recognized As Safe hidroxipropil B ciclodextrina Ingesta Diaria Admisible Joint Expert Committee on Food Additives etil N dodecanoil L arginato The Organisation for Economic Co operation and Development Screening Information Dataset Organizaci n Mundial de la Salud World Health Organization poliamida polietileno propilenglicol poli cido l ctico polipropileno poliestireno polialcohol vin lico relative humidity humedad relativa Scientific Committee on Emerging and Newly Identified Health Risks microscop a electr nica de barrido microscop a electr nica de transmisi n an lisis termogravim trico Uni n Europea European Union United Nations Environment Programme US Department of Agriculture Pr logo La presente Tesi
7. 2009 cultivo plata nano celulosa mel n Piel de ME TVC mes filos y Fern ndez sapo pelado psicr filos LAB Picouet et al kiwi Pseudomonas spp 20102 b pechuga de Enterobacteriaceae Fern ndez et al pollo hongos levaduras 2009 Lloret et carne de al 2012 vacuno plata nano celulosa medio de B subtilis St aureus Pinto et al 2009 cultivo Klebsiella pneumoniae plata nano celulosa medio de E coli Tankhiwale y cultivo Bajpai 2009 plata nano celulosa quitosano medio de St aureus E coli B cereus Yoksan y cultivo Chirachanchai 2009 2010 plata nano celulosa acetato medio de St aureus E coli Chou et al 2005 cultivo plata nano celulosa acetato medio de St aureus E coli Son et al 2004 cultivo Klebsiella pneumoniae Ps Son et al 2006 aeruginosa plata PLA zeolitas medio de St aureus E coli Fern ndez cultivo Soriano et al 2010 plata nano HPMC PVOH medio de St aureus E coli De Moura et al cultivo 2012 plata nano quitosano lactato medio de E coli Tankhiwale y cultivo Bajpai 2010 plata quitosano zeolitas medio de St aureus L Rhim et al 2006 cultivo monocytogenes S plata nano Typhimurium coli O157 H7 300 Anexos plata nano plata nano plata nano xido de zinc nano plata nano plata plata nano zinc xido de zinc nano xido de zinc nano xido de zinc nano AE albahaca quitosano quitosano
8. As en disoluci n cida pH lt 6 los grupos amino en la posici n C2 del pol mero se protonan resultando un polisac rido soluble cargado positivamente Agull et al 2003 Rinaudo 2006 El CS debido a la presencia de grupos amino posee una gran capacidad quelante por lo que puede adsorber iones met licos formar micelas con tensioactivos unirse a prote nas o a ADN Rinaudo 2006 11 Introducci n gt Desechos ate industria alimentaria reducci n tamafio de part cula descalcificaci n disoluci n de HCl desproteinizaci n disoluci n de NaOH decoloraci n disoluci n de KMnO H 0 HOOC COOH 6 KCIO 5 lavado o H NHH P no H o 0 H oH quitina desacetilaci n disoluci n de NaOH 6 enzimas lavado CH O0 Hi uen H NHH NAN 9 lHo7 27H H H HO H VY Lo o NH H 2H H Soy x y COS quitosano hidr lisis total hidr lisis parcial N acetilglucosamina glucosamina quitooligosac ridos quitioligosac ridos acetilaci n esce a gt desacetilacion acetilacion Figura 1 2 Obtenci n de la quitina CS y sus derivados Figura basada en Shahidi et al 1999 Estas cargas positivas del CS tambi n le confieren su actividad antimicrobiana frente a bacterias hongos y levaduras Agull et al 2003 No et al 2007 Shahidi et al 1999 El mecanismo de acci n de la actividad antimicrobiana del CS es diferente seg n se trate de bacterias gram positivas o negativas debido a
9. CD films largely plasticized by G and water could be occurring by mechanisms other than formation of inclusion complexes with HP BCDs It has been reported that CDs are able to self assemble to form nanosized complex aggregates and aggregation happens rapidly with CD concentration Messner et al 2010 Given that the composition of the biocomposites is CS BCDs 1 1 HP BCDs occupy a considerable volume in the polymer matrix which could be forming nanoparticles Furthermore a great increase in the free volume of the film is expected as a result of the presence of G and water 4 Conclusions Films having good transparency and moderate mechanical properties and permeability to water and oxygen have been developed incorporating 1 1 HP BCDs into a CS matrix These hydrophilic films are capable of retaining different amounts of the non polar volatile compound carvacrol Sorption depends on the degree of plasticization of the film by G and water thus it is possible to tailor the amount of the volatile agent in the film Due to the antimicrobial properties of carvacrol the film developed could be used as a sustained release device in food packaging pharmaceutical and agrochemistry applications Acknowledgments Authors thank the financial support of the Spanish Ministry of Science and Innovation projects AGL2009 08776 JAE program from CSIC L H fellowship Generalitat Valenciana J P C fellowship 192 Quitosano Carvacrol Referen
10. Esta variabilidad y heterogeneidad hacen necesario el uso de AE estandarizados o preparados o bien componentes de AE aislados y purificados Delaquis et al 2002 Los constituyentes de los AE pertenecen de manera casi exclusiva a dos grupos caracterizados por or genes biogen ticos distintos el grupo de los terpenoides y el grupo de los compuestos arom ticos derivados del fenilpropano que son mucho menos frecuentes Dos componentes de los AE que destacan por su elevada capacidad antimicrobiana y que han sido empleados en este trabajo son el carvacrol y el cinamaldeh do 33 Introducci n 1 6 3 1 Carvacrol El carvacrol 2 metil 5 1 metiletil fenol como se muestra en la Figura 1 8 es un compuesto fen lico alcohol monoterp nico monoc clico constituyente de diversos AE como los de or gano Origanum vulgare Thymus capitatus Lippia graveolens y tomillo Thymus vulgaris De Vincenzi et al 2004 Se ha demostrado la bioactividad del carvacrol frente a un amplio espectro de microorganismos pat genos incluyendo bacterias gram positivas y negativas hongos y levaduras par sitos Suntres et al 2015 El modo de acci n del carvacrol no se conoce completamente pero su car cter hidr fobo permite su acumulaci n en la membrana citoplasm tica aumentando la fluidez y permeabilidad desintegrando la membrana externa de bacterias gram negativas liberando lipopolisac rido y aumentando la permeabilidad de la memb
11. Ruckman et al 2004 On top of that LAE shows chemical stability and antimicrobial activity in a range of pH 3 7 Asker et al 2011 333 Anexos JOURNAL o AGRICULTURAL AND FOOD CHEMISTRY pubs acs org JAFC Silver lons Release from Antibacterial Chitosan Films Containing in Situ Generated Silver Nanoparticles Gracia L pez Carballo Laura Higueras Rafael Gavara and Pilar Hern ndez Mu oz Institute of Agrochemistry and Food Technology CSIC Avenida Agust n Escardino 7 46980 Paterna Valencia Spain ABSTRACT This study aims to develop antimicrobial films consisting of chitosan and silver nanoparticles that are homogeneously distributed throughout the polymer matrix Nanoparticles were generated in situ during the neutralization of the chitosan acetate film with sodium hydroxide The temperature of neutralization and the concentration of silver in the film were crucial determinants of the shape and size of the nanoparticles Neutralized films exhibited antimicrobial activity against Escherichia coli and Staphylococcus aureus in liquid growth media However the effectiveness of the films was considerably greater in diluted growth media Furthermore no significant differences were found either in the antimicrobial capacities of films incorporating different amounts of silver or in the amount of silver that migrated into the liquid media after 18 h of immersion of the film Neutralized films maintained their ac
12. W 7 HP were provided by Wacker Fine Chemicals S L Barcelona Spain Maltodextrins MDs from maize starch Biochemika 10 was supplied by Sigma Aldrich Madrid Spain 2 2 Film preparation A flowchart with the preparation of chitosan hydroxypropyl f cyclodextrin CS CD films is shown in Figure 4 4 3 CS 1 5 w w was solubilised in 0 596 w w acetic acid solution and filtrated to eliminate impurities Films were prepared by casting pouring a suitable amount of the film forming solution into a flat PS tray and allowing it to dry under controlled environmental conditions 36 h 40 0 1 5 C and 20 9 RH CS CD films were obtained by adding HP BCDs to the CS solution in 1 1 weight ratio of HP BCDs to CS and G at 096 CS CD 0G 2096 CS CD 20G and 3596 CS CD 35G g G 100 g dry matter was added to the film forming solution while stirring at 1500 rpm and 37 C until complete dissolution Films were obtained by casting as described above After peeling the films from the tray they were plasticized with different amounts of water for which purpose samples 550 mm in diameter and 55 5 mm in thickness were stored in glass desiccators with phosphorus pentoxide to achieve humidities close to 0 to dry the films or at humidities of 52 9 0 2 and 75 3 0 1 RH using saturated salt solutions ASTM 2007 in a temperature controlled room at 25 1 C until moisture equilibrium was reached Films were named as CS CD xxGxxRH depending on the am
13. 10 m s range much higher than the results obtained in this work as should be expected from the high size of the LAE molecule Ouattara et al 2000a LAE diffusion in hydrophilic EVOH was estimated in the 10 m s range in agreement with these results Gavara data not published 3 3 LAE antimicrobial activity MICs and MBCs of LAE against bacteria yeasts and fungi determined on solid and liquid media were included in Table 4 1 3 MIC values were 8 pug mL for Gram positive bacteria and 16 g mL for Gram negative bacteria either on solid or in liquid medium MIC values for yeasts ranged from 4 to 16 pg mL in liquid medium and 32 to 104 ug mL on solid medium MIC values for fungi were found between 24 and 120 ug mL on solid medium Similar MIC values have also been reported in the literature Infante et al 1997 Oh and Marshall 1992 Rodriguez et al 2004 LAE MBC values were16 Table 4 1 2 Values of the parameters describing LAE release from CS matrices into water according to the Eq 1 T C 4 C 28 C LAE 96 10 D m s R 10 D m s R 1 8 9 0 8 0 97 134215 0 89 5 7 1 0 5 0 99 11 7 0 7 0 98 10 54 0 4 0 99 10 6 0 8 0 97 Different letters in the same column indicate a statistically significant difference P 0 05 comparing the different amounts of LAE in the matrix 81 Cap tulo 1 Art culo cient fico 1 Table 4 1 3 Minimum inhibitory concentration MIC pg mL and minimum bio
14. 180 160 4 160 140 4 140 120 4 120 100 4 100 80 4 80 60 4 60 40 4 p 40 Sorption equilibrium of carvacrol dry film wl Ap juajuo9 aln siow uinugiinb3 20 4 p 20 0 T 75 90 100 Relative humidity Figure 4 4 13 Effect of RH on equilibrium moisture content and sorption equilibrium of carvacrol in CS CD 35PG 260 Quitosano Carvacrol 25 00 7 Carvacrol sorption in PVOH Carvacrol sorption in PVOH CD Eq moisture content in PVOH Eq moisture content in PVOH CD Bd r 20 Sorption equilibrium of carvacrol dry film uy Ap juajuo9 eunisiou uinuqiinb3 0 20 35 Glycerol dry film Figure 4 4 14 Effect of incorporating HP BCD and G on equilibrium moisture content and sorption equilibrium of carvacrol in PVOH films conditioned at 7596 RH In the studies carried out with PVOH it is noteworthy that sorption was negligible when CDs were not incorporated in the films Figure 4 4 14 there are studies reporting that the addition of B CDs enhances the solubility of xylene and benzene in PVOH membranes Chen et al 2000 Peng et al 2006 Figure 4 4 14 shows that the content of plasticizer exerts a favourable effect on the sorption capacity of the films This effect was also observed with the water content in the films Figure 4 4 15 as described for CS however a high water content in the film decreased its sorption capacity Sorption of cumenol is
15. C After reaching equilibrium water sorption the films were immersed in carvacrol the necessary time to achieve equilibrium 2 11 Sorption of carvacrol The analysis of the concentration of carvacrol retained in the materials was performed by thermal desorption coupled to gas chromatography using a Dynatherm Thermal Desorber Model 890 891 Supelco Teknokroma Barcelona Spain connected in series to the column of an HP5890 gas chromatograph Series II Plus Agilent Technologies Barcelona Spain via a heated transfer line A cut piece of the film was cleaned with a paper tissue 181 Cap tulo 4 Art culo cient fico 4 to remove any excess of volatile compound on the film surface and then inserted into an empty desorption tube 11 5 cmx0 39 cm 1 D The tube was placed in the desorber chamber which was immediately sealed Conditions for desorption were as follows desorption temperature 210 C transfer line 230 C desorption time 7 min He desorption flow 8 15 mL min The GC was equipped with a TRB5 30 m 0 32 mm 0 25 um column Teknokroma Barcelona Spain and a flame ionization detector The chromatographic conditions were 260 C detector temperature 7 min at 45 C heating ramp to 220 C at 18 C min and 1 min more at 220 C After the analysis the film sample was recovered from the desorption tube and weighed on an analytical balance Voyager V11140 model Ohaus Europe Greifensee Switzerland 2 12 Data analys
16. En general la sorci n de monoterpenos fen licos gt 12 fue superior a la sorci n del resto de monoterpenos 1 4 por parte de las pel culas Esta mayor afinidad de los compuestos fen licos puede deberse a las interacciones de los dobles enlaces del anillo de benceno con los grupos polares de la matriz de CS que no se presentan en las estructuras con ciclohexano o ac clicas Los grupos alquilo en el anillo de fenol del carvacrol e isoeugenol pudieron aumentar la hidrofobicidad respecto a los cumeoles por lo que estos ltimos tendr an mayor afinidad por la matriz hidr fila de CS y justifica su mayor sorci n En el caso del isoeugenol la menor capacidad de retenci n de las pel culas puede deberse adem s al impedimento est rico producido por el grupo propileno Por su parte el guayacol aunque presente la mayor polaridad no produjo la mayor capacidad de sorci n de los vol tiles Esto probablemente puede atribuirse a la formaci n de enlaces de hidr geno intramoleculares en la mol cula de guayacol que podr an competir con la formaci n de los puentes de hidr geno entre la matriz y el sorbato disminuyendo la sorci n de ste De igual forma se probaron las pel culas de CS con CD con un 50 de G que tuvieron que ser acondicionadas al 65 de RH para que el contenido en humedad fuera en torno al 31 Un contenido similar de agua e igual de CD en las pel culas permiti determinar la influencia de la cantidad de G en el sis
17. Google Patents Makwana S Choudhary R Dogra N Kohli P Haddock J 2014 Nanoencapsulation and immobilization of cinnamaldehyde for developing antimicrobial food packaging material Lwt Food Science and Technology 57 470 476 Marin L Ailincai D Mares M Paslaru E Cristea M Nica V Simionescu B C 2015 Imino chitosan biopolymeric films Obtaining self assembling surface and antimicrobial properties Carbohydrate Polymers 117 762 770 Marin L Morariu S Popescu M C Nicolescu A Zgardan C Simionescu B C Barboiu M 2014 Out of water constitutional self organization of chitosan cinnamaldehyde dynagels Chemistry a European Journal 20 4814 4821 Marin L Simionescu B Barboiu M 2012 Imino chitosan biodynamers Chemical Communications 48 8778 8780 Marin L Stoica L Mares M Dinu V Simionescu B C Barboiu M 2013 Antifungal vanillin imino chitosan biodynameric films Journal of Materials Chemistry B 1 3353 3358 Mild R M Joens L A Friedman M Olsen C W McHugh T H Law B Ravishankar S 2011 Antimicrobial edible apple films inactivate antibiotic resistant and susceptible Campylobacter jejuni strains on chicken breast Journal of Food Science 76 M163 M168 132 Quitosano Cinamaldeh do Mohamed R R Fekry A 2011 Antimicrobial and anticorrosive activity of adsorbents based on chitosan Schiff s base Int J Electrochem Sci 6 2488 2508 Moo
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19. Inhibition of Penicillium commune by edible whey protein films incorporating lactoferrin lactoferrin hydrolysate and lactoperoxidase systems Journal of Food Science 70 M87 M94 Ming X T Weber G H Ayres J W Sandine W E 1997 Bacteriocins applied to food packaging materials to inhibit Listeria monocytogenes on meats Journal of Food Science 62 413 415 Mitrakas G E Koutsoumanis K P Lazarides H N 2008 Impact of edible coating with or without anti microbial agent on microbial growth during osmotic dehydration and refrigerated storage of a model plant material Innovative Food Science amp Emerging Technologies 9 550 555 Mohamed C Clementine K A Didier M Gerard L Noelle D C M 2013 Antimicrobial and physical properties of edible chitosan films enhanced by lactoperoxidase system Food Hydrocolloids 30 576 580 Moreira S P de Carvalho W M Alexandrino A C de Paula H C B Rodrigues M D P de Figueiredo R W Maia G A de Figueiredo E M A T Brasil I M 2014 Freshness retention of minimally processed melon using different packages and multilayered edible coating containing microencapsulated essential oil International Journal of Food Science and Technology 49 2192 2203 Murillo Mart nez M M Tello Sol s S R Garc a S nchez M A Ponce Alquicira E 2013 Antimicrobial activity and hydrophobicity of edible whey protein isolate films formulated with nisin and or glucose oxidase Journ
20. Jang S A Shin Y J Song K B 2011 Effect of rapeseed protein gelatin film containing grapefruit seed extract on Maehyang strawberry quality International Journal of Food Science and Technology 46 620 625 Jasour M S Ehsani A Mehryar L Naghibi S S 2014 Chitosan coating incorporated with the lactoperoxidase system an active edible coating for fish preservation Journal of the Science of Food and Agriculture n a n a Jiang T J Feng L F Zheng X L 2012 Effect of chitosan coating enriched with thyme oil on postharvest quality and shelf life of shiitake mushroom Lentinus edodes Journal of Agricultural and Food Chemistry 60 188 196 Jin T 2010 Inactivation of Listeria monocytogenes in skim milk and liquid egg white by antimicrobial bottle coating with polylactic acid and nisin Journal of Food Science 75 M83 M88 Jin T Gurtler J B 2011 Inactivation of Salmonella in liquid egg albumen by antimicrobial bottle coatings infused with allyl isothiocyanate nisin and zinc oxide nanoparticles Journal of Applied Microbiology 110 704 712 Jin T Liu L S Zhang H Hicks K 2009 Antimicrobial activity of nisin incorporated in pectin and polylactic acid composite films against Listeria monocytogenes International Journal of Food Science and Technology 44 322 329 318 Anexos Jin T Zhang H 2008 Biodegradable polylactic acid polymer with nisin for use in antimicrobial food packaging Journal of F
21. Koehler J A Brune B J Payne G F 1998 Selective adsorption of methoxyphenol positional isomers Industrial amp Engineering Chemistry Research 37 3685 3690 Goodship V Jacobs D 2009 Polyvinyl alcohol materials processing and applications Smithers Rapra Technology Higueras L L pez Carballo G Cerisuelo J P Gavara R Hern ndez Mu z P 2013 Preparation and characterization of chitosan HP p cyclodextrins composites with high sorption capacity for carvacrol Carbohydrate Polymers 97 262 268 Hildebrand J H Scott R L 1949 Solubility of non electrolytes Reinhold Publishing Corp New York Islam A Yasin T 2012 Controlled delivery of drug from pH sensitive chitosan poly vinyl alcohol blend Carbohydrate Polymers 88 1055 1060 Jiang X C Jiang T Gan L L Zhang X F Dai H Zhang X 2012 The plasticizing mechanism and effect of calcium chloride on starch poly vinyl alcohol films Carbohydrate Polymers 90 1677 1684 Kellner W Kober W 1954 Possibilities of the use of ethereal oils for room disinfection Arzneimittelforschung 4 319 325 Knauth P Sabbah R 1990 Energetics of intra and intermolecular bonds in c alkanediols Structural Chemistry 1 43 46 Kuhn J Considine T Singh H 2006 Interactions of milk proteins and volatile flavor compounds Implications in the development of protein foods Journal of Food Science 71 R72 R82 Kumar M N V R 2000 A revie
22. M E Malcata F X 2012 Evaluation of antimicrobial edible coatings from a whey protein isolate base to improve the shelf life of cheese Journal of Dairy Science 95 6282 6292 Ramos O L Santos A C Le o M V Pereira J O Silva S L Fernandes J C Franco MIL Pintado M E Malcata F X 2012 Antimicrobial activity of edible coatings prepared from whey protein isolate and formulated with various antimicrobial agents International Dairy Journal 25 132 141 Ramos O L Silva S L Soares J C Fernandes J C Pocas M F Pintado M E Malcata F X 2012 Features and performance of edible films obtained from whey protein isolate formulated with antimicrobial compounds Food Research International 45 351 361 Ravishankar S Jaroni D Zhu L B Olsen C McHugh T Friedman M 2012 Inactivation of Listeria monocytogenes on ham and bologna using pectin based apple carrot and hibiscus edible films containing carvacrol and cinnamaldehyde Journal of Food Science 77 M377 M382 Ravishankar S Zhu L B Olsen C W McHugh T H Friedman A 2009 Edible apple film wraps containing plant antimicrobials inactivate foodborne pathogens on meat and poultry products Journal of Food Science 74 M440 M445 Raybaudi Massilia R M Mosqueda Melgar J Mart n Belloso O 2008 Edible alginate based coating as carrier of antimicrobials to improve shelf life and safety of fresh cut melon International Journal of Food Mi
23. ME TVC mes filos y psicr filos bacterias sulfuro productoras LAB Enterobacteriaceae E coli O157 H7 ME TVC mes filos y psicr filos hongos levaduras E coli O157 H7 S Enteritidis ME TVC mes filos y psicr filos hongos levaduras A niger P digitatum Lb lactis Ps putida Streptococcus agalactiae E coli L monocytogenes B subtilis C albicans E coli O157 H7 S enterica L monocytogenes L monocytogenes St aureus S Enteriditis Ps aeruginosa E coli Lb sakei L monocytogenes Lb plantarum Ps fluorescens ME TVC mes filos y psicr filos P citrinum E coli St aureus A niger Botrytis cinerea Rhizopus stolonifer Rhizopus stolonifer Lb acidophilus E coli Ps fluorescens L innocua ME TVC Ps fluorescens Shewanella putrefaciens Photobacterium phosphoreum L innocua Gonz lez Ch fer et al 2011 Ch fer et al 2012 S nchez Gonz lez Ch fer et al 2010 S nchez Gonz lez Pastor et al 2011 Ahmad Benjakul Prodpran et al 2012 Ahmad Benjakul Sumpavapol et al 2012 Raybaudi Massilia Rojas Gra et al 2008 Rojas Gra et al 2006 Rojas Gra Avena Bustillos et al 2007 Raybaudi Massilia Mosqueda Melgar et al 2008 vila Sosa et al 2010 vila Sosa et al 2012 Bahram et al 2014 Du Olsen Avena Bustillos McHugh Levin y Friedman 2009 Hosseini et al 20
24. Ng P K W 2006 Preparation and characterization of chitosan based nanocomposite films with antimicrobial activity Journal of Agricultural and Food Chemistry 54 5814 5822 S nchez Valdes S Ortega Ortiz H Valle L F R D Medell n Rodr guez F J Guedea Miranda R 2009 Mechanical and antimicrobial properties of multilayer films with a polyethylene silver nanocomposite layer Journal of Applied Polymer Science 111 953 962 Sanpui P Murugadoss A Prasad P V D Ghosh S S Chattopadhyay A 2008 The antibacterial properties of a novel chitosan Ag nanoparticle composite International Journal of Food Microbiology 124 142 146 Sau T K Rogach A L 2010 Nonspherical noble metal nanoparticles colloid chemical synthesis and morphology control Advanced Materials 22 1781 1804 Sharma V K Yngard R A Lin Y 2009 Silver nanoparticles green synthesis and their antimicrobial activities Advances in Colloid and Interface Science 145 83 96 Sheikh F A Barakat N A M Kanjwal M A Chaudhari A A Jung I H Lee J H Kim H Y 2009 Electrospun antimicrobial polyurethane nanofibers containing silver nanoparticles for biotechnological applications Macromolecular Research 17 688 696 Tankhiwale R Bajpai S K 2010 Silver nanoparticle loaded chitosan lactate films with fair antibacterial properties Journal of Applied Polymer Science 115 1894 1900 Thomas V Yallapu M M Sreedhar B Bajpai S K
25. Referencias bibliogr ficas Appendini P Hotchkiss J H 2002 Review of antimicrobial food packaging nnovative Food Science amp Emerging Technologies 3 113 126 Catal R Gavara R 2001 Nuevos envases De la protecci n pasiva a la defensa activa de los alimentos envasados Arbor CLXVIII 661 109 127 Coma V 2008 Bioactive packaging technologies for extended shelf life of meat based products Meat Science 78 90 103 Fern ndez lvarez M 2000 Revisi n envasado activo de los alimentos Review active food packaging Food Science and Technology International 6 97 108 Han J H 2003 Antimicrobial food packaging in Ahvenainen R Ed Novel food packaging techniques 1 ed Woodhead Publishing Limites Cambridge pp 50 70 59 4 CAP TULOS Quitosano LAE 4 1 Cap tulo 1 Desarrollo de pel culas de quitosano con etil N dodecanoil L arginato y su aplicaci n en el envasado activo antimicrobiano de alimentos 4 1 1 Estado de la cuesti n El LAE es una novedosa mol cula empleada como aditivo conservante que ha sido aceptada por diferentes instituciones sanitarias oficiales El LAE es un tensioactivo que destaca por su elevada efectividad a bajas concentraciones y amplio espectro antimicrobiano Este compuesto es metabolizado por el organismo generando arginina y ornitina por lo que se considera un producto seguro que no presenta toxicidad en humanos El LAE es hidr filo estable y activo a tempe
26. Rezaei amp Farzi 2012 Altiok Altiok amp Tihminlioglu 2010 However load ing of volatiles in a chitosan matrix presents several difficulties Its incorporation as an additive into the water based chitosan film forming solution is challenging because of general limited aqueous solubility of volatiles and the inevitable partial loss of the com pound by evaporation during the casting and drying of the film In order to overcome these problems Presence of cyclodextrins in the chitosan matrix could improve compatibility between the polymer matrix and the agent Moreover the volatile could be loaded after film casting to avoid agent losses during the film drying step Cyclodextrins CDs are donut like oligosaccharides with hydrophobic cavities and hydrophilic outer surface They are widely used as excipients in pharmacy to solubilize lipophilic drugs Anexos journal homepage www elsevier com locate foodhyd Food Hydrocolloids 43 2015 603 611 Contents lists available at ScienceDirect 3 Food Hydrocolloids Food Hydrocolloids IM Incorporation of hydroxypropyl P cyclodextrins into chitosan films to tailor loading capacity for active aroma compound carvacrol Q CrossMark Laura Higueras Gracia L pez Carballo Rafael Gavara Pilar Hernandez Munoz Instituto de Agroqu mica y Tecnolog a de Alimentos CSIC Avenida Agust n Escardino 7 46980 Paterna Valencia Spain ARTICLE INFO Article history Received 3 M
27. Rinaudo 2006 CS and PVOH can be modified to improve the drug loading capacity of the carrier and to modulate the release kinetics of entrapped compounds These polymers have been blended with other natural or synthetic polymers of various polarities and molecular weights nanofibres and nanoclays mixed with plasticizers and crosslinked in order to control the kinetics of the release Islam and Yasin 2012 Jiang et al 2012 Kumar 2000 Lavorgna et al 2010 Muhd Julkapli et al 2011 Rahman et al 2010 Yu et al 2011 Zuber et al 2013 In the design of polymer systems capable of retaining and releasing antimicrobial volatile organic compounds it is necessary that a minimal amount of volatile be entrapped in the film to provide it with antimicrobial properties The major drawback in the development of these systems is that a high percentage of the active agent is evaporated or inactivated during film processing The alternative method of absorption for loading the volatile into the formed film has low efficacy This is currently due to the fact that most organic volatile compounds are hydrophobic and thus have low compatibility with hydrophilic films Balaguer et aL 2012 Kurek et al 2012 recently studied how the composition of the film forming solution and process parameters affect the retention of volatile liquid carvacrol They found that G and gum arabic were the most effective additives to improve retention of carvacrol whereas t
28. S Copinet A Salin F Deschamps A 2002 Edible antimicrobial films based on chitosan matrix Journal of Food Science 67 1162 1169 161 Cap tulo 3 Art culo cient fico 3 Damodaran S Kinsella J E 1980 Flavor protein interactions Binding of carbonyls to bovine serum albumin Thermodynamic and conformational effects Journal of Agricultural and Food Chemistry 28 567 571 Dos Santos J E Dockal E R Cavalheiro E T G 2005 Synthesis and characterization of Schiff bases from chitosan and salicylaldehyde derivatives Carbohydrate Polymers 60 277 282 Doyle M P Beuchat L R 2007 Food microbiology fundamentals and frontiers ASM Press Washington D C Fleming D W Cochi S L MacDonald K L Brondum J Hayes P S Plikaytis B D Holmes M B Audurier A Broome C V Reingold A L 1985 Pasteurized milk as a vehicle of infection in an outbreak of listeriosis New England Journal of Medicine 312 404 407 Foster L J R Butt J 2011 Chitosan films are NOT antimicrobial Biotechnology Letters 33 417 421 G llstedt M Hedenqvist M S 2006 Packaging related mechanical and barrier properties of pulp fiber chitosan sheets Carbohydrate Polymers 63 46 53 Gill A O Holley R A 2004 Mechanisms of bactericidal action of cinnamaldehyde against Listeria monocytogenes and of eugenol against L monocytogenes and Lactobacillus sakei Applied and Environmental Microbiology 70 5750 5755
29. Su preparaci n y aplicaci n est n descritas como patente espa ola Garc a Dom nguez et al 1983 y como patente europea Contijoch Mestres et al 2001 Urgell Beltr n y Seguer Bonaventura 2003a b c El LAE est siendo empleado como un innovador conservante en alimentaci n productos farmac uticos y cosm ticos Bakal y D az 2005 Infante et al 1997 Su baja toxicidad alta actividad antimicrobiana y el hecho de no modificar las caracter sticas organol pticas del producto original convierten al LAE en una valiosa herramienta para controlar o prevenir el crecimiento microbiano en los alimentos El empleo de LAE como conservante de alimentos es de hasta 225 mg kg El LAE ha sido declarado sustancia GRAS para su uso como agente antimicrobiano en m s de 20 alimentos incluyendo aves de corral entre otras carnes FDA 20053 La EFSA 2007 ha establecido para el LAE una IDA 0 0 5 mg kg de peso corporal M s recientemente el Comit mixto FAO OMS 2009 evalu el LAE INS n 243 estableciendo una IDA de 0 4 mg kg peso corporal Las posibles aplicaciones de LAE son semiconservas de carne y pescado embutidos bebidas no alcoh licas zumos quesos legumbres y cereales procesados platos preparados aperitivos postres l cteos salsas y condimentos rellenos y coberturas etc En el Anexo A se recogen algunos ejemplos de bioenvases antimicrobianos con LAE estudiados en alimentos 1 5 2 Nanopart culas de plata El car
30. Table 4 4 13 Minimum amount of volatile liquid required to produce growth inhibition in vapour phase against S aureus and E coli Compound mg m cumenol 1 4 o cumenol 1 4 carvacrol 1 0 guaiacol 22 0 isoeugenol 2 6 myrtenol 312 nerol 24 carveol 24 dihydrocarveol 2 0 isopulegol 8 0 compounds with a similar structure but with a cyclohexanol rather than phenol group such as menthol which has a very weak antimicrobial activity Ben Arfa et aL 2006 The presence of acetate carvacryl acetate or methyl ether carvacrol methyl ether groups instead of the hydroxyl group in carvacrol makes the molecule a proton acceptor but incapacitates the release of a proton from these groups so they are inefficient as antimicrobials Like carvacrol cumenol isomers contain a hydroxyl group and a system of delocalized electrons in the benzene ring possessing a strong antimicrobial activity Compared with carvacrol the absence of the methyl group in cumenol isomers decreased its antimicrobial activity A decrease in the hydrophobicity of the molecule resulting from the removal of aliphatic substituents may affect its interaction with protein and lipid components of the bacterial membrane Log P of cumenols is considerably lower than that of carvacrol 2 82 vs 3 28 In spite of its high vapour pressure and the presence of the phenol group the very low antimicrobial activity of guaiacol can be attributed to its lower hydrophobicity compared to cumenol
31. and films incorporating 2096 G and conditioned at 7596 RH the sorption of carvacrol in these films was 216 3 22 1 133 3 16 9 and 56 8 3 5 g carvacrol g dry film respectively In spite of having lower water content Figure 4 4 4 films incorporating 3596 G and conditioned at 5396 RH absorbed a greater amount of carvacrol than films incorporating 2096 G and conditioned at 7596 RH When G was not incorporated in the CS CD matrix sorption of carvacrol did not exceed 0 4596 g carvacrol g dry film regardless of the RH at which they were conditioned whereas the maximum amount of carvacrol sorbed by films stored under dry conditions was 4 596 corresponding to films plasticized with 3596 G These observations highlight the fact that G plays a crucial role in the sorption of carvacrol Thus the use of HP BCD together with G and moisture is required to control the loading of carvacrol in a CS matrix It is worth pointing out that previous studies Higueras et al 2013 showed that CS films without HP BCD did not 207 Cap tulo 4 Art culo cient fico 5 retain more than 196 of carvacrol whatever the amount of G and water incorporated in the matrix Incorporation of HP BCD in the CS matrix slightly increased the sorption of carvacrol compared with plain CS films and the use of water and G as coadjuvants was necessary to control the loading of the films with carvacrol Kurek et al 2014 studied the effect of various additives on the retent
32. attractive applications in several industrial areas Chitosan produces highly transparent grease resistant films with excel lent barrier properties to gases and aroma compounds These properties make chitosan a good candidate for use in food packaging applications providing a barrier layer to other polymer films and porous materials such as fibre based paper Gallstedt and Hedenqvist 2006 In addition amino and hydroxyl groups of chitosan can be used to modify the poly mer chemically in order to provide it with new properties and widen its technological applications Moreover chitosan has been extensively studied as a carrier and system for sustained release of active compounds and in this regard it has been applied in a great variety of technological areas such as agrochemistry pharmacy biomedicine textiles and active food packaging Sashiwa and Aiba 2004 The development of antimicrobial materials and their application in the design ofactive packaging is arousing considerable expectation in the food industry since food safety is an area of great concem Regarding chitosan films many studies in the literature have endeavoured to use the antimicrobial properties of protonated chitosan polymer to form films for active food packaging applications However protonated films are water soluble which limits their applications whereas neutralized films lack a Springer 335 Anexos Carbohydrate Polymers 97 2013 262 268 Co
33. culas formuladas despu s de su inmersi n en carvacrol frente a Escherichia coli y Staphylococcus aureus as como su capacidad antibacteriana en el tiempo en funci n del carvacrol remanente en la pel cula Por otro lado se evaluaron las cin ticas de sorci n y desorci n de las pel culas que retuvieron un mayor contenido en carvacrol As como el efecto de la cantidad y tipo la dextrina incorporada a la formulaci n en la capacidad de sorci n de carvacrol Todo ello se recoge en el art culo cient fico Incorporation of hydroxypropyl B cyclodextrins into chitosan films to tailor loading capacity for active aroma compound carvacrol Antimicrobial packaging of chicken fillets based on the release of carvacrol from chitosan cyclodextrin films constituye el tercer art culo de este cap tulo En l la formulaci n que present una mayor sorci n de carvacrol se incluy en un sistema de envasado de filetes de pollo fresco para el control del crecimiento microbiano Para ello los filetes se envasaron en barquetas de polipropileno copol mero de etileno y alcohol vin lico PP EVOH PP selladas con una tapa de aluminio autoadhesivo Diferentes tamafios de la pel cula desarrolladas se adhirieron en la cara interna de la tapa y se almacenaron a 4 C durante 9 d as Se estudi la cantidad de carvacrol presente en la bandeja tapa espacio de cabeza y alimento as como el carvacrol remanente en la pel cula Se evalu la capacidad antim
34. do Por ltimo se desarrollaron pel culas de CS introduciendo en su formulaci n CD y plastificantes con el objetivo de incrementar su capacidad para retener carvacrol La incorporaci n de carvacrol se realiz una vez obtenida la pel cula mediante su inmersi n en el l quido vol til evitando de esta forma las p rdidas de agente durante el secado Seguidamente se estudi la capacidad para liberar el compuesto retenido y el efecto de la RH ambiental en la cin tica de liberaci n Previamente a estos estudios de sorci n y liberaci n se estudi el efecto de incorporar CD en las propiedades funcionales de las pel culas de CS Este estudio se extendi a otros monoterpenos fen licos y no fen licos con el objetivo de estudiar el efecto de la estructura qu mica del compuesto en la capacidad de sorci n La incorporaci n de CD en la matriz de CS redujo la elongaci n a la rotura de las pel culas mientras que su resistencia a la rotura no se vio afectada Por otro lado la permeabilidad al vapor de agua disminuy y la permeabilidad al ox geno experiment un incremento En el estudio morfol gico mediante SEM no se observ separaci n de fases pero el TGA s mostr dos temperaturas de degradaci n correspondientes al CS y a las 281 Discusi n general CD ambas temperaturas sufrieron una alteraci n en varios grados respecto a las correspondientes a los materiales originales revelando cierta interacci n entre ambos Por
35. do Las pel culas iminoderivadas aumentaron la seguridad microbiol gica de la leche La leche con el cinamaldeh do liberado fue aceptada sensorialmente El ltimo m todo empleado se bas en la incorporaci n de monoterpenos antimicrobianos principalmente carvacrol en pel culas de quitosano con coadyuvantes mediante la inmersi n de las pel culas preformadas en el agente vol til en estado l quido La presencia conjunta de hidroxipropil PB ciclodextrinas glicerol y agua en las pel culas de quitosano dio lugar a una elevada capacidad de sorci n de carvacrol de car cter lip filo La liberaci n del carvacrol presente en las pel culas compuestas fue activada por la humedad relativa ambiental Este carvacrol liberado de las pel culas present efectividad antimicrobiana en fase vapor cuando fue incorporado en un sistema de envasado para pechugas de pollo fresco La capacidad de retenci n de las pel culas se pudo modular en funci n de su formulaci n y de la estructura qu mica del monoterpeno sorbido Las pel culas con dichos monoterpenos presentaron capacidad antimicrobiana en fase vapor in vitro y pueden emplearse en el dise o de envases activos adaptados al alimento aplicado CSIC CONSEJO SUPERIOR DE INVESTIGACIONES CIENT FICAS VNIVERSITAT gt D VALENCIA E
36. envases Sin embargo se prev que el mercado de envases activos e inteligentes en los EEUU supere el 896 anual del mercado total de envases y los 3 5 millones de en 2017 Freedonia 2014 Una forma de envases activos que est n siendo objeto de diversos estudios a nivel tecnol gico y suscitando gran inter s industrial son los envases activos antimicrobianos los cuales est n destinados a controlar el crecimiento microbiano en el alimento envasado En el Anexo A se recogen algunos ejemplos de compuestos antimicrobianos incorporados en diversas matrices polim ricas aplicadas en el envasado activo de alimentos 1 1 1 Envasado activo antimicrobiano Las infecciones e intoxicaciones alimentarias son enfermedades ocasionadas por la ingesta de alimentos en mal estado debido a la presencia de microorganismos pat genos o sus toxinas La presencia de estos microorganismos en los alimentos no suelen ser detectables en apariencia pero pueden producir trastornos gastrointestinales graves En 2012 en Europa se notificaron 5363 brotes de origen alimentario siendo Espafia el quinto pa s que m s brotes comunic afectando a 55453 personas resultando 5118 hospitalizaciones y 41 muertes EFSA 2014 Adem s del problema de salud p blica que pueden suponer algunos microorganismos al desarrollarse en los alimentos es importante el control del crecimiento microbiano ya que es una de las principales causas del deterioro de los alimentos lo que supo
37. mandarins Lwt Food Science and Technology 44 2342 2348 Valencia Chamorro S A P rez Gago M B Del R o M A Palou L 2009a Curative and preventive activity of hydroxypropyl methylcellulose lipid edible composite coatings containing antifungal food additives to control citrus postharvest green and blue molds Journal of Agricultural and Food Chemistry 57 2770 2777 Valencia Chamorro S A P rez Gago M B Del R o M A Palou L 2009b Effect of antifungal hydroxypropyl methylcellulose HPMC lipid edible composite coatings on postharvest decay development and quality attributes of cold stored Valencia oranges Postharvest Biology and Technology 54 72 79 Valencia Chamorro S A P rez Gago M B Del R o M A Palou L 2010 Effect of antifungal hydroxypropyl methylcellulose lipid edible composite coatings on Penicillium decay development and postharvest quality of cold stored Ortanique mandarins Journal of Food Science 75 S418 S426 Vasconez M B Flores S K Campos C A Alvarado J Gerschenson L N 2009 Antimicrobial activity and physical properties of chitosan tapioca starch based edible films and coatings Food Research International 42 762 769 Vimala K Mohan Y M Sivudu K S Varaprasad K Ravindra S Reddy N N Padma Y Sreedhar B MohanaRaju K 2010 Fabrication of porous chitosan films impregnated with silver nanoparticles a facile approach for superior antibacterial applica
38. n y evaporaci n del disolvente que fueron secadas a 60 C y neutralizadas con hidr xido de sodio Sin embargo la etapa de formaci n de nanopart culas de plata se realiza a elevada temperatura y en disoluci n mientras que en nuestro caso la m xima temperatura empleada es de 40 C y las nanopart culas de plata se forman principalmente durante la neutralizaci n de la pel cula ya conformada etapa necesaria para la obtenci n de pel culas de CS insolubles Un trabajo similar es el de Tankhiwale y Bajpai 2010 en el que sumergen la pel cula de CS en la soluci n de nitrato de plata y posteriormente en una soluci n de citrato tris dico y en el que las nanopart culas de plata se formaron en la propia pel cula Otro similar es el de Li et al 2010 aunque en este caso se incluy xido de zinc en la composici n y la sonicaci n como etapa adicional necesaria para la obtenci n del nanocompuesto En la presente Tesis Doctoral las nanopart culas de plata se han generado in situ en pel culas de CS incluyendo nitrato de plata como 94 Quitosano Nanopart culas de plata precursor en la formulaci n de las pel culas Las nanopart culas se generaron durante la neutralizaci n de las pel culas con hidr xido de sodio Adem s el CS es un agente reductor d bil en condiciones alcalinas lo que refuerza la acci n reductora del hidr xido Esa metodolog a constituye una alternativa a las empleadas hasta ahora donde com nmente
39. nas o polisac ridos Los polioles glicerol G etilenglicol propilenglicol PG dietilenglicol y trietilenglicol se han estudiado como plastificantes para estos pol meros biodegradables Rahman y Brazel 2004 Vieira et aL 2011 Entre los plastificantes m s empleados destaca el G con la ventaja de ser considerado como GRAS por la FDA y aditivo alimentario con INS n 422 por la GSFA Existen numerosos estudios que lo consideran un excelente plastificante de pel culas hidr filas dada su gran capacidad para retener agua que a su vez tambi n act a de plastificante universal y ubicuo Los plastificantes en general y el G en particular mejoran la extensibilidad de las pel culas y la capacidad de tomar agua sin embargo reducen su resistencia mec nica elasticidad y propiedades barrera al vapor de agua ox geno aromas y grasas aceites y migra a menudo a partir de las pel culas con alta sensibilidad frente a la RH Gontard et al 1993 Rahman y Brazel 2004 Srinivasa et al 2007 Hay autores que apuntan que otro mecanismo de acci n de los plastificantes es facilitar la incorporaci n de agua y que es sta la que ejerce la acci n Karbowiak et aL 2006 Sin embargo su acci n es a corto plazo puesto que el agua se evapora con facilidad Sothornvit y Krochta 2005 Igualmente el PG es bastante empleado como agente plastificante Smits et al 2003 Suyatma et al 2005 Vanin et al 2005 considerado GRAS y aditivo alimentario
40. nisina diacetato de sodio nisina lactato de sodio nisina sorbato de potasio nisina nisina nisina nisina nisina EDTA nisina ED semilla de uva nisina ED semilla de uva EDTA nisina EDTA nisina c c trico nisina c l ctico nisina c m lico nisina c tart rico 302 celulosa bacteriana galactomanano gelatina gelatina gelatina ze na glucomanano de konjac quitosano gluten de trigo HPMC HPMC HPMC metilcelulosa HPMC metilcelulosa HPMC quitosano metilcelulosa LDPE pectina PLA PLA PLA prote na de soja prote nas de soja loncheados salchichas de Frankfurt queso ricota mortadela de pavo jam n cocido mortadela medio de cultivo medio de cultivo mortadela de pavo medio de cultivo medio de cultivo salchichas de Frankfurt salm n ahumado leche entera salchicha de Frankfurt medio de cultivo yema de huevo zumo de naranja medio de cultivo zumo de naranja clara de huevo cruda leche desnatada medio de cultivo medio de cultivo medio de cultivo L monocytogenes ME vo L monocytogenes L monocytogenes Brochothrix thermosphacta E coli O157 H7 Lb sakei Leuconostoc mesenteroides L monocytogenes S Typhimurium L monocytogenes E coli St aureus L monocytogenes B cereus L monocytogenes L innocua St aureus L monocytogenes L innocua St aureus L monocytogenes L m
41. one comprising spherical nanoparticles of 5 10 nm in size and a second population of round shaped anisotropic nanoparticles with diameters ranging from 30 to 50 nm Films neutralized at 37 C incorporating different amounts of silver ranging from 0 2 to 1 596 released similar amounts of total silver to the culture medium after 18 h of immersion and 122 Quitosano Nanopart culas de plata showed similar antimicrobial activity The release of silver to the culture medium from films containing 0 2 silver neutralized at 37 C was monitored for 360 h and showed an increase in the release of silver after the first 10 h of contact However the antimicrobial activity did not change This could be explained by conversion of ionic silver to elemental silver or interaction with the proteins present in the culture medium It has been shown that CS is capable of acting as a carrier of silver nanoparticles allowing slow extended release of silver ions in a liquid medium for 30 days and maintaining their antimicrobial activity Moreover films with a higher silver concentration can exert their antimicrobial activity for longer as they have a larger reservoir of silver ions in the form of silver based nanoparticles The developed films could be used in several fields such as medicine pharmacy and food packaging when a long term antimicrobial effect is desired Author information Corresponding Author E mail phernan iata csic es Phone 34 96390
42. quidos vol tiles naturales con propiedades antimicrobianas hacen referencia a la cantidad de compuesto que se incorpora inicialmente en la disoluci n film gena y no a la cantidad real remanente en las pel culas ya elaboradas y secas y la concentraci n alcanzada en el espacio de cabeza del envase Algunos autores han evaluado la acci n antimicrobiana de diversos AE y sus principales constituyentes en fase vapor estableciendo las concentraciones m nimas inhibitorias en el espacio de cabeza contenido en el recipiente de ensayo Tyagi et al 2012 Otros grupos de investigaci n han estudiado minuciosamente la cin tica y los par metros de equilibrio del carvacrol en filetes de salm n envasado en bandejas selladas t rmicamente con una tapa de PP EVOH PP con carvacrol en su n cleo proponiendo un modelo matem tico para describir y simular el funcionamiento del sistema de envasado y predecir su comportamiento bajo diferentes condiciones Cerisuelo et aL 2013 Por otra parte Guti rrez et al 2010 demuestran in vitro que el comportamiento cin tico de los AE y sus componentes es de gran relevancia en las propiedades antimicrobianas del envase activo Sin embargo no se han encontrado trabajos en los que se estudie en profundidad el reparto de la cantidad de vol til a adido inicialmente en cada uno de los componentes del sistema de envasado que protege al alimento S lo estudios parciales como el de Chi et al 2006 en el cual determinaro
43. rnicos Guo Jin Wang et al 2014 Guo Jin y Yang 2014 Otero et al 2014 desarrollaron una pel cula de politereftalato de etileno recubierta con LAE y demostraron su capacidad para inhibir el crecimiento de Escherichia coli O157 H7 en queso En un trabajo posterior de Muriel Galet et al 2015 aplicaron pel culas de EVOH con LAE en caldo de pollo y derivados de pescado demostrando su eficacia frente a la carga total microbiana y bacterias pat genas inoculadas previamente Adem s existen algunas patentes que incluyen el LAE como agente antimicrobiano en matrices extruidas para su aplicaci n en productos alimenticios como carne roja fresca Ebner 2012 Sin embargo debido a la escasez de trabajos existentes en relaci n a la aplicaci n de LAE en pel culas de CS uno de los objetivos de esta Tesis Doctoral fue estudiar el comportamiento de este biopol mero como matriz portadora de dicho agente activo Cabe decir que el empleo de pel culas de CS incorporando el tensioactivo LAE ambos de car cter cati nico evita interacciones electrost ticas acomplejaci n posible formaci n de precipitados en la soluci n formadora de pel cula adem s de la p rdida de actividad antimicrobiana de la pel cula resultante debido a la incapacidad de liberar el LAE aunque estas hip tesis deben de ser demostradas En este sentido se han realizado diversos estudios con polisac ridos ani nicos y LAE en soluci n acuosa observ ndose una p
44. tartare Journal of Food Protection 70 2127 2132 Voidarou C Vassos D Rozos G Alexopoulos A Plessas S Tsinas A Skoufou M Stavropoulou E Bezirtzoglou E 2011 Microbial challenges of poultry meat production Anaerobe 17 341 343 Wehr H M 1982 Attitudes and policies of governmental agencies on microbial criteria for foods An update Food technology 36 45 54 Zi Xuan L Zhong Su M Jing W Huan L 2012 Preparation and characterization of immobilized lysozyme and evaluation of its application in edible coatings Process Biochemistry 47 201 208 242 4 4 5 Art culo cient fico 7 Effect of HP B cyclodextrins and coadjuvants on the sorption capacity of hydrophilic polymer films for naturally occurring antimicrobial monoterpene alcohols Laura Higueras Gracia L pez Carballo Rafael Gavara Pilar Hern ndez Mufioz Submitted to Carbohydrate Polymers Quitosano Carvacrol Resumen Las pel culas de quitosano CS con hidroxipropil B ciclodextrinas HP BCD en proporci n 1 1 en peso y plastificadas con glicerol G o polietilenglicol PG fueron preparadas mediante extensi n y evaporaci n del solvente y acondicionadas a diferentes humedades relativas RH para alcanzar un contenido de agua similar Se estudiaron las propiedades de sorci n de las pel culas de diversos monoterpenos con estructura fen lica o no fen lica lineal o ciclohexilalcohol despu s de su inmersi n en los l qui
45. y levaduras y hongos y monitoriz durante el almacenamiento Se observ una inhibici n microbiana general aumentando con el tama o del dispositivo activo La inhibici n con un dispositivo de 24 cm oscil desde 0 3 reducciones logar tmicas frente a bacterias de cido l cticas a las 1 8 logs frente a levaduras y hongos Sin embargo la gran cantidad de antimicrobiano que fue absorbido o reaccionado con el filete provoc un deterioro sensorial inaceptable Estos altos valores de sorci n se deben probablemente a la gran compatibilidad qu mica entre las prote nas de pollo y carvacrol Palabras clave Quitosano hidroxipropil B ciclodextrinas carvacrol envasado activo antimicrobiano liberaci n controlada pollo 225 Cap tulo 4 Art culo cient fico 6 Abstract Chitosan cyclodextrin films CS CD incorporating carvacrol were obtained by casting and conditioned at 23 C and 75 relative humidity prior to being immersed in liquid carvacrol until they reached sorption equilibrium In a previous work the in vitro antimicrobial activity of these films was studied In this work active films were used to inhibit microbial growth in packaged chicken breast fillets Samples of CS CD films loaded with carvacrol of different sizes and thus with different quantities of antimicrobial agent were stuck to the aluminium lid used to seal PP EVOH PP cups containing 25 g of chicken fillets These samples were stored for 9 days at 4 C
46. 0005 g of LAE previously measured in weight and thickness 55 5 um were immersed in 10 mL of MilliQ water and conditioned at 28 C or 4 C The amount of LAE released from film was determined by using an Agilent 1100 HPLC equipped with a UV detector 204 16 nm using a Zorbax Eclipse XDB C18 column 4 6 x 150 mm 5 um Agilent Barcelona Spain a mobile phase of acetonitrile water acidified with TFA 0 1 50 50 at 1 0 mL min and 10 pL injection volume All samples were conditioned at 28 1 C before injection 24 Strains The microorganisms tested were Escherichia coli CECT Spanish Type Culture Collection Valencia 434 Staphylococcus aureus MIM Microbiologia Industriale Milano 178 Listeria monocytogenes CECT 934 Salmonella enterica CECT 4300 and Pseudomonas putida ATCC American Type Culture Collection 12633 Candida utilis CCY Czeck Collection of Yeast 29 38 1 Saccharomyces cerevisiae var ellipsoideus NCYC National Collection of Yeats Cultures 2959 and Torulopsis pinus IMAP Istituto Microbiologia Agraria Perugia 4543 Aspergillus niger MIM 28 Penicillium chrysogenum MIM 29 and Cladosporium cladosporioides MIM 259 Tryptic soy agar TSA and malt extract agar MEA were employed as solid media and peptone water PW 0 196 tryptone soy broth TSB and malt extract broth MEB as liquid media All media were supplied by Scharlab Barcelona Spain Bacteria strains were stored in TSB with 2096 glycerol at 80 C until
47. 004 58404 59404 929403 zo 9560 9 035 006 59404 59404 934 04 919 06 3 76 0 07 247 09 244412 987 0 4 m immersion in carvacrol 7596 RH 96 8 04 0 23 0 05 5 7 0 5 5720 5 925045 93 6 0 8 303 023 196209 198206 99 2 0 5 35 913 08 4 51 0 14 272 06 27 5 0 5 99 5 0 5 Different letters in the same column indicated significant differences P lt 0 05 when comparing films incorporating different content of G and conditioned at the same RH Different letters in the same column indicated significant differences P lt 0 05 when comparing films conditioned at different RH prior immersion in carvacrol and incorporating the same amount of G did not significantly P 0 05 modify the colour parameters of the films when compared with those prepared without G Moreover there were no significant differences in colour parameters P gt 0 05 of G plasticized films conditioned at different RH data not shown After immersion in carvacrol films plasticized with G at 20 or 35 and conditioned at 75 RH and films plasticized with 35 G and conditioned at 53 RH acquired a vivid yellow green colour increasing their chroma and hue and slightly decreasing their lightness These changes are related to the amount of carvacrol that the films are capable of retaining Figure 4 4 4 shows the sorption equilibrium of carvacrol in films incorporating different percentages of G and conditioned a
48. 2010 Congreso internacional p ster G L pez Carballo L Higueras V Muriel Galet R Gavara P Hern ndez Mufioz Long lasting antimicrobial properties of biological plastics based on silver nanotechnology 18 IAPRI World Packaging Conference Book of Abstracts 408 414 California USA 17 21 de junio de 2012 332 Anexos International Journal of Food Microbiology 165 2013 339 345 Contents lists available at SciVerse ScienceDirect International Journal of Food Microbiology journal homepage www elsevier com locate ijfoodmicro Development of a novel antimicrobial film based on chitosan with LAE ethyl N dodecanoy l 1 arginate and its application to fresh chicken Me Laura Higueras Gracia L pez Carballo Pilar Hern ndez Mu oz Rafael Gavara Manuela Rollini gt Institute of Agrochemistry and Food Technology CSIC Avda Agust n Escardino 7 46980 Paterna Valencia Spain DEFENS Department of Food Environmental and Nutritional Sciences Universit degli Studi di Milano Via G Celoria 2 20133 Milano Italy ARTICLE INFO ABSTRACT Chitosan CS films incorporating the antimicrobial compound ethyl N dodecanoyl 1 arginate LAE were developed for food packaging applications Cast chitosan films were made with 1 5 or 10 LAE and 20 glycerol in the film forming solution Optical properties release of LAE and antimicrobial activity of developed Article history Received 21 February
49. 2013 http water epa gov drink contaminants secondarystandards cfm EuropeanBioplastics 2013 Bioplastics market growth above average between 2012 and 2017 http en european bioplastics org wp content uploads 2013 12 EuBP market data 2012 pdf FAO 2012 P rdidas y desperdicio de alimentos en el mundo Alcance causas y prevenci n FAO 2014 Appropriate food packaging solutions for developing countries FAO OMS 1995 Evaluation of certain food additives and contaminants 44th report of the Joint FAO WHO Expert Committee on Food Additives WHO Technical Report Series nr 859 G nova P g 54 FAO OMS 2001a Evaluation of certain food additives and contaminants 55th report of the Joint FAO WHO Expert Committee on Food Additives WHO Technical Report Series nr 901 G nova P g 26 FAO OMS 2001b Evaluation of certain food additives and contaminants 55th report of the Joint FAO WHO Expert Committee on Food Additives WHO Technical Report Series nr 901 G nova P g 48 FAO OMS 2004 Evaluation of certain food additives and contaminants 61th report of the Joint FAO WHO Expert Committee on Food Additives WHO Technical Report Series nr 922 G nova P g 35 FAO OMS 2009 Evaluation of certain food additives and contaminants 69th report of the Joint FAO WHO Expert Committee on Food Additives WHO Technical Report Series nr 952 G nova P g 27 FDA 2001 FDA agency response letter GRAS Notice nr GRN 000074 W
50. 2013 Received in revised form 6 June 2013 pois s ss du 2013 films was determined The minimum inhibitory concentration MIC and the minimum biocide concentration MBC of LAE were determined CS films with LAE were transparent and uniform without discontinuities or Keywords visible particles and no visual differences could be perceived between CS and CS LAE films When in contact Chitosan films with an aqueous food simulant the agent was fully released following a Fickian behavior in a few hours at LAE 4 and 28 C Antimicrobial activity of films against mesophiles psychrophiles Pseudomonas spp colifoms Antimicrobial packaging lactic acid bacteria hydrogen sulfide producing bacteria yeast and fungi was evaluated at two six and Active packaging eight days for its application on chicken breast fillets Films were active against bacteria yeasts and fungi in Antimicrobial release liquid and solid media CS films evidenced antimicrobial activity in the range 0 47 2 96 log reductions Poultry packaging while CS 5 LAE film produced 1 78 5 81 log reduction Results highlighted that LAE incorporation in a chitosan based packaging structure may provide a relevant antimicrobial activity that could improve the stability of fresh poultry products 2013 Published by Elsevier B V 1 Introduction prevention of surface contamination of foods Marcos et al 2007 The incidence of foodborne diseases associated with microbial pathogens is widesprea
51. 3 2 g o 1 2 24 o ll 0 E Figure 4 1 2 Growth reduction of indigenous spoilage microbiota in chicken breast fillets at 2 6 and 8 d of storage and 4 C in absence and presence of CS and CS 5 LAE films 86 Quitosano LAE References Asker D Weiss J McClements D J 2011 Formation and stabilization of antimicrobial delivery systems based on electrostatic complexes of cationic non ionic mixed micelles and anionic polysaccharides Journal of Agricultural and Food Chemistry 59 1041 1049 Aymerich T Picouet P A Monfort J M 2008 Decontamination technologies for meat products Meat Science 78 114 129 Bodart M De Penaranda R Deneyer A Flamant G 2008 Photometry and colorimetry characterisation of materials in daylighting evaluation tools Building and Environment 43 2046 2058 Bonnaud M Weiss J McClements D J 2010 Interaction of a food grade cationic surfactant lauric arginate with food grade biopolymers pectin carrageenan xanthan alginate dextran and chitosan Journal of Agricultural and Food Chemistry 58 9770 9777 Brandt A L Castillo A Harris K B Keeton J T Hardin M D Taylor T M 2010 Inhibition of Listeria monocytogenes by food antimicrobials applied singly and in combination Journal of Food Science 75 M557 M563 Cagri A Ustunol Z Ryser E T 2004 Antimicrobial edible films and coatings Journal of Food Protection 67 833 848 Canillac N Mou
52. 50 1 14 16 11 1 22 74 15 0 76f 25 39 237 10 43 01 1 28 7 50 0 09 21 00 0 85 22 30 0 89 70 35 1 14 53 73 131 15 4143 197 10 31 1 72 26 00 1 73f 27 97 1 98f 68 38 0 58 57 17 142 Control 00 94 27 0 82 0 61 0 05 7102071 7 13 0 71 94 91 0 60 0 1 86 06 0 36 0 13 0 15 8212081 8 21 0 81 89 08 1 04 8 32 0 43 0 2 64 06 0 83 2 75 0 83 15 90 0 47 16 14 0 49 80 19 1 35 3165 0 91 Films neutralized at 37 C 0 5 55 37 192 4 81 0 55 14 00 0 695 14 80 0 71 71 04 2 02 39 88 1 85 10 3132 092 2 00 0 53 603 048 6 35 0 51 71 64 1 99f 63 02 0 91 1 5 26 66 0 74f 1 00 0 12f 4 18 0 31f 429 030 76 53 2 01 67 69 0 73 af Values within a column followed by a different lower case letter are significantly different from each other comparing different amounts of silver in the films neutralized at 22 C or 37 C Tukey s adjusted analysis of variance P lt 0 05 112 Quitosano Nanopart culas de plata dm dT b 5 5 b j A MA o Control 01 0 8 1 02 f 05 gt 10 y 1 5 mun ado Cans cuni RC lee IM enl 1 0 i r 200 400 600 T CC Figure 4 2 3 Effect of silver content in 37 C neutralized CS films on the first derivative of weight loss DTG curves dm dT first derivative of weight loss vs time shifted to a higher temperature indicating that silver b
53. 52 17 0 17 Different letters in the same column indicate a statistically significant difference P lt 0 05 different treatments the DS values experienced a reduction which was more acute for films treated at greater temperatures It is noteworthy that even after a retorting like process more than 5096 of bonded cinnamaldehyde remained in the film implying a large reservoir of cinnamaldehyde 3 1 2 Optical properties Table 4 3 4 shows the film colour coordinates L a and b and the chroma Cap and hue ha of CS and CScin films and also CScin films subjected to different temperature time treatments The formation of an unsaturated Schiff base system C C C N from aj unsaturated cinnamaldehyde caused the films to acquire a vivid yellow colour as shown by a decrease in the hue and an increase in the chroma values This has also been reported for other a B unsaturated aldehydes such as citral Jin et aL 2009 The hue of the CScin films decreased significantly when subjected to thermal treatments of 95 C for 10 min and 121 C for 5 min acquiring an orange red colour These changes in colour can be explained by the formation of Maillard reaction products when CS with amine and ketone Table 4 3 4 Colour parameters of films Films Es a b Cas hab cs 915 0 2 19 01 90 0 2 9 2 0 2 101 8 0 1 CScin 742 04 134401 866412 876 12 812 0 1 CScin 4 C 30min 735 02 144 03 877 01 888 403 80 7 0 2 CS
54. 812 Zinoviadou K G Koutsoumanis K P Biliaderis C G 2009 Physico chemical properties of whey protein isolate films containing oregano oil and their antimicrobial action against spoilage flora of fresh beef Meat Science 82 338 345 Zinoviadou K G Koutsoumanis K P Biliaderis C G 2010 Physical and thermo mechanical properties of whey protein isolate films containing antimicrobials and their effect against spoilage flora of fresh beef Food Hydrocolloids 24 49 59 Zivanovic S Chi S Draughon A F 2005 Antimicrobial activity of chitosan films enriched with essential oils Journal of Food Science 70 M45 M51 330 Anexos 7 2 Anexo B Publicaciones La presente Tesis Doctoral dio lugar a siete publicaciones cient ficas en revistas internacionales e L pez Carballo G Higueras L Gavara R Hern ndez Mu oz P 2013 Silver ions release from antibacterial chitosan films containing in situ generated silver nanoparticles Journal of Agricultural and Food Chemistry 61 260 267 e Higueras L L pez Carballo G Hern ndez Mu oz P Gavara R Rollini M 2013 Development of a novel antimicrobial film based on chitosan with LAE ethyl N dodecanoyl L arginate and its application to fresh chicken International Journal of Food Microbiology 165 339 345 Higueras L L pez Carballo Cerisuelo J P G Gavara R Hern ndez Mu oz P 2013 Preparation and characterization of chitosan HP f
55. 9 time days time days Figure 4 4 11 Portion of carvacrol percentage present in each phase of the product package system for the three device sizes and the empty tray with the medium device a small b medium c large and d empty tray in the chicken fillet would be 200 20 900 100 and 4500 200 mg Kg for the small medium and large devices respectively To check the actual concentration in chicken meat samples were cut from different areas of the fillet and analysed by thermal desorption and GC as described in the experimental section Large differences were observed between samples taken from the upper surface and those taken from the fillet bulk The concentration in the upper surface was between 5 and 10 fold higher than in the bulk or in the surface in contact with the tray bottom Carvacrol is widely used as an active agent in food packaging Ben Arfa et al 2007 Du et al 2008 Kavoosi et al 2013 L pez Mata et al 2013 Nostro et al 2012 Ramos et al 2012 This may be because carvacrol was recognized as safe by the Joint FAO WHO 2001 as a food additive permitted for direct addition to food for human consumption synthetic flavouring substances and adjuvants with no safety concern and it has recently been included in the list of flavouring substances EFSA 2012 235 Cap tulo 4 Art culo cient fico 6 3 2 Antimicrobial activity of CS CD Active films on chicken breast fillets Fresh poultry product
56. A R Samad A A 2010 Thermal behaviour and interactions of cassava starch filled with glycerol plasticized polyvinyl alcohol blends Carbohydrate Polymers 81 805 810 Rinaudo M 2006 Chitin and chitosan properties and applications Progress in Polymer Science 31 603 632 Ultee A Bennik M H J Moezelaar R 2002 The phenolic hydroxyl group of carvacrol is essential for action against the food borne pathogen Bacillus cereus Applied and Environmental Microbiology 68 1561 1568 Villaluenga J P G Tabe Mohammadi A 2000 A review on the separation of benzene cyclohexane mixtures by pervaporation processes Journal of Membrane Science 169 159 174 Yu QA Song Y N Shi X M Xu CY Bin Y Z 2011 Preparation and properties of chitosan derivative poly vinyl alcohol blend film crosslinked with glutaraldehyde Carbohydrate Polymers 84 465 470 Zuber M Zia K Barikani M 2013 Chitin and chitosan based blends composites and nanocomposites in Thomas S Visakh P M Mathew A P Eds Advances in Natural Polymers Springer Heidelberg Berlin pp 55 119 269 5 DISCUSI N GENERAL Discusi n general La presente Tesis Doctoral ha tenido como objetivo principal el desarrollo y caracterizaci n de pel culas de CS en las que se incorporaron agentes antimicrobianos para su aplicaci n en el envasado activo de alimentos Esta memoria ha sido estructurada en cuatro cap tulos correspondiendo cada uno de el
57. CS cinnamaldehyde Schiff base films intended for use in antimicrobial food packaging were developed CS was selected as a support matrix for the covalent binding of cinnamaldehyde via nucleophilic addition to free amino groups The degree of substitution achieved was ca 7096 b b b b b b b abab bbbb a a a a al aaa a Int day 3 Pref day 3 Int day 6 Pref day 6 Int day 9 Pref day 9 200 EN contro A 4 C 30 min EX 65 C 30 min MA 72 C 15 min EH 95 C 10 min 150 bbbp 100 y Sum of scores 50 74 Figure 4 3 7 Values of the parameters of odour intensity and preference 160 Quitosano Cinamaldeh do CS derived Schiff base can be hydrolyzed under several temperature time treatments in different liquid media The amount of cinnamaldehyde released and the antimicrobial activity of the films depended on the treatment applied and the liquid medium composition The effectiveness of the films increased as the temperature of the treatment increased and thus the amount of cinnamaldehyde released Although the release of the agent caused a perceptible cinnamon aroma in milk the sensory panel considered this effect as positive treated milk being preferred to the control sample The present study shows that CS Schiff base polymer has great potential for use in the development of stimuli responsive active volatile compound release systems These systems can be applied in the design of new active pack
58. Development and evaluation of a novel biodegradable film made from chitosan and cinnamon essential oil with low affinity toward water Food Chemistry 122 161 166 Ojagh S M Rezaei M Razavi S H Hosseini S M H 2010b Effect of chitosan coatings enriched with cinnamon oil on the quality of refrigerated rainbow trout Food Chemistry 120 193 198 Ou C Y Tsay S F Lai C H Weng Y M 2002 Using gelatin based antimicrobial edible coating to prolong shelf life of tilapia fillets Journal of Food Quality 25 213 222 Ouattara B Sabato S F Lacroix M 2001 Combined effect of antimicrobial coating and gamma irradiation on shelf life extension of pre cooked shrimp Penaeus spp International Journal of Food Microbiology 68 1 9 Ouattara B Simard R E Piette G Begin A Holley R A 2000 Inhibition of surface spoilage bacteria in processed meats by application of antimicrobial films prepared with chitosan International Journal of Food Microbiology 62 139 148 Oussalah M Caillet S Salmieri S Saucier L Lacroix M 2004 Antimicrobial and antioxidant effects of milk protein based film containing essential oils for the preservation of whole beef muscle Journal of Agricultural and Food Chemistry 52 5598 5605 Oussalah M Caillet S Salmieri S Saucier L Lacroix M 2006 Antimicrobial effects of alginate based film containing essential oils for the preservation of whole beef muscle Journal of F
59. F Y G Korel F Yemenicio lu A 2009 Antimicrobial activity of lactoperoxidase system incorporated into cross linked alginate films Journal of Food Science 74 M73 M79 Yildirim M G lec F Bayram M Yildirim Z 2006 Properties of Kashar cheese coated with casein as a carrier of natamycin Italian Journal of Food Science 18 127 138 Yoksan R Chirachanchai S 2009 Silver nanoparticles dispersing in chitosan solution preparation by y ray irradiation and their antimicrobial activities Materials Chemistry and Physics 115 296 302 Yoksan R Chirachanchai S 2010 Silver nanoparticle loaded chitosan starch based films fabrication and evaluation of tensile barrier and antimicrobial properties Materials Science amp Engineering C Materials for Biological Applications 30 891 897 Zhang B C Luo Y C Wang Q 2010 Development of silver zein composites as a promising antimicrobial agent Biomacromolecules 11 2366 2375 Zhu L B Olsen C McHugh T Friedman M Jaroni D Ravishankar S 2014 Apple carrot and hibiscus edible films containing the plant antimicrobials carvacrol and cinnamaldehyde inactivate Salmonella Newport on organic leafy greens in sealed plastic bags Journal of Food Science 79 M61 M66 Zhuang R Beuchat L R Chinnan M S Shewfelt R L Huang Y W 1996 Inactivation of Salmonella Montevideo on tomatoes by applying cellulose based edible films Journal of Food Protection 59 808
60. Fick s law considering that the film was immersed in a homogenous media so that the agent was released by both surfaces and that any release by the film edges could be neglected was used According to Crank 1975 the equation that describes the evolution of release of an agent from a film of uniform thickness and constant diffusion coefficient D is Mt _ Q4 8 yo 1 2n 1 Dt Mo 1 n Liza 2n 1 2 Exp 2 1 results and the diffusion coefficient values were estimated by curve fitting to the experimental values using the Regression Wizard Tool of the SigmaPlot 80 Quitosano LAE 10 0 software The D values and the theoretical curves have been included in Table 4 1 2 and Figure 4 1 1 As can be seen there was a good agreement between theoretical data and experimental results as revealed by the regression coefficient values also included in figure Results evidenced that the release of LAE accelerates with temperature as expected since molecular diffusion was an activated process Also the values obtained showed an effect of LAE concentration on the release kinetics The higher the concentration the slower process which could be attributed to an antiplasticizing effect of the agent on the matrix This effect has been observed in other mass transport processes when the polymer substance interactions improve polymer cohesion L pez Rubio et al 2006 In absolute values the diffusion of acetic and propionic acids in CS was in the
61. Granda X Mate J I 2014 Antimicrobial efficiency of edible coatings on the preservation of chicken breast fillets Food Control 36 69 75 Fern ndez Pan I Mendoza M Mat JI 2013 Whey protein isolate edible films with essential oils incorporated to improve the microbial quality of poultry Journal of the Science of Food and Agriculture 93 2986 2994 Fern ndez Pan L Royo M Mat J I 2012 Antimicrobial activity of whey protein isolate edible films with essential oils against food spoilers and foodborne pathogens Journal of Food Science 77 M383 M390 Fern ndez A Picouet P Lloret E 2010a Cellulose silver nanoparticle hybrid materials to control spoilage related microflora in absorbent pads located in trays of fresh cut melon International Journal of Food Microbiology 142 222 228 Fern ndez A Picouet P Lloret E 2010b Reduction of the spoilage related microflora in absorbent pads by silver nanotechnology during modified atmosphere packaging of beef meat Journal of Food Protection 73 2263 2269 Fern ndez A Soriano E Hern ndez Mu oz P Gavara R 2010 Migration of antimicrobial silver from composites of polylactide with silver zeolites Journal of Food Science 75 E186 E193 Fern ndez A Soriano E L pez Carballo G Picouet P Lloret E Gavara R Hern ndez Mu oz P 2009 Preservation of aseptic conditions in absorbent pads by using silver nanotechnology Food Research Intern
62. Inactivation of Salmonella on whole cantaloupe by application of an antimicrobial coating containing chitosan and allyl isothiocyanate International Journal of Food Microbiology 155 165 170 Chiu P E Lai L S 2010 Antimicrobial activities of tapioca starch decolorized hsian tsao leaf gum coatings containing green tea extracts in fruit based salads romaine hearts and pork slices International Journal of Food Microbiology 139 23 30 Chou W L Yu D G Yang M C 2005 The preparation and characterization of silver loading cellulose acetate hollow fiber membrane for water treatment Polymers for Advanced Technologies 16 600 607 Da Silva M A Iamanaka B T Taniwaki M H Kieckbusch T G 2013 Evaluation of the antimicrobial potential of alginate and alginate chitosan films containing potassium sorbate and natamycin Packaging Technology and Science 26 479 492 Daoud W A Xin J H Zhang Y H 2005 Surface functionalization of cellulose fibers with titanium dioxide nanoparticles and their combined bactericidal activities Surface Science 599 69 75 314 Anexos Datta S Janes M E Xue Q G Losso J La Peyre J E 2008 Control of Listeria monocytogenes and Salmonella Anatum on the surface of smoked salmon coated with calcium alginate coating containing oyster lysozyme and nisin Journal of Food Science 73 M67 M71 Dawson P L Carl G D Acton J C Han LY 2002 Effect of lauric acid and nisin impre
63. Jim nez A 2014 Functional properties of sodium and calcium caseinate antimicrobial active films containing carvacrol Journal of Food Engineering 121 94 101 vila Sosa R Hern ndez Zamoran E L pez Mendoza L Palou E Jim nez Mungu a M T Nev rez Moorill n G V L pez Malo A 2010 Fungal inactivation by mexican oregano Lippia berlandieri Schauer essential oil added to amaranth chitosan or starch edible films Journal of Food Science 75 M127 M133 vila Sosa R Palou E Munguia M T J Nev rez Moorillon G V Cruz A RN L pez Malo A 2012 Antifungal activity by vapor contact of essential oils added to amaranth chitosan or starch edible films International Journal of Food Microbiology 153 66 72 Ayana B Turhan K N 2009 Use of antimicrobial methylcellulose films to control Staphylococcus aureus during storage of kasar cheese Packaging Technology and Science 22 461 469 Bahram S Rezaei M Soltani M Kamali A Ojagh S M Abdollahi M 2014 Whey protein concentrate edible film activated with cinnamon essential oil Journal of Food Processing and Preservation 38 1251 1258 Balaguer M P Fajardo P Gartner H G mez Estaca J Gavara R Almenar E Hern ndez Mu oz P 2014 Functional properties and antifungal activity of films based on gliadins containing cinnamaldehyde and natamycin International Journal of Food Microbiology 173 62 71 Balaguer M P L pez Carbal
64. Latinoamericano sobre Higiene y Calidad en la Agricultura y Alimentaci n Book of proceedings Iquique I Regi n de Tarapac Chile 4 6 de noviembre de 2009 Congreso internacional p ster L Higueras G L pez Carballo R Gavara P Hern ndez Mu oz Chitosan Matrices as Carriers for the Delivery of Natural Volatile Antimicrobials BioMicroWorld 2009 III International Conference on Environmental Industrial and Applied Microbiology Book of Abstracts 277 Lisboa Portugal 2 4 de diciembre de 2009 Congreso internacional p ster G L pez Carballo L Higueras A Fern ndez R Gavara P Hern ndez Mufioz Antimicrobial Properties of Nanostructured Chitosan Silver Membranes BioMicroWorld 2009 III International Conference on Environmental Industrial and Applied Microbiology Book of Abstracts 264 Lisboa Portugal 2 4 de diciembre de 2009 Congreso internacional p ster G L pez Carballo L Higueras R Gavara P Hern ndez Mufioz Development of hybrid nanocomposites based on chitosan and silver nanoparticles for active packaging applications SLIM 2010 Shelf Life International Meeting Book of Abstracts 89 Zaragoza Espa a 23 25 de junio de 2010 Congreso internacional p ster L Higueras G L pez Carballo M P Balaguer R Gavara P Hern ndez Mufioz Active packaging technologies based on chitosan cyclodextrin biocomposites IFT10 Annual Meeting amp Food Expo Chicago IL USA 17 20 de Julio de
65. P ez M Cerda E Az car I Rabagliati F M 2011 Nanocomposites based on polyethylene and nanosilver particles produced by metallocenic in situ polymerization synthesis characterization and antimicrobial behavior European Polymer Journal 47 1541 1549 99 4 2 2 Art culo cient fico 2 Silver ions release from antibacterial chitosan films containing in situ generated silver nanoparticles Gracia L pez Carballo Laura Higueras Rafael Gavara Pilar Hern ndez Mufioz Journal of Agricultural and Food Chemistry 2013 61 260 267 Quitosano Nanopart culas de plata Resumen Este estudio tiene como objetivo desarrollar pel culas antimicrobianas que consisten en quitosano y nanopart culas de plata homog neamente distribuidas en la matriz polim rica Las nanopart culas se generaron in situ durante la neutralizaci n de las pel culas de acetato de quitosano con hidr xido de sodio La temperatura de neutralizaci n y la concentraci n de plata en la pel cula fueron dos factores cruciales de la forma y el tamafio de las nanopart culas Las pel culas neutralizadas presentaron actividad antimicrobiana frente a Escherichia coli y Staphylococcus aureus en medio de cultivo l quido Sin embargo la eficacia de las pel culas fue considerablemente mayor en los medios de cultivo diluidos Adem s no se encontraron diferencias significativas en la capacidad antimicrobiana de pel culas que incorporan diferentes cantidades de pla
66. Park K 2009 Engineered polymers for advanced drug delivery European Journal of Pharmaceutics and Biopharmaceutics 71 420 430 Kong M Chen X G Xing K Park H J 2010 Antimicrobial properties of chitosan and mode of action a state of the art review International Journal of Food Microbiology 144 51 63 Krajewska B 2005 Membrane based processes performed with use of chitin chitosan materials Separation and Purification Technology 41 305 312 Kurita K 2006 Chitin and chitosan functional biopolymers from marine crustaceans Marine Biotechnology 8 203 226 Kurkov S V Loftsson T 2013 Cyclodextrins International Journal of Pharmaceutics 453 167 180 Lambert RJ W Skandamis P N Coote P J Nychas GJ E 2001 A study of the minimum inhibitory concentration and mode of action of oregano essential oil thymol and carvacrol Journal of Applied Microbiology 91 453 462 Leistner L Gorris L G M 1995 Food preservation by hurdle technology Trends in Food Science amp Technology 6 41 46 Marsh K Bugusu B 2007 Food packaging roles materials and environmental issues Journal of Food Science 72 R39 R55 Messner M Kurkov S V Jansook P Loftsson T 2010 Self assembled cyclodextrin aggregates and nanoparticles International Journal of Pharmaceutics 387 199 208 42 Introducci n No H K Meyers S P Prinyawiwatkul W Xu Z 2007 Applications of chitosan for improvement of
67. Potassium sorbate permeability of polysaccharide films chitosan methylcellulose and hydroxypropyl methylcellulose Journal of Food Process Engineering 12 33 48 Weber CJ Haugaard V Festersen R Bertelsen G 2002 Production and applications of biobased packaging materials for the food industry Food Additives and Contaminants 19 172 177 Wei D W Sun W Y Qian W P Ye Y Z Ma X Y 2009 The synthesis of chitosan based silver nanoparticles and their antibacterial activity Carbohydrate Research 344 2375 2382 46 2 OBJETIVOS Objetivos 2 1 Objetivo general El objetivo principal de la presente Tesis Doctoral es el estudio del biopol mero quitosano como matriz portadora y liberadora de agentes activos para el desarrollo de pel culas antimicrobianas y su aplicaci n como envase activo antimicrobiano 2 2 Objetivos espec ficos Para lograr este objetivo general se plantean los siguientes objetivos espec ficos Obtener pel culas de quitosano mediante la t cnica de extensi n y evaporaci n del solvente con o sin coadyuvante y en las que se incluyan diferentes agentes antimicrobianos incorporados en la matriz empleando diferentes metodolog as Caracterizar el efecto de la incorporaci n de los agentes activos y en su caso de los coadyuvantes en las propiedades funcionales m s relevantes de las pel culas activas desarrolladas e Estudiar los diferentes mecanismos de liberaci n de los diverso
68. R 2008 The antimicrobial efficacy of plant essential oil combinations and interactions with food ingredients International Journal of Food Microbiology 124 91 97 Hawkins D R Rocabayera X Ruckman S A Segret R Shaw D 2009 Metabolism and pharmacokinetics of ethyl N lauroyl L arginate hydrochloride in human volunteers Food and Chemical Toxicology 47 2711 2715 He Y F Fu P Shen X H Gao H C 2008 Cyclodextrin based aggregates and characterization by microscopy Micron 39 495 516 Healy M G Romo CR Bustos R 1994 Bioconversion of marine crustacean shell waste Resources Conservation and Recycling 11 139 147 Hoare T R Kohane D S 2008 Hydrogels in drug delivery progress and challenges Polymer 49 1993 2007 Holley R A Patel D 2005 Improvement in shelf life and safety of perishable foods by plant essential oils and smoke antimicrobials Food Microbiology 22 273 292 Infante M R Pinazo A Seguer J 1997 Non conventional surfactants from amino acids and glycolipids structure preparation and properties Colloids and Surfaces a Physicochemical and Engineering Aspects 123 49 70 Karbowiak T Hervet H Leger L Champion D Debeaufort F Voilley A 2006 Effect of plasticizers water and glycerol on the diffusion of a small molecule in iota carrageenan biopolymer films for edible coating application Biomacromolecules 7 2011 2019 Kim S Kim J H Jeon O Kwon I C
69. R Almenar E Hern ndez Mu oz P 2014 Functional properties and antifungal activity of films based on gliadins containing cinnamaldehyde and natamycin International Journal of Food Microbiology 173 62 71 Balaguer M P G mez Estaca J Gavara R Hern ndez Mufioz P 2011a Biochemical properties of bioplastics made from wheat gliadins cross linked with cinnamaldehyde Journal of Agricultural and Food Chemistry 59 13212 13220 Balaguer M P G mez Estaca J Gavara R Hern ndez Mu oz P 2011b Functional properties of bioplastics made from wheat gliadins modified with cinnamaldehyde Journal of Agricultural and Food Chemistry 59 6689 6695 Balaguer M P L pez Carballo G Catal R Gavara R Hern ndez Mu oz P 2013 Antifungal properties of gliadin films incorporating cinnamaldehyde and application in active food packaging of bread and cheese spread foodstuffs International Journal of Food Microbiology 166 369 377 Ben Arfa A Preziosi Belloy L Chalier P Gontard N 2007 Antimicrobial paper based on a soy protein isolate or modified starch coating including carvacrol and cinnamaldehyde Journal of Agricultural and Food Chemistry 55 2155 2162 Brasil IM Gomes C Puerta G mez A Castell P rez M E Moreira R G 2012 Polysaccharide based multilayered antimicrobial edible coating enhances quality of fresh cut papaya Lwt Food Science and Technology 47 39 45 Dos Santos J E Dockal E R C
70. RH these composites retained 21696 carvacrol g 100 g dry matter These results indicate that inclusion of carvacrol in the films could be occurring by mechanisms other than formation of inclusion complexes Keywords Chitosan hydroxypropyl B cyclodextrins biocomposite functional properties sorption of carvacrol 176 Quitosano Carvacrol 1 Introduction CS has been widely studied as a polymer for the design of reservoir delivery systems for the slow release of active compounds over an extended period of time These regulated delivery systems are effective in minimizing the amount of compound used for a specific application and thus improve efficacy and reduce possible side effects associated with the use of large amounts of bioactives Pedro et al 2009 Sivakumar et al 2002 CS employed as a delivery system can find applications in a variety of technological areas such as agrochemistry pharmacy biomedicine textiles and food packaging The development of antimicrobial materials and their application in the design of active packaging is creating considerable expectation in the food industry since food safety is an area of great concern Although there are many studies in the literature that focus on the use of CS films as antimicrobials in contact with food the use of CS films for the release of active compounds has received much less attention Because of their antimicrobial properties many plant extracts and essential oils
71. Soares N D F 2014 Sliced bread preservation through oregano essential oil containing sachet Journal of Food Process Engineering 37 53 62 Scramlin S Newman M Cox R Sepe H Alderton A O Leary J Mikel W 2010 Effects of oregano oil brine enhancement on quality attributes of beef Longissimus dorsi and Semimembranosus muscles from various age animals Journal of Food Science 75 S89 94 289 6 CONCLUSIONES Conclusiones From the results obtained in the development of the present Doctoral Thesis it is possible to highlight the following conclusions 1 Chitosan films capable of incorporating and releasing various antimicrobial agents using different methodologies were obtained by the solvent casting technique and developed for active food packaging applications 2 Chitosan films with LAE were obtained by incorporating the antimicrobial agent into the film forming solution Release of LAE from the chitosan matrix in aqueous medium was completed in 10 to 15 h depending on the temperature The inherent antimicrobial activity of the chitosan film was significantly increased by the presence of 5 LAE which satisfactorily reduced the microbial load present in fresh chicken Therefore these films can be incorporated in the design of an antimicrobial packaging system to extend the shelf life of chicken breasts 3 Chitosan films with silver nanoparticles were obtained using a novel method that complies with the princi
72. Sostenible La incorporaci n de nitrato de plata produjo pel culas de CS con diferente color tras la neutralizaci n stas presentaron una mayor coloraci n marr n rojiza con el incremento de la concentraci n de plata incluida en las pel culas y de la temperatura de neutralizaci n de las mismas De los resultados obtenidos se deduce que la temperatura de neutralizaci n de la pel cula es un factor importante De las temperaturas de neutralizaci n probadas 22 y 37 C se seleccion la mayor temperatura ya que se observ una mayor conversi n del precursor nitrato de plata a nanopart culas de plata pasando a ser estas pel culas neutralizadas a 37 C el objeto principal del estudio Mediante TEM espectroscop a UV visible y difracci n de rayos X se evidenci la formaci n de nanopart culas de plata en las pel culas de CS neutralizadas a 37 C Mientras que el TGA indic que las pel culas de CS con nanopart culas de plata neutralizadas a 37 C son m s termoestables que las pel culas de CS Otro factor a tener en cuenta es la concentraci n de plata en las pel culas Se observ mediante TEM y espectroscop a UV visible que las pel culas de CS neutralizadas a 37 C con menor concentraci n de plata a adida presentaron part culas esf ricas de lt 5 nm y homog neamente distribuidas en el matriz polim rica A mayores concentraciones se observaron dos poblaciones de part culas de distinto di metro Una pobl
73. T Brasil ILM 2014 Freshness retention of minimally processed melon using different packages and multilayered edible coating containing microencapsulated essential oil International Journal of Food Science and Technology 49 2192 2203 Muriel Galet V Cerisuelo J P L pez Carballo G Lara M Gavara R Hern ndez Mufioz P 2012 Development of antimicrobial films for microbiological control of packaged salad International Journal of Food Microbiology 157 195 201 Muriel Galet V Cerisuelo J R L pez Carballo G Aucejo S Gavara R Hernandez Mufioz P 2013 Evaluation of EVOH coated PP films with oregano essential oil and citral to improve the shelf life of packaged salad Food Control 30 137 143 Nedorostova L Kloucek P Kokoska L Stolcova M Pulkrabek J 2009 Antimicrobial properties of selected essential oils in vapour phase against foodborne bacteria Food Control 20 157 160 Nostro A Marino A Blanco A R Cellini L Di Giulio M Pizzimenti F Roccaro A S Bisignano G 2009 In vitro activity of carvacrol against staphylococcal preformed biofilm by liquid and vapour contact Journal of Medical Microbiology 58 791 797 171 Cap tulo 4 Estado de la cuesti n Ouattara B Simard R E Piette G Begin A Holley R A 2000 Diffusion of acetic and propionic acids from chitosan based antimicrobial packaging films Journal of Food Science 65 768 773 Ou draogo I W K De Winte
74. The medium and large devices released 9096 after three days and slightly above 9596 after 9 days without differences between samples In contrast the control sample only released 3596 after 3 days and this release percentage was maintained b ps bd ES ps T 2 a 001 Small Medium sendy Large amp Medium empty Equation 1 Carvacrol concentration g g CS Active o Carvacrol concentration in HS g L 0 3 6 9 0 3 6 9 c d P e Carvacrol concentration g g PP 3 A Carvacrol concentration g cm Lid g l o 3 6 o time days time days Figure 4 4 10 Evolution of carvacrol concentration during product storage for the three device sizes and for control packages without chicken in the different package parts a CS CD Active b headspace HS c package lid and d the PP inner layer of the tray Values are expressed as mean and standard deviation n 9 232 Quitosano Carvacrol throughout the 9 day storage This large difference between the sample without chicken and food packaged samples is obviously due to the presence of the meat fillet A great chemical compatibility between chicken proteins and carvacrol could explain the showed differences Figure 4 4 10b shows the concentration of carvacrol in the headspace As can be seen the concentration in the sample without chicken is nearly 5 10
75. V http dxdoiorg 10 1016 j ijfoodmicro 2013 06 003 Aymerich et al 2008 In this case it is possible to reduce the amount of active agent required satisfying the demand of consumers for fewer additives One of the most innovative antimicrobial agent is ethyl N dodecanoyl i arginate hydrochloride LAE It is a synthetically deriv ative of lauric acid L arginine and ethanol Infante et al 1997 2004 Ruckman et al 2004 which is notable for its antimicrobial effective ness resulting from its chemical structure and surfactant properties Pinazo et al 1999 Rodriguez 2004 Brandt et al 2010 LAE s anti microbial properties are due to its action as cationic surfactant on cytoplasmic membrane and the outer membrane of Gram negative and cell membrane and cytoplasm of Gram positive denaturation proteins These changes produce disturbances in membrane poten tial resulting cell growth inhibition and loss of viability Kanazawa et al 1995 Infante et al 1997 Tattawasart et al 2000 Rodriguez 2004 Luchansky et al 2005 LAE is characterized by a broad spectrum Infante et al 1997 and high antimicrobial efficiency against Gram negatives Gram positives fungi and yeasts with a low dose application Infante et al 1984 Rodriguez 2004 In addition LAE has a low oil water equilibrium partition coefficient Kow lt 0 1 which means that it tends to con centrate in the aqueous phase where most bacterial action occurs
76. and Food Chemistry 56 3082 3088 EFSA 2012 Scientific Opinion on the safety and efficacy of phenol derivatives containing ring alkyl ring alkoxy and side chains with an oxygenated functional group chemical group 25 when used as flavourings for all species EFSA Panel on Additives and Products or Substances used in Animal Feed FEEDAP FAO WHO J 2001 Expert Committee on Food Additives Fifty seventh meeting Rome 5 14 http www leffingwell com Summary 20and 20Conclusions 200f 20the 20Fifty seventh 20meeting pdf accessed on July 24 2014 Gavara R Catal R 2002 Mass transfer in food plastic packaging systems in Engineering and Food for the 21st Century in Welti Chanes J Barbosa Canovas G Aguilera J M Eds Food Preservation Technology Series CRC Press Washington D C Guti rrez J Barry Ryan C Bourke R 2008 The antimicrobial efficacy of plant essential oil combinations and interactions with food ingredients International Journal of Food Microbiology 124 91 97 240 Quitosano Carvacrol Guti rrez L Escudero A Batlle R Ner n C 2009 Effect of mixed antimicrobial agents and flavors in active packaging films Journal of Agricultural and Food Chemistry 57 8564 8571 Higueras L L pez Carballo G Cerisuelo J P Gavara R Hern ndez Mu z P 2013 Preparation and characterization of chitosan HP p cyclodextrins composites with high sorption capacity for carvacrol Carbohyd
77. assays in milk The antimicrobial activity of the films was tested in commercial pasteurized cows milk For this purpose the procedure described in n vitro antimicrobial activity of CScin films against S aureus and E coli section was followed using milk instead of MHB and inoculating L monocytogenes in exponential phase Sterilized tubes with 10 mL of milk were inoculated in sterilized conditions with 100 uL of L monocytogenes in exponential phase 10 CFU mL The tubes were then kept at 4 C for 12 days and antimicrobial assays were performed on days 3 6 and 12 Serial dilutions with peptone water were made and plated in PALCAM Listeria selective agar Merck Darmstadt Germany Plates were incubated at 37 C for 48 h All experiments were carried out in triplicate 2 6 Sensory analysis Sensory tests on commercial pasteurized cow milk that had been exposed to the films and subjected to the time temperature treatments were carried out on the 3rd 6th and 12th days by an untrained panel 44 judges The tests were done in a standardized test room ISO 8589 2007 Samples of milk were placed in hermetic sealed transparent tubes and identified by three digit codes The panel members were asked to smell the sample and describe the intensity of the perceived cinnamon aroma and preference in terms of smell The odour intensity was indicated on a 1 to 5 scale in which 1 was the lowest cinnamon odour intensity and 5 the most intense For the
78. benzoato de sodio sorbato de potasio nisina diacetato de sodio nisina lactato de sodio nisina sorbato de potasio nisina benzoato de sodio sorbato de potasio benzoato de sodio propionato de sodio sorbato de potasio benzoato de sodio sorbato de potasio diacetato de sodio diacetato de sodio nisina lactato de sodio lactato de sodio sorbato de potasio propionato de sodio sorbato de potasio sorbato de potasio sorbato de potasio sorbato de potasio sorbato de potasio sorbato de potasio sorbato de potasio leche quitosano gluten de trigo ze na prote na de suero de leche cera candelilla alginato almid n K carrageenano pectina gelatina HPMC metilcelulosa celulosa HPMC metilcelulosa quitosano ze na prote nas de suero de leche caseinato de sodio celulosa acetato v ginato de calcio v ginato de calcio almid n de boniato v mid n de guisante mid n de patata goma guar v v mid n de tapioca mid n de tapioca goma xantana v v mid n de tapioca celulosa y derivados almid n pavo masa de hojaldre fresca ma z dulce medio de cultivo mortadela salchicha salm n ahumado manzana y patata cortadas medio de cultivo naranjas Valencia mandarina h brida Ortanique mandarina clementina Clemenules medio de cultivo salchicha de pavo carne de vacuno medio de cultivo pan en rebanadas patata queso mozz
79. biologically active molecules into a specific environment have become of increasing interest in recent years These systems are able to reduce the amount of active agent required for treatment by maintaining an effective concentration in the system applied over a certain period of time Ouattara Simard Piette Begin amp Holley 2000 There is a great need for these de vices in diverse technological applications encompassing multi disciplinary areas such as biomedicine pharmacology agriculture packaging food technology textiles and the cosmetic industry for the entrapment and delivery of drugs enzymes nutraceuticals agrochemicals flavours and fragrances biocides etc Currently most of the recently developed delivery systems consist of natural and synthetic polymers polymer blends and composites of organic and inorganic materials that form membranes capsules or micelles depending on the application required Issues concerning biode gradability biocompatibility and non toxicity of the materials used for the development of carrier systems need to be considered Renewable polymers are being widely investigated as delivery Corresponding author Tel 34 96 3900022 fax 34 96 3636301 E mail address phernan iata csic es P Hern ndez Mu oz http dx doi org 10 1016 j foodhyd 2014 07 017 0268 005X 2014 Elsevier Ltd All rights reserved 2014 Elsevier Ltd All rights reserved vehicles because most of them f
80. chitosan films and these films subject ed to different treatments were also evaluated The antimicro bial properties of chitosan Schiff base films were tested in vitro against Staphylococcus aureus and Escherichia coli and in milk inoculated with Listeria monocytogenes The antimicro bial activity varied depending on the treatment applied and consequently the degree of imino bond hydrolysis achieved and cinnamaldehyde released Films of Schiff base chitosan derivative subjected to different time temperature treatments inhibited the growth of L monocytogenes for 12 days under refrigeration conditions which may extend the microbiological shelf life of such products Sensory analysis of milk in contact with the films showed that a cinnamon smell does not cause any rejection among potential consumers These novel films L Higueras G L pez Carballo R Gavara P Hern ndez Mu oz E3 Institute of Agrochemistry and Food Technology IATA CSIC Avenida Agust n Escardino 7 46980 Paterna Valencia Spain e mail pheman a iata csic es Published online 17 October 2014 could be used in the design of antimicrobial food packaging and in various other technological areas where sustained release systems are required Keywords Chitosan Cinnamaldehyde Schiff base Hydrolysis Release Antimicrobial films Introduction Chitosan is a natural biocompatible biodegradable biorenewable and biofunctional polysaccharide that is finding
81. con INS n 1520 Es razonable suponer que los plastificantes empleados como coadyuvantes en biopol meros deben ser tambi n preferentemente biodegradables Rahman y Brazel 2004 Vieira et aL 2011 As existe un gran inter s en el desarrollo y mejora de plastificantes biodegradables al igual que se desarrollan materiales biodegradables y a partir de recursos renovables As tanto el G es biodegradable bajo condiciones aer bicas y anaer bicas de acuerdo con OECD 301 OECD SIDS 2002 como el PG OECD SIDS 2001 21 Introducci n 1 42 Ciclodextrinas Las ciclodextrinas CD son una familia de oligosac ridos c clicos constituidas habitualmente por seis siete u ocho unidades de D glucopiranosa unidas por enlaces a 14 denomin ndose a CD B CD y y CD respectivamente Las CD se obtienen durante la degradaci n enzim tica del almid n mediante la enzima ciclomaltodextrin glucosiltransferasa Del Valle 2004 Kurkov y Loftsson 2013 Szejtli 1998 Las CDs presentan una estructura molecular troncoc nica hueca con una cavidad interior de volumen espec fico Las principales propiedades de las CDs son el di metro de su cavidad y su solubilidad En la Tabla 1 2 se muestra la estructura y caracter sticas de las CDs naturales En la mol cula de CD en torno al borde superior y m s ancho se hallan los hidroxilos secundarios de C2 y C3 en el borde inferior se halla el hidroxilo primario de C6 que es m s estrecho debido a
82. cter bactericida y bacteriost tico de la plata ha sido conocido emp ricamente desde tiempos remotos Su uso fue disminuyendo por la aparici n de los antibi ticos a pesar de continuar siendo el principal tratamiento para quemaduras e infecciones cut neas Sin embargo actualmente se est recuperando el uso de la plata como agente antimicrobiano debido al aumento de las resistencias de ciertos 27 Introducci n microorganismos pat genos a los antibi ticos Rai et aL 2009 Wei et al 2009 La plata i nica posee un amplio espectro biocida mientras que en estado met lico es inerte Los iones de plata son capaces de unirse a los grupos funcionales donadores de electrones como fosfatos hidroxilos imidazoles indoles aminas y tioles principalmente de 1 prote nas y enzimas alterando la estructura y funci n de diferentes org nulos de los microorganismos como la pared celular y membrana externa por tanto la cadena respiratoria y la permeabilidad e integridad celular 2 ADN ARN y prote nas ribosomales interfiriendo en la traducci n de prote nas e inhibiendo la s ntesis de ATP y 3 catalizan especies reactivas de ox geno Dallas et al 2011 De Azeredo 2013 Gibbins y Warner 2005 Rai et al 2009 Por otra parte el empleo abusivo de ste y todos los biocidas puede llevar al desarrollo de resistencias en los microorganismos Y se han descrito resistencias a metales pesados como la plata aunque raras y espor dicas y
83. de inclusi n Los puntos azules representan mol culas de agua el valo naranja la mol cula hidr foba hu sped 24 Figura 1 7 Estructura qu mica del LAE 26 Figura 1 8 Estructuras de a carvacrol b timol c mentol y d ster met lico del carvacrol 34 Figura 1 9 Estructura de a cinamaldeh do b cido cin mico c acetato de cinamilo d 4 hidroxibenzaldeh do y e 3 fenilpropionaldeh do 35 Figure 4 1 1 Normalized release of LAE MyM from CS films into water versus time of exposition at 4 and 28 C Dots are experimental data and lines represent theoretical curves obtained with Eq 1 and D values included in Table 4 1 2 80 Figure 4 1 2 Growth reduction of indigenous spoilage microbiota in chicken breast fillets at 2 6 and 8 d of storage and 4 C in absence and presence of CS and CS 5 LAE films 86 Figura 4 2 1 Esquema de la generaci n de nanopart culas de plata 92 Figure 4 2 2 CS silver based nanoparticle films with different concentrations of silver A neutralized at 22 C B neutralized at 37 C 112 Figure 4 2 3 Effect of silver content in 37 C neutralized CS films on the first derivative of weight loss DTG curves dm dT first derivative of weight loss vs time 113 Figure 4 2 4 TEM images of films containing A 0 2 and B 1 5 of silver and neutralized at 37 C 113 XXI ndice de tablas y figuras Figure 4 2 5 UV visible spectrum of CS films with 0 0 1 0 2 0 5 1 and 1 5 of silv
84. de las pel culas y la liberaci n de iones plata en el tiempo para demostrar si las pel culas en medio l quido liberan gradualmente plata i nica suministrando una acci n antimicrobiana mantenida en el tiempo Referencias bibliogr ficas An J S Zhang M Wang S J Tang J 2008 Physical chemical and microbiological changes in stored green asparagus spears as affected by coating of silver nanoparticles PVP Lwt Food Science and Technology 41 1100 1107 Anastas P Eghbali N 2010 Green chemistry principles and practice Chemical Society Reviews 39 301 312 95 Cap tulo 2 Estado de la cuesti n Basri H Ismail A F Aziz M 2011 Polyethersulfone PES silver composite UF membrane Effect of silver loading and PVP molecular weight on membrane morphology and antibacterial activity Desalination 273 72 80 Basri H Ismail A F Aziz M Nagai K Matsuura T Abdullah M S Ng B C 2010 Silver filled polyethersulfone membranes for antibacterial applications Effect of PVP and TAP addition on silver dispersion Desalination 261 264 271 Boschetto D L Lerin L Cansian R Pergher S B C Di Luccio M 2012 Preparation and antimicrobial activity of polyethylene composite films with silver exchanged zeolite Y Chemical Engineering Journal 204 210 216 Dallas P Sharma V K Zboril R 2011 Silver polymeric nanocomposites as advanced antimicrobial agents classification synthetic paths applica
85. due to condensation of the volatile on the walls and septum of the vial and even cinnamaldehyde sorption in the film caused by a change in the partition equilibrium constant of cinnamaldehyde with temperature Another important feature is the large difference in the amount of cinnamaldehyde released in each liquid medium Other authors have 154 Quitosano Cinamaldeh do 80 4 a 4 C 30 min o Y a 65 C 30 min m 72 C 15 min e 60 4 o E Es D EE o F4 les E co 404 g 3 E 2 c 9 4004 E o 3 t 20 4 E 5 200 4 a oj _ e e a N 5 ie o 10 20 30 40 50 60 70 time min o a T T T 0 20 time h 40 60 Figure 4 3 4 Cinnamaldehyde released from films into MHB during different temperature time treatments empty symbols and time evolution of cinnamaldehyde released into ethanol 5096 at 23 C after applying preservation treatments full symbols reported the importance of the solvent along with the temperature with regard to the reversibility of the Schiff base Huang et al 2001 Mohamad 2013 Muhamad 2011 MHB is an aqueous medium buffered at pH 7 which causes film swelling and therefore increases the diffusion rate of any substance through the matrix This effect explains the higher concentration of agent observed in MHB at 4 C However after the thermal treatments 65 C and above the release into the water ethanol medium
86. en el envasado activo antimicrobiano de alimentos 63 4 1 1 Estado de la cuesti n 63 4 1 2 Art culo cient fico 1 Development of a novel antimicrobial film based on chitosan with LAE ethyl N dodecanoyl L arginate and its application to fresh chicken 69 ndice de contenidos 4 2 Cap tulo 2 Desarrollo de pel culas h bridas de quitosano con nanopart culas de plata formadas in situ 91 4 2 1 Estado de la cuesti n 91 4 2 2 Art culo cient fico 2 Silver ions release from antibacterial chitosan films containing in situ generated silver nanoparticles 101 4 3 Cap tulo 3 Desarrollo de pel culas de quitosano con compuestos vol tiles antimicrobianos anclados mediante un enlace covalente reversible y su aplicaci n en el envasado activo de alimentos 127 4 3 1 Estado de la cuesti n 127 4 3 2 Art culo cient fico 3 Reversible covalent immobilization of cinnamaldehyde on chitosan films via Schiff base formation and their application in active food packaging 135 4 4 Cap tulo 4 Desarrollo de pel culas de quitosano con hidroxipropil B ciclodextrinas y otros coadyuvantes con adaptable capacidad de sorci n y liberaci n de compuestos vol tiles antimicrobianos y su aplicaci n en el envasado activo de alimentos 165 4 4 1 Estado de la cuesti n 165 4 4 2 Art culo cient fico 4 Preparation and characterization of chitosan HP B cyclodextrins composites with high sorption capacity for carvacrol 173 44 3 Art culo c
87. et al 1999 All the CScin films subjected to the various preservation treatments showed antimicrobial activity against the two microorganisms that were tested Generally Gram negative bacteria are more resistant to essential oils than Gram positive bacteria The outer membrane of Gram negative bacteria is highly impermeable to hydrophobic molecules like those found in essential oils and they can work on different targets on the bacteria Nazzaro et al 2013 However the mechanisms of action of essential oil components and thus their antimicrobial activity against Gram positive and Gram negative bacteria depend on their chemical structure Antibacterial effects of essential oils are commonly associated to interactions with the cell MM S aureus Emm E coli Bacterial count Log CFU mL o N o o a NS SY SY SY SY SY SS N por BO E WN Treatment Figure 4 3 5 Antimicrobial activity of CS CScin and CS Schiff base films subjected to various combinations of temperature time treatments against S aureus and E coli 156 Quitosano Cinamaldeh do membrane although the precise mechanisms remain unclear The experimental pieces of evidence on the action mechanisms of cinnamaldehyde are contradictory and support both membrane interaction and inhibition of specific cellular processes or enzymes Gill and Holley 2004 CScin and CScin films subjected to a storage temperature of 4 C for 30 min showed reduced antimicro
88. ethylene glycol measured by differential scanning calorimetry and solid state NMR spectroscopy Carbohydrate Polymers 53 409 416 Song J H Murphy R J Narayan R Davies G B H 2009 Biodegradable and compostable alternatives to conventional plastics Philosophical Transactions of the Royal Society B Biological Sciences 364 2127 2139 Sothornvit R Krochta J M 2005 Plasticizers in edible films and coatings in Han J H Ed Innovations in food packaging Academic Press London pp 403 433 Srinivasa P C Ramesh M N Tharanathan R N 2007 Effect of plasticizers and fatty acids on mechanical and permeability characteristics of chitosan films Food Hydrocolloids 21 1113 1122 Srinivasa P C Tharanathan R N 2007 Chitin chitosan safe ecofriendly packaging materials with multiple potential uses Food Reviews International 23 53 72 Suntres Z E Coccimiglio J Alipour M 2015 The bioactivity and toxicological actions of carvacrol Critical Reviews in Food Science and Nutrition 55 304 318 Suyatma N E Tighzert L Copinet A 2005 Effects of hydrophilic plasticizers on mechanical thermal and surface properties of chitosan films Journal of Agricultural and Food Chemistry 53 3950 3957 Szejtli J 1998 Introduction and general overview of cyclodextrin chemistry Chemical Reviews 98 1743 1753 Szente L Szejtli J 1988 Stabilization of flavors by cyclodextrins Acs Symposium Series 370 148 1
89. films based on CS or PVOH possess an excellent capacity to release the retained 264 Quitosano Carvacrol Table 4 4 14 Antimicrobial activity of films 25 mm diameter surface against S aureus and E coli after loading with monoterpenes measured by the microatmosphere method CS CD 35G 75RH CS CD 50G 65RH CS CD 35PG 90RH PVOH CD 35G 84RH S aureus Amount Zone of Amount Zone of Amount Zone of Amount Zone of mg inhibition mm CuT inhibition mm Gui inhibition mm e inhibition mm m cumenol 327 1 gt 85 275 4 gt 85 226 2 gt 85 169 3 gt 85 o cumenol 347 5 gt 85 243 7 gt 85 222 8 gt 85 101 5 gt 85 carvacrol 146 5 gt 85 184 9 gt 85 194 9 gt 85 147 9 gt 85 guaiacol 106 0 71 121 3 79 96 4 70 194 2 80 isoeugenol 18 7 54 24 1 63 22 2 59 27 0 67 myrtenol S7 44 3 9 10 4 5 29 85 10 nerol 5 4 40 3 8 30 3 0 12 3 2 30 carveol 3 5 27 44 34 43 32 4 6 35 dihydrocarveol 3 0 24 2 8 20 34 20 4 0 40 isopulegol 1 9 0 8 2 9 157 CS CD 35G 75RH CS CD 50G 65RH CS CD 35PG 90RH PVOH CD 35G 84RH E coli Amount Zone of Amount Zone of Amount Zone of Amount Zone of Cite inhibition mm Crile inhibition mm Gui inhibition mm CT inhibition mm m cumenol 282 9 gt 85 297 1 gt 85 244 0 gt 85 206 7 gt 85 o cumenol 272 2 gt 85 2m3 gt 85 193 2 gt 85 182 8 gt 85 carvacrol 120 2 gt 85 208 3 gt 85 219 6 gt 85 152 0 gt 85 guaiacol 136 0 64 144 2 81 114 6 72 190 1 79 isoeugenol 20 6 50 27 5 65 254 63 17 9 51 myrtenol
90. from the 111 200 220 and 311 planes As Figure 4 2 6 shows the XRD pattern of the neutralized CS film has a broad peak at 20 of 20 indicating low levels of crystallinity and this peak was also observed in films neutralized at 37 C Only one diffraction peak of g E amp 3 z 2 2 MAE Y Y v y ARS a Intensity AU 20 60 80 40 20 degrees Figure 4 2 6 X ray diffraction spectra a silver oxide b silver c CS neutralized at 37 C d h CS neutralized at 37 C with 0 196 d 0 296 e 0 596 f 1 g and 1 5 h of silver 115 Cap tulo 2 Art culo cient fico 2 low intensity at 20 38 04 was found in films containing 1 5 of silver This diffraction peak might be associated with the 111 plane of metallic silver or the 200 plane corresponding to powdered silver oxide because their positions are very close However the greatest intensity peak at 2K 32 7 corresponding to the 111 reflection plane of powdered silver oxide was not observed In fact faint diffuse peaks associated with the crystalline structure of metallic silver can hardly be seen The shape of these peaks suggests the presence of small crystalline silver nanoparticles 3 7 Antimicrobial activity of films neutralized at 37 C The antimicrobial capacity of the resulting films neutralized at 37 C was evaluated against S aureus and E coli It is well known that CS with deprotonated amino g
91. g L This concentration is close to that at saturation 6 10 g L This high concentration could be expected since the original device obtained by immersion in carvacrol should have activity 1 The samples containing chicken showed much lower headspace concentrations the smaller the device the lower the carvacrol concentration differences being significant between samples The concentration measured in the sample with the smallest active film was ca 1 5 10 g L that is the carvacrol activity in the vapour phase was below 0 03 Figure 4 4 10c plots the evolution of carvacrol concentration in the package lid Initially no scalping due to the lid was expected since it consists mainly of aluminium foil However analysis revealed that the adhesive coating was absorbing considerable amounts of carvacrol As the figure shows the surface concentration increased with the device size and with the time of exposure Finally Figure 4 4 10d shows the average concentration measured in the tray walls Even though sampling was made by cutting the tray and analysing the complete PP EVOH PP the results are expressed as if all the carvacrol was accumulated in the internal 200 um PP layer The high barrier imposed by EVOH and the short exposure period validate this hypothesis As in the other regions of the package the carvacrol concentration increased with the size of the active device The mass transport of carvacrol through PP was fully characterized i
92. growth After this time the media were diluted with 0 5 HNOz and the concentration of silver in the samples was quantified by graphite furnace atomic absorption spectroscopy GFAAS with a longitudinal AC Zeeman Analyst 600 Perkin Elmer Madrid Spain equipped with a transversely heated graphite atomizer and a built in fully computer controlled AS 800 autosampler Perkin Elmer The furnace program temperature C ramp time s hold time S employed for silver determination was as follows drying 90 C 10 s 20 s 120 C 10 s 20 s 130 C 5 s 40 s 300 C 5 s 5 s pyrolysis 500 C 10 s 20 s cooling 20 C 10 s 20 s atomization 1400 C 0 s 5s cleaning 2450 C 1 s 5 s Twenty microliters of sample with 10 uL of matrix modifier 0 05 mg of Pd and 0 003 mg of Mg NO3 was injected Triplicate analyses of three independent samples were performed for each defined time 3 Results and discussion 3 1 In situ synthesis of silver based nanoparticles in a chitosan film Films were successfully developed based on silver nitrate as a nanoparticle precursor and CS acting as a polymer carrier and mild silver ion reducing agent The amino groups of CS serve as ligands to complex silver ions at near neutral pH To a lesser extent this polymer can also form complexes with hydroxyl groups This makes CS a chelating polymer with excellent adsorption capacities for silver ions in the preparation for the formation of silver nanoparticles Th
93. had on the amount released from the film A further aim was to ascertain whether after the temperature time treatment the films continued releasing cinnamaldehyde to the medium The results of the two experiments are presented together in Figure 4 3 4 In the first test films were immersed in MHB liquid culture medium and subjected to several preservation treatments Immediately afterwards the films were removed and the liquid was analysed by UV vis spectroscopy Five cinnamaldehyde solutions in MHB were also analysed for calibration As can be seen in Figure 4 3 4 the concentrations of cinnamaldehyde in the cs CScin 49C 30min 659 C 30min 729C 15min 95 C 10min CIN Schiff CIN libre 121 C 5min Figure 4 3 3 ATR FTIR spectra of CS and CScin films after the different preservation treatments 153 Cap tulo 3 Art culo cient fico 3 liquid medium increased with the treatment temperature applied to the films At refrigeration temperature the release was significantly lower than when moderate thermal treatments were applied No differences were observed between samples processed at 65 C for 30 min and at 95 C for 10 min The severe retorting process resulted in a greater release of the agent into the liquid medium The second experiment was carried out on a fatty food stimulant ethanol 5096 which simulates alcoholic foods with an alcohol content of above 20 and oil in water emulsions in accordance with
94. have found applications as natural preservatives In this regard carvacrol 5 isopropyl 2 methylphenol is a constituent of essential oils of oregano and thyme with known antifungal insecticidal antitoxigenic and antiparasitic activities Burt 2004 Veldhuizen et al 2006 Carvacrol is categorized as GRAS Generally Regarded as Safe by the FDA Food and Drug Administration for food Volatile active compounds can be added to films to achieve a more effective and rational use of them This step is especially problematic since a large amount of the compounds is lost or inactivated during processing and the remaining amount in the polymer is not enough to exert its effects on the food In addition the active compound must be chemically compatible with the polymer matrix to allow good dispersion in the film but not inhibit its release Chalier et al 2007 Kurek et al 2012 CS films have been loaded with active volatile compounds for several purposes Abdollahi et al 2012 Altiok et al 2010 However loading of volatiles in a CS matrix presents several difficulties Its incorporation as an additive into the water based CS film forming solution is challenging because of general limited aqueous solubility of volatiles and the inevitable partial loss of the compound by evaporation during the casting and drying of the film In order to overcome these problems Presence of cyclodextrins 177 Cap tulo 4 Art culo cient fico 4 in the CS m
95. in the CS matrix compared with the fresh films that were tested giving rise to a greater migration of silver ions The third study was conducted to verify the long term antimicrobial capacity of the films after immersion in liquid medium 0 2 and 1 596 silver films neutralized at 37 C were immersed in an excess of sterile distilled water at a temperature of 22 C The water was replaced with fresh water every 3 days The antimicrobial capacity of the films was tested at day 1 and after 1 month and the results are shown in Table 4 2 5 The antimicrobial capacity of the films after 1 month of immersion in water remained constant for films containing 0 296 silver whereas the films with a higher silver concentration showed a slight increase in activity These results are similar to 121 Cap tulo 2 Art culo cient fico 2 Table 4 2 5 Antimicrobial effect of films neutralized at 37 C with 0 2 and 1 5 silver against S aureus tested in diluted MHB 1 125 after different immersion times in distilled water Bacterial count Log CFU mL Control 7 39 0 27 Time days 0 2 Ag 1 5 Ag 1 4 50 0 17 4 28 0 21 30 4 46 0 23 3 31 0 29 aP Values within a column followed by a different lower case letter are significantly different from each other Tukey s adjusted analysis of variance P lt 0 05 those obtained in the experiment described above for the antimicrobial activity of 0 2 and 1 596 silver films after immersi
96. in triplicate Finally the following films with the same moisture contents were obtained CS CD films with 3596 of G conditioned at 7596 RH CS CD 35G 75RH CS CD films incorporating 5096 of G conditioned at 6596 RH CS CD 50G 65RH CS CD films with 3596 of PG conditioned at 9096 RH CS CD 35PG 90RH and PVOH CD films with 3596 of G conditioned at 84 RH PVOH CD 35G 84RH 250 Quitosano Carvacrol 2 3 Study of miscibility of monoterpene compounds with the plasticizers G and PG Pyrex tubes were filled with 3 mL of volatile liquid and 3 mL of the plasticizer studied and the solution was homogenized and allowed to stand at room temperature 23 C The solutions were observed for liquid liquid phase separation The experiments were done in triplicate 2 4 Sorption method for loading monoterpenes into CS and PVOH films Films with different matrix compositions as described in section 2 2 were immersed in different pure volatile liquids at 23 C and the amount of the compound sorbed in the film was measured over time until sorption equilibrium was reached 2 5 Determination of monoterpene sorbed in a film The amount of volatile liquid in a film was determined by thermal desorption coupled to gas chromatography using a Dynatherm Thermal Desorber Model 890 891 Supelco Teknokroma Barcelona Spain connected in series to the column of an HP5890 gas chromatograph Series II Plus Agilent Technologies Barcelona Spain via a heated trans
97. inicial de carvacrol de 230 40 adheridas en la cara interna de la tapa y conservadas a 4 C En este caso las pel culas de CS no presentaron actividad antimicrobiana puesto que no mantuvieron contacto directo con el alimento y la efectividad de las pel culas de CS con carvacrol dependi de la cantidad de carvacrol aportada por las pel culas y del tiempo de almacenamiento en refrigeraci n Para observar un efecto antimicrobiano significativo al menos se precisaron pel culas de tama os de 4 8 cm con tiempos de exposici n largos Mientras que las pel culas de 24 cm produjeron efectos antimicrobianos significativos durante todo el periodo de conservaci n Se observa que los niveles de bacterias aerobias mes filas se mantuvieron entre 5x10 y 5x10 CFU g de carne en todos los casos en el d a indicado como la fecha de caducidad por el proveedor Pero nicamente los sistemas de envasado con pel culas de 4 8 y 24 cm mantuvieron estos niveles hasta 3 d as despu s de dicha fecha En el caso de Pseudomonas y 285 Discusi n general bacterias psicr filas en general todas las muestras ensayadas se caracterizaron por tener recuentos m s bajos de los esperados Ciertamente las muestras ensayadas no presentaron en ning n caso olores desagradables ni limo como consecuencia del crecimiento microbiano Debido a las propiedades de alta barrera del envase el crecimiento de Pseudomonas pudo verse desfavorecido en beneficio de
98. isomers and carvacrol moreover the presence of a methoxyl group in ortho position gives rise to intramolecular hydrogen bonding with the hydroxyl group Knauth and Sabbah 1990 which could depress its capacity to release its proton and thus inhibit its antimicrobial activity The presence of the propenyl chain in isoeugenol increased its hydrophobicity with respect to guaiacol and it had a higher log P 2 45 vs 1 19 and greater antimicrobial activity The antimicrobial capacity of isoeugenol was lower than that of molecules with an isopropylphenol structure and also than that of dihydrocarveol carveol and nerol In a comparison of monoterpenes with 263 Cap tulo 4 Art culo cient fico 7 similar structures but possessing a cyclohexanol or phenol group molecules with a methylisopropylphenol structure cumenol isomers and carvacrol showed higher activity than compounds with a methylisopropenylcyclohexa nol dihydrocarveol isopulegol or methylisopropenylcyclohexenol carveol structure This highlights the above mentioned importance of the phenol group in the antimicrobial activity of the molecule Isoeugenol nerol and carveol showed similar antimicrobial activity whereas it was slightly higher for dihydrocarveol and lower for myrtenol With regard to the compounds derived from para methylisopropenylcyclo hexanol isopulegol and dihydrocarveol the microorganisms presented lower sensitivity against isopulegol than against dihydro
99. la celulosa con el grupo hidroxilo en posici n C2 sustituido por un grupo acetamido Dutta et aL 2004 Kurita 2006 Anualmente alrededor de 10 toneladas de quitina son producidas por la biomasa Tharanathan y Kittur 2003 La quitina se encuentra como componente estructural y de soporte en el exosqueleto de animales invertebrados como los artr podos insectos ar cnidos o crust ceos an lidos moluscos cefal podos celent reos o braqui podos en paredes celulares de hongos micelios y esporas y de levaduras Kurita 2006 Rinaudo 2006 Tharanathan y Kittur 2003 aunque la principal fuente industrial de la quitina son las gambas langostinos y cangrejos La obtenci n del CS se muestra en la Figura 1 2 El proceso industrial de obtenci n del CS m s empleado es el m todo qu mico resultando cadenas de diferentes pesos moleculares y grados de desacetilaci n que determinan las propiedades y funcionabilidad del CS Shahidi y Abuzaytoun 2005 As se puede adquirir comercialmente CS de bajo 50000 190000 Da medio 190000 310000 Da y alto peso molecular 310000 375000 Da En la presente Tesis Doctoral se ha trabajado con CS de bajo peso molecular con un 75 85 de desacetilaci n A diferencia de la quitina el CS es soluble en soluciones cidas de cidos org nicos con una solubilidad limitada en cidos inorg nicos y casi insoluble a pH alcalino y neutro Su pK se encuentra en torno a 6 3 pK 6 2 6 8 seg n autores
100. la rotaci n libre de este grupo que reduce el di metro efectivo de la cavidad Astray et al 2009 Del Valle 2004 El tomo de ox geno del grupo hidroxilo del C2 de la unidad glucopiran sica forma un enlace por puente de hidr geno con el prot n del grupo hidroxilo del C3 de la unidad adyacente Szejtli 1998 El Tabla 1 2 Estructura qu mica y caracter sticas de las CDs naturales Del Valle 2004 Szejtli 1998 a CD Propiedades N mero de unidades de 6 7 8 glucopiranosa Peso molecular g mol 972 1135 1297 Solubilidad en agua a 25 C 145 0 4 1 85 0 4 23 2 0 4 9 100mL 5 0 85 0 3 Forma del cristal paralelogramos prismas placas hexagonales ae monoclinicos cuadrangular Altura A 7 9 01 ASO 7 95 0H Di metro exterior 14 6 154 17 5 Di metro interior 4 7 5 3 6 0 6 5 7 5 8 3 Volumen interior A 174 262 427 Hidr lisis por a amilasa de one Tun EH A oryzae Mol culas de agua en 6 11 17 interior de cavidad 22 Introducci n interior del cono est revestido por los hidr genos de C3 y C5 y los ox genos glucos dicos Los pares electr nicos no compartidos de los puentes de ox geno glucos dicos hacen que haya una elevada densidad electr nica y le confieren cierto car cter de base de Lewis al interior de la CD Por todo ello las CDs se caracterizan por tener una cavidad interior relativamente apolar y un exterior polar como se muestra en la Figura 1 5 Del Valle 2004 Szejtl
101. lado las nanopart culas de plata constituyen un dep sito de iones plata que pueden ser liberados con el tiempo a trav s de la oxidaci n de la plata met lica de la superficie de la nanopart cula en presencia de humedad Gibbins y Warner 2005 Wei et al 2009 Algunas aplicaciones de las nanopart culas de plata se han resumido en la Tabla 1 3 y diferentes estudios que incorporan nanopart culas de plata en envases biopolim ricos alimentarios pueden observarse en el Anexo A Sin embargo el uso de la nanotecnolog a en el sector alimentario es restringido El riesgo potencial de los nanomateriales en la salud del hombre as como en el medio ambiente es algo desconocido Dowling 2004 Sharma et aL 2009 Y hay que tener presente que adem s de estas fuentes intencionales o sistemas nanotecnol gicos descritos las nanopart culas se encuentran en 1 las fuentes naturales como el polvo volc nico en la mayor a de las aguas naturales 0 2 0 3 ug L suelos y sedimentos alimentos 10 100 pg kg en menor medida en el aire ng m y 2 las fuentes accidentales como subproductos de combusti n y cocci n emisi n de veh culos pilas de combustibles y diferentes procesos industriales OMS 2003 Smita et al 2012 Actualmente no existe una legislaci n espec fica sobre la nanotecnolog a salvo en algunos colorantes filtros UV en productos Tabla 1 3 Aplicaciones de las nanopart culas de plata Rai et al 2009 Sharma et al 20
102. lo que un disefio adecuado y atractivo es fundamental como reclamo publicitario Asimismo es el medio de comunicaci n entre la empresa productora y el consumidor ya que contiene mucha informaci n denominaci n lista de ingredientes al rgenos cantidad neta del alimento fecha de duraci n m nima o fecha de caducidad condiciones especiales de conservaci n y o de utilizaci n nombre o la raz n social y la direcci n del operador o del importador modo de empleo e informaci n nutricional obligada por la legislaci n UE 2011d La trazabilidad es otra funci n primordial del envase ya que se han desarrollado diversos sistemas para el seguimiento de la distribuci n y compra como son c digo de producto universal los c digos de barras o los c digos QR Brody et al 2008 Marsh y Bugusu 2007 Sin embargo en los ltimos a os debido al desarrollo tecnol gico y los cambios en el estilo de vida de los consumidores se han desarrollado sistemas de envasado de alimentos con nuevas funciones como es el envase activo 1 1 Envasado activo Los sistemas convencionales de envasado de alimentos est n dise ados para contener y proteger a los alimentos de forma pasiva es decir que no interact an con el alimento actuando nicamente como una barrera f sica e inerte entre el alimento y su entorno y tratando de retrasar o bloquear los efectos negativos del entorno en el alimento Sin embargo desde hace algunos afios ha surgido un nuevo c
103. obtained for these films after 10 h of immersion in the medium the amount of total silver both ionic and elemental in the medium increased with time levels of 58 60 57 135 170 256 and 354 pg L silver were found in the growth medium after 1 2 10 24 48 168 and 360 h respectively However the antimicrobial capacities of the films did not change These results could be explained by the fact that the amount of free silver ions available in the medium would be lower than the amount of total silver Some of the migrating silver ions might be reduced to elemental silver over time or might not be available because of their interaction with proteins present in the medium In the second study films with 0 2 and 1 596 silver were immersed in 1 125 diluted MHB the medium being replaced with fresh medium every 24 h to avoid possible saturation of the system The medium was collected at 1 3 5 10 15 and 30 days and the antimicrobial activity was evaluated against S aureus After 30 days the antimicrobial activity of the films was also evaluated Table 4 2 4 shows that the antimicrobial capacity of the culture medium where 0 2 or 1 5 silver films were immersed produced a reduction of 3 5 log and this activity was maintained throughout the 30 days of the 120 Quitosano Nanopart culas de plata Table 4 2 4 Antimicrobial activity of diluted MHB 1 125 which has been in contact with films containing 0 2 or 1 596 silver for 24 h being
104. on fresh chicken breasts This antimicrobial activity was increased by the incorporation of LAE in the matrix successfully reducing common food spoilage microorganisms to control microbial deterioration of packaged fresh chicken Silver nitrate precursor was incorporated into the film forming chitosan solution to generate silver nanoparticles in situ during neutralization of the chitosan acetate films The films showed in vitro antibacterial activity maintained over time Silver nanoparticles could act as silver ion reservoirs for sustained release of silver ions in an aqueous environment The third mechanism for incorporating the antimicrobial agent was reversible anchorage of volatile cinnamaldehyde to preformed chitosan films by the formation of a Schiff base This covalent bond was hydrolysed after application of various common food preservation temperature time treatments in an aqueous medium releasing the cinnamaldehyde The imino chitosan films increased the microbiological safety of milk Milk with the released cinnamaldehyde was sensorially accepted The last method employed was based on the incorporation of antimicrobial monoterpenes especially carvacrol in chitosan films with adjuvants by immersing the preformed films in the volatile liquid agent The combined presence of hydroxypropyl B cyclodextrin glycerol and water in the chitosan films resulted in a high capacity for sorption of lipophilic carvacrol Release of the carvacrol prese
105. preference test the samples were ordered from 1 to 5 1 to assign the greatest acceptance of sample and 5 the lowest Data analysis was 145 Cap tulo 3 Art culo cient fico 3 performed with the Compusense five program release 5 0 Compusense Inc Guelph Ontario Canada 2 7 Data analyses Statistical tests were performed using the SPSS Statistics computer program version 19 0 SPSS Inc Chicago IL USA One way analysis of variance was carried out Differences between pairs of means were assessed on the basis of confidence intervals using the Tukey b test Moreover comparisons between two samples were analysed by Student s t test The level of significance was P lt 0 05 The data are represented as average standard deviation The data were analysed and plotted using the SigmaPlot 10 0 software Systat Software Inc Richmond CA USA 3 Results and discussion The unmodified CS films were transparent without discontinuities and with an average thickness of 55 5 um Figure 4 3 1 shows the formation of CS cinnamaldehyde Schiff base Covalent bonding of the volatile aldehyde to the backbone of the polymer stabilizes the molecule avoiding losses during processing and storage of the polymer film Since the compound is reversibly attached to the polymer it can be liberated through hydrolysis of the imino bond Bond cleavage in aqueous or humid media can be promoted by several factors such as light pH temperature etc The
106. quality and shelf life of foods a review Journal of Food Science 72 R87 R100 OECD SIDS 2001 SIDS Initial Assessment Report for 11th SIDS Initial Assessment Meeting 1 2 dihydroxypropane UNEP Publications OECD SIDS 2002 SIDS Initial Assessment Report for 14th SIDS Initial Assessment Meeting Glycerol UNEP Publications OMS 1999 Cortex Cinnamomi In WHO Monographs on selected medicinal plants World Health Organization G nova pp 95 104 OMS 2003 Silver in drinking water Background document for development of WHO Guidelines for Drinking water Quality Ginebra Suiza WHO SDE WSH 03 04 14 OMS 2008 Guidelines for drinking water quality 3 edici n incorporando la 1 y 2 edici n en anexos Volumen 1 Recomendaciones G nova Ouattara B Simard R E Piette G Begin A Holley R A 2000 Inhibition of surface spoilage bacteria in processed meats by application of antimicrobial films prepared with chitosan International Journal of Food Microbiology 62 139 148 Petersen K Vaeggemose Nielsen P Bertelsen G Lawther M Olsen M B Nilsson N H Mortensen G 1999 Potential of biobased materials for food packaging Trends in Food Science amp Technology 10 52 68 PlasticsEurope 2013 Plastics the Facts 2013 An analysis of European latest plastics production demand and waste data Consultic marketing and industrieberatung GmbH Prashanth K V H Tharanathan R N 2007 Chitin chitosan
107. quitosano quitosano di xido de titanio celulosa ze na quitosano alginato de calcio celulosa acetato prote na de pescado gelatina de piel de pescado medio de cultivo medio de cultivo medio de cultivo medio de cultivo medio de cultivo medio de cultivo pollo RTE agua medio de cultivo E coli E coli Bacillus Klebsiella pneumoniae B subtilis E coli St aureus Penicillium spp Aspergillus spp Rhizopus spp levaduras St aureus E coli St aureus E coli E coli Ps aeruginosa Proteus mirabilis S Enteritidis Enterobacter aerogenes St aureus St epidermidis Corynebacterium spp Enterococcus faecalis C albicans C parapsilosis S Typhimurium St aureus E coli L monocytogenes Ps aeruginosa Sanpui et al 2008 Vimala et al 2010 Li et al 2010 Xiao et al 2013 Zhang et al 2010 Wang et al 2004 Akbar y Anal 2014 Chaurasia et al 2010 Arfat et al 2014 p ptidos antimicrobianos bacteriocinas dermaseptin S4 enterocina lactocinas nisina nisina nisina nisina lisozima nisina EDTA lisozima nisina nisina natamicina nisina nisina nisina nisina lacticina 3147 almid n de ma z alginato ze na gluten de trigo agar alginato de calcio alb mina de huevo gluten de trigo prote nas de suero de leche prote nas de soja alginato de calcio alginato de calcio alginato de
108. sodio K carragenato almid n de tapioca almid n de tapioca caseinato de sodio caseinato de sodio K carragenano HPMC metilcelulosa quitosano celulosa pepino medio de cultivo jam n cocido loncheado medio de cultivo salchicha vienesa piel de pollo medio de cultivo carne de vacuno salm n ahumado medio de cultivo medio de cultivo queso Port Salut medio de cultivo queso Mini Babybel medio de cultivo jamon cocido queso Cheddar ME TVC hongos L monocytogenes L innocua Lb plantarum S Typhimurium L monocytogenes Brochothrix thermosphacta L monocytogenes S Anatum Micrococcus luteus L innocua S Enteritidis E coli St aureus L innocua Saccharomyces cerevisiae L innocua L monocytogenes L innocua Micrococcus luteus L innocua St aureus ME TVC LAB Miltz et al 2006 Marcos et al 2007 2010 Massani Botana et al 2014 Massani Molina et al 2014 Natrajan y Sheldon 2000 Ko et al 2001 Cutter y Siragusa 1996 1997 Datta et al 2008 Cha et al 2002 Sanjurjo et aL 2006 Resa et al 2014 Kristo et al 2008 Cao Hoang et al 2010 Cha et al 2003 Scannell et al 2000 301 Anexos nisina nisina nisina nisina lisozima EDTA nisina nisina nisina nisina nisina nisina nisina nisina benzoato de sodio nisina benzoato de sodio sorbato de potasio
109. solution was added to 25 mm diameter sterilized filter paper Blanks prepared by adding 10 mL of DMSO to 25 mm diameter sterile filter disks showed that the DMSO did not have any effect against any of the microorganisms tested Once the Petri dishes and lids had been assembled 204 Quitosano Carvacrol the units were sealed with Parafilm to reduce leakage of the volatile agent and incubated at 37 C for 24 h At the end of the incubation period the antimicrobial activity was determined by measuring the diameter in millimetres of the zone below the filter papers where there was no microbial growth The minimum inhibitory concentration MIC is defined as the lowest concentration of active compound that yields inhibition of microorganism Delaquis et al 2002 Hammer et al 1999 Each assay was performed in triplicate 2 6 3 Antimicrobial activity of the films The procedure to determine the antimicrobial activity of the films was similar to that described above In this case films of the same size as the filter papers and loaded with carvacrol were placed in the lid of the inoculated Petri dishes Each assay was performed in triplicate 2 7 Data analysis Statistical analysis of the results was performed with SPSS commercial software SPSS Inc Chicago Illinois USA A two way analysis was applied to compare the effect of different amounts of G in the same CS or CS CD matrix Additionally one way analysis of variance was carried
110. solutions with different silver nitrate concentrations were prepared corresponding to silver concentrations of 0 1 0 2 0 5 1 and 1 596 g 100 g CS Films were formed by casting on PS plates and dried at 37 C with a RH of 2296 for 48 h The CS acetate films were neutralized with sodium hydroxide to make them insoluble in water at a pH above the pK of CS Hydroxyl ions also accelerate the reduction reaction of silver ions and the formation of silver based nanoparticles by increasing the reducing power of CS 106 Quitosano Nanopart culas de plata For this purpose the films were immersed in a solution of 0 1 M sodium hydroxide for 20 h in a thermostatic chamber and protected from light The effect of the neutralization temperature on the formation of nanoparticles was studied by assaying two temperatures 22 and 37 C After neutralization the films were washed with deionized water and dried in an oven at 37 C Finally the films were stored in a glass desiccator at 22 C and 096 RH prior to use 2 2 Characterization of chitosan silver based nanoparticle films 2 2 1 Film colour The colour of neutralized CS film was measured using a Konica Minolta CM 3500d spectrophotometer set to D65 illuminant 10 observer Film specimens were measured against the surface of a standard white plate and the CIELAB colour space was used to obtain the colour coordinates L lightness black 0 to white 100 a green to red and
111. sulfate and lauric arginate and stored at 4 C Meat Science 71 92 99 Ma Q M Davidson P M Zhong Q X 2013 Antimicrobial properties of lauric arginate alone or in combination with essential oils in tryptic soy broth and 296 reduced fat milk International Journal of Food Microbiology 166 77 84 Martin E M Griffis C L Vaughn K L S O Bryan C A Friedly E C Marcy J A Ricke S C Crandall P G Lary R Y 2009 Control of Listeria monocytogenes by lauric arginate on frankfurters formulated with or without lactate diacetate Journal of Food Science 74 M237 M241 Muriel Galet V L pez Carballo G Gavara R Hern ndez Mu oz P 2012 Antimicrobial food packaging film based on the release of LAE from EVOH nternational Journal of Food Microbiology 157 239 244 Muriel Galet V L pez Carballo G Gavara R Hern ndez Mu oz P 2015 Antimicrobial effectiveness of lauroyl arginate incorporated into ethylene vinyl alcohol copolymers to extend the shelf life of chicken stock and surimi sticks Food and Bioprocess Technology 8 208 217 Nair D V T Nannapaneni R Kiess A Mahmoud B Sharma C S 2014 Antimicrobial efficacy of lauric arginate against Campylobacter jejuni and spoilage organisms on chicken breast fillets Poultry Science 93 2636 2640 66 Quitosano LAE Oladunjoye A Soni K A Nannapaneni R Schilling M W Silva J L Mikel B Bailey R H Mahmoud B S M Sharma C S 2
112. the medium replaced with fresh medium every 24 h for a total period of 30 days and antimicrobial activity of these films after 30 days Bacterial count Log CFU mL Control 7 39 0 27 LEUTE GE VE 0 2 Ag 1 5 6 Ag 1 4 53 0 31 4 28 0 21 3 4 84 0 26 3 88 0 13 5 4 52 0 41 3 54 0 65 10 4 95 0 95 4 14 0 76 15 4 62 0 45 4 02 0 01 30 474 0 35 4 28 0 33 30 film 3 63 0 24 2 87 0 04 Values within a column followed by a different lower case letter are significantly different from each other Tukey s adjusted analysis of variance P lt 0 05 test Films with 0 2 and 1 5 silver were tested after 30 days and produced an inhibition of 3 8 and 4 5 log respectively The antimicrobial activity of the films was slightly higher than that of the liquid This could be explained by assuming that the films exert antimicrobial activity by the release of silver ions to the medium but also by direct contact of the film surface containing ionic silver with the microorganism It is worth noting that the antimicrobial activity of 1 596 silver films which were in contact with the medium for 30 days was almost 1 log higher than that of fresh films This result shows that the release of silver ions from the film had not slowed after 30 days In addition the immersion of 1 596 film in liquid medium for 30 days might promote the formation of a large amount of silver ions on the surface of the nanoparticles embedded
113. through a hermetically closed 500 mL container where the film sample was placed The release of carvacrol from the films was calculated by analysing the amount of carvacrol remaining in the film by thermal desorption 2 5 Analysis of carvacrol in a film The amount of carvacrol in a film was determined by thermal desorption coupled to gas chromatography using a Dynatherm Thermal Desorber Model 890 891 Supelco Teknokroma Barcelona Spain connected in series to the column of an HP5890 gas chromatograph Series II Plus Agilent Technologies Barcelona Spain via a heated transfer line The desorption tube containing the film sample was placed in the desorber chamber which was immediately sealed Conditions for desorption were as follows desorption temperature 210 C transfer line 230 C desorption time 7 min He desorption flow 8 15 mL min The GC was equipped with a TRB5 30 m 0 32 mm 0 25 mm column Teknokroma Barcelona Spain and a flame ionization detector The chromatographic conditions were 260 C 203 Cap tulo 4 Art culo cient fico 5 detector temperature 7 min at 45 C heating ramp to 220 C at 18 C min and 1 min more at 220 C After the analysis the film sample was recovered from the desorption tube and weighed on an analytical balance Voyager V11140 model Ohaus Europe Greifensee Switzerland 2 6 Antimicrobial assays 2 6 1 Bacterial strains and growth conditions Two model microbial strains were
114. was greater This might be caused by the higher solubility of cinnamaldehyde in this simulant The release results show that the films were activated by temperatures 265 C reaching high concentrations of cinnamaldehyde in the medium Films stored at refrigeration temperature produce a sustained release over time The data obtained indicated that the films developed in this work can be used as a reservoir capable of sustained release of cinnamaldehyde over time and as a coadjuvant of preservation treatments 3 3 Antimicrobial activity 3 3 1 In vitro study The antimicrobial activity of the films was studied against a Gram positive bacterium S aureus and a Gram negative bacterium E coli First 155 Cap tulo 3 Art culo cient fico 3 the in vitro effectiveness of the films exposed to various preservation treatments in MHB liquid medium pH 7 was determined Figure 4 3 5 shows the antimicrobial activity of CScin films before and after preservation treatments CS is a known antimicrobial agent positively charged amino groups interact with the negatively charged membrane of bacteria altering the permeability and disrupting DNA replication Coma et al 2002 Zivanovic et al 2005 However the results showed that the prepared CS film did not present substantial antimicrobial activity as was to be expected since the CS films were neutralized and subsequently the amino groups were not protonated Foster and Butt 2011 Shahidi
115. 0 20 13327 16 93 216 00 22 009 E Different letters a c in the same column indicate a statistically significant difference P lt 0 05 comparing different amounts of G in the same matrix CS or CS CD Different letters x and y in the same column indicate a statistically significant difference P lt 0 05 comparing CS and CS CD with the same G content Different letters m o in the same rows indicate a statistically significant difference P lt 0 05 comparing different RHs of conditioning for the same matrix and G content positive effect on the sorption of carvacrol these compounds act as plasticizers decreasing polymer polymer interactions and increasing chain mobility and free volume in the polymer matrix facilitating sorption of carvacrol However the presence of a high water content in the polymer matrix makes the films more polar reducing their affinity for the non polar phenolic compound carvacrol It is worth noting that only CS CD films without G could be conditioned at 9096 RH since when G was added the films conditioned at that humidity were very sticky and could not be handled In general water and G have a similar effect in CS matrices incorporating HP BCDs However retention of carvacrol in the composites ranged from 0 2696 for non plasticized dry film to 21696 for films containing 3596 G and conditioned at 7596 RH the sorption amount being dependent on the water and G content in the films It was expected th
116. 00 Minolta Chroma meter Minolta Camera Co Ltd Osaka Japan The film samples were placed on a white standard plate the results were expressed in accordance with the CIELAB system with reference 202 Quitosano Carvacrol to illuminant D65 and a visual angle of 109 The measurements were performed through a 6 4 mm diameter diaphragm containing an optical glass monitoring L a b chroma po a b and hue ha arctan b a The samples were measured in triplicate by eight measurements in different locations for each film sample 2 4 Loading and release of carvacrol After being conditioned at different RH to achieved the desired water content film samples with various matrix compositions as described in Film preparation section were immersed in liquid carvacrol at 25 C and the amount of the compound sorbed in the film was measured over time until sorption equilibrium was reached For that after a determined period of time depending of the film composition a piece of the film was cleaned with a paper tissue to remove any excess of carvacrol on the film surface and then inserted into an empty desorption tube 11 5 x 0 39 cm LD for thermal desorption The release of carvacrol from the films was evaluated as a function of time at 25 C and at three RH 43 2 0 4 52 9 0 2 and 90x 296 For this purpose a flow of air of 200 mL min was bubbled in a saturated salt solution to provide the desired RH ASTM 2007 and passed
117. 00 uL of the exponential phase culture of each microorganism Only films neutralized at 37 C were tested Previously a set of films were first autoclaved to study the effect of moist heat at gt 100 C on their antimicrobial activity This study was done because some silver ions could remain in the matrix after the formation of nanoparticles and hydrothermal treatment could lead to chemical reduction and possibly modify their antimicrobial activity Samples weighing 0 25 g were then cut into 1 5 cm pieces and added to each tube A control film of 108 Quitosano Nanopart culas de plata neutralized CS without the incorporation of silver nitrate was used as a blank in each experiment The tubes were then incubated at 37 C for 18 h Depending on the turbidity of the tubes serial dilutions with peptone water were made and plated in Petri dishes with 15 mL of TSA culture medium Colonies were counted after incubation at 37 C for 18 h 2 4 Antimicrobial activity of films neutralized at 37 C in liquid media over time Three sets of experiments were carried out with films having 0 2 and or 1 596 w w of silver and neutralized at 37 C to study the antimicrobial capacity of the films over time The first experiment studied how the immersion time of the film in the growth medium prior to inoculation with bacteria affects its antimicrobial activity For this purpose 0 25 g of film was immersed in 10 mL of 1 125 diluted MHB for 0 2 10 24
118. 0022 Fax 34 963636301 Funding We thank the Spanish MICINN for financial support through Projects AGL 2009 08776 and Ingenio Consolider CSD2007 00063 and the JAE program from CSIC L H fellowship Notes The authors declare no competing financial interest Acknowledgments We thank Dr Dinoraz V lez for technical assistance with graphite furnace atomic absorption spectroscopy References Ershov B G 1999 Short lived metal clusters in aqueous solutions formation identification and properties Russian Chemical Bulletin 48 1 15 Feng Q L Wu J Chen G Q Cui F Z Kim T N Kim J O 2000 A mechanistic study of the antibacterial effect of silver ions on Escherichia coli and Staphylococcus aureus Journal of Biomedical Materials Research 52 662 668 Guerra R Lima E Viniegra M Guzm n A Lara V 2012 Growth of Escherichia coli and Salmonella Typhi inhibited by fractal silver nanoparticles supported on zeolites Microporous and Mesoporous Materials 147 267 273 123 Cap tulo 2 Art culo cient fico 2 Huang J Arthanareeswaran G Zhang K S 2012 Effect of silver loaded sodium zirconium phosphate nanoAgZ nanoparticles incorporation on PES membrane performance Desalination 285 100 107 Kim J K Ahn H 2008 Fabrication and characterization of polystyrene gold nanoparticle composite nanofibers Macromolecular Research 16 163 168 Kong H Jang J 2008 Antibacterial properties of nov
119. 005 De modo que la utilizaci n de los AE debe ser evaluada y controlada para no alcanzar los niveles que puedan dar lugar a estos problemas Todo ello hace que el empleo de los AE se contemple en alimentos tradicionalmente especiados y de composici n sencilla o se emplee en la tecnolog a de barreras o hurdle technology Bagamboula et al 2004 Burt 2004 Tiwari et al 2009 As hay casos citados en los que los AE empleados a muy bajas concentraciones son efectivos microbiol gicamente y aceptados sensorialmente Chi et al 2006 Du et al 2012 La FDA 2014b en la ltima revisi n mantiene que las especias y condimentos y sus AE y componentes de stos pueden considerarse sustancias GRAS 21CFR182 10 21CFR182 20 21CFR182 40 21CFR182 50 En Europa las sustancias aromatizantes ya est n legisladas CE 2008b y existe una lista de estas sustancias que no representan un riesgo para la salud de los consumidores UE 2012 2013 2014 Los AE son mezclas muy complejas que presentan una gran variabilidad en calidad cantidad y composici n en funci n de factores clim ticos y estacionales composici n del suelo tejido del que se extrae la edad ciclo vegetativo y estr s de la planta Bakkali et aL 2008 Burt 2004 Los AE pueden componerse de m s de sesenta componentes individuales y los componentes principales pueden constituir hasta el 85 mientras que otros componentes est n presentes s lo como trazas Burt 2004
120. 009 Kong et al 2010 Por otra parte existen diferentes propuestas para el mecanismo antimicrobiano frente a hongos interfiriendo en su crecimiento activando respuestas defensivas acumulaci n de quitinasas s ntesis de inhibidores de peptidasas lignificaci n etc quelando diversas sustancias e inhibiendo la esporulaci n y la germinaci n Agull et al 2003 Dutta et al 2009 Kong et al 2010 Como se observa en la Figura 1 1 adem s del grupo amino el CS posee un grupo hidroxilo secundario en la posici n C3 y un grupo hidroxilo primario en la posici n C6 haciendo de ste un pol mero muy vers til Prashanth y Tharanathan 2007 Shahidi et aL 1999 De este modo los grupos hidroxilo originan reacciones inespec ficas generalmente eterificaci n y esterificaci n sulfataci n mientras que el grupo amino causa reacciones espec ficas como la N carboxialquilaci n formaci n de bases de Schiff sustituci n enzim tica etc As se pueden introducir diversos grupos para dise ar un derivado del CS para una determinada aplicaci n Kurita 2006 Prashanth y Tharanathan 2007 Rinaudo 2006 La versatilidad y peculiares caracter sticas del CS y sus derivados permiten su amplia aplicaci n en diversas reas tecnol gicas Tabla 1 1 medicina farmacia diet tica cosm tica biotecnolog a e investigaci n agricultura y medio ambiente tratamiento de aguas e industria de los alimentos papelera textil fotogr fica
121. 011 Development of edible films and coatings with antimicrobial activity Food and Bioprocess Technology 4 849 875 Catal R Gavara R 2001 Nuevos envases De la protecci n pasiva a la defensa activa de los alimentos envasados Arbor CLXVIII 661 109 127 CE 2004 Reglamento CE n 1935 2004 del Parlamento Europeo y del Consejo de 27 de octubre de 2004 sobre los materiales y objetos destinados a entrar en contacto con alimentos y por el que se derogan las Directivas 80 590 CEE y 89 109 CEE CE 2006 Reglamento CE n 1907 2006 del Parlamento Europeo y del Consejo de 18 de diciembre de 2006 relativo al registro la evaluaci n la autorizaci n y la restricci n de las sustancias y preparados qu micos REACH por el que se crea la Agencia Europea de Sustancias y Preparados Qu micos se modifica la Directiva 1999 45 CE y se derogan el Reglamento CEE n 793 93 del Consejo y el Reglamento CE n 1488 94 de la Comisi n as como la Directiva 76 769 CEE del Consejo y las Directivas 91 155 CEE 93 67 CEE 93 105 CE y 2000 21 CE de la Comisi n Diario Oficial de la UE 2006 L396 CE 2008a Reglamento CE n 1333 2008 del Parlamento Europeo y del Consejo de 16 de diciembre de 2008 sobre aditivos alimentarios CE 2008b Reglamento CE n 1334 2008 del Parlamento Europeo y del Consejo de 16 de diciembre de 2008 sobre los aromas y determinados ingredientes alimentarios con propiedades aromatizantes utilizados en los al
122. 013 Synergistic activity between lauric arginate and carvacrol in reducing Salmonella in ground turkey Poultry Science 92 1357 1365 Otero V Becerril R Santos J A Rodr guez Calleja J M Ner n C Garc a L pez M L 2014 Evaluation of two antimicrobial packaging films against Escherichia coli O157 H7 strains in vitro and during storage of a Spanish ripened sheep cheese Zamorano Food Control 42 296 302 Porto Fett A C S Campano S G Smith J L Oser A Shoyer B Call J E Luchansky J B 2010 Control of Listeria monocytogenes on commercially produced frankfurters prepared with and without potassium lactate and sodium diacetate and surface treated with lauric arginate using the sprayed lethality in container SLIC R delivery method Meat Science 85 312 318 Serio A L pez C C Paparella A 2012 Effectiveness of surface treatments with Mirenat N against Listeria monocytogenes in Taleggio and Caciotta cheeses talian Journal of Food Science 24 141 144 Sharma C S Ates A Joseph P Nannapaneni R Kiess A 2013 Reduction of Salmonella in skinless chicken breast fillets by lauric arginate surface application Poultry Science 92 1419 1424 Sharma C S Ates A Joseph P Soni K A Schilling M W Kiess A 2013 Evaluation of antimicrobial effects of lauric arginate on reduction of Salmonella spp in ground chicken International Journal of Food Science and Technology 48 1410 1415 Sommer
123. 02 HO Figura 1 9 Estructura de a cinamaldeh do b cido cin mico c acetato de cinamilo d 4 hidroxibenzaldeh do y e 3 fenilpropionaldeh do Introducci n La elevada electronegatividad del grupo aldeh do conjugado con un doble enlace carbono carbono Figura 1 9 parece ser la principal responsable de la actividad antimicrobiana del cinamaldeh do Dorman y Deans 2000 Holley y Patel 2005 Tales compuestos electronegativos pueden interferir en los procesos biol gicos que involucran la transferencia de electrones y reaccionar con compuestos nitrogenados como las prote nas y cidos nucleicos e inhibir el crecimiento de los microorganismos Dorman y Deans 2000 La relevancia de este grupo aldeh do se ve reflejada en que el cinamaldeh do tiene mayor efecto antimicrobiano que el cido cin mico y el acetato de cinamilo Figura 1 9 mientras que la importancia de la deslocalizaci n electr nica en la cadena carbonada m s larga se demuestra porque el cinamaldeh do es m s eficaz que el 4 hidroxibenzaldeh do y 3 fenilpropionaldeh do Figura 1 9 Chang et al 2001 El AE de canela es com nmente utilizado en la industria de alimentos y fragancias debido a su aroma caracter stico pero ha sido en los ltimos afios cuando ha despertado un gran inter s por su actividad antimicrobiana Por ello el cinamaldeh do tambi n se ha empleado como agente activo para la obtenci n de pel culas antimicrobianas para aliment
124. 03 Antimicrobial properties of basil and its possible application in food packaging Journal of Agricultural and Food Chemistry 51 3197 3207 Vaara M 1992 Agents that increase the permeability of the outer membrane Microbiological Reviews 56 395 411 Valencia Chamorro S A Palou L Del Rio M A P rez Gago M B 2011 Antimicrobial edible films and coatings for fresh and minimally processed fruits and vegetables a review Critical Reviews in Food Science and Nutrition 51 872 900 220 Quitosano Carvacrol Van de Manakker F Vermonden T Van Nostrum C F Hennink W E 2009 Cyclodextrin based polymeric materials synthesis properties and pharmaceutical biomedical applications Biomacromolecules 10 3157 3175 Zaika L L 1988 Spices and herbs their antimicrobial activity and its determination Journal of Food Safety 9 97 118 Zhang X G Wu Z M Gao XJ Shu SJ Zhang HJ Wang Z Li C X 2009 Chitosan bearing pendant cyclodextrin as a carrier for controlled protein release Carbohydrate Polymers 77 394 401 221 4 4 4 Art culo cient fico 6 Antimicrobial packaging of chicken fillets based on the release of carvacrol from chitosan cyclodextrin films Laura Higueras Gracia L pez Carballo Pilar Hern ndez Mufioz Ram n Catal Rafael Gavara International Journal of Food Microbiology 2014 188 53 59 Quitosano Carvacrol Resumen Las peliculas de quitosano ciclodextrina CS CD co
125. 05 comparing different amounts of G in the same matrix CS or CS CD Different letters in the same column x y indicate a statistically significant difference P lt 0 05 comparing CS and CS CD Different letters in the same rows m n indicate a statistically significant difference P lt 0 05 comparing different RHs of conditioning for the same matrix and G content 189 Cap tulo 4 Art culo cient fico 4 property at 53 and 7596 RH Apparently the integrity of the matrices is damaged when films are elongated more than 5096 of their initial length G and humidity produced a similar effect on the tensile strength and modulus of elasticity of both CS and CS CD films However the decline of these properties was more pronounced in the CS CD composites indicating a greater plasticizing capacity It is worth highlighting that the amount of G related to the CS polymer in the composite was double the amount in the plain film 3 7 Sorption of carvacrol Samples of CS and CS CD composite films prepared with and without G and conditioned at various RHs were immersed in carvacrol for three months After this prolonged exposure to carvacrol the films did not break or lose their integrity they were easy to handle and showed an apparently good mechanical resistance Differences within samples were evident by visual inspection since initially colourless composites acquired a yellow green colour depending on the amount of carvacrol sorbed Figure
126. 09 Ojagh et al 2010a b Xing et al 2011 Peng y Li 2014 Perdones et al 2014 G mez Estaca et al 2009 G mez Estaca et al 2010 Anexos AE clavo AE clavo AE or gano AE clavo AE or gano AE c rcuma AE AE hierba lim n hierba lim n AE or gano AE AE aure ima AE tomillo AE ima AE tomillo AE AE ED im n pomelo semilla de pomelo AE or gano AE or gano AE or gano AE or gano AE or gano AE or gano AE tomillo ED c tricos AE or gano prote nas de girasol prote nas de suero de leche quitosano caseinato de calcio almid n de sag alginato de sodio alginato de calcio pur de manzana gelatina goma de mezquite quitosano quitosano alginato de sodio alginato de calcio almid n de boniato almid n quitosano gelatina de pescado pez gato gelatina de pescado carpa plateada quitosano gelatina metilcelulosa gelatina metilcelulosa muc lago de semilla de membrillo pescado dorado hamburguesa de sardina pechugas de pollo queso kashar zanahoria medio de cultivo manzana Fuji troceada trucha Arco iris papaya tomate fresa uva de mesa medio de cultivo medio de cultivo medio de cultivo pescado pez gato carpa china medio de cultivo medio de cultivo trucha Arco iris E coli Lb acidophilus ME TVC bac
127. 09 Silver et al 2006 Tambi n se puede consultar http www nanotechproject org inventories silver medicina y farmacia liberaci n sostenida a partir de materiales m dicos e resinas en odontolog a e recubrimiento de cat teres e instrumental quir rgico injertos vasculares e vendajes y ap sitos para quemaduras Acticoat by Smith amp Nephew cosm tica protectores solares desodorantes industria alimentaria recipientes y utensilios de cocina antimicrobianos superficies antimicrobianas en las plantas de procesado de alimentos preparados higienizantes de frutas y verduras Microdyn amp M jico desinfecci n de agua y filtros agua ej Brita Company Australia industria del envasado aplicado en pollo industria textil e calcetines ropa y calzado deportivo alfombras e textiles para hospitales electrodom sticos e lavadoras Sharp Jap n Samsung Electronics fabricadas por Nanogist EEUU Daewoo Electronics Chile frigor ficos Daewoo Electronics fabricado por Nanogist lavavajillas Hitachi amp Jap n otras e superficies antimicrobianas en inodoros Toto Jap n filtros de aire 29 Introducci n cosm ticos y medicamentos que s tienen en cuenta el tama o de part cula CE 2012 El REACH CE 2006 no contiene disposiciones que se refieran de manera expl cita a las nanopart culas La EFSA 2011 public una Gu a de Orientaci n para evaluar los riesgos de las aplicaci
128. 2004 Cyclodextrins and their uses a review Process Biochemistry 39 1033 1046 Delaquis P J Stanich K Girard B Mazza G 2002 Antimicrobial activity of individual and mixed fractions of dill cilantro coriander and eucalyptus essential oils International Journal of Food Microbiology 74 101 109 DeMerlis C C Schoneker D R 2003 Review of the oral toxicity of polyvinyl alcohol PVA Food and Chemical Toxicology 41 319 326 Donhowe 1 G Fennema O 1994 Edible films and coatings characteristics formation definitions and testing methods Edible coatings and films to improve food quality 1 24 Dorigato A Pegoretti A 2012 Biodegradable single polymer composites from polyvinyl alcohol Colloid and Polymer Science 290 359 370 Dorman H J D Deans S G 2000 Antimicrobial agents from plants antibacterial activity of plant volatile oils Journal of Applied Microbiology 88 308 316 Dowling A P 2004 Development of nanotechnologies Materials Today 7 30 35 Du W X Avena Bustillos R J Woods R Breksa A P McHugh T H Friedman M Levin C E Mandrell R 2012 Sensory evaluation of baked chicken wrapped with antimicrobial apple and tomato edible films formulated with cinnamaldehyde and carvacrol Journal of Agricultural and Food Chemistry 60 7799 7804 39 Introducci n Dutta P K Dutta J Tripathi V S 2004 Chitin and chitosan chemistry properties and applications Jo
129. 2008 Microencapsulation of cinnamon leaf Cinnamomum zeylanicum and garlic Allium sativum oils in B cyclodextrin Journal of Inclusion Phenomena and Macrocyclic Chemistry 60 359 368 169 Cap tulo 4 Estado de la cuesti n Becerril R G mez Lus R Goni P L pez P Ner n C 2007 Combination of analytical and microbiological techniques to study the antimicrobial activity of a new active food packaging containing cinnamon or oregano against E coli and S aureus Analytical and Bioanalytical Chemistry 388 1003 1011 Ben Arfa A Combes S Preziosi Belloy L Gontard N Chalier P 2006 Antimicrobial activity of carvacrol related to its chemical structure Letters in Applied Microbiology 43 149 154 Brasil IM Gomes C Puerta G mez A Castell P rez M E Moreira R G 2012 Polysaccharide based multilayered antimicrobial edible coating enhances quality of fresh cut papaya Lwt Food Science and Technology 47 39 45 Buonocore G G Del Nobile M A Panizza A Corbo M R Nicolais L 2003 A general approach to describe the antimicrobial agent release from highly swellable films intended for food packaging applications Journal of Controlled Release 90 97 107 Burt S A Fledderman M J Haagsman H P Van Knapen F Veldhuizen E J A 2007 Inhibition of Salmonella enterica serotype Enteritidis on agar and raw chicken by carvacrol vapour International Journal of Food Microbiology 119 346 350 C
130. 2009 Fabrication characterization of chitosan nanosilver film and its potential antibacterial application Journal of Biomaterials Science Polymer Edition 20 2129 2144 Tolaymat T M El Badawy A M Genaidy A Scheckel K G Luxton T P Suidan M 2010 An evidence based environmental perspective of manufactured silver nanoparticle in syntheses and applications a systematic review and critical appraisal of peer reviewed scientific papers Science of the Total Environment 408 999 1006 Triebel C Vasylyev S Damm C Stara H Ozpinar C Hausmann S Peukert W Munstedt H 2011 Polyurethane silver nanocomposites with enhanced silver ion release using multifunctional invertible polyesters Journal of Materials Chemistry 21 4377 4383 Twu Y K Chen Y W Shih C M 2008 Preparation of silver nanoparticles using chitosan suspensions Powder Technology 185 251 257 Wei D W Qian W P 2008 Facile synthesis of Ag and Au nanoparticles utilizing chitosan as a mediator agent Colloids and Surfaces B Biointerfaces 62 136 142 Wei D W Sun WY Qian W P Ye Y Z Ma X Y 2009 The synthesis of chitosan based silver nanoparticles and their antibacterial activity Carbohydrate Research 344 2375 2382 98 Quitosano Nanopart culas de plata Yeo S Y Jeong S H 2003 Preparation and characterization of polypropylene silver nanocomposite fibers Polymer International 52 1053 1057 Zapata P A Tamayo L
131. 4 Quitosano Nanopart culas de plata exhibit more than one band depending on their shape Pal et al 2007 Spherical metallic nanoparticles give a symmetrical intense band close to 400 nm whereas the presence of silver oxide in metallic nanoparticles gives broader less intense bands that shift to red Yin et al 2002 In this work the absence of well defined bands could also be associated with the formation of silver oxide during neutralization with sodium hydroxide The typical band associated with the SPR of silver nanoparticles is not observed in the UV visible spectra of films with 0 1 and 0 2 of silver which is probably due to the formation of small nanoparticles greatly dispersed in the matrix 3 6 Structural characterization The crystal structure of CS films neutralized at 37 C was determined by XRD Figure 4 2 6 shows the XRD patterns of films incorporating different amounts of silver and neutralized at 37 C and also the powder diffraction pattern of metallic silver and silver oxide The typical XRD pattern of commercial silver nanoparticle powder diameter 100 nm includes four diffraction peaks at 20 of 38 4 44 5 64 7 and 77 6 corresponding respectively to the 111 200 220 and 311 planes of the facecentered cubic fcc structure of silver The powdered silver oxide sample possesses a simple cubic structure with diffraction peaks at 20 of 32 7 38 54 7 and 65 2 assigned to the reflections
132. 4 4 2 Table 4 4 5 shows the sorption of carvacrol by CS and CS CD films plasticized with different amounts of G 0 20 and 3596 and conditioned at various RHs dry film 53 75 and 90 For the CS films the sorption of carvacrol did not exceed 196 reflecting the low affinity of this hydrophilic polymer for carvacrol The presence of water and G in the CS matrix significantly affected the carvacrol retention capacity It was observed that in the films conditioned at a given RH and thus having a fixed water content sorption of carvacrol increased when the G content increased from 0 to 35 g 100 g dry matter G unplasticized films and films with 3596 G conditioned at 9096 RH retained less carvacrol than those conditioned at 53 and 7596 RH On the one hand water and G have a Figure 4 4 2 CS CD 35G composites conditioned at 7596 RH before left and after right in contact with carvacrol for three months 190 Quitosano Carvacrol Table 4 4 5 Sorption equilibrium of carvacrol in CS and CS CD films at 23 1 C Materials films Sorption of carvacrol g 100 g dry matter 0 RH 53 RH 75 RH 90 RH cs 0 08 0 017 017 0 01 0 19 0 03 0 11 0 01 CS 20G 0 09 0 02 0 23 0 03 0 36 0 01 0 27 0 03 CS 35G 047 0 07 0 96 0 04 0 92 0 077 0 68 0 07 CS CD 0 26 0 02 040 0 01 0 43 E 0 03 9 97 1 19 CS CD 20G 0 34 0 02 643 0 3957 56 84 3 525 CS CD 35G 450
133. 48 168 or 360 h after which bacteria were inoculated in the tubes and the antimicrobial activity was evaluated as described above In the second experiment we determined the antimicrobial activity of the culture medium in which the film was previously immersed To do this 0 25 g of film was immersed in 10 mL of 1 125 diluted MHB the old culture medium being replaced every 24 h with fresh medium The antimicrobial capacity of the replaced culture medium was assayed at 1 3 5 10 15 and 30 days After 30 days the antimicrobial capacity of the film was also tested in fresh culture medium as described above In the third experiment films were immersed in 200 mL of sterile distilled water for a month During this period the water was periodically refreshed to avoid microbial contamination The antimicrobial activity of the films was evaluated at 1 and 30 days films were put in tubes with 10 mL of 1 125 diluted MHB and inoculated with S aureus The antimicrobial activity of the films was studied as described previously 2 5 Release of silver into the liquid culture medium Migration of silver from films neutralized at 37 C to the culture growth medium was studied by immersion of 0 25 g of films of different silver concentrations comprising 0 1 0 2 0 5 1 or 1 596 g 100 g CS in 10 mL of 1 125 diluted MHB for a period of time at 37 C incubation conditions 109 Cap tulo 2 Art culo cient fico 2 resembling those of microorganism
134. 5 2006 del Parlamento Europeo y del Consejo y por el que se derogan la Directiva 87 250 CEE de la Comisi n la Directiva 90 496 CEE del Consejo la Directiva 1999 10 CE de la Comisi n la Directiva 2000 13 CE del Parlamento Europeo y del Consejo las Directivas 2002 67 CE y 2008 5 CE de la Comisi n y el Reglamento CE n 608 2004 de la Comisi n UE 2012 Reglamento de Ejecuci n UE n 872 2012 de la Comisi n de 1 de octubre de 2012 por el que se adopta la lista de sustancias aromatizantes prevista en el Reglamento CE n 2232 96 del Parlamento Europeo y del Consejo se incluye dicha lista en el anexo I del Reglamento CE n 1334 2008 del Parlamento Europeo y del Consejo y se derogan el Reglamento CE n 1565 2000 de la Comisi n y la Decisi n 1999 217 CE de la Comisi n UE 2013 Reglamento UE n 985 2013 de la Comisi n de 14 de octubre de 2013 por el que se modifica y corrige el anexo I del Reglamento CE n 1334 2008 del Parlamento Europeo y del Consejo en lo que se refiere a determinadas sustancias aromatizantes UE 2014 Reglamento UE n 246 2014 de la Comisi n de 13 de marzo de 2014 por el que se modifica el anexo I del Reglamento CE n 1334 2008 del Parlamento Europeo y del Consejo en lo relativo a la retirada de diversas sustancias aromatizantes de la lista de la Uni n UNEP 2011 Towards a green economy pathways to sustainable development and poverty eradication www unep org greeneconomy 286 32
135. 57 44 Introducci n Tharanathan R N Kittur F S 2003 Chitin The undisputed biomolecule of great potential Critical Reviews in Food Science and Nutrition 43 61 87 Tiwari B K Valdramidis V P O Donnell C P Muthukumarappan K Bourke P Cullen P J 2009 Application of natural antimicrobials for food preservation Journal of Agricultural and Food Chemistry 57 5987 6000 Trotta F Zanetti M Camino G 2000 Thermal degradation of cyclodextrins Polymer Degradation and Stability 69 373 379 UE 2011a Recomendaci n 2011 696 UE de la Comisi n de 18 de octubre de 2011 relativa a la definici n de nanomaterial Correcci n de errorres la Recomendaci n 2011 696 UE de la Comisi n de 18 de octubre de 2011 relativa a la definici n de nanomaterial 15 01 2013 UE 2011b Reglamento UE n 10 2011 de la Comisi n de 14 de enero de 2011 sobre materiales y objetos pl sticos destinados a entrar en contacto con alimentos UE 2011c Reglamento UE n 1129 2011 de la Comisi n de 11 de noviembre de 2011 por el que se modifica el anexo II del Reglamento CE n 1333 2008 del Parlamento Europeo y del Consejo para establecer una lista de aditivos alimentarios de la Uni n UE 2011d Reglamento UE n 1169 2011 del Parlamento Europeo y del Consejo de 25 de octubre de 2011 sobre la informaci n alimentaria facilitada al consumidor y por el que se modifican los Reglamentos CE n 1924 2006 y CE n 192
136. 6 3 40 3 6 20 3 6 20 32 8 nerol 54 37 3 8 24 2 9 8 3 0 25 carveol 4 7 22 4 4 29 0 5 3 4 28 dihydrocarveol 5 0 24 3 2 17 3 9 20 3 2 36 isopulegol 1 8 0 8 2 9 12 265 Cap tulo 4 Art culo cient fico 7 volatile liquid in the agar medium The antimicrobial activity of the films observed against S aureus and E coli was similar The films with cumenols and carvacrol produced complete inhibition of bacterial growth against both microorganisms Films loaded with guaiacol produced almost complete microbial inhibition in spite of its low antimicrobial activity this result is in accordance with the greater amount of compound sorbed compared with isoeugenol and non phenolic monoterpenes Isoeugenol produced inhibition zones covering about 60 8096 of the surface of the Petri dish Although this compound presented antimicrobial capacity similar to nerol carveol dihydrocarveol and myrtenol it was sorbed in greater amounts than them The antimicrobial activity of isopulegol was lower than that of all the other compounds studied except guaiacol and it had the lowest sorption values of all the films tested Consequently films with isopulegol did not show antimicrobial activity a higher amount of compound in the films would be needed to produce an inhibitory effect and in the present case the isopulegol content in the films was less than 8 mg the amount of compound required to produce an inhibition effect Table 4 4 13 Except for
137. 7 with 20 35 and 5096 G respectively Therefore an increase in the plasticization level of the film facilitates the entry of sorbate molecules However this behaviour was not observed for CS CD films when the G content increased from 35 to 5096 but it must be taken into account that in the present case the water content of the films was much higher 34 vs 1596 approx thus making the films much more hydrophilic When 3596 G was replaced with PG in the CS CD films maintaining the same water content the sorption of cumenol isomers carvacrol and guaiacol decreased whereas the sorption of the other compounds was not modified Table 4 4 12 Owing to the miscibility of PG with monoterpenes one might expect greater sorption of the volatile liquids The lower capacity of PG to plasticize the polymer matrix compared with G might explain these results As stated above it was demonstrated that sorption was favoured when the polymer film was plasticized by G but an excess of water used as plasticizer caused an increase in film hydrophilicity reducing affinity for less hydrophilic compounds In this regard Figure 4 4 13 shows the effect of water on the sorption of carvacrol in CS CD films plasticized with 3596 PG Water acts as a plasticizer for hydrophilic CS based films favouring the entry of sorbates However an excess of water suppressed sorption of carvacrol 200 200 EE Carvacrol sorption 180 4 e Eq moisture content
138. 7 Antimicrobial paper based on a soy protein isolate or modified starch coating including carvacrol and cinnamaldehyde Journal of Agricultural and Food Chemistry 55 2155 2162 Burt S 2004 Essential oils their antibacterial properties and potential applications in foods A review International Journal of Food Microbiology 94 223 253 Cerisuelo J P Muriel Galet V Bermudez J M Aucejo S Catal R Gavara R Hern ndez Mu oz P 2012 Mathematical model to describe the release of an antimicrobial agent from an active package constituted by carvacrol in a hydrophilic EVOH coating on a PP film Journal of Food Engineering 110 26 37 Conte A Buonocore G G Sinigaglia M L pez L C Favia P D Agostino R Del Nobile M A 2008 Antimicrobial activity of immobilized lysozyme on plasma treated polyethylene films Journal of Food Protection 71 119 125 Crank J 1975 The mathematics of diffusion 2nd ed Clarendon Press Oxford England Chi S Zivanovic S Penfield M P 2006 Application of chitosan films enriched with oregano essential oil on bologna active compounds and sensory attributes Food Science and Technology International 12 111 117 Du W X Olsen C W Avena Bustillos R T Mchugh T H Levin C E Friedman M 2008 Storage stability and antibacterial activity against Escherichia coli O157 H7 of carvacrol in edible apple films made by two different casting methods Journal of Agricultural
139. 85 0 08 1 91 0 20 2 05 0 27 isopulegol 0 30 0 74 0 52 0 03 0 28 0 01 0 92 0 19 isopropyl group hindering hydrogen bonding through hydroxyl groups of o cumenol and the hydrophilic matrix of CS or PVOH In fact swelling and loss of dimensional stability were observed in CS CD 35G films loaded with m cumenol With regard to carvacrol the incorporation of a methyl group in the 3 isopropylphenol structure increased the hydrophobicity of the molecule as given by log P see Table 4 4 11 decreasing its affinity for the film and its absorption in the bulk matrix Substitution of isopropyl in o cumenol by the methoxyl group in guaiacol produced a decrease in sorption values in spite of having a lower log P than carvacrol and ortho and meta cumenol on the basis of which greater affinity for the highly hydrophilic matrices would be expected As in the case of o cumenol it might be thought that the methoxyl group in the ortho position in phenol could hinder formation of hydrogen bridges with polar groups of the polymer matrix However although the isopropyl substituent in o cumenol is bulkier than the methoxyl group in guaiacol the former is sorbed to a greater extent Guaiacol has the ability to form intramolecular hydrogen bonds thus competing with the formation of hydrogen bonds with the polymer matrix and limiting its retention in the polymer Glemza et al 1998 observed suppression in the uptake of o methoxyphenol in acrylic est
140. 9 UNEP 2013 Guidelines for national waste management strategies moving from challenges to opportunities http www unep org ietc Portals 136 Publications Waste 20Management UNEP 20NWMS 20Englis h pdf Urgell Beltran J B Seguer Bonaventura J 2003a New preservatives systems and their use in cosmetic preparations Urgell Beltran J B Seguer Bonaventura J 2003b Use of cationic preservative in food products 45 Introducci n Urgell Beltr n J B Seguer Bonaventura J 2003c Use of cationic surfactants in cosmetic preparations Usta J Kreydiyyeh S Bajakian K Nakkash Chmaisse H 2002 In vitro effect of eugenol and cinnamaldehyde on membrane potential and respiratory chain complexes in isolated rat liver mitochondria Food and Chemical Toxicology 40 935 940 Vanin F M Sobral P J A Menegalli F C Carvalho R A Habitante A M Q B 2005 Effects of plasticizers and their concentrations on thermal and functional properties of gelatin based films Food Hydrocolloids 19 899 907 Veldhuizen EJ A Tjeerdsma Van Bokhoven J L M Zweijtzer C Burt S A Haagsman H P 2006 Structural requirements for the antimicrobial activity of carvacrol Journal of Agricultural and Food Chemistry 54 1874 1879 Vieira M G A Da Silva M A Dos Santos L O Beppu M M 2011 Natural based plasticizers and biopolymer films a review European Polymer Journal 47 254 263 Vojdani F Torres J A 1990
141. 9 CScin 4 C 30min 82 63 2 56 CScin 65 C 30min 81 17 2 12 CScin 72 C 15min 81 06 0 89 CScin 95 C 10min 79 54 0 78 CScin 121 C 5min 78 18 2 54 2B Different letters in the same column indicate a statistically significant difference P lt 0 05 151 Cap tulo 3 Art culo cient fico 3 were considerably modified This difference may be due to the nature of the measurements made Polymer chain relaxation due to water intake occurs during swelling Chemical cinnamaldehyde or physical temperature crosslinking gives rise to a more compact polymer matrix and from a physical point of view water molecules will be more difficult to enter By contrast CA only provides information about the hydrophobicity of the film surface 3 1 5 ATR FTIR ATR FTIR spectra were recorded from the various films obtained in this work Figure 4 3 2 shows the FTIR ATR spectra of samples of CS and CScin before and after the washing procedure The spectra have been maximized with respect to the CS peak at 1 025 cm In comparison with the CS spectra the CScin film spectra presented distinctive features at 690 751 1451 and 1492 cm which correspond to the phenolic group of cinnamaldehyde The 1660 cm peak corresponding to the stretching of the C O bond shows that the aldehyde group is present in the unwashed sample However this band appears as a shoulder in the washed sample indicating that the free cinnamaldehyde is pr
142. 9 Russell 2003 3 4 Antimicrobial activity of LAE CS films Antimicrobial activity of LAE CS films was tested against the selected microorganisms in liquid and solid media Table 4 1 4 presents antimicrobial effectiveness of 1 cm of LAE films against bacteria and yeasts in liquid media It is noteworthy that there was some inhibition of bacteria ca 0 3 log units by CS film due the cationic nature of the non neutralized polysaccharide film Shahidi et al 1999 Films with 1 of LAE increased the bacterial growth inhibition to 1 1 5 log reductions Relevant reductions were 82 Quitosano LAE observed on samples containing CS 5 LAE and CS 10 LAE films The growth of Gram negative bacteria was reduced 2 5 logs by CS 5 LAE films and 4 8 logs by CS 1096LAE films The inhibition caused in the growth of Gram positive bacteria was even larger 4 and 6 log reductions for the CS 596LAE and CS 10 LAE films respectively In the case of yeast CS film produces a large inhibition 5 logs whereas the presence of LAE in films produced total growth inhibition There was probably a synergistic effect of LAE and CS both known to be yeasts inhibitors Dai et al 2010 Shahidi et al 1999 Table 4 1 5 shows antimicrobial activity of 1 cm of LAE films against bacteria yeasts and fungi on solid media It is noteworthy to mention that CS films inhibition only occurred in the area of direct contact with the film since there was no diffusion of antimic
143. 90 Li B Kennedy J F Peng J L Yie X Xie B J 2006 Preparation and performance evaluation of glucomannan chitosan nisin ternary antimicrobial blend film Carbohydrate Polymers 65 488 494 319 Anexos Li B Peng J L Yie X Xie BJ 2006 Enhancing physical properties and antimicrobial activity of konjac glucomannan edible films by incorporating chitosan and nisin Journal of Food Science 71 C174 C178 Li L H Deng J C Deng H R Liu Z L Li X L 2010 Preparation characterization and antimicrobial activities of chitosan Ag ZnO blend films Chemical Engineering Journal 160 378 382 Lian Z X Ma Z S Wei J Liu H 2012 Preparation and characterization of immobilized lysozyme and evaluation of its application in edible coatings Process Biochemistry 47 201 208 Lim G O Hong Y H Song K B 2008 Incorporating grapefruit seed extract into Gelidium corneum whey protein isolate blend packaging film increases the shelf life of fish paste Research note Journal of Food Science and Nutrition 13 370 374 Lim G O Hong Y H Song K B 2010 Application of Gelidium corneum edible films containing carvacrol for ham packages Journal of Food Science 75 C90 C93 Lim G O Jang S A Bin Song K 2010 Physical and antimicrobial properties of Gelidium corneum nano clay composite film containing grapefruit seed extract or thymol Journal of Food Engineering 98 415 420 Liu L S Fin
144. Aquesta activitat antimicrobiana es va incrementar en incorporar el LAE a la matriu reduint satisfactoriament els microorganismes presents en l aliment per al control de la deterioraci microbiana de pollastre fresc envasat En segon lloc es va incorporar el precursor nitrat de plata a la soluci film gena de quitosa per a generar in situ nanopart cules de plata durant la neutralitzaci de les pel licules d acetat de quitos Les pell cules desenvolupades van presentar una activitat antibacteriana in vitro mantinguda en el temps Les nanopart cules de plata van poder actuar com a reservoris d ions de plata alliberats sostingudament en un entorn aqu s El tercer mecanisme per incorporar l agent antimicrobi va ser l ancoratge reversible del volatil cinamald hid a pel licules preformades de quitos mitjancant la formaci d una base de Schiff Aquest enllac covalent va ser hidrolitzat despr s de l aplicaci de diversos tractaments de temperatura temps habituals en la conservaci d aliments en un medi aqu s alliberant se el cinamald hid Les pel l cules iminoderivades van augmentar la seguretat microbiol gica de la llet La llet amb el cinamald hid alliberat va ser acceptada sensorialment L ltim m tode emprat es va basar en la incorporaci de monoterpens antimicrobians principalment carvacrol en pel licules de quitos amb coadjuvants mitjan ant la immersi de les pel licules preformades en l agent vol til en estat liqui
145. CS CD Active films produced an antimicrobial effect that depended on the size of the film and the storage time the larger the active device the greater the microbial inhibition observed which became more evident at longer exposure times However an increase in the device size produced an increase in the concentration of carvacrol retained in the chicken which affected sensory attributes hampering its application for this product in the present form Further studies are needed to optimize the films developed such as its application with other hurdle technologies such as modified atmosphere packaging or with other food products Acknowledgments The authors are grateful for financial support from the Spanish Ministry of Science and Innovation project AGL2012 39920 C03 01 EU Nafispack project 212544 and CSIC European Social Fund JAEPredoc L H fellowship and to Mr Karel Clapshaw translation services 239 Cap tulo 4 Art culo cient fico 6 References Appendini P Hotchkiss J H 2002 Review of antimicrobial food packaging nnovative Food Science amp Emerging Technologies 3 113 126 ASTM 2007 Standard practice for maintaining constant relative humidity by means of aqueous solutions ASTM International West Conshohocken PA Aymerich T Picouet P A Monfort J M 2008 Decontamination technologies for meat products Meat Science 78 114 129 Ben Arfa A Preziosi Belloy L Chalier P Gontard N 200
146. CS films was analysed by determination of water CA The water CA of neutralized CS films was 78 3 2 1 Table 4 3 6 similar to the result reported by Vallapa et al 2011 The CA is defined as the angle between the surface of a liquid in this work water and the tangent line at the point of contact with the substrate The value of the CA depends mainly on the relationship between the adhesive forces between the liquid and the solid and the liquid cohesive forces CS films were modified with a hydrophobic molecule cinnamaldehyde and therefore higher CA and lower wettability would be expected After grafting of cinnamaldehyde to the films the surface became more hydrophobic since the CA experienced a slight increase with a value of 82 73 0 59 but the differences were not significant The CA of films subjected to thermal treatments experienced a reduction which increased slightly with treatment temperature Thus films treated at 121 C for 5 min presented CA values close to those of neutralized CS films This was probably because as the temperature of the treatment increases more cinnamaldehyde is lost from the film surface and it becomes more hydrophilic However the CA differences were not statistically significant It should be pointed out that covalently anchored cinnamaldehyde to CS films barely changed the CA values but swelling properties of the films Table 4 3 6 Contact angle of films CONNECT cs 78 03 2 01 CScin 82 73 0 5
147. EDTA lisozima EDTA quitosano ze na ze na ze na alb mina de garbanzos alb mina de suero bovino cultivo queso mozzarella medio de cultivo medio de cultivo medio de cultivo hamburguesa de vacuno medio de cultivo Ps fluorescens hongos levaduras E coli O157 H7 L monocytogenes Lb plantarum B subtilis E coli L monocytogenes S Typhimurium coli O157 H7 ME TVC coliformes B subtilis E coli Duan et al 2007 Brown et al 2008 Mecitoglu et al 2006 nalan et al 2011 Mecitoglu et al 2007 aceites esenciales e AE ajedrea AE canela AE or gano AE ajo AE ajo AE clavo AE or gano AE ajo AE or gano AE ajo AE or gano AE pimienta de Jamaica AE ajo AE ajo xido de zinc nano AE albahaca AE alcaravea AE alga roja Gelidium corneum AE rbol del t AE cilantro AE laurel AE romero AE tomillo blanco AE rbol del t AE rbol del t EO prop leos resveratrol AE rbol del t alginato de calcio alginato de calcio prote nas de desechos de merluza prote na de suero de leche pur de tomate quitosano quitosano prote na de pescado gelatina de piel de pescado pululano prote na de harina de semilla de girasol prote na de suero de leche quitosano quitosano quitosano carne de vacuno mortadela jam n fileteados medio de cultivo medio de cultivo
148. Gu H Y 2008 Construction application and biosafety of silver nanocrystalline chitosan wound dressing Burns 34 623 628 Murugadoss A Chattopadhyay A 2008 A green chitosan silver nanoparticle composite as a heterogeneous as well as micro heterogeneous catalyst Nanotechnology 19 Nassar M A Youssef A M 2012 Mechanical and antibacterial properties of recycled carton paper coated by PS Ag nanocomposites for packaging Carbohydrate Polymers 89 269 274 Perkas N Amirian G Dubinsky S Gazit S Gedanken A 2007 Ultrasound assisted coating of nylon 6 6 with silver nanoparticles and its antibacterial activity Journal of Applied Polymer Science 104 1423 1430 Pinto RJ B Fernandes S C M Freire C S R Sadocco P Causio J Neto C P Trindade T 2012 Antibacterial activity of optically transparent nanocomposite films based on chitosan or its derivatives and silver nanoparticles Carbohydrate Research 348 77 83 97 Cap tulo 2 Estado de la cuesti n Potara M Jakab E Damert A Popescu O Canpean V Astilean S 2011 Synergistic antibacterial activity of chitosan silver nanocomposites on Staphylococcus aureus Nanotechnology 22 Regiel A Irusta S Kyziot A Arruebo M Santamar a J 2013 Preparation and characterization of chitosan silver nanocomposite films and their antibacterial activity against Staphylococcus aureus Nanotechnology 24 Rhim J W Hong SI Park H M
149. H Release of carvacrol from the films was greatly affected by RH The films showed antimicrobial activity against Staphylococcus aureus and Escherichia coli after 20 days of storage at 25 C and 43 environmental RH These films could be useful in the design of systems for delivering active volatiles Keywords Chitosan hydroxypropyl B cyclodextrins carvacrol loading and release antimicrobial films 198 Quitosano Carvacrol 1 Introduction Delivery systems based on polymers capable of carrying and delivering a continuous supply of biologically active molecules into a specific environment have become of increasing interest in recent years These systems are able to reduce the amount of active agent required for treatment by maintaining an effective concentration in the system applied over a certain period of time Ouattara et al 2000 There is a great need for these devices in diverse technological applications encompassing multidisciplinary areas such as biomedicine pharmacology agriculture packaging food technology textiles and the cosmetic industry for the entrapment and delivery of drugs enzymes nutraceuticals agrochemicals flavours and fragrances biocides etc Currently most of the recently developed delivery systems consist of natural and synthetic polymers polymer blends and composites of organic and inorganic materials that form membranes capsules or micelles depending on the application required Issues conc
150. H and 25 C 214 Table 4 4 9 Antimicrobial activity produced by carvacrol in vapour phase against S aureus and E coli 215 Table 4 4 10 Antimicrobial activity of CS CD films loaded with carvacrol against S aureus and E coli 216 Table 4 4 11 Physico chemical properties and molecular structure of liquid volatiles plasticizers and polymers 255 Table 4 4 12 Sorption capacity of films based on CS or PVOH incorporating HP BCD in 1 1 weight ratio and plasticized with G or PG for monoterpenes at 23 C 96 g 100 g dry film 258 Table 4 4 13 Minimum amount of volatile liquid required to produce growth inhibition in vapour phase against S aureus and E coli 263 XX ndice de tablas y figuras Table 4 4 14 Antimicrobial activity of films 25 mm diameter surface after loading with monoterpenes measured by the microatmosphere method against S aureus and E coli 265 Tabla 5 1 Pel culas antimicrobianas de CS 273 Tabla 5 2 Propiedades del CS en cada uno de los sistemas de envasado antimicrobiano desarrollado 288 NDICE DE FIGURAS Figura 1 1 Estructura qu mica del CS 11 Figura 1 2 Obtenci n de la quitina CS y sus derivados 12 Figura 1 3 Obtenci n de PVOH 18 Figura 1 4 Efecto del grado de hidr lisis y peso molecular sobre propiedades de inter s en el PVOH 19 Figura 1 5 Representaci n esquem tica de la estructura qu mica de las CDs 23 Figura 1 6 Representaci n esquem tica de la formaci n de complejos
151. H J 2003 Release of nisin from various heat pressed and cast films Lebensmittel Wissenschaft Und Technologie Food Science and Technology 36 209 213 Cha D S Choi J H Chinnan M S Park H J 2002 Antimicrobial films based on Na alginate and kappa carrageenan Lebensmittel Wissenschaft Und Technologie Food Science and Technology 35 715 719 Chafer M S nchez Gonz lez L Gonzalez Martinez C Chiralt A 2012 Fungal decay and shelf life of oranges coated with chitosan and bergamot thyme and tea tree essential oils Journal of Food Science 77 E182 E187 Chalier P Ben Arfa A Preziosi Belloy L Gontard N 2007 Carvacrol losses from soy protein coated papers as a function of drying conditions Journal of Applied Polymer Science 106 611 620 Chaurasia V Chand N Bajpai S K 2010 Water sorption properties and antimicrobial action of zinc oxide nanoparticles loaded cellulose acetate films Journal of Macromolecular Science Part a Pure and Applied Chemistry 47 309 317 Chen M C Yeh G H C Chiang B H 1996 Antimicrobial and physicochemical properties of methylcellulose and chitosan films containing a preservative Journal of Food Processing and Preservation 20 379 390 Chen M J Weng Y M Chen W L 1999 Edible coating as preservative carriers to inhibit yeast on Taiwanese style fruit preserves Journal of Food Safety 19 89 96 Chen W X Jin T Z Gurtler J B Geveke D J Fan X T 2012
152. J Barron F H Bunn J M Testin R F 2000 Water vapor transmission rates and sorption behavior of chitosan films Journal of Food Science 65 1175 1179 194 4 4 3 Art culo cient fico 5 Incorporation of hydroxypropyl f cyclodextrins into chitosan films to tailor loading capacity for active aroma compound carvacrol Laura Higueras Gracia L pez Carballo Rafael Gavara Pilar Hern ndez Mufioz Food Hydrocolloids 2015 43 603 611 Quitosano Carvacrol Resumen Se han desarrollado pel culas de quitosano CS con hidroxipropil B ciclodextrinas HP BCD y glicerol G capaces de modular la capacidad de carga y la liberaci n de carvacrol Las pel culas fueron obtenidas por extensi n y evaporaci n del disolvente y acondicionadas a diferentes humedades relativas RH antes de su inmersi n en carvacrol l quido La incorporaci n de las ciclodextrinas en la matriz de quitosano aument ligeramente sorci n de carvacrol y fue necesario utilizar glicerol y agua como coadyuvantes para el control de carga de las pel culas con el vol til Se encontr buena correspondencia entre la retenci n de carvacrol y plastificaci n de la pel cula de glicerol y agua Se evalu la cin tica de sorci n carvacrol de las pel culas se evalu a 25 C Los coeficientes de difusi n de sorci n variaron desde 0 011x10 m s para las pel culas que incorporaban el 35 de glicerol acondicionadas al 0 HR a 19x10 m s para las pel culas q
153. J B Marques P A A P Neto C P Trindade T Daina S Sadocco P 2009 Antibacterial activity of nanocomposites of silver and bacterial or vegetable cellulosic fibers Acta Biomaterialia 5 2279 2289 Pires A C D Soares N D F De Andrade NJ Do Silva L H M Camilloto G P Bernardes P C 2008 Development and evaluation of active packaging for sliced mozzarella preservation Packaging Technology and Science 21 375 383 Pranoto Y Rakshit S K Salokhe V M 2005 Enhancing antimicrobial activity of chitosan films by incorporating garlic oil potassium sorbate and nisin Lwt Food Science and Technology 38 859 865 Pranoto Y Salokhe V M Rakshit S K 2005 Physical and antibacterial properties of alginate based edible film incorporated with garlic oil Food Research International 38 267 272 Qi L F Xu ZR Jiang X Hu CH Zou X F 2004 Preparation and antibacterial activity of chitosan nanoparticles Carbohydrate Research 339 2693 2700 324 Anexos Ramos Garc a M Bosquez Molina E Hern ndez Romano J Zavala Padilla G Terr s Rojas E Alia Tejacal I Barrera Necha L Hern ndez L pez M Bautista Ba os S 2012 Use of chitosan based edible coatings in combination with other natural compounds to control Rhizopus stolonifer and Escherichia coli DH5a in fresh tomatoes Crop Protection 38 1 6 Ramos O L Pereira J O Silva SI Fernandes J C Franco MI Lopes da Silva J A Pintado
154. LA Violet Red Bile Agar coliforms MRS agar lactic acid bacteria and Lyngby Iron agar hydrogen sulfide producing bacteria Colonies were counted after incubation at 30 C for 24 h for mesophiles 10 C for 10 d for psychrophiles 30 C for 5 d for yeasts and fungi 25 C for 24 h for Pseudomonas 37 C for 24 h for coliforms 25 C for 5 d for lactic acid bacteria and 20 C for 3 d for hydrogen sulfide producing bacteria Tests were performed in triplicate 2 8 Data analysis Statistical analysis of the results was performed with SPSS commercial software SPSS Inc Chicago IL USA A one way analysis of variance was carried out for data Differences between means were assessed on the basis of confidence intervals using the Tukey b test at a level of significance of P lt 0 05 Data were represented as average standard deviations Data were 78 Quitosano LAE plotted using the SigmaPlot 10 0 software Systat Software Inc Richmond CA The release of LAE data was analyzed using the Regression Wizard Tool of the SigmaPlot 10 0 software 3 Results and discussion 3 1 Optical properties CS films with LAE were transparent flexible and uniform without discontinuities or visible particles and with an average thickness of 50 3 um As shown in Table 4 1 1 the presence of LAE in the polymer matrix increased significantly the chroma of films with respect to the control sample the concentration of LAE having a non significant e
155. LECULAR Disminuci n Aumento HIDR LISIS E de la solubilidad de la flexibilidad 9 dela resistencia al agua a la sensibilidad al agua de la resistencia a la tracci n del poder de dispersi n de la resistencia a disolventes de la adhesi n a superficies hidrof bicas de la adhesi n a superficies hidrofilicas Figura 1 4 Efecto del grado de hidr lisis y peso molecular sobre propiedades de inter s en el PVOH El PVOH es un pol mero semicristalino y posee excelentes propiedades adhesivas emulsionantes y film genas Presenta fuertes fuerzas intermoleculares creadas por puentes de hidr geno entre los grupos hidroxilo de diferentes cadenas lo que confiere un elevado grado de cohesi n y una reducci n del volumen libre Las pel culas de PVOH pueden ser extra das u obtenidas por disoluci n en agua casting o extensi n y evaporaci n del solvente presentan gran flexibilidad y resistencia mec nica qu micamente son resistentes a grasas aceites y solventes org nicos y solubles en agua Las pel culas de PVOH son excelentes barrera al ox geno en seco consecuencia de su mencionada cristalinidad y cohesi n interna Sin embargo estas propiedades dependen de la humedad que act a como plastificante reduciendo su resistencia mec nica y barrera al ox geno Dorigato y Pegoretti 2012 Goodship y Jacobs 2005 Su principal uso se encuentra en la industria de pl sticos adhesivos pinturas textil agricultura y farmac utica
156. N is the carbon nitrogen ratio The degree of acetylation of CS was 20 396 which was in agreement with the value given by the supplier for low molecular weight CS 15 2596 degree of acetylation The degree of substitution DS defined as the ratio of Schiff base units in relation to free amino groups in CS was estimated following the work of Inukai et al 1998 CAJA a n where C N is the carbon to nitrogen ratio of the CS derivative C N g is the carbon to nitrogen ratio of CS and n is the number of carbon introduced into the modified CS Table 4 3 3 shows the DS in percentage of CS films after Schiff base linkage and of those films after being subjected to different temperature time treatments The DS value indicated a high conversion of amino groups into imine linkages After the films had been subjected to 147 Cap tulo 3 Art culo cient fico 3 Table 4 3 3 Elemental analysis and substitution degree of films Substitution Films N C NOS degree cs 7 33 0 07 40 22 0 07 748 0 04 CScin 6 03 0 04 53 93 0 08 6 64 0 01 7241 0 20 CScin 4 C 30min 5 58 0 30 51 48 0 19 6 77 0 04 65 46 0 59 CScin 65 C 30min 5 76 0 27 52 73 0 41 6 80 0 05 65 09 0 78 CScin 72 C 15min 5 97 0 09 53 07 0 11 6 75 0 03 59 32 0 28 CScin 95 C 10min 6 15 0 16 51 82 0 45 6 92 0 06 53 55 0 66 CScin 121 C 5min 6 29 0 06 53 37 0 03 6 95 0 09
157. Octubre de 2015 Dra Pilar Hern ndez Mu oz Cient fica Titular del Instituto de Agroqu mica y Tecnolog a de los Alimentos Dra Gracia L pez Carballo Colaboradora I D I del Instituto de Agroqu mica y Tecnolog a de los Alimentos Dr Rafael Gavara Clemente Profesor de Investigaci n del Instituto de Agroqu mica y Tecnolog a de los Alimentos CERTIFICAN Que D Laura Higueras Contreras licenciada en Ciencia y Tecnolog a de los Alimentos ha realizado bajo nuestra direcci n en el Instituto de Agroqu mica y Tecnolog a de los Alimentos IATA CSIC el trabajo titulado Quitosano como matriz biopolim rica para el desarrollo de envases activos antimicrobianos de alimentos y presenta esta memoria que constituye su Tesis para optar al t tulo de Doctor por la Universitat de Val ncia Y para que conste a efectos oportunos firman el presente certificado en Paterna el de de 2015 DEAR Firmado Dra Pilar B Firmado Dr Rafael Gavara Clemente A mis padres A vosotros Investigar es ver lo que todo el mundo ya ha visto y pensar lo que nadie ha pensado todav a Albert Szent Gy rgyi 1893 1986 Agradecimientos La realizaci n de una Tesis Doctoral puede ser un largo camino en el que vamos intentando superar problemas y dificultades todo ello impl cito en la labor investigadora Sin embargo afortunadamente tambi n resulta ser un camino de encuentro con personas que te ayudan y anim
158. P BCDs to the films resulted in higher transmittance for wavelengths between 190 and 600 nm and a decrease in opacity as the values of these parameters in Table 4 4 1 show These results correlated well with the L values and were indicative of the previously mentioned dilution effect caused by the addition of HP BCDs to the film matrix 3 2 Morphology In a visual inspection the CS CD composite films were homogeneous with no observed phase separation and with smooth surfaces and high transparency SEM images not shown indicate compact surfaces smooth and homogeneous without pores or discontinuities indicating a good miscibility of the three components biopolymer oligosaccharide and plasticizer No differences were observed between samples which indicates 183 Cap tulo 4 Art culo cient fico 4 high dispersion and solubilisation of the HP BCDs in the CS matrix Films formed without plasticizer showed a similar morphology without pores or cracks 3 3 Thermogravimetric analysis Dry samples were analyzed by TGA to determine the thermal stability of the samples and to detect potential interactions between components Figure 4 4 1 shows the weight loss with temperature dm dT derivative for selected samples including pure HP BCD and CS As can be seen CS CD composites presented two main features at temperatures close to those of their two components The addition of HP BCDs to CS appeared to produce a delay of ca 6 C in CS
159. Pan J S Tangpasuthadol V Kiatkamjornwong S Hoven V P 2011 Enhancing antibacterial activity of chitosan surface by heterogeneous quaternization Carbohydrate Polymers 83 868 875 Wang D An J Luo Q Li X Yan L 2012 Synthesis characterization and application of silver based antimicrobial nanocomposites Nano Antimicrobials Springer pp 47 84 163 Cap tulo 3 Art culo cient fico 3 Zivanovic S Chi S Draughon A F 2005 Antimicrobial activity of chitosan films enriched with essential oils Journal of Food Science 70 M45 M51 164 Quitosano Carvacrol 4 4 Cap tulo 4 Desarrollo de pel culas de quitosano con hidroxipropil B ciclodextrinas y otros coadyuvantes con adaptable capacidad de sorci n y liberaci n de compuestos vol tiles antimicrobianos y su aplicaci n en el envasado activo de alimentos 4 4 1 Estado de la cuesti n Existe una creciente demanda por parte de los consumidores por alimentos m s saludables y libres de aditivos artificiales Una alternativa es el empleo de conservantes de origen natural como son los extractos vegetales y AE En la actualidad se conocen aproximadamente 3000 tipos diferentes de AE de los cuales m s de 300 son empleados como aromatizantes Las especias y hierbas arom ticas se han empleado emp ricamente en remedios caseros y en la conservaci n de alimentos desde la antig edad Actualmente se sabe que diferentes extractos vegetales y AE poseen activid
160. Rydlo T Mor A Polyakov V 2006 Potency evaluation of a dermaseptin S4 derivative for antimicrobial food packaging applications Packaging Technology and Science 19 345 354 Min B J Han LY Dawson P L 2010 Antimicrobial gelatin films reduce Listeria monocytogenes on turkey bologna Poultry Science 89 1307 1314 Min BJ Oh J H 2009 Antimicrobial activity of catfish gelatin coating containing origanum Thymus capitatus oil against Gram negative pathogenic bacteria Journal of Food Science 74 M143 M148 Min S Harris LJ Han J H Krochta J M 2005 Listeria monocytogenes inhibition by whey protein films and coatings incorporating lysozyme Journal of Food Protection 68 2317 2325 Min S Harris L J Krochta J M 2005a Antimicrobial effects of lactoferrin lysozyme and the lactoperoxidase system and edible whey protein films incorporating the lactoperoxidase system against Salmonella enterica and Escherichia coli O157 H7 Journal of Food Science 70 M332 M338 Min S Harris LJ Krochta J M 2005b Listeria monocytogenes inhibition by whey protein films and coatings incorporating the lactoperoxidase system Journal of Food Science 70 M317 M324 Min S Harris L J Krochta J M 2006 Inhibition of Salmonella enterica and Escherichia coli O157 H7 on roasted turkey by edible whey protein coatings incorporating the lactoperoxidase system Journal of Food Protection 69 784 793 Min S Krochta J M 2005
161. S y LAE mantuvieron estos niveles por debajo de 10 incluso tras 8 d as desde la recepci n de la materia prima en el comercio alargando as la vida til del producto Por otra parte los principales microorganismos causantes de alteraci n pertenecen al g nero Pseudomonas siendo las pseudomonas psicr trofas la flora predominante sobre su superficie en canales de aves almacenadas a baja temperatura Cuando su n mero llega a 105 10 UFC g de carne aparecen 274 Discusi n general los olores desagradables y cuando sobrepasan de 10 UFC g se forma el limo F bregas y Toca 1997 ICMSF 2005 Las muestras de pollo control alcanzaron estos niveles el d a indicado como la fecha de caducidad Mientras que las muestras de pollo envasadas con pel culas de CS produjeron una cierta reducci n del recuento de bacterias psicr trofas y Pseudomonas disminuy ndolo por debajo de 10 cuando las pel culas incorporaron LAE esta disminuci n fue mucho m s significativa 10 Asimismo las Enterobacteriaceae coliformes y E coli valoran el grado de contaminaci n de origen fecal F bregas y Toca 1997 estableci ndose legislativamente niveles entre 50 500 CFU de E coli por gramo de carne separada mec nicamente CE 2005 Se observa que las muestras de pollo en el d a de recepci n en el comercio no sobrepasaron estos niveles tal como se exige en la legislaci n La acci n de las pel culas de CS mantuvo los recuentos de coliformes en ni
162. Se emplea como excipiente de medicamentos lentes de contacto blandas y emoliente oft lmico hilo quir rgico bolsas para la ropa sucia industrial y hospitalaria guantes para la protecci n qu mica etiquetas y sellos rehumedecibles espesante de pinturas y lacas adhesivo y apresto en telas pelotas de golf formador de pel culas para qu micos fertilizantes herbicidas desinfectantes tintes colorantes escaladores cosm ticos etc 19 Introducci n Sin embargo debido a su dif cil procesado y su extrema sensibilidad al agua el PVOH tiene pocas aplicaciones como material de envase Se emplea como recubrimientos en papel o pel culas solubles en agua siendo esta ltima la principal aplicaci n en el campo de envases de alimentos Este pol mero est autorizado por la FDA para su uso en contacto con alimentos El PVOH con un grado de hidr lisis de 86 5 a 89 096 est aceptado como aditivo alimentario con INS n 1203 Codex Alimentarius FAO OMS 2004 El PVOH est aprobado como un diluyente en mezclas de aditivos colorantes para la coloraci n de los huevos con c scara 21 CFR 73 1 b 2 para su uso en productos de embalaje de carne y en el envasado de productos de aves de corral por la Divisi n de Inspecci n de Carnes y Aves del USDA DeMerlis y Schoneker 2003 1 4 Coadyuvantes para la obtenci n de las pel culas En el desarrollo de sistemas de liberaci n sostenida a partir de hidrogeles suelen emplearse coady
163. The packages were hermetically sealed and it was confirmed that they provided an infinite barrier to carvacrol The partition of the antimicrobial agent within the food packaging system was analysed The antimicrobial devices rapidly released a large percentage of the agent load amounts that were gained by the adhesive coating of the lid and especially by the chicken fillets The latter were the main sorbent phase with average concentrations ranging between 200 and 5000 mg Kg during the period of storage The microbiota of the packaged fresh chicken fillets mesophiles psychrophiles Pseudomonas spp enterobacteria lactic acid bacteria and yeasts and fungi were analysed and monitored during storage A general microbial inhibition was observed increasing with the size of the active device Inhibition with a 24 cm device ranged from 0 3 log reductions against lactic acid bacteria to 1 8 logs against yeasts and fungi However the large amount of antimicrobial that was sorbed or that reacted with the fillet caused an unacceptable sensory deterioration These high sorption values are probably due to a great chemical compatibility between chicken proteins and carvacrol Keywords Chitosan films hydroxypropyl B cyclodextrins carvacrol antimicrobial active packaging controlled release chicken 226 Quitosano Carvacrol 1 Introduction Antimicrobial packaging systems have received special attention in recent years because they can provi
164. VC Salmonella spp ME TVC hongos levaduras B cereus L innocua L monocytogenes Ayana y Turhan 2009 Hong et al 2009a b Pastor et al 2011 Lim Jang et al 2010 Lim et al 2008 Shin et al 2012 Cha et al 2002 Jang et al 2011 Song et al 2012 Corrales et al 2009 Sivarooban et al 2008 Theivendran et al 2006 Gadang et al 2008 Xu et al 2007 Chiu y Lai 2010 Kim et al 2006 Siripatrawan y Noipha 2012 componentes de aceites esenciales Jin y Gurtler 2011 Gamage et al 2009 Chen et al 2012 Soliman et al 2014 309 Anexos benzaldeh do cinamaldeh do carvacro carvacro metilcinamato carvacro Carvacro Carvacro Carvacro cinamaldeh do Carvacro Carvacro Carvacro Carvacro cinamaldeh do cinamaldeh do citral carvacrol cinamaldeh do c m lico citral c m lico eugenol c m lico cinamaldeh do eugenol cinamaldeh do cinamaldeh do cinamaldeh do cinamaldeh do cinamaldeh do AE tomillo cinamaldeh do c ac tico cinamaldeh do c propi nico cinamaldeh do cinamaldeh do 310 alga roja Gelidium corneum alginato caseinato de calcio caseinato de sodio gelatina prote nas de soja pur de manzana pur de hibisco pur de zanahoria pur de manzana pur de manzana pur de tomate quitosano alginato de calc
165. a hecho que se piense en ellos para el desarrollo de envases activos basados en la liberaci n de sustancias En el Anexo A se sefialan diversos ejemplos de envases activos antimicrobianos que emplean este tipo de biopol meros Adem s de por las ventajas ya descritas estos pol meros naturales se caracterizan por una amplia disponibilidad baja toxicidad biocompatibilidad y su susceptibilidad a la degradaci n enzim tica Entre este tipo de biopol meros destacan los glicopol meros como el quitosano que presenta una baja inmunogenicidad frente a otros biopol meros de origen proteico Bhattarai et al 2010 1 2 1 Quitosano El quitosano CS Figura 1 1 es una familia de aminopolisac ridos cati nicos lineales basados mayoritariamente en unidades de glucosamina 2 amino 2 desoxi B D glucosa unidas mediante enlaces glicos dicos B 124 y obtenido de la N desacetilaci n parcial de la quitina El grado de acetilaci n suele ser inferior al 30 4096 aunque para que el CS sea considerado como tal debe alcanzar al menos un grado de desacetilaci n del 5096 Rinaudo 2006 Shahidi y Abuzaytoun 2005 La quitina el segundo pol mero natural m s abundante de la tierra despu s de la celulosa es un biopol mero lineal de alto peso molecular de 10 Introducci n Figura 1 1 Estructura qu mica del CS poli B 1 gt 4 2 acetamido 2 desoxi B D glucosa N acetil D glucosamina La estructura qu mica de la quitina es la misma que
166. a puede ser activada mediante diferentes est mulos como la temperatura y la RH Chalier et aL 2009 Kurek et al 2014 As hidrogeles como el CS podr a hincharse al sorber el agua presente en el espacio de cabeza del envase plastificarse y facilitar la difusi n y liberaci n de mol culas atrapadas en la matriz como podr an ser los agentes vol tiles Buonocore et al 2003 Mascheroni et al 2011 Por otra parte cabe destacar que el carvacrol posee una elevada actividad antimicrobiana en estado vapor Becerril et al 2007 Ben Arfa et al 2006 Burt et al 2007 Nostro et al 2009 Existen diversos trabajos publicados en los que el carvacrol act a en estado vapor tras ser incorporado en distintas matrices como PP Ramos et al 2013 Ramos et al 2012 PP y PE EVOH Guti rrez et al 2010 L pez et al 2007 y EVOH 167 Cap tulo 4 Estado de la cuesti n Cerisuelo et al 2013 Cerisuelo et al 2012 Muriel Galet et al 2012 Muriel Galet et al 2013 No obstante hasta lo que se ha podido indagar nicamente vila Sosa et al 2012 y Kurek et al 2013 incorporan el AE de or gano o carvacrol en pel culas de CS y prueban su eficacia en estado vapor frente a microorganismos alterantes o pat genos aunque s lo realizan estudios in vitro no lo aplican en el envasado activo de alimentos Es de destacar que la mayor parte de los trabajos existentes en la bibliograf a relacionados con la incorporaci n de l
167. aci n con una base Vojdani y Torres 1990 Por este motivo las pel culas de CS as elaboradas pierden su capacidad antimicrobiana ya que los grupos amino no se encuentran protonados y no interacciona con los microorganismos Foster y Butt 2011 Ouattara et al 2000 Las pel culas de CS est n siendo ampliamente estudiadas en el desarrollo de sistemas portadores y de liberaci n controlada de agentes activos antimicrobianos en el envasado activo de alimentos Anexo A IZ Introducci n 1 32 Polialcohol vin lico El polialcohol vin lico PVOH es un pol mero sint tico e hidr filo proveniente de la hidr lisis controlada total o parcial del poliacetato de vinilo PVA en presencia de un catalizador alcalino tal como se observa en la Figura 1 3 Dependiendo de la cantidad de grupos acetato capaces de ser hidrolizados el PVOH se clasifica comercialmente como de alta 99 media 88 y baja 78 hidr lisis siendo ste el porcentaje molar de unidades repetitivas con grupos hidroxilo Este grado de hidr lisis propiedad m s importante del PVOH define sus propiedades qu micas y f sicas como son la solubilidad en agua y la actividad superficial Dado que la solubilidad en agua es debida a los grupos hidroxilo el grado de solubilidad se puede modificar mediante el control del grado de hidr lisis Tambi n resulta de importancia el peso molecular del pol mero el cual se suele relacionar con la viscosidad de las soluc
168. aci n se caracteriz por ser esf rica con di metro de 5 10 nm mientras que otra fue algo m s irregular con di metro de 30 50 nm Por otra parte se prob la capacidad antimicrobiana de las pel culas de CS con diferentes cantidades de plata y neutralizadas a 37 C en medio 276 Discusi n general de cultivo l quido diluido y sin diluir Puesto que las pel culas de CS fueron neutralizadas y sus grupos amino desprotonados stas perdieron su car cter antimicrobiano Por tanto en este caso la matriz de CS no contribuye a la capacidad antimicrobiana actuando nicamente como matriz La actividad antimicrobiana de las pel culas con nanopart culas de plata es debida a la acci n del agente activo incorporado Se observa que todas las pel culas probadas presentaron la misma capacidad antimicrobiana independientemente de la concentraci n de plata que incorporasen excepto las de menor concentraci n en medio de cultivo sin diluir ya que se prob que las cantidades de plata liberadas por las pel culas ensayadas al medio de contacto fueron iguales Probablemente la plata migrada se encuentre como iones de plata ya que la difusi n de las nanopart culas de plata a trav s de la matriz es dif cil debido a su confinamiento en el pol mero Tal vez se generen iones de plata en la superficie de las nanopart culas Esto se ver a favorecido por el ambiente acuoso en el que las pel culas se encuentran inmersas Adem s el hinchamie
169. acrol measured independently by gravimetry The same procedure was used to determine the carvacrol concentration during chicken storage within each phase of the food package system Briefly samples of tray lid film or chicken were cut and desorbed following the previous procedure Every package was tested three times and three packages were analysed per sample The carvacrol concentration in the package headspace was analysed by sampling 500 uL of gas with a gas tight Hamilton syringe through an 229 Cap tulo 4 Art culo cient fico 6 adhesive septum stuck to the package wall The gas sample was immediately analysed by using a Model 6850 GC Agilent Technologies equipped with a semi capillary RTX 1301 30 m 0 53 mm 0 5 gm column Restek Teknokroma Barcelona Spain The chromatographic conditions were 3 8 mL min He oven at 150 C for 11 min injector and detector temperatures 220 C and 250 C respectively The GC was previously calibrated by injections of a known amount of carvacrol in hexane 2 5 Antimicrobial activity of carvacrol CS films on chicken breast fillets At selected times chicken samples were transferred aseptically and weighed in a sterile Stomacher bag diluted with 25 mL of 0 196 peptonated water Scharlab Barcelona Spain and blended in a Stomacher IUL S L Barcelona for 6 min Ten fold dilution series of the suspensions obtained were made in peptonated water and plated using the following selectiv
170. actically eliminated after washing In both washed and unwashed CScin films there is a strong band at 1633 cm which is assigned to the stretching of the imine group C N of the Schiff base Also part of the cinnamaldehyde bonded to the CS is also released probably because of a partial reversion of the Schiff base reaction A 05 Y z CScin SL CScin CIN Schiff CIN libre gt 7 CIN Figure 4 3 2 ATR FTIR spectra of CS and CS modified with cinnamaldehyde before CScin SL and after washing CScin 152 Quitosano Cinamaldeh do After washing the films were exposed to different temperature time treatments Figure 4 3 3 compares the ATR FTIR spectra for the various samples including pure CS using the 1025 cm band as reference During the treatments there is a partial release of cinnamaldehyde because of the reversibility of the reaction Nevertheless there is a large percentage of cinnamaldehyde still anchored to the CS matrix even after the most severe treatment 121 C for 5 min 3 2 Release of cinnamaldehyde Two experiments were performed to evaluate the cinnamaldehyde released by the films exposed to the different treatments a release to MHB during the treatments and b release to 5096 ethanol during treatments and until 1 h afterwards The main aim of the second experiment was to study the effect that changing the medium to one more compatible with cinnamaldehyde
171. ad antimicrobiana y podr an emplearse como antimicrobianos naturales en los alimentos Entre estos compuestos destaca el carvacrol monoterpeno fen lico y componente del AE del or gano o del tomillo que posee una elevada actividad antimicrobiana de amplio espectro Por ello los AE o sus componentes activos son interesantes en el desarrollo de envases antimicrobianos Los biopol meros como el CS han sido ampliamente utilizados como matrices portadoras de stos Anexo A No obstante la incorporaci n libre de estos compuestos en la matriz polim rica genera elevadas p rdidas durante el procesado de la pel cula mayoritariamente durante la etapa de secado debido a su elevada volatilidad siendo necesario adicionar altas concentraciones de agente en la soluci n formadora de pel cula para que una vez procesada la pel cula quede una cantidad residual capaz de conferir actividad antimicrobiana a la misma As en diversos trabajos se han encontrado p rdidas del 39 9996 del carvacrol adicionado inicialmente en funci n de los distintos m todos de obtenci n de las pel culas de CS y de las condiciones en las que se procesan Chi et aL 2006 Kurek et al 2012 Kurek et al 2013 L pez Mata et al 2013 Perdones et al 2014 Estudios previos llevados a cabo en nuestro laboratorio demostraron p rdidas similares 99 8 98 6 y 9896 cuando se adicion a la disoluci n de CS un 100 de carvacrol respecto a la materia seca en pel culas pl
172. ages to increase food safety Acknowledgments The authors wish to thank the financial support provided by the Spanish Ministry of Science and Innovation project AGL2012 39920 C03 01 and Spanish Research Council CSIC JAEPredoc L H fellowship References Abreu F O M S Oliveira E F Paula H C B de Paula R C M 2012 Chitosan cashew gum nanogels for essential oil encapsulation Carbohydrate Polymers 89 1277 1282 Balaguer M P G mez Estaca J Gavara R Hern ndez Mufioz P 2011a Biochemical properties of bioplastics made from wheat gliadins cross linked with cinnamaldehyde Journal of Agricultural and Food Chemistry 59 13212 13220 Balaguer M P G mez Estaca J Gavara R Hern ndez Mu oz P 2011b Functional properties of bioplastics made from wheat gliadins modified with cinnamaldehyde Journal of Agricultural and Food Chemistry 59 6689 6695 Belletti N Lanciotti R Patrignani F Gardini F 2008 Antimicrobial efficacy of citron essential oil on spoilage and pathogenic microorganisms in fruit based salads Journal of Food Science 73 M331 M338 Burt S 2004 Essential oils their antibacterial properties and potential applications in foods A review International Journal of Food Microbiology 94 223 253 Cocchiara J Letizia C S Lalko J Lapczynski A Api A M 2005 Fragrance material review on cinnamaldehyde Food and Chemical Toxicology 43 867 923 Coma V Martial Gros A Garreau
173. aggregates could enhance the loading of amphiphilic molecules such as carvacrol by plasticized CS films that otherwise present a very low sorption potential Thus formation of CD aggregates could explain the high carvacrol sorption values found for some of the films developed in the present work Moreover the effect of HP BCDs concentration in a CS film on carvacrol sorption capacity was also studied Figure 4 4 5 shows the sorption equilibrium of carvacrol in CS films plasticized with 3596 G and conditioned at 7596 RH and incorporating HP BCDs in CS CD weight ratios of 1 0 1 0 25 1 0 5 1 1 and 1 2 The sorption of carvacrol greatly depended on the HP 208 Quitosano Carvacrol 350 300 4 250 4 200 4 150 4 100 4 Sorption equilibrium of carvacrol dry weight o HR 1 11 1 0 5 1 0 25 CS CD weight ratio Figure 4 4 5 Effect of HP BCD content in a CS CD 35G 75RH film on the sorption capacity of carvacrol at 25 C BCDs content in the film The highest sorption value was obtained for the 1 2 formulation However although 1 2 films were plasticized with G and water they were very brittle and difficult to handle because of the high CD content incorporated into the CS matrix which exerts an anti plasticizing effect Maltodextrins MDs are non cyclic oligosaccharides consisting of linear and branched amylose and amylopectin degradation products These starch derivatives can form complexes wi
174. al 2009 Sharma et al 2010 In this regard increasing attention is being paid to the incorporation of silver nanoparticles in polymer matrices for the development of antimicrobial films and coatings The antimicrobial capacity of the composites formed will depend on the physical and chemical properties of the nanoparticles and also the silver ion release properties of the carrier matrix There is also great interest in the use of synthetic and natural hydrophilic polymers and hydrogels as carriers of silver These materials absorb large amounts of water owing to the presence in their structure of polar groups OH NH CONH COOH Hydrophilic polymers are capable of swelling in a moist environment thus facilitating the diffusion of the active agent through the polymer matrix and its release to the medium in contact with the polymer CS is a biodegradable and biocompatible polymer obtained from biomass and possesses excellent film forming properties which have made it of great interest for technological applications in several areas such as pharmacy medicine agrochemistry and packaging The hydrophilic nature of CS has aroused interest in its use as a sustained release carrier when the release of the retained active compound is required in a moist environment 105 Cap tulo 2 Art culo cient fico 2 Inclusion of silver based nanoparticles as an antimicrobial nanofiller in a CS matrix could be done directly or by using silver nitr
175. al 9596 v v y acidificada en la que estaba disuelto el aldeh do El grado de sustituci n 96 del grupo amino por cinamaldeh do fue pr ximo al 7096 La espectroscop a de infrarrojos por transformada de Fourier con reflexi n total atenuada ATR FTIR revel la formaci n de la base de Schiff entre el quitosano y el cinamaldeh do La hidr lisis del enlace imino y posterior liberaci n del cinamaldeh do se estudiaron despu s que las pel culas fueron sometidas a diferentes tratamientos de temperatura tiempo simulando los m todos de conservaci n de alimentos La cantidad del aldeh do que se mantuvo unido covalentemente a las pel culas se monitoriz por ATR FTIR y el grado de sustituci n se determin mediante an lisis elemental Tambi n se evaluaron ngulo de contacto y los par metros de color de las pel culas de cinamaldeh do imina quitosano y de stas sometidas a los diferentes tratamientos Las propiedades antimicrobianas de pel culas de quitosano base de Schiff se probaron in vitro frente a Staphylococcus aureus y Escherichia coli y en leche inoculada con Listeria monocytogenes La actividad antimicrobiana vari dependiendo del tratamiento aplicado y por tanto del grado de hidr lisis del enlace imino y cinamaldeh do liberado Las pel culas de quitosano base Schiff sometidas a diferentes tratamientos de tiempo temperatura inhibieron el crecimiento de L monocytogenes durante 12 d as bajo condiciones de refrigeraci n y
176. al of Food Science 78 M560 M566 Nam S Scanlon M G Han J H Izydorczyk M S 2007 Extrusion of pea starch containing lysozyme and determination of antimicrobial activity Journal of Food Science 72 E477 E484 322 Anexos Nassiri R Nafchi A M 2013 Antimicrobial and barrier properties of bovine gelatin films reinforced by nano TiO Journal of Chemical Health Risks 3 Natrajan N Sheldon B W 2000 Inhibition of Salmonella on poultry skin using protein and polysaccharide based films containing a nisin formulation Journal of Food Protection 63 1268 1272 Neetoo H Mahomoodally F 2014 Use of antimicrobial films and edible coatings incorporating chemical and biological preservatives to control growth of Listeria monocytogenes on cold smoked salmon Biomed Research International Neetoo H Ye M Chen H Q 2010 Bioactive alginate coatings to control Listeria monocytogenes on cold smoked salmon slices and fillets International Journal of Food Microbiology 136 326 331 Nguyen V T Gidley MJ Dykes G A 2008 Potential of a nisin containing bacterial cellulose film to inhibit Listeria monocytogenes on processed meats Food Microbiology 25 471 478 Norajit K Ryu G H 2011 Preparation and properties of antibacterial alginate films incorporating extruded white ginseng extract Journal of Food Processing and Preservation 35 387 393 Ojagh S M Rezaei M Razavi S H Hosseini S M H 2010a
177. aldeh do buffer were purchased from Scharlab Barcelona Spain Water was obtained from a Milli Q Plus purification system Millipore Molsheim France 2 2 Film preparation 2 2 1 Chitosan films A 1 596 CS w w solution was solubilised in 0 596 w w acetic acid aqueous solution and filtered to eliminate impurities CS acetate films with 55 5 um average thickness were obtained by casting on PS plates dried at 37 C for 48 h and 22 RH CS acetate films were neutralized with 0 1 M sodium hydroxide for 24 h at 37 C to make them insoluble in water After neutralization the CS films were washed with deionised water and dried at 37 C The films were cut into 1 7x1 7 cm samples and stored in amber glass desiccators prior to use at 23 C and with P20 to achieve a RH close to zero in order to avoid the presence of moisture since this can promote Schiff base hydrolysis during the storage and consequently the release of cinnamaldehyde from the films 2 2 2 Schiff base formation A cinnamaldehyde solution was prepared by adding 4 g of cinnamaldehyde to 75 mL of ethanol 9696 v v Then neutralized CS films 2 9 were immersed in the solution and kept in a shaking bath at 60 C for 24 h After that time the samples were washed by dipping and shaking them in ethanol 9696 v v for 24 h at room temperature and this process was performed three times Finally chitosan cinnamaldehyde Schiff base polymer films CScin were stored in an ambe
178. aldehyde Schiff base was determined For this purpose immediately after each treatment the liquid medium was transferred to a quartz cuvette 143 Cap tulo 3 Art culo cient fico 3 and the amount of cinnamaldehyde was measured by UV vis spectroscopy at 221 nm using an Agilent 8453 UV visible spectrophotometer Agilent Barcelona Spain Additionally the release of cinnamaldehyde was determined by measuring specific migration from the polymer into ethanol 5096 a fatty food simulant specified in European law EC Regulation 10 2011 A 1 7x1 7 cm film sample was placed in a glass vial with 7 mL of ethanol 5096 and the vial was closed tightly with a PTFE septum and aluminium caps to constitute a sample After the various treatments Treatment of CScin films at different combinations of temperature time treatments section yielding three vial samples per treatment and exposure time 5 10 15 and 30 min and 1 8 24 and 48 h the vials were opened and the content of cinnamaldehyde in the liquid quantified by UV vis spectroscopy at 221 nm Three vial samples per treatment and exposure time were analysed 2 5 Antimicrobial assays 2 5 1 Culture strains Staphylococcus aureus CECT 86 Escherichia coli CECT 434 and Listeria monocytogenes CECT 934 were obtained from the Spanish Type Culture Collection Valencia Spain Strains were stored in Tryptone Soy Broth TSB Scharlab Barcelona Spain with 2096 G at 80 C until needed For
179. also interact with thiol groups of proteins causing bacterial enzyme inactivation Liau et al 1997 The entry of silver ions through cell walls can cause deposition of proteins in cells Feng et al 2000 Other authors claim that silver ions affect only the membrane surface activating a bacterial defence mechanism Morones et al 2005 The presence of silver ions has an antimicrobial effect but some authors also suggest that small nanoparticles might be bactericidal Kong and Jang 2008 Lok et aL 2006 Sondi and Salopek Sondi 2004 The mechanism of action by which silver nanoparticles have an antimicrobial effect is mainly due to their adhesion to the cell membrane altering its permeability and attacking the respiratory chain Kvitek et al 2008 but they can also penetrate inside bacteria and release silver ions which interact with thiol groups and or phosphates of compounds such as bacterial DNA or protein Silver nanoparticles show a clear antimicrobial capacity compared with silver salts because of their high specific surface which allows a greater area of contact 118 Quitosano Nanopart culas de plata with microorganisms Studies show that the antibacterial effect of silver nanoparticles depends on their size with those between 1 and 10 nm presenting a more direct interaction with bacteria Morones et al 2005 In this work we did not expect migration of nanoparticles from the film given their confinement in the CS mat
180. also shown in Figure 4 4 8 The rate of carvacrol release largely depended on the RH to which the films were exposed It is known that the diffusion of small molecules in hydrophilic polymers such as CS strongly depends on the moisture content of the matrix Chalier et al 2009 Mascheroni et al 2011 at intermediate and high RH hydrophilic materials absorb moisture which triggers the release of volatiles entrapped in their structure Moisture acts as a plasticizer thereby governing the rate of volatile loss The release of carvacrol was greatly accelerated when films were exposed to high RH conditions 9096 compared to those exposed to intermediate RH 43 and 5396 All the desorption curves had a two step pattern In the first step there was a fast release of carvacrol which was more acute for films conditioned at 9096 RH followed by a slower release of the remaining volatile Films conditioned at 9096 RH released 96 of the carvacrol in the first 34 h and took approx 8 days to release 9996 of the initial amount Films conditioned at 5396 RH had released 9496 of the initial amount of carvacrol after 15 days and 1 596 still remained in the film after 23 days Films exposed to 4396 RH released 9596 of the carvacrol content in 22 days and after 62 days the residual amount of volatile was 2 696 Carvacrol diffusion coefficients were calculated for films exposed to different RH by fitting experimental release kinetics data to Equation 2 and the v
181. alues obtained are shown in Table 4 4 8 Increasing the RH at which the 10 43 RH m 53 RH 9096 RH 0 8 o a Carvacrol ratio e P 0 2 4 m VA 004 a cA aA M B m ami am wam m WaW o 5000 10000 15000 20000 25000 30000 Time minutes Figure 4 4 8 Experimental symbols and theoretical continuous line normalized time desorption curves of carvacrol from CS CD 35G 75RH film measured at 25 C as a function of environmental RH 213 Cap tulo 4 Art culo cient fico 5 Table 4 4 8 Diffusion coefficient of carvacrol in CS CD 35G 75RH film at different RH and 25 C 96 RH D m s 10 R 43 1 21 0 04 0 998 53 145 0 08 0 988 90 25 3 2 1 0 968 Different letters in the same column indicated significant differences P lt 0 05 desorption was carried out led to an increase in diffusivity of carvacrol Films exposed to 9096 RH experienced a rapid loss of carvacrol having a diffusion coefficient one order of magnitude greater than that obtained for films exposed to intermediate RH Films conditioned at 43 or 5396 RH had diffusion coefficients with the same order of magnitude Diffusion coefficient values were of the same order of magnitude as those obtained by Mascheroni et al 2011 at 30 C in wheat gluten coated paper containing 1596 wt of carvacrol where the diffusion coefficients ranged from 0 143 x 10 to 2 8 x 10 m s for RH ranging between 60 an
182. ameter of the inhibition zone of S aureus and E coli It is worth mentioning that the antimicrobial activity of carvacrol loaded CS films having different 215 Cap tulo 4 Art culo cient fico 5 amounts of G and water incorporated into the matrix but without the incorporation of HP BCDs was also evaluated The retention capacity of these films was less than 196 carvacrol and none of them exerted antimicrobial activity in vapour phase The addition of HP BCDs to the CS matrix increased sorption of carvacrol and therefore changed the antimicrobial capacity No inhibition zone was observed for carvacrol loaded 1 1 CS CD films without G and conditioned at 0 53 and 7596 RH prior to immersion in carvacrol since the amount of carvacrol in the film did not reach the minimum inhibitory dose of 1 mg However there was observed a reduction in growth density across the Petri dish for films without G and conditioned at 53 and 7596 RH the amount of carvacrol retained by these films was next to 0 6 mg Dry CS CD films plasticized with 2096 G retained less than 0 4 mg of carvacrol thus they did not also experience any antimicrobial activity CS CD films plasticized with 3596 G and conditioned at 7596 and 5396 RH and CS CD films plasticized with 2096 G and conditioned at 7596 RH possessed the greatest content in carvacrol 60 mg and produced complete inhibition of bacterial growth in the Petri dish 85 mm diameter whereas the inhibition halo was red
183. an y a los que hoy tengo que agradecer su amistad y su afecto En primer lugar gracias a mis directores de Tesis que han contribuido a ella de manera decisiva Pilar gracias por brindarme la oportunidad de disfrutar en primer lugar de las pr cticas de empresa y luego del doctorado en el IATA Gracias por ofrecerme la posibilidad de participar en este gran equipo Rafa excelente investigador y mejor persona gracias por encontrar siempre la soluci n m s inteligente a cualquier problema que surja sacar lo mejor de cada uno y ser un ejemplo a seguir Ram n gracias por tu incesante disponibilidad pasi n gusto por el orden y las cosas bien hechas Y c mo no gracias Gracia No hay suficientes palabras que expresen mi gratitud por todo lo que has hecho por m Gracias por tu esfuerzo tiempo y entusiasmo Gracias por ser un espejo en el que mirarme por tu aliento incondicional y sobre todo regalarme tu amistad Siempre tengo la gran suerte de encontrarme buena gente y en Italia encontr la mejor El tiempo de estancias fue breve pero suficiente como para consolidar una enorme amistad Gracias a la Prof Manzoni Manu y Ali Gracias por abrirme literalmente las puertas de vuestras casas por ayudarme tanto en ESE momento tan dif cil y por vuestra presencia en nuestra vida desde entonces Tambi n agradezco su dedicaci n y espacio a Anna y la gente de Bioqu mica Tambi n te agradezco M Jesus tu continuo apoyo esta vez por ser mi tut
184. an en rebanadas medio de cultivo medio de cultivo productoras LAB Enterobacteriaceae L monocytogenes ME TVC Pseudomonas spp L innocua S Enteritidis St aureus E coli O157 H7 Pseudomonas spp ME TVC LAB coliformes Staphylococcus spp Pseudomonas spp L monocytogenes E coli O157 H7 ME TVC LAB Brochothrix thermosphacta Enterobacteriaceae Pseudomonas spp hongos levaduras Rhizopus stolonifer Aspergillus niger Ps aeruginosa St aureus E coli E coli O157 H7 L monocytogenes S Typhimurium Lb plantarum B cereus Yersinia enterocolitica Vibrio cholera Shewanella putrefaciens L monocytogenes E coli Streptococcus agalactiae E coli St aureus Klebsiella pneumoniae Ps aeruginosa L innocua Serratia marcenscens Aeromonas hydrophila Achromobacter denitrificans Alcaligenes faecalis ME TVC mes filos Pseudomonas spp LAB Enterobacteriaceae Brochothrix thermosphacta hongos y levaduras ME TVC mes filos y psicr filos Pseudomonas spp hongos y levaduras Ps putida ME TVC Colletotrichum musae Fusarium spp Lasiodiplodia theobromae hongos levaduras E coli St epidermidis E coli B subtilis Ps aeruginosa L monocytogenes S Typhimurium Tammineni et al 2013 Zinoviadou et al 2009 Royo et al 2010 Oussalah et al 2004 Emiroglu et al 2010 Zivanovic et al 2005 Petrou et al 2012 Dos Santos e
185. and transparency parameters obtained from transmittance T in the UV visible region COLOUR L at Materials films b C h ab lab cs 945 0 17 1 31 0 08 109 04 109405 96 9 0 2 CS 20G 94 3 E08 ll als SO lala Qu 97 1 0 4 CS 35G 947 0 4 1 32 0 09 10 7 0 4 10 8 0 4 97 0 0 3 CS CD 2585202 02 2008 5920 50203 92 7 0 5 CS CD 20G 955 02 0 33 0 05 59 02 259203 93 3 0 3 CS CD 35G 953 03 0 48 0 07 624503 62 03 934204 TRANSPARENCY Materials films Opacity T 96 T 96 Average T AU x nm at 280 nm at 325 nm 400 800nm cs 31 8 1 6 127 14 229 1 8 83 4 0 8 CS CD 26 6 17 36 5 1 7 44 9 1 3 85 8 0 3 No statistically significant differences between means P gt 0 05 were found by Tukey s test when comparing different amounts of G in the same matrix CS or CS CD Different letters x and y in the same column indicate a statistically significant difference P lt 0 05 comparing CS and CS CD Different letters m and n in the same column indicate a statistically significant difference Px0 05 comparing CS and CS CD observed with higher values of L and lower values of a b C and h These results suggest that HP BCDs dilute the CS matrix reducing the colour and increasing the lightness of the composite films Both films had high transmittance greater than 8096 in the visible region indicative of transparent films The addition of H
186. ando la reacci n se lleva a cabo entre el aldeh do y el CS en forma de pel cula y no como disoluci n formadora de pel cula Las pel culas de CS tras reaccionar con el cinamaldeh do adquirieron un color amarillo debido a la formaci n de la base de Schiff insaturada constituyendo el crom foro C2 C CzN A continuaci n estas pel culas se sometieron a diferentes combinaciones de temperatura y tiempo caracter sticas de diferentes tratamientos para la conservaci n de alimentos Estos se realizaron en medio acuoso para promover la hidr lisis de la base de Schiff formada Las pel culas sometidas a tratamientos m s suaves no sufrieron cambios mientras que a mayores temperaturas se tornaron naranja rojizo puede que debido al desarrollo de productos de la reacci n de Maillard formados entre los grupos amino del CS y los grupos carbonilo a elevadas temperaturas El grado de sustituci n y por tanto el contenido en cinamaldeh do en la pel cula fue disminuyendo conforme aument la temperatura de los tratamientos aplicados a las pel culas lo que indica una mayor hidr lisis de la base de Schiff Sin embargo cabe resaltar que incluso con temperaturas de esterilizaci n se mantuvo m s del 50 de los enlaces imino en las pel culas lo que indica un gran reservorio de cinamaldeh do Por otra parte se realizaron estudios de liberaci n en un medio de cultivo l quido durante el tratamiento de las pel culas con diferentes combinacio
187. antidades de sales de plata por lo que aumenta el coste econ mico y ambiental del material En la actualidad las nanopart culas de plata suponen una excelente alternativa y est n siendo ampliamente estudiadas y empleadas en la fabricaci n de pol meros nanocompuestos para el desarrollo de nuevos materiales con propiedades espec ficas Aunque las nanopart culas de plata son de por s antimicrobianas stas tambi n pueden actuar como reservorios de iones plata permitiendo su liberaci n sostenida en el tiempo De este modo la superficie de las nanopart culas de plata met lica se oxida a iones de plata en medio acuoso y con el tiempo las nanopart culas se disuelven liberando los iones de plata antimicrobianos en el medio l quido Por lo tanto otra manera de crear materiales nanocompuestos polim ricos 91 Cap tulo 2 Estado de la cuesti n antimicrobianos es la inmovilizaci n de nanopart culas en una matriz polim rica capaz de liberar los iones de plata oxidada Existen numerosas investigaciones patentes y aplicaciones comerciales en relaci n con las nanopart culas de plata incluidas en matrices polim ricas Sin embargo su aplicaci n en el desarrollo de envases antimicrobianos alimentarios est restringida debido a cuestiones legislativas por la cantidad de plata que puede migrar Generalmente la formaci n de las nanopart culas de plata en soluci n requiere una sal precursora y soluble un agente reductor y un agen
188. arch 2014 Accepted 15 July 2014 Available online 7 August 2014 Keywords Chitosan Hydroxypropyl B cyclodextrins Carvacrol Loading and release Antimicrobial films ABSTRACT Chitosan incorporating hydroxypropyl B cyclodextrins and glycerol films capable of modulating loading capacity and release of carvacrol have been developed Films were obtained by casting and conditioned at different relative humidities RH prior to immersion in liquid carvacrol Incorporation of cyclodextrins in the chitosan matrix slightly increased sorption of carvacrol and it was necessary to use glycerol and water as coadjuvants to control loading of the films with the volatile Good agreement was found be tween carvacrol retention and plasticization of the film by glycerol and water The kinetics of carvacrol sorption by the films was evaluated at 25 C Diffusion coefficients of sorption varied from 0 011 x 10 m s for films incorporating 35 glycerol and conditioned at 0 RH to 19 x 10 m s for films incorporating 35 glycerol and conditioned at 75 RH Release of carvacrol from the films was greatly affected by RH The films showed antimicrobial activity against Staphylococcus aureus and Escherichia coli after 20 days of storage at 25 C and 43 environmental RH These films could be useful in the design of systems for delivering active volatiles 1 Introduction Delivery systems based on polymers capable of carrying and delivering a continuous supply of
189. ared and filtered to eliminate impurities LAE 0 1443 g of Mirenat D was diluted in 50 mL of Milli Q water to obtain a stock of 2000 g mL Different amounts of this stock were added to the film forming solution to obtain films with 1 5 or 1096 LAE g of LAE 100 g of dry CS Considering purity actual LAE concentrations were 0 69396 3 46596 and 6 93096 respectively Also solutions without LAE were prepared and used to produce control films All films were plasticized with 20 G g G 100 g of dry CS Films were prepared by casting on a flat polystyrene PS tray under controlled environmental conditions 36 h 40 0 1 5 C and 20 9 RH 2 2 Optical properties The colour of the CS films was measured with a CR 300 Minolta Chroma meter Minolta Camera Co Ltd Osaka Japan Film samples were placed on a white standard plate results were expressed in accordance with the CIELAB system with reference to illuminant D65 and a visual angle of 10 The measurements were performed through a 6 4 mm diameter diaphragm containing an optical glass monitoring L a b chroma C a a b 5 hue ha arctan b a and total colour difference AE AL Aary Ab 517 Tests were done in triplicate in eight different positions for each film sample 75 Cap tulo 1 Art culo cient fico 1 2 3 LAE release from films Samples of 1 cm of CS film with 1 average weight 0 0110 0 0005 g 5 0 0115 0 0005 g and 1096 0 0120 0
190. arella medio de cultivo manzana pepino tomate medio de cultivo calabaza en conserva fresa O157 H7 S Typhimurium ME hongos levaduras L monocytogenes L monocytogenes E coli O157 H7 S Typhimurium ME TVC LAB hongos levaduras L monocytogenes ME TVC y anaerobios ME TVC P digitatum P italicum P notatum Rhodotorula rubra L monocytogenes ME TVC LAB Pseudomonas spp L monocytogenes ME hongos ME TVC ME TVC LAB lactococos Pseudomonas spp Enterobacteriaceae E coli P expansum Cl herbarum A niger P oxalicum CL cucumerinum P expansum Cl fulvum Zygosaccharomyces bailii ME TVC mes filos LAB hongos levaduras ME TVC mes filos y psicr filos hongos levaduras 2008 Rivero et al 2013 Carlin et al 2001 Cagri et al 2003 Cagri et al 2001 2002 Neetoo y Mahomoodally 2014 Neetoo et al 2010 Baldwin et al 1996 Valencia Chamorro et al 2008 2011 Valencia Chamorro et al 2009a b 2010 Chen et al 1996 Lungu y Johnson 2005a Zinoviadou et al 2010 Kristo et al 2008 Soares et al 2002 Mitrakas et al 2008 Mastromatteo et al 2014 Shen et al 2010 Mehyar et al 2011 Arismendi et al 2013 Flores et al 2007 Flores et al 2010 Garc a et al 2008 Garc a et al 1998 2001 299 Anexos sorbato de potasio quitosano medio de E c
191. arriga M 2010 Physical performance of biodegradable films intended for antimicrobial food packaging Journal of Food Science 75 E502 E507 Martins J T Cerqueira M A Souza B W S Avides M D Vicente A A 2010 Shelf life extension of ricotta cheese using coatings of galactomannans from nonconventional sources incorporating nisin against Listeria monocytogenes Journal of Agricultural and Food Chemistry 58 1884 1891 Mary G Bajpai S K Chand N 2009 Copper II ions and copper nanoparticles loaded chemically modified cotton cellulose fibers with fair antibacterial properties Journal of Applied Polymer Science 113 757 766 Massani M B Botana A Eisenberg P Vignolo G 2014 Development of an active wheat gluten film with Lactobacillus curvatus CRL705 bacteriocins and a study of its antimicrobial performance during ageing Food Additives and Contaminants Part a Chemistry Analysis Control Exposure amp Risk Assessment 31 164 171 Massani M B Molina V Sanchez M Renaud V Eisenberg P Vignolo G 2014 Active polymers containing Lactobacillus curvatus CRL705 bacteriocins effectiveness assessment in wieners nternational Journal of Food Microbiology 178 7 12 Mastromatteo M Conte A Faccia M Del Nobile M A Zambrini A V 2014 Combined effect of active coating and modified atmosphere packaging on prolonging the shelf life of low moisture Mozzarella cheese Journal of Dairy Science 97 36 45
192. arrollo de pel culas h bridas de quitosano con nanopart culas de plata formadas in situ 4 2 1 Estado de la cuesti n Desde la antig edad la plata i nica ha sido empleada como biocida debido a su elevada efectividad y amplio espectro de acci n Actualmente el inter s por la plata est en auge dada la mayor frecuencia de microorganismos cada vez m s resistentes al extenso conjunto de agentes antimicrobianos disponibles sobre todo en el mbito de la medicina y de la conservaci n de alimentos Actualmente los iones de plata se est n incorporando en materiales inorg nicos siendo el intercambio i nico el m todo m s empleado Generalmente se utilizan materiales porosos como por ejemplo las zeolitas Demirci et aL 2014 que liberan lentamente iones de plata y pueden incorporarse a otros materiales formando superficies antimicrobianas La FDA acepta varios de estos dispositivos como sustancias seguras para entrar en contacto con los alimentos Asimismo estos portadores cargados con plata se pueden incorporar en materiales polim ricos a partir de los cuales liberarse zeolitas en poliuretano Kamisoglu et aL 2008 polietileno PE Boschetto et al 2012 o PLA Fern ndez et al 2010 filosilicatos en policaprolactona o ze na Incoronato et aL 2010 o CS Lavorgna et al 2014 y fosfato de sodio y circonio en policaprolactona Duan et aL 2007 o polietersulfona Huang et aL 2012 Sin embargo esta tecnolog a precisa elevadas c
193. ased bioactive films Food Chemistry 168 341 347 Gniewosz M Krasniewska K Woreta M Kosakowska O 2013 Antimicrobial activity of a pullulan caraway essential oil coating on reduction of food microorganisms and quality in fresh baby carrot Journal of Food Science 78 M1242 M1248 G mez Estaca J L pez de Lacey A G mez Guillien M C L pez Caballero M E Montero P 2009 Antimicrobial activity of composite edible films based on fish gelatin and chitosan incorporated with clove essential oil Journal of Aquatic Food Product Technology 18 46 52 G mez Estaca J L pez de Lacey A L pez Caballero M E G mez Guillen M C Montero P 2010 Biodegradable gelatin chitosan films incorporated with essential oils as antimicrobial agents for fish preservation Food Microbiology 27 889 896 Guo M M Jin T Z Wang L X Scullen O J Sommers C H 2014 Antimicrobial films and coatings for inactivation of Listeria innocua on ready to eat deli turkey meat Food Control 40 64 70 317 Anexos Guo M M Jin T Z Yang RJ 2014 Antimicrobial polylactic acid packaging films against Listeria and Salmonella in culture medium and on ready to eat meat Food and Bioprocess Technology 7 3293 3307 Hoffman K L Han LY Dawson P L 2001 Antimicrobial effects of corn zein films impregnated with nisin lauric acid and EDTA Journal of Food Protection 64 885 889 Hong Y H Lim G O Song K B 2009a Antimicro
194. ased nanoparticles increase the thermal stability of the films 3 4 Transmission electron microscopy studies TEM was used to study the size and shape of the nanoparticles generated in films with 0 1 0 2 0 5 1 and 1 5 of silver Panels A and B of Figure 4 2 4 show TEM images of neutralized CS films with silver concentrations of 0 2 and 1 5 respectively The formation of nanoparticles with a spherical morphology and sizes lt 5 nm distributed homogeneously throughout the polymer matrix was observed for films having 0 1 0 5 of silver Films possessing silver concentrations of 1 and 1 5 displayed the formation of spherical nanoparticles of 5 10 nm in size and a second population of round shaped anisotropic nanoparticles with diameters ranging from 30 to 50 nm These larger nanoparticles are probably aggregates formed from smaller ones Figure 4 2 4 TEM images of films containing A 0 2 and B 1 5 of silver and neutralized at 37 C 113 Cap tulo 2 Art culo cient fico 2 3 5 UV visible analysis Figure 4 2 5 shows the UV visible spectra of CS films incorporating various concentrations of silver The figure depicts two spectral bands at 355 and 454 nm Under conditions in which one electron reduction of metal ions occurs predominantly the subsequent aggregation of the resulting atoms and ions gives more or less complex small clusters and then quasimetallic particles These species are associated with the presence of
195. ashington DC US Food and Drug Administration octubre 40 Introducci n DA 2002 FDA agency response letter GRAS Notice nr GRN 000073 Washington DC US Food and Drug Administration enero DA 2005a FDA agency response letter GRAS Notice nr GRN 000164 Washington DC US Food and Drug Administration septiembre FDA 2005b FDA agency response letter GRAS Notice nr GRN 000170 Washington DC US Food and Drug Administration octubre DA 2011 FDA agency response letter GRAS Notice nr GRN 000397 Washington DC US Food and Drug Administration diciembre FDA 2013 FDA agency response letter GRAS Notice nr GRN 000443 Washington DC US Food and Drug Administration febrero DA 2014a FDA Code of Federal Regulations CFR Title 21 Food and Drugs Vol 3 Sec 172 515 Synthetic flavoring substances and adjuvants revisado 01 04 2014 FDA 2014b FDA Code of Federal Regulations CFR Title 21 Food and Drugs Vol 3 Sec 182 10 Spices and other natural seasonings and flavorings 182 20 Essential oils oleoresins solvent free and natural extractives including distillates 182 40 Natural extractives solvent free used in conjunction with spices seasonings and flavorings y 182 50 Certain other spices seasonings essential oils oleoresins and natural extracts revisado 01 04 2014 FDA 2014c FDA Code of Federal Regulations CFR Title 21 Food and Drugs Vol 3 Sec 182 60 Synthetic flavorin
196. astificadas con 0 20 y 3596 de G respectivamente y 165 Cap tulo 4 Estado de la cuesti n secadas en c mara 36h 40 04 1 5 C y 20 9 HR Por lo que hay que recurrir a otras t cnicas para incluir el vol til en la matriz o a coadyuvantes para un uso m s racional y eficaz de estos agentes antimicrobianos En el presente trabajo los vol tiles se incluyen despu s de la formaci n de la pel cula de CS por inmersi n en el l quido vol til para evitar p rdidas del compuesto antimicrobiano durante la etapa de secado de la pel cula Hasta la fecha no se han descrito trabajos similares que empleen dicha t cnica Sin embargo Ou draogo et al 2013 presentaron un estudio con algunas similitudes al desarrollado en la presente Tesis Doctoral ya que determinaron el efecto de la estructura de los monoterpenos a pineno citronelal carvona y terpinen 4 ol en su retenci n en distintas matrices celul sicas tras la impregnaci n de las matrices con los monoterpenos diluidos en acetona o hexano Misharina et al 2006 examinaron las interacciones de diferentes compuestos arom ticos en agua con CS carragenano y almid n Por otro lado es limitado el porcentaje de compuestos ligeramente hidr fobos como los componentes de AE que se puede incorporar en pel culas hidr filas como las formadas de CS Asimismo hay que tener presente que exista cierta afinidad qu mica entre el compuesto a incorporar y la matriz polim rica para favorece
197. at HP BCDs would promote the sorption of carvacrol owing to their ability to form inclusion complexes with non polar molecules Theoretically assuming the formation of 1 1 or 1 2 B CD carvacrol complexes Locci et aL 2004 Ravi and Divakar 2001 the carvacrol content of the composites would range between 5 and 1096 g carvacrol 100 g dry matter As shown in Table 4 4 5 HP BCDs enhanced the retention of carvacrol in the biocomposites compared to plain films Biocomposites without G conditioned at 53 and 7596 RH or in a dry environment and dry biocomposites containing 2096 G presented significant increases in carvacrol gain with respect to CS films but carvacrol retention was below 196 191 Cap tulo 4 Art culo cient fico 4 Biocomposites containing 3596 G and conditioned at 53 or 7596 RH and those having 2096 G and conditioned at 5396 RH retained high levels of carvacrol 133 216 and 5796 carvacrol g 100 g dry matter respectively carvacrol sorption in these biocomposites being much greater than the theoretical sorption expected Assuming that carvacrol is able to form 1 1 and 2 1 inclusion complexes guest HP BCD the theoretical amount of carvacrol expected to be sorbed by the film would be 1096 g carvacrol 100 g dry matter Since amounts of carvacrol above 1096 were found in some films an alternative mechanism to the formation of inclusion complexes must be taking place These results suggest that sorption of carvacrol in CS
198. at pH 7 was reduced by approx 3396 with respect to the same films immersed in acidic solution whereas surface area was reduced by approx 1796 Table 4 3 5 The decrease in water uptake is due to the unprotonated state of the amino groups pH gt pK of CS When cinnamaldehyde was attached to amino groups of CS films water uptake and surface area were reduced by approx 75 and 3596 respectively compared with the values for CS films immersed in the same buffer solution It is worth noting that several authors have also shown the properties of cinnamaldehyde as a crosslinking agent for proteins increasing the strength and reducing the moisture barrier and water holding properties of the films obtained Balaguer et aL 2011a b No significant differences were observed between water sorption and dimensional stability of CScin films and those subjected to thermal treatments The results showed that at pH 7 hydrolysis is not favoured and 150 Quitosano Cinamaldeh do primary amino groups forming imino bonds are not available for interaction with water molecules or thermal crosslinking It is worth mentioning that this study was also conducted at 48 h with relevant results At pH 3 the integrity of the films was lost and therefore the samples could not be handled or measured When the experiment was carried out at pH 7 no differences were found between films immersed in buffer solution for 24 or 48 h 3 1 4 CA Hydrophobicity of modified
199. ata M A Ruiz Cruz S Silva Beltr n N P Ornelas Paz J D Zamudio Flores P B Burruel Ibarra S E 2013 Physicochemical antimicrobial and antioxidant properties of chitosan films incorporated with carvacrol Molecules 18 13735 13753 L pez P S nchez C Batlle R Ner n C 2005 Solid and vapor phase antimicrobial activities of six essential oils susceptibility of selected foodborne bacterial and fungal strains Journal of Agricultural and Food Chemistry 53 6939 6946 L pez P S nchez C Batlle R Ner n C 2007 Development of flexible antimicrobial films using essential oils as active agents Journal of Agricultural and Food Chemistry 55 8814 8824 Mascheroni E Guillard V Gastaldi E Gontard N Chalier P 2011 Antimicrobial effectiveness of relative humidity controlled carvacrol release from wheat gluten montmorillonite coated papers Food Control 22 1582 1591 Messner M Kurkov S V Jansook P Loftsson T 2010 Self assembled cyclodextrin aggregates and nanoparticles International Journal of Pharmaceutics 387 199 208 Misharina T A Terenina M B Krikunova N I Kalinchenko M A 2006 Sorption of components from a mixture of odorants by polysaccharides of starch chitosan and carrageenan Applied Biochemistry and Microbiology 42 111 115 Moreira S P de Carvalho W M Alexandrino A C de Paula H C B Rodrigues M D P de Figueiredo R W Maia G A de Figueiredo E M A
200. ate as a precursor CS is soluble in aqueous solution and has the ability to bind many metal ions including silver via chelation with the amine groups CS has also been reported to be a mild reducing agent used for reduction of silver ions and it is frequently employed as an ion capping agent to control the growth of nanoparticles and avoid their aggregation Murugadoss and Chattopadhyay 2008 The aim of this study therefore was to develop films for the slow sustained release of silver ions consisting of CS polymer as the carrier matrix filled with silver based nanoparticles synthesized in situ The resulting films were characterized and the antimicrobial activity was tested in liquid growth media The ability of the carrier system to exert antimicrobial activity over time was also studied 2 Materials and methods 2 1 Synthesis of chitosan silver based nanoparticle films Low molecular weight chitosan CS MW 50 190 kDa 75 8596 deacetylated from shrimp shells sodium hydroxide ACS reagent 297 096 pellets and silver nitrate ACS reagent 299 096 were obtained from Aldrich Chemical Co Inc Milwaukee WI USA A 1 596 w w CS solution was prepared in 0 596 w w acetic acid and stirred at 40 C for 1 h After the solution had cooled to room temperature silver nitrate previously diluted with a small amount of distilled water was added to the solution and left shaking protected from light until complete dissolution Several CS
201. ational 42 1105 1112 Flores S Haedo A S Campos C Gerschenson L 2007 Antimicrobial performance of potassium sorbate supported in tapioca starch edible films European Food Research and Technology 225 375 384 Flores S K Costa D Yamashita F Gerschenson L N Grossmann M V 2010 Mixture design for evaluation of potassium sorbate and xanthan gum effect on properties of tapioca starch films obtained by extrusion Materials Science amp Engineering C Materials for Biological Applications 30 196 202 316 Anexos Franklin N B Cooksey K D Getty KJ K 2004 Inhibition of Listeria monocytogenes on the surface of individually packaged hot dogs with a packaging film coating containing nisin Journal of Food Protection 67 480 485 Gadang V P Hettiarachchy N S Johnson M G Owens C 2008 Evaluation of antibacterial activity of whey protein isolate coating incorporated with nisin grape seed extract malic acid and EDTA on a turkey frankfurter system Journal of Food Science 73 M389 M394 Gamage GR Park HJ Kim KM 2009 Effectiveness of antimicrobial coated oriented polypropylene polyethylene films in sprout packaging Food Research International 42 832 839 Garc a M Bifani V Campos C Martino M N Sobral P Flores S Ferrero C Bertola N Zaritzky N E Gerschenson L Ramirez C Silva A Ihl M Menegalli F 2008 Edible coating as an oil barrier or active system in Gut
202. atrix could improve compatibility between the polymer matrix and the agent Moreover the volatile could be loaded after film casting to avoid agent losses during the film drying step Cyclodextrins CDs are donut like oligosaccharides with hydrophobic cavities and hydrophilic outer surface They are widely used as excipients in pharmacy to solubilise lipophilic drugs by means of inclusion complex However non inclusion based aspects of CDs are being studied and their importance to solubilisation by formation of self assemble aggregates or surfactant like effects The aim of this work was to develop CS films with a selective carvacrol loading capacity For this purpose biocomposites based on the addition of HP BCD a highly water soluble CD derivative into the CS film forming solution were prepared and their physico chemical properties studied including morphology and optical mechanical and barrier properties The carvacrol loading capacity of the films was also studied as a function of the water and G content 2 Materials and methods 2 1 Materials Carvacrol kosher 9896 and low molecular weight chitosan CS were supplied by Sigma Barcelona Spain Hydroxypropyl B cyclodextrin HP BCD CAVASOL W7 HP was supplied by Wacker Ib rica Barcelona Spain Glycerol G and acetic acid were purchased from Aldrich Barcelona Spain 2 2 Film preparation First a 1 596 CS w w solution in an aqueous 0 5 w w acetic acid solution
203. ature and the decomposition temperature was measured from the first derivative of weight per cent versus temperature DTGA 2 7 Moisture content Samples 0 4 0 5 g of each film were cut into pieces and placed on aluminium plates They were placed in desiccators containing saturated solutions of magnesium nitrate 6 hydrate Sigma Barcelona Spain sodium chloride Scharlau Barcelona Spain and barium chloride 2 hydrate Fluka Madrid Spain in a chamber conditioned at 23 1 C in order to maintain a RH of 53 0 0 5 75 0 0 5 and 90 0 3 0 respectively ASTM 2007 These values were confirmed by direct RH measurements with hygrometers Hygrodynamics Newport Scientific Inc Jessup MD USA After reaching weight equilibrium in approximately 2 weeks they were weighed and placed in desiccators with phosphorus pentoxide Sigma Barcelona Spain for dehydration for 2 more weeks The tests were done in triplicate 2 8 Barrier properties 2 8 1 Water vapour permeability WVP WVP tests were carried out at two RH gradients 0 5396 and 0 7596 and 23 1 C using permeability cups Elcometer Manchester England in accordance with ASTM E96 E96M 10 for flexible films ASTM 2010c To ensure the necessary RH the cups were stored in desiccators containing salt solutions magnesium nitrate 6 hydrate and sodium chloride for 5396 and 7596 RH respectively The cups were weighed daily and the plot of the weight increase vs time provided the wate
204. avalheiro E T G 2005a Synthesis and characterization of Schiff bases from chitosan and salicylaldehyde derivatives Carbohydrate Polymers 60 277 282 Dos Santos J E Dockal E R Cavalheiro E T G 2005b Thermal behavior of Schiff bases from chitosan Journal of Thermal Analysis and Calorimetry 79 243 248 Gamage GR Park H J Kim KM 2009 Effectiveness of antimicrobial coated oriented polypropylene polyethylene films in sprout packaging Food Research International 42 832 839 Guinesi L S Cavalheiro E T G 2006a Influence of some reactional parameters on the substitution degree of biopolymeric Schiff bases prepared from chitosan and salicylaldehyde Carbohydrate Polymers 65 557 561 Guinesi L S Cavalheiro E T G 2006b Influence of the degree of substitution in biopolymeric Schiff bases on the kinetic of thermal decomposition by non isothermal procedure Thermochimica Acta 449 1 7 Guo Z Y Xing RE Liu S Zhong Z M Ji X Wang L Li P C 2007 Antifungal properties of Schiff bases of chitosan N substituted chitosan and quaternized chitosan Carbohydrate Research 342 1329 1332 Han Y P Lin Q 2012 Synthesis characterization and antibacterial activity of quaternized of N aromatic chitosan derivatives Applied Mechanics and Mechanical Engineering li Pts 1 and 2 138 139 1202 1208 131 Cap tulo 3 Estado de la cuesti n Hirano S Hayashi H 2003 Some fragrant fibres and yarns based
205. b blue to yellow The colour was expressed using the polar coordinates L C h and AE where L is the same as above C is chroma h is hue angle and AE AL Aa Ab 7 Eight measurements were taken of each sample and three samples of each film were measured 2 2 2 Thermogravimetric analysis TGA TGA of CS films neutralized at 37 C was carried out using a Mettler Toledo TGA SDTA 851 Samples of approximately 10 mg were heated from room temperature to 800 C at 10 C min and held at an isotherm for 3 min Nitrogen was used as the carrier gas The TGA data were plotted as the weight percentage versus temperature and the decomposition temperature was obtained from the first derivative of weight loss curve DTG 2 2 3 UV Visible spectroscopy The particles generated in CS films neutralized at 37 C were characterized by testing their optical absorption with an Agilent 8453 UV vis diode array spectrophotometer 2 2 4 Transmission electron microscopy TEM The morphology of the nanoparticles generated in CS films neutralized at 37 C was studied using a JEOL 1200 EX transmission electron 107 Cap tulo 2 Art culo cient fico 2 microscope The morphology of the nanoparticles generated in films neutralized at 37 C was studied using a JEOL 1200 EX transmission electron microscope at an acceleration voltage of 100 kV TEM specimens were cut from films using a Porter Blum MT 2B ultramicrotome equ
206. bands around 350 nm Ershov 1999 The conduction electrons on the surface of metallic nanoparticles undergo a collective oscillation when stimulated with incident light This oscillation is known as a surface plasmon resonance SPR and results in strong scattering and absorption properties Silver nanoparticles have a characteristic surface plasmon band around 400 nm in the UV visible spectrum SPR of spherical silver nanoparticles is responsible for the band appearing at 454 nm in Figure 4 2 5 for films neutralized at 37 C It can be observed that this band became stronger with a large asymmetrical broadening as the silver concentration in the film increased This band shape has been associated with the deviation of nanoparticles from a perfect spherical shape and an increase in size distribution which has been previously observed in TEM studies The presence of a shoulder at 570 nm can be observed a phenomenon that has also been reported by other authors According to Mie s theory small spherical nanoparticles should exhibit a single surface plasmon band whereas anisotropic particles should 4 Control 0 1 Ag 37 C _ 454 nm LE 0 2 Ag 37 C E 0 5 Ag 37 C 1 096 Ag 37 C een 1 596 Ag Absorbance UA N T T T T 200 400 600 800 1000 Wavelenght nm Figure 4 2 5 UV visible spectrum of CS films with 0 0 1 0 2 0 5 1 and 1 5 of silver neutralized at 37 C 11
207. bergamota AE hierba lim n AE canela c m lico AE hierba lim n c m lico AE clavo c m lico AE canela AE hierba lim n AE or gano AE canela c m lico AE hierba lim n c m lico AE palmarosa c m lico AE canela AE hierba lim n AE or gano AE canela AE canela AE clavo AE pimienta de Jamaica AE canela AE clavo AE tomillo AE canela AE canela AE canela AE canela AE clavo AE clavo 306 HPMC quitosano quitosano HPMC gelatina de pescado lija Aluterus monoceros alginato de calcio alginato de calcio pur de manzana pur de manzana alginato de calcio amaranto almid n de ma z quitosano prote nas de suero de leche pur de manzana quitosano quitosano quitosano quitosano quitosano cido oleico gelatina gelatina quitosano cultivo naranja Navel Powell medio de cultivo uva Moscatel medio de cultivo lubina manzana Fuji troceada medio de cultivo mel n Piel de sapo pelado medio de cu tivo medio de cu tivo medio de cu tivo medio de cu tivo medio de cu trucha Arco tivo iris azufaifo medio de cultivo medio de cultivo fresa medio de cultivo salm n en rodajas medio de cultivo monocytogenes P italicum P italicum ME TVC mes filos hongos levaduras St aureus E coli L monocytogenes S Typhimurium
208. bial activity 1 log reduction These results are in agreement with the release study described in the previous section Figure 4 3 4 Films not activated by thermal treatment released cinnamaldehyde very slowly owing to the slow reversibility of the Schiff base at neutral pH accompanied by low temperature After treatment at 65 C for 30 min CScin films showed a large log reduction of 5 66 0 04 against S aureus and of 4 76 0 02 against E coli It was also observed that the films treated at 72 C for 15 min 95 C for 10 min and 121 C for 5 min produced a bactericidal effect Therefore the antimicrobial activity is related to the active agent released during the different treatments Films subjected to higher temperatures presented a higher degree of Schiff base hydrolysis and consequently released more cinnamaldehyde increasing their antimicrobial capacity The results of the antimicrobial study show that these films can be very effective when subjected to a thermal treatment At low temperatures cinnamaldehyde imino chitosan films presented extended stability with very slow agent release At mild and sustained heat treatments hot filling or mild pasteurization the release is high enough to produce substantial inhibition of microbial growth More severe heat treatments for short times are much more effective with the films even providing a bactericidal effect 3 3 2 Study of the antimicrobial capacity of CScin films applied to inoculat
209. bial activity of a Gelidium corneum gelatin blend film containing fermented pollen extract and its application in the packaging of pork loins Korean Journal for Food Science of Animal Resources 29 391 395 Hong Y H Lim G O Song K B 2009b Physical properties of Gelidium corneum gelatin blend films containing grapefruit seed extract or green tea extract and its application in the packaging of pork loins Journal of Food Science 74 C6 C10 Hosseini M H Razavi S H Mousavi M A 2009 Antimicrobial physical and mechanical properties of chitosan based films incorporated with thyme clove and cinnamon essential oils Journal of Food Processing and Preservation 33 727 743 Iturriaga L Olabarrieta I Mart nez de Mara n I 2012 Antimicrobial assays of natural extracts and their inhibitory effect against Listeria innocua and fish spoilage bacteria after incorporation into biopolymer edible films International Journal of Food Microbiology 158 58 64 Jagannath J H Nanjappa C Das Gupta D Bawa A S 2006 Studies on the stability of an edible film and its use for the preservation of carrot Daucus carota International Journal of Food Science and Technology 41 498 506 Janes M E Kooshesh S Johnson M G 2002 Control of Listeria monocytogenes on the surface of refrigerated ready to eat chicken coated with edible zein film coatings containing nisin and or calcium propionate Journal of Food Science 67 2754 2757
210. blished December 5 2012 dx doi org 10 1021 304006y 1 Agric Food Chem 2013 61 260 267 Anexos Food Bioprocess Technol DOI 10 1007 511947 014 1421 8 Reversible Covalent Immobilization of Cinnamaldehyde on Chitosan Films via Schiff Base Formation and Their Application in Active Food Packaging Laura Higueras Gracia L pez Carballo Rafael Gavara Pilar Hern ndez Mu oz Received 6 May 2014 Accepted 6 October 2014 Springer Science Business Media New York 2014 Abstract In this work cinnamaldehyde was reversibly an chored to chitosan films via imino covalent bonding The Schiff base was synthesized in solid phase employing neutral ized chitosan films immersed in acidified 95 v v ethanolic solution in which the aldehyde was dissolved The substitution degree of cinnamaldehyde to the amine group was close to 70 Attenuated total reflectance Fourier transform infrared spectroscopy ATR FTIR analysis revealed the formation of the chitosan cinnamaldehyde Schiff base The hydrolysis of the imino bond and subsequent release of cinnamaldehyde were studied after the films had been subjected to different combinations of temperature time treatments simulating food preservation methods The amount of aldehyde that remained covalently attached to the films was monitored by ATR FTIR and the substitution degree was determined by elemental anal ysis Surface contact angle and colour parameters of cinnamaldehyde imine
211. ca y aseo personal para 1 la estabilizaci n de aromas vitaminas o aceites esenciales 2 la eliminaci n de aromas sabores indeseables u otros compuestos como grasas 3 la solubilizaci n de colorantes y vitaminas y 4 la liberaci n controlada de ciertos constituyentes alimentarios Astray et al 2009 Del Valle 2004 Las CDs son productos biocompatibles seminaturales producidos a partir de un material natural y renovable como es el almid n por una conversi n enzim tica relativamente simple Se producen en miles de toneladas por afio mediante tecnolog as respetuosas con el medio ambiente Por lo que el coste inicialmente alto de las CDs ha descendido hasta niveles aceptables para la mayor a de aplicaciones industriales Szejtli 1998 Las CDs son consideradas at xicas por v a oral No se absorben en el tracto intestinal alto lt 3 debido a su exterior hidrof lico sino que se degradan parcialmente debido al cido estomacal y la mayor parte de las CDs ingeridas o los subproductos de la acci n de la a amilasa pancre tica son metabolizadas por la microbiota bacteriana del colon Kurkov y Loftsson 2013 Entre las CDs la B CD sustancia GRAS 2001 aceptada por la FAO OMS 1995 como aditivo alimentario con INS n 459 es la m s accesible la m s barata y generalmente la m s utilizada en las industrias farmac utica y alimentaria con una ingesta diaria admisible IDA de 5 mg kg d a en alimentos Del Valle 2004 Kurko
212. caci n en el envasado activo antimicrobiano de alimentos consta del art culo cient fico Development of a novel antimicrobial film based on chitosan with LAE ethyl N dodecanoyl L arginate and its application to fresh chicken En este trabajo se han desarrollado pel culas de CS mediante la t cnica de extensi n y evaporaci n del solvente e incorporando LAE al 1 5 y 1096 Su liberaci n se monitoriz en un simulante alimentario acuoso a 4 y 28 C Previamente al estudio de la actividad antimicrobiana de las pel culas se determin la concentraci n m nima inhibitoria y la concentraci n m nima biocida del agente antimicrobiano frente a determinados microorganismos modelo bacterias hongos y levaduras en medio l quido y s lido Finalmente se evalu la eficacia de las pel culas en el envasado de pechugas de pollo fileteadas estudi ndose su actividad frente a bacterias mes filas psicr filas Pseudomonas spp coliformes cido l cticas sulfuro productoras hongos y levaduras tras 2 6 y 8 d as de almacenamiento en condiciones de refrigeraci n El cap tulo 2 Desarrollo de pel culas h bridas de quitosano con nanopart culas de plata formadas in situ comprende el art culo cient fico denominado Silver ions release from antibacterial chitosan films containing in situ generated silver nanoparticles En este cap tulo se hace uso del conocimiento sobre el empleo de nanopart culas de plata como agentes portadores y libe
213. cal structure Letters in Applied Microbiology 43 149 154 Burt S 2004 Essential oils their antibacterial properties and potential applications in foods A review International Journal of Food Microbiology 94 223 253 Crank J 1975 The mathematics of diffusion 2nd ed Clarendon Press Oxford England Chalier P Ben Arfa A Guillard V Gontard N 2009 Moisture and temperature triggered release of a volatile active agent from soy protein coated paper effect of glass transition phenomena on carvacrol diffusion coefficient Journal of Agricultural and Food Chemistry 57 658 665 Delaquis P J Stanich K Girard B Mazza G 2002 Antimicrobial activity of individual and mixed fractions of dill cilantro coriander and eucalyptus essential oils International Journal of Food Microbiology 74 101 109 Dorman H J D Deans S G 2000 Antimicrobial agents from plants antibacterial activity of plant volatile oils Journal of Applied Microbiology 88 308 316 Dutta J Tripathi S Dutta P K 2012 Progress in antimicrobial activities of chitin chitosan and its oligosaccharides a systematic study needs for food applications Food Science and Technology International 18 3 34 Garnero C Aloisio C Longhi M 2013 Ibuprofen maltodextrin interaction study of enantiomeric recognition and complex characterization Pharmacology amp Pharmacy 4 18 30 Griffin S Wyllie S G Markham J Leach D 1999 The ro
214. can be classified as Fickian Case I or non Fickian anomalous Case II and Super Case II depending on the solvent diffusion and polymer relaxation rates Diffusion categories can be distinguished by the shape of the sorption uptake curve of a polymer penetrant system M kt 1 where M is the mass of solute uptake at time t and M is the mass of solute uptake when the polymer has reached equilibrium k is a constant and n a diffusion exponent which denotes the type of diffusion mechanism If the exponent n is equal to 0 5 the diffusion is Fickian and the solvent diffusion rate is slower than the polymer relaxation rate n equal to 1 refers to Case II type diffusion with the solvent diffusion rate faster than the polymer relaxation process A value of n between 0 5 and 1 refers to anomalous diffusion which happens when the diffusion and relaxation rates are comparable Super Case diffusion occurs for n 1 Experimental sorption uptake curves plotted in Figure 4 4 7 were fitted to the power law described by Equation 1 and the mass transport mechanism was evaluated by calculating parameter n The values of n were between 0 5 and 0 6 Table 4 4 7 Therefore the mathematical model based on the one dimensional solution of Fick s second law of diffusion in a plane sheet was applied to the experimental sorption uptake data This model considers the diffusion coefficient independently of the concentration of the sorbed compound Assuming the ini
215. carveol the only difference between them being the hydroxyl group position Thus 8 0 mg of isopulegol was needed compared with 2 0 mg of dihydrocarveol to produce inhibition of bacterial growth Lipophilicity has been used as the descriptor with the strongest influence on antimicrobial activity owing to the great affinity of lipophobic compounds for cell membrane Dambolena et al 2012 This property is specially considered for compounds with log P between 3 and 4 However in the present study monoterpenes with similar log P values exerted different antimicrobial activities These results indicate that other factors besides hydrophobicity are involved such as the presence and hydrogen donating ability of compounds with a phenol chemical structure 3 5 Antimicrobial capacity of films loaded with monoterpenes Table 4 4 14 shows the antimicrobial capacity in vapour phase against S aureus and E coli of CS CD films plasticized with G or PG and PVOH CD films plasticized with G after reaching sorption equilibrium in the volatile liquids Antimicrobial activity was measured as the inhibition growth halo produced on the agar surface A clear positive relationship was found between the bacterial growth inhibition zone and the amount of volatile retained in the film All the films that retained a greater amount of antimicrobial compound than that corresponding to the minimal inhibition dose exerted antimicrobial activity This shows that the formulated
216. cate a statistically significant difference P lt 0 05 comparing different amounts of G in the same matrix CS or CS CD Different letters in the same column x y indicate a statistically significant difference P lt 0 05 comparing CS and CS CD Different letters in the same rows m n indicate a statistically significant difference P lt 0 05 comparing different RHs of analysis for the same matrix and G content Table 4 4 4 Tensile strength modulus of elasticity and elongation at break of CS and CS CD films with different G contents at 53 and 75 RH and 23 1 C Tensile strength Elongation at Modulus of Tensile strength Modulus of Materials films MPa break elasticity MPa MPa elasticity MPa Elongation at break 7596 RH 53 RH cs 5747 3 25 16 39 4 23 1635 54 150 63 46 45 3 32 40 58 7 01 1297 0 121 7 CS 20G 40 00 4 74 57 04 7 59 609 00 70 06 25 46 3 46 54 15 3 53 149 2 38 004 CS 35G 31 00 7 24 56 92 5 27 175 95 52 61 19 40 3 26 53 52 5 21 gt 28 7 4 6 CS CD 58 00 3 88 5 09 0 79 1855 28 160 00 44 32 3 27 6 19 0 73 1440 8 128 5 CS CD 20G 1991 1 19 55 21 4 27 198 00 61 00 13 21 1 29 54 41 5 249 46 6 7 49 CS CD 35G Ws DGA 56 61 7 39 14 36 2 10 5 00 1 91 55 33 5 08 Egi s 077 Different letters in the same column a c indicate a statistically significant difference P lt 0
217. ce inhibiting or retarding microbial growth Buonocore et al 2003 Cutter 2002 The release of the volatile from the polymer matrix can be triggered by various stimuli such as the humidity present in the headspace of the package with the hydrophilic nature of the polymer and the level of humidity of the headspace being major factors that control the release kinetics of the agent In this respect hydrogels are hydrophilic polymers of either natural or synthetic origin with a great potential to be used as sustained release carriers of active molecules Hydrogels are responsive to changes in environmental humidity depending on their composition they can absorb a certain amount of moisture and consequently swell to different extents facilitating release of the entrapped molecule Thus these polymers can 247 Cap tulo 4 Art culo cient fico 7 retain the volatile compound in a dry environment and release it when they are moisture activated CS and PVOH are biodegradable hydrophilic polymers with excellent film forming properties CS is a natural linear cationic biopolymer produced by partial deacetylation of chitin PVOH is a synthetic polymer obtained from controlled hydrolysis of polyvinyl acetate Solid dispersions based on CS and PVOH polymers have been extensively employed in the form of thin films nanoparticles capsules tablets etc as carrier systems for sustained release of drugs Goodship and Jacobs 2009 Kumar et aL 2004
218. cenamiento del producto envasado Son varios los factores que pueden desencadenar la actividad del pol mero El agente activo puede ser no vol til o vol til en el primer caso es necesario el contacto del pol mero con el alimento Mientras que los agentes activos vol tiles pueden liberarse al espacio de cabeza del envase sin la necesidad de que exista contacto directo lo cual es muy adecuado a la hora de envasar alimentos s lidos Cabe decir que existen otros tipos de pol meros antimicrobianos aquellos cuya capacidad antimicrobiana es inherente debido a su estructura qu mica o pol meros que han sido modificados y en los cuales se ha inmovilizado de forma irreversible la mol cula antimicrobiana En la presente Tesis Doctoral se han desarrollado sistemas activos basados en matrices de CS capaces de liberar agentes antimicrobianos de diferente naturaleza 1 un s lido como el LAE previamente incorporado a la formaci n de la pel cula 2 iones plata que se liberan desde la matriz y proceden de nanopart culas de plata creadas 54 Justificaci n y esquema de tesis previamente durante el proceso de formaci n de la pel cula 3 vol tiles anclados de forma reversible una vez obtenida la pel cula y 4 vol tiles incorporados en la pel cula tras su procesado De acuerdo con ello la Tesis se ha dividido en cuatro cap tulos El cap tulo 1 titulado Desarrollo de pel culas de quitosano con etil N dodecanoil L arginato y su apli
219. ces Abdollahi M Rezaei M Farzi G 2012 Improvement of active chitosan film properties with rosemary essential oil for food packaging International Journal of Food Science and Technology 47 847 853 Altiok D Altiok E Tihminlioglu F 2010 Physical antibacterial and antioxidant properties of chitosan films incorporated with thyme oil for potential wound healing applications Journal of Materials Science Materials in Medicine 21 2227 2236 ASTM 2007 Standard practice for maintaining constant relative humidity by means of aqueous solutions ASTM International West Conshohocken PA ASTM 2010a ASTM D882 10 Standard test method for tensile properties of thin plastic sheeting ASTM International West Conshohocken PA ASTM 2010b Standard test method for oxygen gas transmission rate through plastic film and sheeting using a coulometric sensor ASTM International West Conshohocken PA ASTM 2010c Standard test methods for water vapor transmission of materials ASTM International West Conshohocken PA Bell L N Labuza T P 2000 Moisture sorption practical aspects of isotherm measurement and use 2 ed Saint Paul ed Eagan Press Brown C D Kreilgaard L Nakakura M Caram Lelham N Pettit D K Gombotz W R Hoffman A S 2001 Release of PEGylated granulocyte macrophage colony stimulating factor from chitosan glycerol films Journal of Controlled Release 72 35 46 Burt S 2004 Essential oils
220. cheroni E Guillard V Gastaldi E Gontard N Chalier P 2011 Antimicrobial effectiveness of relative humidity controlled carvacrol release from wheat gluten montmorillonite coated papers Food Control 22 1582 1591 Messner M Kurkov S V Jansook P Loftsson T 2010 Self assembled cyclodextrin aggregates and nanoparticles International Journal of Pharmaceutics 387 199 208 Ouattara B Simard R E Piette G Begin A Holley R A 2000 Diffusion of acetic and propionic acids from chitosan based antimicrobial packaging films Journal of Food Science 65 768 773 Prabaharan M Mano J F 2006 Chitosan derivatives bearing cyclodextrin cavities as novel adsorbent matrices Carbohydrate Polymers 63 153 166 Ramya R Venkatesan J Kim S K Sudha P N 2012 Biomedical applications of chitosan an overview Journal of Biomaterials and Tissue Engineering 2 100 111 Ravi P Divakar S 2001 Stereoselective hydrogenation of thymol over Rh alumina in the presence of p cyclodextrin and its derivatives Journal of Inclusion Phenomena and Macrocyclic Chemistry 39 27 33 Ribeiro L S S Veiga F J B Ferreira D 2003 Physicochemical investigation of the effects of water soluble polymers on vinpocetine complexation with B cyclodextrin and its sulfobutyl ether derivative in solution and solid state European Journal of Pharmaceutical Sciences 20 253 266 Suppakul P Miltz J Sonneveld K Bigger S W 20
221. chitosan International Journal of Food Microbiology 62 139 148 Pinazo A Wen X Y P rez L Infante M R Franses EIL 1999 Aggregation behavior in water of monomeric and gemini cationic surfactants derived from arginine Langmuir 15 3134 3142 Rodr guez E Seguer J Rocabayera X Manresa A 2004 Cellular effects of monohydrochloride of L arginine N lauroyl ethylester LAE on exposure to Salmonella Typhimurium and Staphylococcus aureus Journal of Applied Microbiology 96 903 912 88 Quitosano LAE Ruckman S A Rocabayera X Borzelleca J F Sandusky C B 2004 Toxicological and metabolic investigations of the safety of N lauroyl L arginine ethyl ester monohydrochloride LAE Food and Chemical Toxicology 42 245 259 Russell A D 1995 Mechanisms of bacterial resistance to biocides International Biodeterioration amp Biodegradation 36 247 265 Russell A D 2003 Similarities and differences in the responses of microorganisms to biocides Journal of Antimicrobial Chemotherapy 52 750 763 Shahidi F Arachchi J K V Jeon Y J 1999 Food applications of chitin and chitosans Trends in Food Science amp Technology 10 37 51 Siragusa G R Cutter C N Willett J L 1999 Incorporation of bacteriocin in plastic retains activity and inhibits surface growth of bacteria on meat Food Microbiology 16 229 235 Soni K A Nannapaneni R Schilling M W Jackson V 2010 Bactericidal activi
222. ci n Crecimiento Agente A B Y WD reductor 4 7 OA OG 9 A2 3 Ag NO gt G Q LR H O te LAAD 2 x Q Atomos de Disoluci n coloidal de plata libres nanopart culas de plata Figura 4 2 1 Esquema de la generaci n de nanopart culas de plata 92 Quitosano Nanopart culas de plata Los agentes estabilizadores m s comunes son los tensioactivos y los pol meros que ejercen su acci n al unirse a la superficie de la part cula Sharma et al 2009 Existen diferentes m todos tanto f sicos como qu micos para preparar dichos compuestos polim ricos siendo el m s com n la dispersi n de nanopart culas preparadas previamente s ntesis ex situ en la matriz polim rica en soluci n Sin embargo este m todo puede producir una distribuci n y tama o no homog neos de las part culas en el pol mero debido a su aglomeraci n en el medio acuoso Para evitar esto se pueden generar las nanopart culas met licas in situ mediante la disoluci n y la reducci n de las sales met licas en el pol mero Otra alternativa para conseguir una dispersi n homog nea de las nanopart culas en la matriz polim rica ser a mediante la polimerizaci n y la reducci n del metal de forma simult nea De Azeredo 2013 Sharma et al 2009 Se han publicado diversos estudios sobre la incorporaci n de nanopart culas de plata libres en diversas matrices polim ricas con el objeto de conferirles actividad antimicrobiana como en PA Damm
223. cide concentration MBC g mL of LAE against the selected microorganisms in liquid and solid media Liquid medium Solid medium MIC MBC MIC MBC Microorganisms Species Gram positives Listeria monocytogenes 8 16 8 16 Staphylococcus aureus 8 16 8 16 o Escherichia coli 16 24 16 24 Pseudomonas putida 16 24 16 24 Salmonella enterica 16 24 16 24 Candida utilis 16 24 104 120 Yeasts Saccharomyces cerevisiae 16 24 104 120 Torulopsis pinus 4 8 32 48 Aspergillus niger 24 320 Fungi Cladosporium cladosporioides 24 80 Penicillium chrysogenum 120 280 g mL for Gram positive bacteria and 24 ug mL for Gram negative bacteria either on solid or in liquid medium These values were 8 24 pg mL in liquid medium and 48 120 g mL on solid medium for yeasts and 80 320 ug mL for fungi on solid medium Results highlighted a slightly higher sensitivity of Gram positive bacteria than Gram negative bacteria This behaviour observed for other antimicrobial compounds Canillac and Mourey 2001 Delaquis et al 2002 was mainly due to the presence of an outer membrane surrounding cell wall in Gram negative bacteria that restricts the diffusion of hydrophobic compounds through its lipopolysaccharide covering among other defence mechanisms Nikaido and Vaara 1985 Russell 1995 Furthermore in general these values show a greater sensibility of bacteria than yeasts and fungi against biocides also reported in many references McDonnell and Russell 199
224. cience and Technology 51 9 15 133 Cap tulo 3 Estado de la cuesti n Soliman E A Khalil A A Deraz S F El Fawal G Abd Elrahman S 2014 Synthesis characterization and antibacterial activity of biodegradable films prepared from Schiff bases of zein Journal of Food Science and Technology Mysore 51 2425 2434 Stroescu M Stoica Guzun A Isopencu G Jinga S L Parvulescu O Dobre T Vasilescu M 2015 Chitosan vanillin composites with antimicrobial properties Food Hydrocolloids Tirkistani F A A 1998 Thermal analysis of some chitosan Schiff bases Polymer Degradation and Stability 60 67 70 Wang J T Lian Z R Wang H D Jin X X Liu Y J 2012 Synthesis and antimicrobial activity of Schiff base of chitosan and acylated chitosan Journal of Applied Polymer Science 123 3242 3247 134 4 3 2 Art culo cient fico 3 Reversible covalent immobilization of cinnamaldehyde on chitosan films via Schiff base formation and their application in active food packaging Laura Higueras Gracia L pez Carballo Rafael Gavara Pilar Hern ndez Mufioz Food and Bioprocess Technology 2015 8 526 538 Quitosano Cinamaldeh do Resumen En este trabajo el cinamaldeh do fue reversiblemente anclado a pel culas de quitosano mediante la formaci n de un grupo imino Esta base de Schiff se sintetiz en fase s lida empleando pel culas de quitosano neutralizado inmersas en una soluci n etan lica
225. cin 65 C 30min 74 0409 145 1 0 8824155 894413 807 07 CScin 72 C 30min 722415 162205 9044115 9194105 79 9 1 0 CScin 95 C 10min 69 1 0 8 20214 940 11 962 08 779209 CScin 121 C Smin 5320 85 387206 733502 829201 622204 Different letters in the same column indicate a statistically significant difference P lt 0 05 148 Quitosano Cinamaldeh do groups is exposed to high temperatures Regarding the chroma values no differences were observed between the CScin films and those subjected to low temperature treatments 4 and 65 C The C of films treated at higher temperatures 72 C for 15 min 95 C for 10 min and 121 C for 5 min increased significantly Films treated at 72 and 95 C showed a higher colour intensity than CScin films However this parameter decreased for films treated at 121 C for 5 min This behaviour could be related to a higher release of cinnamaldehyde from the film during the treatment as a consequence of Schiff base hydrolysis in combination with Maillard formation products The L lightness parameter was used to measure the transparency of the films CS films have high transparency which was significantly reduced after Schiff base formation This decrease became greater as the temperature of the treatment increased A decrease in the L parameter was also observed after heating CS films which had reduced lightness and acquired a vivid yellow colour Retorted CS film presen
226. cipalmente CS del que ya se han descrito sus principales caracter sticas y propiedades y polialcohol vin lico 1 3 1 Quitosano El CS es la base de muchos materiales polim ricos y puede presentarse formando pel culas recubrimientos hidrogeles fibras c psulas o nanopart culas y encontrarse entrecruzado o no solo o mezclado formando materiales compuestos etc Dutta et al 2009 Prashanth y Tharanathan 2007 Rinaudo 2006 Los mecanismos del CS implicados en la conservaci n de los alimentos que permiten su uso como pel cula comestible son varios 1 capacidad antimicrobiana cuando se encuentra protonado 2 antioxidante 3 control de la transferencia de humedad entre el alimento y el medio debido a que presenta valores moderados de permeabilidad al agua 4 reducci n de la presi n parcial de ox geno por ser una excelente barrera a la transmisi n de ste cuando existe baja RH 5 control de la tasa de respiraci n 6 control de la maduraci n enzim tica en frutas por retardar la producci n de etileno 7 control de la liberaci n de sustancias 16 Introducci n antimicrobianas antioxidantes nutrientes y aromas y finalmente 8 inversi n osm tica Shahidi et al 1999 Las pel culas obtenidas a partir de CS se caracterizan por ser transparentes ligeramente amarillentas de suave superficie flexibles cohesivas de gran resistencia mec nica comparable a las de muchos pol meros comerciales hidr filas ino
227. city for sorption of carvacrol The kinetics of release in ideal conditions were also measured and the in vitro antimicrobial activity against Staphylococcus aureus and Escherichia coli was tested Higueras et al 2013 Higueras et al 2015 In this work a package containing this device was designed and used as an active package containing fresh chicken fillets The partition of carvacrol among the different components of the food active package environment system was studied and the effect of the active packaging on the chicken microbiota was analysed 227 Cap tulo 4 Art culo cient fico 6 2 Materials and methods 2 1 Materials Carvacrol kosher gt 98 glycerol G acetic acid and low molecular weight chitosan CS were supplied by Sigma Barcelona Spain hydroxypropyl B cyclodextrin HP BCD CAVASOL W7 HP were provided by Wacker Fine Chemicals S L Barcelona Spain 2 2 Film preparation A 1 596 CS w w solution in an aqueous 0 5 w w acetic acid solution was prepared and filtered to eliminate impurities G plasticized chitosan hydroxypropyl B cyclodextrin films were obtained by adding HP BCD to the CS solution in a 1 1 proportion w w with respect to CS and G at 35 g G 100 g dry matter and stirring at 1500 rpm and 37 C until complete dissolution Films were obtained by casting i e pouring a suitable amount of the solution into a flat PS tray and allowing it to dry under controlled environmental condi
228. cohols did not increase sorption properties On the contrary sorption values were lower for phenolic compounds This indicated that the plasticizer plays a more important role in swelling the polymer matrix than in increasing film affinity for sorbates When PVOH was employed as the polymer matrix the sorption behaviour of the films for monoterpenes was similar but the sorption values achieved for phenolic compounds were lower than those of CS CD films In spite of its less polar nature the less pronounced plasticizing effect of G on PVOH and its crystallinity might be responsible for these findings All the monoterpene alcohols presented antimicrobial activity against the bacteria assayed whereas the activity of the loaded films was directly related to the volatile content The films that were developed could be used for various purposes including acting as carriers and systems for release of naturally occurring compounds for active packaging applications Acknowledgments The authors acknowledge financial support from the Spanish Ministry of Economy and Competitiveness MINECO AGL2012 39920 C03 01 project Laura Higueras thanks the Spanish National Research Council for a JAE PreDoc Fellowship References ASTM 2007 Standard practice for maintaining constant relative humidity by means of aqueous solutions ASTM International West Conshohocken PA Balaguer M P Gavara R Hern ndez Mufioz P 2012 Food aroma mass transport prope
229. crobial activity that could improve the stability of fresh poultry products Keywords Chitosan films LAE antimicrobial packaging active packaging antimicrobial release poultry packaging 72 Quitosano LAE 1 Introduction The incidence of foodborne diseases associated with microbial pathogens is widespread and represents a threat to public health and a challenge for the food industry L pez Carballo et aL 2008 Considerable efforts are underway to find effective treatments to control recontamination of meat and poultry products to enhance their safety and quality In this sense applications of antimicrobial films and coatings to food have received considerable attention in recent years because they can act as protective barriers against microbiological contamination Cagri et al 2004 Suppakul et al 2003 Direct addition of antimicrobial agents into meat formulations may result in partial inactivation of the active substances by interaction with product constituents and is therefore expected to have only limited effect on surface microbiota Siragusa et al 1999 Torres et al 1985 Also direct application of antimicrobial agents onto meat surfaces by dipping or spraying has been shown to be inefficient due to the rapid diffusion of the active substances within the bulk of food Ouattara et al 2000b Siragusa et al 1999 Torres et al 1985 In contrast an antimicrobial active film can be developed to deliver a co
230. crobiology 121 313 327 Raybaudi Massilia R M Rojas Gra M A Mosqueda Melgar J Mart n Belloso O 2008 Comparative study on essential oils incorporated into an alginate based edible coating to assure the safety and quality of fresh cut Fuji apples Journal of Food Protection 71 1150 1161 Resa C P O Gerschenson L N Jagus R J 2013 Effect of natamycin on physical properties of starch edible films and their effect on Saccharomyces cerevisiae activity Food and Bioprocess Technology 6 3124 3133 Resa C P O Gerschenson L N Jagus RJ 2014 Natamycin and nisin supported on starch edible films for controlling mixed culture growth on model systems and Port Salut cheese Food Control 44 146 151 Rhim J W Hong SL Park H M Ng P K W 2006 Preparation and characterization of chitosan based nanocomposite films with antimicrobial activity Journal of Agricultural and Food Chemistry 54 5814 5822 Rivero S Giannuzzi L Garc a M A Pinotti A 2013 Controlled delivery of propionic acid from chitosan films for pastry dough conservation Journal of Food Engineering 116 524 531 Rojas Gra M A Avena Bustillos R J Friedman M Henika P R Mart n Belloso O Mchugh T H 2006 Mechanical barrier and antimicrobial properties of apple puree edible films containing plant essential oils Journal of Agricultural and Food Chemistry 54 9262 9267 Rojas Gra M A Avena Bustillos R J Olsen C Friedma
231. ctivas Kurkov y Loftsson 2013 Messner et al 2010 Van de Manakker et al 2009 Adem s con el prop sito de mejorar el procesado y manipulaci n de las pel culas se puede incluir un plastificante hidr filo compatible con la matriz como el G que es un aditivo alimentario y biodegradable Asimismo se ha demostrado que el G facilita una mayor retenci n de estos compuestos vol tiles debido a que es capaz de interponerse en las cadenas interrumpiendo las fuerzas intercatenarias Kurek et al 2012 Igualmente se podr a pensar que otros plastificantes con el mismo mecanismo de acci n como el agua realizar an una acci n similar El objetivo de crear pel culas antimicrobianas con agentes vol tiles que conformen el envase o parte de l es que pueda difundir hacia el espacio de cabeza donde se acumule en estado vapor y ejerza su acci n antimicrobiana en la superficie del alimento e incluso difundir en el alimento y realizar una acci n en profundidad sin necesidad de contactar con el alimento Idealmente la matriz polim rica act a como reservorio desde el que se libera de forma sostenida el agente De esta manera se asegura una concentraci n eficaz de antimicrobiano en el espacio de cabeza que impida la proliferaci n de microorganismos en la superficie del alimento precis ndose cantidades menores de agente activo Buonocore et al 2003 Ouattara et al 2000 Adem s la liberaci n del vol til desde la matriz polim ric
232. ctivity Shahidi et al 1999 Similar results were observed 83 Cap tulo 1 Art culo cient fico 1 Table 4 1 4 Antimicrobial effectiveness of LAE films against the selected microorganisms in liquid medium at 28 C for 24 h Values expressed as logarithm of colony forming units log CFU mL and log reduction value LRV in brackets Microorganisms Species Control CS LRV CS 1 LAE LRV CS 5 LAE LRV CS 10 LAE LRV Listeria monocytogenes 90 01 87 401 0 3 81401 09 49401 41 27201 6 3 Staphylococcus aureus 9 0 01 86 03 04 804015 10 45 01 45 29 010 6 1 Gram positives Escherichia coli 892017 86 01 03 75 01 14 63 01 26 41 01 48 Gram negatives Pseudomonas putida 912047 87 01 04 754015 16 65 03 26 44 01 47 Salmonella enterica 892017 86 02 03 74 01 15 65 01 24 48 01 41 Candida utilis 77 01 25 01 52 Totalinhibition Totalinhibition Total inhibition Yeasts Saccharomyces cerevisiae 7 8 0 1 27 02 51 Total inhibition Total inhibition Total inhibition Torulopsis pinus 764015 23 01 53 Total inhibition Total inhibition Total inhibition 4 Different letters in the same row indicate a statistically significant difference P lt 0 05 comparing the control samples and the different amounts of LAE in the matrix Different letters in the same column indicate a statistically significant difference P lt 0 05 comparing the inhibition effectivity o
233. ctos plantas Los AE m s destacados por su elevada efectividad son los procedentes de clavo or gano romero tomillo albahaca hierba lim n o citronela salvia mostaza canela y vainilla Bakkali et al 2008 Burt 2004 Holley y Patel 2005 Sin embargo su mecanismo de acci n no 31 Introducci n est bien definido Considerando la gran variedad de compuestos qu micos presentes en los AE es muy probable que su actividad antimicrobiana no sea atribuible a un mecanismo espec fico sino a la acci n combinada de varios de ellos sobre distintas localizaciones de la c lula Burt 2004 Por una parte la hidrofobicidad de estos AE permite su uni n a los l pidos de la membrana celular y mitocondrial bacteriana distorsionando las estructuras y haci ndolas m s permeables provocando la salida de iones y el contenido celular por lo que se inhibe el transporte de electrones la translocaci n de prote nas o la cascada de fosforilaci n Adem s los componentes de los AE pueden interaccionan con las ATPasas presentes en la membrana citoplasm tica Burt 2004 Tiwari et aL 2009 Por otra parte los componentes de los AE a bajas concentraciones afectan a enzimas para la producci n energ tica mientras que a concentraciones mayores pueden causar la desnaturalizaci n proteica Igualmente pueden actuar sobre las prote nas de la membrana citoplasm tica e incluso atravesar las porinas y acceder al periplasma de las bacterias gram
234. cuas biocompatibles biodegradables Butler et al 1996 Shahidi et al 1999 Tharanathan y Kittur 2003 y adecuadas para distintos grupos de alimentos No et al 2007 Como otras pel culas basadas en polisac ridos estas propiedades se ven mermadas cuando las pel culas de CS se someten a elevada RH ya que interaccionan con el agua al ser tan hidr filas Dutta et aL 2009 No et al 2007 Las pel culas de CS se pueden obtener mediante distintos m todos Donhowe y Fennema 1994 Krajewska 2005 siendo el del casting o extensi n y evaporaci n del solvente el m s empleado para la obtenci n de pel culas comestibles a base de biopol meros Campos et aL 2011 Dutta et al 2009 Para ello en primer lugar se disuelve el pol mero en una disoluci n acuosa a pH ligeramente cido pH 6 5 para despu s reformar la cadena polim rica en una matriz o hidrogel Esto se logra por extensi n de la soluci n film gena y evaporaci n del disolvente cre ndose enlaces de hidr geno e hidrof licos intercatenarios y o entrecruzamiento electrol tico o i nico Butler et al 1996 Estos entrecruzamientos y la presencia de los grupos hidroxilo y amino permiten al CS formar hidrogeles Bhattarai et aL 2010 Una vez evaporado el disolvente la pel cula generada est constituida por la sal de CS por lo que para estabilizar la pel cula en agua evitar su disoluci n y la p rdida de sus propiedades debe ser sometida a un proceso de neutraliz
235. cyclodextrins composites with high sorption capacity for carvacrol Carbohydrate Polymers 97 262 268 Higueras L L pez Carballo G Hern ndez Mufioz P Catala R Gavara R 2014 Antimicrobial packaging of chicken fillets based on the release of carvacrol from chitosan cyclodextrin films International Journal of Food Microbiology 188 53 59 Higueras L L pez Carballo G Gavara R Hern ndez Mu oz P 2015 Incorporation of hydroxypropyl B cyclodextrins into chitosan films to tailor loading capacity for active aroma compound carvacrol Food Hydrocolloids 43 603 611 Higueras L L pez Carballo G Gavara R Hern ndez Mufioz P 2015 Reversible covalent immobilization of cinnamaldehyde on chitosan films via schiff base formation and their application in active food packaging Food and Bioprocess Technology 1 13 e Higueras L L pez Carballo G Gavara R Hern ndez Mufioz P Effect of HP B cyclodextrins and coadjuvants on the sorption capacity of hydrophilic polymer films for naturally occurring antimicrobial monoterpene alcohols Submitted to Carbohydrate Polymers 331 Anexos Adem s de las siguientes contribuciones a congresos Congreso nacional p ster G L pez Carballo L Higueras A Fern ndez M J Galotto A Guarda R Gavara P Hern ndez Mu oz Desarrollo y Propiedades Antimicrobianas de Pel culas Compuestas de Quitosano Nanopart culas de Plata II Congreso del Caribe y I
236. d La pres ncia conjunta de hidroxipropil Resum ciclodextrines glicerol i aigua en les pel l cules de quitos va donar lloc a una elevada capacitat de sorci de carvacrol de caracter lipofil L alliberament del carvacrol present en les pel licules compostes va ser activada per la humitat relativa ambiental Aquest carvacrol alliberat de les pel l cules va presentar efectivitat antimicrobiana en fase vapor quan va ser incorporat en un sistema d envasat per a pits de pollastre fresc La capacitat de retenci de les pel licules es va poder modular en funci de la seua formulaci i de l estructura qu mica del monoterp absorbit Les pel licules amb aquests monoterpens van presentar capacitat antimicrobiana en fase vapor in vitro i poden emprar se en el disseny d envasos actius adaptats a l aliment aplicat XIV Abstract This PhD dissertation focuses on the development of renewable active chitosan films with a variety of antimicrobial agents for active food packaging applications Chitosan films were obtained by the solvent casting technique Bioactive substances were incorporated using various methodologies The solid ethyl N dodecanoyl L arginate LAE was incorporated prior to the formation of the chitosan film The LAE was completely released by the films in an aqueous food simulant over several hours at various temperatures The chitosan films which were not neutralized showed antimicrobial activity against microorganisms
237. d 100 Chalier et al 2009 studied the diffusivity of carvacrol at 30 C in soy protein coated paper where the diffusion coefficients ranged from 0 02 to 14 x 10 m s7 for RH varying between 60 and 10096 Kurek et al 2014 evaluated the diffusion coefficient of carvacrol in CS films measured at RH 9696 finding values of 3 8 x 10 at 20 C and 5 5 x 10 at 37 C 3 4 Antimicrobial activity 3 4 1 Antimicrobial activity of carvacrol vapour against S aureus and E coli The antimicrobial activity of carvacrol was tested against S aureus and E coli using the microatmosphere method as described in Materials and Methods and the MIC minimal inhibitory concentration was determined Table 4 4 9 shows that the minimum dose of carvacrol found to produce inhibition zones on agar was 1 mg The inhibition zone diameters produced by disks with carvacrol were 15 and 13 mm for S aureus and E coli respectively The inhibition zones increased as the amount of carvacrol added to the filter paper disk increased thus 5 mg of carvacrol produced an inhibition zone of 44 mm against S aureus and E coli 214 Quitosano Carvacrol Table 4 4 9 Antimicrobial activity produced by carvacrol in vapour phase against S aureus and E coli S aureus E coli caracolimg Inhibition zone mm Inhibition zone mm 5 00 44 44 2 00 29 30 1 50 20 21 1 25 18 18 1 00 15 13 0 75 0 50 Several studies regarding the antimicrobial act
238. d and represents a threat to public health and a challenge for the food industry L pez Carballo et al 2008 Considerable efforts are underway to find effective treatments to con trol recontamination of meat and poultry products to enhance their safety and quality In this sense applications of antimicrobial films and coatings to food have received considerable attention in recent years because they can act as protective barriers against microbiolog ical contamination Suppakul et al 2003 Cagri et al 2004 Direct addition of antimicrobial agents into meat formulations may result in partial inactivation of the active substances by interaction with product constituents and is therefore expected to have only limited effect on surface microbiota Torres et al 1985 Siragusa et al 1999 Also direct application of antimicrobial agents onto meat sur faces by dipping or spraying has been shown to be inefficient due to the rapid diffusion of the active substances within the bulk of food Torres et al 1985 Siragusa et al 1999 Ouattara et al 20004 In contrast an antimicrobial active film can be developed to deliver a continued and gradual release of an antimicrobial agent during the storage and distribution of food packaging thus providing an effective Corresponding author Tel 34 963900022 fax 34 963636301 E mail address rgavara iata csic es R Gavara 0168 1605 5 see front matter 2013 Published by Elsevier B
239. d at 0 53 and 75 RH prior to immersion in carvacrol 3 2 2 Kinetics of sorption of carvacrol in CS CD films Figure 4 4 7 shows the kinetics of sorption of carvacrol in 1 1 CS CD films plasticized with 35 G and conditioned at 0 53 or 75 RH and films plasticized with 20 G and conditioned at 75 RH The plots represent the sorption of liquid carvacrol into the films versus time It can be observed that the equilibrium times varied among films depending on their G and water content Sorption equilibrium was achieved faster for films which presented a greater level of plasticization i e films incorporating 20 or 35 G and conditioned at 7596 RH whereas it took longer to reach sorption equilibrium for films conditioned under dry conditions or at 5396 RH before being immersed in carvacrol 1 2 Carvacrol ratio Y CS CD 35G OHR 0 2 CS CD 35G 75HR 4 CS CD 20G 75HR s CSICD 35G 53HR 0 0 o 2000 4000 6000 8000 10000 12000 14000 16000 18000 Time minutes Figure 4 4 7 Comparison between experimental sorption curves symbols of liquid carvacrol in CS CD films with a 1 1 w w blend ratio at 25 C and modelled curves continuous line obtained from Equation 2 Inset graph Experimental data and predicted values line for CS CD 35G ORH films 210 Quitosano Carvacrol According to Alfrey et al 1966 the diffusion of a sorbate in a polymer sheet
240. d borne pathogens Holley and Patel 2005 However the organoleptic effect of essential oils is one of the most important factors that limit their application as antimicrobial agents to real food products even though their antimicrobial efficiency has been widely described in in vitro tests Belletti et aL 2008 Therefore any food application of this agent should consider the potential sensory impact which could result in non acceptance by the consumer The aim of this study was firstly to obtain and characterize homogeneous transparent cinnamaldehyde imino chitosan films and to study the release of cinnamaldehyde via hydrolysis of the imino bond as affected by several combinations of time temperature treatments The antimicrobial properties of the films were then studied in vitro and in vivo against foodborne pathogens The sensory impact of the films when applied to a food product was also evaluated 2 Materials and methods 2 1 Materials Low molecular weight chitosan with a degree of acetylation of 15 25 and sodium peroxide were supplied by Sigma Barcelona Spain Trans cinnamaldehyde and acetic acid were provided by Aldrich Steinheim Germany Sodium hydroxide and ethanol 96 v v were purchased from Panreac Barcelona Spain and hydrochloric acid 37 from Merck Darmstadt Germany Ortho phosphoric acid sodium hydroxide pH 3 buffer and potassium dihydrogen phosphate disodium hydrogen phosphate pH 7 140 Quitosano Cinam
241. d for composites incorporating 35 Biocomposite glycerol and conditioned at 75 these composites retained 216 carvacrol g 100g dry matter These Functional properties results indicate that inclusion of carvacrol in the films could be occurring by mechanisms other than Sorption of carvacrol formation of inclusion complexes 2013 Elsevier Ltd All rights reserved 1 Introduction antifungal insecticidal antitoxigenic and antiparasitic activities Chitosan has been widely studied as a polymer for the design of reservoir delivery systems for the slow release of active com pounds over an extended period of time These regulated delivery systems are effective in minimizing the amount of compound used for a specific application and thus improve efficacy and reduce possible side effects associated with the use of large amounts of bioactives Pedro Cabral Albuquerque Ferreira amp Sarmento 2009 Sivakumar Manjubala amp Rao 2002 Chitosan employed as a deliv ery system can find applications in a variety of technological areas such as agrochemistry pharmacy biomedicine textiles and food packaging The development of antimicrobial materials and their application in the design of active packaging is creating consider able expectation in the food industry since food safety is an area of great concern Although there are many studies in the literature that focus on the use of chitosan films as antimicrobials in con tact with food the use o
242. d the effect of the humidity and the concentration of G in the films were evaluated As shown the addition of HP BCDs to the CS matrix produced changes in its mechanical properties In the absence of G the tensile strength and modulus of elasticity of CS and CS CD films did not differ significantly at any of the humidities tested However the elongation at break was significantly reduced P lt 0 05 by the incorporation of HP BCDs at both 5396 and 7596 RH This reduction was more acute in films conditioned at 7596 RH Thus HP BCDs act as fillers for the CS matrix decreasing the strain of the films It has been reported that fillers produce a rapid decrease in the elongation at break of polymers especially if there is good adhesion between the phases Regarding the effect of moisture both matrices CS and CS CD showed a decrease in tensile strength and modulus of elasticity when films were conditioned at 7596 RH which can be explained by the plasticizing effect of water on the polymer matrix The elongation at break of CS films rose with humidity but the effect of humidity on the elongation at break of the CS CD composites was much less acute G produced a decrease in tensile strength and modulus of elasticity in both CS and CS CD matrices and higher values of elongation effects which were more marked for films having a greater content of G All these effects were predictable since G acts in any of these matrices as a plasticizer reducin
243. da S J 1998 Selective separation of germanium IV by 2 3 dihydroxypropylchitosan resin Analytica Chimica Acta 371 187 193 Ji C Shi J 2013 Thermal crosslinked porous chitosan scaffolds for soft tissue engineering applications Materials Science and Engineering C 33 3780 3785 162 Quitosano Cinamaldeh do Jin T 2010 Inactivation of Listeria monocytogenes in skim milk and liquid egg white by antimicrobial bottle coating with polylactic acid and nisin Journal of Food Science 75 M83 M88 Jin X Wang J Bai J 2009 Synthesis and antimicrobial activity of the Schiff base from chitosan and citral Carbohydrate Research 344 825 829 Junttila J R Niemela S L Hirn J 1988 Minimum growth temperatures of Listeria monocytogenes and non haemolytic listeria Journal of Applied Bacteriology 65 321 327 Kasaai M R Arul J Chin S L Charlet G 1999 The use of intense femtosecond laser pulses for the fragmentation of chitosan Journal of Photochemistry and Photobiology A Chemistry 120 201 205 Kirdant A S Shelke V A Shankarwar S G Shankarwar A G Chondhekar T K 2011 Kinetic study of hydrolysis of N salicylidene m methyl aniline spectrophotomerically Journal of Chemical and Pharmaceutical Research 3 4 790 796 Kuhn J Considine T Singh H 2006 Interactions of milk proteins and volatile flavor compounds Implications in the development of protein foods Journal of Food Science 71 R72 R82
244. de c m lico nisina leche c l ctico nisina c c trico c c trico nisina c l ctico c l ctico nisina c m lico c m lico nisina c tart rico c tart rico nisina c l ctico prote na de suero de natamicina leche c l ctico natamicina quitooligosac ridos prote nas de soja c l ctico prote na de suero de c propi nico leche c l ctico prote na de suero de quitooligosac ridos leche c l urico prote na de soja c l urico nisina c l urico ze na c l urico EDTA c l urico nisina c l urico ze na c l urico nisina c m lico alginato de calcio c m lico cinamaldeh do c m lico citral c m lico eugenol c m lico c m lico citral c m lico eugenol c m lico geraniol alginato de calcio c m lico prote nas de suero de c m lico nisina leche c m lico nisina natamicina c m lico EDTA ED prote na de suero de 298 carne de vacuno magra dientes de ajo jam n cocido mortadela pastrami pescado mojarra ciruela estilo taiwan s medio de cultivo medio de cultivo queso Saloio medio de cultivo queso modelo experimental mortadela de pavo loncheada medio de cultivo medio de cultivo manzana Fuji troceada mel n Piel de sapo pelado medio de cultivo salchichas de L monocytogenes S Typhimurium col
245. de an important obstacle in the so called hurdle technologies that are being implemented to commercialize fresher food products In these systems interactions between the food the surrounding headspace and the package walls or independent devices are responsible for growth inhibition or death of pathogenic and or spoilage microorganisms Appendini and Hotchkiss 2002 Although exceptionally this beneficial activity can be carried out by direct contact of the microorganisms with the package surface Conte et aL 2008 Muriel Galet et al 2013 Zi Xuan et al 2012 the mechanism of activity in most packaging systems is based on mass transport processes through the packaging food interface and especially on the release of antimicrobial substances The active agent can be incorporated within a suitable polymeric matrix from which it is released following diffusion mechanisms and accumulated into the food package system following thermodynamic principles Therefore compatibility between the agent and the various system components and diffusion from the polymeric vehicles are key parameters and the efficiency and validity of an active packaging has to be tested for the specific product Recently a biopolymeric device consisting of a combination of two biodegradable renewable materials CS and HP BCD and a known natural volatile antimicrobial compound carvacrol was developed and characterized In that report the film demonstrated a great capa
246. de efecto invernadero causadas por su producci n 596 son emisiones evitables FAO 2012 UNEP 2011 2013 Los alimentos se deterioran con el tiempo fundamentalmente por la acci n de organismos vivos bacterias levaduras hongos insectos roedores o el propio hombre la acci n f sico qu mica del entorno temperatura humedad relativa RH ox geno radiaciones y la actividad biol gica del propio alimento enzimas respiraci n estr s o dafio f sico Este deterioro implica la merma de las caracter sticas organol pticas del valor nutritivo y seguridad microbiol gica del alimento llevando finalmente a la p rdida del mismo Por ello a lo largo de la historia el hombre ha desarrollado una serie de tecnolog as y procesos de diferente naturaleza y complejidad para la conservaci n de alimentos como la modificaci n de la temperatura cocci n refrigeraci n la conservaci n qu mica basada en la disminuci n del pH o de la actividad de agua acidificaci n adici n de az cares salazones curado ahumado secado al sol conservaci n biol gica fermentaciones inmersi n en grasa orza o aceite o adici n de especias Para proteger contener y almacenar estos alimentos tratados se desarrollaron los envases Los primeros envases se elaboraron con materiales naturales disponibles en el momento como hojas calabazas o conchas y m s tarde cestas de ca a botas de vino cajas y barriles de madera jarrones y nforas de barr
247. deh do sometidas a temperaturas de refrigeraci n produjeron una reducci n logar tmica de 1 34 a los 3 d as 0 81 a los 6 d as y 0 52 a los 12 d as Las pel culas de CS con cinamaldeh do tratadas con temperaturas suaves de 65 C durante 30 minutos presentaron 2 40 2 16 y 346 reducciones logar tmicas a los mismos tiempos Mientras que las pel culas desarrolladas sometidas a 95 C durante 10 minutos mostraron 4 15 3 41 y 3 87 reducciones logar tmicas respectivamente Las pel culas no se pudieron probar a 121 C durante 5 minutos ya que la leche coagul debido probablemente por la acci n coagulante y floculante del CS o bien por la uni n del cinamaldeh do a las prote nas l cteas Estas diferencias entre la actividad antimicrobiana en las pruebas in vitro y cuando se aplica al alimento pueden deberse al efecto protector de la matriz alimentaria sobre el microorganismo la uni n del cinamaldeh do a las prote nas o la diluci n de agente activo en la matriz alimentaria Este sistema presenta una gran ventaja y es que la liberaci n se producir a al aplicar el tratamiento t rmico al alimento o durante el llenado en caliente del envase con el alimento De existir una contaminaci n en ese momento sta coincidir a con la liberaci n del agente activo antimicrobiano As al inicio del crecimiento del microorganismo en el alimento el cinamaldeh do ya se habr a liberado en el medio en parte de manera que 280 Discusi n g
248. ding blocks for the development of a wide variety of polymeric networks and assemblies with a higher drug loading capacity They have been blended with polymers in the design of nano microparticles and micelles for the sustained release and targeted delivery of bioactive substances Van de Manakker et al 2009 The aim of this work was to develop novel films by blending CS with HP BCD a water soluble CD derivative in order to improve the capacity of CS film to be loaded with carvacrol a naturally occurring phenolic volatile with antimicrobial properties which is a major component of thyme and oregano essential oils The loading capacity and sorption kinetics of carvacrol were studied as a function of film formulation and the release of carvacrol was evaluated at different RH The antimicrobial activity of the films was tested in vitro in vapour phase against pathogen bacteria Staphylococcus 200 Quitosano Carvacrol aureus and Escherichia coli Finally the antimicrobial activity of the films was monitored over time 2 Materials and methods 2 1 Materials Carvacrol kosher gt 98 dimethyl sulfoxide 99 996 ACS Reagent grade phosphorus pentoxide magnesium nitrate 6 hydrate glycerol G acetic acid and low molecular weight chitosan CS were supplied by Sigma Barcelona Spain Sodium chloride potassium carbonate and barium chloride 2 hydrate were supplied by Fluka Madrid Spain Hydroxypropyl B CDs HP BCDs CAVASOL
249. ding silver via chelation with the amine groups Chitosan has also been reported to be a mild reducing agent used for reduction of silver ions and it is frequently employed as an ion capping agent to control the growth of nanoparticles and avoid their aggregation The aim of this study therefore was to develop films for the slow sustained release of silver ions consisting of chitosan polymer as the carrier matrix filled with silver based nano particles synthesized in situ The resulting films were characterized and the antimicrobial activity was tested in liquid growth media The ability of the carrier system to exert antimicrobial activity over time was also studied W MATERIALS AND METHODS Synthesis of Chitosan Silver Based Nanoparticle Films Low molecular weight chitosan MW 50 190 kDa 75 85 deacetylated from shrimp shells sodium hydroxide ACS reagent 297 096 pellets and silver nitrate ACS reagent 299 096 were obtained from Aldrich Chemical Co Inc Milwaukee WI USA A 1 5 w w chitosan solution was prepared in 0 5 w w acetic acid and stirred at 40 C for 1 h After the solution had cooled to room temperature silver nitrate previously diluted with a small amount of distilled water was added to the solution and left shaking protected from light until complete dissolution Several chitosan solutions with different silver Received September 17 2012 Revised December 4 2012 Accepted December 5 2012 Pu
250. dos productos alimenticios con valor a adido fertilizante biopesticida descontaminaci n pel culas y recubrimientos comestibles agente activo antimicrobiano y antioxidante transmisi n de genes por v as no v ricas biosensores inmovilizaci n enzim tica cromatograf a floculante quelante de metales pesticidas y bifenilos policlorados adsorci n de colorantes industria textil agente antiest tico industria papelera aditivo fotograf a agente fijador de color bater as conductor permitiendo su uso como aditivo alimentario directo secundario en la producci n de bebidas alcoh licas Adem s ha habido diversos intentos fallidos por parte de la empresa Primex Ingredients ASA para aprobar el CS procedente de camar n Pandalus borealis como aditivo alimentario en carnes y aves de corral FDA 2002 2005b 2013 aunque ya se comercializa 14 Introducci n como un suplemento diet tico en m s de 50 pa ses en todo el mundo incluida la UE los EEUU y Jap n Seg n el informe Chitin and chitosan a global strategic business report de Global Industry Analysts Inc tras la aprobaci n de todas las patentes pendientes el mercado mundial de quitina y CS crecer muy r pidamente llegando a 63000 y 21000 millones de respectivamente en 2015 La glucosamina seguida del CS son los derivados m s reclamados de la quitina La principal aplicaci n del CS es el tratamiento de agua seguido de su aplicaci n e
251. dos vol tiles En general las pel culas presentaron una considerable capacidad de retenci n de los compuestos monofen licos con valores que van desde 43096 para el m cumenol al 10696 para el guayacol dependiendo de la formulaci n de la pel cula Estos valores fueron dos rdenes de magnitud mayores que los de los compuestos sin el grupo fenol La afinidad por los compuestos monofen licos disminuy de las pel culas plastificadas con el hidr filo PG mientras que no se observaron cambios en la retenci n de los monoterpenos no fen licos La retenci n de compuestos monofen licos disminuy considerablemente cuando se sustituy el CS por el alcohol polivin lico PVOH excepto el caso del isoeugenol Por ltimo se evalu la actividad antimicrobiana de los monoterpenos y de las pel culas con ellos in vitro mediante el m todo de la microatm sfera frente E coli y S aureus El presente estudio muestra que las HP BCD y el nivel de plastificaci n alcanzado por pel culas hidr filas se pueden utilizar para regular selectivamente la capacidad de carga y de sorci n de compuestos antimicrobianos origen natural Palabras clave Quitosano alcohol polivin lico hidroxipropil B ciclodextrinas pel culas sorci n selectiva actividad antimicrobiana 245 Cap tulo 4 Art culo cient fico 7 Abstract Chitosan CS films filled with hydroxypropyl B cyclodextrins HP BCD at a 1 1 weight ratio and plasticized with glycerol G
252. e como en los casos anteriores la formaci n del enlace imino se lleva a cabo en disoluci n En el presente trabajo se desarrollan y caracterizan matrices activas basadas en el anclaje de cinamaldeh do en una pel cula de CS preformada mediante la formaci n de una base de Schiff El agente activo antimicrobiano puede ser liberado como respuesta a diferentes condiciones debido a la reversibilidad de la base de Schiff Las pel culas desarrolladas se aplican como envase activo en leche y se estudia su efecto a nivel sensorial Referencias bibliogr ficas Babu V N Kannan S 2012 Enhanced delivery of baicalein using cinnamaldehyde cross linked chitosan nanoparticle inducing apoptosis nternational Journal of Biological Macromolecules 51 1103 1108 Badawy M E L Rabea EIL 2013 Synthesis and structure activity relationship of N cinnamyl chitosan analogs as antimicrobial agents nternational Journal of Biological Macromolecules 57 185 192 Badawy M E L Rabea EIL 2014 Synthesis and antifungal property of N aryl and quaternary N aryl chitosan derivatives against Botrytis cinerea Cellulose 21 3121 3137 130 Quitosano Cinamaldeh do Balaguer M P Borne M Chalier P Gontard N Morel M H Peyron S Gavara R Hern ndez Mu oz P 2013 Retention and release of cinnamaldehyde from wheat protein matrices Biomacromolecules 14 1493 1502 Balaguer M P Fajardo P Gartner H G mez Estaca J Gavara
253. e media Scharlab Barcelona Spain and culture conditions tryptone soy agar for total aerobic bacteria incubated at 30 C for 48 h and also for total aerobic psychrophiles incubated at 10 C for 10 days King B agar for Pseudomonas spp incubated at 25 C for 48 h violet red bile glucose agar for enterobacteria incubated at 37 C for 24 h MRS agar for lactic acid bacteria incubated at 25 C for 5 days and malt extract agar for yeasts and fungi incubated at 28 C for 5 days Tests were performed in triplicate 2 6 Data analysis Statistical analysis of the results was performed with SPSS commercial software SPSS Inc Chicago Illinois USA Differences between means were assessed on the basis of confidence intervals using the Tukey b test at a level of significance of P lt 0 05 The data are represented as averagetstandard deviation 3 Results and discussion CS CD Control samples were prepared first They were transparent practically colourless and without discontinuities After the immersion in carvacrol for 3 weeks the samples were transparent and had an intense yellow colour The initial carvacrol content of the samples was 2 3 0 4 g g of dried film HP BCD and CS These high uptakes were in agreement with 230 Quitosano Carvacrol previous studies Higueras et al 2013 Nevertheless the carvacrol content in each sample was determined individually prior to use 3 1 Quantification of carvacrol concentration in
254. e and Technology 51 9 15 Tyagi A K Malik A Gottardi D Guerzoni M E 2012 Essential oil vapour and negative air ions a novel tool for food preservation Trends in Food Science amp Technology 26 99 113 Van de Manakker F Vermonden T Van Nostrum C F Hennink W E 2009 Cyclodextrin based polymeric materials synthesis properties and pharmaceutical biomedical applications Biomacromolecules 10 3157 3175 172 4 4 2 Art culo cient fico 4 Preparation and characterization of chitosan HP B cyclodextrins composites with high sorption capacity for carvacrol Laura Higueras Gracia L pez Carballo Josep P Cerisuelo Rafael Gavara Pilar Hern ndez Mufioz Carbohydrate Polymers 2013 97 262 268 Quitosano Carvacrol Resumen El objetivo de este trabajo fue dise ar nuevos sistemas basados en pol meros que presentan una capacidad de carga de carvacrol ajustable dependiendo de la formulaci n de la pel cula Para este prop sito se desarrollaron pel culas biocompuestas empleando quitosano CS como matriz polim rica e hidroxipropil B ciclodextrinas HP BCDs como un adyuvante para aumentar la sorci n de carvacrol en la matriz polim rica Se investigaron la morfolog a propiedades pticas mec nicas y de barrera de las pel culas obtenidas y se evalu su capacidad de sorci n de carvacrol Los biocompuestos resultaron muy transparentes y de elevada resistencia mec nica y barrera a la humedad La sorc
255. e film containing origanum Thymus capitatus oil Journal of Food Science 76 C178 C184 315 Anexos Emiro lu Z K Yemi G P Co kun B K Cando an K 2010 Antimicrobial activity of soy edible films incorporated with thyme and oregano essential oils on fresh ground beef patties Meat Science 86 283 288 Eswaranandam S Hettiarachchy N S Johnson M G 2004 Antimicrobial activity of citric lactic malic or tartaric acids and nisin incorporated soy protein film against Listeria monocytogenes Escherichia coli O157 H7 and Salmonella Gaminara Journal of Food Science 69 M79 M84 Fagundes C P rez Gago M B Monteiro A R Palou L 2013 Antifungal activity of food additives in vitro and as ingredients of hydroxypropylmethylcellulose lipid edible coatings against Botrytis cinerea and Alternaria alternata on cherry tomato fruit International Journal of Food Microbiology 166 391 398 Fajardo P Martins J T Fucinos C Pastrana L Teixeira J A Vicente A A 2010 Evaluation of a chitosan based edible film as carrier of natamycin to improve the storability of Saloio cheese Journal of Food Engineering 101 349 356 Fayaz A M Balaji K Girilal M Kalaichelvan P T Venkatesan R 2009 Mycobased synthesis of silver nanoparticles and their incorporation into sodium alginate films for vegetable and fruit preservation Journal of Agricultural and Food Chemistry 57 6246 6252 Fern ndez Pan I Carri n
256. e hsian tsao Mesona procumbens prote na de soja quitosano PLA prote nas de soja quitosano ze na queso kashar lomo de cerdo uva de mesa medio de cultivo pescado triturado queso panceta medio de cultivo medio de cultivo fresa salm n medio de cultivo lomo de cerdo medio de cultivo salchichas de pavo salchichas de pavo uva Redglobe ensalada con fruta carne de cerdo cortada corazones de lechuga Romana medio de cultivo salchichas clara de huevo brotes de alfalfa r bano y br coli mel n Cantalupo medio de cultivo St aureus E coli O157 H7 L monocytogenes ME TVC mes filos hongos levaduras E coli O157 H7 L monocytogenes E coli O157 H7 L monocytogenes S Typhimurium E coli O157 H7 L monocytogenes Micrococcus luteus L innocua S Enteritidis E coli St aureus L monocytogenes E coli O157 H7 ME TVC hongos levaduras E coli O157 H7 L monocytogenes L monocytogenes E coli St aureus Enterococcus faecalis Enterococcus faecium S Typhimurium B thermosphacta L monocytogenes E coli O157 H7 S Typhimurium L monocytogenes L monocytogenes E coli O157 H7 S Typhimurium B cinerea ME TVC mes filos y psicr filos hongos levaduras B cereus L monocytogenes St aureus St aureus Streptococcus mutans ME TVC mes filos LAB hongos levaduras Salmonella spp ME T
257. e of action of cationic biocides morphological effect on antibacterial activity The Journal of applied bacteriology 78 55 60 L pez Carballo G Cava D Lagar n J M Catal R Gavara R 2005 Characterization of the interaction between two food aroma components a pinene and ethyl butyrate and ethylene vinyl alcohol 87 Cap tulo 1 Art culo cient fico 1 copolymer EVOH packaging films as a function of environmental humidity Journal of Agricultural and Food Chemistry 53 7212 7216 L pez Carballo G Hern ndez Mufioz R Gavara R Ocio MJ 2008 Photoactivated chlorophyllin based gelatin films and coatings to prevent microbial contamination of food products International Journal of Food Microbiology 126 65 70 L pez Rubio A Gavara R Lagar n J M 2006 Unexpected partial crystallization of an amorphous polyamide as induced by combined temperature and humidity Journal of Applied Polymer Science 102 1516 1523 L pez de Dicastillo C Ner n C Alfaro P Catal R Gavara R Hern ndez Mu oz P 2011 Development of new antioxidant active packaging films based on ethylene vinyl alcohol copolymer EVOH and green tea extract Journal of Agricultural and Food Chemistry 59 7832 7840 Luchansky J B Call J E Hristova B Rumery L Yoder L Oser A 2005 Viability of Listeria monocytogenes on commercially prepared hams surface treated with acidic calcium sulfate and lauric arginate and
258. ed food Once the in vitro effectiveness of the CScin films had been verified their antimicrobial activity was examined in refrigerated and pasteurized whole milk with a fat content of 3 696 CScin films were immersed in milk and subjected to different preservation treatments after which the milk was inoculated with L monocytogenes and then kept at 4 C for 12 days and the antimicrobial assays were performed on days 3 6 and 12 L monocytogenes 157 Cap tulo 3 Art culo cient fico 3 is a microorganism that is able to grow at low temperatures Doyle and Beuchat 2007 Previous studies on the thermal behaviour of L monocytogenes in foods showed that the mean minimum growth temperature was 1 1 C Junttila et al 1988 and that this microorganism can survive pasteurization Fleming et al 1985 Lovett et al 1987 The characteristics of refrigerated milk pH close to neutrality large presence of nutrients might also have favoured an increase in the viable counts of L monocytogenes Muriel Galet et al 2012 As Figure 4 3 6 shows the more severe the temperature treatment was the greater was the reduction of bacterial growth in good correlation with the data obtained in the in vitro assays and in the release tests CScin films treated at 4 C for 30min yielded a log reduction of 1 34 at 3 days 0 81 at 6 days and 0 52 at 12 days Activation of the films by higher temperatures resulted in more efficient antimicrobial activity Thu
259. edio l quido transfiri ndose a otro medio para evitar la posible saturaci n del medio Se determin la capacidad antimicrobiana tanto del medio retirado como de esa pel cula tras los 30 d as del experimento Los medios de cultivo presentaron la misma actividad antimicrobiana 3 5 reducciones logar tmicas independientemente del d a de ensayo Por otra parte las pel culas presentaron mayor actividad que los medios de cultivos en los que estuvieron inmersas Por lo que se deduce que las pel culas actuaron en parte mediante la liberaci n del agente activo y en parte por contacto de las pel culas con los microorganismos Y tambi n se prueba que las pel culas son capaces de liberar iones de plata tras su inmersi n en medios acuosos y mantener su efectividad a lo largo del tiempo Para verificar esto en el tercer estudio se opera de modo similar al anterior con vol menes de medio fresco elevados y las pel culas se probaron a los 30 d as En las pel culas con baja concentraci n de plata la actividad se mantuvo tras los 30 d as mientras que en las de mayor concentraci n presentaron mayor actividad tras los 30 d as As se corrobora que las pel culas de CS con plata son efectivas a lo largo del tiempo y las pel culas con mayores cantidades de plata podr an actuar como matrices de liberaci n sostenida para las nanopart culas de plata siendo grandes reservorios de iones plata El tercer mecanismo para incorporar el agente a
260. el poly methyl methacrylate nanofiber containing silver nanoparticles Langmuir 24 2051 2056 Kvitek L Panacek A Soukupova J Kolar M Vecerova R Prucek R Holecova M Zboril R 2008 Effect of surfactants and polymers on stability and antibacterial activity of silver nanoparticles NPs Journal of Physical Chemistry C 112 5825 5834 Liau S Y Read D C Pugh W J Furr J R Russell A D 1997 Interaction of silver nitrate with readily identifiable groups relationship to the antibacterial action of silver ions Letters in Applied Microbiology 25 279 283 Lok C N Ho C M Chen R He Q Y Yu W Y Sun HZ Tam P K H Chiu J F Che C M 2006 Proteomic analysis of the mode of antibacterial action of silver nanoparticles Journal of Proteome Research 5 916 924 Lu W B Liao F Luo Y L Chang G H Sun XP 2011 Hydrothermal synthesis of well stable silver nanoparticles and their application for enzymeless hydrogen peroxide detection Electrochimica Acta 56 2295 2298 Morones J R Elechiguerra J L Camacho A Holt K Kouri J B Ramirez J T Yacaman M J 2005 The bactericidal effect of silver nanoparticles Nanotechnology 16 2346 2353 Murugadoss A Chattopadhyay A 2008 A green chitosan silver nanoparticle composite as a heterogeneous as well as micro heterogeneous catalyst Nanotechnology 19 Musarrat J Dwivedi S Singh B R Al Khedhairy A A Azam A Naqvi A 2010 Prod
261. eleased carvacrol activated by environmental relative humidity The antimicrobial effectiveness of the carvacrol released by the films was shown in in vitro assays employing the microatmosphere test The films were effective when incorporated in the prototype design of a package for fresh chicken breasts The volatile exerted its antimicrobial action in vapour phase and without contact between the film and the food However because of its high compatibility large amounts of volatile were retained in the chicken causing unacceptable sensory deterioration Tailored amounts of lipophilic antimicrobial compounds were retained in hydrophilic polymer films of chitosan or polyvinyl alcohol depending on the film formulation and chemical structure of the sorbate The new films presented higher retention capacity when they were formulated with chitosan instead of PVOH and employing glycerol as plasticizer rather than propylene glycol Phenolic monoterpenes were sorbed in greater amounts than cyclohexyl and linear monoterpene alcohols Films loaded with monoterpenes presented antimicrobial capacity in vapour phase and could be applied in the design of active food packages The results obtained demonstrate that chitosan can be a versatile multifunctional matrix applicable to the development of antimicrobial active packaging 7 ANEXOS Anexos 7 1 Anexo A Ejemplos de materiales biopolim ricos para el envasado antimicrobiano de alimentos
262. en menor medida que frente a antibi ticos Rai et al 2009 Silver 2003 Silver et aL 2006 El mecanismo de acci n inespec fico y con diferentes dianas de la plata hace menos probable que los microorganismos desarrollen resistencias como ocurre con los antibi ticos convencionales ya que implicar a mutaciones simult neas Gibbins y Warner 2005 La plata i nica como agente antimicrobiano ha sido empleada tradicionalmente en forma de sal como el nitrato de plata Sin embargo una alternativa son las nanopart culas de plata met lica ya que se ha comprobado que son m s eficaces contra bacterias virus y otros microorganismos eucariotas que sus hom logas de cobre titanio magnesio u oro Rai et al 2009 Un nanomaterial es un material natural accidental o fabricado que contenga part culas sueltas o formando un agregado o aglomerado y en el que el 5096 o m s de las part culas en la granulometr a num rica presente una o m s dimensiones externas en el intervalo de tama os comprendido entre 1 nm y 100 nm UE 20113 Se piensa que las nanopart culas met licas presentan un mecanismo de acci n similar al de los iones de plata pero con mayor efecto antimicrobiano debido a su elevada superficie espec fica y mayor penetrabilidad Dallas et al 2011 Ciertos autores sefialan como fundamental la presencia de iones plata en la superficie de la nanopart cula para ejercer su actividad antimicrobiana Por 28 Introducci n otro
263. eneral podr a alargar la fase lag de los microorganismos Posteriormente la liberaci n mantenida del cinamaldeh do desde las pel culas coincidir a con la fase exponencial de aquellos microorganismos que superen la fase anterior Ambas fases del crecimiento microbiano son m s susceptibles a los agentes qu micos que la fase estacionaria por lo que este sistema resulta muy conveniente Todos estos resultados ponen de manifiesto que las pel culas desarrolladas pueden aumentar la seguridad de alimentos susceptibles de contaminarse con Listeria monocytogenes Asimismo es de esperar que las pel culas con cinamaldeh do sean efectivas para otros microorganismos por lo que se podr a aumentar la vida til de la leche pasteurizada que se encuentra limitada a 3 d as bajo refrigeraci n El inconveniente de emplear aceites esenciales o sus componentes activos como agentes antimicrobianos es el posible impacto en las propiedades organol pticas de los alimentos en los que se aplican por lo que se llev a cabo un an lisis sensorial de la leche tras el contacto con las pel culas de CS durante 3 6 y 12 d as de almacenamiento en condiciones de refrigeraci n El estudio se realiz con catadores no entrenados que percibieron como olor a canela el cinamaldeh do migrado de las pel culas en las muestras de leche Los resultados tambi n demostraron una mayor aceptaci n de las muestras con cinamaldeh do que las muestras control sin cinamaldeh
264. ent fico 1 Yeast suspension 300 pL was pour plated in 15 mL MEA Preweighed films were placed on the surface of solidified medium and plates incubated 24 h at 28 C Tests were performed in triplicate As regards fungi 10 uL of the previous spore suspension was spread on the surface of 15 mL of solidified MEA plates Pre weighed films were placed on the inoculated plates and incubated for 5 days at 28 C Tests were performed in triplicate 2 7 Antimicrobial activity of LAE CS films on chicken breast fillets Skinless chicken breast fillet was purchased in a local store Slices ca 25 g and 63 cm of surface were individually prepared for each tested periods i e day of receipt to after 2 days t2 the suggested day as the expiration date ts and 2 days after expiration date tg Slices were wrapped with CS or CS 596LAE films to achieve intimate contact between film and meat and externally wrapped with PE film A negative control sample for each time was also prepared without CS wrapping Samples were stored at 4 C At appropriate times samples were transferred aseptically and weighed in a sterile Stomacher bag diluted with 25 mL PW Scharlab Barcelona Spain and blended in Stomacher IUL S L Barcelona for 6 min Ten fold dilution series in PW of the obtained suspensions were made and plated on selective solid media TSA mesophiles and psychrophiles MEA yeasts and fungi Pseudomonas agar base Pseudomonas spp VRB
265. ent of antimicrobial films and coatings The antimicrobial capacity of the composites formed will depend on the physical and chemical properties of the nanoparticles and also the silver ion release properties of the carrier matrix There is also great interest in the use of synthetic and natural hydrophilic polymers and hydrogels as carriers of silver These materials absorb large amounts of water owing to the presence in their structure of polar groups COH NH CONH COOH Hydrophilic polymers are capable of swelling in a moist environment thus facilitating the diffusion of the active agent through the polymer matrix and its release to the medium in contact with the polymer Chitosan is a biodegradable and biocompatible polymer obtained from biomass and possesses excellent film forming properties which have made it of great interest for technological applications in several areas such as pharmacy e ACS Publications 2012 American Chemical Society 334 260 medicine agrochemistry and packaging The hydrophilic nature of chitosan has aroused interest in its use as a sustained release carrier when the release of the retained active compound is required in a moist environment Inclusion of silver based nanoparticles as an antimicrobial nanofiller in a chitosan matrix could be done directly or by using silver nitrate as a precursor Chitosan is soluble in aqueous solution and has the ability to bind many metal ions inclu
266. er neutralized at 37 C 114 Figure 4 2 6 X ray diffraction spectra a silver oxide b silver c CS neutralized at 37 C d h CS neutralized at 37 C with 0 1 d 0 2 e 0 5 f 1 g and 1 5 h of silver 115 Figure 4 3 1 Nucleophilic addition of amino group of CS backbone to carbonyl group of cinnamaldehyde and formation of Schiff base 147 Figure 4 3 2 ATR FTIR spectra of CS and CS modified with cinnamaldehyde before CScin SL and after washing CScin 152 Figure 4 3 3 ATR FTIR spectra of CS and CScin films after the different preservation treatments 153 Figure 4 3 4 Cinnamaldehyde released from films into MHB during different temperature time treatments empty symbols and time evolution of cinnamaldehyde released into ethanol 50 at 23 C after applying preservation treatments full symbols 155 Figure 4 3 5 Antimicrobial activity of CS CScin and CS Schiff base films subjected to various combinations of temperature time treatments against S aureus and E coli 156 Figure 4 3 6 Antimicrobial effect of CS film modified with cinnamaldehyde and subjected to different preservation treatments against L monocytogenes in pasteurized milk 158 Figure 4 3 7 Values of the parameters of odour intensity and preference 160 Figure 4 4 1 Temperature derivative of sample mass obtained by TGA for selected composites and components 184 Figure 4 4 2 CS CD 35G composites conditioned at 7596 RH before left and after rig
267. er sorbent compared with the meta and para isomers owing to the formation of intramolecularly hydrogen bonded species With respect to isoeugenol the presence of the propylene substituent in the 2 methoxyphenol structure engenders the hydrophobicity of the molecule which has a log P close to isopropylphenol however its sorption in the polymers was low an order of magnitude lower than the other phenols studied 258 Quitosano Carvacrol A remarkable depletion was observed in the sorption of cyclic and linear non phenolic monoterpenes with values ranging from 0 25 to 3 596 g 100 g dry matter Bicyclic myrtenol and linear nerol with a log P similar to carvacrol together with the more hydrophilic carveol had the highest sorption values among the non phenolic compounds whereas isopulegol had the lowest sorption among all the monoterpenes studied Positional isomers dihydrocarveol and isopulegol have a slightly higher log P than carveol 2 92 vs 2 55 The cyclohexene ring in carveol increased polarity and sorption affinity for the films compared with the cyclohexane ring of dihydrocarveol whereas the sorption of positional isomer isopulegol decreased considerably which might be due to the isopropenyl substituent next to the hydroxyl group It can be concluded that the presence of a benzene group in the molecular structure of monoterpenes allowed high sorption values and plasticization of the films Plasticization by benzene of hydroph
268. erisuelo J P Berm dez J M Aucejo S Catal R Gavara R Hern ndez Mu oz P 2013 Describing and modeling the release of an antimicrobial agent from an active PP EVOH PP package for salmon Journal of Food Engineering 116 352 361 Cerisuelo J P Muriel Galet V Bermudez J M Aucejo S Catal R Gavara R Hern ndez Mu oz P 2012 Mathematical model to describe the release of an antimicrobial agent from an active package constituted by carvacrol in a hydrophilic EVOH coating on a PP film Journal of Food Engineering 110 26 37 Ciobanu A Mallard I Landy D Brabie G Nistor D Fourmentin S 2013 Retention of aroma compounds from Mentha piperita essential oil by cyclodextrins and crosslinked cyclodextrin polymers Food Chemistry 138 291 297 Chalier P Ben Arfa A Guillard V Gontard N 2009 Moisture and temperature triggered release of a volatile active agent from soy protein coated paper effect of glass transition phenomena on carvacrol diffusion coefficient Journal of Agricultural and Food Chemistry 57 658 665 Chalier P Ben Arfa A Preziosi Belloy L Gontard N 2007 Carvacrol losses from soy protein coated papers as a function of drying conditions Journal of Applied Polymer Science 106 611 620 Chi S Zivanovic S Penfield M P 2006 Application of chitosan films enriched with oregano essential oil on bologna active compounds and sensory attributes Food Science and Technolo
269. erning biodegradability biocompatibility and non toxicity of the materials used for the development of carrier systems need to be considered Renewable polymers are being widely investigated as delivery vehicles because most of them fulfil the aforementioned requirements CS polymer poly B 1 4 N acetyl D glucosamine has been intensively studied during recent decades Dutta et al 2012 L pez Carballo et aL 2013 Valencia Chamorro et al 2011 It is a natural cationic linear aminopolysaccharide obtained from partial N deacetylation of chitin CS is receiving a great deal of attention in biomedicine and pharmacology for the delivery of drugs Ramya et al 2012 CS can also act as a carrier for sustained release and delivery of compounds other than drugs which are of interest in foods personal care agriculture etc Kumar et al 2004 Prabaharan and Mano 2006 Zhang et al 2009 Antimicrobial carriers are of great interest in the area of food packaging and great efforts are being made to develop effective antimicrobial food packaging systems Appendini and Hotchkiss 2002 Suppakul et al 2003 Antimicrobial food packaging technologies which are based on the incorporation of active volatiles in polymer matrices do not require the film be in contact with the food product to be active In this case 199 Cap tulo 4 Art culo cient fico 5 the volatile is released to the headspace of the package and exerts its activity when co
270. et al 2002 Dos Santos et al 2005a b Guinesi y Cavalheiro 2006a b vainillina y derivados Sashikala y Syed Shafi 2014 salicilaldehido para su aplicaci n en distintos campos tales como filtros de aire cosm ticos y textiles Sin embargo en estos trabajos el aldeh do se a adi a la disoluci n de CS lo que conduce en muchos casos a la gelificaci n o la disminuci n de la capacidad film gena del CS Muzzarelli y Ilari 1994 Incluso Marin et al 2012 apuntan que el rendimiento de la formaci n de la imina en la disoluci n acuosa cida es bajo 1 12 y r pidamente reversible y obtienen elevados rendimientos de formaci n de la base de Schiff entre el CS en forma de hidrogel y el cinamaldeh do 9096 Por esta raz n en la presente Tesis Doctoral se ha desarrollado una metodolog a en la que inicialmente se obtienen pel culas de CS que posteriormente se sumergen en una disoluci n etan lica del aldeh do para que se forme el iminoderivado con la finalidad de obtener rendimientos elevados en la formaci n de la base de Schiff Moore y Roberts 1981 impregnaron pel culas de CS con los aldeh dos serie hom loga de acetaldeh do a decanal isobutiraldeh do isovaleraldeh do pivalaldeh do benzaldeh do salicilaldeh do o m y p tolualdeh do o y p nitrobenzaldeh do y p dimetilaminobenzaldeh do Pero no estudiaron la potencial capacidad antimicrobiana de los biocompuestos y su aplicaci n para la co
271. et al 2007 Damm et aL 2008 Perkas et aL 2007 polietersulfona Basri et aL 2011 Basri et al 2010 PP Yeo y Jeong 2003 PE Dehnavi et al 2013 S nchez Valdes et al 2009 Zapata et al 2011 PS Nassar y Youssef 2012 poliuretano Jain y Pradeep 2005 Sheikh et al 2009 Triebel et al 2011 polivinilpirrolidona An et aL 2008 o bien en PVOH Galya et al 2008 Hong et aL 2006 As como tambi n en matrices biopolim ricas como se recogen en el Anexo A La actividad antimicrobiana de las pel culas nanocompuestas con iones o nanopart culas de plata depender de varios factores tales como la cristalinidad y la capacidad de hinchamiento en agua del pol mero ya que la matriz tiene que estar plastificada para facilitar la difusi n Otros factores est n relacionados con el tipo de nanomaterial empleado para soportar los iones o nanopart culas as la liberaci n de iones en zeolitas o de nanopart culas met licas ser diferente Por ejemplo las nanopart culas de plata ofrecen m s estabilidad y una liberaci n de iones de plata m s lenta mientras que las zeolitas con plata tiene un efecto antimicrobiano superior en tiempos cortos Tambi n hay que considerar el tamafio y la forma de la part cula Las part culas m s pequefias tienen una mayor superficie espec fica para liberar iones de plata Por su parte las nanopart culas de plata esf ricas 93 Cap tulo 2 Estado de la cuesti n son m s reacti
272. experimental use the stock cultures were maintained by regular subculture on agar Tryptone Soy Agar TSA Scharlab Barcelona Spain slants at 4 C and transferred monthly 2 5 2 In vitro antimicrobial activity of CScin films against S aureus and E coli Before analysis a loopful of each strain was transferred to 10 mL of TSB and incubated at 37 C for 18 h to obtain early stationary phase cells Cell cultures of each microorganism in stationary phase with an optical density of 0 9 at 600 nm were diluted in TSB and incubated at 37 C until exponential phase with an optical density of 0 2 at 600 nm 10 colony forming units CFU mL A sample of 0 25 g of CScin film was placed in contact with 10 mL of MHB and subjected to different temperature time 144 Quitosano Cinamaldeh do treatments as described above Neutralized CS films were used as controls and also underwent these treatments After each treatment the liquid medium was recovered and was allowed to reach room temperature Then 100 pL of cell culture in exponential phase 10 CFU mL was added and the tubes were incubated at 37 C for 18 h Depending on the turbidity of the tubes serial dilutions with peptone water were carried out and plated in Petri dishes with 15 mL of TSA culture medium Colonies were counted after incubation at 37 C for 18 h The result was expressed in log of CFU per millilitre All analyses were carried out in triplicate 2 5 3 Antimicrobial
273. f a given sample against the different microorganisms Table 4 1 5 Antimicrobial activity of LAE films against the selected microorganisms on solid medium at 28 C and for 24 h for bacteria and yeasts and 5 d for fungi Zones of growth inhibition mm showing antimicrobial activity film size 10 mm plate diameter 90 mm Values for zone of growth inhibition are presented as mean SD n 3 Microorganisms Species CS 1 LAE CS 5 LAE CS 10 LAE Gram positives Listeria monocytogenes 10 1 0 1 238 03 269 02 27 5 0 5 Staphylococcus aureus 10 0 0 1 214 04 255 04 256 04 Gram negatives Escherichia coli 10 0 0 1 123 0 2 141 03 143205 Pseudomonas putida 10 0 0 1 115 0 2 140 0 3 146 03 Salmonella enterica 10 0 01 119202 141202 142 04 Yeasts Candida utilis 10 0 0 1 10 0 0 1 135 09 14 5 0 7 Saccharomyces cerevisiae 10 0 0 1 10 0 0 1 110 208 14 0 0 9 Torulopsis pinus 10 1 0 1 101 01 153 05 18 8 0 8 Fungi Aspergillus niger 10 0 0 1 10 0 0 1 10 0 0 1 10 1 0 1 Cladosporium cladosporioides 10 0 0 1 100 01 10 0 0 1 101 01 Penicillium chrysogenum 10 0 0 1 10 0 0 1 101 01 10 1 0 1 4 Different letters in the same row indicate a statistically significant difference P lt 0 05 comparing the control samples and the different amounts of LAE in the matrix Different letters in the same column indicate a statistically significant difference P lt 0 05 c
274. f around 60 mm After 12 days of storage the carvacrol content retained in the film was 4 5 mg and the inhibition zones produced were 57 and 56 mm in diameter for E coli and S aureus respectively After 20 days of storage the amount of carvacrol remaining in the film did not change greatly this was found to be 4mg and the inhibition zone against the bacteria tested was similar to the 12th day It is worth pointing out that the inhibition zone created by the film disk containing 4 5 mg of carvacrol was slightly greater than that produced by a filter paper of similar dimensions and incorporating 5 mg of carvacrol when the two samples film and filter paper were submitted to the same microatmosphere test conditions 37 C for 24 h and gt 99 RH Because of the high RH reached in the Petri dish and the temperature used in the antimicrobial experiment it was expected that the small amount of carvacrol incorporated in the paper 5 mg and remaining in the film 4 5 mg would be released in 24 h Slight differences in inhibition could be related to carvacrol release behaviour from these two materials during the antimicrobial test which could affect microbial growth 4 Conclusions This work shows that it is possible to control the loading of hydrophobic antimicrobial carvacrol in hydrophilic CS For this purpose the polymer was blended with HP BCDs and G and water were incorporated as coadjuvants Depending on the amount of these compounds in t
275. f chitosan films for the release of active compounds has received much less attention Because of their antimicrobial properties many plant extracts and essential oils have found applications as natural preserva tives In this regard carvacrol 5 isopropyl 2 methylphenol is a constituent of essential oils of oregano and thyme with known Corresponding author Tel 34 963900022 fax 34 963636301 E mail address phernan iata csic es P Hern ndez Mu oz 0144 8617 see front matter 2013 Elsevier Ltd All rights reserved http dx doi org 10 1016 j carbpol 2013 04 007 336 Burt 2004 Veldhuizen Tjeerdsma Van Bokhoven Zweijtzer Burt amp Haagsman 2006 Carvacrol is categorized as GRAS Generally Regarded as Safe by the FDA Food and Drug Administration for food Volatile active compounds can be added to films to achieve a more effective and rational use of them This step is especially prob lematicsince a large amount ofthe compounds is lost or inactivated during processing and the remaining amount in the polymer is not enough to exert its effects on the food In addition the active com pound must be chemically compatible with the polymer matrix to allow good dispersion in the film but not inhibit its release Chalier Ben Arfa Preziosi Belloy amp Gontard 2007 Kurek Descours Galic Voilley amp Debeaufort 2012 Chitosan films have been loaded with active volatile compounds for several purposes Abdollahi
276. factoriamente el crecimiento de la carga microbiana por lo que se recurre a la estrategia de combinar distintos agentes antimicrobianos Otra estrategia ser a emplear el LAE como agente antimicrobiano incorporado en materiales polim ricos que permitan su liberaci n sostenida en la superficie del alimento y su empleo en el dise o de envases activos En este sentido ya se han nombrado las ventajas de incorporar el agente antimicrobiano en el material que conforma el envase respecto a su aplicaci n directa en la superficie del alimento Sin embargo la informaci n disponible sobre la aplicaci n de LAE en pel culas polim ricas y la evaluaci n de su efectividad en alimentos es limitada Muriel Galet et al 2012 han demostrado con xito la efectividad de incorporar LAE en pel culas de EVOH stas inhibieron el crecimiento de L monocytogenes y Salmonella enterica en una leche infantil almacenada en condiciones de refrigeraci n Theinsathid et al 2012 aplicaron un recubrimiento de PLA con LAE en jam n cocido loncheado confirmando su actividad antimicrobiana frente a L monocytogenes y Salmonella Typhimurium Jin et al 2013 desarrollaron exitosamente recubrimientos de PLA o CS con LAE para la c scara de huevo y frente a Salmonella Posteriormente se ha desarrollado una pel cula de PLA 64 Quitosano LAE recubierta de CS incorporando LAE y un recubrimiento de CS con LAE siendo eficaces frente a Listeria y Salmonella en derivados c
277. fer line A cut piece of the film was cleaned with a paper tissue to remove any excess of volatile compound on the film surface and then inserted into an empty desorption tube 11 5 x 0 39 cm 1 D The tube was placed in the desorber chamber which was immediately sealed Conditions for desorption were as follows desorption temperature 210 C transfer line 230 C desorption time 7 min He desorption flow 8 15 mL min The GC was equipped with a TRB5 30 m 0 32 mm 0 25 um column Teknokroma Barcelona Spain and a flame ionization detector The chromatographic conditions were 260 C detector temperature 7 min at 45 C heating ramp to 220 C at 18 C min and 1 min more at 220 C After the analysis the film sample was recovered from the desorption tube and weighed on an analytical balance Voyager V11140 model Ohaus Europe Greifensee Switzerland 251 Cap tulo 4 Art culo cient fico 7 2 6 Antimicrobial assays 2 6 1 Bacterial strains and growth conditions Two model microbial strains were obtained from the Spanish Type Culture Collection CECT Valencia Spain the Gram positive bacterium Staphylococcus aureus CECT 86 and the Gram negative bacterium Escherichia coli CECT 434 The strains were stored in Mueller Hinton Broth MHB Scharlab Barcelona Spain with 2096 G at 80 C until needed For experimental use the stock cultures were maintained by regular subculture on TSA slants at 4 C and transferred monthly In
278. ffect However the hue was not significantly different So the differences were not visually perceptible between control CS film and LAE incorporated films since AE lt 1 colour differences were not obvious for the human eye Bodart et al 2008 3 2 LAE release from films LAE release from the tested films into water was studied at 28 C temperature selected for the antimicrobial activity tests as well as at 4 C habitual temperature for refrigerated storage Figure 4 1 1 shows the results obtained at both temperatures For better comparison results were presented as the ratio between the amount of LAE released into the aqueous media at time t and that obtained at equilibrium M M which in both cases was in coincidence with the theoretical value estimated considering full extraction As can be seen in the figure both tests provided similar profiles which could be described as that of an exponential function growing to a maximum which was achieved at ca 10 h at 28 C and at 15 h at 4 C Table 4 1 1 Colour parameter values of CS films with different amounts of LAE and plasticized with 2096 G LAE 96 L a b Ce has INP 0 877 03 24401 107 405 110505 1028504 1 876505 26 01 116 09 119 09 1028206 094056 5 878203 26 01 115 05 1172055 1026 0 2 09203 10 88 0 0 3 26 01 120207 123207 1024 04 07 05 Different letters in the same column indicate a statistically significant difference P lt 0 05 comparin
279. films were prepared too 2 4 Quantification of carvacrol concentration The analysis of the initial concentration of carvacrol retained in the materials was performed by thermal desorption coupled to gas chromatography using a Dynatherm Thermal Desorber Model 890 891 Supelco Teknokroma Barcelona Spain connected in series to the column of an HP5890 gas chromatograph Series II Plus Agilent Technologies Barcelona Spain via a heated transfer line A sample of the CS CD Active film was cleaned with a paper tissue to remove any excess of volatile compound on the film surface and then immediately inserted into an empty desorption tube 11 5 x 0 39 cm 1 D The tube was placed in the desorber chamber which was then closed Conditions for desorption were as follows desorption temperature 210 C transfer line 230 C desorption time 7 min He desorption flow 8 15 mL min The GC was equipped with a TRB5 30 m 0 32 mm 0 25 um column Teknokroma Barcelona Spain and a flame ionization detector The chromatographic conditions were 260 C detector temperature 7 min at 45 C heating ramp to 220 C at 18 C min and 1min more at 220 C After the analysis the film sample was recovered from the desorption tube and weighed on an analytical balance Voyager model V11140 Ohaus Europe Greifensee Switzerland The desorber GC was calibrated by measuring other polymeric polyethylene and polypropylene film samples with known amounts of carv
280. foodborne pathogens on meat and poultry products Journal of Food Science 74 M440 M445 Raybaudi Massilia R M Rojas Gra M A Mosqueda Melgar J Mart n Belloso O 2008 Comparative study on essential oils incorporated into an alginate based edible coating to assure the safety and quality of fresh cut Fuji apples Journal of Food Protection 71 1150 1161 Rojas Gra M A Avena Bustillos R J Olsen C Friedman M Henika P R Mart n Belloso O Pan Z L McHugh T H 2007 Effects of plant essential oils and oil compounds on mechanical barrier and antimicrobial properties of alginate apple puree edible films Journal of Food Engineering 81 634 641 Sanla Ead N Jangchud A Chonhenchob V Suppakul P 2012 Antimicrobial activity of cinnamaldehyde and eugenol and their activity after incorporation into cellulose based packaging films Packaging Technology and Science 25 7 17 Sashikala S Syed Shafi S 2014 Synthesis and characterization of chitosan schiff base derivatives Der Pharmacia Lettre 6 90 97 Singh A Narvi S S Dutta P K D Pandey N 2006 External stimuli response on a novel chitosan hydrogel crosslinked with formaldehyde Bulletin of Materials Science 29 233 238 Sipahi R E Castell P rez M E Moreira R G Gomes C Castillo A 2013 Improved multilayered antimicrobial alginate based edible coating extends the shelf life of fresh cut watermelon Citrullus lanatus Lwt Food S
281. for 24 h Values expressed as logarithm of colony forming units log CFU mL and log reduction value LRV in brackets 84 Table 4 1 5 Antimicrobial activity of LAE films against the selected microorganisms on solid medium at 28 C and for 24 h for bacteria and yeasts and 5 d for fungi Zones of growth inhibition mm showing antimicrobial activity film size 10 mm plate diameter 90 mm Values for zone of growth inhibition are presented as mean SD n 3 84 Table 4 2 1 Colour parameters of CS films with different concentrations of silver neutralized at 22 C and 37 C 112 Table 4 2 2 Antimicrobial activity of non autoclaved and autoclaved AUTO CS films neutralized at 37 C against S aureus and E coli in Mueller Hinton Broth MHB and diluted MHB 1 125 117 Table 4 2 3 Antimicrobial activity of diluted MHB 1 125 which has been in contact with films incorporating 0 2 of silver for several times 120 Table 4 2 4 Antimicrobial activity of diluted MHB 1 125 which has been in contact with films containing 0 2 or 1 5 silver for 24 h being the medium replaced with fresh medium every 24 h for a total period of 30 days and antimicrobial activity of these films after 30 days 121 ndice de tablas y figuras Table 4 2 5 Antimicrobial effect of films neutralized at 37 C with 0 2 and 1 5 silver against S aureus tested in diluted MHB 1 125 after different immersion times in distilled water 122 Tabla 4 3 1 Diversos bio
282. forming part of the aroma profile of fruits presenting 337 Anexos International Journal of Food Microbiology 188 2014 53 59 Contents lists available at ScienceDirect International Journal of Food Microbiology journal homepage www elsevier com locate ijfoodmicro Antimicrobial packaging of chicken fillets based on the release of carvacrol from chitosan cyclodextrin films CrossMark Laura Higueras Gracia L pez Carballo Pilar Hern ndez Mu oz Ram n Catal Rafael Gavara Instituto de Agroqu mica y Tecnolog a de Alimentos IATA CSIC Avenida Agustin Escardino 7 46980 Paterna Valencia Spain ARTICLE INFO ABSTRACT Article history Chitosan cyclodextrin films CS CD incorporating carvacrol were obtained by casting and conditioned at 23 C Received 1 May 2014 and 75 relative humidity prior to being immersed in liquid carvacrol until they reached sorption equilibrium In Received in revised form 15 July 2014 a previous work the in vitro antimicrobial activity of these films was studied In this work active films were used Jic a Pa m to inhibit microbial growth in packaged chicken breast fillets Samples of CS CD films loaded with carvacrol of y different sizes and thus with different quantities of antimicrobial agent were stuck to the aluminium lid used Keywords to seal PP EVOH PP cups containing 25 g of chicken fillets These samples were stored for 9 days at 4 C The pack Chitosan films ages we
283. g njol hierba lim n citronela o lemongrass dorado o lampuga lisozima o muramidasa mojarra o tilapia natamicina o pimaricina ovotransferrina o conalb mina pez gato bagre o siluro Referencias bibliogr ficas Abdollahi M Rezaei M Farzi G 2012 Improvement of active chitosan film properties with rosemary essential oil for food packaging International Journal of Food Science and Technology 47 847 853 Ahmad M Benjakul S Prodpran T Agustini T W 2012 Physico mechanical and antimicrobial properties of gelatin film from the skin of unicorn leatherjacket incorporated with essential oils Food Hydrocolloids 28 189 199 Ahmad M Benjakul S Sumpavapol P Nirmal N P 2012 Quality changes of sea bass slices wrapped with gelatin film incorporated with lemongrass essential oil International Journal of Food Microbiology 155 171 178 Akbar A Anal AK 2014 Zinc oxide nanoparticles loaded active packaging a challenge study against Salmonella Typhimurium and Staphylococcus aureus in ready to eat poultry meat Food Control 38 88 95 Aloui H Khwaldia K S nchez Gonz lez L Muneret L Jeandel C Hamdi M Desobry S 2014 Alginate coatings containing grapefruit essential oil or grapefruit seed extract for grapes preservation International Journal of Food Science and Technology 49 952 959 Sul Anexos Alparslan Y Baygar T Baygar T Hasanhocaoglu H Metin C 2014 Effects of gela
284. g interchain interactions and cohesion The addition of this plasticizer had a secondary effect which was the increment in water content of the matrices at any humidity because of the high hydrophilicity of G as already shown in Table 4 4 2 Since both water and G produced the same effect on the matrices their effect on mechanical properties was cumulative the materials being less brittle and more deformable for a greater content in either G or humidity Regarding the effect of G in the elongation at break of CS or CS CD films Table 4 4 4 shows that contents exceeding 2096 G did not affect this 188 Quitosano Carvacrol Table 4 4 3 Water vapour permeability of CS and CS CD films obtained with 0 53 and 0 75 humidity gradient at 23x 1 C and oxygen permeability of CS and CS CD films obtained at 50 and 7596 RH at 23 1 C Water vapour permeability VEO 27 0 53 RH 0 75 RH Oxygen permeability Materials films cc m m s Pa 50 RH 75 RH cs 249 0 137 5772037 2 80 0 12 10 9 27 0 06 104 CS 20G 2 57 012 693 0 62 498 0 0510 5 40 0 9010 P CS 35G 529 0225 111120392 115 40 01 10 1 25 0 16 10 CS CD 2 16 0 03 349 04 451 0 1310 104 0 1010 v CS CD 20G 2 35 0 06 596 038 798 01510 717 07910 CS CD 35G 6 61 0 10 10 37 38 243 009105 145 0 2510 Different letters in the same column a c indi
285. g substances and adjuvants revisado 01 04 2014 Fernandez Alvarez M 2000 Revision envasado activo de los alimentos Review active food packaging Food Science and Technology International 6 97 108 Foster L J R Butt J 2011 Chitosan films are NOT antimicrobial Biotechnology Letters 33 417 421 Freedonia 2014 Active and intelligent packaging The Freedonia Group Inc http www freedoniagroup com industry category pack packaging htm Frejo M T D az M J Lobo M Garcia J Cap M 2011 Nanotoxicolog a ambiental retos actuales Medicina balear 26 36 46 Garcia Dominguez J J Infante M R Erra P Julia M R 1983 N alpha acil L alkylaminoguanidinic acids and their salts as surfactants with antimicrobial action ES 512643 A1 Spanish patent Gibbins B Warner L 2005 The role of antimicrobial silver nanotechnology Medical Device amp Diagnostic Industry Magazine 1 1 2 Gontard N Guilbert S Cuq J L 1993 Water and glycerol as plasticizers affect mechanical and water vapor barrier properties of an edible wheat gluten film Journal of Food Science 58 206 211 Goodship V Jacobs D 2005 Polyvinyl alcohol materials processing and applications Rapra Review Reports 16 1 131 Gupta P Vermani K Garg S 2002 Hydrogels from controlled release to pH responsive drug delivery Drug Discovery Today 7 569 579 41 Introducci n Guti rrez J Barry Ryan C Bourke
286. g the different amounts of LAE in the matrix 79 Cap tulo 1 Art culo cient fico 1 4C 28 C 10 j x 0 8 8 8 06 s E 7 Re z z 0 4 02 CS A LAE CS 1 LAE CS S LAE CS 5 LAE CS TO LAE CS 10 LAE 0 0 0 0 time min time min Figure 4 1 1 Normalized release of LAE M M from CS films into water versus time of exposition at 4 and 28 C Dots are experimental data and lines represent theoretical curves obtained with Eq 1 and D values included in Table 4 1 2 When exposed to wet environments CS as an hydrophilic polymer is able to gain a large amount of water that swells the polymer increases chain flexibility reduces interchain cohesion energy and consequently increases the kinetics of mass transport processes within the polysaccharidic matrix As a consequence of the plasticizing process the diffusion of substances within the CS matrixes is altered and the process might not follow Fickian behaviour Nevertheless the size of the water molecule is so small compared to LAE s molecule that the water sorption in the matrix and its relaxation can be considered to occur before any relevant amount of LAE has been released This assumption has been successfully applied in the mass transport of other substances from hydrophilic polymeric materials L pez Carballo et al 2005 L pez de Dicastillo et al 2011 To characterize the kinetics of LAE release from CS films the solution to
287. gente activo para la obtenci n de pel culas antimicrobianas para su aplicaci n en envases alimentarios Esta gran cantidad de trabajos tiene que ver con el reconocimiento del carvacrol como sustancia segura por la FAO OMS 2001b n de la JECFA 710 como aditivo alimentario permitido para adici n directa a alimentos de consumo humano 21CFR172 515 por la FDA 2014a y su reciente inclusi n en la lista de sustancias aromatizantes UE 2012 1 6 3 2 Cinamaldeh do El cinamaldeh do 3 fenil 2 propenal cuya estructura qu mica se muestra en la Figura 1 9 es un compuesto fenilpropanoide constituyente del AE de la corteza del rbol de la canela y otras especies de su g nero Cinnamomum verum C cassia encontr ndose generalmente en un 65 9596 de la composici n total del AE y dot ndolo de su sabor y olor caracter sticos Cocchiara et al 2005 OMS 1999 El cinamaldeh do ha demostrado tener un amplio espectro de acci n antimicrobiana Bickers et al 2005 Debido a su estructura e hidrofobicidad el cinamaldeh do puede pasar a trav s de las porinas de la membrana externa de las bacterias gram negativas sin la desintegraci n de la membrana ni la depleci n del ATP Holley y Patel 2005 El cinamaldeh do act a inhibiendo la producci n de enzimas intracelulares lo que provoca el deterioro de la pared Holley y Patel 2005 Al acceder al citoplasma puede afectar a diferentes org nulos como las mitocondrias Usta et al 20
288. ght chitosan CS was supplied by Sigma Barcelona Spain Polyvinyl alcohol PVOH Gohsenol type AH 17 saponification degree 97 98 5 mol and viscosity 25 30 mPa s was obtained from The Nippon Synthetic Chemical Co Osaka Japan Hydroxypropyl B cyclodextrins HP BCD CAVASOL W 7 HP were supplied by Wacker Ib rica Barcelona Spain Carvacrol kosher gt 98 L carveol gt 95 mixture of cis and trans dihydrocarveol kosher gt 96 isopulegol 9996 isoeugenol gt 98 mixture of cis and trans nerol kosher gt 97 guaiacol and dimethyl sulfoxide 99 9 ACS reagent DMSO were supplied by Sigma Barcelona Spain meta Cumenol 29796 and ortho cumenol 29896 were purchased from Fluka Madrid Spain R Myrtenol 9596 glycerol G propylene glycol PG and acetic acid were obtained from Aldrich Barcelona Spain Sodium nitrite sodium chloride potassium chloride and barium chloride dehydrate were supplied by Sigma Aldrich Madrid Spain Peptone Water PW 0 196 Tryptone Soy Agar TSA and Tryptone Soy Broth TSB were supplied by Scharlau Barcelona Spain 249 Cap tulo 4 Art culo cient fico 7 2 2 Film preparation Films based on CS were prepared from 1 596 w w CS solution dissolved in 0 596 w w acetic acid stirred at 40 C for 1 h and filtered to eliminate impurities For films based on PVOH a 496 w w PVOH solution was prepared in distilled water and stirred at 85 90 C for 2 h CDs were added to the film f
289. gnated soy based films on the growth of Listeria monocytogenes on turkey bologna Poultry Science 81 721 726 De Moura M R Mattoso L H C Zucolotto V 2012 Development of cellulose based bactericidal nanocomposites containing silver nanoparticles and their use as active food packaging Journal of Food Engineering 109 520 524 De Oliveira T M Soares N D F Pereira R M Fraga K D 2007 Development and evaluation of antimicrobial natamycin incorporated film in Gorgonzola cheese conservation Packaging Technology and Science 20 147 153 Del Nobile M A Conte A Incoronato A L Panza O 2008 Antimicrobial efficacy and release kinetics of thymol from zein films Journal of Food Engineering 89 57 63 D az Visurraga J Mel ndrez M F Garc a A Paulraj M C rdenas G 2010 Semitransparent chitosan TiO2 nanotubes composite film for food package applications Journal of Applied Polymer Science 116 3503 3515 Dos Santos N S T Aguiar A J A A De Oliveira C E V De Sales C V Silva S D E Da Silva R S Stamford T C M De Souza E L 2012 Efficacy of the application of a coating composed of chitosan and Origanum vulgare L essential oil to control Rhizopus stolonifer and Aspergillus niger in grapes Vitis labrusca L Food Microbiology 32 345 353 Du W X Olsen C W Avena Bustillos R J McHugh T H Levin C E Friedman M 2008 Antibacterial activity against E coli O157 H7 physical prope
290. gy International 12 111 117 Guti rrez L Batlle R S nchez C Ner n C 2010 New approach to study the mechanism of antimicrobial protection of an active packaging Foodborne Pathogens and Disease 7 1063 1069 Kayaci F Uyar T 2012 Encapsulation of vanillin cyclodextrin inclusion complex in electrospun polyvinyl alcohol PVA nanowebs prolonged shelf life and high temperature stability of vanillin Food Chemistry 133 641 649 Kfoury M Auezova L Fourmentin S Greige Gerges H 2014 Investigation of monoterpenes complexation with hydroxypropyl B cyclodextrin Journal of Inclusion Phenomena and Macrocyclic Chemistry 80 51 60 170 Quitosano Carvacrol Kurek M Descours E Galic K Voilley A Debeaufort F 2012 How composition and process parameters affect volatile active compounds in biopolymer films Carbohydrate Polymers 88 646 656 Kurek M Guinault A Voilley A Galic K Debeaufort F 2014 Effect of relative humidity on carvacrol release and permeation properties of chitosan based films and coatings Food Chemistry 144 9 17 Kurek M Moundanga S Favier C Galic K Debeaufort F 2013 Antimicrobial efficiency of carvacrol vapour related to mass partition coefficient when incorporated in chitosan based films aimed for active packaging Food Control 32 168 175 Kurkov S V Loftsson T 2013 Cyclodextrins International Journal of Pharmaceutics 453 167 180 L pez M
291. hams shortly after vacuum packaging by spray application of lauric arginate Journal of Food Protection 72 2517 2523 Taormina P J Dorsa W J 2009b Short term bactericidal efficacy of lauric arginate against Listeria monocytogenes present on the surface of frankfurters Journal of Food Protection 72 1216 1224 67 Cap tulo 1 Estado de la cuesti n Theinsathid P Visessanguan W Kruenate J Kingcha Y Keeratipibul S 2012 Antimicrobial activity of lauric arginate coated polylactic acid films against Listeria monocytogenes and Salmonella Typhimurium on cooked sliced ham Journal of Food Science 77 M142 M149 Woodcock N H Hammond B H Ralyea R D Boor KJ 2009 N Lauroyl L arginine ethylester monohydrochloride reduces bacterial growth in pasteurized milk Journal of Dairy Science 92 4207 4210 68 4 1 2 Art culo cient fico 1 Development of a novel antimicrobial film based on chitosan with LAE ethyl N dodecanoy l L arginate and its application to fresh chicken Laura Higueras Gracia L pez Carballo Pilar Hern ndez Mu oz Rafael Gavara Manuela Rollini International Journal of Food Microbiology 2013 165 339 345 Quitosano LAE Resumen Se han desarrollado pel culas de quitosano CS con el agente antimicrobiano etil N dodecanoil L arginato LAE para su aplicaci n en el envasado de alimentos Las pel culas fueron obtenidas por extensi n y evaporaci n de la soluci n film gena
292. he CS matrix the films can be loaded with different input amounts of carvacrol Moreover the release rate of carvacrol from the films depends greatly on the environmental RH The films developed have antimicrobial properties and can be active for an extended period of time They could be applied in the design of active packages to inhibit microbial growth on the surface of solid foods Because of the volatile properties of carvacrol direct contact of the film with the food would not be required since the moisture present in the package triggers and controls release of the compound entrapped in the film 218 Quitosano Carvacrol Acknowledgments The authors acknowledge financial support from the Spanish Ministry of Economy and Competitiveness MINECO AGL2012 39920 C03 01 project Laura Higueras thanks the Spanish National Research Council for a JAE PreDoc Fellowship References Alfrey T Gurnee E F Lloyd W G 1966 Diffusion in glassy polymer Journal of Polymer Science Part C Polymer Symposia 12 249 261 Appendini P Hotchkiss J H 2002 Review of antimicrobial food packaging nnovative Food Science amp Emerging Technologies 3 113 126 ASTM 2007 Standard practice for maintaining constant relative humidity by means of aqueous solutions ASTM International West Conshohocken PA Ben Arfa A Combes S Preziosi Belloy L Gontard N Chalier P 2006 Antimicrobial activity of carvacrol related to its chemi
293. he effect of nanoclays and emulsifiers was weak B CDs are cyclic oligosaccharides composed of seven glucopyranose units with a truncated cone shape characterized by a hydrophilic external surface and a hydrophobic cavity This unique structure enables CDs to form inclusion complexes entrapping all or part of a guest molecule inside their 248 Quitosano Carvacrol cavities and presenting potential interest as agents to retain or release entrapped substances However enhancement of the solubility of hydrophobic compounds by non inclusion aspects of CDs is currently being studied such as solubilisation by formation of self assembled aggregates or surfactant like effects Messner et al 2010 The present work studies the effect of incorporating water soluble derivatives of CDs HP BCD and low molecular weight hydrophilic plasticizers G water and PG on the sorption capacity of films based on CS polymer for several naturally occurring monoterpenes Sorption properties of those films which absorbed a greater amount of sorbate were compared with those of films formed by replacing CS with synthetic PVOH as the polymer matrix Previously the antibacterial activity of the volatile liquids was tested against S aureus and E coli using the microatmosphere method of Kellner and Kober 1954 Finally the antimicrobial activity of the resulting films was tested by the same method 2 Materials and methods 2 1 Chemicals Low molecular wei
294. hese properties make CS a good candidate for use in food packaging applications providing a barrier layer to other polymer films and porous materials such as fibre based paper G llstedt and Hedenqvist 2006 In addition amino and hydroxyl groups of CS can be used to modify the polymer chemically in order to provide it with new properties and widen its technological applications Moreover CS has been extensively studied as a carrier and system for sustained release of active compounds and in this regard it has been applied in a great variety of technological areas such as agrochemistry pharmacy biomedicine textiles and active food packaging Sashiwa and Aiba 2004 The development of antimicrobial materials and their application in the design of active packaging is arousing considerable expectation in the food industry since food safety is an area of great concern Regarding CS films many studies in the literature have endeavoured to use the antimicrobial properties of protonated CS polymer to form films for active food packaging applications However protonated films are water soluble which limits their applications whereas neutralized films lack antimicrobial properties Other studies have focused on the development of antimicrobial films for food packaging applications by incorporating antimicrobial compounds in the film forming solution prior to film formation Usually the active molecule is incorporated during film formation but
295. ht in contact with carvacrol for three months 190 Figure 4 4 3 Flowchart of film preparation 202 Figure 4 4 4 Sorption equilibrium of carvacrol in CS CD films with a 1 1 w w blend ratio incorporating several percentages of G and conditioned at different RH prior to immersion in the solvate and measured at 25 C Figure also shows the equilibrium moisture content of the films at 25 C before being immersed in carvacrol 207 Figure 4 4 5 Effect of HP BCD content in a CS CD 35G 75RH film on the sorption capacity of carvacrol at 25 C 209 Figure 4 4 6 Carvacrol sorption capacity by films of CS incorporating MDs 1 1 weight ratio Films were plasticized with 3596 G and conditioned at 0 53 and 7596 RH prior to immersion in carvacrol 210 Figure 4 4 7 Comparison between experimental sorption curves symbols of liquid carvacrol in CS CD films with a 1 1 w w blend ratio at 25 C and modelled curves continuous line obtained from Equation 2 Inset graph Experimental data and predicted values line for CS CD 35G ORH films 210 XXII ndice de tablas y figuras Figure 4 4 8 Experimental symbols and theoretical continuous line normalized time desorption curves of carvacrol from CS CD 35G 75RH film measured at 25 C as a function of environmental RH 213 Figure 4 4 9 Carvacrol remaining in CS CD 35G 75RH film at 25 C and 43 RH and inhibition halo produced against S aureus and E coli over time 217 Figure 4 4 10 E
296. hysical mechanical and structural properties of chitosan films Food Hydrocolloids 36 287 293 Perdones A S nchez Gonz lez L Chiralt A Vargas M 2012 Effect of chitosan lemon essential oil coatings on storage keeping quality of strawberry Postharvest Biology and Technology 70 32 41 Perdones A Vargas M Atares L Chiralt A 2014 Physical antioxidant and antimicrobial properties of chitosan cinnamon leaf oil films as affected by oleic acid Food Hydrocolloids 36 256 264 Peretto G Du W X Avena Bustillos R J Berrios J D J Sambo P McHugh T H 2014 Optimization of antimicrobial and physical properties of alginate coatings containing carvacrol and methyl cinnamate for strawberry application Journal of Agricultural and Food Chemistry 62 984 990 Petrou S Tsiraki M Giatrakou V Savvaidis LN 2012 Chitosan dipping or oregano oil treatments singly or combined on modified atmosphere packaged chicken breast meat nternational Journal of Food Microbiology 156 264 271 Pintado C M B S Ferreira M A S S Sousa I 2009 Properties of whey protein based films containing organic acids and nisin to control Listeria monocytogenes Journal of Food Protection 72 1891 1896 Pintado C M B S Ferreira M A S S Sousa L 2010 Control of pathogenic and spoilage microorganisms from cheese surface by whey protein films containing malic acid nisin and natamycin Food Control 21 240 246 Pinto R
297. i 1998 Las CDs son estables en disoluciones neutras y b sicas pero se degradan lentamente en pH cido Del Valle 2004 Kurkov y Loftsson 2013 El punto de fusi n de las CDs nativas no est bien definido pero en general comienzan a descomponerse a partir de 197 C y en atm sfera inerte 252 400 C Trotta et al 2000 Debido a la presencia de estos grupos hidroxilo en los extremos de la cavidad las CDs son solubles en agua y son insolubles en la mayor a de los solventes org nicos comunes y su solubilidad es el resultado de la capacidad de interacci n de dichos grupos hidroxilo con el medio acuoso En el caso de la B CD se establecen siete enlaces por puentes de hidr geno formando un cintur n en la cara secundaria y origina una estructura r gida estable y dif cil de solvatar lo que explica en cierta medida la menor solubilidad en agua Astray et al 2009 Szejtli 1998 La solubilidad de las CDs naturales especialmente de la B CD se puede aumentar por la formaci n de derivados mediante ataque nucleof lico a los grupos hidroxilo Del Valle 2004 En el presente trabajo se han empleado las hidroxipropil B CDs HP BCD que presentan una mayor solubilidad en agua que su CD nativa la B CD Debido a su peculiar estructura c nica hueca de superficie externa hidr fila y cavidad interior hidrof bica las CDs pueden hospedar distintos compuestos formando complejos de inclusi n Figura 1 6 Una o dos mol culas hu sped p
298. i O157 H7 ME TVC mes filos hongos Lb sakei Serratia liquefaciens ME LAB Enterobacteriaceae ME TVC y anaerobios Zygosaccharomyces rouxii Zygosaccharomyces mellis L monocytogenes L monocytogenes E coli O157 H7 S Gaminara ME TVC mes filos Lactococcus spp Lactobacillus spp Staphylococcus spp Pseudomonas spp Enterobacteriaceae hongos levaduras E coli St aureus Yarrowia lipolytica ME Staphylococcus spp Pseudomonas spp Enterobacteriaceae hongos levaduras L monocytogenes S Enteriditis L monocytogenes Lb plantarum E coli O157 H7 ME TVC mes filos y psicr filos hongos levaduras S Enteritidis ME TVC mes filos y psicr filos hongos levaduras L monocytogenes Penicillium spp Ps aeruginosa Yarrowia lipolytica L monocytogenes E coli Siragusa y Dickson 1992 1993 Geraldine et al 2008 Ouattara et al 2000 Ou et al 2002 Chen et al 1999 Pintado et al 2009 Eswaranandam et al 2004 Ramos Pereira et al 2012 Ramos Silva et al 2012 Ramos Santos et al 2012 Dawson et al 2002 Hoffman et al 2001 Padgett et al 2000 Raybaudi Massilia Rojas Gra et al 2008 Raybaudi Massilia Mosqueda Melgar et al 2008 Pintado et al 2010 Gadang et al Anexos semilla de uva nisina c propi nico c s rbico c s rbico PABA benzoato de sodio nisina
299. i n de carvacrol se vio muy afectada por el contenido de humedad y glicerol G de las biocompuestos Los valores m s altos de sorci n se lograron para materiales compuestos con un 35 de glicerol y acondicionados a 7596 RH reteniendo un 21696 carvacrol g 100 g de materia seca Estos resultados indican que la inclusi n de carvacrol en las pel culas puede estar ocurriendo por mecanismos distintos de la formaci n de complejos de inclusi n Palabras clave Quitosano hidroxipropil B ciclodextrinas biocompuesto propiedades funcionales sorci n de carvacrol 175 Cap tulo 4 Art culo cient fico 4 Abstract The aim of this work was to design new polymer based systems exhibiting an adjustable loading capacity of carvacrol depending on the film formulation For this purpose biocomposite films were developed employing chitosan CS as the polymer matrix and hydroxypropyl B cyclodextrins HP BCDs as an adjuvant to improve the sorption of carvacrol in the polymer matrix The morphology optical mechanical and barrier properties of the resulting films were investigated and the sorption capacity of carvacrol evaluated Biocomposites resulted highly transparent with higher mechanical resistance and moisture barrier properties Sorption of carvacrol was greatly affected by the humidity and glycerol G content of the biocomposites The highest sorption values were achieved for composites incorporating 3596 glycerol and conditioned at 75
300. i n de complejos Esta liberaci n se vio incrementada con el aumento de temperatura y menores concentraciones de LAE Adem s la liberaci n del LAE pudo extenderse 10 h a 28 C y 15 h a 4 C hasta el agotamiento de la pel cula a lo cual influy directamente el elevado tama o de la mol cula de LAE Asimismo se prob la eficacia antimicrobiana de las pel culas de CS con 596 LAE para el envasado activo de pollo fresco En este caso se evidenci la capacidad antimicrobiana inherente del pol mero ya que las pel culas de CS no fueron neutralizadas siendo de esperar que parte de sus grupos amino se mantuvieran protonados despu s del procesado de la pel cula La actividad antimicrobiana se vio incrementada significativamente por la presencia de LAE en la matriz biopolim rica El recuento de bacterias aerobias mes filas se utiliza como ndice de la calidad higi nica de la carne con niveles aceptables entre 5x10 y 5x10 CFU de aerobios por gramo de carne separada mec nicamente CE 2005 F bregas y Toca 1997 En las muestras control de pechugas de pollo analizadas el recuento de bacterias mes filas se encontr por debajo de este nivel hasta el d a de fecha de caducidad sexto d a indicada por el proveedor a partir de la cual ya se sobrepas Por su parte las muestras envasadas con las pel culas de CS efectuaron una ligera inhibici n del recuento microbiano total Sin embargo las muestras envasadas con las pel culas con C
301. i rrez L pez G Barbosa Canovas G Welti Chanes J Parada Arias E Eds Food Engineering Integrated Approaches Springer New York pp 225 241 Garc a M A Martino M N Zaritzky N E 1998 Plasticized starch based coatings to improve strawberry Fragaria ananassa quality and stability Journal of Agricultural and Food Chemistry 46 3758 3767 Garc a M A Martino M N Zaritzky N E 2001 Composite starch based coatings applied to strawberries Fragaria ananassa Nahrung Food 45 267 272 Gemili S Yemenicioglu A Altinkaya S A 2009 Development of cellulose acetate based antimicrobial food packaging materials for controlled release of lysozyme Journal of Food Engineering 90 453 462 Geraldine R M Soares N D F Botrel D A Goncalves L D 2008 Characterization and effect of edible coatings on minimally processed garlic quality Carbohydrate Polymers 72 403 409 Giatrakou V Ntzimani A Savvaidis IN 2010 Combined chitosan thyme treatments with modified atmosphere packaging on a ready to cook poultry product Journal of Food Protection 73 663 669 Gill A O Holley R A 2000 Surface application of lysozyme nisin and EDTA to inhibit spoilage and pathogenic bacteria on ham and bologna Journal of Food Protection 63 1338 1346 Giteru S G Coorey R Bertolatti D Watkin E Johnson S Fang Z 2015 Physicochemical and antimicrobial properties of citral and quercetin incorporated kafirin b
302. ial packaging systems have received special attention in recent years because they can provide an important obstade in the so called hurdle technologies that are being implemented to commercial ize fresher food products In these systems interactions between the food the surrounding headspace and the package walls or independent devices are responsible for growth inhibition or death of pathogenic and or spoilage microorganisms Appendini and Hotchkiss 2002 Although exceptionally this beneficial activity can be carried out by direct contact of the microorganisms with the package surface Conte et al 2008 Muriel Galet et al 2013 Zi Xuan et al 2012 the mecha nism of activity in most packaging systems is based on mass transport processes through the packaging food interface and especially on the release of antimicrobial substances The active agent can be incorporated within a suitable polymeric matrix from which itis released following dif fusion mechanisms and accumulated into the food package system following thermodynamic principles Therefore compatibility between the agent and the various system components and diffusion from the Corresponding author Tel 34 963900022 fax 34 963636301 E mail address rgavara iata csic es R Gavara http dx doi org 10 1016 j ijfoodmicro 2014 07 018 0168 1605 6 2014 Elsevier BV All rights reserved 338 Recently a biopolymeric device consisting of a combination of two bi
303. iberar el agente activo que contiene al medio externo Existen diferentes mecanismos en respuesta a determinados est mulos ambientales f sicos temperatura ultrasonidos luz electricidad 15 Introducci n tensi n mec nica qu micos pH fuerza i nica y biol gicos enzimas biomol culas Bajpai et al 2008 Gupta et al 2002 Kim et al 2009 Los hidrogeles tanto sint ticos polialcohol vin lico como naturales polisac ridos como el CS han sido estudiados para el desarrollo de veh culos portadores y liberadores de agentes activos basados en sistemas polim ricos Bhattarai et al 2010 Gupta et al 2002 Los hidrogeles son redes polim ricas tridimensionales que presentan la caracter stica de absorber grandes cantidades de agua debido a la presencia en su estructura de grupos polares OH NH2 CONH COOH As son capaces de contener el agente activo en seco y liberarlo en respuesta a un ambiente h medo ya que la humedad provoca el hinchamiento del pol mero debido a un incremento en su volumen en agua lo que facilita la difusi n del agente activo a trav s de la matriz polim rica Bajpai et al 2008 Bhattarai et aL 2010 Gupta et aL 2002 Asimismo los hidrogeles se pueden formular bajo diversa formas f sicas incluyendo micro y nanopart culas recubrimientos y pel culas Bhattarai et aL 2010 En la presente Tesis Doctoral se ha trabajado con dos matrices capaces de formar hidrogeles prin
304. ible adaptar la capacidad de sorci n de las matrices en funci n de la matriz polim rica de la cantidad y cualidad del oligosac rido y grado de plastificaci n por parte del G y el agua As como tambi n se puede seleccionar el agente vol til para las distintas aplicaciones Con este sistema se consigue una elevada incorporaci n y liberaci n de agentes antimicrobianos de car cter lip filo en una matriz hidr fila De los estudios de liberaci n de carvacrol y de la capacidad antimicrobiana de las pel culas se deduce que las pel culas desarrolladas pueden ser aplicadas en el dise o de envases activos para el control microbiano durante el tiempo de conservaci n de los alimentos Las pel culas podr an formar parte de un sistema de envasado para alimentos con contenido en agua medio o alto La humedad del espacio de cabeza proveniente del alimento asegurar a una liberaci n adecuada del vol til desde la pel cula que ejercer a su acci n en fase vapor por lo que no ser a necesario el contacto directo de la pel cula con el alimento Con este objetivo las pel culas con mayor capacidad de sorci n CS CD 35G 75RH de carvacrol se integraron en un sistema de envasado para pechugas de pollo fresco fileteadas As las muestras de pechuga de pollo se colocaron en el fondo de unas barquetas de PP EVOH PP selladas con una tapa de aluminio autoadhesiva con diferentes tama os 0 24 4 8 y 24 cm de pel culas de CS con un contenido
305. icho aldeh do es incorporado a la disoluci n film gena previamente a la formaci n de la pel cula y debido a su elevada volatilidad se pierde en gran parte durante la etapa de evaporaci n del disolvente Ben Arfa et al 2007 Una estrategia para disminuir las p rdidas de la biomol cula es formar complejos de inclusi n con compuestos coadyuvantes como pueden ser las CDs Brasil et al 2012 Sipahi et al 2013 No obstante este proceso limita la cantidad de principio activo que se puede incluir en los pol meros ya que los complejos de inclusi n suelen presentar una estequiometria 1 1 1 2 Otra forma de reducir las p rdidas del vol til durante el secado de las pel culas es mediante encapsulaci n en nanoliposomas Makwana et al 2014 Sin embargo existen otras estrategias m s ventajosas que consisten en anclar qu micamente el cinamaldeh do a la Tabla 4 3 1 Diversos biopol meros con cinamaldeh do para el envasado antimicrobiano de alimentos Biopol mero Referencia prote nas de soja Ben Arfa et al 2007 Gamage et al 2009 pur de manzana Mild et al 2011 CS Ouattara et al 2000 prote nas de soja prote nas de suero de leche PVOH pur de manzana pur de hibisco pur de zanahoria Ouattara et al 2001 Ravishankar et al 2012 Ravishankar et al 2009 alginato de calcio Raybaudi Massilia et al 2008 alginato de calcio pur de manzana Rojas Gra et al 2007 celulosa Sanla Ead e
306. icrobiana del envase frente a microorganismos end genos bacterias mes filas psicr filas Pseudomonas spp Enterobacteriaceae cido l cticas hongos y levaduras presentes en el pollo Dichos estudios se llevaron a cabo el d a en el que se envasaron las pechugas y tras 3 6 y 9 d as de almacenamiento en condiciones de refrigeraci n En la cuarta publicaci n del presente cap tulo titulada Effect of HP B cyclodextrins and coadjuvants on the sorption capacity of hydrophilic polymer films for naturally occurring antimicrobial monoterpene alcohols se trabaja con las pel culas que mayor capacidad de sorci n de carvacrol han mostrado stas son CS con HP BCD plastificadas con 35 G y 58 Justificaci n y esquema de tesis acondicionadas a 7596 RH y se estudia el efecto de la estructura qu mica en la sorci n de otros monoterpenos c clicos y ac clicos y monoterpenos fen licos de origen natural presentes en AE m cumenol o cumenol carvacrol guayacol isoeugenol mirtenol nerol carveol dehidrocarveol e isopulegol Previamente a los estudios de sorci n se determin la actividad antimicrobiana in vitro en fase vapor de todos los compuestos frente a Escherichia coli y Staphylococcus aureus Se estudi el efecto del pol mero y del plastificante empleado en la capacidad de sorci n para ello se eligi el pol mero PVOH y PG como plastificante siempre manteniendo el mismo contenido en humedad que en las pel culas de CS
307. ido etil N dodecanoil L arginato LAE se incorpor previamente a la formaci n de la pel cula de quitosano Las pel culas con LAE liberaron completamente el agente activo en un simulante alimentario acuoso en varias horas a diferentes temperaturas Las pel culas de quitosano que no fueron neutralizadas presentaron actividad antimicrobiana frente a microorganismos presentes en pechugas de pollo frescas Esta actividad antimicrobiana se increment al incorporar el LAE a la matriz reduciendo satisfactoriamente los microorganismos presentes en el alimento para el control del deterioro microbiano de pollo fresco envasado En segundo lugar se incorpor el precursor nitrato de plata a la soluci n filmogena de quitosano para generar in situ nanopart culas de plata durante la neutralizaci n de las pel culas de acetato de quitosano Las pel culas desarrolladas presentaron una actividad antibacteriana in vitro mantenida en el tiempo Las nanopart culas de plata pudieron actuar como reservorios de iones de plata liberados sostenidamente en un entorno acuoso El tercer mecanismo para incorporar el agente antimicrobiano fue el anclaje reversible del vol til cinamaldeh do a pel culas preformadas de quitosano mediante la formaci n de una base de Schiff Este enlace covalente fue hidrolizado tras la aplicaci n de diversos tratamientos de temperatura tiempo habituales en la conservaci n de alimentos en un medio acuoso liber ndose el cinamaldeh
308. ience 275 177 182 Yakabe Y Sano T Ushio H Yasunaga T 1980 Kinetic studies of the interaction between silver ion and deoxyribonucleic acid Chemistry Letters 373 376 Yamanaka M Hara K Kudo J 2005 Bactericidal actions of a silver ion solution on Escherichia coli studied by energy filtering transmission electron microscopy and proteomic analysis Applied and Environmental Microbiology 71 7589 7593 Yin Y D Li Z Y Zhong Z Y Gates B Xia Y N Venkateswaran S 2002 Synthesis and characterization of stable aqueous dispersions of silver nanoparticles through the Tollens process Journal of Materials Chemistry 12 522 527 Zou J Xu Y Hou B Wu D Sun Y 2007 Controlled growth of silver nanoparticles in a hydrothermal process China Particuology 5 206 212 125 Quitosano Cinamaldeh do 4 3 Cap tulo 3 Desarrollo de pel culas de quitosano con compuestos vol tiles antimicrobianos anclados mediante un enlace covalente reversible y su aplicaci n en el envasado activo de alimentos 4 3 1 Estado de la cuesti n El cinamaldeh do es un aldeh do arom tico a B insaturado componente del AE de canela con un amplio espectro antimicrobiano y aceptado como aromatizante El cinamaldeh do ha sido incluido en matrices biopolim ricas en diferentes estudios como agente activo para el envasado antimicrobiano de distintos grupos de alimentos Anexo A En muchos de estos trabajos Tabla 4 3 1 d
309. ience 71 M141 M145 Brasil ILM Gomes C Puerta G mez A Castell P rez M E Moreira R G 2012 Polysaccharide based multilayered antimicrobial edible coating enhances quality of fresh cut papaya Lwt Food Science and Technology 47 39 45 Brown C A Wang B W Oh J H 2008 Antimicrobial activity of lactoferrin against foodborne pathogenic bacteria incorporated into edible chitosan film Journal of Food Protection 71 319 324 Cagri A Ustunol Z Osburn W Ryser E T 2003 Inhibition of Listeria monocytogenes on hot dogs using antimicrobial whey protein based edible casings Journal of Food Science 68 291 299 Cagri A Ustunol Z Ryser E T 2001 Antimicrobial mechanical and moisture barrier properties of low pH whey protein based edible films containing p aminobenzoic or sorbic acids Journal of Food Science 66 865 870 Cagri A Ustunol Z Ryser E T 2002 Inhibition of three pathogens on bologna and summer sausage using antimicrobial edible films Journal of Food Science 67 2317 2324 Cao Hoang L Gregoire L Chaine A Wache Y 2010 Importance and efficiency of in depth antimicrobial activity for the control of Listeria development with nisin incorporated sodium caseinate films Food Control 21 1227 1233 C rdenas G D az J Mel ndrez M Cruzat C Cancino A G 2009 Colloidal Cu nanoparticles chitosan composite film obtained by microwave heating for food package applications P
310. ient fico 5 Incorporation of hydroxypropyl B cyclodextrins into chitosan films to tailor loading capacity for active aroma compound carvacrol 195 4 44 Art culo cient fico 6 Antimicrobial packaging of chicken fillets based on the release of carvacrol from chitosan cyclodextrin films 223 44 5 Art culo cient fico 7 Effect of HP B cyclodextrins and coadjuvants on the sorption capacity of hydrophilic polymer films for naturally occurring antimicrobial monoterpene alcohols 243 5 DISCUSI N GENERAL 271 6 CONCLUSIONES 291 7 ANEXOS 295 7 1 Anexo A Ejemplos de materiales biopolim ricos para el envasado antimicrobiano de alimentos 297 7 2 Anexo B Publicaciones 331 XVIII ndice de tablas y figuras NDICE DE TABLAS Tabla 1 1 Aplicaciones del CS y sus derivados 14 Tabla 1 2 Estructura qu mica y caracter sticas de las CDs naturales 22 Tabla 1 3 Aplicaciones de las nanopart culas de plata 29 Table 4 1 1 Colour parameter values of CS films with different amounts of LAE and plasticized with 2096 G 79 Table 4 1 2 Values of the parameters describing LAE release from CS matrices into water according to the Eq 1 81 Table 4 1 3 Minimum inhibitory concentration MIC pg mL and minimum biocide concentration MBC ug mL of LAE against the selected microorganisms in liquid and solid media 82 Table 4 1 4 Antimicrobial effectiveness of LAE films against the selected microorganisms in liquid medium at 28 C
311. ilic pervaporation membranes has been reported in the literature Villaluenga and Tabe Mohammadi 2000 In a comparison of the sorption properties of the films formulated with CS CD possessing approximately the same water content and differing in the amount of G greater sorption values were observed for ortho and meta cumenol in CS CD films with 3596 G In a previous study regarding the sorption capacity of CS films blended with CD and different amounts of G and water it was demonstrated that sorption is almost suppressed in unplasticized films requiring the presence of CD and plasticization by G and water to retain carvacrol Higueras et al 2013 In that work it was concluded that G enhances sorption of carvacrol more than water does In the present study it was observed that at a fixed water content of 33 3496 g g dry film increasing the G content from 35 to 5096 did not affect the sorption of phenolic monoterpenes with the exception of ortho and meta cumenol which reduced their sorption This might be related to an excess of film plasticization restricting the very high sorption values of cumenol isomers In an unpublished previous study it was found that for a lower water content in the films around 15 when CS CD films were conditioned at 5396 RH the content of sorbed carvacrol increased with the amount of G in the film giving sorption values of 6 13 133 27 and 300 when plasticized 259 Cap tulo 4 Art culo cient fico
312. imentos y por el que se modifican el Reglamento CEE n 1601 91 del Consejo los Reglamentos CE n 2232 96 y CE n 110 2008 y la Directiva 2000 13 CE CE 2009 Reglamento CE n 450 2009 de la Comisi n de 29 de mayo de 2009 sobre materiales y objetos activos e inteligentes destinados a entrar en contacto con alimentos CE 2012 Comunicaci n del Parlamento Europeo el Consejo y el Comit Econ mico y Social europeo Segunda revisi n regulatoria de los nanomateriales Bruselas 3 de octubre de 2012 Cocchiara J Letizia C S Lalko J Lapczynski A Api A M 2005 Fragrance material review on cinnamaldehyde Food and Chemical Toxicology 43 867 923 Coma V 2008 Bioactive packaging technologies for extended shelf life of meat based products Meat Science 78 90 103 Coma V 2012 Recent developments in chitin and chitosan bio based materials used for food preservation Polysaccharide building blocks John Wiley amp Sons Inc pp 143 175 Contijoch Mestres A Rodriguez Mart nez F J Seguer Bonaventura J 2001 Process for the preparation of cationic surfactants Cosgrove J 2010 The global chitosan market Increasing application prospects are contributing to a continued positive outlook http www nutraceuticalsworld com contents view_online exclusives 2010 12 02 the global chitosan market sthash 4uOFpYOR dpuf 38 Introducci n Chang S T Chen P F Chang S C 2001 Antibacterial act
313. imicrobial agent requiring higher concentrations to achieve the same effect Guti rrez et al 2008 Similar differences between in vivo and in vitro antimicrobial activity of antimicrobial agents and antimicrobial films have been reported previously Belletti et aL 2008 Burt 2004 Muriel Galet et al 2012 L monocytogenes is an important pathogenic microorganism involved in cases of septicaemia and meningitis especially in children the elderly and those with immunosuppression induced by drugs or diseases However there are also cases of listeriosis in apparently healthy children and adults In pregnant women it can cause abortions or premature death of the foetus Therefore the films developed in this work could improve the safety of products susceptible to contamination with microorganisms such as L monocytogenes and could also extend the commercialization period an important advantage for a product with a shelf life of only 3 days under refrigeration 3 4 Sensory analysis The use of essential oils in food may have a significant sensory impact that could result in non acceptance by the consumer For this reason a sensory analysis was carried out by a panel of judges with the aim of determining whether the content of active component migrated to pasteurized milk modifies its aroma appreciably and if so to determine whether this odour is accepted by consumers The tests were conducted on the 3rd 6th and 12th days of refrigerated s
314. inaci n de los grupos amino libre del CS Otros autores han estudiado la s ntesis y caracterizaci n de la base de Schiff a partir de diversos aldeh dos como se observa en la Tabla 4 3 2 No obstante en dichos trabajos la base de Schiff no forma parte de una matriz polim rica ni se estudia su reversibilidad para la liberaci n del agente antimicrobiano Muzzarelli y llari 1994 formaron la base de Schiff entre varios aldeh dos o vainillina vainillina siringaldeh do veratraldeh do 3 4 dihidroxibenzaldeh do y el CS en disoluci n obteniendo a continuaci n pel culas de caracter sticas similares a la lignina al incorporar grupos metoxifenilo Hirano y Hayashi 2003 prepararon mediante la formaci n de una base de Schiff en disoluci n fibras de CS modificado con cinamaldeh do 2 hexilcinamaldeh do jasminal citronelal citral heptanal n nonanal 1 decanal y 10 undecanal como nuevos biomateriales perfumados 128 Quitosano Cinamaldeh do Tabla 4 3 2 Bases de Schiff sintetizadas y caracterizadas a partir de CS y diversos aldeh dos Aldeh do Referencia acetaldehido Kurita et al 2002 4 anisaldehido Tirkistani 1998 benzaldehido y derivados Tirkistani 1998 cinamaldehido Marin et al 2014 4 dimetilaminobenzaldehido Tirkistani 1998 Hirano et al 1977 Kurita et al 2002 formaldehido Singh et al 2006 furfural Hongliang et al 2010 glutaraldehido Hirano et al 1977 pentanal Kurita
315. incorporaci n ala de las Migraci n al No aplicada en alimentos Ancl i Lech CS cinamaldeh do formaci n de una e aT ibn eo VE base de Shiff a la SEM P A temperatura pel cula de CS Mr Migraci n al medio l quido L quido incorporaci n ala Libre en la matriz k nm 5 a Migraci n al pel cula acomplejado con Pechuga de CS carvacrol espacio de compuesta de CS CD o agregados pollo r cabeza mediante de CD inmersi n 273 Discusi n general De entre las diferentes formas de incorporar el agente activo en la matriz polim rica la m s com n es durante la formaci n de la pel cula Este m todo es muy til cuando el agente activo no es vol til ni termol bil de forma que durante la etapa de secado no hay p rdidas Por otro lado la pel cula ha de estar en contacto directo con el alimento envasado para que el agente pueda ser liberado efectivamente Siguiendo este mecanismo de acci n se desarrollaron pel culas de CS con LAE Las pel culas con el agente activo se caracterizaron por ser flexibles y transparentes y sin cambios visibles de color Debido a la capacidad del CS de formar hidrogeles y a la presencia del G las pel culas obtenidas se hincharon en presencia de agua y liberaron el LAE Cabe destacar que el car cter cati nico tanto del CS cuando se encuentra protonado como del LAE permiti la liberaci n del agente tensioactivo ya que no se produjeron interacciones electrost ticas ni formac
316. ing immobilised bacteriocins Lacticin 3147 and Nisaplin International Journal of Food Microbiology 60 241 249 Sebti L Coma V 2002 Active edible polysaccharide coating and interactions between solution coating compounds Carbohydrate Polymers 49 139 144 Sebti I Chollet E Degraeve P Noel C Peyrol E 2007 Water sensitivity antimicrobial and physicochemical analyses of edible films based on HPMC and or chitosan Journal of Agricultural and Food Chemistry 55 693 699 Sebti I Ham Pichavant F Coma V 2002 Edible bioactive fatty acid cellulosic derivative composites used in food packaging applications Journal of Agricultural and Food Chemistry 50 4290 4294 Seol K H Lim D G Jang A Jo C Lee M 2009 Antimicrobial effect of carrageenan based edible film containing ovotransferrin in fresh chicken breast stored at 5 C Meat Science 83 479 483 Seydim A C Sarikus G 2006 Antimicrobial activity of whey protein based edible films incorporated with oregano rosemary and garlic essential oils Food Research International 39 639 644 Seydim A C Sarikus G 2007 Antimicrobial activity of whey protein based edible films incorporated with oregano rosemary and garlic essential oils Food Research International 40 949 949 Shen X L Wu J M Chen Y H Zhao G H 2010 Antimicrobial and physical properties of sweet potato starch films incorporated with potassium sorbate or chitosan Food Hyd
317. io pectina B CD alginato de calcio pur de manzana alginato de calcio celulosa gliadina PLA prote nas de soja prote nas de soja prote nas de soja prote nas de suero de leche PVOH pur de manzana quitosano quitosano pectina B CD jam n fresa medio de cultivo medio de cultivo medio de cultivo pechuga de pollo jam n mortadela vegetales de hoja verde medio de cultivo pechuga de pollo medio de cultivo medio de cultivo sand a cortada medio de cultivo manzana Fuji troceada medio de cultivo queso pan medio de cultivo medio de cultivo brotes de alfalfa r bano y br coli gambas cocidas irradiadas a bajas dosis pechuga de pollo jam n cocido mortadela pastrami papaya troceada Clostridium sporogenes E coli S enterica Yersinia enterocolitica E coli O157 H7 L monocytogenes E coli O157 H7 Botrytis cinerea E coli St aureus Ps aeruginosa E coli St aureus B subtilis Botrytis cinerea E coli S Enteritidis E coli O157 H7 L monocytogenes S Newport E coli O157 H7 Campylobacter jejuni E coli O157 H7 S Typhimurium E coli O157 H7 ME TVC mes filos y psicr filos coliformes hongos levaduras E coli O157 H7 E coli O157 H7 ME TVC mes filos y psicr filos hongos levaduras L monocytogenes St aureus E coli S Enteritidis C albicans Saccharomyces cerevisiae Penicillium spp ME ho
318. ion of carvacrol during the processing of CS films by casting and found that carvacrol retention was directly correlated with the incorporation of G and nanoclays into the matrix moreover retention of carvacrol was also favoured by blending CS with gum arabic because of the formation of coacervates which encapsulate carvacrol and prevent its evaporation from the matrix during the drying of the films CDs are commonly used to solubilise lipophilic molecules B CDs are capable of forming 1 1 and 12 host guest complexes with carvacrol Locci et al 2004 Ravi and Divakar 2001 In this work the maximum percentage of carvacrol that could be held in CS films incorporating HP BCD was 1096 g carvacrol 100 g dry matter When water and G were present the amount of carvacrol retained in the films exceeded this percentage so mechanisms other than the formation of inclusion complexes participate in the sorption of carvacrol In the last few years a growing body of research has shown that CDs can act as building units able to self assemble into aggregates driven by CD CD H bonds and these aggregates can act as solubilizers The size of these aggregates tends to grow with increasing concentration of CDs and aggregates up to several micrometres in diameter have been reported Messner et al 2010 Water soluble polymers contribute to the stabilization of these aggregates through formation of CD polymer hydrogen bonds Ribeiro et aL 2003 Formation of CD
319. iones acuosas que forma Barrera et al 2007 Goodship y Jacobs 2005 El efecto del peso molecular y grado de hidr lisis sobre diferentes propiedades del PVOH se muestra en la Figura 1 4 El PVOH es un pol mero inodoro y no t xico DeMerlis y Schoneker 2003 Goodship y Jacobs 2005 con un gran potencial tecnol gico como material biodegradable Se ha demostrado que varios microorganismos ubicuos en sistemas s pticos vertederos compostaje o suelo son capaces de degradar el PVOH mediante procesos enzim ticos Una combinaci n de actividades oxidasa e hidrolasa degradan el PVOH en cido ac tico En este caso tambi n el grado de hidr lisis y su solubilidad afecta a su tasa de biodegradaci n DeMerlis y Schoneker 2003 Dorigato y Pegoretti 2012 Por otra parte el PVOH se puede obtener a partir de gas natural como materia prima renovable en lugar de derivados del petr leo Dorigato y Pegoretti 2012 a E cd a al O O O OH imerizaci l HC CH Polimerizaci n CH CH J Neon 6H CH GH CH3 n d MeOH Acetato de vinilo Acetato polivin lico Alcohol polivin lico Figura 1 3 Obtenci n de PVOH 18 Introducci n de viscosidad de la resistencia a la tracci n de la resistencia al agua de la resistencia de solvataci n de la resistencia adhesiva de la solubilidad de la flexibilidad a la sensibilidad al agua de la facilidad de solvataci n Aumento Aumento PESO MO
320. ipped with a diamond knife to give around 70 nm thick sections and placed on 300 mesh copper grids Images were obtained with a digital micrograph acquisition software Gatan Inc 2007 2 2 5 X ray diffraction XRD The XRD patterns of CS films neutralized at 37 C were recorded using a Bruker AXS D500 spectrometer with a Bragg Brentano geometry at a wavelength of 1 5406 corresponding to the peak Cu Ko X ray diffractograms were recorded in a diffraction angle 20 range of 5 80 using a step size of 0 02 and an exposure time of 2 s 2 3 Antimicrobial activity of films neutralized at 37 C Staphylococcus aureus ATCC 12600 and Escherichia coli ATCC 25922 were obtained from the Spanish Type Culture Collection Valencia Spain Strains were stored in tryptone soy broth TSB Scharlab Barcelona Spain with 2096 G at 80 C until needed For experimental use the stock cultures were maintained by regular subculture on tryptone soy agar TSA Scharlab slants at 4 C and transferred monthly In a first step a loopful of each strain was transferred to 10 mL of TSB and incubated at 37 C for 18 h to obtain early stationary phase cells Cell cultures of each microorganism in stationary phase with an optical density of 0 9 at 600 nm were diluted in TSB and incubated at 37 C until an optical density of 0 2 at 600 nm 10 CFU mL was reached Tubes with 10 mL of Mueller Hinton broth MHB Scharlab and 1 125 diluted MHB were inoculated with 1
321. is Statistical analysis of the results was performed with SPSS commercial software SPSS Inc Chicago IL USA A two way analysis was applied to compare the effect of different amounts of G in the same matrix CS or CS CD Additionally one way analysis of variance was carried out for the other data Differences between means were assessed on the basis of confidence intervals using the Tukey b test at a level of significance of P lt 0 05 The data are represented as average standard deviations The data were analyzed and plotted using the SigmaPlot 10 0 software Systat Software Inc Richmond CA USA 3 Results and discussion 3 1 Optical properties The influence of the presence or absence of HP BCDs and the different proportions of G added to the film forming solution on the colour parameter values L C Nap a b are presented in Table 4 4 1 In all the materials the high values of L gt 94 are indicative of high lightness while slightly negative values of a and positive b are indicative of a yellow green colour The addition of G did not significantly affect the colour coordinates P 0 05 within the G range tested However when 5096 of the CS content in the film was replaced by HP BCD significant changes in colour parameters were 182 Quitosano Carvacrol Table 4 4 1 Colour parameter values of CS films and chitosan hydroxypropyl B cyclodextrin composite CS CD plasticized with different concentrations 96 of G
322. is polysaccharide has also been described as a weak reducing agent owing to the presence in their structure of the organic compounds aldehyde ketone and alcohol The presence of sodium hydroxide accelerated the reduction rate of silver ions Singh et al 2009 and thus the formation of silver nanoparticles in the film However the coexistence of elementary silver and silver oxide nanoparticles is expected owing to the presence of sodium hydroxide Treatment with sodium hydroxide allows neutralization of amino groups in CS film promoting the integrity of the resulting films in aqueous medium at a pH above the pK of CS This less toxic method for the synthesis of nanoparticles avoids the employment of commonly used toxic reducing agents 110 Quitosano Nanopart culas de plata 3 2 Chitosan silver based nanoparticles films Films neutralized at 37 C were successfully developed with nominal concentrations of silver between 0 1 and 1 596 Higher concentrations of silver greatly increased the viscosity of the film forming solution giving rise to the formation of a gel The films were homogeneous to the naked eye and their thickness ranged between 55 and 65 um Figure 4 2 2 shows a photograph of films with different concentrations of silver neutralized at 22 or 37 C The incorporation of silver nitrate produced films with a different color after the neutralization step It can be observed that transparency decreased and the films acqui
323. isopulegol the amount of antimicrobial in the films was in all cases greater than the amount required to cause a microbial inhibitory effect in vapour phase Apparently the antimicrobial activity of the films with myrtenol nerol dihydrocarveol and carveol was similar owing to the similar sorption values and similar antimicrobial activity of these compounds although slight differences were found For example CS CD 50G films with 3 9 mg of myrtenol produced an inhibition zone of 10 mm while the film with 3 8 mg of nerol produced an inhibition zone of 30 mm the minimal dose necessary to produce inhibition being 3 2 mg for myrtenol and 2 4 mg for nerol 4 Conclusions The present study shows that HP BCD together with low molecular weight plasticizers G and PG and moisture are capable of regulating the sorption capacity of hydrophilic CS films for various monoterpene alcohols The sorption affinity for monoterpene phenolic compounds was dramatically higher than that for compounds possessing a cyclohexanol structure or linear alcohols This was associated with the greater polarity of the benzene 266 Quitosano Carvacrol ring and its affinity for CS polar groups It was also found that sorption properties of positional isomers differed slightly which might be related to phenolic or cyclohexanol substituents impeding interactions between the hydroxyl group and polar groups in CS Increasing the affinity of the plasticizer for monoterpene al
324. ity and amount of the plasticizer added G or PG on the sorption properties of CS films for various monoterpene compounds with phenolic or non phenolic linear or cyclohexyl alcohol structures Moreover in order to investigate the effect of the hydrophilicity of the polymer matrix on the sorption behaviour of the films CS was replaced with PVOH polymer This synthetic polymer presents great affinity for water but is less hydrophilic than CS Prior to immersion in the volatile liquids the various films were conditioned at the appropriate RH to obtain a similar water content in all of them In general the water content of the films studied ranged between 32 and 3596 g g dry film 253 Cap tulo 4 Art culo cient fico 7 CS CD 35G films conditioned at 7596 RH retained 33 9 0 8 of water The water content of CS CD 50G films conditioned at 65 RH was 32 9 1 796 As expected an increase in the G content increased the water retention capacity so the RH at which they were conditioned was lower When G was replaced with PG in the CS CD 35PG films they had to be conditioned at 90 RH to reach a water content of 33 9 1 4 which can be explained by the less hydrophilic nature of PG and its lower capacity to retain water Table 4 4 11 shows some physico chemical parameters of the various plasticizers used in this work They include log P which is the octanol water partition coefficient and provides a quantitative measurement of the hydroph
325. ity values measured for CS and CS CD based films at 23 1 C and 50 and 75 RH CS films provided a high barrier to permeation of oxygen in dry and intermediate humidity conditions but this property worsened with environmental humidity as happens with other polymers with high cohesive energy density but also high affinity for water such as EVOH or PVOH G llstedt and Hedenavist 2006 Kjellgren et al 2006 Mensitieri et al 2011 A similar effect was caused by the addition of G The presence of humidity and G plasticized the polymer matrix of CS and CS CD films and consequently caused a large deterioration in the oxygen permeability Contrasting with the effect observed in water permeability the addition of HP BCDs produced a significant increase in oxygen permeability P lt 0 05 which was noticeable in all composites and conditions The cavities of HP BCDs might be used as channels for diffusion which would explain the results obtained The huge deterioration in the oxygen permeability determined for the CS CD 35G sample is noteworthy the barrier worsened by a factor of 100 000 The obtaining of a loose polymer network owing to the G content might be responsible for this mass transport behaviour 187 Cap tulo 4 Art culo cient fico 4 3 6 Mechanical properties Tensile strength elongation at break and modulus of elasticity were determined Table 4 4 4 The effect on these properties of the presence of HP BCDs in CS films an
326. ium medio de E coli O157 H7 L Lim Jang et al corneum cultivo monocytogenes 2010 timo ze na medio de B cereus C lusitaniae Del Nobile et al cultivo Pseudomonas spp 2008 vainillina alginato de calcio pur manzana Fuji ME TVC psicr filos Rojas Gra de manzana troceada hongos levaduras Raybaudi L innocua Massilia et al 2007 vainillina metilcelulosa quitosano mel n E coli Saccharomyces Sangsuwan et al Cantalupo cerevisiae 2008 pi a vainillina quitosano bizcocho ME hongos Sangsuwan et al 2014 LAE PLA jam n cocido L monocytogenes Theinsathid et al loncheado S Typhimurium 2012 LAE quitosano c scara de S Enteritidis Jin et al 2013 huevo LAE quitosano pavo RTE L monocytogenes Guo Jin Wang et L innocua S al 2014 Guo Typhimurium Jin y Yang 2014 Abreviaturas c cido PABA c p aminobenzoico A Aspergillus AE aceite esencial de B Bacillus C Candida Cl Cladosporium E Escherichia EDTA cido etildiaminotetraac tico ED extracto de HPMC hidroxipropilmetilcelulosa nano tama o 1 100 nm L Listeria LAB bacterias cido l cticas Lb Lactobacillus LAE etil N dodecanoil L arginato ME microbiota end gena P Penicillium PLA cido polil ctico Ps Pseudomonas RTE ready to eat S Salmonella St Staphylococcus TVC recuento de microorganismos aerobios totales Sin nimos alcaravea o comino del prado azufaifo chincho o
327. ive packaging Packaging Technology and Science 15 129 132 Soliman E A Khalil A A Deraz S F El Fawal G Abd Elrahman S 2014 Synthesis characterization and antibacterial activity of biodegradable films prepared from Schiff bases of zein Journal of Food Science and Technology Mysore 51 2425 2434 Son W K Youk J H Lee T S Park W H 2004 Preparation of antimicrobial ultrafine cellulose acetate fibers with silver nanoparticles Macromolecular Rapid Communications 25 1632 1637 Son W K Youk J H Park W H 2006 Antimicrobial cellulose acetate nanofibers containing silver nanoparticles Carbohydrate Polymers 65 430 434 547 Song H Y Shin Y J Song K B 2012 Preparation of a barley bran protein gelatin composite film containing grapefruit seed extract and its application in salmon packaging Journal of Food Engineering 113 541 789 795 Song N B Song H Y Jo W S Song K B 2013 Physical properties of a composite film containing sunflower seed meal protein and its application in packaging smoked duck meat Journal of Food Engineering 116 Tammineni N nl G Min S C 2013 Development of antimicrobial potato peel waste based edible films with oregano essential oil to inhibit Listeria monocytogenes on cold smoked salmon International Journal of Food Science and Technology 48 211 214 Tankhiwale R Bajpai S K 2009 Graft copolymerization onto cellulose based filter paper and it
328. ivity of essential oils against food spoilage microorganisms and foodborne pathogens agree on their slightly greater activity against Gram positive bacteria compared to Gram negative bacteria Burt 2004 This has been attributed to differences in the cell wall of the two types of microorganism since the outer membrane of Gram negative bacteria restricts diffusion of hydrophobic compounds through its lipopolysaccharide covering Vaara 1992 On the other hand some studies suggest that Gram positive bacteria are more resistant than Gram negative bacteria to the antibacterial properties of essential oils Zaika 1988 However other authors have not obtained evidence for a general greater effectiveness of essential oils against Gram positive or Gram negative bacteria and the results found depend on the essential oil tested Dorman and Deans 2000 With regard to the antimicrobial activity of carvacrol the major component of oregano and thyme oil a similar antibacterial activity against E coli and S aureus was found in the present work these results are in agreement with other studies Ben Arfa et al 2006 Griffin et al 1999 3 4 2 Study of the antimicrobial activity of films loaded with carvacrol The antimicrobial properties of carvacrol loaded 1 1 CS CD films plasticized with 20 or 3596 G and conditioned at different RH values were tested against S aureus and E coli Table 4 4 10 shows the carvacrol content of the films and the di
329. ivity of leaf essential oils and their constituents from Cinnamomum osmophloeum Journal of Ethnopharmacology 77 123 127 Chi S Zivanovic S Penfield M P 2006 Application of chitosan films enriched with oregano essential oil on bologna active compounds and sensory attributes Food Science and Technology International 12 111 117 Dainelli D Gontard N Spyropoulos D Zondervan Van den Beuken E Tobback P 2008 Active and intelligent food packaging legal aspects and safety concerns Trends in Food Science amp Technology 19 103 S112 Dallas P Sharma V K Zboril R 2011 Silver polymeric nanocomposites as advanced antimicrobial agents classification synthetic paths applications and perspectives Advances in Colloid and Interface Science 166 119 135 De Azeredo H M C 2013 Antimicrobial nanostructures in food packaging Trends in Food Science amp Technology 30 56 69 De Kruijf N Van Beest M Rijk R Sipilainen Malm T Losada P P De Meulenaer B 2002 Active and intelligent packaging applications and regulatory aspects Food Additives and Contaminants 19 144 162 De Vincenzi M Stammati A De Vincenzi A Silano M 2004 Constituents of aromatic plants carvacrol Fitoterapia 75 801 804 Debeaufort F Quezada Gallo J A Voilley A 1998 Edible films and coatings tomorrow s packagings a review Critical Reviews in Food Science and Nutrition 38 299 313 Del Valle E M M
330. ivo liberaci n de agentes antimicrobianos envasado de aves de corral 71 Cap tulo 1 Art culo cient fico 1 Abstract Chitosan CS films incorporating the antimicrobial compound ethyl N dodecanoyl L arginate LAE were developed for food packaging applications Cast chitosan films were made with 1 5 or 1096 LAE and 2096 glycerol in the film forming solution Optical properties release of LAE and antimicrobial activity of developed films was determined The minimum inhibitory concentration MIC and the minimum biocide concentration MBC of LAE were determined CS films with LAE were transparent and uniform without discontinuities or visible particles and no visual differences could be perceived between CS and CS LAE films When in contact with an aqueous food simulant the agent was fully released following a Fickian behavior in a few hours at 4 and 28 C Antimicrobial activity of films against mesophiles psychrophiles Pseudomonas spp colifoms lactic acid bacteria hydrogen sulfide producing bacteria yeast and fungi was evaluated at two six and eight days for its application on chicken breast fillets Films were active against bacteria yeasts and fungi in liquid and solid media CS films evidenced antimicrobial activity in the range 0 47 2 96 log reductions while CS 596LAE film produced 1 78 5 81 log reduction Results highlighted that LAE incorporation in a chitosan based packaging structure may provide a relevant antimi
331. kenstadt V L Liu C K Jin T Fishman M L Hicks K B 2007 Preparation of poly lactic acid and pectin composite films intended for applications in antimicrobial packaging Journal of Applied Polymer Science 106 801 810 Liu L S Jin T Coffin D R Liu C K Hicks K B 2010 Poly lactic acid membranes containing bacteriocins and EDTA for inhibition of the surface growth of Gram negative bacteria Journal of Applied Polymer Science 117 486 492 Longano D Ditaranto N Cioffi N Di Niso F Sibillano T Ancona A Conte A Del Nobile M A Sabbatini L Torsi L 2012 Analytical characterization of laser generated copper nanoparticles for antibacterial composite food packaging Analytical and Bioanalytical Chemistry 403 1179 1186 L pez Mata M A Ruiz Cruz S Silva Beltr n N P Ornelas Paz J D Zamudio Flores P B Burruel Ibarra S E 2013 Physicochemical antimicrobial and antioxidant properties of chitosan films incorporated with carvacrol Molecules 18 13735 13753 Lungu B Johnson M G 2005a Fate of Listeria monocytogenes inoculated onto the surface of model turkey frankfurter pieces treated with zein coatings containing nisin sodium diacetate and sodium lactate at 4 C Journal of Food Protection 68 855 859 Lungu B Johnson M G 2005b Potassium sorbate does not increase control of Listeria monocytogenes when added to zein coatings with nisin on the surface of full fat turkey frankf
332. l 1 Introduction Nowadays consumers demand natural products that undergo minimal processing and are free of synthetic preservatives However these products often have a short microbiological shelf life which makes it necessary to find new alternatives In this regard hurdle technology employs combined treatments and their synergies to preserve food more efficiently Active packaging technologies combined with the use of naturally occurring preservatives could be an approach to hurdle technology for the preservation of minimally processed foods Among the antimicrobial agents used as food preservatives there is a growing tendency to employ natural compounds from plant extracts and essential oils Burt 2004 Lang and Buchbauer 2012 Essential oils and their components are considered food additives and classified by the JECFA Joint FAO WHO Expert Committee on Food Additives as flavourings These compounds have a great potential to be used as active agents in the development of antimicrobial active packaging technologies which are a complementary method for increasing the microbial safety of packaged foods Antimicrobial food packaging technologies which are based on the incorporation of active volatiles in polymer matrices do not require the film to be in contact with the food product to be active In this case the volatile is released to the headspace of the package and exerts its activity when it comes into contact with the food surfa
333. l 1984 Rodr guez et al 2004 In addition LAE has a low oil water equilibrium partition coefficient Kow b 0 1 which means that it tends to concentrate in the aqueous phase where most bacterial action occurs Ruckman et al 2004 On top of that LAE shows chemical stability and antimicrobial activity in a range of pH 3 7 Asker et al 2011 With these properties LAE used in lower concentrations than other agents presents a significant advantage over other food preservatives created for the same applications on the market Asker et al 2011 Weiss et al 2007 LAE is primarily and rapidly metabolized and hydrolyzed chemically in the human body to natural endogenous compounds present in the human diet i e the amino acid arginine which in turn is metabolized to CO and urea and lauric acid and ornithine Asker et al 2011 Ruckman et al 2004 This property gives LAE an important degree of security As a result LAE has been classified as GRAS generally recognized as safe and approved for food safety and quality the USDA United States Department of Agriculture has approved its use in meat and poultry products but is currently not approved in dairy products OMS 2009 Theinsathid et al 2012 To date the use of LAE as an antimicrobial agent in food products has been well reported by different studies Luchansky et aL 2005 Martin et al 2009 Soni et al 2010 Taormina and Dorsa 2009 Thus due to its low toxicity lo
334. l amount of carvacrol contained in each part of the package was calculated and the results are presented as a percentage of the total amount in Figure 4 4 11 As can be seen the carvacrol release was practically complete after the first three days Interestingly the lid was the package constituent that retained most carvacrol which is especially relevant for the largest size The carvacrol scalping in PP was significant but much reduced with ca 296 of the total amount at the end of the storage period Finally the carvacrol present in the headspace could be negligible 1 per million in all cases when it is compared with the other components of the packaging system As mentioned at the beginning of the discussion section the package was designed to fully retain all the carvacrol initially inserted in the food packaging system This was confirmed when the package without chicken was analysed As can be seen in Figure 4 4 11d the package retained nearly 9796 of the initial content in agreement with the infinite barrier approach Therefore the percentage of carvacrol that is not present in the package component should necessarily be assigned to sorption in the chicken fillet as represented in Figure 4 4 11 Assuming that the agent was homogeneously distributed in the food matrix the concentration of carvacrol 234 Quitosano Carvacrol Carvacrol percentage Carvacrol percentage
335. l the films formulated was that phenolic monoterpenes were sorbed in greater amounts than non phenolic cyclic and linear monoterpenes The presence of the benzene ring increases affinity of the molecule for the film owing to the affinity between the double bonds of benzene and polar groups of the film hydroxyl amino acetamido and carbonyl groups of the chain end Sorption of phenols increased in the following order m cumenol gt o cumenol gt carvacrol gt guaiacol gt isoeugenol m Cumenol was sorbed to a slightly greater extent than o cumenol one possible explanation for which is stearic hindrance of the 257 Cap tulo 4 Art culo cient fico 7 Table 4 4 12 Sorption capacity of films based on CS or PVOH incorporating HP BCD in 1 1 weight ratio and plasticized with G or PG for monoterpenes at 23 C 96 9 100 g dry film CS CD 35G 75RH CS CD 50G 65RH CS CD 35PG 90RH PVOH CD 35G 84RH m cumenol 455 06 18 88 372 38 18 85 269 94 13 38 274 27 44 19 o cumenol 419 67 2199 339 40 17 95 227 28 18 42 226 37 8 68 carvacrol 230 11 18 74 224 80 9 85 179 62 3 09 128 23 2 09 guaiacol IIo SS PIEENINSO 184 89 7 95 106 59 3 16 85 70 7 48 isoeugenol 12 02 6 72 12 59 3 28 11 50 2 04 13 56 2 56 myrtenol 3 48 0 93 2 16 0 58 2 26 0 97 2 27 0 11 nerol 3 09 0 79 2 48 0 06 2 43 0 79 2 39 0 51 carveol 2 37 0 74 2 41 0 08 2 50 0 40 2 39 0 06 dihydrocarveol 1 27 0 09 1
336. las bacterias gram negativas y la membrana celular y el citoplasma de las bacterias gram positivas desnaturalizando las prote nas Estas alteraciones producen perturbaciones en el potencial y permeabilidad de membrana la estructura celular y los procesos metab licos inhibiendo la proliferaci n celular y perdiendo la viabilidad celular Bakal y D az 2005 Rodr guez et al 2004 El LAE se caracteriza por tener un amplio espectro y elevada eficacia antimicrobiana contra bacterias gram negativas gram positivas hongos y levaduras a bajas concentraciones Bakal y D az 2005 Infante et al 1997 Rodr guez et al 2004 El LAE se metaboliza en el tracto gastrointestinal humano r pidamente dando lugar a compuestos habituales en la dieta humana como son el cido l urico y los amino cidos ornitina y arginina que a su vez se metabolizan en CO y urea Esta caracter stica otorga al LAE un importante grado de cido graso CH3 CH 0 Gl Figura 1 7 Estructura qu mica del LAE 26 Introducci n seguridad que ha sido demostrado en diferentes estudios toxicol gicos desarrollados en los ltimos a os Hawkins et al 2009 Ruckman et al 2004 Adem s debido a su estructura simple y natural a partir de fuentes renovables amino cidos y aceites vegetales es biodegradables y de baja ecotoxicidad Infante et al 1997 El LAE fue sintetizado por un grupo de investigaci n del CSIC en 1984 Bakal y D az 2005
337. las part culas se generaban en la soluci n formadora de pel culas mediante la incorporaci n de agentes reductores t xicos como el borohidruto de sodio u otros no t xicos como la glucosa El CS es un excelente quelante de iones plata debido a la presencia en su estructura de grupos amino contribuyendo tambi n a ello aunque en menor medida los grupos hidroxilo Gracias a estos grupos es posible generar nanopart culas controlando su tamafio y evitando su agregaci n Dallas et al 2011 Murugadoss y Chattopadhyay 2008 Twu et al 2008 Esta v a alternativa de obtenci n de las nanopart culas de plata tiene otras ventajas 1 no emplean disolventes org nicos ya que la matriz de CS se disuelve y neutraliza en agua 2 no se generan subproductos t xicos 3 no emplea agentes reductores fuertes ni estabilizadores t xicos 4 se generan en el seno de una matriz biodegradable que proviene del material de desecho de la industria alimentaria renovable y no t xica por lo que se reduce el impacto medioambiental y 5 se emplean moderadas y bajas temperaturas lo que supone menor gasto energ tico Todas ellas cuestiones clave entre los doce principios de la denominada Qu mica Sostenible Anastas y Eghbali 2010 Dallas et al 2011 Sharma et al 2009 En el trabajo realizado en la presente Tesis Doctoral tambi n se han caracterizado las pel culas resultantes y las nanopart culas generadas adem s de estudiarse la actividad antimicrobiana
338. ld decrease the availability of free silver ions to exert antimicrobial activity Although we observed a slight tendency for the antimicrobial activity of the films to increase as their silver concentration increased no significant differences were found in the antimicrobial capacity of films incorporating different amounts of silver ranging from 0 1 to 1 596 in MHB diluted to 1 125 This behaviour might be due to a similar quantity of silver ion migrating to the medium To confirm this hypothesis the silver concentration in diluted MHB after 18 h of being in contact with films incorporating different amounts of silver was evaluated by GFAAS The release of silver proved to be similar in all of the samples The silver concentration in the medium ranged from 135 to 150 ug L and there were no differences P gt 0 05 in migration values between films of different silver concentrations Although the films released a similar amount of silver after 18 h of contact with the test environment the kinetics of silver ion migration may differ between films and thus affect their antimicrobial activity This might explain slight but nonsignificant differences in the antimicrobial effectiveness of the films At present the mechanism of action of silver is not clear When silver ions are inside the bacterial cell this causes condensation of the DNA molecule which loses its ability to replicate thus affecting cell viability Feng et al 2000 The silver ions
339. le of structure and molecular properties of terpenoids in determining their antimicrobial activity Flavour and Fragrance Journal 14 322 332 Hammer K A Carson C F Riley T V 1999 Antimicrobial activity of essential oils and other plant extracts Journal of Applied Microbiology 86 985 990 219 Cap tulo 4 Art culo cient fico 5 Higueras L L pez Carballo G Cerisuelo J P Gavara R Hern ndez Mu z P 2013 Preparation and characterization of chitosan HP p cyclodextrins composites with high sorption capacity for carvacrol Carbohydrate Polymers 97 262 268 Kumar M N V R Muzzarelli R A A Muzzarelli C Sashiwa H Domb A J 2004 Chitosan chemistry and pharmaceutical perspectives Chemical Reviews 104 6017 6084 Kurek M Guinault A Voilley A Galic K Debeaufort F 2014 Effect of relative humidity on carvacrol release and permeation properties of chitosan based films and coatings Food Chemistry 144 9 17 Locci E Lai S Piras A Marongiu B Lai A 2004 13C CPMAS and H NMR Study of the inclusion complexes of p cyclodextrin with carvacrol thymol and eugenol prepared in supercritical carbon dioxide Chemistry amp Biodiversity 1 1354 1366 L pez Carballo G Higueras L Gavara R Hern ndez Mu oz P 2013 Silver ions release from antibacterial chitosan films containing in situ generated silver nanoparticles Journal of Agricultural and Food Chemistry 61 260 267 Mas
340. llo fresco envasado En segundo lugar se incorpor el precursor nitrato de plata a la soluci n film gena de quitosano para generar in situ nanopart culas de plata durante la neutralizaci n de las pel culas de acetato de quitosano Las pel culas desarrolladas presentaron una actividad antibacteriana in vitro mantenida en el tiempo Las nanopart culas de plata pudieron actuar como reservorios de iones de plata liberados sostenidamente en un entorno acuoso El tercer mecanismo para incorporar el agente antimicrobiano fue el anclaje reversible del vol til cinamaldeh do a pel culas preformadas de quitosano mediante la formaci n de una base de Schiff Este enlace covalente fue hidrolizado tras la aplicaci n de diversos tratamientos de temperatura tiempo habituales en la conservaci n de alimentos en un medio acuoso liber ndose el cinamaldehido Las pel culas iminoderivadas aumentaron la seguridad microbiol gica de la leche La leche con el cinamaldeh do liberado fue aceptada sensorialmente El ltimo m todo empleado se bas en la incorporaci n de monoterpenos antimicrobianos principalmente carvacrol en pel culas de quitosano con coadyuvantes mediante la inmersi n de las pel culas Resumen preformadas en el agente vol til en estado l quido La presencia conjunta de hidroxipropil B ciclodextrinas glicerol y agua en las pel culas de quitosano dio lugar a una elevada capacidad de sorci n de carvacrol de car cter lip fi
341. lo G Catal R Gavara R Hern ndez Mu oz P 2013 Antifungal properties of gliadin films incorporating cinnamaldehyde and application in active food packaging of bread and cheese spread foodstuffs International Journal of Food Microbiology 166 369 377 Baldwin E A Nisperos M O Chen X Hagenmaier R D 1996 Improving storage life of cut apple and potato with edible coating Postharvest Biology and Technology 9 151 163 312 Anexos Ben Arfa A Preziosi Belloy L Chalier P Gontard N 2007 Antimicrobial paper based on a soy protein isolate or modified starch coating including carvacrol and cinnamaldehyde Journal of Agricultural and Food Chemistry 55 2155 2162 Benavides S Villalobos Carvajal R Reyes J E 2012 Physical mechanical and antibacterial properties of alginate film effect of the crosslinking degree and oregano essential oil concentration Journal of Food Engineering 110 232 239 Bosquez Molina E Ronquillo de Jes s E Bautista Ba os S Verde Calvo J R Morales L pez J 2010 Inhibitory effect of essential oils against Colletotrichum gloeosporioides and Rhizopus stolonifer in stored papaya fruit and their possible application in coatings Postharvest Biology and Technology 57 132 137 Bower C K Avena Bustillos R J Olsen C W McHugh T H Bechtel P J 2006 Characterization of fish skin gelatin gels and films containing the antimicrobial enzyme lysozyme Journal of Food Sc
342. lo La liberaci n del carvacrol presente en las pel culas compuestas fue activada por la humedad relativa ambiental Este carvacrol liberado de las pel culas present efectividad antimicrobiana en fase vapor cuando fue incorporado en un sistema de envasado para pechugas de pollo fresco La capacidad de retenci n de las pel culas se pudo modular en funci n de su formulaci n y de la estructura qu mica del monoterpeno sorbido Las pel culas con dichos monoterpenos presentaron capacidad antimicrobiana en fase vapor in vitro y pueden emplearse en el disefio de envases activos adaptados al alimento aplicado XII Resum En aquesta Tesi s ha abordat el desenvolupament de pell cules renovables i actives de quitos amb agents antimicrobians de diversa naturalesa per a la seua aplicaci en l envasat actiu d aliments Les pel licules de quitos es van obtindre mitjangant la t cnica d extensi i evaporaci del solvent i les subst ncies bioactives van ser incorporades mitjan ant diferents metodologies En primer lloc el s lid etil N dodecanoil L arginat LAE es va incorporar pr viament a la formaci de la pel cula de quitos Les pel l cules amb LAE van alliberar completament l agent actiu en un simulant alimentari aqu s en diverses hores a diferents temperatures Les pel l cules de quitosa que no van ser neutralitzades van presentar activitat antimicrobiana enfront de microorganismes presents en pits de pollastre fresques
343. los a diferentes mecanismos de incorporaci n y liberaci n de agentes antimicrobianos de diversa naturaleza en una matriz de CS Todo ello se muestra resumido en la Tabla 5 1 La matriz seleccionada para el desarrollo de los sistemas de envasado antimicrobiano fue el CS debido a sus diversas y ventajosas propiedades El CS se caracteriza por su elevada disponibilidad y por ser biodegradable y comestible A causa de su estructura policati nica el CS presenta capacidad antimicrobiana La presencia de grupos funcionales reactivos hacen del CS un pol mero muy vers til y funcional Asimismo tiene capacidad de formar pel culas e hidrogeles por lo que el CS puede ser empleado como matriz biopolim rica para el envasado activo de alimentos donde sea preciso la incorporaci n y liberaci n controlada de sustancias activas desde su seno Tabla 5 1 Pel culas antimicrobianas de CS a Estado del de plata disoluci n film gena de CS L quido anclaje mediante la nanopart culas Libre en la matriz medio l quido Reversi n del enlace covalente en Modo de z P E agente Mecanismo z incorporaci n del d A Pe EIU Sistema antimicrobiano de acci n del Aplicaci n agente i piz A en la matriz de sistema antimicrobiano cs S lido Pp incorporaci n a la Migrate Pechuga de CS LAE i v Libre en la matriz medio l quido disoluci n 2n pollo mah o s lido film gena de CS Precusor s lido Creaci n in situ CS nanopart culas
344. ltivo medio de cultivo tomate Cherry medio de cultivo Micrococcus luteus L innocua S Enteritidis E coli St aureus E coli S Typhimurium St aureus C albicans E coli ME TVC Brochothrix thermosphacta E coli O157 H7 Lb sakei Leuconostoc mesenteroides L monocytogenes S Typhimurium P digitatum P italicum E coli O157 H7 E coli L monocytogenes E coli O157 H7 S Typhimurium L monocytogenes E coli O157 H7 S Typhimurium E coli O157 H7 L monocytogenes S Enteriditis L monocytogenes L plantarum B subtilis E coli B subtilis E coli Alternaria alternata Botrytis cinerea P digitatum P italicum Cha et al 2002 Seol et al 2009 Gill y Holley 2000 Valencia Chamorro et al 2008 Liu et al 2010 Padgett et al 1998 Sivarooban et al 2008 Gadang et al 2008 Brown et al 2008 Hoffman et al 2001 Mecitoglu et al 2006 Mecitoglu et al 2007 Fagundes et al 2013 Valencia Chamorro et al 2008 297 Anexos cidos org nicos y de c ac tico c l ctico alginato de calcio c ac tico agar agar c ac tico c ac tico c l urico c ac tico cinamaldeh do c propi nico c propi nico c l urico c propi nico cinamaldeh do c benzoico quitosano gelatina c benzoico metilcelulosa c c trico nisina prote nas de suero
345. ltzer M Garrigos M C 2012 Characterization and antimicrobial activity studies of polypropylene films with carvacrol and thymol for active packaging Journal of Food Engineering 109 513 519 Rokka M Eerola S Smolander M Alakomi H L Ahvenainen R 2004 Monitoring of the quality of modified atmosphere packaged broiler chicken cuts stored in different temperature conditions Food Control 15 601 607 S nchez J A 2011 Establecimiento de valores microbiol gicos de referencia en un matadero y sala de despiece de aves Eurocarne 199 62 68 Scramlin S Newman M Cox R Sepe H Alderton A O Leary J Mikel W 2010 Effects of oregano oil brine enhancement on quality attributes of beef Longissimus dorsi and Semimembranosus muscles from various age animals Journal of Food Science 75 S89 94 Skrinjar M M Nemet N T 2009 Antimicrobial effects of spices and herbs essential oils Acta Periodica Technologica 195 209 Sofos J N 1994 Microbial growth and its control in meat poultry and fish in Pearson A M Dutson T R Eds Quality attributes and their measurement in meat poultry and fish products Blackie Academic amp Professional London pp 358 403 241 Cap tulo 4 Art culo cient fico 6 Veldhuizen EJ A Creutzberg T O Burt S A Haagsman H P 2007 Low temperature and binding to food components inhibit the antibacterial activity of carvacrol against Listeria monocytogenes in steak
346. luorescens S Typhimurium S enterica E coli O157 H7 L monocytogenes P commune EM TVC Xanthomonas campestris pv mangiferaeindicae Colletotrichum gloeosporioides Lasiodiplodia theobromae ME TVC mes filos y psicr filos Shewanella putrefaciens Ps fluorescens L monocytogenes S Anatum Micrococcus luteus L innocua S Enteritidis E coli St aureus Brochotrix thermosphacta Micrococcus lysodeikticus E coli B amyloliquefaciens E coli B subtilis Streptococcus cremoris Brochothrix thermosphacta E coli O157 H7 Lb sakei Leuconostoc mesenteroides L monocytogenes S Typhimurium Lb plantarum E coli L monocytogenes ME TVC hongos levaduras S Enteriditis ME TVC coliformes hongos levaduras E coli St aureus E coli Streptococcus faecalis L monocytogenes E coli Murillo Mart nez et al 2013 Yener et al 2009 Lee y Min 2013 2014 Min Harris et al 2005a b Min et al 2006 Min y Krochta 2005 Mohamed et al 2013 Jasour et al 2014 Datta et al 2008 Cha et al 2002 Nam et al 2007 Appendini y Hotchkiss 1997 Gemili et al 2009 Bower et al 2006 Gill y Holley 2000 Padgett et al 1998 Min Harris Han et al 2005 Kim et al 2008 Lian et al 2012 Park et al 2004 Duan et al 2008 Anexos lisozima lactoferrina lisozima EDTA lisozima EDTA lisozima
347. m and the 10 um EVOH layer was located in the centre of the structure With this composition it was evident that the EVOH layer was well protected and that in terms of aroma barrier the tray could be considered as an infinite barrier Nevertheless the aroma scalping ability of polyolefins is known and therefore a portion of the added carvacrol was expected to be sorbed in the most internal PP layer of the tray Instead of a thermosealable lid an aluminium foil tape was used to close the package hermetically to guarantee an infinite barrier to the antimicrobial agent and to attach the antimicrobial device that is the CS CD Active film However the adhesive 231 Cap tulo 4 Art culo cient fico 6 coating could also be involved in scalping processes and therefore it was considered in the study Besides the packaging and of course the device carvacrol was also partitioned in the package headspace and in the chicken fillet These two phases were also analysed Figure 4 4 10 presents the concentration of carvacrol in the different parts of the packaging headspace chicken system As can be seen in Figure 4 4 10a the CS CD Active film device partially released the agent to the other packaging phases Large differences were observed between the sample without chicken medium empty and the samples containing chicken After three days the small device had released ca 9896 of the initial content reaching 9996 after the 9 days of storage
348. m semitectum Pintado et al 2010 Fajardo et al 2010 Cong et al 2007 nv quA eee Mal keen Rn eee er glucosa oxidasa glucosa oxidasa nisina lactoperoxidasa lactoperoxidasa lactoperoxidasa lactoperoxidasa actoperoxidasa isozima isozima nisina isozima nisina EDTA isozima isozima isozima isozima isozima nisina EDTA isozima isozima EDTA isozima isozima quitosano isozima isozima isozima 304 prote na de suero de leche alginato de calcio harina de soja desgrasada prote na de suero de leche quitosano quitosano alginato de calcio alginato de sodio K carragenato almid n de guisante celulosa triacetato celulosa acetato gelatina de piel de pescado gelatina prote na de soja ze na prote na de suero de leche prote na de suero de leche prote nas de suero de leche quitosano quitosano quitosano medio de cultivo medio de cultivo medio de cultivo medio de cultivo pavo asado salm n ahumado medio de cultivo trucha Arco iris salm n ahumado medio de cultivo medio de cultivo medio de cultivo medio de cultivo medio de cultivo jam n cocido mortadela medio de cultivo medio de cultivo salm n ahumado huevo duro medio de cultivo medio de cultivo medio de L innocua Brochothrix thermosphacta E coli Enterococcus faecalis E coli L innocua Ps f
349. ma de tesis se produce en el envasado de ste despu s de una potencial contaminaci n de la superficie por la manipulaci n del alimento lo que se suma a la acci n protectora del envase que evita posteriores contaminaciones y 4 se pueden incorporar al envase sustancias activas l biles que entrar an en contacto con el alimento durante el envasado que puede ser posterior al tratamiento agresivo del alimento De esta manera se pueden adicionar menores cantidades de antimicrobiano reducir el posible impacto en las propiedades organol pticas del alimento adem s de aumentar la seguridad microbiol gica del producto y su aceptaci n por el consumidor En la actualidad se est n desarrollando sistemas de envasado basados en matrices polim ricas naturales o artificiales que incorporan agentes activos y que mediante fen menos de transferencia de masa se pueden ceder de manera gradual y controlada a la superficie del alimento durante el tiempo de almacenaje y distribuci n Coma 2008 El dispositivo activo puede formar parte de la pared del envase o bien estar incluido en la etiqueta tapa en forma de almohadilla separadores pero siempre evitando que sea apreciado por el consumidor como un elemento extrafio o que pueda ser ingerido accidentalmente Por otro lado es fundamental tener presente el mecanismo por el cual se produzca la liberaci n del agente activo por parte del pol mero cuando sea requerido normalmente durante el alma
350. mal culture medium for the microorganism in in vitro assays magnifies any effect Burt 2004 The initial working plan included a sensory test with consumers which of course was cancelled because of the unacceptable carvacrol content of 238 Quitosano Carvacrol the product Nevertheless the authors of this work decided to check the aroma of the fresh fillet stored in the small active package after the nine days of storage and in fact the effect on the aroma of the fresh fillets was perceptible but acceptable A further consideration is that the process of cooking would produce an appreciable loss of antimicrobial agent Samples of chicken breast were cooked in the oven 270 C 20 min and tested by the authors The aroma of the baked fillets was acceptable and they could be distinguished from a control sample by their warmly pungent odour as described in the literature an aroma that is readily accepted by consumers Burt 2004 Chi et aL 2006 However in the mouth a bitter flavour and a dry texture were observed Scramlin et al 2010 reported that oregano oil carvacrol is the major component of this essential oil can reduce lipid oxidation and extend the shelf life of meat products but tends to develop off flavours causing lower acceptability 4 Conclusions Materials containing carvacrol were developed as antimicrobial devices for active packaging of food products and were tested in the packaging of fresh chicken
351. maximum decomposition temperature of CS appeared at 288 C whereas for film with silver based nanoparticles the peak 111 Cap tulo 2 Art culo cient fico 2 A The aim of this work has been to develop antimicrobial films ba silver jte AgNPs were produced by reduction of si Tu salt was added to the film forming solution in concentration polymer The morphology of the synthesized nanoparticles microscopy and the antimicrobial activity o pez cp pan fate ied u aureus in liquid media Mueller Hinton broth and in the Same me Muted with o of 0 1 Ag 0 2 Ag 0 5 Ag 1 0 Ag 1 5 Ag i csic c lonic silver has long ga tru its mechanism of inh antimicrobial action in has and proteins altering thei fos hav qa d AT cause structural changes in Envelope a mic mem cter a Ie used 0 196 A 0 296 A 0 56 A 1 0 Ag 1 5 Ag g g g Figure 4 2 2 CS silver based nanoparticle films with different concentrations of silver A neutralized at 22 C B neutralized at 37 C Table 4 2 1 Colour parameters of CS films with different concentrations of silver neutralized at 22 C and 37 C Ag L a b C h AE Control 00 94 27 0 82 0 61 0 05 7102071 7135071 94 91 0 60 0 1 88 71 0 44 0 62 0 03 5 82 0 19 5 85 0 18 96 11 0 44 5 71 0 39 0 2 7510 147 2 28 0 84 12 57 0 96 12 78 1 09 7972x103 20 14 1 66 Films neutralized at 22 C 05 7084 211 4 40 0 86 15
352. mbes S Preziosi Belloy L Gontard N Chalier P 2006 Antimicrobial activity of carvacrol related to its chemical structure Letters in Applied Microbiology 43 149 154 Berger K R 2002 A brief history of packaging Agricultural and Biological Engineering Department Florida Cooperative Extension Service Institute of Food and Agricultural Sciences University of Florida Reviewed by Welt B 2005 Bhattarai N Gunn J Zhang M Q 2010 Chitosan based hydrogels for controlled localized drug delivery Advanced Drug Delivery Reviews 62 83 99 Bickers D Calow P Greim H Hanifin J M Rogers A E Saurat J H Sipes I G Smith R L Tagami H panel R e 2005 A toxicologic and dermatologic assessment of cinnamyl alcohol cinnamaldehyde and cinnamic acid when used as fragrance ingredients Food and Chemical Toxicology 43 799 836 Brody A L Bugusu B Han J H Sand C K Mchugh T H 2008 Innovative food packaging solutions Journal of Food Science 73 R107 R116 37 Introducci n Burt S 2004 Essential oils their antibacterial properties and potential applications in foods A review International Journal of Food Microbiology 94 223 253 Butler B L Vergano P J Testin R F Bunn J M Wiles J L 1996 Mechanical and barrier properties of edible chitosan films as affected by composition and storage Journal of Food Science 61 953 amp Campos C A Gerschenson L N Flores S K 2
353. medio de cultivo medio de cultivo medio de cultivo pan de ajo medio de cultivo zanahoria tierna pato ahumado medio de cultivo medio de cultivo br coli cortado medio de E coli O157 H7 S Typhimurium L monocytogenes St aureus B cereus Brochothrix thermosphacta E coli L innocua L monocytogenes Ps putida S Typhimurium Shewanella putrefaciens E coli O157 H7 St aureus S Enteritidis L monocytogenes Lb plantarum E coli O157 H7 S enterica L monocytogenes E coli St aureus S Typhimurium L monocytogenes B cereus ME hongos L monocytogenes Ps aeruginosa S Enteritidis St aureus Saccharomyces cerevisiae A niger L monocytogenes S Enteritidis Ps fragi L monocytogenes P italicum E coli O157 H7 L monocytogenes ME TVC mes filos y psicr filos E coli St aureus L Oussalah et al 2006 2007 Pranoto Salokhe et al 2005 Teixeira et al 2014 Seydim y Sarikus 2006 2007 Du Olsen Avena Bustillos McHugh Levin Mandrell et al 2009 Pranoto Rakshit et al 2005 Sangsuwan et al 2012 Arfat et al 2014 Gniewosz et al 2013 Song et al 2013 Fern ndez Pan et al 2012 S nchez Gonz lez Gonz lez Mart nez et al 2010 lvarez et al 2013 S nchez 305 Anexos AE bergamota AE lim n AE rbol del t AE bergamota AE tomillo AE bergamota AE
354. mentar m s el volumen libre de la matriz sin llegar a desestructurar las pel culas Asimismo la geometr a molecular y la cantidad de dextrinas presentes en las pel culas resultaron fundamentales en la capacidad de sorci n de las pel culas Por una parte cuando se incluyeron maltodextrinas en lugar de ciclodextrinas la sorci n de carvacrol por las pel culas no super el 1 596 Por otra la sorci n de carvacrol fue mayor cuanto mayor fue el contenido de CD Sin embargo cuando la proporci n de CD super a la de CS las pel culas fueron demasiado quebradizas y dif ciles de manejar Por lo que la proporci n de CS y CD ptima se estableci en 1 1 Por otra parte para determinar el efecto de la estructura qu mica de los agentes naturales vol tiles en la capacidad de sorci n de las matrices se seleccion la pel cula que mostr mayor capacidad de sorci n de carvacrol CS CD 1 1 en peso plastificada con 3596 de G y acondicionada a 7596 de RH En estas condiciones las pel culas compuestas incorporaron alrededor de un 3596 de agua A continuaci n las pel culas fueron inmersas en otros monoterpenos con actividad antimicrobiana como ya se hizo con el 282 Discusi n general carvacrol Los monoterpenos presentes en AE probados fueron monoterpenos ac clicos c clicos y bic clicos mirtenol nerol carveol dehidrocarveol e isopulegol y monoterpenos fen licos m cumenol o cumenol carvacrol guayacol isoeugenol
355. modifications and their unlimited application potential an overview Trends in Food Science amp Technology 18 117 131 Rahman M Brazel C S 2004 The plasticizer market an assessment of traditional plasticizers and research trends to meet new challenges Progress in Polymer Science 29 1223 1248 Rai M Yadav A Gade A 2009 Silver nanoparticles as a new generation of antimicrobials Biotechnology Advances 27 76 83 Restuccia D Spizzirri U G Parisi O L Cirillo G Curcio M Iemma F Puoci F Vinci G Picci N 2010 New EU regulation aspects and global market of active and intelligent packaging for food industry applications Food Control 21 1425 1435 Rinaudo M 2006 Chitin and chitosan properties and applications Progress in Polymer Science 31 603 632 Rodr guez E Seguer J Rocabayera X Manresa A 2004 Cellular effects of monohydrochloride of L arginine N lauroyl ethylester LAE on exposure to Salmonella Typhimurium and Staphylococcus aureus Journal of Applied Microbiology 96 903 912 Rooney M L 1995 Overview of active food packaging in Rooney M L Ed Active food packaging Blackie Academic and Professional Londres pp 1 260 Ruckman S A Rocabayera X Borzelleca J F Sandusky C B 2004 Toxicological and metabolic investigations of the safety of N lauroyl L arginine ethyl ester monohydrochloride LAE Food and Chemical Toxicology 42 245 259 43 Int
356. n M Henika P R Mart n Belloso O Pan Z L McHugh T H 2007 Effects of plant essential oils and oil compounds on mechanical barrier and antimicrobial properties of alginate apple puree edible films Journal of Food Engineering 81 634 641 325 Anexos Rojas Gra M A Raybaudi Massilia R M Soliva Fortuny R C Avena Bustillos R J McHugh T H Mart n Belloso O 2007 Apple puree alginate edible coating as carrier of antimicrobial agents to prolong shelf life of fresh cut apples Postharvest Biology and Technology 45 254 264 Rojhan M Nouri L 2013 Antimicrobial physicochemical mechanical and barrier properties of tapioca starch films incorporated with eucalyptus extract Journal of Chemical Health Risks 3 Romanazzi G Karabulut O A Smilanick J L 2007 Combination of chitosan and ethanol to control postharvest gray mold of table grapes Postharvest Biology and Technology 45 134 140 Royo M Fern ndez Pan I Mat J I 2010 Antimicrobial effectiveness of oregano and sage essential oils incorporated into whey protein films or cellulose based filter paper Journal of the Science of Food and Agriculture 90 1513 1519 Ruiz Navajas Y Viuda Martos M Sendra E P rez lvarez J A Fern ndez L pez J 2013 In vitro antibacterial and antioxidant properties of chitosan edible films incorporated with Thymus moroderi or Thymus piperella essential oils Food Control 30 386 392 Salgado P R L
357. n P 2008 Effect of chitosan methyl cellulose films on microbial and quality characteristics of fresh cut cantaloupe and pineapple Postharvest Biology and Technology 49 403 410 Sangsuwan J Srikok K Duangsawat J Rachtanapun P 2012 Development of chitosan film incorporating garlic oil or potassium sorbate as an antifungal agent for garlic bread Journal of Agricultural Science and Technology B 2 128 136 Sanjurjo K Flores S Gerschenson L Jagus R 2006 Study of the performance of nisin supported in edible films Food Research International 39 749 754 326 Anexos Sanla Ead N Jangchud A Chonhenchob V Suppakul P 2012 Antimicrobial activity of cinnamaldehyde and eugenol and their activity after incorporation into cellulose based packaging films Packaging Technology and Science 25 7 17 Sanpui P Murugadoss A Prasad P V D Ghosh S S Chattopadhyay A 2008 The antibacterial properties of a novel chitosan Ag nanoparticle composite International Journal of Food Microbiology 124 142 146 Santiago Silva P Soares N F F N brega J E J nior M A W Barbosa K B F Volp A C P Zerdas E R M A W rlitzer NJ 2009 Antimicrobial efficiency of film incorporated with pediocin ALTA R 2351 on preservation of sliced ham Food Control 20 85 89 Scannell A G M Hill C Ross R P Marx S Hartmeier W Arendt E K 2000 Development of bioactive food packaging materials us
358. n el carvacrol en pel culas de CS y en las lonchas de mortadela antes y tras 5 d as de contacto con la pel cula colocada como separador Kurek et al 2013 determinaron la capacidad antimicrobiana de pel culas de CS con carvacrol en relaci n a su coeficiente de partici n evaluando la cantidad de carvacrol en las pel culas y en la fase gaseosa pero sin ser aplicado a ning n alimento Passarinho et al 2014 evaluaron la concentraci n de AE de or gano en pan tras ser envasado junto con bolsitas que conten an el agente antimicrobiano 168 Quitosano Carvacrol Rodr guez et al 2008 analizaron la cantidad de AE de canela en el pan envasado con papel recubierto de parafina con dicho AE para el control del crecimiento de Rhizopus stolonifer durante el almacenamiento pero no se realiza un an lisis del AE en el resto del sistema de envasado a lo largo del tiempo Por lo tanto es necesario un conocimiento m s profundo del comportamiento termodin mico de estos activos vol tiles en el sistema de envasado y en el propio alimento para determinar la eficacia y validez de los sistemas activos antimicrobianos en alimentos espec ficos Al igual que el carvacrol otros constituyentes individuales vol tiles de los AE presentan una elevada capacidad antimicrobiana en estado vapor pudi ndose adaptar el vol til al alimento al que se aplica en funci n de su car cter organol ptico para una mayor aceptaci n de los productos envasados y e
359. n a entrar en contacto con el alimento Reglamento 135 2004 CE 2004 que incluye las limitaciones espec ficas y globales de migraci n de las sustancias desde el material Adem s de respetar el Reglamento 450 2009 espec fico de envases activos CE 2009 que completa los principios generales definidos en el reglamento anterior y contiene una lista positiva de sustancias autorizadas que podr n utilizarse en los materiales activos Asimismo deber n cumplir la legislaci n espec fica del material de que se componga el envase Dainelli et al 2008 Restuccia et al 2010 Por otra parte todas las sustancias activas que migren al alimento son consideradas aditivos alimentarios CE 2008a o aromas CE 2008b por lo que deber cumplir la legislaci n vigente as como la referente al etiquetado UE 20110 La comercializaci n de los envases activos comenz en Jap n y Australia en los a os ochenta sin embargo su empleo en Europa y EEUU ha estado m s restringido por una legislaci n m s estricta y un menor Introducci n conocimiento del consumidor de las ventajas y eficacia de estos sistemas y su impacto econ mico y medioambiental De Kruijf et al 2002 Actualmente Am rica del Norte y en menor medida Europa suponen el principal mercado para la tecnolog a de envasado activo En los EEUU en 2010 el envasado en atm sfera modificada y el envasado activo e inteligente conjuntamente representaron el 5 del mercado total de
360. n a recent paper Cerisuelo et al 2012 Considering the partition coefficient for carvacrol between PP and air at 23 C Hppyair and the diffusion coefficient of carvacrol in PP Dc and assuming that the concentration of carvacrol in the headspace was maintained constant Cc the predicted concentration was estimated by using the solution to Fick s laws for a film sample that sorbs a penetrant through one of its surfaces Crank 1975 233 Cap tulo 4 Art culo cient fico 6 B e rJ ot cP 4 eo 5 1 e B 1 E m E 3l y 2 The average concentration of carvacrol in the polymer Cc is a function of time t the carvacrol concentration in the polymer at equilibrium Cc o Hepjai Cc and the diffusion coefficient With all these assumptions the predicted concentration of carvacrol in the tray was calculated and the results are also plotted in Figure 4 4 10d As can be seen the predictions are within the same order of magnitude despite the assumptions just mentioned Although because of the time scale of the figure and the log scale of the Y axis the predicted values appear to be reaching constancy the predicted release after 9 days was less than 2096 of the maximum release that would be expected when equilibrium had been reached This result is in agreement with the hypothesis of a tray with an infinite barrier To have amore visual idea of the partition of carvacrol in the various package regions the tota
361. n agroqu mica El mercado mundial de CS se estim en 13700 toneladas para 2010 y se esperan 21400 toneladas para el a o 2015 Cosgrove 2010 Por una parte 3096 de los pescados y productos marinos se desperdician a lo largo de la cadena FAO 2012 Por otra los residuos de las plantas procesadoras de mariscos suponen un problema medioambiental dada su acumulaci n por su lenta velocidad de degradaci n As actualmente se puede obtener CS en grandes cantidades a partir de quitina procedente de estos desechos y excedentes Healy et al 1994 Srinivasa y Tharanathan 2007 Y la reutilizaci n de estas materias primas para obtener biomateriales destinados a la conservaci n de alimentos puede reducir la acumulaci n de desechos y su impacto medioambiental 1 3 Los hidrogeles como sistemas polim ricos de liberaci n sostenida El desarrollo de sistemas antimicrobianos basados en la liberaci n sostenida del agente activo es un rea de inter s en diversos sectores tecnol gicos aparte del sector alimentario como son el sector agr cola textil m dico y farmac utico En la actualidad se est n realizando grandes esfuerzos en el desarrollo de mecanismos de liberaci n sostenida con el fin de mantener una determinada concentraci n del compuesto activo en el alimento envasado durante un per odo de tiempo determinado Coma 2008 Otro factor a tener presente es el mecanismo por el cual el hidrogel va a desarrollar su actividad es decir l
362. n carvacrol fueron obtenidas por extensi n y evaporaci n del disolvente y acondicionadas a 23 C y una humedad relativa del 75 antes de ser sumergidas en carvacrol l quido hasta que se alcanz el equilibrio de sorci n En un trabajo anterior se estudi la actividad antimicrobiana in vitro de estas pel culas En este trabajo se utilizaron pel culas activas para inhibir el crecimiento microbiano en pechugas de pollo fileteadas envasadas Las muestras de pel culas de CS CD cargadas con carvacrol de diferentes tama os y por tanto con diferentes cantidades de agente antimicrobiano fueron pegadas a la tapa de aluminio empleadas para sellar tarrinas de PP EVOH PP con 25 g de filetes de pollo Estas muestras fueron almacenadas durante 9 d as a 4 C Los envases fueron sellados herm ticamente y se confirm que presentaban una barrera infinita a carvacrol Se analiz el reparto del agente antimicrobiano en el sistema alimentario embalaje Estos dispositivos liberaron r pidamente un gran porcentaje de la carga del antimicrobiano cantidades que fueron obtenidas por el recubrimiento adhesivo de la tapa y especialmente por los filetes de pollo stos ltimos fueron la principal fase sorbente con concentraciones medias que oscilan entre 200 y 5 000 mg kg durante el per odo de almacenamiento Se analiz la microbiota de los filetes de pollo frescos envasados mes filos psicr filos Pseudomonas spp enterobacterias bacterias cido l cticas
363. n coefficients of films conditioned at O and 53 RH differed by one order of magnitude and also of films conditioned at 53 and 75 RH Water has the ability to plasticize and swell hydrophilic polymers such as CS increasing chain mobility and interchain distance As the RH at which the films were conditioned increased they experienced a successive plasticization and swelling by water molecules giving rise to a looser polymer matrix which enhanced the diffusion of carvacrol Films conditioned at 75 RH are expected to be greatly plasticized by water and an increase in the amount of G from 20 to 35 did not greatly affect the diffusion coefficient 3 3 Desorption kinetics of carvacrol The release of carvacrol vapour from 1 1 CS CD films plasticized with 35 G and conditioned at 75 RH CS CD 35G 75RH prior to immersion in the volatile solvent was evaluated at 25 C and under different RH 43 53 and 90 Figure 4 4 8 shows the normalized experimental curves of release of carvacrol vapour from these films As in the sorption process described in 3 2 section the one dimensional solution of Fick s second law of diffusion in a plane sheet for the boundary conditions in a desorption process t 0 0 lt x lt L 0g t gt 0 x 0 x L c 0 The solution under these conditions is 7 8 NX 1 n D 2n 1 2 t 3 Meses 52 lc 1 ex D Jl 212 Quitosano Carvacrol The theoretical curves modelled with Equation 3 for a desorption process are
364. n funci n de riesgo microbiol gico L pez et aL 2005 Nedorostova et al 2009 La capacidad de retenci n de sistemas polim ricos con CD y la actividad antimicrobiana de otros monoterpenos distintos al carvacrol puede estar determinada por la estructura qu mica de estos vol tiles Por todo lo descrito en el presente trabajo se ha desarrollado y caracterizado un sistema pol mero CD plastificante capaz de retener vol tiles naturales antimicrobianos en funci n de la formulaci n de la pel cula Seguidamente se ha estudiado la cin tica de liberaci n del agente como respuesta a est mulos como la RH del entorno para poder ser aplicado en estado vapor en el envasado antimicrobiano de alimentos Posteriormente con las pel culas obtenidas se ha desarrollado y estudiado la eficacia de un envase activo para la conservaci n de pechugas de pollo Referencias bibliogr ficas Aoki N Nishikawa M Hattori K 2003 Synthesis of chitosan derivatives bearing cyclodextrin and adsorption of p nonylphenol and bisphenol A Carbohydrate Polymers 52 219 223 vila Sosa R Palou E Munguia M TJ Nev rez Moorillon G V Cruz A R N L pez Malo A 2012 Antifungal activity by vapor contact of essential oils added to amaranth chitosan or starch edible films International Journal of Food Microbiology 153 66 72 Ayala Zavala J F Soto Valdez H Gonz lez Le n A lvarez Parrilla E Mart n Belloso O Gonz lez Aguilar G A
365. na de las pel culas frente a Escherichia coli y Staphylococcus aureus en medio de crecimiento l quido diluido y sin diluir Asimismo se estudi si la actividad antimicrobiana de las pel culas se manten a a lo largo del tiempo para demostrar si dichas nanopart culas podr an actuar como nanoreservorios de iones plata de liberaci n sostenida La liberaci n se cuantific mediante espectrometr a de absorci n at mica de horno de grafito GFAAS El cap tulo 3 Desarrollo de pel culas de quitosano con compuestos vol tiles antimicrobianos anclados mediante un enlace covalente reversible y su aplicaci n en el envasado activo de alimentos est constituido por el art culo cient fico Reversible covalent immobilization of cinnamaldehyde on chitosan films via Schiff base formation and their application in active food packaging En este cap tulo se desarrollan pel culas de CS en las que se encuentra anclado el compuesto vol til cinamaldeh do a trav s de la formaci n de una base de Schiff creando un enlace covalente el cual es reversible mediante hidr lisis permitiendo la liberaci n de cinamaldeh do La formaci n de la base de Schiff se llev a cabo en una disoluci n etan lica acidificada de cinamaldeh do en la que se introducen las pel culas de CS neutralizadas obtenidas por extensi n y evaporaci n del solvente De todos los estudios realizados anteriormente se eligieron las condiciones de trabajo tiempo temperatura c
366. nas del alimento y la difusi n del carvacrol hacia el seno del alimento Adem s de la acci n protectora que ofrecen las matrices alimentarias a los microorganismos Ello hace que se requieran cantidades elevadas de carvacrol y que el producto pueda ser inaceptable sensorialmente Bitar et al 2008 indicaron que el umbral de olor del carvacrol es de 31 mg kg en aceite de girasol Sin embargo Scramlin et al 2010 apuntaron que el aceite de or gano puede extender la vida til de los productos a base de carne pero tiende a desarrollar mal sabor causando baja aceptabilidad Mientras que Chi et al 2006 comprobaron que la adici n de 45 mg kg o menos de AE de or gano en los derivados c rnicos es aceptado por los consumidores Por su parte las muestras de pollo envasadas con las pel culas de CS con carvacrol de 0 24 4 8 y 24 cm contienen 200 900 y 4500 mg kg respectivamente Tras el horneado gran parte del carvacrol fue liberado desde la matriz alimentaria quedando de 8 12 36 54 y 180 270 mg kg respectivamente Estas muestras 286 Discusi n general tras 9 d as de conservaci n antes y despu s de su horneado presentaron un aroma perceptible pero aceptable como a or gano No obstante las muestras de pollo presentaron un gusto amargo picante y con sensaci n de quemaz n en la lengua y una textura seca tras el horneado Otros autores tambi n comprobaron esta merma de la calidad sensorial al emplear AE de or gan
367. ncrease 49 26 26 cs 2317 16 1199 34 155 8 0 9 99 8 1 9 CScin 2352 62 1167 5 3 36 9 0 9 15 4 1 6 CScin 4 C 30min 237 1 6 1 110 7 5 9 29 1 0 4 15 8 1 1 gt CScin 65 C 30min 166 8 13 6 88 3 10 9 30 1 1 2 17 8 3 6 CScin 72 C 15min 165 6 10 3 81 3 143 314 1 5 17 8 4 1 CScin 95 C 10min 167 3 13 6 78 3 10 4 313 15 177 31 CScin 121 C 5min 915 14 2 524 6 7 321 1 6 17 9 4 6 Different letters in the same column indicate a statistically significant difference P lt 0 05 hydrolysis releasing cinnamaldehyde and providing more free amino groups for protonation These two facts could explain the absence of differences between CS CScin and CScin 4 C for 30 min films CScin films subjected to thermal treatment had reduced water uptake capacity and increased dimensional stability The water uptake and area of films treated at 65 C for 30 min 72 C for 15 min and 95 C for 5 min were reduced by around 30 and 65 respectively whereas films treated at 120 C for 5 min experienced a reduction in water uptake of approx 68 and the area decreased by 79 These results may be due to thermal crosslinking of the CS matrix giving rise to a more compact film structure Ji and Shi 2013 The films presented much lower water sorption values at pH 7 than in acidic conditions Water uptake of neutralized CS films immersed in buffered solution
368. ne un enorme problema econ mico ambiental y tico en todo el mundo Por otra parte en la actualidad los consumidores demandan alimentos f ciles de preparar con mayor informaci n seguros pero que est n libres de aditivos y que hayan sufrido un procesamiento m nimo conservando su frescura y sus propiedades organol pticas y nutritivas Por ello en los ltimos afios se est n desarrollando tecnolog as alternativas a los m todos Introducci n de conservaci n tradicionales m s agresivos como puedan ser los tratamientos t rmicos para la inactivaci n microbiana Estas nuevas tecnolog as pretenden mantener las propiedades originales del alimento ahorrar energ a y ser m s respetuosas con el medioambiente y a la vez ser efectivas contra los microorganismos pat genos y alterantes Sin embargo estas alternativas m s leves suelen ser tambi n menos eficaces y pueden no eliminar todos los microorganismos alterantes y pat genos a diferencia de los tratamientos t rmicos convencionales As para la aplicaci n de estas tecnolog as de procesamiento emergente que pueden no ser suficiente por ellas mismas una estrategia adecuada es su combinaci n en lo que se conoce como tecnolog as de barreras hurdle technology Leistner y Gorris 1995 En este sentido el envasado activo presenta un gran potencial para ser aplicado en combinaci n con otras tecnolog as de conservaci n de alimentos no t rmicas Los envases activos antimicr
369. needed For experimental use stock cultures were maintained by regular subculture on TSA slants at 4 C and transferred monthly Yeast and fungi strains were stored in MEB with 25 30 glycerol at 20 C For experimental use stock cultures were maintained by regular subcultures on MEA slants at 4 C and transferred monthly Cell suspension of O D 600 nm 0 400 0 500 in PW from a fresh slant was made providing a concentration of 10 10 cells mL for bacteria 10 10 cells mL for yeasts and 10 10 spores mL for fungi Measures were taken employing a Jenway Mod 6705 UV Vis Spectrophotometer 76 Quitosano LAE 2 5 LAE antimicrobial activity The minimum inhibitory concentration MIC and minimum biocide concentration MBC of LAE were determined First for the determination of the MIC and MBC for bacteria and yeasts in liquid medium cell suspension of microorganisms 100 pL was inoculated in 10 mL of culture medium with different amounts of LAE and incubated for 24 48 h at 28 C LAE tested concentrations were 0 2 4 8 16 24 32 and 40 pg mL Turbidity was determined after 24 and 72 h of incubation employing a Jenway Mod 6705 UV Vis Spectrophotometer MIC was determined as the lowest LAE concentration able to inhibit microbial growth To determine MBC all tubes showing no growth were subcultured 1 mL on agar MBC is defined as the lowest LAE concentration from which no colonies growth was observed Tests were performed in
370. negativas Bakkali et al 2008 Burt 2004 Dorman y Deans 2000 Lambert et al 2001 Adem s debido a este mecanismo inespec fico no se han descrito resistencias o adaptaciones significativas a los AE a concentraciones biocidas a diferencia de los antibi ticos Bakkali et al 2008 Los AE son muy empleados como aromatizantes en alimentaci n en cosm tica y perfumer a dent fricos loci n para despu s del afeitado perfumes en productos farmac uticos y fitosanitarios preparados para masajes repelente de insectos Burt 2004 Asimismo existe una gran cantidad de estudios dedicados al empleo de los AE y sus componentes como conservantes de alimentos y como agentes activos en el dise o de envases antimicrobianos para alimentos como se puede observar en el Anexo A Sin embargo stos pueden ser t xicos si se emplean en elevadas concentraciones Otros inconvenientes son su impacto en las propiedades organol pticas del alimento y posible rechazo a nivel sensorial por parte del consumidor y el posible enmascaramiento de olores que se desarrollan durante la alteraci n de los alimentos Por otra parte en ocasiones la reducci n de la actividad antimicrobiana de los AE cuando son empleados en alimentos en comparaci n con los resultados obtenidos in vitro hace que 32 Introducci n sea necesario el empleo de niveles superiores a los aceptados organol pticamente Burt 2004 Guti rrez et al 2008 Holley y Patel 2
371. nes de temperatura tiempo y se estudi la actividad 279 Discusi n general antimicrobiana de la pel cula tras cada tratamiento Los resultados mostraron que las pel culas mantenidas a bajas temperaturas como las temperaturas habituales de refrigeraci n fueron muy estables con una lenta liberaci n del agente activo y redujeron el crecimiento microbiano aproximadamente 1 log Las pel culas bajo condiciones de temperaturas suaves y mantenidas como la pasteurizaci n suave o el llenado del envase en caliente presentaron una liberaci n suficiente y mantenida para reducir el crecimiento microbiano en 5 log seg n el microorganismo testado Las pel culas activadas a elevadas temperaturas y tiempos cortos mostraron un efecto bactericida Se realizaron estudios de migraci n espec fica de cinamaldeh do en etanol al 5096 simulante de alimentos grasos tras los distintos tratamientos de temperatura tiempo observ ndose una mayor liberaci n de cinamaldeh do en 50 etanol que en el medio acuoso debido probablemente a la mayor solubilidad del cinamaldeh do en este medio hidroalcoh lico Por lo que las pel culas desarrolladas podr an aplicarse en alimentos grasos o emulsiones como la leche entera entre otros Por ello se comprob la efectividad antimicrobiana de las pel culas con cinamaldeh do en leche entera pasteurizada refrigerada e inoculada con Listeria monocytogenes En la matriz alimentaria las pel culas con cinamal
372. ngos P expansum ME hongos E coli B cereus Botrytis cinerea E coli ME TVC Ps putida ME TVC Campylobacter jejuni Lb sakei Serratia liquefaciens ME LAB Enterobacteriaceae ME TVC mes filos y psicr filos hongos Lim Hong et al 2010 Peretto et al 2014 Arrieta et al 2014 Kavoosi et al 2013 Ben Arfa et al 2007 Chalier et al 2007 Ravishankar et al 2012 Ravishankar et al 2009 Zhu et al 2014 Du Olsen Avena Bustillos Mchugh et al 2008 Mild et al 2011 Du Olsen Avena Bustillos McHugh et al 2008 L pez Mata et al 2013 Sipahi et al 2013 Rojas Gra Avena Bustillos et al 2007 Raybaudi Massilia Rojas Gra et al 2008 Sanla Ead et al 2012 Balaguer et al 2014 Balaguer et al 2013 Makwana et al 2014 Ben Arfa et al 2007 Gamage et al 2009 Ouattara et al 2001 Mild et al 2011 Ouattara et al 2000 Brasil et al 2012 Moreira et al Anexos mel n troceado levaduras 2014 citral c m lico alginato de calcio mel n Piel de S Enteritidis ME TVC Raybaudi eugenol c m lico sapo pelado mes filos y psicr filos Massilia geraniol c m lico hongos levaduras Mosqueda Melgar et al 2008 citra kafirina medio de Ps fluorescens Giteru et al citral quercetina cultivo Campylobacter jejuni L 2015 monocytogenes timo alga roja Gelid
373. nservaci n de alimentos Hirano et al 1999 anclaron formaldeh do glioxal propional butanal benzaldeh do 2 hidroxibenzaldeh do y vainillina a fibras ya preformadas de CS 129 Cap tulo 3 Estado de la cuesti n comprobando que las fibras manten an el aroma tras 6 meses en un ambiente h medo Las fibras resultantes fueron estables en agua y soluciones acuosas alcalinas pero inestables y solubles en soluciones acuosas cidas Sin embargo no determinaron la liberaci n de dichos aldeh dos a partir de las fibras como consecuencia de la hidr lisis de la base de Schiff no estudiaron la actividad antimicrobiana ni lo aplicaron en alimentos Por otra parte se han encontrado varios trabajos en los que se demuestran las propiedades antimicrobianas de las bases de Schiff desarrolladas Guo et al 2007 Jin et al 2009 2010 Kumar et aL 2014 Mohamed y Fekry 2011 Porras et al 2009 Wang et al 2012 Aunque en todos ellos la formaci n de la base de Schiff se produce en disoluci n y los estudios de la capacidad antimicrobiana se realizaron sobre los iminoderivados en forma de polvo y no de pel cula Por otra parte Marin et al 2013 Marin et al 2015 y Stroescu et al 2015 obtuvieron iminoquitosanos a partir de varios aldeh dos benzaldeh do cinamaldeh do citral salicilaldeh do vainillina para desarrollar pel culas antimicrobianas frente Escherichia coli Staphylococcus aureus y Candida albicans aunqu
374. nt with the information gathered from the thermal analysis 3 5 Barrier properties Permeability to gases and vapours is one of the most important properties of materials with potential application in food packaging design In this work permeability to water vapour and to oxygen were measured at 23 1 C and at two RH conditions 3 5 1 Water vapour permeability WVP The effect of the various matrix components and the humidity gradient on the resistance to permeation of water vapour was evaluated for the films and the values are presented in Table 4 4 3 The images show various features First G in the films gave rise to greater values of permeability to water vapour through both CS and CS CD based materials As a plasticizer G reduces the fragility of the polymer material by reducing interchain interactions G interacts via hydrogen bonds with the NH C O and OH substituents of the macromolecules Brown et al 2001 Quijada Garrido et al 2007 thus increasing chain flexibility and mobility which leads to lower resistance to the diffusion of permeants The higher the concentration of G the more noticeable is the increase in water vapour permeability for a given sample and humidity gradient The presence of HP BCDs in the matrix resulted in an improvement in the water vapour barrier provided The presence of HP BCDs appeared to produce an antiplasticizing effect on the film as has been reported after the incorporation of fillers in p
375. nt in the composite film was activated by the environmental relative humidity Carvacrol released from the films presented antimicrobial effectiveness in vapour phase when it was Abstract incorporated into a packaging system for fresh chicken breasts It was possible to tailor the retention capacity of the films by varying the film formulation and the chemical structure of the sorbed monoterpene Films with these monoterpenes showed in vitro antimicrobial activity in vapour phase Thus they can be applied in the design of active packages adapted to food products XVI ndice de contenidos 1 INTRODUCCI N 1 1 1 Envasado activo 4 1 11 Envasado activo antimicrobiano 6 1 2 Biopol meros 8 1 2 1 Quitosano 10 1 3 Los hidrogeles como sistemas polim ricos de liberaci n sostenida 15 1 3 1 Quitosano 16 1 3 2 Polialcohol vin lico 18 1 4 Coadyuvantes para la obtenci n de las pel culas 20 1 4 1 Plastificantes 20 1 4 2 Ciclodextrinas 22 1 5 Agentes antimicrobianos 25 1 5 1 Etil N dodecanoil L arginato 26 1 5 2 Nanopart culas de plata 27 1 5 3 Aceites esenciales y sus componentes 31 1 5 3 1 Carvacrol 34 1 5 3 2 Cinamaldeh do 35 1 6 Referencias bibliogr ficas 37 2 OBJETIVOS 47 2 1 Objetivo general 49 2 2 Objetivos espec ficos 49 3 JUSTIFICACI N Y ESQUEMA DE TESIS 51 4 CAP TULOS 61 4 1 Cap tulo 1 Desarrollo de pel culas de quitosano con etil N dodecanoil L arginato y su aplicaci n
376. ntact with the food surface There is a wide range of volatile compounds derived from plants or forming part of the aroma profile of fruits presenting biocide properties which could be applied in the design of antimicrobial carriers since most of them are generally recognized as safe GRAS and are used as food flavouring or seasoning agents However volatiles can be lost to some extent during entrapment or encapsulation in the polymer matrix which consequently decreases the retention process Therefore it would be of great interest to develop suitable carriers with a high entrapment capacity and sustained release properties for volatile compounds In addition the release of the volatile from the polymer matrix can be triggered by different stimuli such as the moisture present in the headspace of the package in this respect the hydrophilic nature of the polymer and the RH of the headspace are major factors controlling the release kinetics of the agent Cyclodextrins CDs are cyclic oligosaccharides consisting of a three dimensional structure forming a truncated cone with a hydrophobic cavity and a hydrophilic outer surface CDs are widely used as excipients in pharmacy to solubilise lipophilic molecules by means of inclusion complexes However non inclusion aspects of CDs are being studied such as solubilisation by formation of selfassembled aggregates or surfactant like effects In recent years CDs and their derivatives have been used as buil
377. ntents lists available at SciVerse ScienceDirect Carbohydrate Polymers journal homepage www elsevier com locate carbpol Preparation and characterization of chitosan HP B cyclodextrins composites with high sorption capacity for carvacrol Domo Laura Higueras Gracia L pez Carballo Josep P Cerisuelo Rafael Gavara Pilar Hern ndez Mu oz Instituto de Agroqu mica y Tecnolog a de Alimentos IATA CSIC Avenida Agust n Escardino 7 Paterna Valencia 46980 Spain ARTICLE INFO ABSTRACT Article history The aim of this work was to design new polymer based systems exhibiting an adjustable loading capac Received 5 July 2012 ity of carvacrol depending on the film formulation For this purpose biocomposite films were developed Received in revised form 22 March 2013 Accepted 6 April 2013 Available online 12 April 2013 employing chitosan CS as the polymer matrix and hydroxypropyl B cyclodextrins HP BCDs as an adjuvant to improve the sorption of carvacrol in the polymer matrix The morphology optical mechani cal and barrier properties of the resulting films were investigated and the sorption capacity of carvacrol evaluated Biocomposites resulted highly transparent with higher mechanical resistance and moisture barrier properties Sorption of carvacrol was greatly affected by the humidity RH and glycerol G con Hydroxypropyl B cyclodextrins tent of the biocomposites The highest sorption values were achieve
378. ntimicrobiano en la matriz de CS que se emple fue la inmovilizaci n covalente y reversible del 278 Discusi n general agente antimicrobiano de forma que el agente activo es liberado con un determinado est mulo Esta forma de incorporar el agente activo se llev a cabo mediante la formaci n de una base de Schiff entre un aldeh do vol til de origen natural con propiedades antimicrobianas el cinamaldeh do y el CS Este anclaje covalente se realiz sobre el CS previamente procesado en forma de pel cula El enlace covalente consisti en la adici n nucleof lica del grupo amino del CS al grupo carbonilo del cinamaldeh do El enlace imino formado puede revertirse para liberar el cinamaldeh do en presencia de agua y promovido por la temperatura pH del medio o presencia de luz Sin estos est mulos de activaci n estas pel culas fueron estables evitando la p rdida del vol til durante el almacenamiento de la pel cula hasta su uso El cinamaldeh do permaneci anclado a la matriz de CS ya que en un ambiente seco la reversibilidad de la base de Schiff es muy baja A pesar del intenso aroma que caracteriza al cinamaldeh do las pel culas mantenidas en estas condiciones apenas ol an Los resultados del an lisis elemental y la ATR FTIR muestran que el rendimiento de reacci n del cinamaldeh do con las pel culas de CS fue elevado gt 70 tal como se esperaba seg n se se ala en el estado de la cuesti n del cap tulo 3 cu
379. ntinued and gradual release of an antimicrobial agent during the storage and distribution of food packaging thus providing an effective prevention of surface contamination of foods Aymerich et aL 2008 Marcos et al 2007 In this case it is possible to reduce the amount of active agent required satisfying the demand of consumers for fewer additives One of the most innovative antimicrobial agent is ethyl N dodecanoyl L arginate hydrochloride LAE It is a synthetically derivative of lauric acid L arginine and ethanol Infante et aL 2004 Infante et al 1997 Ruckman et al 2004 which is notable for its antimicrobial effectiveness resulting from its chemical structure and surfactant properties Brandt et al 2010 Pinazo et aL 1999 Rodr guez et al 2004 LAE s antimicrobial properties are due to its action as cationic surfactant on cytoplasmic membrane and the outer membrane of Gram negative and cell membrane and cytoplasm of Gram positive denaturation proteins These changes produce disturbances in membrane potential resulting cell growth inhibition and loss of viability Infante et al 1997 Kanazawa et al 1995 Luchansky et al 2005 Rodr guez et al 2004 Tattawasart et al 2000 73 Cap tulo 1 Art culo cient fico 1 LAE is characterized by a broad spectrum Infante et aL 1997 and high antimicrobial efficiency against Gram negatives Gram positives fungi and yeasts with a low dose application Infante et a
380. nto de las pel culas de CS debido a su capacidad de formar hidrogeles en presencia de agua favorecer a la difusi n de estos iones de plata a trav s del pol mero hacia el medio de liberaci n Otro factor que se debe considerar es el medio de cultivo en el que se liberan los iones plata y ejercen su acci n antibacteriana La mayor capacidad antimicrobiana de las pel culas se dio en medios de cultivo diluidos donde existe menor presencia de agentes quelantes como las prote nas y por tanto una mayor disposici n de iones de plata libre Tambi n se puede pensar que en medios con escasos nutrientes y desfavorables aumenta la susceptibilidad de los microorganismos a los agentes antimicrobianos Y todo ello debe tenerse en cuenta a la hora de aplicar estas matrices Parte de las pel culas de CS con nanopart culas de plata neutralizadas a 37 C se autoclavaron previamente al ensayo de la capacidad antimicrobiana para determinar si este tratamiento hidrot rmico produc a m s nanopart culas de plata a partir de los iones de plata remanentes en la matriz de CS No obstante la capacidad antimicrobiana de las pel culas mostrada no vari tras el autoclavado Esto puede deberse bien a que el rendimiento de la reacci n durante la neutralizaci n fue muy elevado no quedando iones de plata libre o bien que el resto de los iones de plata no reaccionados se hubieran eliminado con el lavado con agua destilada tras la neutralizaci n Asimismo se
381. o bolsas tejidas etc Con el tiempo los materiales fueron cada vez m s elaborados como es el caso del vidrio metal y papel o ya de pl stico a partir de principios del siglo XX Berger 2002 En la actualidad el desarrollo de la tecnolog a del envasado es fundamental para reducir las p rdidas de alimentos y para garantizar la seguridad alimentaria as como la mejora del comercio mundial de alimentos que es la clave para el desarrollo econ mico de las diferentes econom as FAO 2014 Marsh y Bugusu 2007 La industria del envase es el tercer mayor sector industrial por detr s nicamente de la industria Introducci n alimentaria y petroqu mica De toda la industria mundial del envase el envase alimentario y de bebidas representa el 6996 FAO 2014 La principal funci n del envase es contener y proteger a los alimentos frente a factores ambientales como microorganismos contaminantes qu micos ox geno y vapor de agua extendiendo su vida til y mejorando su seguridad Adem s el envase tiene otras funciones importantes como comodidad y conveniencia el marketing y la comunicaci n e informaci n al consumidor El envase debe facilitar determinados tratamientos industriales el almacenamiento transporte y distribuci n as como el consumo del alimento envases unitarios familiares Brody et aL 2008 Fern ndez lvarez 2000 Marsh y Bugusu 2007 Por otra parte el envase es la imagen de la empresa productora por
382. o el LAE se muestra eficaz en l cteos y derivados Woodcock et al 2009 Soni et al 2010 Soni et al 2012 Serio et al 2012 y Ma et al 2013 probaron la eficacia del LAE en productos l cteos El LAE tambi n se 63 Cap tulo 1 Estado de la cuesti n ha probado en zumo de manzana como coadyuvante para la reducci n del tiempo y la temperatura aplicados en la pasteurizaci n del zumo por pulsos el ctricos Por otra parte el LAE se ha utilizado en la conservaci n de productos del mar Por ejemplo Guo et al 2013 recubrieron gambas cocidas con CS incorporando LAE antes de su congelaci n para el control del crecimiento de Listeria Kang et al 2014 y Soni et al 2014 a adieron LAE sobre salm n ahumado conservado bajo vac o y refrigeraci n como listericida Incluso ya existen patentes en las que se incluye el LAE en una mezcla para la desinfecci n de superficies de frutas verduras o piezas de carne Coughlin 2010 No obstante pocos trabajos se han encontrado en los que el antimicrobiano sea aplicado en pollo Sharma Ates Joseph Nannapaneni et al 2013 Sharma Ates Joseph Soni et al 2013 y Oladunjoye et al 2013 emplearon el LAE en pechugas y carne picada de pollo o pavo para la reducci n del crecimiento de Salmonella El LAE tambi n redujo el crecimiento de Campylobacter jejuni en pechugas de pollo Nair et al 2014 En muchos de estos estudios la concentraci n probada de LAE no disminuy satis
383. o en otros productos como en el pan Passarinho et al 2014 o la uva Dos Santos et al 2012 A raz n de todo ello una alternativa para no tener que emplear cantidades elevadas de agente activo puede ser aplicar estos sistemas de envasado a otros alimentos con menor contenido en prote nas o en los que habitualmente se condimentan con especias Guti rrez et al 2008 indicaron que 300 mg kg de AE de or gano en zanahorias son organol pticamente aceptables Muriel Galet et al 2012 demostraron que la ensalada m nimamente procesada envasada en PP EVOH con 596 de AE de or gano y atm sfera modificada adem s de presentar una mayor vida til y seguridad fue m s aceptada sensorialmente que la ensalada envasada sin AE Du et al 2012 describieron que las pechugas de pollo recubiertas con pel culas de tomate con carvacrol tras su horneado a 204 C 30 minutos fueron aceptadas organol pticamente mientras que fueron rechazadas en el caso de recubrimientos con pel culas de manzana con carvacrol De manera resumida la Tabla 5 2 recoge las propiedades que presenta el CS en los distintos sistemas de envasado desarrollados en la presente Tesis Doctoral En todos los sistemas el CS result ser una excelente matriz para los agentes antimicrobianos ya que tiene la capacidad de hincharse en presencia de agua favoreciendo la difusi n de peque as mol culas a su trav s Por tanto la humedad propia del alimento puede emplearse como est mulo
384. obianos incorporan agentes antimicrobianos que inhiben o retardan el desarrollo de los microorganismos en la superficie del alimento o del envase Appendini y Hotchkiss 2002 En muchos alimentos la mayor carga microbiana se encuentra en la superficie del mismo debido principalmente a la recontaminaci n o contaminaci n cruzada de la superficie de los alimentos ya tratados Este problema se viene solucionando mediante la aplicaci n de sustancias antimicrobianas por inmersi n o pulverizaci n Sin embargo la aplicaci n directa de estos agentes antimicrobianos en la superficie del alimento presenta el inconveniente de poder ser neutralizados r pidamente interaccionar con componentes del alimento l pidos o prote nas o difundir hacia el interior del alimento precisando mayor cantidad del agente El resultado es el encarecimiento del producto adem s de una posible modificaci n sensorial y consecuente rechazado por el consumidor Por lo que los envases activos antimicrobianos pueden ser una alternativa adecuada al empleo de estos tratamientos en superficie ya que pueden liberar de forma sostenida el agente antimicrobiano en la superficie del alimento manteniendo concentraciones adecuadas en ella y durante per odos prolongados de tiempo Appendini y Hotchkiss 2002 Coma 2008 Ouattara et al 2000 Por todo lo mencionado y dado el gran potencial que presenta el envasado activo de alimentos frente al envasado tradicional la presente In
385. obic nature of a compound it can be observed that PG has a higher log P than G Table 4 4 11 also shows the Hildebrand solubility parameter 5 which is defined as the square root of the cohesive energy density of a substance Hildebrand and Scott 1949 This parameter provides a numerical estimate of the degree of miscibility of components of a system it is expected that greater interactions will take place among components that have closer solubility parameters Since the solubility parameter is related to the heat of vaporization this parameter can be theoretically calculated for non volatile compounds by group contribution methods However these methods have limitations when it comes to predicting interactions among molecules in which hydrogen bonding and electrostatic interactions are predominant In this regard Table 4 4 11 also shows that the value of the Hildebrand solubility parameter of G is greater than that of PG 33 5 vs 30 1 MPa so it would be expected to have a greater affinity for water 5 47 9 MPa When PVOH was used as the polymer matrix in the PVOH CD 35G formulation the films had to be conditioned at a higher RH than the corresponding films made with CS 8496 vs 7596 RH respectively to achieve a similar water content 32 4 1 7 The solubility parameter is frequently used to study compatibility in polymer plasticizer polymer drug and polymer aroma systems With regard to the polymer water system studied in
386. obtained from the Spanish Type Culture Collection CECT Valencia Spain the Gram positive bacterium S aureus CECT 86 and the Gram negative bacterium E coli CECT 434 The strains were stored in Mueller Hinton Broth MHB Scharlab Barcelona Spain with 2096 G at 80 C until needed For experimental use the stock cultures were maintained by regular subculture on Tryptone Soy Agar TSA Scharlab Barcelona Spain slants at 4 C and transferred monthly In the first step a loopful of each strain was transferred to 10 mL of Tryptone Soy Broth TSB Scharlab Barcelona Spain and incubated at 37 C overnight to obtain early stationary phase cells 2 6 2 Determination of minimal inhibitory concentration of carvacrol in vapour phase The microatmosphere method was selected to carry out an antimicrobial test in which no direct contact between the device containing the volatile and the agar medium is necessary for the former to exert its activity In this method the volatile compound migrates from the carrier filter paper film into the headspace of the system thus becoming available to contact the growth medium and the microorganism 100 mL of a bacterium suspension containing approximately 10 CFU mL was spread over the surface of 90 mm diameter Petri dishes containing approximately 15 mL of solid culture TSA medium Decreasing quantities of carvacrol were dissolved in dimethyl sulfoxide DMSO and a final volume of 10 mL of the carvacrol
387. ocua L monocytogenes Debaromyces hansenii P commune P roqueforti Saccharomyces cerevisiae L innocua ME hongos P roqueforti ME Staphylococcus spp TVC psicr filos hongos levaduras A niger P roqueforti E coli St aureus Yarrowia lipolytica ME TVC mes filos Lactococcus spp Lactobacillus spp Staphylococcus spp Pseudomonas spp Enterobacteriaceae hongos levaduras Padgett et al 1998 Dawson et al 2002 Theivendran et al 2006 Gadang et al 2008 Pintado et al 2009 2010 Murillo Mart nez et al 2013 Pranoto Rakshit et al 2005 Lungu y Johnson 2005a b Hoffman et al 2001 Janes et al 2002 Padgett et al 2000 Ming et al 1997 Santiago Silva et al 2009 Trinetta et al 2010 Da Silva et al 2013 Resa et al 2013 2014 Yildirim et al 2006 De Oliveira et al 2007 Pires et al 2008 T re et al 2011 T re et al 2008 Ramos Silva et al 2012 Ramos Pereira et al 2012 303 Anexos natamicina c m lico natamicina nisina natamicina c m lico nisina natamicina natamicina prote nas de suero de leche quitosano quitosano medio de cultivo queso Saloio medio de cultivo mel n Hami L monocytogenes Penicillium spp Ps aeruginosa Yarrowia lipolytica A niger P crustosum P commune P roqueforti Alternaria alternata Fusariu
388. odegradable renewable materials chitosan and HP B cyclodextrins and a known natural volatile antimicrobial compound carvacrol was developed and characterized In that report the film demonstrated a great capacity for sorption of carvacrol The kinetics of release in ideal conditions were also measured and the in vitro antimicrobial activity against Staphylococcus aureus and Escherichia coli was tested Higueras et al 2013 Higueras et al 2014 In this work a package containing this device was designed and used as an active package containing fresh chicken fillets The partition of carvacrol among the different components of the food active package environment system was studied and the effect of the active packaging on the chicken microbiota was analysed 2 Materials and methods 2 1 Materials Carvacrol kosher gt 98 glycerol acetic acid and low molecu lar weight chitosan were supplied by Sigma Barcelona Spain Quitosano como matriz biopolim rica para el desarrollo de envases activos antimicrobianos de alimentos En esta Tesis se ha abordado el desarrollo de peliculas renovables y activas de quitosano con agentes antimicrobianos de diversa naturaleza para su aplicaci n en el envasado activo de alimentos Las pel culas de quitosano se obtuvieron mediante la t cnica de extensi n y evaporaci n del solvente y las sustancias bioactivas fueron incorporadas mediante diferentes metodolog as En primer lugar el s l
389. oduced an inhibition of 0 26 to 0 40 log while after 9 days inhibition was 1 13 to 1 77 log for all groups of microorganisms Again lactic acid bacteria were the microorganism least affected by carvacrol with 0 16 and 0 64 log reduction after 3 and 9 days This is in agreement with the literature since it has been observed that lactic acid bacteria were the most resistant to carvacrol while fungi and yeasts were the most sensitive Holley and Patel 2005 S nchez 2011 reported that values equal to or lower than 4 84 log CFU g of the total count of aerobic bacteria and equal to or lower than 3 70 log CFU g for enterobacteria can be accepted for fresh chicken Wehr 1982 considered the acceptability threshold to be 6 70 and 3 log CFU g for aerobic bacteria and enterobacteria respectively Sofos 1994 indicated as common enterobacteria counts between 3 and 4 log CFU cm on chicken 236 Quitosano Carvacrol e Control 5 Small 6 Medium A Large Mesophiles log CFU g of sample a o T T 0 4 time days 8 Control 2 5 Small 5 6 Y Medium 4 Large 2 b 5 E Y da a ad 5 E B 2 3 2 a ol r 0 4 time days 8 gt Control E Small 8 64 7 Medium 4 Large 2 b gt 3 b a 24 S t s i E E z E y 2 i 0 0 4 time days Yeast and fungi log CFU g of sample Psychrophiles log CFU g
390. of sample Enterobacteria log CFU g of sample E o N 6 4 Large b e Control 4 Small y Medium 0 2 4 6 8 time days 9 Control 9 Small 6 7 Medium A Large a time days Control Small Medium 4 Large on c time days Figure 4 4 12 Evolution of chicken fillet microbiota packaged with control and active packages during 9 days of storage Different letters a b c indicate significant differences among samples at a specific storage time Turkey s adjusted analysis of variance P 0 05 Values are expressed as mean and standard deviation n 3 237 Cap tulo 4 Art culo cient fico 6 surface Differences can be explained by the type of food processing In this work small medium and large CS CD Active films maintained mesophilic bacteria counts within the limit suggested by other authors during storage time Rokka et al 2004 Wehr 1982 Large samples also limited the growth of enterobacteria counts of which remained between log 2 and log 3 during the 9 day storage The small size CS CD Active film did not produce an efficient microbial inhibition even though the total amount of carvacrol present in that device was slightly greater than the observed MIC 1 mg of carvacrol in headspace tests Higueras et al 2015 One important reason is that chicken tends to sorb carvacrol from the heads
391. oli St aureus S Pranoto Rakshit cultivo Typhimurium L et al 2005 monocytogenes B cereus sorbato de potasio quitosano bizcocho ME hongos Sangsuwan et al pan de ajo 2014 Sangsuwan et al 2012 sorbato de potasio quitosano medio de Lactobacillus spp V sconez et al quitosano almid n de cultivo Zygosaccharomyces bailii 2009 tapioca salm n en ME TVC mes filos y rodajas psicr filos sorbato de potasio ze na queso St aureus Torres 1984 EXE xido de cobre celulosa zumo de pi a ME hongos levaduras Llorens et al zumo de mel n 2012 cobre nano Piel de sapo pelado cobre celulosa quitosano medio de E coli Mary et al 2009 cobre nano cultivo cobre nano PLA medio de Ps fluorescens Ps Longano et al cultivo putida 2012 cobre nano quitosano medio de St aureus S C rdenas et al cultivo Typhimurium 2009 cobre nano quitosano medio de E coli S Choleraesuis S Qi et al 2004 cultivo Typhimurium St aureus di xido de titanio celulosa medio de St aureus Daoud et al nano cultivo 2005 di xido de titanio gelatina bovina medio de St aureus E coli Nassiri y Nafchi nano cultivo 2013 di xido de titanio prote nas de suero de medio de E coli Kadam et al nano leche cultivo 2014 ze na di xido de titanio quitosano medio de S Typhimurium E coli St D az Visurraga et nano cultivo aureus al 2010 plata nano alginato de sodio medio de St aureus E coli Fayaz et al
392. olina et al 2010 Ramos Garc a et al 2012 Perdones et al 2012 Aloui et al 2014 Benavides et al 2012 Ehivet et al 2011 Pelissari et al 2009 Min y Oh 2009 Wu et al 2014 Iturriaga et al 2012 Jouki Yazdi Mortazavi y Koocheki 2014 Jouki Yazdi Mortazavi Koocheki et al 2014 307 Anexos AE or gano AE or gano AE or gano AE or gano AE pimiento AE or gano pimiento AE or gano AE tomillo AE tomillo romero AE or gano AE or gano AE or gano AE romero AE romero AE tomillo AE tomillo AE tomillo AE tomillo AE tomillo cinamaldeh do ED canela ED canela ED clavo ED eucalipto ED ginseng 308 piel de patata prote nas de suero de leche prote nas de suero de leche prote na de suero leche caseinato de calcio prote nas de soja quitosano quitosano quitosano muc lago de semilla de membrillo quitosano quitosano quitosano quitosano quitosano prote nas de soja prote nas de suero de leche PVOH quitosano almid n de tapioca almid n de tapioca alginato de calcio salm n ahumado carne de vacuno medio de cultivo carne de vacuno fileteada hamburguesa de vacuno mortadela pechugas de pollo uva medio de cultivo medio de cultivo medio de cultivo medio de cultivo kebab de pollo hongo shiitake gambas cocidas irradiadas a bajas dosis banana p
393. olymer Bulletin 62 511 524 Carlin F Gontard N Reich M Nguyen The C 2001 Utilization of zein coating and sorbic acid to reduce Listeria monocytogenes growth on cooked sweet corn Journal of Food Science 66 1385 1389 Coma V Sebti L Pardon P Deschamps A Pichavant F H 2001 Antimicrobial edible packaging based on cellulosic ethers fatty acids and nisin incorporation to inhibit Listeria innocua and Staphylococcus aureus Journal of Food Protection 64 470 475 Cong F S Zhang Y G Dong W Y 2007 Use of surface coatings with natamycin to improve the storability of Hami melon at ambient temperature Postharvest Biology and Technology 46 71 75 Cooksey K 2005 Effectiveness of antimicrobial food packaging materials Food Additives and Contaminants 22 980 987 313 Anexos Corrales M Han J H Tauscher B 2009 Antimicrobial properties of grape seed extracts and their effectiveness after incorporation into pea starch films International Journal of Food Science and Technology 44 425 433 Cutter C N Siragusa G R 1996 Reduction of Brochothrix thermosphacta on beef surfaces following immobilization of nisin in calcium alginate gels Letters in Applied Microbiology 23 9 12 Cutter C N Siragusa G R 1997 Growth of Brochothrix thermosphacta in ground beef following treatments with nisin in calcium alginate gels Food Microbiology 14 425 430 Cha D S Cooksey K Chinnan M S Park
394. olymers Polymer CD interactions and the structural rigidity of HP BCDs could create steric hindrance and decrease segmental mobility restricting diffusivity of the permeant through the CS matrix Moreover as can be seen in Table 4 4 2 the addition of HP BCDs also reduces the water uptake of the matrices Thus it might be expected that a reduction in diffusion and in the solubility coefficient would result in a decrease in the permeability values of the biocomposites 186 Quitosano Carvacrol Finally increasing the humidity gradient yielded higher permeability values for the plain CS and the CS CD films with or without G This effect can be correlated to the known water plasticization of hydrocolloids Caner et al 1998 Wiles et al 2000 As humidity increases water uptake also increases and so does the plasticization of biopolymer chains The effects of HP BCDs and G on the CS films were also affected by the humidity gradient The barrier improvement caused by the addition of HP BCDs is more noticeable at the greater gradient 7596 RH as could be derived from the lower water content observed in the composite samples in comparison with the content in plain CS films On the other hand the plasticizing effect of G was less perceptible in the WVP values measured at 7596 RH since at high humidity the water uptake plasticizes the polymer film mimicking the G effect 3 5 2 Oxygen permeability Table 4 4 3 shows the oxygen permeabil
395. om the water and drying it The increase was taken as the dimensional stability of the film in buffered solution The experiment was performed in triplicate 2 3 4 Contact angle CA After the films had been neutralized CS films modified CScin films and subjected to different treatments they were left under pressure between two glass sheets for 48 h to increase film flatness and then stored for 48 h in a desiccator with P Os The CA was measured using an OCA 15EC goniometer DataPhysics Instruments GmbH Filderstadt Germany A 2 pL water droplet was dispensed onto the sample surface and the drop image was recorded for 2 min The CA at 60 s was estimated by using the SCA20 embedded software module The experiment was performed in triplicate 2 3 5 Attenuated total reflectance Fourier transform infrared spectroscopy ATR FTIR The films were analysed by ATR FTIR Dry samples were placed in a Golden Gate single reflection diamond ATR accessory Teknokroma Barcelona Spain and the spectra were recorded with a Bruker Tensor 27 FTIR spectrometer Bruker Espa ola S A Barcelona Spain The resolution was 4 cm in the range of 4000 to 600 cm and 128 scans were recorded per test Results were recorded in triplicate and analysed with the OPUS v 2 06 software 2 4 Imino bond hydrolysis and cinnamaldehyde release After each temperature time treatment the amount of cinnamaldehyde released in MHB owing to hydrolysis of the CS cinnam
396. omers carvacrol and guaiacol was lower in the PVOH CD 35G films than in the films with CS as the polymer matrix Owing to its high hydrophilicity G has a greater capacity to plasticize more polar CS compared with PVOH which might explain the lower sorption values obtained for phenolic monoterpenes with the exception of isoeugenol Moreover PVOH has a certain degree of crystallinity which restricts sorption Similar sorption values were found for isoeugenol and non phenolic monoterpenes in the CS CD 35G and PVOH CD 35G films the less hydrophilic nature of PVOH tended to increase values of 4 4 and 2396 for films plasticized with 20 and 3596 G respectively whereas carvacrol sorption in CS films without CD was lower than 196 irrespective of their water and G contents Higueras et al 2013 261 Cap tulo 4 Art culo cient fico 7 e o EXA Sorption equilibrium Eq moisture content o o o mm o 1 NI o e o 1 o o 80 4 S o 60 4 wo o 40 4 Sorption equilibrium of carvacrol 96 dry film nN a o o vay Ap 95 1081009 aiNysiow unugiinb3 20 4 o 0 75 100 Relative humidity Figure 4 4 15 Sorption equilibrium of carvacrol and equilibrium moisture content of PVOH CD 35G films conditioned at different RH 3 4 Antimicrobial capacity of monoterpenes in vapour phase The minimum inhibitory dose of monoterpenes against S aureus and E coli evaluated in vap
397. ompared with the films in diluted MHB might be due to excess proteins in the culture 116 Quitosano Nanopart culas de plata Table 4 2 2 Antimicrobial activity of non autoclaved and autoclaved AUTO CS films neutralized at 37 C against S aureus and E coli in Mueller Hinton Broth MHB and diluted MHB 1 125 Bacterial count Log CFU ml Ag AUTO MHB MHB 1 125 AUTO MHB 1 125 Control 8 34 0 37 8 34 0 37 745 0 23 745 0 23 01 7 65 0 52 7 49 0 21 4 78 0 69 4 97 0 03 0 2 5 74 0 40 6 52 0 17 4 50 0 17 4 74 0 10 Ssaureus 0 5 6 44 0 24 6 32 0 06 4 25 0 26 4 60 0 12 1 0 5 79 0 40 6 35 0 11 4 29 0 13 4 53 0 29 1 5 6 25 0 19 6 20 0 20 4 28 0 21 4 31 0 45 Control 8 16 0 14 8 16 0 14 7 31 0 54 7 31 0 54 01 7 28 0 09 742 0 14 4 90 0 00 5 10 0 25 02 6 78 0 02 6 29 0 03 4 32 0 32 4 28 0 44 Ecol 0 5 6 26 0 05 5 33 0 06 3 57 0 30 4 12 0 30 1 0 6 44 0 08 5 52 0 04 3 79 0 70 3 89 0 05 15 6 02 0 19 6 18 0 36 3 89 0 29 4 08 0 192 ac Values within a column followed by a different lower case letter are significantly different from each other comparing different amounts of silver in the films against S aureus or E coli Tukey s adjusted analysis of variance P 0 05 117 Cap tulo 2 Art culo cient fico 2 media chelating the released silver ions This cou
398. omparing the inhibition effectivity of a given sample against the different microorganisms 84 Quitosano LAE against coliforms and hydrogen sulfide producing bacteria where CS 596LAE film halved growth inhibition caused by CS alone As regards lactic acid bacteria CS films originated 0 93 0 2 log reduction at all tested times while films with 596 LAE evidenced a log reduction range between 1 78 at tz and 3 44 at tg In the case of fungi and yeasts obtained data were not conclusive Results summarize that CS films had antimicrobial activity in the range of 0 47 2 96 log reduction dependent of time and bacterial group studied On the other hand incorporation of LAE in the matrix CS 596LAE film increased antimicrobial activity to 1 78 5 81 log reduction Therefore the composite CS LAE presents excellent potential for packaging of fresh poultry products 4 Conclusions This work provides an example of active food packaging in which 1 5 or 1096 LAE was incorporated in a CS matrix Films were found continuous flexible and transparent and able to release the antimicrobial agent when exposed to highly humid media When films were immersed in water LAE release follows a Fickian process reaching full release within the first 15 h LAE diffusion coefficient in the CS matrix was estimated in the 10 m s range decreasing with temperature and with LAE concentration The addition of LAE to CS significantly increased its antimicrobial acti
399. on chitosan Carbohydrate Polymers 54 131 136 Hirano S Nagamura K Zhang M Kim S K Chung B G Yoshikawa M Midorikawa T 1999 Chitosan staple fibers and their chemical modification with some aldehydes Carbohydrate Polymers 38 293 298 Hirano S Yamaguchi R Matsuda N Miura O Kondo Y 1977 Chitosan aldehyde gel a novel polysaccharide gel produced from chitosan and aldehydes Agricultural and Biological Chemistry 41 1547 1548 Hongliang W Cuncai M Huijuan C Jun Y Kai Y Lei J 2010 Study on synthesis of Schiff base from chitosan and furfural Guangdong Chemical Industry 11 008 Jin X X Wang J T Bai J 2009 Synthesis and antimicrobial activity of the Schiff base from chitosan and citral Carbohydrate Research 344 825 829 Jin XX Wang J T Bai J 2010 Synthesis of Schiff base from chitosan and cinnamaldehyde and its antimicrobial activity Journal of Chemical Engineering of Chinese Universities 4 018 Kumar S Kumari M Dutta P K Koh J 2014 Chitosan biopolymer Schiff base preparation characterization optical and antibacterial activity International Journal of Polymeric Materials and Polymeric Biomaterials 63 173 177 Kurita K Mori S Nishiyama Y Harata M 2002 N Alkylation of chitin and some characteristics of the novel derivatives Polymer Bulletin 48 159 166 Le T C Dupuis C 2009 Amine based and imine based polymers uses and preparation thereof
400. on in 1 125 diluted MHB for 30 days Although the experimental conditions were different both experiments show that films are capable of releasing silver ions after immersion in liquid medium and maintain their effectiveness over time It is noteworthy that films with a greater silver concentration slightly increased their antimicrobial activity after 30 days of immersion in water compared with 0 296 silver films This result might indicate that although initially the films neutralized at 37 C tested had similar antimicrobial activity the films with a greater number of nanoparticles may have a greater number of silver ions available to exert their antimicrobial activity over time These silver ions would be released after oxidation of silver from the surface of the nanoparticles over time In this work a methodology using compounds of low toxicity has been developed to obtain silver based nanoparticles embedded in a CS film Silver nitrate was used as a precursor for the synthesis of nanoparticles and sodium hydroxide accelerated the reduction rate of silver ions and the formation of nanoparticles during the neutralization step However the coexistence of elementary silver and silver oxide is expected owing to the presence of sodium hydroxide TEM images showed the formation of spherical nanoparticles in films incorporating 0 1 and 0 296 of silver and neutralized at 37 C Films with 0 5 to 1 596 silver presented two populations of nanoparticles
401. on los poli steres polihidroxialcanoatos poli 3 hidroxibutirato poli 3 hidroxivalerato o los polisac ridos celulosa bacteriana curdlan goma xantana o pululano As los biopol meros suelen ser biodegradables mientras que todos los materiales biodegradables no necesariamente tienen que ser biomateriales Weber et al 2002 el polialcohol vin lico es biodegradable pero no proviene de la biomasa mientras que la poliamida PA 9 y PA 11 provienen del cido oleico y el aceite de ricino respectivamente pero no son biodegradables Siracusa et aL 2008 Si bien los biopol meros presentan numerosas ventajas tambi n tienen problemas como su rendimiento procesamiento y coste sobre todo los pol meros extra dos directamente de la biomasa Alonso et al 2011 Petersen et al 1999 El coste medio de obtenci n actual de los biopol meros es de 2 5 kg frente a los 1 2 kg de los petropol meros aproximadamente Song et al 2009 La capacidad de producci n mundial de materiales basados en biopol meros ascendi a m s de 1 5 millones de toneladas en 2012 y se prev que aumente a m s de 6 2 millones de toneladas para el a o 2017 EuropeanBioplastics 2013 El incremento en la producci n de estos materiales facilitar la reducci n de su precio Alonso et al 2011 Petersen et aL 1999 Cuando se piensa en el empleo de biopol meros para el envasado de alimentos hay que tener presente que algunos de ellos presentan dive
402. oncentraci n en las que se obtuvo un mayor rendimiento de 56 Justificaci n y esquema de tesis la reacci n cuantificado mediante an lisis elemental La evidencia del anclaje se confirm mediante espectroscop a de infrarrojos por transformada de Fourier con reflexi n total atenuada ATR FTIR Se procedi a la caracterizaci n de las pel culas en relaci n a algunas de sus propiedades funcionales como sorci n de agua ngulo de contacto y propiedades pticas Se estudi el efecto de la temperatura simulando diferentes tratamientos t rmicos de conservaci n en la liberaci n del vol til en medio l quido Se determin la capacidad antimicrobiana de las pel culas en ensayos in vitro en medio l quido frente a dos modelos bacterianos Escherichia coli y Staphylococcus aureus Finalmente se valid su aplicaci n como sistema activo en leche pasteurizada inoculada con Listeria monocytogenes durante su almacenamiento en condiciones de refrigeraci n y su aceptaci n sensorial El cap tulo 4 denominado Desarrollo de pel culas de quitosano con hidroxipropil B ciclodextrinas y otros agentes coadyuvantes con adaptable capacidad de sorci n y liberaci n de compuestos vol tiles antimicrobianos y su aplicaci n en el envasado activo de alimentos consta de cuatro art culos cient ficos En este cap tulo se estudia c mo la presencia de CD junto con plastificantes y humedad pueden modular la capacidad de sorci n de vol tiles antimic
403. oncepto de envase el Introducci n denominado envase activo que adem s de desarrollar las funciones de un envase tradicional tiene la capacidad de interaccionar con el producto y o con su entorno para mejorar uno o m s aspectos de su calidad y o seguridad Catal y Gavara 2001 Rooney 1995 En el envasado activo el sistema alimento envase entorno act a de forma coordinada para prolongar la vida til de los alimentos mejorar la aceptabilidad organol ptica la calidad y seguridad alimentaria y o facilitar su procesado y consumo Catal y Gavara 2001 Fern ndez lvarez 2000 El envase activo est dise ado para incorporar intencionadamente agentes activos que pueden ser liberados en el alimento envasado o en su entorno o pueden absorber sustancias del alimento o de su entorno CE 2004 2009 Las interacciones beneficiosas creadas entre el alimento y el envase pueden basarse en la regulaci n del contenido en gases ox geno di xido de carbono etileno etc en el control de la humedad aditivos antivaho absorbentes etc en la liberaci n de aromas saborizantes y o la retenci n de compuestos no deseados naringina y limonina mercaptanos en la liberaci n de compuestos con propiedades antioxidantes y o antimicrobianas Brody et al 2008 Respecto a los aspectos legislativos referentes al envase activo cabe decir que los envases deben cumplir con la normativa referida a todos aquellos materiales que vaya
404. ones de la nanociencia y de las nanotecnolog as en los alimentos y en la cadena alimentaria Sin embargo actualmente a n no se disponen de los resultados de esa evaluaci n para la regulaci n de la introducci n de estos materiales en el mercado Frejo et al 2011 El Reglamento 1935 2004 y el posterior 10 2011 espec fico para materiales en contacto con alimentos y de envasado no incluye ninguna indicaci n particular para el uso de los compuestos de plata en envases y establece la prohibici n de incorporar nanopart culas a materiales pl sticos que est n en contacto con alimentos debido a la falta de informaci n existente El Reglamento 450 2009 sobre envases activos e inteligentes se ala que el elemento activo tiene que ser identificado con la informaci n adecuada sobre los usos permitidos y su cantidad m xima Y el Reglamento 1169 2011 sobre la informaci n alimentaria facilitada al consumidor incluye la definici n de nanomaterial artificial y obliga a etiquetar todos los ingredientes presentes en forma de nanomateriales artificiales Adem s se ala que los nanomateriales artificiales deber n indicarse claramente en la lista de ingredientes seguidos de la palabra nano entre par ntesis valos et aL 2013 La EPA 1992 y la OMS 2003 2008 sugieren que los niveles de plata en agua potable se sit en por debajo de 0 1 mg L para evitar el problema de salud denominado argiria irreversible coloraci n azul gris cea de piel
405. onocytogenes ME TVC y anaerobios A niger Kocuria rhizophila L monocytogenes Lb plantarum L monocytogenes E coli O157 H7 S Enteriditis L monocytogenes E coli O157 H7 L monocytogenes E coli O157 H7 S Typhimurium L monocytogenes E coli O157 H7 S Gaminara Nguyen et al 2008 Martins et al 2010 Min et al 2010 Gill y Holley 2000 Ku y Bin Song 2007 Li Kennedy et al 2006 Li Peng et al 2006 McCormick et al 2005 Coma et al 2001 Sebti y Coma 2002 Sebti et al 2002 Franklin et al 2004 Neetoo y Mahomoodally 2014 Sebti et al 2007 Cooksey 2005 Jin et al 2009 Liu et al 2007 Jin 2010 Jin y Zhang 2008 Liu et al 2010 Sivarooban et al 2008 Eswaranandam et al 2004 Anexos nisina nisina EDTA nisina nisina c l urico nisina nisina ED semilla de uva nisina ED t verde nisina c m lico EDTA ED semilla de uva nisina c m lico nisina natamicina nisina c m lico natamicina nisina nisina glucosa oxidasa nisina nisina nisina diacetato de sodio nisina nisina EDTA nisina c l urico nisina nisina propionato de calcio nisina c l urico pediocina pediocina sakacina A natamicina natamicina nisina natamicina natamicina natamicina natamicina natamicina natamicina c l ctico natamicina quitooligo
406. ood Protection 69 2364 2369 Oussalah M Caillet S Salmieri S Saucier L Lacroix M 2007 Antimicrobial effects of alginate based films containing essential oils on Listeria monocytogenes and Salmonella Typhimurium present in bologna and ham Journal of Food Protection 70 901 908 Padgett T Han LY Dawson P L 1998 Incorporation of food grade antimicrobial compounds into biodegradable packaging films Journal of Food Protection 61 1330 1335 323 Anexos Padgett T Han LY Dawson P L 2000 Effect of lauric acid addition on the antimicrobial efficacy and water permeability of corn zein films containing nisin Journal of Food Processing and Preservation 24 423 432 Park S I Daeschel M A Zhao Y 2004 Functional properties of antimicrobial lysozyme chitosan composite films Journal of Food Science 69 M215 M221 Pastor C S nchez Gonzalez L Marcilla A Chiralt A Ch fer M Gonz lez Mart nez C 2011 Quality and safety of table grapes coated with hydroxypropylmethylcellulose edible coatings containing propolis extract Postharvest Biology and Technology 60 64 70 Pelissari F M Grossmann M V E Yamashita F Pineda E A G 2009 Antimicrobial mechanical and barrier properties of cassava starch chitosan films incorporated with oregano essential oil Journal of Agricultural and Food Chemistry 57 7499 7504 Peng Y Li Y F 2014 Combined effects of two kinds of essential oils on p
407. ood Science 73 M127 M134 Jin T Z Gurtler J B Li S Q 2013 Development of antimicrobial coatings for improving the microbiological safety and quality of shell eggs Journal of Food Protection 76 779 785 Jouki M Mortazavi S A Yazdi F T Koocheki A 2014 Characterization of antioxidant antibacterial quince seed mucilage films containing thyme essential oil Carbohydrate Polymers 99 537 546 Jouki M Yazdi F T Mortazavi S A Koocheki A 2014 Quince seed mucilage films incorporated with oregano essential oil physical thermal barrier antioxidant and antibacterial properties Food Hydrocolloids 36 9 19 Jouki M Yazdi F T Mortazavi S A Koocheki A Khazaei N 2014 Effect of quince seed mucilage edible films incorporated with oregano or thyme essential oil on shelf life extension of refrigerated rainbow trout fillets International Journal of Food Microbiology 174 88 97 Kadam D M Wang C Wang S Grewell D A Lamsal B P Yu C 2014 Microstructure and antimicrobial functionality of nano enhanced protein based biopolymers Transactions of the ASABE 57 1141 Kavoosi G Dadfar S M M Purfard A M Mehrabi R 2013 Antioxidant and antibacterial properties of gelatin films incorporated with carvacrol Journal of Food Safety 33 423 432 Kechichian V Ditchfield C Veiga Santos P Tadini C C 2010 Natural antimicrobial ingredients incorporated in biodegradable films based on cassava s
408. opol meros para el desarrollo de pl sticos Los biopol meros provienen de recursos renovables y la mayor parte son biodegradables es decir que son susceptibles de ser convertidos en agua di xido de carbono metano compuestos inorg nicos o biomasa incluso emplearse para enriquecer los suelos como compost Siracusa et aL 2008 Song et al 2009 Por otro lado la obtenci n de estos materiales a partir de los excedentes y desechos industriales agr colas o de la pesca podr a abaratar los costes de producci n y facilitar la eliminaci n de residuos procedentes de la industria alimentaria Los pol meros elaborados a partir de biomasa o biopol meros pueden dividirse en tres categor as bas ndose en el m todo de producci n Petersen et al 1999 Srinivasa y Tharanathan 2007 Introducci n 1 Pol meros extra dos directamente a partir de materiales naturales modificados o no como son polisac ridos almid n alginato carragenato celulosa goma guar pectinas quitosano prote nas alb mina case na col geno gelatina gluten de trigo prote na de soja prote nas de suero de leche ze na o bien l pidos cidos grasos acilgliceroles ceras Son de origen fundamentalmente vegetal aunque tambi n marino 2 Pol meros sintetizados qu micamente a partir de mon meros de biomasa como es el poli cido l ctico PLA 3 Pol meros producidos por microorganismos o bacterias modificadas gen ticamente como s
409. or propylene glycol PG were prepared by casting and conditioned at different relative humidities RH to achieve a similar water content Sorption properties of the films for various monoterpene compounds with phenolic or non phenolic linear or cyclohexyl alcohol structures were studied after their immersion in the volatile liquids In general the films presented a considerable capacity to retain monophenolic compounds with values ranging from 43096 for m cumenol to 106 for guaiacol depending on the film formulation these values were two orders of magnitude higher than those of compounds without the phenol group The affinity for monophenolic compounds decreased in films plasticized with hydrophilic PG whereas no changes were Observed in the retention of non phenolic monoterpenes Replacement of CS with polyvinyl alcohol PVOH polymer considerably decreased the retention of monophenolic compounds with the exception of isoeugenol Finally the antimicrobial activity of monoterpenes and films loaded with them was evaluated in vitro by the microatmosphere test against E coli and S aureus The present study shows that HP BCD and the plasticization level achieved by hydrophilic films can be used to regulate loading capacity and sorption selectivity of naturally occurring antimicrobial compounds Keywords Chitosan polyvinyl alcohol hydroxypropyl B cyclodextrins films sorption selectivity antimicrobial activity 246 Quitosano Carvacro
410. ora Igualmente gracias a mis amigos y compafieros pasados presentes y espero que futuros Guada debo agradecerte que me lo ensefiaras todo en mis inicios en el IATA Josep Pasqual mi gratitud va m s all de tus ensefianzas sobre las celdas o la m quina de ensayo universal gracias por tu tiempo de xarrades y confesiones Qu no se te olvide que te mereces lo mejor Gracias tambi n a Carol Poli Jos Antonio Vir Mari Pau Jader Joaqu n Paula Bel n Ana Carolina V Pili Nerea Irene Josep Llu s Ivan Agradecimientos Sonia Alex Marisa Carolina Rosa Luz Felipe Francisca Joan Javi Irene D Mabooh a los vecinos Marta V Marta C Silvia Ren Deni Vicenta Maite Antonio y en general a todos los de la 2 planta Clara Loles Antonio Consuelo Alex Sandra ngela Teresa Ana Susana Inma y sobre todo a Laura L Un gran beso a Alba y a todo el personal de Servicios y a Hortensia Gracias a todos por compartir vuestro tiempo dentro y fuera del IATA comidas y picaetas conmigo y darme nimos cada vez que me veis Aprovecho este fin de etapa para agradeceros a vosotros Amparo y scar referencia y apoyo en mi vida Siempre me hab is demostrado que con trabajo y buen coraz n se puede conseguir cualquier reto Alg n d a me gustar a poder llegar a ser como vosotros Y por supuesto gracias a vosotros Vicen y Diego sois la motivaci n en cada momento para seguir hacia delante Gracias por es
411. orming solution in a 1 1 proportion w w with respect to CS or PVOH and stirred at 37 C until complete dissolution Then G or PG plasticizer was added at the corresponding 96 g plasticizer 100 g dry matter polymer CD to the film forming solution Films were formed by casting on PS plates and dried at 37 C and 4096 relative humidity RH for 36 h Film thickness was measured using a digital micrometer Mitutoyo Manufacturing Co Ltd Tokyo Japan with a sensitivity of 1 um Five measurements were taken for each sample one at the sample centre and four around the perimeter The average thickness of the films was 55 5 um With the aim of comparing sorption properties of films obtained using different plasticizers and polymers but having a similar water content prior to immersion in various volatile liquids various film samples 0 4 0 5 g 550 mm in diameter and 55 5 um in thickness were placed on aluminium plates and stored at 23 1 C in glass desiccators containing saturated salt solutions to achieve the desired water content ASTM 2007 RH values achieved in each desiccator were confirmed by direct RH measurements with hygrometers Hygrodynamics Newport Scientific Inc Jessup MD USA After reaching moisture sorption equilibrium the water content of the films was measured For this purpose the films were weighed and dried with phosphorus pentoxide Sigma Barcelona Spain until constant weight was reached The tests were done
412. orporen un contenido del 3096 de agua La capacidad de sorci n de las pel culas compuestas de PVOH fue menor que las pel culas compuestas de CS para todos los monoterpenos llegando a ser incluso la mitad en el caso de vol tiles con estructura fen lica Por su parte el PG se caracteriza por ser algo m s apolar que el G Por ello hubo que acondicionar las pel culas de CS con CD que incorporan 3596 de PG en lugar de 35 de G al 90 de RH en lugar de al 75 de RH La sustituci n del G por el PG en la formulaci n produjo una disminuci n en la capacidad de retenci n de casi todos los vol tiles por parte las pel culas de CS con CD a pesar de la mayor compatibilidad de los vol tiles con el PG que la existente con el G Por lo que parece ser que la plastificaci n de la matriz es un factor m s preponderante que la compatibilidad entre los componentes del sistema As un mayor grado de plastificaci n implica una mayor retenci n de vol tiles hasta alcanzar un nivel de plastificaci n por el plastificante y el agua que provoquen un entorno demasiado hidr filo como se ve a en el caso de pel culas compuestas con el 5096 de G o que las pel culas pierdan su integridad f sica o maniobrabilidad como en el caso de pel culas de CS con CD con 3596 de G acondicionadas al 9096 de RH La capacidad antibacteriana de las pel culas compuestas con los distintos vol tiles dependi de la cantidad de agente activo retenido por las matrices y su p
413. os como se muestra en el Anexo A El cinamaldeh do fue reconocido como sustancia segura n de la JECFA 656 por la FAO OMS 20013 La dosis sin efecto adverso observable se ha establecido en 620 mg kg de peso corporal y d a mientas que la estimaci n de su ingesta como aromatizante es de 42 pg kg de peso corporal por lo que no existe preocupaci n en cuanto a la seguridad en los niveles actuales de ingesti n como aditivo alimentario FAO OMS 20013 El cinamaldeh do se metaboliza en el organismo a cido cin mico y se elimina por v a urinaria como glicina glucuronato del cido cin mico o minoritariamente como conjugado del glutati n para formar derivados del cido mercapt rico Bickers et al 2005 La FDA 2014c considera que el cinamaldeh do es sustancia GRAS para su uso como sustancia aromatizante sint tica y adyuvante 21CFR182 60 y ha sido incluida en la lista de sustancias aromatizantes UE 2012 36 Introducci n 1 6 Referencias bibliogr ficas Agull E Rodr guez M S Ramos V Albertengo L 2003 Present and future role of chitin and chitosan in food Macromolecular Bioscience 3 521 530 Alonso R Figueroa P Fuci os C Pastrana L L pez Vilari o J M Sartal A Teller a J A 2011 Bioforo tendencias y soluciones biotecnol gicas para la innovaci n y el empendimiento en Galicia Biopl sticos envases y embalajes activos e inteligentes Informe de conclusiones de la mesa de exper
414. osterior liberaci n y de la capacidad antimicrobiana y volatilidad de los componentes presentes en el AE La capacidad antimicrobiana in vitro de los monoterpenos probados en fase vapor fue carvacrol gt o cumenol m cumenol gt dehidrocarveol gt nerol carveol gt isoeugenol gt mirtenol gt isopulegol gt guayacol As todas las matrices polim ricas con CD que incorporaron cumenoles y carvacrol produjeron una elevada inhibici n bacteriana en las pruebas de capacidad antimicrobiana en fase vapor debido a la mayor cantidad de agente antibacteriano en las pel culas como consecuencia de su elevada capacidad de sorci n En el caso del guayacol la inhibici n producida fue menor que en los casos anteriores debido en parte a la menor cantidad retenida de este monoterpeno fen lico que no llega a alcanzar la cantidad m nima inhibitoria en fase vapor Las pel culas con isoeugenol y los monoterpenos c clicos y ac clicos mirtenol nerol carveol y dehidrocarveol presentaron una actividad antimicrobiana intermedia dependiendo de la pel cula de la que se trate y por tanto de su capacidad de retenci n de los vol tiles y de la capacidad antimicrobiana del 284 Discusi n general compuesto activo Mientras que todas las pel culas compuestas con isopulegol no presentaron capacidad antibacteriana ya que la cantidad retenida en las matrices compuestas fue menor a la cantidad m nima inhibitoria en fase vapor De este modo es pos
415. otros microorganismos menos ox geno dependiente Las pel culas de 4 8 y 24 cm a los 6 y 9 d as de exposici n presentaron un intervalo de reducciones logar tmicas de bacterias psicr filas y Pseudomonas de 0 62 a 1 16 Respecto a las Enterobacteriaceae nicamente las pel culas de 24 cm mantuvieron un recuento microbiano en torno a 3 log Debido a la mayor resistencia de las bacterias cido l cticas a la acci n antimicrobiana del carvacrol la reducci n de stas fue menor en todos los casos Solamente las pel culas de 24 cm tras 9 d as presentaron reducciones logar tmicas mayores de 0 5 Por su parte a causa de la mayor sensibilidad de las levaduras y hongos al carvacrol las peliculas de CS con carvacrol de tama o de 4 8 y 24 cm presentaron 0 78 y 1 77 reducciones logar tmicas tras 9 d as de envasado Globalmente se obtuvo una actividad antimicrobiana menor a la esperada ya que la cantidad de carvacrol presente en todas las pel culas de CS probadas exced a la cantidad m nima inhibitoria de carvacrol determinada en fase vapor en la pruebas in vitro Una causa puede ser que la mayor parte del carvacrol de las pel culas acab siendo sorbido por el pollo disminuyendo la concentraci n de carvacrol en el espacio de cabeza por debajo de esta cantidad m nima inhibitoria De manera que la cantidad de carvacrol libre y disponible en la superficie del pollo pudo verse disminuida por la interacci n del carvacrol con las prote
416. ount of G and the RH at which they were conditioned prior to being loaded with carvacrol 201 Cap tulo 4 Art culo cient fico 5 solubilisation in 0 596 w w acetic acid Y filtration Y 1 5 w w chitosan solution HP BCD 0 20 or 35 G stirring 1500 rpm 37 C y film forming solution casting 40 0 1 5 C 20 9 RH 36h v CS CD G films cut 2 550 mm thickness selection 55 5 um conditioning 0 53 or 75 RH 25 1 C CS CD G RH films Figure 4 4 3 Flowchart of film preparation To study how the amount of HP BCDs blended with CS affects sorption of carvacrol by the resulting films specimens were prepared at 1 2 1 0 5 and 1 0 25 weight ratio and compared with 1 1 CS CD proportion film samples of 550 mm in diameter and 55 5 mm in thickness were plasticized with 3596 G and conditioned at 7596 RH at room temperature as described above prior to immersion in carvacrol CS film samples of similar size and thickness as mentioned above and plasticized with 35 G were also mixed with MDs in a 1 1 w w proportion and conditioned at three different humidities as described above and carvacrol sorption properties studied 2 3 Optical properties of the films before and after immersion in carvacrol The colour of the films before and after immersion in carvacrol was measured with a CR 3
417. our phase is given in Table 4 4 13 Carvacrol showed the greatest antimicrobial activity against both microorganisms with 0 9 mg of carvacrol being needed to produce clear inhibition of growth on agar its sorption ability for more hydrophobic compounds as compared with CS In fact Figure 4 4 14 shows that the affinity between the PVOH films without CD and carvacrol was greater than for CS films reaching carvacrol sorption plates followed by ortho and meta cumenol However it was necessary to use more than 1 mg of these compounds to see a clear effect and the inhibition halo created by o cumenol was larger 29 vs 19 mm The greater vapour pressure of o cumenol compared with that of m cumenol Table 4 4 11 might explain the higher activity in vapour phase the greater antimicrobial activity of ortho and meta isomers compared with para isomers of some drugs is reported in the literature Biava et al 1999 Phenolic compounds present in essential oils have been found to be good inhibitors of target bacteria Kuhn et al 2006 Ultee et al 2002 stated that the capacity of the hydroxyl group in phenol to release its proton and the presence of a delocalized electron system corresponding to the benzene ring which to some extent stabilizes the phenoxide ion formed is responsible for the high antimicrobial activity of this molecule These features give carvacrol greater antimicrobial activity compared with other 262 Quitosano Carvacrol
418. our phase of films submitted to desorption at 25 C and 43 RH was monitored for 20 days every 2 days for 2 weeks and then after 8 days Figure 4 4 9 shows the effect of time on the amount of carvacrol remaining in the film and the inhibition zone created against E coli and S aureus As expected the inhibition halo experienced a reduction as the carvacrol content in the film decreased over time The initial amount of carvacrol in the film was 146 mg producing total inhibition of bacterial growth A rapid reduction of carvacrol in the films was observed during the first week of storage giving rise to almost total growth inhibition on the agar plate Thus the amount of carvacrol in the film was reduced by 93 after 8 days of storage and being the inhibition zone reduced to 64 and 66 mm in diameter for S aureus and E coli respectively After that film experienced a slow release of carvacrol at 160 100 EE S aureus EA E coli 140 4 e Carvacrol content in the film 120 4 100 4 60 40 20 0 500 Residual carvacrol mg o Inhibition zone mm o o 1 S B o 1 N m o L o Time hours Figure 4 4 9 Carvacrol remaining in CS CD 35G 75RH film at 25 C and 43 RH and inhibition halo produced against S aureus and E coli over time 217 Cap tulo 4 Art culo cient fico 5 the middle of the storage period the carvacrol content in the film was 6 5 mg having inhibition zones o
419. out for the other data Differences between means were assessed on the basis of confidence intervals using the Tukey b test at a significance level of Px0 05 The data are represented as average standard deviation The data were analysed and plotted using the SigmaPlot 10 0 software Systat Software Inc Richmond California USA 3 Results and discussion CS CD films prepared at a weight ratio of 1 1 were homogeneous with no phase separation visible to the naked eye easy to handle and highly transparent After immersion in carvacrol the films maintained their integrity and did not break when handled 3 1 Colour properties Table 4 4 6 shows colour parameters of films incorporating 0 20 and 3596 G and conditioned at different RH before and after being immersed in carvacrol Before immersion in carvacrol the incorporation of G at 20 or 3596 205 Cap tulo 4 Art culo cient fico 5 Table 4 4 6 Colour parameters of CS CD 1 1 weight ratio films Glycerol a b Cs hab Prior to immersion in carvacrol 0 955202 0 27 0 06 5 9 0 3 5920 3 927 0 5 m 95 5 0 27 0 33 0 05 5 9 0 2 5 9 0 3 93 3 0 3 95 3 03 048 0 07 6 2 0 3 62203 93 4 0 4 m immersion in carvacrol 096 RH 95 6 0 8 0 32 0 03 59403 59z03 932 03 95 8 0 7 032 003 57 04 57404 931 04 35 95 6 0 5 0 33 002 59 05 59206 931r06 After immersion in carvacrol 5396 RH 0 95 5 0 5 0 29
420. pace keeping the actual headspace concentration below the MIC Actually the carvacrol activity was 40 times lower than that at saturation Moreover the role of food proteins in protecting microorganisms from carvacrol action has been reported Guti rrez et al 2008 Therefore the antimicrobial efficiency of essential oils and their components is diminished by their interaction with proteins and only the free fraction of carvacrol would exert antimicrobial activity Veldhuizen et al 2007 In this regard only the CS CD Active films of medium and large size would provide a sufficient amount of free carvacrol to produce inhibition and consequently the growth of microorganisms would be more affected by the exposure time However large quantities of carvacrol would be required to obtain this effective quantity of carvacrol in the headspace probably exceeding the acceptable sensory threshold for the consumer which may limit its use Burt 2004 Guti rrez et aL 2009 Skrinjar and Nemet 2009 Previous studies carried out in in vitro conditions showed that medium CS CD Active film stuck to the lid of a Petri dish produced a bactericidal effect against S aureus and E coli Higueras et al 2015 The lower antimicrobial activity of the films observed for chicken compared with the activity assayed in laboratory conditions may be due to the complexity of the food matrix and interaction of components with carvacrol in addition the use of the opti
421. para la liberaci n controlada de agentes activos a partir estos sistemas de envasado Por el contrario en algunos casos esta misma propiedad supone un inconveniente Las pel culas de CS se muestran muy sensibles a la humedad mermando algunas de sus propiedades por lo que es necesaria su neutralizaci n Y la p rdida de sus cargas conlleva la p rdida de sus propiedades antimicrobianas Por este motivo en estos casos se requiere la inclusi n de sustancias bioactivas en la matriz biopolim rica para su aplicaci n en envases antimicrobianos En la presente Tesis Doctoral nicamente las pel culas con LAE no fueron neutralizadas y puestas en contacto con el alimento por ello son las nicas que presentan capacidad antimicrobiana inherente En el resto de sistemas desarrollados los grupos amino del CS han sido neutralizados por lo que la actividad antimicrobiana es atribuible a las sustancias antimicrobianas incorporadas 287 Discusi n general Tabla 5 2 Propiedades del CS en cada uno de los sistemas de envasado antimicrobiano desarrollado Sistema Propiedades del CS Soporte ES LAE Antimicrobiano Matriz compatible liberaci n controlada Soporte Quelante estabilizante de nanopart culas Reductor formador de nanopart culas Reservorio liberaci n controlada Soporte Versatilidad anclaje del vol til al grupo amino Liberaci n controlada reversi n de la base de Schiff frente a un est mulo Soporte Liberaci n con
422. paraci n de bebidas y alimentos Sin embargo estos compuestos debido a su capacidad antimicrobiana y antioxidante presentan gran potencial como conservantes naturales atendiendo as la demanda de los consumidores por productos saludables sin aditivos artificiales a adidos Burt 2004 Dorman y Deans 2000 Los AE son sintetizados como metabolitos secundarios por algunos vegetales como respuesta a situaciones de estr s siendo almacenados en c lulas secretoras cavidades canales c lulas epid rmicas o tricomas glandulares Bakkali et al 2008 Holley y Patel 2005 Se caracterizan por ser l quidos vol tiles a temperatura ambiente generalmente con pesos moleculares por debajo de 300 Da y poseer un fuerte aroma Su densidad suele ser inferior a la del agua excepto los AE de sasafr s clavo o canela Son liposolubles y solubles en los disolventes org nicos habituales Bakkali et al 2008 Los AE se extraen habitualmente por el m todo de destilaci n por arrastre con vapor o extracci n mec nica por presi n en frio Tambi n se pueden emplear fluidos supercr ticos disolventes org nicos o grasas pirogenaci n o microondas pero muchos autores consideran que al producto de extracci n por estos ltimos m todos no se le puede denominar AE Bakkali et al 2008 Burt 2004 Generalmente los AE poseen notables propiedades antimicrobianas frente a un amplio espectro de organismos como bacterias hongos y levaduras virus inse
423. pez Caballero M E G mez Guill n M C Mauri A N Montero M P 2013 Sunflower protein films incorporated with clove essential oil have potential application for the preservation of fish patties Food Hydrocolloids 33 74 84 S nchez Gonz lez L Ch fer M Chiralt A Gonz lez Mart nez C 2010 Physical properties of edible chitosan films containing bergamot essential oil and their inhibitory action on Penicillium italicum Carbohydrate Polymers 82 277 283 S nchez Gonz lez L Ch fer M Hern ndez M Chiralt A Gonz lez Mart nez C 2011 Antimicrobial activity of polysaccharide films containing essential oils Food Control 22 1302 1310 S nchez Gonz lez L Gonz lez Mart nez C Chiralt A Ch fer M 2010 Physical and antimicrobial properties of chitosan tea tree essential oil composite films Journal of Food Engineering 98 443 452 S nchez Gonz lez L Pastor C Vargas M Chiralt A Gonz lez Mart nez C Ch fer M 2011 Effect of hydroxypropylmethylcellulose and chitosan coatings with and without bergamot essential oil on quality and safety of cold stored grapes Postharvest Biology and Technology 60 57 63 Sangsuwan J Rattanapanone N Pongsirikul L 2014 Development of active chitosan films incorporating potassium sorbate or vanillin to extend the shelf life of butter cake International Journal of Food Science amp Technology 1 8 Sangsuwan J Rattanapanone N Rachtanapu
424. ples of Green Chemistry Silver nitrate was incorporated as a precursor to the film forming solution and nanoparticles were generated in situ during the chitosan film neutralization step The neutralization temperature and the concentration of silver in the film were crucial in determining the shape and size of the nanoparticles The films showed antimicrobial activity and maintained their effectiveness releasing silver ions over time Silver nanoparticles synthesized in chitosan films behaved as nanoreservoirs and systems for sustained release of silver ions in liquid media 4 The naturally occurring antimicrobial cinnamaldehyde was covalently attached to preformed chitosan films via reversible Schiff base The reaction yield was quite high Various temperature time food preservation treatments in aqueous media successfully produced hydrolysis of the imine bond and cinnamaldehyde release The films that were developed can be used to improve the microbiological safety of foodstuffs susceptible to contamination with Listeria monocytogenes such as milk Moreover these products were well accepted by the sensory panel 5 HP B cyclodextrin was incorporated in chitosan films to modify the capacity for sorption of the naturally occurring antimicrobial carvacrol 293 Conclusiones 294 Plasticization by glycerol and water was required to improve and tailor sorption properties of the films immersed in the volatile liquid agent Loaded films r
425. pol meros con cinamaldeh do para el envasado antimicrobiano de alimentos 127 Tabla 4 3 2 Bases de Schiff sintetizadas y caracterizadas a partir de CS y diversos aldeh dos 129 Table 4 3 3 Elemental analysis and substitution degree of films 148 Table 4 3 4 Colour parameters of films 148 Table 4 3 5 Water uptake and dimensional stability of films 150 Table 4 3 6 Contact angle of films 151 Table 4 4 1 Colour parameter values of CS films and chitosan hydroxypropyl B cyclodextrin composite CS CD plasticized with different concentrations 96 of G and transparency parameters obtained from transmittance T in the UV visible region 183 Table 4 4 2 Water vapour uptake by films and components at 23 1 C and various RH 96 185 Table 4 4 3 Water vapour permeability of CS and CS CD films obtained with 0 5396 and 0 75 humidity gradient at 23 1 C and oxygen permeability of CS and CS CD films obtained at 50 and 75 RH at 23 1 C 189 Table 4 4 4 Tensile strength modulus of elasticity and elongation at break of CS and CS CD films with different G contents at 53 and 75 RH and 23 1 C 189 Table 4 4 5 Sorption equilibrium of carvacrol in CS and CS CD films at 23 1 C 191 Table 4 4 6 Colour parameters of CS CD 1 1 weight ratio films 206 Table 4 4 7 Power law parameters and diffusion coefficients of carvacrol in CS CD films at 25 C 212 Table 4 4 8 Diffusion coefficient of carvacrol in CS CD 35G 75RH film at different R
426. pplication of a coating composed of chitosan and Origanum vulgare L essential oil to control Rhizopus stolonifer and Aspergillus niger in grapes Vitis labrusca L Food Microbiology 32 345 353 Du W X Avena Bustillos R J Woods R Breksa A P McHugh T H Friedman M Levin C E Mandrell R 2012 Sensory evaluation of baked chicken wrapped with antimicrobial apple and tomato edible 288 Discusi n general films formulated with cinnamaldehyde and carvacrol Journal of Agricultural and Food Chemistry 60 7799 7804 F bregas X Toca M F 1997 Control higi nico sanitario de carnes frescas de aves de corral Eurocarne n 59 pp 1 9 Guti rrez J Rodr guez G Barry Ryan C Bourke P 2008 Efficacy of plant essential oils against foodborne pathogens and spoilage bacteria associated with ready to eat vegetables Antimicrobial and sensory screening Journal of Food Protection 71 1846 1854 ICMSF 2005 Microorganisms in foods 6 microbial ecology of food commodities International Commission on Microbiological Specifications for Foods 2 ed Kluwer Academic Plenum Publishers pp 1 763 Muriel Galet V Cerisuelo J P L pez Carballo G Lara M Gavara R Hernandez Mufioz P 2012 Development of antimicrobial films for microbiological control of packaged salad International Journal of Food Microbiology 157 195 201 Passarinho A T P Dias N F Camilloto G P Cruz R S Otoni C G Moraes A R F
427. pueden extender la vida til microbiol gica de estos productos El an lisis sensorial de la leche en contacto con las pel culas mostr que el olor a canela no causa ning n rechazo entre los consumidores potenciales Estas nuevas pel culas podr an ser utilizadas en el dise o de envases antimicrobianos alimentarios y en otras reas tecnol gicas donde se requieran sistemas de liberaci n sostenida Palabras clave Quitosano cinamaldeh do base de Schiff hidr lisis liberaci n pel cula antimicrobiana 137 Cap tulo 3 Art culo cient fico 3 Abstract In this work cinnamaldehyde was reversibly anchored to chitosan CS films via imino covalent bonding The Schiff base was synthesized in solid phase employing neutralized chitosan films immersed in acidified 9596 v v ethanolic solution in which the aldehyde was dissolved The substitution degree 96 of cinnamaldehyde to the amine group was close to 7096 Attenuated total reflectance Fourier transform infrared spectroscopy ATR FTIR analysis revealed the formation of the chitosan cinnamaldehyde Schiff base The hydrolysis of the imino bond and subsequent release of cinnamaldehyde were studied after the films had been subjected to different combinations of temperature time treatments simulating food preservation methods The amount of aldehyde that remained covalently attached to the films was monitored by ATR FTIR and the substitution degree was determined by elemental analy
428. que conten a 1 5 o 1096 de LAE y 2096 de glicerol Se determinaron las propiedades pticas la liberaci n del LAE y la capacidad antimicrobiana de las pel culas desarrolladas Tambi n se determin la concentraci n m nima inhibitoria MIC y la concentraci n m nima biocida MBC del LAE Se obtuvieron pel culas de CS con LAE transparentes y uniformes sin discontinuidades ni part culas visibles y sin diferencias visuales con las pel culas de CS sin LAE En contacto con un simulante alimentario acuoso este agente se liber completamente en unas pocas horas a 4 y 28 C siguiendo un comportamiento fickiano La actividad antimicrobiana de las pel culas frente a mes filos psicr filos Pseudomonas spp coliformes bacterias cido l cticas y bacterias sulfuro productoras hongos y levaduras fue evaluada a los dos seis y ocho d as para su aplicaci n en pechugas de pollo fileteadas Las pel culas fueron activas frente a bacterias hongos y levaduras en medio l quido y s lido Las pel culas de CS originaron una reducci n logar tmica entre 0 47 2 96 mientras que las pel culas CS 5 LAE produjeron 1 78 5 81 reducciones logar tmicas Estos resultados indican que la incorporaci n de LAE en sistemas de envasado basados en CS contribuye relevantemente a la capacidad antimicrobiana para mejorar la estabilidad de los productos de aves de corral frescos Palabras clave Pel culas de quitosano LAE envasado antimicrobiano envasado act
429. r J Gerbaux P Bonzi Coulibaly Y L 2013 Volatility profiles of monoterpenes loaded onto cellulosic based materials Industrial Crops and Products 51 100 106 Passarinho A T P Dias N F Camilloto G P Cruz R S Otoni C G Moraes A R F Soares N D F 2014 Sliced bread preservation through oregano essential oil containing sachet Journal of Food Process Engineering 37 53 62 Perdones A Vargas M Atares L Chiralt A 2014 Physical antioxidant and antimicrobial properties of chitosan cinnamon leaf oil films as affected by oleic acid Food Hydrocolloids 36 256 264 Ramos M Beltr n A Vald s A Peltzer M Jim nez A Garrig s M Zaikov G 2013 Active packaging for fresh food based on the release of carvacrol and thymol Chemistry amp Chemical Technology 295 303 Ramos M Jim nez A Peltzer M Garrigos M C 2012 Characterization and antimicrobial activity studies of polypropylene films with carvacrol and thymol for active packaging Journal of Food Engineering 109 513 519 Rodr guez A Ner n C Batlle R 2008 New cinnamon based active paper packaging against Rhizopus stolonifer food spoilage Journal of Agricultural and Food Chemistry 56 6364 6369 Sipahi R E Castell P rez M E Moreira R G Gomes C Castillo A 2013 Improved multilayered antimicrobial alginate based edible coating extends the shelf life of fresh cut watermelon Citrullus lanatus Lwt Food Scienc
430. r sticas intr nsecas suelen ser multifuncionales a diferencia de los pl sticos convencionales que suelen ser m s inertes y con ciclos de vida m s largos Entre estos biopol meros destaca el CS principalmente debido a su elevada disponibilidad y su car cter no t xico y biodegradable Entre las diferentes propiedades del CS es de destacar su gran versatilidad y la presencia de distintos grupos funcionales que le confieren la capacidad de desarrollar diferentes sistemas portadores y liberadores de agentes activos Asimismo este material pseudonatural de naturaleza polisac rida y comestible tiene capacidad film gena de manera que puede aplicarse como pel cula o recubrimiento en el envasado activo de alimentos Adem s cuando sus grupos amino se encuentran protonados el CS tiene propiedades quelantes y antimicrobianas La incorporaci n de agentes antimicrobianos en recubrimientos y pel culas para el envasado de alimentos as como en las superficies de procesado de alimentos presenta ventajas frente a su aplicaci n directa sobre el alimento o en su seno 1 se reduce la p rdida de agente activo por neutralizaci n interacci n con componentes del alimento o diluci n en el seno del alimento 2 presenta una elevada superficie de contacto debido a que la contaminaci n microbiana se produce principalmente en la superficie de los alimentos 3 el contacto del agente activo y la superficie del alimento 53 Justificaci n y esque
431. r gain at all the humidities tested Plain CS films had higher water affinity than HP BCDs The CS CD composite films had intermediate values which are in agreement with the additivity principle G was the component with the highest water uptake and therefore the plasticized films had higher water uptake values than the corresponding unplasticized samples as shown in the table This effect was more noticeable at 75 and 9096 RH Compared with the results derived from the application of the additivity principle water sorption by the plasticized composites was below the theoretical value in all cases This is interpreted as a result of Table 4 4 2 Water vapour uptake by films and components at 23 1 C and various RH 96 Materials films 53 RH 75 RH 90 RH Glycerol G 27 00 82 00 215 00 Hydroxypropyl B cyclodextrin HP BCD 9 30 0 07 11304030 28 70 x 0 13 cs 16 98 0 60 28 60 0 30 45 95 0 50 CS 20G 18 10 0 20 33 10 0 90 64 20 1 50 CS 35G 18 90 0 60 37 70 1 60 75 50 0 40 CS CD 1225 025 19 30 0 20 39 04 0 30 CS CD 20G 13 20 0 30 28 80 0 10 61 00 0 40 CS CD 35G 1370 0 30 34804020 70 00 0 35 Data obtained from Bell and Labuza 2000 185 Cap tulo 4 Art culo cient fico 4 interaction between the G and the film matrix especially with CS which reduces the ability of the compound to retain water and limits the accessibility of water molecules in the matrix This result is also in agreeme
432. r glass desiccator with P Os at 23 C until use The film thickness was measured individually with a digital Mitutoyo micrometre Metrotec San Sebastian Spain 22 3 Treatment of CScin films at different combinations of temperature time treatments With the purpose of promoting hydrolysis of the imino bond in the CScin films and thus the release of cinnamaldehyde films were subjected to different combinations of temperature time treatments simulating different food preservation processes For this purpose film samples 0 25 g were 141 Cap tulo 3 Art culo cient fico 3 placed in a glass vial with 10 mL of Mueller Hinton broth MHB Scharlab Barcelona Spain buffer solution at pH 7 or pasteurized whole milk The vials were then subjected to different treatments a 30 min at 4 C in a cooling chamber to simulate refrigeration conditions b 30 min at 65 C 15 min at 72 C or 10 min at 95 C in a thermostatic bath with agitation to simulate pasteurization treatments and c 5 min at 121 C in an autoclave to simulate retorting processes 2 3 Characterization of CScin films before and after imino bond hydrolysis 2 3 1 Elemental analysis The substitution degree of cinnamaldehyde to the amino group in the CS films was determined by measuring the C N ratio of dry samples The analysis was performed with a CHNS O elemental analyser CE Instruments EA 1110 Thermo Fisher Scientific Waltham MA USA Samples were anal
433. r partition coefficients and estimated using software ACD Labs 12 0 ChemSketch Hildebrand solubility parameter estimated according to Fedors in van Krevelen and te Nijenhuis 2009 Solubility parameter of water from Grulke 1989 d Literature experimental Hildebrand solubility parameter values in van Krevelen and te Nijenhuis 2009 255 Cap tulo 4 Art culo cient fico 7 account is that PVOH is a semicrystalline polymer which also limits its water sorption capacity 3 2 Miscibility studies Plasticizers are low molecular compounds chemically compatible with the polymer to be plasticized which at appropriate concentrations impart flexibility and facilitate film handling In a plasticization process the plasticizer molecules are accommodated in the polymer matrix by disrupting intermolecular forces between polymer chains spacing them apart and increasing the free volume thus acting as diluents Plasticization of hydrophilic polymer matrices is commonly carried out by polyols among which G and PG are commonly used for this purpose Most plasticizers employed in polysaccharide films such as polyols are frequently employed in the flavouring industry as solvents and liquid supports for flavours The presence of hydroxyl groups in G make it a good solvent for many ingredients used in pharmaceutical preparations and flavour compounds G can behave as a binder of relatively polar volatile compounds such as alcohols aldehydes ke
434. r una buena dispersi n en la pel cula pero sin que inhiba su liberaci n Chalier et aL 2007 Kurek et al 2012 Como se expone en la Introducci n de la presente Tesis Doctoral el uso de diversos coadyuvantes como las CD pueden aumentar la estabilidad y compatibilidad entre el CS y los agentes activos lip filos As las CD se pueden emplear para disminuir las p rdidas de estas sustancias inestables y vol tiles mediante la formaci n de complejos de inclusi n De esta manera estas peque as biomol culas pueden microencapsularse en las CD y stas a su vez se pueden incluir libremente en la matriz polim rica Ayala Zavala et al 2008 Brasil et al 2012 Kayaci y Uyar 2012 Moreira et al 2014 Sipahi et al 2013 o bien anclar al pol mero para que se d un efecto acumulativo en la capacidad de formaci n de complejos de inclusi n Aoki et al 2003 Debido a la reversibilidad del complejo estas mol culas arom ticas se puede liberar de forma controlada desde el sistema Ciobanu et al 2013 Kfoury et al 2014 Sin embargo en estos casos la cantidad incluida en la matriz es peque a ya que est limitada al volumen interior total de las CD Por ello se puede recurrir a otros mecanismos distintos a la formaci n de complejos de 166 Quitosano Carvacrol inclusi n como los agregados autoensamblados que se producen con elevadas concentraciones de CD para aumentar la capacidad de retenci n de estas sustancias bioa
435. r vapour transmission rate These 180 Quitosano Carvacrol values were then divided by the water pressure gradient and multiplied by the sample thickness to obtain the water vapour permeability value 2 8 2 Oxygen permeability The oxygen permeation rates of the materials were determined at 50 and 75 RH and 23 1 C using an OXTRAN Model 2 21 ML Mocon Lippke Neuwied Germany based on the ASTM standard ASTM 2010b The film samples were previously conditioned at the RH of the experiment After conditioning the samples in the OXTRAN cells for 6 h the transmission values were determined every 45 min until constant 2 9 Mechanical properties A Mecmesin MultiTest 1 universal test machine Landes Poli Ib rica S L Barcelona Spain equipped with a 100 N static load cell was used to evaluate the maximum tensile strength Gm percentage of elongation at break j and Young s modulus E of the films according to ASTM D882 09 18 ASTM 20103 Films were conditioned at 53 and 75 RH for one week before testing Sample films were cut into 25 4 mm x 130 mm strips Grip separation was set at 100 mm and cross head speed at 25 mm min Twenty replicates from each sample were tested 2 10 Conditioning and immersion in carvacrol Circular film samples 55 mm in diameter were stored in glass desiccators at O with phosphorus pentoxide to dry films 53 0 0 5 75 0 0 5 and 90 0 3 0 RH ASTM 2007 in a temperature controlled room at 23 1
436. radores de iones plata las propiedades acomplejantes y la capacidad reductora del pol mero CS Con ello se desarrolla una metodolog a en la que las nanopart culas son creadas in situ en la pel cula de CS El nitrato de plata se emplea como precursor de las nanopart culas incorpor ndose en la disoluci n film gena Las nanopart culas se obtienen durante la neutralizaci n de las pel culas de acetato de CS con hidr xido de sodio La ligera capacidad reductora y 55 Justificaci n y esquema de tesis quelante de iones del CS sumadas a la acci n del ani n hidr xido favorecen la reducci n de la plata y la formaci n de las nanopart culas sin tener que recurrir a reductores fuertes y m s t xicos As se afiadieron a la disoluci n de CS diferentes concentraciones de nitrato de plata empleado como precursor 0 1 0 2 0 5 1 y 1 596 de plata Las pel culas elaboradas por extensi n y evaporaci n del disolvente se neutralizaron con hidr xido de sodio a dos temperaturas 22 y 37 C para determinar el efecto de la temperatura de neutralizaci n en la forma y tamafio de las nanopart culas La evidencia de la formaci n de nanopart culas de plata se evalu mediante microscop a electr nica de transmisi n TEM espectroscop a UV visible y difracci n de rayos X Las pel culas nanocompuestas se caracterizaron mediante la determinaci n de su color y an lisis termogravim trico TGA Se determin la actividad antimicrobia
437. rana citoplasm tica al ATP El carvacrol interact a con la membrana celular de las bacterias gram positivas y negativas distorsion ndola e incrementando su permeabilidad Se produce la disrupci n de la fuerza prot n motriz alterando el flujo de electrones provocando el desacople de protones y la salida de K alterando el transporte activo inhibiendo la generaci n de ATP y produciendo la muerte celular Burt 2004 Holley y Patel 2005 Lambert et al 2001 Seg n algunos autores la estructura fen lica del carvacrol y del timol Figura 1 8 es responsable de sus propiedades antibacterianas de modo que podr an actuar como transportador de H al interior celular y de K al exterior Se ha demostrado que si se encuentra bloqueado el grupo hidroxilo del grupo fen lico del carvacrol como ocurre en el ster met lico o el anillo benc nico se encuentra saturado como en el mentol Figura 1 8 su actividad disminuye considerablemente Ben Arfa et al 2006 Burt 2004 Holley y Patel 2005 Sin embargo otros autores apuntan que el grupo _CH a OH p OH CH g 9H CH d 9 CH3 CH3 CH3 CH H3C H3C H3C H3C 3 CH3 CH3 Figura 1 8 Estructuras de a carvacrol b timol c mentol y d ster met lico del carvacrol 34 Introducci n hidroxilo s lo contribuye a esta acci n biocida pero que no es esencial Veldhuizen et al 2006 Como se puede observar en el Anexo A son diversos los estudios que emplean el carvacrol como a
438. rate Polymers 97 262 268 Higueras L L pez Carballo G Gavara R Hern ndez Mu z P 2015 Incorporation of hydroxypropyl B cyclodextrins into chitosan films to tailor loading capacity for active aroma compound carvacrol Food Hydrocolloids 43 603 611 Holley R A Patel D 2005 Improvement in shelf life and safety of perishable foods by plant essential oils and smoke antimicrobials Food Microbiology 22 273 292 Kavoosi G Dadfar S M M Purfard A M Mehrabi R 2013 Antioxidant and antibacterial properties of gelatin films incorporated with carvacrol Journal of Food Safety 33 423 432 L pez Mata M A Ruiz Cruz S Silva Beltr n N P Ornelas Paz J D Zamudio Flores P B Burruel Ibarra S E 2013 Physicochemical antimicrobial and antioxidant properties of chitosan films incorporated with carvacrol Molecules 18 13735 13753 Muriel Galet V Talbert J N Hern ndez Mu oz P Gavara R Goddard J M 2013 Covalent immobilization of lysozyme on ethylene vinyl alcohol films for nonmigrating antimicrobial packaging applications Journal of Agricultural and Food Chemistry 61 6720 6727 Nostro A Scaffaro R D Arrigo M Botta L Filocamo A Marino A Bisignano G 2012 Study on carvacrol and cinnamaldehyde polymeric films mechanical properties release kinetics and antibacterial and antibiofilm activities Applied Microbiology and Biotechnology 96 1029 1038 Ramos M Jim nez A Pe
439. raturas por debajo de 107 C y pH comprendido entre 3 7 ste puede ser aplicado en diferentes alimentos sin alterar sus propiedades organol pticas En los ltimos afios se ha demostrado la elevada efectividad del LAE frente a microorganismos pat genos y alterantes siendo mayoritariamente aplicado en la superficie de productos c rnicos As Bakal y D az 2005 demostraron la efectividad del agente activo frente a pat genos como Listeria monocytogenes y a bacterias cido l cticas alterantes de las caracter sticas organol pticas en jam n curado y cocido El conservante fue empleado en soluci n acuosa para la inmersi n de los productos el resultado fue un aumento de su vida til en condiciones de refrigeraci n El efecto antimicrobiano del LAE frente a L monocytogenes tambi n ha sido estudiado pulveriz ndolo en una soluci n acuosa en la superficie del alimento o del envase justo antes del envasado al vacio de diversos alimentos como jam n Luchansky et al 2005 Stopforth et al 2010 Taormina y Dorsa 2009a o salchichas Martin et al 2009 Porto Fett et al 2010 Taormina y Dorsa 2009b conservados a baja temperatura Sommers et al 2010 testaron el LAE en salchichas frente a otros pat genos como Salmonella y Staphylococcus aureus adem s de frente a L monocytogenes y posteriormente en L innocua Sommers et al 2012 Benli et al 2011 estudiaron su efectividad en carcasas de aves frente a Salmonella enterica Asimism
440. rdida de la actividad antimicrobiana de este ltimo Loeffler et aL 2014 y formaci n de agregados insolubles dependiendo del tipo de biopol mero Bonnaud et al 2010 En la presente Tesis Doctoral se han obtenido pel culas de CS y LAE estudiado su liberaci n y eficacia sobre un alimento c rnico fresco como son los filetes de pechuga de pollo ya que son muy limitados los estudios realizados sobre la eficacia del LAE en este tipo de producto Referencias bibliogr ficas Bakal G D az A 2005 The lowdown on lauric arginate food antimicrobial eats at plasma membrane disrupting a pathogen s metabolic process Food Quality 12 54 61 Benli H S nchez Plata M X Keeton J T 2011 Efficacy of epsilon polylysine lauric arginate or acidic calcium sulfate applied sequentially for Salmonella reduction on membrane filters and chicken carcasses Journal of Food Protection 74 743 750 65 Cap tulo 1 Estado de la cuesti n Bonnaud M Weiss J McClements D J 2010 Interaction of a food grade cationic surfactant lauric arginate with food grade biopolymers pectin carrageenan xanthan alginate dextran and chitosan Journal of Agricultural and Food Chemistry 58 9770 9777 Coughlin M F 2010 Lauric arginate as a contact antimicrobial Ebner C L 2012 Antimicrobial packaging material and methods of making and using the same Guo M M Jin T Z Scullen O J Sommers C H 2013 Effects of antimicrobial coa
441. re G K Roberts G A F 1981 Reactions of chitosan 3 Preparation and reactivity of Schiff s base derivatives of chitosan International Journal of Biological Macromolecules 3 337 340 Muzzarelli RA A Ilari P 1994 Chitosans carrying the methoxyphenyl functions typical of lignin Carbohydrate Polymers 23 155 160 Ouattara B Sabato S F Lacroix M 2001 Combined effect of antimicrobial coating and gamma irradiation on shelf life extension of pre cooked shrimp Penaeus spp International Journal of Food Microbiology 68 1 9 Ouattara B Simard R E Piette G Begin A Holley R A 2000 Inhibition of surface spoilage bacteria in processed meats by application of antimicrobial films prepared with chitosan International Journal of Food Microbiology 62 139 148 Porras G Esquivel M Ballesteros M R S Madrigal Carballo S Calvo M 2009 Quitosano N acilado con cinamaldeh do un potencial bioplaguicida contra agentes pat genos en el campo agr cola Revista Iberoamericana de Pol meros 10 170 176 Ravishankar S Jaroni D Zhu L B Olsen C McHugh T Friedman M 2012 Inactivation of Listeria monocytogenes on ham and bologna using pectin based apple carrot and hibiscus edible films containing carvacrol and cinnamaldehyde Journal of Food Science 77 M377 M382 Ravishankar S Zhu L B Olsen C W McHugh T H Friedman A 2009 Edible apple film wraps containing plant antimicrobials inactivate
442. re hermetically sealed and it was confirmed that they provided an infinite barrier to carvacrol The Hydroxypropyl B cydodextrins partition of the antimicrobial agent within the food packaging system was analysed The antimicrobial devices Carvacrol rapidly released a large percentage of the agent load amounts that were gained by the adhesive coating of the Antimicrobial active packaging lid and especially by the chicken fillets The latter were the main sorbent phase with average concentrations Controlled release ranging between 200 and 5000 mg Kg during the period of storage The microbiota ofthe packaged fresh chicken Chicken fillets mesophiles psychrophiles Pseudomonas spp enterobacteria lactic acid bacteria and yeasts and fungi were analysed and monitored during storage A general microbial inhibition was observed increasing with the size of the active device Inhibition with a 24 cm device ranged from 0 3 log reductions against lactic acid bacteria to 1 8 logs against yeasts and fungi However the large amount of antimicrobial that was sorbed or that reacted with the fillet caused an unacceptable sensory deterioration These high sorption values are probably due to a great chemical compatibility between chicken proteins and carvacrol 2014 Elsevier B V All rights reserved 1 Introduction polymeric vehicles are key parameters and the efficiency and validity of an active packaging has to be tested for the specific product Antimicrob
443. realizaron ensayos de actividad antimicrobiana a lo largo del tiempo Con ellos se demuestra que las pel culas de CS con 0 296 de plata neutralizadas mantuvieron la misma capacidad antimicrobiana al menos los 15 d as que se mantuvieron inmersas en el medio acuoso As la 277 Discusi n general capacidad antimicrobiana fue independiente del tiempo de inmersi n de las pel culas aunque la cantidad de plata migrada al medio acuoso se increment con este tiempo cuando se superaron las 10 h de inmersi n Esto puede explicarse ya que la t cnica empleada para determinar la liberaci n de plata GFAAS cuantifica globalmente plata i nica y elemental As que es posible que la cantidad de plata i nica disponible en el medio sea baja por la reducci n de sta o por su interacci n con las prote nas del medio Por otro lado se observa que estas pel culas originan una concentraci n de plata total migrada durante tiempos cortos 10 h que se encuentra alrededor de 0 06 mg L valores muy pr ximos a los l mites de migraci n m xima de plata 0 05 mg L en agua y con capacidad antimicrobiana elevada superior a 3 reducciones logar tmicas Por lo que estas pel culas nanocompuestas podr an ser potencialmente empleadas para el envasado activo de alimentos En un segundo estudio las pel culas fueron inmersas en un medio acuoso renovado cada 24 h Cada d a del mes en que se llev a cabo el ensayo las pel culas se extrajeron del m
444. red a reddish brown tone as the concentration of silver and the neutralization temperature increased Colour coordinates of the films are shown in Table 4 2 1 CS silver nitrate films neutralized at 22 C experienced an increase in chroma C and colour difference AE and a decrease in hue angle h and lightness L compared to un neutralized films As the concentration of silver increased the films experienced a decrease in C h lightness and AE These changes were more acute for films neutralized at 37 C indicating a greater conversion of silver nitrate into silver based nanoparticles The C values of the films neutralized at 37 C also increased with silver however C values of 1 and 1 596 silver films were lower than the control and films became opaque Because a higher neutralization temperature ensures a greater conversion of silver nitrate into silver based nanoparticles studies of the thermal stabilities of the films and their antimicrobial activities along with characterization of the nanoparticles formed in the films were undertaken only with those neutralized at 37 C 3 3 Thermogravimetric analysis Figure 4 2 3 shows the effect of silver content in 37 C neutralized CS films on the first derivative of weight loss curves DTG The initial thermal decomposition of films neutralized at 37 C happened at a slightly higher temperature than the film prepared without silver It can be seen in the DTGmax curves that the
445. refore when the film is used to package food the packaged food processing treatments and or the conditions inside the package during storage can activate or extend the release of the molecule to exert its effect In this connection Schiff base can act as a stimuli responsive linker providing a means of obtaining antimicrobial release systems with potential applications in active packaging of foods Synthesis of Schiff base derivative was confirmed by ATR FTIR and the substitution degree was evaluated To the naked eye the films maintained their transparency and acquired a light yellow colour owing to the presence of a conjugated double bond after Schiff base formation CScin films were subjected to various food preservation processes consisting of different temperature time combinations after which the films were characterized and the cinnamaldehyde released was evaluated 146 Quitosano Cinamaldeh do Figure 4 3 1 Nucleophilic addition of amino group of CS backbone to carbonyl group of cinnamaldehyde and formation of Schiff base 3 1 Characterization of chitosan films modified with cinnamaldehyde 3 1 1 Elemental analysis The elemental composition of CS films before and after Schiff base formation and after the various preservation processes is shown in Table 4 3 3 The degree of acetylation DA for the untreated sample was calculated with the following equation Kasaai et al 1999 DA NL ELM 2 6 861 5 145 where C
446. rey A 2001 Antibacterial activity of the essential oil of Picea excelsa on Listeria Staphylococcus aureus and coliform bacteria Food Microbiology 18 261 268 Crank J 1975 The mathematics of diffusion 2nd ed Clarendon Press Oxford England Dai Y M Normand M D Weiss J Peleg M 2010 Modeling the efficacy of triplet antimicrobial combinations yeast suppression by lauric arginate cinnamic acid and sodium benzoate or potassium sorbate as a case study Journal of Food Protection 73 515 523 Delaquis P J Stanich K Girard B Mazza G 2002 Antimicrobial activity of individual and mixed fractions of dill cilantro coriander and eucalyptus essential oils International Journal of Food Microbiology 74 101 109 Infante M R Garc a Dom nguez J J Erra P Juli M R Prats M 1984 Surface active molecules preparation and properties of long chain N acyl L a amino o guanidine alkyl acid derivatives International Journal of Cosmetic Science 6 275 282 Infante M R P rez L Pinazo A Clap s P Mor n M C Angelet M Garc a M T Vinardell M P 2004 Amino acid based surfactants Comptes Rendus Chimie 7 583 592 Infante M R Pinazo A Seguer J 1997 Non conventional surfactants from amino acids and glycolipids structure preparation and properties Colloids and Surfaces a Physicochemical and Engineering Aspects 123 49 70 Kanazawa A Ikeda T Endo T 1995 A novel approach to mod
447. rix and the difficulty for them to diffuse through the polymer However generation of silver ions on the surface of the nanoparticles embedded in the CS matrix is expected Diffusion of silver ions through the polymer matrix has been probed this being encouraged by the hydrophilic nature of CS Swelling of CS by water facilitates the mobility of the polymer chains and therefore the transport of ions through the matrix to the release media Table 4 2 2 shows that the films had a slightly higher antimicrobial capacity against E coli than against S aureus Studies by TEM energy dispersive X ray showed that the morphological changes that occur in the internal structure of the cell are similar in both types of bacteria such that it loses its ability to replicate and proteins are inactivated by interaction with silver ions Feng et al 2000 However although similar morphological changes were observed in both organisms they were less marked in S aureus which is attributed to differences in the cell wall of the two microorganisms In this case S aureus has a more effective defence system than E coli because Gram positive cell walls have a thicker peptidoglycan layer than Gram negative cell walls It has been reported in the bibliography that the peptidoglycan cell wall has a greater capacity to protect the cell from the penetration of silver ions and small nanoparticles into the cytoplasm Rai et al 2009 3 8 Antimicrobial activity of films ne
448. robial agents from film while those with LAE evidenced a halo of growth inhibition around the film due to LAE migration Significantly LAE films produced a greater inhibition against Gram positive than Gram negative bacteria A slight inhibitory effect against yeasts was observed for CS 596LAE and CS 10 LAE films nevertheless the amount of LAE migrated from CS 196 LAE films could have not been enough to produce inhibition No growth inhibition against fungi was observed around any of the tested films because the migrated concentration could have not been sufficient As can be seen in Table 4 1 5 there was no difference between the halos produced by 5 to 1096 For this reason film with CS 596LAE was selected for studying its antimicrobial activity in food 3 5 Antimicrobial activity of LAE CS films on chicken breast fillets Figure 4 1 2 shows the efficacy of CS and CS 5 LAE on microorganisms present in fresh chicken breast fillets In general there was an increase in microbial growth as time increases until it reached the expiry date stated on the product itself At to counts were low as expected in samples of chicken breast fillets without skin A large growth reduction 4 log for mesophiles psychrophiles and Pseudomonas spp after 2 6 and 8 days was evident in chicken wrapped with CS 596LAE film A much lower reduction was produced by control CS films from 0 5 log after 2 days and 1 70 log at 8 days presumably due to CS antimicrobial a
449. robianos naturales procedentes de AE en pel culas de CS En este cap tulo tambi n se estudia la liberaci n de dichos compuestos simulando el espacio de cabeza de un envase y diferentes RH y finalmente la aplicaci n de uno de los sistemas generados En el primer art culo cient fico de este apartado Preparation and characterization of chitosan HP B cyclodextrins composites with high sorption capacity for carvacrol se detalla el desarrollo de las pel culas compuestas a partir de materiales procedentes de recursos renovables y con car cter biodegradable como son el CS y las CD Para ello se fija el contenido en HP BCD respecto al CS en 1 1 p p y se var a el contenido en G 0 20 y 3596 g 100 g de CS Las pel culas se obtuvieron por extensi n y evaporaci n del disolvente y se caracterizaron f sico qu micamente propiedades pticas mec nicas barrera a gases y vapores sorci n de vapor de agua morfol gicas y TGA Tras la caracterizaci n se estudi el efecto de las HP BCD G y contenido en agua en la capacidad de sorci n de carvacrol 57 Justificaci n y esquema de tesis para ello las muestras se acondicionaron a diferentes RH 53 75 y 90 y se sumergieron en el l quido vol til a 23 C hasta alcanzar el equilibrio de sorci n El contenido en carvacrol se determin mediante desorci n t rmica acoplada a cromatograf a de gases Asimismo se determin la capacidad antimicrobiana en fase vapor de todas las pel
450. rocolloids 24 285 290 Shin Y J Song H Y Seo Y B Song K B 2012 Preparation of red algae film containing grapefruit seed extract and application for the packaging of cheese and bacon Food Science and Biotechnology 21 225 231 Sipahi R E Castell P rez M E Moreira R G Gomes C Castillo A 2013 Improved multilayered antimicrobial alginate based edible coating extends the shelf life of fresh cut watermelon Citrullus lanatus Lwt Food Science and Technology 51 9 15 Siragusa G R Dickson J S 1992 Inhibition of Listeria monocytogenes on beef tissue by application of organic acids immobilized in a calcium alginate gel Journal of Food Science 57 293 296 Siragusa G R Dickson J S 1993 Inhibition of Listeria monocytogenes Salmonella Typhimurium and Escherichia coli O157 H7 on beef muscle tissue by lactic or acetic acid contained in calcium alginate gels Journal of Food Safety 13 147 158 327 Anexos Siripatrawan U Noipha S 2012 Active film from chitosan incorporating green tea extract for shelf life extension of pork sausages Food Hydrocolloids 27 102 108 Sivarooban T Hettiarachchy N S Johnson M G 2008 Physical and antimicrobial properties of grape seed extract nisin and EDTA incorporated soy protein edible films Food Research International 41 781 785 Soares N F F Rutishauser D M Melo N Cruz R S Andrade NJ 2002 Inhibition of microbial growth in bread through act
451. roducci n SCENIHR 2014 Opinion on nanosilver safety health and environmental effects and role in antimicrobial resistance Shahidi F Abuzaytoun R 2005 Chitin chitosan and co products chemistry production applications and health effects Advances in food and nutrition research 49 93 135 Shahidi F Arachchi J K V Jeon Y J 1999 Food applications of chitin and chitosans Trends in Food Science amp Technology 10 37 51 Sharma V K Yngard R A Lin Y 2009 Silver nanoparticles green synthesis and their antimicrobial activities Advances in Colloid and Interface Science 145 83 96 Silver S 2003 Bacterial silver resistance molecular biology and uses and misuses of silver compounds Fems Microbiology Reviews 27 341 353 Silver S Phung L T Silver G 2006 Silver as biocides in burn and wound dressings and bacterial resistance to silver compounds Journal of Industrial Microbiology amp Biotechnology 33 627 634 Siracusa V Rocculi P Romani S Dalla Rosa M 2008 Biodegradable polymers for food packaging a review Trends in Food Science amp Technology 19 634 643 Smita S Gupta S K Bartonova A Dusinska M Gutleb A C Rahman Q 2012 Nanoparticles in the environment assessment using the causal diagram approach Environmental Health 11 Smits A L M Kruiskamp P H Van Soest J J G Vliegenthart J F G 2003 Interaction between dry starch and plasticisers glycerol or
452. roups does not exhibit antimicrobial activity Shahidi et al 1999 In this work neutralized CS films without silver acted as a control Thus the antimicrobial properties of the films were expected to be due to the release of silver ions from the nanoparticles embedded in the CS matrix which acted as a support for silver based nanoparticles Table 4 2 2 shows the antimicrobial capacity of films neutralized at 37 C with a silver concentration ranging from 0 1 to 1 596 against the pathogen microorganisms S aureus and E coli using MHB or 1 125 diluted MHB as culture medium It is noteworthy that the antimicrobial capacity of the films did not change after autoclaving Some authors have reported that hydrothermal treatments such as autoclaving can produce nanoparticles from silver ions Lu et al 2011 Zou et al 2007 In our experiment nanoparticles were supposed to be generated during the neutralization step without free silver ions remaining in the matrix When MHB was used films containing 0 1 silver produced reductions of 0 62 log for S aureus and 0 88 against E coli whereas for higher silver concentrations the growth of microorganisms was reduced by about 2 log The antimicrobial activity of the films increased significantly when microbiological tests were carried out in 1 125 diluted MHB giving an approximate reduction of 3 log cycles of viable cells for each microorganism tested The lower antimicrobial capacity of the films in MHB c
453. rsas desventajas respecto a sus propiedades funcionales frente a los pol meros convencionales As por ejemplo los polisac ridos y prote nas son en general materiales muy hidr filos y como consecuencia sus propiedades mec nicas y barrera var an en gran medida con la RH adem s de presentar grandes Introducci n dificultades a la hora de ser procesados por v a seca mediante las t cnicas usuales empleadas en pl sticos convencionales Si bien estos materiales podr an emplearse en aplicaciones espec ficas como en alimentos con baja actividad de agua o que no necesitan un envase de alta barrera o incluso convertir este inconveniente en una ventaja en el caso de alimentos frescos que se benefician del intercambio gaseoso Siracusa et al 2008 Weber et al 2002 O bien se pueden desarrollar sistemas multicapa con materiales que suplan sus carencias Weber et al 2002 o dise ar derivados de los biopol meros que mejoren estas caracter sticas Petersen et al 1999 Muchos de estos biopol meros hidrogeles como polisac ridos y prote nas debido a su capacidad film gena y solubilidad en medios acuosos y o etan licos son los principales constituyentes de las pel culas y recubrimientos comestibles Campos et al 2011 Debeaufort et al 1998 o recubrimientos de otros materiales como papel cart n u otros pol meros demostrando ser excelentes sistemas portadores y liberadores de compuestos activos Esta ltima propiedad h
454. rties and storage stability of novel carvacrol containing edible tomato films Journal of Food Science 73 M378 M383 Du W X Olsen C W Avena Bustillos R J McHugh T H Levin C E Friedman M 2009 Effects of allspice cinnamon and clove bud essential oils in edible apple films on physical properties and antimicrobial activities Journal of Food Science 74 M372 M378 Du W X Olsen C W Avena Bustillos R J McHugh T H Levin C E Mandrell R Friedman M 2009 Antibacterial effects of allspice garlic and oregano essential oils in tomato films determined by overlay and vapor phase methods Journal of Food Science 74 M390 M397 Du W X Olsen C W Avena Bustillos R T Mchugh T H Levin C E Friedman M 2008 Storage stability and antibacterial activity against Escherichia coli O157 H7 of carvacrol in edible apple films made by two different casting methods Journal of Agricultural and Food Chemistry 56 3082 3088 Duan J Kim K Daeschel M A Zhao Y 2008 Storability of antimicrobial chitosan lysozyme composite coating and film forming solutions Journal of Food Science 73 M321 M329 Duan J Park S L Daeschel M A Zhao Y 2007 Antimicrobial chitosan lysozyme CL films and coatings for enhancing microbial safety of Mozzarella cheese Journal of Food Science 72 M355 M362 Ehivet F E Min B Park M K Oh J H 2011 Characterization and antimicrobial activity of sweetpotato starch based edibl
455. rties in renewable hydrophilic polymers Food Chemistry 130 814 820 Ben Arfa A Combes S Preziosi Belloy L Gontard N Chalier P 2006 Antimicrobial activity of carvacrol related to its chemical structure Letters in Applied Microbiology 43 149 154 Biava M Fioravanti R Porretta G C Sleiter G Deidda D Lampis G Pompei R 1999 Antimycobacterial activity of new ortho meta and para toluidine derivatives Farmaco 54 721 727 267 Cap tulo 4 Art culo cient fico 7 Buonocore G G Del Nobile M A Panizza A Bove S Battaglia G Nicolais L 2003 Modeling the lysozyme release kinetics from antimicrobial films intended for food packaging applications Journal of Food Science 68 1365 1370 Burt S 2004 Essential oils their antibacterial properties and potential applications in foods A review International Journal of Food Microbiology 94 223 253 Cutter C N 2002 Microbial control by packaging a review Critical Reviews in Food Science and Nutrition 42 151 161 Chen H L Wu L G Tan J Zhu C L 2000 PVA membrane filled B cyclodextrin for separation of isomeric xylenes by pervaporation Chemical Engineering Journal 78 159 164 Dambolena J S Lopez A G Meriles J M Rubinstein H R Zygadlo J A 2012 Inhibitory effect of 10 natural phenolic compounds on Fusarium verticillioides A structure property activity relationship study Food Control 28 163 170 Glemza AJ
456. s C Mackay W Geveke D Lemmenes B Pulsfus S 2012 Inactivation of Listeria innocua on frankfurters by flash pasteurization and lauric arginate ester Journal of Food Processing amp Technology 3 147 Sommers C H Scullen O J Sites J E 2010 Inactivation of foodborne pathogens on frankfurters using ultraviolet light and GRAS antimicrobials Journal of Food Safety 30 666 678 Soni K A Desai M Oladunjoye A Skrobot F Nannapaneni R 2012 Reduction of Listeria monocytogenes in queso fresco cheese by a combination of listericidal and listeriostatic GRAS antimicrobials International Journal of Food Microbiology 155 82 88 Soni K A Nannapaneni R Schilling M W Jackson V 2010 Bactericidal activity of lauric arginate in milk and queso fresco cheese against Listeria monocytogenes cold growth Journal of Dairy Science 93 4518 4525 Soni K A Shen Q Nannapaneni R 2014 Reduction of Listeria monocytogenes in cold smoked salmon by bacteriophage P100 nisin and lauric arginate singly or in combinations International Journal of Food Science and Technology 49 1918 1924 Stopforth J D Visser D Zumbrink R Van Dijk L Bontenbal E W 2010 Control of Listeria monocytogenes on cooked cured ham by formulation with a lactate diacetate blend and surface treatment with lauric arginate Journal of Food Protection 73 552 555 Taormina P J Dorsa W J 2009a Inactivation of Listeria monocytogenes on
457. s CScin films treated at 95 C for 10 min showed a log reduction of 4 15 0 02 at 3 days 3 41 0 02 at 6 days and 3 87 0 07 after 12 days It was not possible to inoculate the samples treated at 121 C for 5 min because the milk was coagulated after treatment There are two possible reasons for this effect It is documented that certain aromatic compounds such as cinnamaldehyde may cause conformational changes in proteins by binding Damodaran and Kinsella 1980 Kuhn et al 2006 Combinations of temperature and the aromatic aldehyde can lead to denaturation of milk proteins and subsequent unfolding and aggregation forming a gel This 10 ENH day 3 EE day 6 EMI day 12 Bacterial count Log CFU mL o N A o o hz Treatment Figure 4 3 6 Antimicrobial effect of CS film modified with cinnamaldehyde and subjected to different preservation treatments against L monocytogenes in pasteurized milk 158 Quitosano Cinamaldeh do treatment could also produce hydrolysis of CS and migration to the medium causing milk coagulation due to the coagulation and flocculation properties of CS Renault et al 2009 The lower antimicrobial activity of the films observed when applied to milk Figure 4 3 6 compared with that in MHB medium Figure 4 3 5 can be explained because the use of the optimal culture medium for the microorganism magnifies any effect Moreover milk is a complex food matrix which may interfere with the ant
458. s agentes activos incorporados en las pel culas e Estudiar la capacidad antimicrobiana in vitro de los agentes activos y las pel culas obtenidas frente a microorganismos pat genos modelo Evaluar la eficacia de las pel culas desarrolladas en alimentos reales y su posible empleo en el dise o de envases activos antimicrobianos para alimentos 49 3 JUSTIFICACI N Y ESQUEMA DE TESIS Justificaci n y esquema de tesis Actualmente el inter s por los sistemas de envasado antimicrobiano de alimentos est aumentando Por una parte el sistema f sicamente a sla el alimento y evita la contaminaci n con nuevos microorganismos Por otra la presencia de agentes antimicrobianos permite la inhibici n o retardo del crecimiento de los microorganismos Estas acciones conjuntas permiten el control de microorganismos pat genos que pueden suponer un problema de salud p blica y de microorganismos alterantes responsables del deterioro de los alimentos Por otra parte se pretende el desarrollo de materiales m s sostenibles a partir de recursos renovables y a partir de excedentes y residuos de la industria alimentaria agr cola y de la pesca Con ello el prop sito es reducir la acumulaci n de desechos aport ndoles un valor afiadido Entre estos materiales destacan los biopol meros que pueden emplearse en el envasado activo de alimentos Asimismo estos biopol meros adem s de ser muchos de ellos comestibles debido a sus caracte
459. s Doctoral se estructura en siete secciones En la secci n INTRODUCCI N se presenta el contexto de esta Tesis describiendo los aspectos fundamentales sobre los que se va a trabajar En la secci n OBJETIVOS se proponen esquem ticamente los objetivos generales y los diferentes objetivos espec ficos de esta Tesis Doctoral A continuaci n en la secci n JUSTIFICACI N Y ESQUEMA DE TESIS se expone una breve y cohesiva descripci n de los cap tulos y los art culos que los conforman La siguiente secci n denominada CAP TULOS est constituida por cuatro apartados que incluyen los antecedentes y el estado de la cuesti n y las publicaciones cient ficas que ha originado esta investigaci n En la secci n DISCUSI N GENERAL se examinan e integran los resultados de cada apartado resaltando los m s notables En la sexta secci n se presentan las CONCLUSIONES m s relevantes Y finalmente en la secci n ANEXOS se adjunta una extensa revisi n bibliogr fica de materiales biopolim ricos para el envasado antimicrobiano de alimentos y las siete publicaciones a las que esta Tesis Doctoral ha dado lugar 1 INTRODUCCI N Introducci n Alrededor de un tercio de los alimentos producidos en el mundo para el consumo humano aproximadamente 1300 millones de toneladas anuales se pierde o desperdicia Esto significa que enormes cantidades de los recursos destinados a la producci n de alimentos se utilizan en vano y que las emisiones de gases
460. s are very perishable because of their rich nutritional composition high pH 5 5 6 5 and water activity 0 98 0 99 Thus these products are very susceptible to microbial contamination and development of a wide variety of spoilage microorganisms and food borne pathogens Aymerich et al 2008 In order to solve this problem four types of packages were prepared and filled with 25 g of chicken breast small medium and large samples with different sizes of CS CD Active and a medium sample of CS Control without active component Figure 4 4 12 shows the effect of the different packages on chicken breast microbiota There was an increase in microbial growth as time increased until it reached the expiry date stated on the product The present mesophile enterobacteria and lactic acid bacteria counts are in agreement with those reported by other authors Rokka et al 2004 Voidarou et al 2011 In general the small film did not produce a relevant microbial growth reduction especially at shorter exposure times After 9 days of exposure the growth of fungi and yeasts mesophiles and enterobacteria was reduced by 0 38 0 37 and 0 33 log g respectively The medium film did not produce relevant inhibition at short times The growth reduction improved with exposure time reaching a log reduction ranging between 0 63 and 0 89 for all microorganisms after 9 days except for lactic acid bacteria where the reduction was 0 33 The large film after 3 days pr
461. s further development as silver nanoparticles loaded antibacterial food packaging material Colloids and Surfaces B Biointerfaces 69 164 168 Tankhiwale R Bajpai S K 2010 Silver nanoparticle loaded chitosan lactate films with fair antibacterial properties Journal of Applied Polymer Science 115 1894 1900 Teixeira B Marques A Pires C Ramos C Batista I Saraiva J A Nunes M L 2014 Characterization of fish protein films incorporated with essential oils of clove garlic and origanum physical antioxidant and antibacterial properties Lwt Food Science and Technology 59 533 539 Theinsathid P Visessanguan W Kruenate J Kingcha Y Keeratipibul S 2012 Antimicrobial activity of lauric arginate coated polylactic acid films against Listeria monocytogenes and Salmonella Typhimurium on cooked sliced ham Journal of Food Science 77 M142 M149 Theivendran S Hettiarachchy N S Johnson M G 2006 Inhibition of Listeria monocytogenes by nisin combined with grape seed extract or green tea extract in soy protein film coated on turkey frankfurters Journal of Food Science 71 M39 M44 Torlak E Nizamlioglu M 2011 Antimicrobial effectiveness of chitosan essential oil coated plastic films against foodborne pathogens Journal of Plastic Film amp Sheeting 27 235 248 328 Anexos Torres J A 1984 Exploration of the stability of intermediate moisture foods Massachusetts Inst of Technology Cambridge
462. s were performed through a 6 4 mm diameter diaphragm containing an optical glass monitoring L a b chroma C a b 7 and hue ha arctan b a The samples were measured in triplicate by eight measurements in different locations for each film sample The apparent opacity was evaluated Agilent 8453 UV visible spectrophotometer Agilent Barcelona Spain as the integrated area under the curve which was calculated using UV WIN Lab software and expressed as the product of absorbance value A and wavelength nm Samples were measured in triplicate 2 5 Morphology Films were fractured under liquid nitrogen and the cross section surface morphology studied by field emission FE scanning electron microscopy SEM using a HITACHI S 4100 unit equipped with a secondary electron SE detector and an EMIP 3 0 image capture system HITACHI Madrid Spain Samples were coated under vacuum with gold palladium in a 179 Cap tulo 4 Art culo cient fico 4 sputter coating unit and their fracture surface was investigated Images were captured at 10 kV at a distance of 14 cm with 1000x magnification 2 6 Thermogravimetric analysis TGA TGA of films was carried out using a Mettler Toledo TGA SDTA 851 Columbus OH USA Samples of approximately 10 mg were heated from room temperature to 900 C at 10 C min and held at an isotherm for 3 min under a nitrogen atmosphere The TGA data were plotted as weight per cent versus temper
463. sac ridos natamicina quitooligosac ridos c l ctico prote na de soja ze na prote nas de soja prote na de soja prote na de suero de leche prote nas de suero de leche prote na de suero de leche quitosano ze na ze na ze na ze na celulosa celulosa acetato pululano alginato alginato quitosano almid n de tapioca case na celulosa celulosa gluten de trigo metilcelulosa prote na de suero de leche prote na de suero de leche medio de cultivo mortadela de pavo loncheado salchichas de pavo salchichas de pavo medio de cultivo medio de cultivo medio de cultivo salchicha de pavo medio de cultivo pollo cocido medio de cultivo jam n carne de vacuno pechuga de pavo jam n loncheado pechuga de pavo RTE medio de cultivo queso Port Salut queso kashar fresco queso gorgonzola queso mozzarella loncheada medio de cultivo queso kashar fresco medio de cultivo queso Saloio Lb plantarum E coli L monocytogenes L monocytogenes L monocytogenes E coli O157 H7 S Typhimurium L monocytogenes Penicillium spp Ps aeruginosa Yarrowia lipolytica L innocua Brochothrix thermosphacta E coli Enterococcus faecalis E coli St aureus S Typhimurium L monocytogenes B cereus L monocytogenes S Enteriditis L monocytogenes L monocytogenes Lb plantarum L monocytogenes L inn
464. sis Surface contact angle and colour parameters of cinnamaldehyde imine chitosan films and these films subjected to different treatments were also evaluated The antimicrobial properties of chitosan Schiff base films were tested in vitro against Staphylococcus aureus and Escherichia coli and in milk inoculated with Listeria monocytogenes The antimicrobial activity varied depending on the treatment applied and consequently the degree of imino bond hydrolysis achieved and cinnamaldehyde released Films of Schiff base chitosan derivative subjected to different time temperature treatments inhibited the growth of L monocytogenes for 12 days under refrigeration conditions which may extend the microbiological shelf life of such products Sensory analysis of milk in contact with the films showed that a cinnamon smell does not cause any rejection among potential consumers These novel films could be used in the design of antimicrobial food packaging and in various other technological areas where sustained release systems are required Keywords Chitosan cinnamaldehyde Schiff base hydrolysis release antimicrobial films 138 Quitosano Cinamaldeh do 1 Introduction CS is a natural biocompatible biodegradable biorenewable and biofunctional polysaccharide that is finding attractive applications in several industrial areas CS produces highly transparent grease resistant films with excellent barrier properties to gases and aroma compounds T
465. stema antimicrobiano desarrollado consisti en la formaci n de pel culas de CS con nanopart culas de plata En este caso se emple una sal inorg nica de plata nitrato de plata como agente precursor de las nanopart culas de plata Esta sal se incorpor en la soluci n formadora de las pel culas y las nanopart culas se obtuvieron durante la neutralizaci n 275 Discusi n general de las pel culas de CS con hidr xido de sodio La matriz de CS actu como agente quelante de los iones plata de la sal como agente reductor d bil en condiciones alcalinas estabilizante soporte y portador de las nanopart culas formadas y gener la liberaci n sostenida en el tiempo de los iones plata que se formaron en la superficie de las nanopart culas tras su oxidaci n La formaci n de las nanopart culas en la matriz de CS permiti una liberaci n m s lenta de los iones plata Cabe resaltar que el hidr xido de sodio empleado como agente reductor es el habitualmente utilizado en este proceso de neutralizaci n de las pel culas de CS en el que se aumenta el pH del medio por encima del pK del CS y se estabilizan las pel culas en medios acuosos Por lo que se evita recurrir a otros reductores m s t xicos empleados com nmente en la s ntesis de nanopart culas Esta cuesti n entre otras expuestas en el estado de la cuesti n del cap tulo 2 hace que esta s ntesis in situ de nanopart culas de plata se enmarque en la denominada Qu mica
466. stored at 4 C Meat Science 71 92 99 Marcos B Aymerich T Monfort J M Garriga M 2007 Use of antimicrobial biodegradable packaging to control Listeria monocytogenes during storage of cooked ham International Journal of Food Microbiology 120 152 158 Martin E M Griffis C L Vaughn K L S O Bryan C A Friedly E C Marcy J A Ricke S C Crandall P G Lary R Y 2009 Control of Listeria monocytogenes by lauric arginate on frankfurters formulated with or without lactate diacetate Journal of Food Science 74 M237 M241 McDonnell G Russell A D 1999 Antiseptics and disinfectants activity action and resistance Clinical Microbiology Reviews 12 147 Nikaido H Vaara M 1985 Molecular basis of bacterial outer membrane permeability Microbiological Reviews 49 1 32 Oh D H Marshall D L 1992 Effect of pH on the minimum inhibitory concentration of monolaurin against Listeria monocytogenes Journal of Food Protection 55 449 450 OMS 2009 Safety evaluation of certain food additives WHO food additives series 60 39 84 Ouattara B Simard R E Piette G Begin A Holley R A 2000a Diffusion of acetic and propionic acids from chitosan based antimicrobial packaging films Journal of Food Science 65 768 773 Ouattara B Simard R E Piette G Begin A Holley R A 2000b Inhibition of surface spoilage bacteria in processed meats by application of antimicrobial films prepared with
467. su parte como era de esperar la presencia de G y agua en las pel culas de CS aument los valores de permeabilidad al vapor de agua y al ox geno y de elongaci n mientras que disminuy su resistencia a la rotura La incorporaci n de CD increment ligeramente la sorci n de carvacrol Sin embargo cuando la pel cula adem s de incorporar CD se plastific con G y agua se produjo un elevado incremento en la sorci n Las pel culas que presentaron una mayor capacidad de sorci n fueron las plastificadas con 20 35 de G y acondicionadas a 75 de RH y aquellas con un 3596 G acondicionadas a 5096 RH con valores de 57 216 13396 g 100 g de pel cula seca respectivamente Mientras que todas las pel culas de CS sin CD retienen menos del 1 de carvacrol independientemente del G y del agua que contengan Te ricamente el contenido de carvacrol en las pel culas con CD puede ser entre 5 1096 asumiendo la formaci n de complejos de inclusi n 1 1 o 1 2 Puesto que diversas pel culas presentaron valores de sorci n mayores al 1096 de carvacrol se deduce que se estar an produciendo otros mecanismos diferentes a la formaci n de complejos de inclusi n como la formaci n de complejos supramoleculares de ciclodextrinas El agua y el G incrementar an la movilidad de las cadenas polim ricas y por tanto el volumen libre de las pel culas permitiendo la autoagregaci n de las ciclodextrinas Esta disposici n de las ciclodextrinas podr a au
468. sus diferencias estructurales Kong et al 2010 aunque el mecanismo de acci n a n hoy es confuso e incluso contradictorio seg n el estudio de diversos autores El CS interact a electrost ticamente con las cargas negativas de la membrana plasm tica celular bacteriana desestabiliz ndola alterando su permeabilidad seguido de su adhesi n al ADN celular inhibiendo la s ntesis de ARNm y prote nas e impidiendo su replicaci n Coma 2012 Dutta et al 2009 Shahidi et al 1999 En el caso de las gram positivas puede que el CS forme una membrana polim rica en la superficie celular inhibiendo la entrada de nutrientes a la c lula bacteriana o interaccione con las cargas negativas de los grupos fosforilo de cidos teicoicos y teicur nico Cuando se trata de gram negativas el CS puede 12 Introducci n competir con el lipopolisac rido y prote nas de la superficie por los cationes divalentes que estabilizan la membrana externa alterando la funci n celular o provocando incluso la lisis celular aunque actualmente se acepta m s que el CS tiene actividad bacteriost tica m s que bacteriocida Adem s el CS de menor peso molecular penetra en la c lula bacteriana por difusi n alterando el metabolismo celular Dutta et al 2009 Kong et al 2010 No et al 2007 Existen varios factores que afectan a la capacidad antimicrobiana del CS su grado de desacetilaci n peso molecular concentraci n y el pH del medio Dutta et aL 2
469. t al 2012 Jouki Mortazavi et al 2014 Abdollahi et al 2012 Altiok et al 2010 Ruiz Navajas et al 2013 Giatrakou et al 2010 Jiang et al 2012 Ouattara et al 2001 Win et al 2007 Kechichian et al 2010 Rojhan y Nouri 2013 Norajit y Ryu 2011 Anexos ED hoja de olivo ED polen fermentado ED semilla de pomelo ED t verde ED prop leos ED semilla de pomelo ED semilla de pomelo ED semilla de pomelo ED semilla de pomelo ED semilla de pomelo EDTA ED semilla de pomelo ED semilla de pomelo ED semillas de uva ED semilla de uva EDTA ED semilla de uva nisina ED semilla de uva EDTA nisina ED semilla de uva nisina ED t verde nisina ED semilla de uva c m lico EDTA nisina ED semilla de uva ED t verde ED t verde ED t verde alil isotiocianato alil isotiocianato nisina alil isotiocianato alil isotiocianato alil isotiocianato nisina benzaldeh do 4 dimetilamino metilcelulosa alga roja Gelidium corneum gelatina HPMC alga roja Gelidium corneum alga roja Gelidium corneum prote nas del suero de leche alga roja Gelidium corneum alginato de sodio K carragenato gelatina prote na de semilla de colza gelatina prote na de salvado de cebada almid n de guisante prote na de soja prote na de soja prote na de suero de leche quitosano almid n de tapioca goma de hoja d
470. t al 2012 127 Cap tulo 3 Estado de la cuesti n matriz mejorando las propiedades del biopol mero a la vez que le confieren capacidad antimicrobiana como se observa en los trabajos de Balaguer et al 2011a b Balaguer L pez Carballo et al 2013 Balaguer Borne et al 2013 y Balaguer et al 2014 en los que se modifican qu micamente gliadinas con cinamaldeh do o en el de Soliman et al 2014 en el que se modifica ze na con el mismo fin El cinamaldeh do debido a su estructura presenta una elevada reactividad Su grupo carbonilo C 0 proporciona un lugar de reacci n para la adici n nucleof lica debido principalmente a la capacidad del ox geno para aceptar una carga negativa Por ello es posible lograr la uni n covalente del cinamaldeh do con el CS dando como resultado la formaci n de compuestos iminoderivados del CS del tipo N acilado mediante la formaci n de la base de Schiff En la bibliograf a se han descrito diversas bases de Schiff formadas entre el CS y diferentes aldeh dos y m s concretamente entre el CS y el cinamaldeh do Babu y Kannan 2012 Badawy y Rabea 2013 2014 Han y Lin 2012 Le y Dupuis 2009 Sin embargo en la mayor parte de estos casos la base de Schiff se emplea como puente entrecruzante producto intermedio a cuaternizar grupo protector en alguna etapa intermedia de otro proceso modificaci n para desarrollar derivados quelantes para la sorci n de metales en la determ
471. t the end of the incubation period the antimicrobial activity of the volatile liquids was determined by measuring the diameter of the zone on the surface of the agar where there was no microbial growth The minimum inhibitory dose is 252 Quitosano Carvacrol defined as the lowest amount of active compound that yields inhibition of microbial growth on the agar surface Each assay was performed in triplicate 2 6 3 Antimicrobial activity of the films The procedure for determining the antimicrobial activity of the films was similar to that described above In this case films which were of the same size as the filter papers and loaded with the compound were placed on the centre of the Petri lid After the incubation period the diameter of the resulting inhibition zone was measured Each assay was performed in triplicate 2 7 Data analysis The data are represented as average standard deviation The data were graphically plotted with SigmaPlot software Systat Software Inc Richmond CA USA 3 Results and discussion Films based on CS or PVOH as the polymer matrix incorporating CD at a 1 1 weight ratio and plasticized with G or PG were successfully obtained by casting To the naked eye the films were homogeneous with smooth surfaces very transparent and easy to handle The thickness of the films was about 55 5 um 3 1 Equilibrium moisture content of the films A study was made of the effect of incorporating CD and the polar
472. t there was complete miscibility of the various volatile liquids in PG resulting in a single liquid layer Complete miscibility was also observed for G and o cumenol m cumenol and guaiacol Carvacrol and G were partially miscible and complete immiscibility was observed for the remaining compounds The miscibility studies showed that PG has greater compatibility with all the compounds tested whereas the greater polarity of G limits its compatibility with them This feature could modify the sorption properties of the films depending on the plasticizer used Table 4 4 11 shows the solubility parameters of monoterpenes It is expected that the closer the parameters for a sorbent and a polymer the greater the sorption affinity will be Hydrophilic components of the films CS PVOH G PG and water 38 25 8 29 1 33 5 30 1 and 47 9 respectively had high values of 6 compared with those of the monoterpenes This means that phenolic monoterpenes with 5 values closer to those of the film components ranging from 23 6 to 26 5 MPa are expected to be retained in the films to a greater extent than cyclic and linear non phenolic monoterpene alcohols with lower solubility parameters comprised between 19 3 and 21 MPa Absorption of volatile liquids in a film will be affected by chemical affinity between the sorbate and the film components Sorption properties of the films for the various compounds are shown in Table 4 4 12 A common feature for al
473. t various RH According to the results obtained changes in colour parameters were not significant for films retaining less than 6 of carvacrol 3 2 Sorption properties 3 2 1 Sorption equilibrium of carvacrol in films As Figure 4 4 4 shows the amount of carvacrol retained in the films depended on the level of plasticization by G and moisture Carvacrol 206 Quitosano Carvacrol 1000 40 z gt r z z 5 30 100 2 ES 5 ES 8 S El 5 20 5 E 2 23 5 o 2 Lio E 2 E 17 2 3 un a S NS AS S S SU So E E ge E ARS AS amp co e e e o 5 ed oS Se S 2 Ko Ke QC S gt gt C o OU dO eO GO ux PO uu uc Film Figure 4 4 4 Sorption equilibrium of carvacrol in CS CD films with a 1 1 w w blend ratio incorporating several percentages of G and conditioned at different RH prior to immersion in the solvate and measured at 25 C Figure also shows the equilibrium moisture content of the films at 25 C before being immersed in carvacrol retention in films with a fixed amount of G increased as the RH at which they were conditioned increased water acts as a plasticizer for CS CD films enhancing their carvacrol loading capacity It can also be observed that at a fixed RH carvacrol sorption increased with the G content in the film The highest carvacrol loading capacity was achieved by films plasticized with 3596 G and conditioned at 7596 RH followed by the same films conditioned at 5396 RH
474. ta o en la cantidad de plata que emigr en los medios l quidos despu s de 18 h de inmersi n de la pel cula Las pel culas neutralizadas mantuvieron su actividad tras 1 mes inmersas en agua desionizada lo cual puede atribuirse a la liberaci n sostenida de iones de plata y por lo tanto la eficacia a lo largo del tiempo Palabras clave S ntesis nanopart culas de plata quitosano actividad antimicrobiana 103 Cap tulo 2 Art culo cient fico 2 Abstract This study aims to develop antimicrobial films consisting of chitosan CS and silver nanoparticles that are homogeneously distributed throughout the polymer matrix Nanoparticles were generated in situ during the neutralization of the chitosan acetate film with sodium hydroxide The temperature of neutralization and the concentration of silver in the film were crucial determinants of the shape and size of the nanoparticles Neutralized films exhibited antimicrobial activity against Escherichia coli and Staphylococcus aureus in liquid growth media However the effectiveness of the films was considerably greater in diluted growth media Furthermore no significant differences were found either in the antimicrobial capacities of films incorporating different amounts of silver or in the amount of silver that migrated into the liquid media after 18 h of immersion of the film Neutralized films maintained their activity after 1 month of immersion in deionized water which can be attribu
475. tar a mi lado siempre brind ndome vuestra alegr a carifio paciencia y comprensi n Os quiero con locura a los cuatro Gracias a los cuatro por apoyarme escucharme y ayudarme en la toma de decisiones y confiar ciegamente en m Y perdonarme por robaros tanto tiempo Gracias a todos por estar ah por concederme vuestro apoyo para conseguir una meta m s Os doy las gracias a todos porque todos hac is un pedacito de m Resumen En esta Tesis se ha abordado el desarrollo de pel culas renovables y activas de quitosano con agentes antimicrobianos de diversa naturaleza para su aplicaci n en el envasado activo de alimentos Las pel culas de quitosano se obtuvieron mediante la t cnica de extensi n y evaporaci n del solvente y las sustancias bioactivas fueron incorporadas mediante diferentes metodolog as En primer lugar el s lido etil N dodecanoil L arginato LAE se incorpor previamente a la formaci n de la pel cula de quitosano Las pel culas con LAE liberaron completamente el agente activo en un simulante alimentario acuoso en varias horas a diferentes temperaturas Las pel culas de quitosano que no fueron neutralizadas presentaron actividad antimicrobiana frente a microorganismos presentes en pechugas de pollo frescas Esta actividad antimicrobiana se increment al incorporar el LAE a la matriz reduciendo satisfactoriamente los microorganismos presentes en el alimento para el control del deterioro microbiano de po
476. tarch Lwt Food Science and Technology 43 1088 1094 Kim K M Lee B Y Kim Y T Choi S G Lee J Cho S Y Choi W S 2006 Development of antimicrobial edible film incorporated with green tea extract Food Science and Biotechnology 15 478 481 Kim K W Daeschel M Zhao Y 2008 Edible coatings for enhancing microbial safety and extending shelf life of hard boiled eggs Journal of Food Science 73 M227 M235 Ko S Janes M E Hettiarachchy N S Johnson M G 2001 Physical and chemical properties of edible films containing nisin and their action against Listeria monocytogenes Journal of Food Science 66 1006 1011 Kristo E Koutsoumanis K P Biliaderis C G 2008 Thermal mechanical and water vapor barrier properties of sodium caseinate films containing antimicrobials and their inhibitory action on Listeria monocytogenes Food Hydrocolloids 22 373 386 Ku K Bin Song K 2007 Physical properties of nisin incorporated gelatin and corn zein films and antimicrobial activity against Listeria monocytogenes Journal of Microbiology and Biotechnology 17 520 523 Lee H Min S C 2013 Antimicrobial edible defatted soybean meal based films incorporating the lactoperoxidase system Lwt Food Science and Technology 54 42 50 Lee H Min S C 2014 Development of antimicrobial defatted soybean meal based edible films incorporating the lactoperoxidase system by heat pressing Journal of Food Engineering 120 183 1
477. te estabilizador La elecci n de cada uno de ellos as como del m todo de obtenci n determinar las caracter sticas propiedades y estabilidad de las nanopart culas de plata que se generen Si bien es posible la formaci n de nanopart culas de plata mediante la reducci n de su forma i nica a trav s de m todos foto sono y electroqu micos f sicos y biol gicos etc el m todo m s utilizado es la v a h meda mediante reducci n qu mica con agentes reductores tales como borohidruro de sodio citrato dimetilformida hidracina ascorbato hidr geno elemental etc Siendo el nitrato de plata la sal precursora m s empleada Sau y Rogach 2010 Sharma et al 2009 Tolaymat et al 2010 Inicialmente se reduce el ion de plata Ag que permite la formaci n de tomos de Ag libres A continuaci n se agregan en pequefios grupos de 2 10 tomos clusters en los que coexisten tomos e iones plata Finalmente estos n cleos crecen se reducen completamente y pueden dar lugar a la formaci n de part culas coloidales de plata Ershov 1999 Sau y Rogach 2010 Esto en la bibliograf a se describe como el enfoque bottom up en el que las unidades m s peque as iones tomos y mol culas se ensamblan para formar estructuras nanom tricas Figura 4 2 1 en contraposici n al enfoque top down que parte de entidades m s grandes y reduce su tama o hasta obtener el rango nanom trico Sau y Rogach 2010 Tolaymat et al 2010 Nuclea
478. ted the following colour parameters L 88 6 0 8 a 2 38 0 1 b 23 65 1 2 Cop 23 7 1 1 and Ngp 84 2 0 4 results not included in Table 4 3 4 3 1 3 Water uptake and dimensional stability CS is a hydrophilic polymer that presents a high water retention capacity Therefore the effect of grafting cinnamaldehyde on CS films on the water sorption and dimensional stability of the resulting films and also of films subjected to preservation treatments were studied For this purpose films were immersed in two buffered media at pH 3 and 7 for 24 h The results are shown in Table 4 3 5 The pK of CS is 6 5 6 3 at lower pH values protonation of amino groups increases water uptake and swelling properties of the film until complete protonation of amino groups is achieved and the film dissolves As the results in Table 4 3 5 show CS films absorb a great amount of water at pH 3 since high ionization of amino groups produces electrostatic repulsion between polymer segments allowing film swelling and large water gain which subsequently practically doubled their surface area Grafting of cinnamaldehyde to CS films did not modify water uptake and dimensional stability when parameters were measured at pH 3 At this pH a high degree of protonation is achieved Acidic pHs favour Schiff base 149 Cap tulo 3 Art culo cient fico 3 Table 4 3 5 Water uptake and dimensional stability of films pH 3 pH 7 Water uptake Area increase Water uptake Area i
479. ted to the slow sustained release of silver ions and thus efficacy over time Keywords Synthesis silver based nanoparticles chitosan antimicrobial activity 104 Quitosano Nanopart culas de plata 1 Introduction Silver ions have long been recognized as an effective biocide against a broad spectrum of microorganisms their inhibition mechanism being the subject of considerable research Musarrat et al 2010 Petrus et al 2011 Yamanaka et al 2005 Ionic silver can exert its antimicrobial action in several ways It has been reported to complex with the thiol groups of enzymes and proteins altering their structure and function Liau et aL 1997 Silver ions have also been found to bind with DNA and cause structural changes in the cell envelope and cytoplasmic membrane of bacteria Kim and Ahn 2008 Yakabe et al 1980 Ionic silver has been used as an antimicrobial in the form of salts and more recently it has been incorporated into inorganic materials such as zeolites zirconium phosphate and glass Guerra et al 2012 Huang et al 2012 A new approach is the development of silver nanoparticles that can act as nanoreservoirs for the delivery of silver ions ensuring their availability in the substrate over time There is a great variety of chemical and physical processes for the synthesis of silver nanoparticles most of them involving the formation of colloidal nanoparticles or their incorporation in other materials Rai et
480. tema Las pel culas de CS con CD y 50 de G tambi n fueron capaces de retener elevada cantidad de vol tiles naturales aunque algo menos que las pel culas que incorporaron el 35 de G Esto puede ser debido a que una mayor cantidad de G un componente hidr filo en la matriz propicie un entorno m s hidr filo y menos compatible con los vol tiles de car cter lip filo Asimismo se comprob si el sistema desarrollado era factible con otros hidrogeles hidrof licos y otros plastificantes Para ello se estudi la capacidad de sorci n de los diferentes vol tiles por parte de hidrogeles como el CS o PVOH con CD y con G o PG Todos los sistemas fueron formulados y acondicionados a una determinada RH para que captaran 30 35 de agua En este caso un contenido similar de agua e igual de CD en las pel culas permiti observar el efecto del tipo de matriz y plastificante en la capacidad de sorci n de componentes presentes en AE Se escogi el PVOH por ser al igual que el CS un pol mero no t xico biocompatible biodegradable y con excelente capacidad film gena Adem s se trata de un pol mero sint tico y comercializado ya que est 283 Discusi n general aprobado por la FDA como aditivo alimentario indirecto para productos que est n en contacto con alimentos Debido a la semicristalinidad y elevada energ a de cohesi n del PVOH fue necesario acondicionar las pel culas de PVOH con CD y 3596 de G a 8496 de RH para que inc
481. terias sulfuro productoras luminiscentes LAB Pseudomonas spp Enterobacteriaceae ME TVC TVC mes filos bacterias sulfuro productoras luminiscentes LAB Pseudomonas spp Enterobacteriaceae ME TVC mes filos y psicr filos LAB Pseudomonas spp Enterobacteriaceae E coli O157 H7 L monocytogenes St aureus ME TVC coliformes hongos levaduras E coli O157 H7 S Enteriditis St aureus L innocua ME TVC psicr filos hongos levaduras ME TVC mes filos y psicr filos LAB Enterobacteriaceae Colletotrichum gloeosporioides Rhizopus stolonifer Rhizopus stolonifer E coli Botrytis cinerea P digitatum E coli S Enteritidis St aureus L monocytogenes E coli O157 H7 L monocytogenes S Enteritidis E coli St aureus B cereus S Enteriditis S Typhimurium E coli O157 H7 ME TVC L innocua Ps fluorescens Aeromonas hydrophila caviae L monocytogenes S Typhimurium B cereus Yersinia enterocolitica Ps aeruginosa St aureus E coli E coli O157 H7 Shewanella putrefaciens Vibrio cholera Lb plantarum ME TVC mes filos y psicr filos Pseudomonas spp bacterias sulfuro Salgado et al 2013 Fern ndez Pan et al 2014 Fern ndez Pan et al 2013 Torlak y Nizamlioglu 2011 Jagannath et al 2006 Maizura et al 2008 Maizura et al 2007 Rojas Gra Raybaudi Massilia et al 2007 Alparslan et al 2014 Bosquez M
482. th hydrophobic molecules host guest complexation being the most common MDs conformation goes from flexible coil to helix as the dextrose equivalent DE number decreases The inside of the helical structure is hydrophobic as in CDs but more flexible than the cavity of them which means less steric hindrance As in the case of CDs in addition to hydrophobic interactions MDs participate in hydrogen bonding with guest molecules Garnero et al 2013 In an attempt to establish the effect of the molecular shape of oligosaccharides on the carvacrol sorption capacity of CS films non cyclic oligosaccharide MDs were incorporated in the CS matrix as an alternative to HP BCDs and carvacrol sorption properties were studied Figure 4 4 6 shows the sorption equilibrium of carvacrol in films formulated with CS and MDs at a ratio of 1 1 by weight plasticized with 3596 G and conditioned at 0 53 and 7596 RH prior to immersion in carvacrol In no case did sorption of carvacrol exceed 1 596 These results show the effect of the molecular geometry of low molecular weight starch derivatives incorporated in CS films on the carvacrol loading capacity 209 Cap tulo 4 Art culo cient fico 5 0 8 4 0 6 4 0 4 4 Sorption equilibrium of carvacrol 26 0 2 4 0 0 o 53 75 Relative humidity Figure 4 4 6 Carvacrol sorption capacity by films of CS incorporating MDs 1 1 weight ratio Films were plasticized with 35 G and conditione
483. the packaging headspace chicken system Fresh chicken breast fillets are commonly packaged in trays closed with a thermosealable lid and distributed and commercialized under refrigeration The materials used for the design of these trays are variable polyethylene terephthalate PET expanded polystyrene PS or PP laminated or coextruded to a polyamide polyethylene PA PE or EVOH PP but since the product is normally preserved in a modified atmosphere materials that provide a medium to high barrier to oxygen and carbon dioxide are commonly employed These materials present this barrier because of a combination of properties that might include high crystallinity low free volume adequate chain rigidity and high interchain cohesive energy With these properties the materials also provide a high to very high barrier to organic compounds such as food aroma components vitamins fats or organic acids Gavara and Catal 2002 To resemble real behaviour a very high barrier package consisting of a tray and a lid was selected for this study The thermoformed tray was obtained from a PP EVOH PP coextruded sheet with an initial thickness of 750 um as measured in the top flat sealing area To assess the structure and dimensions of the layers along the tray profile a sample was cut diagonally immersed in an aqueous solution of cresol red to tint the EVOH layer and observed under magnifying lenses The average thickness of the PP layers was 200 20 u
484. the conditions set out in Regulation 10 2011 EC Commission Regulation EU of 14 January 2011 on plastic materials and articles intended to come into contact with food After temperature time treatments the films were stored at room temperature and liquid aliquots were extracted at several times during 1 h 48 h for the refrigerated sample As Figure 4 3 4 reveals the release profile of cinnamaldehyde over time depended on the treatment CScin films processed at low temperature present a very low release ca 1 mg L after the treatment but the amount released increases with time reaching ca 70 mg L after 48 h The treatments at 65 72 and 95 C yielded much higher release values 280 mg L without differences between treatments It should also be noted that the cinnamaldehyde released does not change significantly with time during the storage period studied indicating that probably all the free cinnamaldehyde present in the matrix owing to hydrolysis of the imino bond was released during the treatments The films subjected to the sterilization treatment released the highest concentration of cinnamaldehyde with values of ca 700 mg L after treatment However the concentration of the agent presented a decreasing trend during storage Since the measured concentration indicates the cinnamaldehyde molecules that had already moved out of the film a rebuild of the Schiff base is certainly unexpected Most probably the decrease in concentration was
485. the first step a loopful of each strain was transferred to 10 mL of TSB and incubated at 37 C overnight to obtain early stationary phase cells 2 6 2 Determination of minimal inhibitory dose of each volatile liquid in vapour phase The microatmosphere method was selected to carry out an antimicrobial test in which no direct contact between the device containing the volatile and the agar medium is necessary for the former to exert its activity In this method the volatile compound migrates from the carrier filter paper film to the headspace of the Petri dish thus becoming available to contact the growth medium and the microorganism For this purpose 100 uL of a bacterium suspension containing approximately 10 colony forming units CFU mL was spread over the surface of 90 mm diameter Petri dishes containing approximately 15 mL of solid culture TSA medium Decreasing quantities of each volatile liquid were dissolved in DMSO and a final volume of 10 uL of the solution was added to 25 mm diameter sterilized filter paper which was placed in the centre of the Petri lid Blanks were prepared by adding 10 uL of DMSO to 25 mm diameter sterile filter disks having previously checked that the DMSO did not have any antibacterial effect against any of the microorganisms tested Once the Petri dishes and lids had been assembled the units were sealed with Parafilm to reduce leakage of the volatile agent and incubated upside down at 37 C for 24 h A
486. the present work the experimental Hildebrand solubility parameter of PVOH ranges from 25 8 to 29 1 MPa compared with 38 MPa for CS whereas 6 for water is 47 9 MPa which is in accordance with the lower moisture sorption of PVOH films and the greater RH required to make their water content equal to that of CS films A further factor to be taken into 254 Quitosano Carvacrol Table 4 4 11 Physico chemical properties and molecular structure of volatile liquids plasticizers and polymers Vapour Molecular Molecular structure Log P pressure 5 MPa mass Pa OH TN b m cumenol A ch 136 19 2 82 6 67 24 1 CH3 OH CH o cumenol TR CH HET 2 82 14 93 241 icd OH CH3 carvacrol m 150 22 3 28 4 00 23 6 CH OH 5 OCHs b guaiacol es 124 14 1 19 23 86 26 5 xa OH OCH E isoeugenol d 164 20 245 0 67 24 3 d CHa OH myrtenol 49 152 23 3 22 2 40 21 0 Ho CH3 b nerol 154 25 3 28 1 73 20 9 HaC CHa CHa A OH carveol 152 23 2 55 1 60 19 7 HCW SCH CHa OH dihydrocarveol 154 25 2 92 2 40 19 3 HaC CH CH isopulegol Sr sen 154 25 2 92 13 20 19 3 HaC CH glycerol albo de 92 09 1 84 lt 0 01 33 5 CH pedos Ho j 76 09 105 27 20 30 1 glycol water 18 02 1 38 3263 01 47 9 50 190 b CS KDa 0 01 38 from 25 8 prod 5 i to 29 1 Note molar volume and log P predicted from ACD Labs log P hydrophobicity of the molecule expressed as the logarithm of octanol wate
487. their antibacterial properties and potential applications in foods A review International Journal of Food Microbiology 94 223 253 Caner C Vergano P J Wiles J L 1998 Chitosan film mechanical and permeation properties as affected by acid plasticizer and storage Journal of Food Science 63 1049 1053 Chalier P Ben Arfa A Preziosi Belloy L Gontard N 2007 Carvacrol losses from soy protein coated papers as a function of drying conditions Journal of Applied Polymer Science 106 611 620 G llstedt M Hedenqvist M S 2006 Packaging related mechanical and barrier properties of pulp fiber chitosan sheets Carbohydrate Polymers 63 46 53 Kjellgren H G llstedt M Engstr m G J rnstr m L 2006 Barrier and surface properties of chitosan coated greaseproof paper Carbohydrate Polymers 65 453 460 Kurek M Descours E Galic K Voilley A Debeaufort F 2012 How composition and process parameters affect volatile active compounds in biopolymer films Carbohydrate Polymers 88 646 656 Locci E Lai S Piras A Marongiu B Lai A 2004 13C CPMAS and H NMR Study of the inclusion complexes of B cyclodextrin with carvacrol thymol and eugenol prepared in supercritical carbon dioxide Chemistry amp Biodiversity 1 1354 1366 Mensitieri G Di Maio E Buonocore G G Nedi L Oliviero M Sansone L lannace S 2011 Processing and shelf life issues of selected food packaging materials and s
488. thermal degradation and an advance of 10 C in HP BCD degradation These observations are indicative of a certain degree of interaction between the two components The addition of 3596 G to the CS dm dT 200 400 600 800 T C Figure 4 4 1 Temperature derivative of sample mass obtained by TGA for selected composites and components 184 Quitosano Carvacrol matrix reduced the temperature at which maximum weight loss was measured from 289 to 282 C 285 C with 20 G thermogram not shown A previous transition at 212 C is due to the degradation of G Finally the composite films plasticized with 2096 G showed the three transitions which could be expected from the addition rule In contrast the sample with 3596 G presented only two transitions as can be seen in Figure 4 4 1 The transition assigned to the HP BCDs appeared at the same temperature as in the unplasticized film However a new intermediate feature appeared between those of CS and G indicating that the two compounds degrade and volatilize together 3 4 Moisture content The properties and behaviour of the composites were expected to be dependent on environmental humidity as occurs with most hydrocolloid based films The water gained by the composites was measured at three RHs 53 75 and 9096 Table 4 4 2 resumes the results for various films and components including bibliographic data for G Bell and Labuza 2000 HP BCDs showed the lowest wate
489. this process has certain drawbacks when working with volatile compounds because of the loss of volatiles during film processing In order to overcome this drawback recent studies have reported the incorporation in CS of previously encapsulated essential oils Abreu et aL 2012 Higueras et al 2013 Hosseini et al 2013 There are some studies devoted to the synthesis of Schiff base from CS and the potential antimicrobial activity of the derivatives obtained Dos Santos et al 2005 Guinesi and Cavalheiro 2006 Guo et al 2007 Jin 2010 Wang et al 2012 However none of these studies focus on the reversibility of the Schiff base and its effect on the antimicrobial results obtained Hydrolysis of the imino 139 Cap tulo 3 Art culo cient fico 3 bond C N formed in the Schiff base can be promoted by temperature UV light pH etc or combinations of them Huang et al 2001 Kirdant et aL 2011 Li et al 2013 The formation of a reversible Schiff base in the backbone of polymer films could have great potential in the design of sustained release systems Cinnamaldehyde is a naturally occurring aromatic o B unsaturated aldehyde derived from cinnamon and approved by the FAO WHO Expert Committee on Food Additives JECFA for use as a food flavouring agent and it is the main component of cinnamon bark extract Cocchiara et al 2005 Cinnamaldehyde is a well known natural antimicrobial compound active against a wide spectrum of foo
490. tial boundary conditions t 0 0O lt x lt L C C t gt 0 x 0 x L C CS where c is the initial concentration of sorbate in the polymer cy 0 and c is the concentration of the sorbate in both surfaces of the plane sheet which is assumed to be constant throughout the experiment the solution under these conditions is e 2 B 8 1 n D 2n 1 t nes 1 7 22 lc 1 exp D Hy where L is the thickness of the film m and D the diffusion coefficient m s Crank 1975 Figure 4 4 7 shows that sorption uptake data were well fitted 211 Cap tulo 4 Art culo cient fico 5 Table 4 4 7 Power law parameters and diffusion coefficients of carvacrol in CS CD films at 25 C K min 10 R POVO 10 R CS CD 35G ORH 0 26 0 09 0 52 0 04 0 988 0 0110 0 0005 0 990 CS CD 35G 53RH 0 98 0 119 0 56 0 03 0 987 0 22 0 02 0 992 CS CD 35G 75RH 3 37 0 82 0 55 0 05 0 951 1 90 0 08 0 988 CS CD 20G 75RH 2 86 0 85 0 59 0 06 0 988 2 10 0 13 0 981 Different letters in the same column indicated significant differences P lt 0 05 by Equation 2 Table 4 4 7 shows values of diffusion coefficients for films plasticized with 35 G and conditioned at 0 53 and 75 RH prior to immersion in carvacrol and films plasticized with 20 G and conditioned at 75 RH The diffusion coefficient of carvacrol in films incorporating 35 G and conditioned at different RH increased as the water content increased Diffusio
491. tin based edible films enriched with laurel essential oil on the quality of rainbow trout Oncorhynchus mykiss fillets during refrigerated storage Food Technology and Biotechnology 52 325 333 Altiok D Altiok E Tihminlioglu F 2010 Physical antibacterial and antioxidant properties of chitosan films incorporated with thyme oil for potential wound healing applications Journal of Materials Science Materials in Medicine 21 2227 2236 lvarez M V Ponce A G Moreira M D 2013 Antimicrobial efficiency of chitosan coating enriched with bioactive compounds to improve the safety of fresh cut broccoli Lwt Food Science and Technology 50 78 87 Appendini P Hotchkiss J H 1997 Immobilization of lysozyme on food contact polymers as potential antimicrobial films Packaging Technology and Science 10 271 279 Arfat Y A Benjakul S Prodpran T Sumpavapol P Songtipya P 2014 Properties and antimicrobial activity of fish protein isolate fish skin gelatin film containing basil leaf essential oil and zinc oxide nanoparticles Food Hydrocolloids 41 265 273 Arismendi C Chillo S Conte A Del Nobile M A Flores S Gerschenson L N 2013 Optimization of physical properties of xanthan gum tapioca starch edible matrices containing potassium sorbate and evaluation of its antimicrobial effectiveness Lwt Food Science and Technology 53 290 296 Arrieta M P Peltzer M A L pez J Garrigos M D Valente AJ M
492. tings and cryogenic freezing on survival and growth of Listeria innocua on frozen ready to eat shrimp during thawing Journal of Food Science 78 M1195 M1200 Guo M M Jin T Z Wang L X Scullen O J Sommers C H 2014 Antimicrobial films and coatings for inactivation of Listeria innocua on ready to eat deli turkey meat Food Control 40 64 70 Guo M M Jin T Z Yang RJ 2014 Antimicrobial polylactic acid packaging films against Listeria and Salmonella in culture medium and on ready to eat meat Food and Bioprocess Technology 7 3293 3307 Jin T Z Gurtler J B Li S Q 2013 Development of antimicrobial coatings for improving the microbiological safety and quality of shell eggs Journal of Food Protection 76 779 785 Kang J Stasiewicz MJ Murray D Boor KJ Wiedmann M Bergholz T M 2014 Optimization of combinations of bactericidal and bacteriostatic treatments to control Listeria monocytogenes on cold smoked salmon International Journal of Food Microbiology 179 1 9 Loeffler M McClements D J McLandsborough L Terjung N Chang Y Weiss J 2014 Electrostatic interactions of cationic lauric arginate with anionic polysaccharides affect antimicrobial activity against spoilage yeasts Journal of Applied Microbiology 117 28 39 Luchansky J B Call J E Hristova B Rumery L Yoder L Oser A 2005 Viability of Listeria monocytogenes on commercially prepared hams surface treated with acidic calcium
493. tion Colloids and Surfaces B Biointerfaces 76 248 258 Wang X Du Y M Liu H 2004 Preparation characterization and antimicrobial activity of chitosan Zn complex Carbohydrate Polymers 56 21 26 Win N K K Jitareerat P Kanlayanarat S Sangchote S 2007 Effects of cinnamon extract chitosan coating hot water treatment and their combinations on crown rot disease and quality of banana fruit Postharvest Biology and Technology 45 333 340 329 Anexos Wu J Ge S Liu H Wang S Chen S Wang J Li J Zhang Q 2014 Properties and antimicrobial activity of silver carp Hypophthalmichthys molitrix skin gelatin chitosan films incorporated with oregano essential oil for fish preservation Food Packaging and Shelf Life 2 7 16 Xiao W Xu J B Liu X Y Hu Q L Huang J G 2013 Antibacterial hybrid materials fabricated by nanocoating of microfibril bundles of cellulose substance with titania chitosan silver nanoparticle composite films Journal of Materials Chemistry B 1 3477 3485 Xing Y G Xu Q L Che Z M Li X H Li W T 2011 Effects of chitosan oil coating on blue mold disease and quality attributes of jujube fruits Food amp Function 2 466 474 Xu W T Huang K L Guo F Qu W Yang J J Liang Z H Luo Y B 2007 Postharvest grapefruit seed extract and chitosan treatments of table grapes to control Botrytis cinerea Postharvest Biology and Technology 46 86 94 Yener
494. tions 36 h 40 0 1 5 C and 20 9 RH After peeling the films from the tray samples measuring 550 mm in diameter and 5545 pum in thickness using a digital micrometer Mitutoyo Manufacturing Co Ltd Tokyo Japan were stored in glass desiccators at 75 0 0 5 RH ASTM 2007 in a temperature controlled room at 23 1 C for at least two weeks the time required by the samples to reach moisture equilibrium Some of these films were used as control samples CS CD Control Others CS CD Active were immersed in carvacrol for at least 3 weeks the time required to reach equilibrium The analysis of the carvacrol sorbed by the samples was performed by thermal desorption and subsequent gas chromatographic analysis as described below A more detailed description of the manufacturing procedure can be found elsewhere 2 3 Packaging of chicken breast fillets Samples of chicken breast 25 g were placed at the bottom of polypropylene PP ethylene vinyl alcohol copolymer EVOH PP trays measuring 156 cm The trays were sealed with adhesive aluminium foil Miarco Valencia Spain and stored at 4 C for 0 3 6 and 9 days CS CD 228 Quitosano Carvacrol Active films measuring 0 24 4 8 and 24 cm were stuck to the centre of the tray lid and constituted the small medium and large samples respectively A negative control sample for each time was also prepared with CS Control Packages without chicken containing a medium size CS CD Active
495. tions and perspectives Advances in Colloid and Interface Science 166 119 135 Damm C M nsted H R sch A 2007 Long term antimicrobial polyamide 6 silver nanocomposites Journal of Materials Science 42 6067 6073 Damm C M nstedt H R sch A 2008 The antimicrobial efficacy of polyamide 6 silver nano and microcomposites Materials Chemistry and Physics 108 61 66 De Azeredo H M C 2013 Antimicrobial nanostructures in food packaging Trends in Food Science amp Technology 30 56 69 Dehnavi A S Aroujalian A Raisi A Fazel S 2013 Preparation and characterization of polyethylene silver nanocomposite films with antibacterial activity Journal of Applied Polymer Science 127 1180 1190 Demirci S Ustaoglu Z Yilmazer G A Sahin F Bac N 2014 Antimicrobial properties of zeolite X and zeolite A ion exchanged with silver copper and zinc against a broad range of microorganisms Applied Biochemistry and Biotechnology 172 1652 1662 Duan Y Y Jia J Wang S H Yan W Jin L Wang Z Y 2007 Preparation of antimicrobial poly s caprolactone electrospun nanofibers containing silver loaded zirconium phosphate nanoparticles Journal of Applied Polymer Science 106 1208 1214 Ershov B G 1999 Short lived metal clusters in aqueous solutions formation identification and properties Russian Chemical Bulletin 48 1 15 FDA Inventory of Effective Food Contact Substance notifications Food contact s
496. tivity after 1 month of immersion in deionized water which can be attributed to the slow sustained release of silver ions and thus efficacy over time KEYWORDS synthesis silver based nanoparticles chitosan antimicrobial activity W INTRODUCTION Silver ions have long been recognized as an effective biocide against a broad spectrum of microorganisms their inhibition mechanism being the subject of considerable research Ionic silver can exert its antimicrobial action in several ways It has been reported to complex with the thiol groups of enzymes and proteins altering their structure and function Silver ions have also been found to bind with DNA and cause structural changes in the cell envelope and cytoplasmic membrane of bacteria Tonic silver has been used as an antimicrobial in the form of salts and more recently it has been incorporated into inorganic materials such as zeolites zirconium phosphate and glass A new approach is the development of silver nanoparticles that can act as nanoreservoirs for the delivery of silver ions ensuring their availability in the substrate over time There is a great variety of chemical and physical processes for the synthesis of silver nanoparticles most of them involving the formation of colloidal nanoparticles or their incorporation in other materials In this regard increasing attention is being paid to the incorporation of silver nanoparticles in polymer matrices for the developm
497. tones and organic acids by forming hydrogen bonds even acetals ketals and esters PG is used as a plasticizer to improve the flexibility and processability of polymers and is extensively used in the food industry as a flavour solvent carrier PG is miscible with water alcohol and many flavour compounds and can also interact chemically or physically with them As commented above Table 4 4 11 shows some physico chemical parameters of the plasticizers used in this work including 6 and log P It can be observed that PG has a higher log P than G whereas the 6 of G is higher than that of PG These two parameters indicate the greater hydrophilicity and capacity to retain water of G a ubiquitous plasticizer for hydrophilic polymers whereas owing to its less hydrophilic nature PG would be expected to have a higher affinity to the hydrophobic volatile liquids present in essential oils 3 3 Sorption of monoterpenes in the films Chemical compatibility between volatile liquids and the film polymer matrix will determine sorption properties and the presence in the film of low molecular weight compounds such as residual solvents and plasticizers can 256 Quitosano Carvacrol also affect the sorption behaviour Therefore before studying the effect of G and PG on the sorption properties of the films for the volatile liquids appearing in Table 4 4 11 miscibility studies between the plasticizers and those compounds were carried out It was found tha
498. torage at 4 C The samples were evaluated by a minimum of 40 random non expert judges Friedman analysis indicated significant differences in the intensity of cinnamon odour perceived and acceptability since in all cases the value of F exceeded the threshold level of significance of P lt 0 001 159 Cap tulo 3 Art culo cient fico 3 According to the intensity of cinnamon odour after 3 days of storage there were no significant differences among the milk samples treated with CScin films but the differences with respect to the control sample were significant Figure 4 3 7 The same results were obtained in the tests carried out on the 6th and 12th days Preference was evaluated with a score from 1 to 5 5 being the worst score Samples with cinnamaldehyde were preferred to the control without any significant differences among them after 3 and 12 days of storage Sensory analysis showed that panellists perceived the presence of cinnamaldehyde in the milk exposed to CScin films Nevertheless the panellists preferred the milk samples in contact with CScin films at the 3 days tested Fresh pasteurized milk is a product whose shelf life is very short 2 3 days once opened The use of the films developed may not only increase the safety of such products and subsequently lengthen their shelf life owing to their antimicrobial activity but additionally provide a flavour that has high acceptance by the consumer 4 Conclusions In this work
499. tos Appendini P Hotchkiss J H 2002 Review of antimicrobial food packaging nnovative Food Science amp Emerging Technologies 3 113 126 Astray G Gonz lez Barreiro C Mejuto J C Rial Otero R Simal Gandara J 2009 A review on the use of cyclodextrins in foods Food Hydrocolloids 23 1631 1640 Avalos A Haza A Morales P 2013 Nanopart culas de plata aplicaciones y riesgos t xicos para la salud humana y el medio ambiente Revista Complutense de Ciencias Veterinarias 7 1 23 Bagamboula C F Uyttendaele M Debevere J 2004 Inhibitory effect of thyme and basil essential oils carvacrol thymol estragol linalool and p cymene towards Shigella sonnei and S flexneri Food Microbiology 21 33 42 Bajpai A K Shukla S K Bhanu S Kankane S 2008 Responsive polymers in controlled drug delivery Progress in Polymer Science 33 1088 1118 Bakal G D az A 2005 The lowdown on lauric arginate food antimicrobial eats at plasma membrane disrupting a pathogen s metabolic process Food Quality 12 54 61 Bakkali F Averbeck S Averbeck D Waomar M 2008 Biological effects of essential oils a review Food and Chemical Toxicology 46 446 475 Barrera J E Rodr guez J A Perilla J E Algecira N A 2007 Estudio de la degradaci n t rmica de poli alcohol vin lico mediante termogravimetr a y termogravimetr a diferencial ngenier a e Investigaci n 27 100 105 Ben Arfa A Co
500. triplicate On the other hand to determine the MIC and MBC of LAE for bacteria yeasts and fungi in solid medium 12 mL TSA or MEA was pour plated in presence of 3 mL of serial LAE dilutions made up in sterile Milli Q water from a 2000 pg mL LAE stock LAE tested concentrations were 0 2 and 4 and from 8 to 360 g mL in a stepwise of 8 g mL Solidified culture media were spread surface inoculated with 3 uL of cell suspension and incubated at 28 C 24 48 h for bacteria and yeasts and 5 days for fungi Tests were performed in triplicate 2 6 Antimicrobial activity of LAE chitosan films Antimicrobial activity of films against bacteria and yeasts in liquid medium was also determined A 1 cm film sample pre weighed and 100 uL of the microorganism suspension were added to 10 mL of culture medium and incubated at 28 C for 24 h Ten fold dilution series were made in peptone water and plated on solid medium Colonies were counted after incubation at 37 C for 48 h Tests were performed in triplicate Moreover the antimicrobial activity of the films against bacteria yeasts and fungi on agar was tested Bacteria suspension 100 pL was plated on TSA 1 cm pre weighed films of CS 196 LAE CS 596 LAE and CS 1096 LAE were placed on the medium surface Plates were incubated at 28 C for 24 h and the diameter of the resulting bacterial inhibition zone around the film was measured Tests were performed in triplicate FA Cap tulo 1 Art culo ci
501. troducci n Tesis Doctoral se ha focalizado en el desarrollo y caracterizaci n de materiales polim ricos con actividad antimicrobiana para su aplicaci n en el disefio de envases activos para alimentos 1 2 Biopol meros El gran uso de los pl sticos convencionales ha hecho que se planteen serios problemas como 1 incremento en el consumo de derivados del petr leo ya que m s del 9996 de los pl sticos provienen de recursos f siles cada vez m s escasos caros y con precios variables seg n el mercado 2 el elevado consumo de energ a en su fabricaci n 3 el agotamiento de los vertederos y 4 la contaminaci n del medio ambiente porque el 62 296 de los residuos pl sticos son envases PlasticsEurope 2013 y hay acumulaci n de materiales dif ciles de gestionar acumulaci n de los aditivos empleados en su fabricaci n que permanecen largo tiempo en el entorno y producci n de productos t xicos por su combusti n Esto no quiere decir que el agotamiento del petr leo significar el fin de los pl sticos convencionales ya que s lo el 296 del consumo del petr leo se utiliza para la producci n de pl sticos EIA 2013 y algunos como las poliolefinas ya pueden ser sintetizadas a partir de recursos renovables Sin embargo como respuesta a todos problemas mencionados y a la demanda de los consumidores y de las autoridades reguladoras de unos materiales mas sostenibles alternativamente se pueden emplear siempre que sea posible bi
502. trolada CS nanopart culas de plata CS cinamaldeh do CS carvacrol Tambi n cabe resaltar otras funciones del CS como la de reductor en condiciones alcalinas o quelante de iones actuando como reductor d bil y estabilizante en la formaci n de nanopart culas de plata respectivamente O bien gracias a sus grupos amino reactivos se pueden anclar mol culas vol tiles como el cinamaldeh do estabiliz ndolas hasta su uso As este trabajo muestra la versatilidad y multifuncionalidad del CS y la aplicabilidad de los envases activos antimicrobianos que pueden ser disefiados a medida de las necesidades del alimento que protegen Referencias bibliogr ficas Bitar A Ghaddar T Malek A Haddad T Toufeili I 2008 Sensory thresholds of selected phenolic constituents from thyme and their antioxidant potential in sunflower oil Journal of the American Oil Chemists Society 85 641 646 CE 2005 Reglamento CE n 2073 2005 de la Comisi n de 15 de noviembre de 2005 relativo a los criterios microbiol gicos aplicables a los productos alimenticios Chi S Zivanovic S Penfield M P 2006 Application of chitosan films enriched with oregano essential oil on bologna active compounds and sensory attributes Food Science and Technology International 12 111 117 Dos Santos N S T Aguiar AJ A A De Oliveira C E V De Sales C V Silva S D E Da Silva R S Stamford T C M De Souza E L 2012 Efficacy of the a
503. tructures from renewable resources Trends in Food Science amp Technology 22 72 80 193 Cap tulo 4 Art culo cient fico 4 Messner M Kurkov S V Jansook P Loftsson T 2010 Self assembled cyclodextrin aggregates and nanoparticles International Journal of Pharmaceutics 387 199 208 Pedro A S Cabral Albuquerque E Ferreira D Sarmento B 2009 Chitosan an option for development of essential oil delivery systems for oral cavity care Carbohydrate Polymers 76 501 508 Quijada Garrido I Iglesias Gonz lez V Maz n Arechederra J M Barrales Rienda J M 2007 The role played by the interactions of small molecules with chitosan and their transition temperatures Glass forming liquids 1 2 3 propantriol glycerol Carbohydrate Polymers 68 173 186 Ravi P Divakar S 2001 Stereoselective hydrogenation of thymol over Rh alumina in the presence of p cyclodextrin and its derivatives Journal of Inclusion Phenomena and Macrocyclic Chemistry 39 27 33 Sivakumar M Manjubala L Rao K P 2002 Preparation characterization and in vitro release of gentamicin from coralline hydroxyapatite chitosan composite microspheres Carbohydrate Polymers 49 281 288 Veldhuizen EJ A Tjeerdsma Van Bokhoven J L M Zweijtzer C Burt S A Haagsman H P 2006 Structural requirements for the antimicrobial activity of carvacrol Journal of Agricultural and Food Chemistry 54 1874 1879 Wiles J L Vergano P
504. ty of lauric arginate in milk and queso fresco cheese against Listeria monocytogenes cold growth Journal of Dairy Science 93 4518 4525 Suppakul P Miltz J Sonneveld K Bigger S W 2003 Active packaging technologies with an emphasis on antimicrobial packaging and its applications Journal of Food Science 68 408 420 Taormina P J Dorsa WJ 2009 Inactivation of Listeria monocytogenes on hams shortly after vacuum packaging by spray application of lauric arginate Journal of Food Protection 72 2517 2523 Tattawasart U Hann A C Maillard J Y Furr J R Russell A D 2000 Cytological changes in chlorhexidine resistant isolates of Pseudomonas stutzeri The Journal of antimicrobial chemotherapy 45 145 152 Theinsathid P Visessanguan W Kruenate J Kingcha Y Keeratipibul S 2012 Antimicrobial activity of lauric arginate coated polylactic acid films against Listeria monocytogenes and Salmonella Typhimurium on cooked sliced ham Journal of Food Science 77 M142 M149 Torres J A Motoki M Karel M 1985 Microbial stabilization of intermediate moisture food surfaces I Control of surface preservative concentration Journal of Food Processing and Preservation 9 75 92 Weiss J McClements D J Decker E A 2007 Stabilized antimicrobial compositions and related methods of preparation Patent Application Publication US 2007 0082018 A1 23 89 Quitosano Nanopart culas de plata 4 2 Cap tulo 2 Des
505. ubstance notification FCN mio 47 193 270 351 535 697 773 1235 http www accessdata fda gov scripts fdcc index cfm set FCN amp sort FCN_No amp order DESC amp star trow 501 amp type basic amp search revisado 20 02 2015 Fern ndez A Soriano E Hern ndez Mufioz P Gavara R 2010 Migration of antimicrobial silver from composites of polylactide with silver zeolites Journal of Food Science 75 E186 E193 Galya T Sedlarik V Kuritka L Novotny R Sedlarikova J Saha P 2008 Antibacterial poly vinyl alcohol film containing silver nanoparticles preparation and characterization Journal of Applied Polymer Science 110 3178 3185 96 Quitosano Nanopart culas de plata Hoang V T Lam D T Cham T B Hoang D V Thinh N N Dien G P Phuc X N 2010 Synthesis characterization antibacterial and antiproliferative activities of monodisperse chitosan based silver nanoparticles Colloids and Surfaces a Physicochemical and Engineering Aspects 360 32 40 Hong K H Park J L Sul IH Youk J H Kang T J 2006 Preparation of antimicrobial poly vinyl alcohol nanofibers containing silver nanoparticles Journal of Polymer Science Part B Polymer Physics 44 2468 2474 Huang H Z Yuan Q Yang X R 2004 Preparation and characterization of metal chitosan nanocomposites Colloids and Surfaces B Biointerfaces 39 31 37 Huang J Arthanareeswaran G Zhang K S 2012 Effect of silver loaded sodium
506. uced to 79 mm and 76 mm against S aureus and E coli respectively for dry films plasticized with 3596 G and around 19 mg carvacrol content An inhibition halo of 67 mm was observed for CS CD films plasticized with 2096 G conditioned at 5396 RH and having around 11 mg of carvacrol In conclusion the major antimicrobial activity was exerted by CS CD films Table 4 4 10 Antimicrobial activity of CS CD films loaded with carvacrol against S aureus and E coli S aureus E coli ETAETA ge Inhibition zone Carvacrol Inhibition zone Gils mm Gils mm CS CD 0G 0RH 0 124 0 124 slight decrease slight decrease CDS L nd in ran density Osel in uen density CS CD 0G 75RH 0 626 marked decrease 0 605 marked decrease in growth density in growth density CS CD 20G 0RH 0 315 0 340 CS CD 20G 53RH 11 6 67 111 67 CS CD 20G 75 RH 614 85 611 85 CS CD 35G 0RH 19 3 79 184 76 CS CD 35G 53RH 84 3 85 80 8 85 CS CD 35G 75RH 153 85 146 85 216 Quitosano Carvacrol possessing adequate combination of G content and RH conditioning which allowed to be loaded with a higher content of carvacrol 260 mg and produce bactericide effect against Gram positive and Gram negative bacteria 3 4 3 Antimicrobial activity of films during long term storage CS CD 35G 75RH films which showed the greatest capacity for sorption of carvacrol were chosen to follow their antimicrobial capacity over time For this purpose the antimicrobial activity in vap
507. uction of antimicrobial silver nanoparticles in water extracts of the fungus Amylomyces rouxii strain KSU 09 Bioresource Technology 101 8772 8776 Pal S Tak Y K Song J M 2007 Does the antibacterial activity of silver nanoparticles depend on the shape of the nanoparticle A study of the Gram negative bacterium Escherichia coli Applied and Environmental Microbiology 73 1712 1720 Petrus E Tinakumari S Chai L Ubong A Tunung R Elexson N Chai L Son R 2011 A study on the minimum inhibitory concentration and minimum bactericidal concentration of nano colloidal silver on food borne pathogens nternational Food Research Journal 18 55 66 Rai M Yadav A Gade A 2009 Silver nanoparticles as a new generation of antimicrobials Biotechnology Advances 27 76 83 Shahidi F Arachchi J K V Jeon Y J 1999 Food applications of chitin and chitosans Trends in Food Science amp Technology 10 37 51 Sharma V K Yngard RA Lin Y Zboril R 2010 Silver nanoparticles green synthesis approaches Abstracts of Papers of the American Chemical Society 239 124 Quitosano Nanopart culas de plata Singh M Sinha L Mandal R K 2009 Role of pH in the green synthesis of silver nanoparticles Materials Letters 63 425 427 Sondi I Salopek Sondi B 2004 Silver nanoparticles as antimicrobial agent a case study on E coli as a model for Gram negative bacteria Journal of Colloid and Interface Sc
508. ue incorporan 35 de glicerol acondicionadas al 75 de RH La liberaci n de carvacrol de las pel culas se ve muy afectada por RH Las pel culas mostraron actividad antimicrobiana contra Staphylococcus aureus y Escherichia coli despu s de 20 d as de almacenamiento a 25 C y 43 de RH ambiental Estas pel culas podr an ser empleadas en el dise o de sistemas de liberaci n de vol tiles activos Palabras clave Quitosano hidroxipropil B ciclodextrinas carvacrol carga y liberaci n pel culas antimicrobianas 197 Cap tulo 4 Art culo cient fico 5 Abstract Chitosan CS incorporating hydroxypropyl B cyclodextrins HP BCD and glycerol G films capable of modulating loading capacity and release of carvacrol have been developed Films were obtained by casting and conditioned at different relative humidities RH prior to immersion in liquid carvacrol Incorporation of cyclodextrins in the chitosan matrix slightly increased sorption of carvacrol and it was necessary to use glycerol and water as coadjuvants to control loading of the films with the volatile Good agreement was found between carvacrol retention and plasticization of the film by glycerol and water The kinetics of carvacrol sorption by the films was evaluated at 25 C Diffusion coefficients of sorption varied from 0 011x10 m s for films incorporating 3596 glycerol and conditioned at 0 RH to 1 9x10 m s for films incorporating 35 glycerol and conditioned at 75 R
509. ueden ser atrapadas por una dos o tres CDs siendo la 6 OH gt Ho CH Exterior gt hidrof lico Xan OH Figura 1 5 Representaci n esquem tica de la estructura qu mica de las CDs 23 Introducci n E ESE A e rE A Figura 1 6 Representaci n esquem tica de la formaci n de complejos de inclusi n Los puntos azules representan mol culas de agua el valo naranja la mol cula hidr foba hu sped estequiometr a m s usual la 1 1 Esta inclusi n puede ser completa o parcial si la mol cula sustrato es de mayor dimensi n respecto al interior de la CD Por una parte la formaci n del complejo de inclusi n depende de la compatibilidad est rica del sustrato y la cavidad interna de la CD Por otra depende de las interacciones termodin micas entre los diferentes componentes del sistema CD sustrato solvente Las principales fuerzas impulsoras de la formaci n de complejos son el desplazamiento de mol culas de agua de elevada entalp a de la cavidad apolar de la CD la formaci n de puentes de hidr geno entre las mol culas de agua que salen de la CD y el resto de mol culas de agua del medio disminuci n de la repulsi n entre el sustrato apolar y el medio acuoso aumento de las interacciones hidrof bicas entre el sustrato y la cavidad de CD y disminuci n de la tensi n en el anillo de CD Las fuerzas de Van der Waals interacciones hidrof bicas y los puentes de hidr geno mantienen la CD y el sustrato
510. ulfil the aforementioned requirements Chitosan polymer poly f 1 4 N acetyl D glucosamine has been intensively studied during recent decades Dutta Tripathi amp Dutta 2012 Lopez Carballo Higueras Gavara amp Hernandez Munoz 2013 Valencia Chamorro Palou del Rio amp Perez Gago 2011 It is a natural cationic linear aminopolysaccharide obtained from partial N deacetylation of chitin Chitosan is receiving a great deal of attention in biomedicine and pharmacology for the delivery of drugs Ramya Venkatesan Kim amp Sudha 2012 Chitosan can also act as a carrier for sustained release and delivery of compounds other than drugs which are of interest in foods personal care agriculture etc Kumar Muzzarelli Muzzarelli Sashiwa amp Domb 2004 Prabaharan amp Mano 2006 Zhang et al 2009 Antimicrobial carriers are of great interest in the area of food packaging and great efforts are being made to develop effective antimicrobial food packaging systems Appendini amp Hotchkiss 2002 Suppakul Miltz Sonneveld amp Bigger 2003 Antimicrobial food packaging technologies which are based on the incorporation of active volatiles in polymer matrices do not require the film be in contact with the food product to be active In this case the volatile is released to the headspace of the package and exerts its activity when contact with the food surface There is a wide range of volatile compounds derived from plants or
511. unidos Astray et al 2009 Del Valle 2004 Szejtli 1998 Cabe resaltar la estabilidad de los complejos formados por las B CDs con los mono y sesquiterpenos Szente y Szejtli 1988 Debido a su capacidad para establecer enlaces covalentes o no covalentes con otras CDs las CDs en disoluci n pueden autoagregarse y formar complejos supramoleculares como catenanos rotaxanos polirotaxanos y tubos Del Valle 2004 He et al 2008 agregados tipo micelar Del Valle 2004 He et al 2008 Messner et al 2010 perlas nanofibras o nanoc psulas Kurkov y Loftsson 2013 Por otra parte se pueden formar complejos ternarios pol mero hidrosoluble CD principio activo que pueden aumentar la estequiometr a 1 1 entre la CD y el principio activo Kurkov y Loftsson 2013 Las CDs han sido muy estudiadas y empleadas desde su descubrimiento en 1891 por A Villiers Del Valle 2004 Kurkov y Loftsson 2013 Szejtli 1998 La formaci n de los complejos de inclusi n provoca 24 Introducci n variaciones reversibles en las propiedades fisicoqu micas del sustrato solubilizaci n o precipitaci n de principios activos estabilizaci n de sustancias vol tiles protecci n contra la oxidaci n luz o calor Por ello las CDs y sus derivados son ampliamente empleadas en biotecnolog a protecci n ambiental qu mica anal tica industria textil etc Y destaca su aplicaci n en la tecnolog a de los alimentos y envasado farmacolog a cosm ti
512. urnal of Scientific amp Industrial Research 63 20 31 Dutta P K Tripathi S Mehrotra G K Dutta J 2009 Perspectives for chitosan based antimicrobial films in food applications Food Chemistry 114 1173 1182 EFSA 2006 Opinion of the scientific panel on food additives flavourings processing aids and materials in contact with food on a request related to a 12th list of substances for food contact materials 26 y 27 de septiembre de 2006 The EFSA Journal 2006 395 401 1 21 EFSA 2007 Opinion of the scientific panel on food additives flavourings processing aids and materials in contact with food on a request from the commission related to an application on the use of ethyl lauroyl arginate as a food additive 17 de abril de 2007 The EFSA Journal 511 1 27 EFSA 2011 Opini n cient fica Gu a para la evaluaci n de riesgos de las aplicaciones de nanociencia y nanotecnolog a artificiales en alimentos y piensos The EFSA Journal 9 5 2140 EFSA 2014 The UE summary report on trends and sources of zoonoses zoonotic agents and food borne outbreaks in 2012 19 de febrero de 2014 252 300 The EFSA Journal 12 2 3547 EIA 2013 What are the products and uses of petroleum http www eia gov tools fags faq cfm id 418 t 6 EPA 1992 6 ed Secondary drinking water regulations guidance for nuisance chemicals US EPA Office of Ground Water amp Drinking Water Washington D C ltima actualizaci n 31 de mayo de
513. urter pieces in a model system at 4 C Journal of Food Science 70 M95 M99 Llorens A Lloret E Picouet P Fern ndez A 2012 Study of the antifungal potential of novel cellulose copper composites as absorbent materials for fruit juices International Journal of Food Microbiology 158 113 119 Lloret E Picouet P Fern ndez A 2012 Matrix effects on the antimicrobial capacity of silver based nanocomposite absorbing materials Lwt Food Science and Technology 49 333 338 Maizura M Fazilah A Norziah M Karim A 2008 Antibacterial activity of modified sago starch alginate based edible film incorporated with lemongrass Cymbopogon citratus oil International Food Research Journal 15 233 236 320 Anexos Maizura M Fazilah A Norziah M H Karim A A 2007 Antibacterial activity and mechanical properties of partially hydrolyzed sago starch alginate edible film containing lemongrass oil Journal of Food Science 72 C324 C330 Makwana S Choudhary R Dogra N Kohli P Haddock J 2014 Nanoencapsulation and immobilization of cinnamaldehyde for developing antimicrobial food packaging material Lwt Food Science and Technology 57 470 476 Marcos B Aymerich T Monfort J M Garriga M 2007 Use of antimicrobial biodegradable packaging to control Listeria monocytogenes during storage of cooked ham International Journal of Food Microbiology 120 152 158 Marcos B Aymerich T Monfort J M G
514. utralized at 37 C in liquid media over time Three studies were carried out to determine the transfer of silver to the media In the first study we studied how the immersion time of the film in the growth medium prior to inoculation with bacteria affects its antimicrobial activity For this purpose films with a silver concentration of 0 296 were immersed in 1 125 diluted MHB and stored in an incubator at 37 C for 1 2 10 24 48 168 and 360 h prior to inoculation of the microorganism Table 4 2 3 shows the antimicrobial activity of diluted MHB 119 Cap tulo 2 Art culo cient fico 2 Table 4 2 3 Antimicrobial activity of diluted MHB 1 125 which has been in contact with films incorporating 0 2 96 of silver for several times S aureus Bacterial count Log CFU mL 0 7 02 0 30 1 3 63 0 13 2 3 70 0 71 10 3 22 0 17 24 3 50 0 06 48 3 50 0 16 168 3 36 0 09 360 3 19 0 32 ab Values within a column followed by a different lower case letter are significantly different from each other Tukey s adjusted analysis of variance P lt 0 05 1 125 against S aureus after being in contact with 0 2 silver films neutralized at 37 C as a function of the immersion time As can be seen the antimicrobial effect of the films did not vary over time After 24 and 360 h in contact with the culture medium films showed log reductions of 3 38 and 3 80 log respectively According to the migration values
515. uvantes con la finalidad de conseguir una mayor capacidad de carga y modular su posterior liberaci n Entre dichos coadyuvantes cabe destacar los plastificantes Karbowiak et al 2006 y las ciclodextrinas Hoare y Kohane 2008 1 4 1 Plastificantes Los plastificantes son aditivos cuya funci n principal es mejorar la extensibilidad y procesabilidad de pol meros mediante la reducci n de la temperatura de transici n v trea La Uni n Internacional de Qu mica Pura y Aplicada IUPAC defini plastificante como una sustancia o material incorporado en un material por lo general un pl stico o un elast mero para aumentar su flexibilidad manejabilidad o distensibilidad Los plastificantes son resinas O l quidos de bajo peso molecular no vol tiles que intercal ndose entre las cadenas de los pol meros forman enlaces secundarios reducen las interacciones intermoleculares pol mero pol mero y la cohesi n de la matriz polim rica proporcionando un mayor volumen libre y movilidad de las cadenas resultando una estructura f cilmente deformable y menos fr gil Las interacciones entre el plastificante y el pol mero son de car cter d bil porque algunos plastificantes pueden volatilizarse o migrar de 20 Introducci n la matriz polim rica Rahman y Brazel 2004 Sothornvit y Krochta 2005 Vieira et al 2011 El uso de plastificantes es necesario para mejorar las propiedades mec nicas de pel culas formadas a partir de prote
516. v y Loftsson 2013 Aunque de momento no se ha encontrado nada especificado por parte de la FDA y FAO OMS para derivados como las HP BCD 1 5 Agentes antimicrobianos En la presente Tesis Doctoral se ha trabajado con los siguientes agentes antimicrobianos etil N dodecanoil L arginato nanopart culas de plata y componentes de aceites esenciales como son el carvacrol y el cinamaldeh do 25 Introducci n 1 5 1 Etil N dodecanoil L arginato El monohidrocloruro de etil N dodecanoil L arginato etil lauroil arginato o ster et lico de N lauroil L arginina LAE es un derivado del cido l urico L arginina y etanol con propiedades tensioactivas y actividad antimicrobiana Bakal y Diaz 2005 Rodr guez et aL 2004 La estructura qu mica del LAE se muestra en la Figura 1 7 Se trata de un s lido blanco que se puede disolver en agua a 20 C m s de 247 g kg con un punto de fusi n entre 50 5 y 58 C y se descompone a temperaturas superiores a 107 C Su coeficiente de reparto aceite agua es menor a 0 1 por lo que la mol cula se localiza principalmente en la fracci n acuosa m s susceptible a la contaminaci n microbiana El LAE muestra estabilidad qu mica a un pH entre 3 y 7 y mantiene su actividad antimicrobiana dentro de este intervalo Bakal y D az 2005 Las propiedades antimicrobianas del LAE se deben a su acci n como tensioactivo cati nico De este modo el LAE altera la membrana externa y citoplasm tica de
517. vas debido a su alta densidad at mica y por tanto una liberaci n de iones m s r pida Existen diversos trabajos en los que se desarrollan pel culas nanocompuestas de nanopart culas de plata y CS Algunos trabajos incluyen nanopart culas de plata en una matriz de CS Huang et al 2011 Lu et aL 2008 Pinto et al 2012 Potara et al 2011 Rhim et al 2006 pero en estos casos la obtenci n de las nanopart culas son generadas previamente Otros autores han obtenido nanopart culas de plata en la disoluci n de CS pero empleando agentes reductores fuertes como el borohidruro de sodio Huang et al 2004 Por otra parte existen diversos ejemplos de m todos de obtenci n de nanopart culas de plata en el seno de la matriz de CS en una sola etapa y en condiciones suaves respetando el medio ambiente As Murugadoss y Chattopadhyay 2008 y Sanpui et al 2008 desarrollaron un m todo de obtenci n de nanocompuestos de CS plata en el que el CS act a como reductor de la plata i nica en presencia de hidr xido de sodio a 95 C en medio acuoso De forma similar procedieron Hoang et al 2010 Regiel et al 2013 Thomas et al 2009 Wei y Qian 2008 y Wei et al 2009 ya que afiadieron la disoluci n de nitrato de plata a la disoluci n ac tica de CS empleando una temperatura de entre 30 100 C para la formaci n de las nanopart culas Algunos de stos adem s obtuvieron pel culas a partir de esta disoluci n mediante extensi
518. veles aceptables y las pel culas de CS con LAE los disminuyeron por debajo de los esos niveles Por otra parte la pel cula de PE de baja densidad con la que se envolvi externamente entorpecer a la entrada de ox geno lo que podr a justificar el predominio de Pseudomonas En envasados m s impermeables se acumula CO inhibiendo el crecimiento de Pseudomonas y desplazando el crecimiento mayoritario hacia una carga microbiana menos ox geno dependiente como las bacterias cido l cticas y las sulfuro productoras predominando Shewanella putrefaciens entre stas ltimas ICMSF 2005 Las pel culas probadas redujeron los niveles tanto de las bacterias cido l cticas como de las sulfuro productoras por lo que este sistema de envasado podr a tambi n aplicarse en sistemas de envasado al vac o o con materiales menos permeables En el caso de hongos y levaduras los datos obtenidos no son concluyentes Estos resultados demuestran que las pel culas de CS con LAE pueden emplearse potencialmente en el dise o del envasado activo antimicrobiano de pollo Adem s probablemente pueda ser empleado para otros alimentos ya que como se expone en el estado de la cuesti n del cap tulo 1 el LAE ya ha sido satisfactoriamente aplicado frente a diversos microorganismos y en distintos grupos de alimentos puesto que se trata de un aditivo de baja toxicidad y que no altera las propiedades organol pticas del producto en el que se aplica El siguiente si
519. vity towards diverse microorganisms Films of CS 596LAE reduced satisfactorily the microbiota commonly found in fresh chicken therefore presenting excellent potential for packaging of fresh poultry products Future work will indicate whether these antimicrobial packaging systems will be effective to improve the safety and to extend the shelf life of other raw and processed food products Acknowledgments Authors thank the financial support of the Spanish Ministry of Science and Innovation projects AGL2009 08776 AGL2012 39920 C03 01 EU Nafispack project 212544 and JAE program from CSIC L H fellowship and Mr Karel Clapshaw translation services 85 Cap tulo 1 Art culo cient fico 1 10 10 EE Control Mesophiles EE Control Psychrophiles Gam CS EE CS a CS 5 LAE a E CS 5 LAE o H E S 5 264 5 o o S 3 5 LL 5 o O 4 44 2 3 2 2 2 I d 0 0 Time days Time days 10 10 HE Control Pseudomonas spp WEB Control Coliform bacteria mum cs mH CS e CS 5 LAE a E CS 5 LAE 2 2 a a E de 30 5 5 2 2 5 5 2 2 2 24 0 I 0 I 0 Time days bi Time days 10 10 EE Control Lactic acid bacteria EHE Control Hydrogen sulfide producing bacteria mmm cs mEmH CS a EE CS 5 LAE sj CS 5 LAE 2 o 2 2 E E S s S 64 5 El 5 gt 5 3 3 l _ kh o o i 8 Time days Time days 10 ENEH Control js Yeasts and fungi a m CS 5 LAE o a E 64 E E
520. volution of carvacrol concentration during product storage for the three device sizes and for control packages without chicken in the different package parts a CS CD Active b headspace HS c package lid and d the PP inner layer of the tray Values are expressed as mean and standard deviation n 9 232 Figure 4 4 11 Portion of carvacrol percentage present in each phase of the product package system for the three device sizes and the empty tray with the medium device a small b medium c large and d empty tray 235 Figure 4 4 12 Evolution of chicken fillet microbiota packaged with control and active packages during 9 days of storage Different letters a b c indicate significant differences among samples at a specific storage time Turkey s adjusted analysis of variance P 0 05 Values are expressed as mean and standard deviation n 3 237 Figure 4 4 13 Effect of RH on equilibrium moisture content and sorption equilibrium of carvacrol in CS CD 35PG 260 Figure 4 4 14 Effect of incorporating HP BCD and G on equilibrium moisture content and sorption equilibrium of carvacrol in PVOH films conditioned at 7596 RH 261 Figure 4 4 15 Sorption equilibrium of carvacrol and equilibrium moisture content of PVOH CD 35G films conditioned at different RH 262 XXIII Abreviaturas y s mbolos ADN AE ARN ARNm ATR FTIR CD CE CEE CS Da EIA EFSA EPA EVOH FAO FDA G GFAAS GSFA GRAS HP BCD IDA JECFA LAE OECD SIDS
521. w modification of food organoleptic characteristics and considerable antimicrobial activity LAE is becoming a product widely used in the field of conservation of pharmaceuticals cosmetics and food However limited information is available related to its antimicrobial activity when LAE is applied via packaging system Bonnaud et al 2010 CS a biodegradable and biocompatible polymer obtained from biomass with excellent film forming properties has attracted great interest for technological applications in several areas such as pharmacy medicine agrochemistry and packaging The hydrophilic nature of CS has aroused interest as a sustained carrier when the release of the retained active compound is required under a moisture environment 74 Quitosano LAE The aim of this study therefore was to prepare and characterize LAE CS antimicrobial films for food packaging applications LAE release from CS films was also assessed The efficacy of the antimicrobial films was monitored both by in vitro microbiological tests and by performing storage trials on samples of chicken breast fillets 2 Materials and methods Chitosan CS acetic acid and glycerol G were supplied by Sigma Barcelona Spain LAE 69 396 purity in maltodextrin commercialized as Mirenat D was gently provided by Vedeqsa Grupo LAMIRSA Barcelona Spain 2 1 Films preparation First a 1 596 CS w w solution in an aqueous 0 596 w w acetic acid solution was prep
522. w of chitin and chitosan applications Reactive amp Functional Polymers 46 1 27 Kumar M N V R Muzzarelli R A A Muzzarelli C Sashiwa H Domb A J 2004 Chitosan chemistry and pharmaceutical perspectives Chemical Reviews 104 6017 6084 Kurek M Descours E Galic K Voilley A Debeaufort F 2012 How composition and process parameters affect volatile active compounds in biopolymer films Carbohydrate Polymers 88 646 656 268 Quitosano Carvacrol Lang G Buchbauer G 2012 A review on recent research results 2008 2010 on essential oils as antimicrobials and antifungals A review Flavour and Fragrance Journal 27 13 39 Lavorgna M Piscitelli F Mangiacapra P Buonocore G G 2010 Study of the combined effect of both clay and glycerol plasticizer on the properties of chitosan films Carbohydrate Polymers 82 291 298 Messner M Kurkov S V Jansook P Loftsson T 2010 Self assembled cyclodextrin aggregates and nanoparticles International Journal of Pharmaceutics 387 199 208 Muhd Julkapli N Akil H M D Ahmad Z 2011 Preparation properties and applications of chitosan based biocomposites blend materials a review Composite Interfaces 18 449 507 Peng F Jiang Z Hu C Wang Y Lu L Wu H 2006 Pervaporation of benzene cyclohexane mixtures through poly vinyl alcohol membranes with and without B cyclodextrin Desalination 193 182 192 Rahman W A W A Sin L T Rahmat
523. was prepared and filtrated to eliminate impurities Pure CS films were prepared by casting that is pouring a suitable amount of the solution into a flat PS tray and allowing it to dry under controlled environmental conditions 36 h 40 0 1 5 C and 20 9 RH G plasticized films were produced by adding G at 2096 or 3596 g G 100 g dry matter to the film forming solution Chitosan hydroxypropyl B cyclodextrin composites CS CD were obtained by adding HP BCD to the CS solution in a 1 1 proportion w w with 178 Quitosano Carvacrol respect to CS stirring at 1500 rpm and 37 C until complete dissolution and submitting the solution to the casting process Plasticized CS CD films were prepared by adding G at 2096 or 3596 g G 100 g dry matter to the film forming solution 2 3 Film thickness The film thickness of each sample was individually measured using a digital micrometre Mitutoyo Manufacturing Co Ltd Tokyo Japan with a sensitivity of 1 um Five readings were taken for each sample one at the sample centre and four measurements around the perimeter Average thickness of the films was 55 5 um 2 4 Optical properties The colour of the films was measured with a CR 300 Minolta Chroma meter amp Minolta Camera Co Ltd Osaka Japan The film samples were placed on a white standard plate the results were expressed in accordance with the CIELAB system with reference to illuminant D65 and a visual angle of 10 The measurement
524. y mucosas La EFSA 2006 y SCENIHR 2014 Comit Cient fico asesor de la CE concluyeron que no hay problemas de seguridad para el consumidor si la migraci n total de iones de plata no supera el l mite espec fico de migraci n de 0 05 mg kg en alimentos 0 05 mg L en agua aunque no se especifica para la nanoplata La plata es considerada aditivo alimentario UE 20110 pero se trata de un colorante restringido a coberturas de confiter a y reposter a chocolate para decoraci n y licores Sin embargo en la actualidad la aplicaci n de nanopart culas de plata en materiales nanocompuestos para generar superficies antimicrobianas es de gran inter s puesto que presenta varias ventajas frente a otros antimicrobianos La incorporaci n de nanopart culas de plata genera 30 Introducci n pol meros m s estables puesto que no se evaporan como otros aditivos org nicos resisten altas temperaturas no suelen afectar a las propiedades intr nsecas de los pol meros les confieren un amplio espectro antimicrobiano y puede aportar propiedades conductoras a los nanomateriales 1 5 3 Aceites esenciales y sus componentes En las ltimas d cadas se ha puesto de relieve el potencial de los extractos vegetales y en especial de algunos aceites esenciales AE como agentes antimicrobianos naturales Tradicionalmente los AE y los componentes o derivados de stos han sido utilizados en la industria alimentaria como aromatizantes en la pre
525. y de bater as El CS fue aprobado como producto GRAS en EEUU para la alimentaci n animal 1983 y para la purificaci n del agua En Jap n el CS fue aprobado como aditivo alimentario en 1983 y como ingrediente funcional en 1992 y en Corea como aditivo alimentario en 1995 Kong et al 2010 No et aL 2007 Shahidi y Abuzaytoun 2005 La FDA 2011 determin el estado GRAS nicamente para el CS producido por el hongo Aspergillus niger 13 Introducci n Tabla 1 1 Aplicaciones del CS y sus derivados Dutta et al 2004 Rinaudo 2006 Shahidi y Abuzaytoun 2005 Srinivasa y Tharanathan 2007 medicina cosm tica farmacia diet tica y nutrici n industria alimentaria agroqu mica industria del envasado biotecnolog a e investigaci n tratamiento de aguas otras anticoagulante antitromb tico piel artificial reparaci n de cart lago membranas de di lisis hemost tico lentes de contacto implantes dentales cremas geles lociones esmalte de ufias dent fricos champ es y acondicionadores de cabello diluyente inmunoadyuvante sistemas de liberaci n de principios activos microc psulas microesfera nanopart culas anticolesterolemiante hipotrigliceridemiante fibra soluble y prebi tico espesante emulsionante estabilizante de color y aroma antioxidante y antimicrobiano clarificaci n y desacidificaci n de zumos y bebidas fermentadas floculaci n y recuperaci n de residuos s li
526. ysed in triplicate Results are expressed as average value standard deviation 2 3 2 Optical properties Colour coordinates of the various CS based films were measured with a CR 300 Minolta Chroma meter Minolta Camera Co Ltd Osaka Japan The film samples were placed on a standard white plate the results were expressed in accordance with the CIELAB system with reference to illuminant D65 and a visual angle of 10 The measurements were performed through a 6 4 mm diameter diaphragm containing an optical glass monitoring L lightness a b and calculating chroma C gp a b and hue hap arctan b a Films were measured in triplicate and eight measurements were taken at different locations of each sample 2 3 3 Water uptake and dimensional stability Film specimens 1 7x1 7 cm comprising CS and CScin films subjected to different treatments were immersed in aqueous solution buffered at pH 3 or pH 7 for 24 h at 23 C After that time the films were removed from the water wiped off with a paper towel and immediately weighed final wet 142 Quitosano Cinamaldeh do weight w The samples were replaced in the desiccator until they reached a constant weight final dry weight w The percentage of water uptake AW was calculated as follows f f AW 96 Ww Va 100 wwe 1 The dimensional stability of the films was calculated similarly by measuring the film surface area after removing the film fr
527. zirconium phosphate nanoAgZ nanoparticles incorporation on PES membrane performance Desalination 285 100 107 Huang L Y Dai T H Xuan Y Tegos G P Hamblin M R 2011 Synergistic combination of chitosan acetate with nanoparticle silver as a topical antimicrobial efficacy against bacterial burn infections Antimicrobial Agents and Chemotherapy 55 3432 3438 Incoronato A L Buonocore G G Conte A Lavorgna M Del Nobile M A 2010 Active systems based on silver montmorillonite nanoparticles embedded into bio based polymer matrices for packaging applications Journal of Food Protection 73 2256 2262 Jain P Pradeep T 2005 Potential of silver nanoparticle coated polyurethane foam as an antibacterial water filter Biotechnology and Bioengineering 90 59 63 Kamisoglu K Aksoy E A Akata B Hasirci N Bac N 2008 Preparation and characterization of antibacterial zeolite polyurethane composites Journal of Applied Polymer Science 110 2854 2861 Lavorgna M Attianese I Buonocore G G Conte A Del Nobile M A Tescione F Amendola E 2014 MMT supported Ag nanoparticles for chitosan nanocomposites Structural properties and antibacterial activity Carbohydrate Polymers 102 385 392 Li L H Deng J C Deng H R Liu Z L Li X L 2010 Preparation characterization and antimicrobial activities of chitosan Ag ZnO blend films Chemical Engineering Journal 160 378 382 Lu S Y Gao W J

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