Innovative Systems for the Delivery of Naturally Occurring Antimicrobial Volatiles in Active Food-Packaging Technologies for Fresh and Minimally Processed Produce: Stimuli-Responsive Materials.

Certain naturally occurring volatile organic compounds are able to mitigate food spoilage caused by microbial growth. Their considerable vapor pressure enables them to create an antimicrobial atmosphere within a package, and this property can be used for the development of active food-packaging technologies. The volatility of these molecules, however, makes their stabilization difficult and limits their effectiveness. Whilst much research is being undertaken on the use of natural antimicrobial volatiles for inhibiting microbial growth in food, less attention has been paid to the design of controlled-release mechanisms that permit the efficient application of these compounds. Most studies to date either spray the volatile directly onto the fresh product, immerse it in a solution containing the volatile, or embed the volatile in a paper disc to create a vapor in the headspace of a package. More sophisticated alternatives would be delivery systems for the sustained release of volatiles into the package headspace. Such systems are based on the encapsulation of a volatile in organic or inorganic matrices (cyclodextrins, electrospun non-wovens, polymer films, micelles, molecular frameworks, etc.). However, most of these devices lack an efficient triggering mechanism for the release of the volatile; most are activated by humidity. All of these techniques are revised in the present work, and the most recent and innovative methods for entrapping and releasing volatiles based on reversible covalent bonds are also discussed.

Agro-Industrial Protein Waste and Co-Products Valorization for the Development of Bioplastics: Thermoprocessing and Characterization of Feather Keratin/Gliadin Blends.

Biopolymers based on plant and animal proteins are interesting alternatives in the development of films with future prospects as food packaging. Considering that in recent years there has been an increasing interest in the valorization of agro-industrial residues and by-products and that the blending of polymers can lead to materials with improved properties, in this work, keratin-rich feather fibers and gliadins were blended at different ratios in order to develop sustainable and biodegradable films. Control gliadin G100, feather F100 films, and their blends at 3:1 (G75F25), 2:2 (G50F50), and 1:3 (G25F75) ratios were successfully developed through thermoprocessing. The physical properties were differentiated as a function of the concentration of both polymeric matrices. Although gliadins showed higher hydrophilicity as confirmed by their highest swelling degree, films with high gliadin ratios exhibited lower water vapor permeability values at low and medium relative humidities. On the other hand, the feather fiber-based films displayed the highest Young's modulus values and provided an oxygen barrier to the blends, principally at the highest relative humidity. In conclusion, the blend of these protein-based polymers at different ratio resulted in interesting composites whose physical properties could be adjusted.

ZIF-8 encapsulation improves the antifungal activity of benzaldehyde and methyl anthranilate in films.

In this work, two ZIF-8-based biocomposites were obtained by entrapping the biomolecules benzaldehyde and methyl anthranilate via direct impregnation with fast encapsulation kinetics and high molecule payloads were achieved. The obtained biocomposites exhibit an enhanced antifungal activity against Penicilium expansum after integration in biopolymeric zein films in comparison with the action of free molecules, making these biomaterials promising candidates for food preservation and packaging applications.

Designing an Oxygen Scavenger Multilayer System Including Volatile Organic Compound (VOC) Adsorbents for Potential Use in Food Packaging.

Oxygen scavengers are valuable active packaging systems because several types of food deterioration processes are initiated by oxygen. Although the incorporation of oxygen scavenger agents into the polymeric matrices has been the trend in recent years, the release of volatile organic compounds (VOC) as a result of the reaction between oxygen and oxygen scavenger substances is an issue to take into account. This is the case of an oxygen scavenger based on a trans-polyoctenamer rubber (TOR). In this work, the design of an oxygen scavenger multilayer system was carried out considering the selection of appropriate adsorbents of VOCs to the proposed layer structure. Firstly, the retention of some representative organic compounds by several adsorbent substances, such as zeolites, silicas, cyclodextrins and polymers, was studied in order to select those with the best performances. A hydrophilic silica and an odor-adsorbing agent based on zinc ricinoleate were the selected adsorbing agents. The principal VOCs released from TOR-containing films were carefully identified, and their retention first by the pure adsorbents, and then by polyethylene incorporated with the selected compounds was quantified. Detected concentrations decreased by 10- to 100-fold, depending on the VOC.

pH modulates antibacterial activity of hydroxybenzaldehyde derivatives immobilized in chitosan films via reversible Schiff bases and its application to preserve freshly-squeezed juice.

Antimicrobial food grade hydroxybenzaldehyde derivatives were immobilized on the surface of chitosan films by means of reversible Schiff bases. Spectroscopy and elemental analysis evidenced the different ability of the aldehydes to form Schiff bases with chitosan. Chitosan films modified with Schiff bases of aldehydes exerted antimicrobial properties against E. coli under mild acidic environments. The efficacy of the films lied on the reversibility of synthetized imine bonds and release of the aldehydes which was promoted in mildly acid aqueous solutions. Besides acidity, imine bond reversibility depended on the chemical structure of the aldehyde covalently bonded. Films carrying salicylaldehyde presented the highest in vitro antimicrobial performance and thus, they were chosen to evaluate their effectivity in inhibiting E. coli proliferation in freshly-squeezed carrot-orange juice. Films were successfully activated by the acid environment of the juice and reduced the population of the inoculated pathogen. Salicylaldehyde migrated to the juice did not exert toxic effects on Caco-2 cells.

New Isolated Metschnikowia pulcherrima Strains from Apples for Postharvest Biocontrol of Penicillium expansum and Patulin Accumulation.

Wild yeasts isolated from the surface of apples were screened for antagonistic activity against Penicillium expansum, the main producer of the mycotoxin patulin. Three antagonistic yeasts (Y33, Y29 and Y24) from a total of 90 were found to inhibit P. expansum growth. Identification by ITS region sequence and characterization showed that three selected isolates of yeast should be different strains of Metschnikowia pulcherrima. Several concentrations of the selected yeasts were used to study their in vitro antifungal effectivity against P. expansum on Petri dishes (plates with 63.6 cm2 surface) whereas their potential activity on patulin reduction was studied in liquid medium. Finally, the BCA that had the best in vitro antifungal capacity against P. and the best patulin degradation capacity was selected to be assessed directly on apples. All the selected strains demonstrated antifungal activity in vitro but the most efficient was the strain Y29. Isolated strains were able to reduce patulin content in liquid medium, Y29 being the only strain that completely reduced patulin levels within 120 h. The application of Y29 as biocontrol agent on the surface of apples inoculated with P. expansum, inhibited fungal growth and patulin production during storage. Therefore, the results shown that this yeast strain could be used for the reduction of P. expansum and its mycotoxin in apples or apple-based products by adapting the procedure application.

Development of Biodegradable Films Loaded with Phages with Antilisterial Properties.

The inhibitory and bactericidal capacity of Listex P100 bacteriophage has been studied against different concentrations of Listeria monocytogenes in stationary and exponential phases. Three different matrices were employed to developed films incorporating Listex P100: (1) sodium caseinate, (2) sodium alginate mixed with gelatin, and (3) polyvinyl alcohol (PVOH). All the films were successfully developed by casting at room temperature. These active biodegradable films were optical, structural, and thermally characterized, and their antimicrobial capacities against L. monocytogenes were studied. The incorporation of phages did not affect the morphology, colour, opacity, and thermal stability of polymers. The antimicrobial analysis revealed the bacteriophage presented a high antimicrobial capacity against L. monocytogenes in the stationary phase (4.40 and 6.19 log reduction values or bactericide effect depending on the initial inoculum of the pathogen). Developed films showed antimicrobial capacity close to 1 log after 24 h of incubation at 30 °C. The effectiveness of PVOH films was greater under refrigeration conditions, reaching 2 log reduction after eight days of incubation. The use of these films as a coating in a food or as part of a packaging could improve food safety against the growth of pathogenic microorganisms such as Listeria monocytogenes.

Broadening the antimicrobial spectrum of nisin-producing Lactococcus lactis subsp. Lactis to Gram-negative bacteria by means of active packaging.

Cast films obtained from polyvinyl alcohol (PVOH) blended with casein hydrolysates (HCas) in a weight ratio of 1:1 were employed to carry nisin-producing L. lactis and phytic acid in order to broaden the antimicrobial spectrum of L. lactis to Gram-positive and Gram-negative spoilage and pathogen bacteria. For this purpose, the effect of the antimicrobial activity of various film formulations and combinations of films on the growth of E. coli at 37 °C for 24 h was studied. The film system that showed antimicrobial activity against Gram-negative bacteria consisted of phytic acid and L. lactis incorporated in separate films. When the active agents were in the same film the viability of L. lactis decreased considerably and it did not exert antimicrobial activity against the bacterium. Therefore, the combination of L. lactis and phytic acid in separate films was chosen as the reliable system, and the effect of its activity on the growth of Gram-negative bacteria (E. coli, Salmonella enterica, and Pseudomonas fluorescens) and Gram-positive bacteria (Listeria monocytogenes) in liquid culture medium was tested at refrigeration temperature (4 °C), and with simulated breaks in the cold chain (14 °C and 24 °C). The survival of L. lactis in coexistence with these bacteria was also studied. The film system exerted an antimicrobial effect against the Gram-negative bacteria tested, and the activity depended on the bacteria and the temperature assayed. With regard to the antimicrobial activity against L. monocytogenes, phytic acid improved the antimicrobial capacity of L.lactis. The survival of L. lactis was maintained at 7-8 log (CFU/mL) culture in liquid medium throughout the storage period. The films developed were intended to be used as coatings in the design of a double-sided active bag for a non-fermented dairy product. The bags were filled with homemade preservative-free pastry cream, and the microbiological shelf life and evolution of pH of the packaged ready-to-eat food stored at 4 °C was studied for 20 days. The results showed a reduction in the growth of spoilage bacteria and therefore an increase in the shelf life of the packaged product. The films developed could be applied in the design of packages for perishable dairy foods in order to increase their microbiological shelf life.

PVOH/protein blend films embedded with lactic acid bacteria and their antilisterial activity in pasteurized milk.

PVOH-based polymer matrices in the form of films were evaluated as carriers of living Lactococcus lactis subsp. Lactis. These lactic acid bacteria are capable of producing nisin, which is an effective antilisterial peptide. A low percentage (1:0.125 w/w) of yeast extract, gelatin, sodium caseinate, gelatin, or casein hydrolysates was incorporated in PVOH matrices with the aim of increasing the viability of bacteria in the film. The films were obtained by casting after incorporating L. lactis. Then they were evaluated for antilisterial activity in liquid medium at 37 °C for 24 h, and also at 4 °C for 21 days in order to simulate the storage of liquid foods in refrigeration conditions. The survival of the lactic acid bacteria was also evaluated at both temperatures during the experiment. L. lactis remained viable in all the films tested at 37 and 4 °C. The antimicrobial activity of the films was greater at 4 °C than at 37 °C. With regard to the effect of the film composition, the activity of the films was higher when protein hydrolysates and sodium caseinate were incorporated in the formulation. Films supplemented with protein hydrolysates or sodium caseinate inhibited growth of the pathogen during the 21 days of storage at 4 °C. At 37 °C, after 24 h the films had slowed the growth of the inoculated pathogen by between 2 and 4 log CFU/mL. Finally, as the films developed are intended to be used in the design of active packaging of foods, they were tested in pasteurized milk inoculated with 4 log CFU/mL of Listeria monocytogenes and stored at 4 °C for 21 days. The pathogen began to grow after the second day of storage with or without film, but when the films were added to the medium the growth of the pathogen was slowed down, without reaching >6 log CFU, whereas the control reached a maximum growth of 8.5 log CFU. The pH of the milk was monitored throughout the experiment, and it decreased with time. This was due to the generation of organic acids by the lactic bacteria. Buffering the food stabilized the pH without modifying the activity of the films. Thus, the current study shows that PVOH films supplemented with nutrients can act as carriers of L. lactis, and they can help to increase the safety of refrigerated dairy beverages and sauces.

Chromatic Sensor to Determine Oxygen Presence for Applications in Intelligent Packaging.

A chromatic sensor has been designed for the detection of oxygen in package headspace. The sensor is based on the redox change of methylene blue (MB) to its leuco form. Its formulation includes the pigment, glycerol, as a sacrificial electron donor, TiO2, as a photocatalyst and ethylene-vinyl alcohol copolymer (EVOH), as a structural polymer matrix. The final sensor design that allows its manufacture by conventional printing and laminating technologies consists of the sensing polymer matrix (MB-EVOH) sandwiched in a suitable transparent multilayer structure. The outer layers protect the sensor from the external atmosphere and allow visualization of the colour. The inner layer is sufficiently opaque to facilitate sensor reading from the outside, is thick enough to avoid direct contact with food (functional barrier), and is oxygen-permeable to expose the sensing material to the internal package atmosphere. In the absence of oxygen, the sensor becomes white by irradiation with halogen lamps in less than 60 s. All components are substances permitted for food contact except the pigment, but specific migration analysis showed no trace of migration thanks to the functional barrier included in the design.

Antimicrobial packaging based on a LAE containing zein coating to control foodborne pathogens in chicken soup.

In this study, zein coatings containing Lauroyl-l-arginine ethyl ester monohydrochloride (LAE) were developed to be applied on polypropylene films and manufacture an active food packaging. The concentration of LAE and the addition of a suitable plasticizer (glycerol or oleic acid (OA)) were the main variables considered. Active plasticized zein films, with glycerol or oleic acid were characterized in terms of release kinetics, mechanical, barrier, optical, and antimicrobial properties. Results showed that active agent concentration, (5 and 10%), had no-significant effect on mechanical and WVP properties of the plasticized films. Films plasticized with OA presented greater water resistance, UV-light opacity, and water barrier properties than glycerol-plasticized films. On the contrary, the latter had better antimicrobial properties. The analysis of LAE release kinetics from films to different food simulants revealed different behaviours, depending on both film formulation and food simulant. Despite the lower water resistance of coatings containing glycerol, bags based on polypropylene/glycerol plasticized zein containing 10% of LAE presented a great antimicrobial activity in tests with chicken soup (real food system) contaminated with pathogen bacteria, concretely, the films showed 3.21 Log reduction against Listeria monocytogenes and 3.07 log reductions against Escherichia coli. These results suggest a promising strategy on the use of LAE-containing zein in active food packaging to control foodborne pathogens.

Development and optimization of antifungal packaging for sliced pan loaf based on garlic as active agent and bread aroma as aroma corrector.

The aim of the present work was the development of antimicrobial films containing garlic extract to be applied as active packaging for preservative-free sliced pan loaf, with the goal of extending its shelf-life. First, the antimicrobial capacity of garlic extract, a compound used as active agent, was tested against Penicillium expansum by the disc diffusion method. The extract showed high antimicrobial activity, 0.1 µL per Petri dish being the minimum inhibitory amount, and 0.25 µL the minimum fungicidal amount. Bread aroma was also used to mask the pungent odour of garlic and it was confirmed to have no antimicrobial activity. Subsequently, polyethylene (PE) aqueous emulsion and ethylene-vinyl alcohol copolymer (EVOH) and zein hydroalcoholic solutions containing 0.25 and 0.5% (w/w per dry polymer) of garlic extract and bread aroma were used to coat PE films, producing PE/PE, PE/EVOH and PE/zein active films. The antimicrobial capacity of the films was studied in vitro against Penicillium expansum, and in vivo with natural sliced bread. The results showed that all the films presented some antimicrobial activity, PE film coated with zein containing 0.5% of garlic extract and bread aroma being the film presenting the best results, maintaining bread free of mould infection for 30 days. Sensory tests showed that the addition of 1% of bread aroma improved the sensory experience of consumers and also revealed good purchase intention.

Chilean berry Ugni molinae Turcz. fruit and leaves extracts with interesting antioxidant, antimicrobial and tyrosinase inhibitory properties.

The knowledge of the biological properties of fruits and leaves of murta (Ugni molinae Turcz.) has been owned by native Chilean culture. The present study investigated the phenolic content, the antioxidant, antimicrobial and anti-tyrosinase activities of different murta fruit and leaves extracts to approach their uses on future food, pharmaceutical and cosmetic applications. Extractions of murta fruit and leaves were carried out under water, ethanol and ethanol 50%. Phenolic content of these extracts was measured through Folin Ciocalteu test and the antioxidant power by four different antioxidant systems (ORAC, FRAP, DPPH and TEAC assays) owing to elucidate the main mechanism of antioxidant. Some flavonoids, such as rutin, isoquercitrin and quercitrin hydrate were identified and quantified through HPLC analysis. Antimicrobial activity was determined measuring minimum inhibition concentration (MIC) and minimum bactericidal concentration (MBC) values against Escherichia coli and Listeria monocytogenes, and the effect of these extracts on L. monocytogenes was confirmed by flow cytometry. Highest contents of polyphenol compounds were obtained in hydroalcoholic extracts (28±1mggallicacid/g dry fruit, and 128±6mggallicacid/g dry leaves). The same trend was found for the values of biological properties: hydroalcoholic extracts showed the strongest activities. Leaves presented higher antioxidant, antimicrobial and anti-tyrosinase properties than murta fruit. Highest antioxidant activity values according to ORAC, FRAP, TEAC and DPPH were 80±8mgTrolox/g, 70±2mgTrolox/g, 87±8mgTrolox/g and 110±12mgTrolox/g, respectively, for murta fruit samples, and 280±10mgTrolox/g, 192±4mgTrolox/g, 286±13mgTrolox/g and 361±13mgTrolox/g, respectively, for murta leaves. These activities were confirmed by HPLC analysis that revealed highest presence of analyzed compounds on leaves hydroalcoholic extract. Regarding to antimicrobial analysis, hydroalcoholic leaves extract presented the highest activity presenting the lowest MIC value for L. monocytogenes (0.07mg/mL). This extract also performed the highest anti-tyrosinase activity (CE50 values of 1.6±0.3 (g/L) and 8.9±1.2 (g/L) for leaves and fruit, respectively).

Effect of hydroxypropyl-ß-cyclodextrin and coadjuvants on the sorption capacity of hydrophilic polymer films for monoterpene alcohols.

Chitosan films filled with hydroxypropyl-ß-cyclodextrin at a 1:1 weight ratio and plasticized with 35 or 50% glycerol or 35% propylene glycol were prepared by casting and conditioned at different relative humidities to achieve a similar water content. Sorption properties of the films for various monoterpene compounds with phenolic, or with 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 sorption values ranging from 455% for meta-cumenol to 193% for guaiacol, for chitosan films with the same 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 propylene glycol, whereas no changes were observed in the retention of non-phenolic monoterpenes. Replacement of chitosan with polyvinyl alcohol 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 Escherichia coli and Staphylococcus aureus. The present study shows that hydroxypropyl-ß-cyclodextrin and the plasticization level achieved by hydrophilic films can be used to regulate loading capacity and sorption selectivity of naturally occurring antimicrobial compounds.

Antimicrobial packaging of chicken fillets based on the release of carvacrol from chitosan/cyclodextrin films.

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 25g of chicken fillets. These samples were stored for 9days at 4°C. 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 5000mg/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 24cm(2) device ranged from 0.3 log reductions against lactic acid bacteria to 1.8logs 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.

Antifungal properties of gliadin films incorporating cinnamaldehyde and application in active food packaging of bread and cheese spread foodstuffs.

Gliadin films incorporating 1.5, 3 and 5% cinnamaldehyde (g/100g protein) were tested against food-spoilage fungi Penicillium expansum and Aspergillus niger in vitro, and were employed in an active food packaging system for sliced bread and cheese spread. Gliadin films incorporating cinnamaldehyde were highly effective against fungal growth. P. expansum and A. niger were completely inhibited after storage in vitro for 10 days in the presence of films incorporating 3% cinnamaldehyde. Indeed 1.5% cinnamaldehyde was sufficient in the case of P. expansum. The amount of cinnamaldehyde retained in films after storage for 45 days at 20 °C and 0% RH was also sufficient in most cases to prevent fungal growth in vitro. Active food packaging with gliadin films incorporating 5% cinnamaldehyde increased the shelf-life of both sliced bread and cheese spread. Mold growth was observed on sliced bread after 27 days of storage at 23 °C with active packaging, whereas in the control bread packaged without the active film fungal growth appeared around the fourth day. In the cheese spread, no fungi were observed after 26 days of storage at 4 °C when the product was packaged with the active film. However, growth of fungi was observed in control packaged cheese after 16 days of storage. This work demonstrates a noteworthy potential of these novel bioplastics incorporating natural antimicrobial compounds as innovative solutions to be used in active food packaging to extend shelf-life of food products.

Preparation and characterization of chitosan/HP-ß-cyclodextrins composites with high sorption capacity for carvacrol.

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-ß-cyclodextrins (HP-ßCDs) 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 (RH) and glycerol (G) content of the biocomposites. The highest sorption values were achieved for composites incorporating 35% glycerol and conditioned at 75% these composites retained 216% 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.

Development of a novel antimicrobial film based on chitosan with LAE (ethyl-N(α)-dodecanoyl-l-arginate) and its application to fresh chicken.

Chitosan (CS) films incorporating the antimicrobial compound ethyl-N(α)-dodecanoyl-l-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 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-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.

Active films based on cocoa extract with antioxidant, antimicrobial and biological applications.

Novel films of ethylene-vinyl alcohol copolymer (EVOH) containing flavonoid-rich cocoa were developed. To understand their potential application as active packaging material, antioxidant and antimicrobial properties of the films were determined as well as the antioxidant activity of the release compounds in Caco-2 human epithelial colorectal adenocarcinoma cells. Exposure of the films to aqueous food simulant showed antioxidant capacity. The release of cocoa extract components was dependent on the antioxidant concentration incorporated in the film and on temperature. Cocoa extract and the fraction obtained after in vitro gastrointestinal digestion presented antioxidant activity against oxidative stress induced by hydrogen peroxide in Caco-2 cells. Films with 10%, 15%, and 20% cocoa extract produced bactericidal effect against Staphylococcus aureus, Listeria monocytogenes, Escherichia coli and Salmonella enterica. The application of films to an infant milk formula, previously inoculated with L. monocytogenes, inhibited the growth of bacteria 1.5 log units the first day and showed sustained release, inhibiting 0.52 and 0.76 log units, respectively, by the sixth day, while cocoa powder added directly did not produce any effect.

Silver ions release from antibacterial chitosan films containing in situ generated silver nanoparticles.

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 activity 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.