Characterization and antimicrobial analysis of chitosan-based films

Chitosan-based films for food packaging applications were prepared by casting and dried at room temperature or heat-treated in order to study functional properties and antimicrobial activity. In all cases, films were flexible and transparent, regardless of chitosan molecular weight, glycerol content, and temperature. Regarding antimicrobial activity, chitosan film forming solutions showed antimicrobial behaviour against Escherichia coli and Lactobacillus plantarum. It was also observed that the bacteriostatic property of chitosan-based films against bacteria employed in this study was notably affected by temperature. Moreover, temperature produced significant variation in the functional properties of chitosan-based films, such as colour, wettability, resistance against UV light and mechanical properties. In good agreement with this behaviour, total soluble matter (TSM), fourier transform infrared (FTIR) spectroscopy, thermo-gravimetric analysis (TGA) and X-ray diffraction (XRD) results suggested a change in the chemical structure of chitosan films, possibly due to Maillard reaction when heat treatment was used.     

Chitosan Polymer as Bioactive Coating and Film against Aspergillus niger Contamination

ABSTRACT: The inhibitory activity of chitosan-based edible coatings and films was assessed against the Aspergillus niger food pathogen and deterioration microorganism. Spore-counting assays showed an almost total inhibition of A. niger growth when either film-forming solution or film were used at a low concentration of chitosan (0.1% w/v). Epifluorescence microscopic results showed the action of chitosan on the relative proportion of RNA compared with DNA. The water vapor permeability (WVP) of chitosan film was relatively low compared with the poor moisture barrier of some polysaccharide films. Moreover, a coating with chitosan film on an agar gel, used as a food model, induced a 30% reduction in water loss. These results showed potential applications of chitosan-based films as bioactive packaging with properties to limit the food dehydration phenomenon.     

Physical and antimicrobial properties of chitosan–tea tree essential oil composite films

Antimicrobial films were prepared by incorporating different concentrations of tea tree essential oil (TTO) into chitosan (CH) films. Film-forming dispersions (FFD) were characterized in terms of rheological properties, particle size distribution and ζ-potential. In order to study the impact of the incorporation of TTO into the CH matrix, the water vapour permeability (WVP), mechanical and optical properties of the dry films were evaluated. The properties of the films were related with their microstructure, which was observed by SEM. Furthermore, the antimicrobial effectiveness of CH–TTO composite films against Listeria monocytogenes and Penicillium italicum was studied.     

Physicochemical and Engineering Properties of Nanocomposite Films Based on Chitosan and Pseudoboehmite Alumina

Chitosan-based nanocomposite plastic films were developed by adding 0, 1, 3, and 5 % boehmite alumina (BAH) nanoparticles as a percentage of chitosan powder weight. The films were cast via solution. The effect of BAH content on the physicochemical and engineering properties of the resultant films were determined. The swelling, water vapor adsorption capacity, and transparency were significantly reduced with increased BAH content. The stability of the films against microbial degradation under high relative humidity also increased with BAH content. The water vapor permeability (WVP) reduced with increased temperature giving rise to negative activation energy values, which ranged between 2.08 and 3.36 kJ/mol. However, at constant temperature, inclusion of BAH did not have significant effect on water vapor permeability (WVP). WVP was predicted to high accuracy (r ²?=?0.984) using a full quadratic regression model. All the films had similar tensile and thermal behaviors. The implications of the findings are discussed based on prospective applications of the biodegradable film especially for fresh produce packaging.     

Effects of Interactions between the Constituents of Chitosan-Edible Films on Their Physical Properties

The main objective of this work was to evaluate the effect of chitosan and plasticizer concentrations and oil presence on the physical and mechanical properties of edible films. The effect of the film constituents and their in-between interactions were studied through the evaluation of permeability, opacity and mechanical properties. The effects of the studied variables (concentrations of chitosan, plasticizer and oil) were analysed according to a 2³ factorial design. Pareto charts were used to identify the most significant factors in the studied properties (water vapour, oxygen and carbon dioxide permeability; opacity; tensile strength; elongation at break and Young’s modulus). When addressing the influence of the interactions between the films’ constituents on the properties above, results show that chitosan and plasticizer concentrations are the most significant factors affecting most of the studied properties, while oil incorporation has shown to be of a great importance in the particular case of transport properties (gas permeability), essentially due to its hydrophobicity. Water vapour permeability values (ranging from 1.62?×?10^?11 to 4.24?×?10^?11 g m^?1 s^?1 Pa^?1) were half of those reported for cellophane films. Also the mechanical properties (tensile strength values from 0.43 to 13.72 MPa and elongation-at-break values from 58.62% to 166.70%) were in the range of those reported for LDPE and HDPE. Based on these results, we recommend the use of 1.5% (w/w) chitosan concentration to produce films, where the oil and plasticizer proportions will have to be adjusted in a case-by-case basis according to the use intended for the material. This work provides a useful guide to the formulation of chitosan-based film-forming solutions for food packaging applications.     

Antimicrobial Polylactic Acid Packaging Films against Listeria and Salmonella in Culture Medium and on Ready-to-Eat Meat

The contamination of Listeria monocytogenes and Salmonella spp. in ready-to-eat (RTE) meat products has been a concern for the meat industry. In this study, edible chitosan-acid solutions incorporating lauric arginate ester (LAE), sodium lactate (NaL), and sorbic acid (SA) alone or in combinations were developed and coated on polylactic acid (PLA) packaging films. Antimicrobial effects of coated PLA films on the growth of Listeria innocua, L. monocytogenes, and Salmonella Typhimurium in a culture medium (tryptic soy broth, TSB) and on the surface of meat samples were investigated. Antimicrobial PLA films containing 1.94 mg/cm² of chitosan and 1.94 μg/cm² of LAE were the most effective against both Listeria and Salmonella in TSB and reduced them to undetectable level (<0.69 log CFU/ml). The same PLA films with LAE significantly (p?L. innocua, L. monocytogenes, and S. Typhimurium on RTE meat during 3 and 5 weeks’ storage at 10 °C, achieving 2–3 log reduction of Listeria and 1–1.5 log reduction of Salmonella as compared with controls. PLA films coated with 1.94 mg/cm² of chitosan, 0.78 mg/cm² of NaL, and 0.12 mg/cm² of SA significantly reduced the growth of L. innocua but were less effective against Salmonella. The combination of NaL (0.78 mg/cm²) and SA (0.12 mg/cm²) with LAE (1.94 μg/cm²) did not generate additional or synergetic antimicrobial effect against Listeria or Salmonella on the meat surface. L. innocua had a similar sensitivity to the film treatments as L. monocytogenes, suggesting that L. innocua may be used as a surrogate of L. monocytogenes for further scaleup and validation studies. The film treatments were more effective against the microorganisms in TSB culture medium than in RTE meat, which suggests that in vivo studies are a necessary step to develop antimicrobial packaging for applications in foods.     

Effects of Vacuum Packaging and Wrapping with Chitosan-Based Edible Film on the Extension of the Shelf Life of Sea Bass (Dicentrarchus labrax) Fillets in Cold Storage (4 °C)

In the present study, we assessed the possibility of improving the shelf life of fresh sea bass (Dicentrarchus labrax) fillets by using vacuum packaging and wrapping with chitosan-based edible films during cold storage at 4 °C. Sea bass fillet samples were periodically evaluated to assess chemical (pH, trimethylamine and total volatile basic nitrogen) and microbiological (presence of mesophilic aerobic bacteria and psychrotrophic bacteria) quality. Chemical spoilage (trimethylamine and total volatile basic nitrogen) and growth of microorganisms (total mesophilic and total psychrophilic aerobic bacterial counts) were significantly reduced (P?≤?0.05) in vacuum-packaged chitosan film wrapping during cold storage at 4 °C. The results showed that the shelf life of the control and vacuum-packaged groups ended within 5 days, whereas that of vacuum-packaged chitosan film-wrapped samples ended at 25 to 30 days. Therefore, the shelf life of sea bass fillets wrapped in chitosan was prolonged by about 20 days.     

Chitosan-gelatin composites and bi-layer films with potential antimicrobial activity

The aims of this work were to develop composite (Chi-Ge) and bi-layer (Chi/Ge) edible and biodegradable films based on gelatin and chitosan. physico-chemical properties such as water resistance, transparency and color were analyzed. Composite and bi-layer systems were uniform, homogeneous and thin; they showed a compact structure indicating a good compatibility between components, which could interact by strong hydrogen bonding, as was confirmed by FTIR. Water vapor permeability (WVP) was determined. Both, bi-layer and laminated systems resulted effective alternatives to reduce WVP of chitosan control film. The tensile strength of composite and bi-layer system did not differ significantly (P > 0.05), but elongation at break of composite films was 40% higher (P < 0.05) than that of bi-layer film. Antimicrobial activity of the films was analyzed. The results indicated that both E. coli and L. monocytogenes showed sensitivity to all the films forming solutions. The inhibition halos of both pathogens to the solutions of Chi and Chi-Ge showed to be extremely sensitive. Results obtained with edible films indicated that only E. coli was sensitive to the combination Chi-Ge and Chi/Ge. Neat Chi film did not induce significant inhibition halos for none of the pathogens, which was quite surprising and still under study.     

Microbial competition: effect of Pseudomonas fluorescens on the growth of Listeria monocytogenes

Listeria monocytogenes Scott A was cultured alone and in coculture with Pseudomonas fluorescens ATCC 33231 to characterize quantitatively the effects of microbial competition on the growth of this psychrotrophic pathogen. The bacteria were cultured in brain–heart infusion broth (BHI), using a 3×3×3×2 complete factorial design to assess the impact of temperature (4, 12, 19°C), initial pH (5·0, 6·0, 7·0), and sodium chloride content (5, 25, 45 gl⁻¹) on the interaction between the two micro-organisms. Samples were periodically plated on BHI agar and Vogel Johnson agar to obtain total counts and L. monocytogenes counts, respectively. Growth curves were generated by fitting the data to the Gompertz equation, and the derived growth kinetics were compared. WhenP. fluorescens did influence the growth of L. monocytogenes, the primary effect was a suppression of the maximum population density (MPD) reached by the pathogen. Suppression of L. monocytogenes was generally associated with low incubation temperatures (4°C) and sodium chloride levels (5 and 25 gl⁻¹). Slight increases (<1·0 log cfu ml⁻¹) in the MPD attained by L. monocytogenes were observed when grown in the presence of P. fluorescens at higher temperatures (12 and 19°C) and sodium chloride levels (25 and 45 gl⁻¹) when the pH was 5·0. The current study supports earlier work that indicates that reliance on microbial competition as a barrier to control L. monocytogenes in refrigerated foods will require detailed knowledge of how the interaction between the pathogen and the microflora is affected by environmental and food characteristics such as storage temperature, pH, and water activity.     

Preparation and Characterization of Antimicrobial Films Based on Chitosan for Active Food Packaging Applications

The aim of this paper was to characterize chitosan samples from the shrimp shells for the later development of antimicrobial active systems. These systems include 100 % chitosan-based films obtained by casting, polyamide films with 5 and 10 % of chitosan obtained by extrusion and polyethylene/polyethylene terephthalate films with a coating of 0.6 % of chitosan. For that purpose, several analytical techniques including IR, ¹H NMR, GPC, and microscopic techniques (scanning electron microscopy and transmission electron microscopy) were used. Within the studied samples, C1 showed the lowest DA and MW and consequently presented the most suitable properties for the development of an active packaging. Additionally, mechanical properties were performed. The effectiveness of the developed systems was evaluated by means of microbiological assays. The tested films showed antimicrobial capacity against coliform enterobacteria, mesophilic aerobic microorganism, and yeast and moulds.     

Biodegradable gelatin–chitosan films incorporated with essential oils as antimicrobial agents for fish preservation

Essential oils of clove (Syzygium aromaticum L.), fennel (Foeniculum vulgare Miller), cypress (Cupressus sempervirens L.), lavender (Lavandula angustifolia), thyme (Thymus vulgaris L.), herb-of-the-cross (Verbena officinalis L.), pine (Pinus sylvestris) and rosemary (Rosmarinus officinalis) were tested for their antimicrobial activity on 18 genera of bacteria, which included some important food pathogen and spoilage bacteria. Clove essential oil showed the highest inhibitory effect, followed by rosemary and lavender. In an attempt to evaluate the usefulness of these essential oils as food preservatives, they were also tested on an extract made of fish, where clove and thyme essential oils were the most effective. Then, gelatin–chitosan-based edible films incorporated with clove essential oil were elaborated and their antimicrobial activity tested against six selected microorganisms: Pseudomonas fluorescens, Shewanella putrefaciens, Photobacterium phosphoreum, Listeria innocua, Escherichia coli and Lactobacillus acidophilus. The clove-containing films inhibited all these microorganisms irrespectively of the film matrix or type of microorganism. In a further experiment, when the complex gelatin–chitosan film incorporating clove essential oil was applied to fish during chilled storage, the growth of microorganisms was drastically reduced in gram-negative bacteria, especially enterobacteria, while lactic acid bacteria remained practically constant for much of the storage period. The effect on the microorganisms during this period was in accordance with biochemical indexes of quality, indicating the viability of these films for fish preservation.     

Influence of α-tocopherol on physicochemical properties of chitosan-based films

Chitosan has been exploited as a material for the development of edible films, and additionally can be used as a carrier of functional compounds such as α-tocopherol. The aim of this work was to evaluate the effects of the incorporation of α-tocopherol in chitosan-based films. FTIR and thermal analyses were performed and showed that the incorporation of α-tocopherol affects the chemical structure of chitosan-based films with the establishment of new chemical bonds and the decrease of crystallinity. Results also showed that the increase of α-tocopherol concentration promotes a decrease of water content (from 12.6 to 11.4%) of the films. The addition of α-tocopherol to chitosan films leads to a significant reduction (p < 0.05) of tensile strength from 34.06 to 16.24 MPa, and elongation-at-break from 53.84 to 23.12%. Film opacity values (ranging from 4.74 to 7.83%) increased when α-tocopherol was incorporated into the film. Antioxidant capacity of chitosan-based films was evaluated and was enhanced when α-tocopherol was present in the film matrix. Results showed that α-tocopherol can be successfully added to the chitosan films enhancing the final quality and shelf-life extension of food products.     

Effects of gelatin origin,bovine-hide and tuna-skin,on the properties of compound gelatin–chitosan films

With the purpose to improve the physico-chemical performance of plain gelatin and chitosan films, compound gelatin–chitosan films were prepared. The effect of the gelatin origin (commercial bovine-hide gelatin and laboratory-made tuna-skin gelatin) on the physico-chemical properties of films was studied. The dynamic viscoelastic properties (elastic modulus G′, viscous modulus, G″ and phase angle) of the film-forming solutions upon cooling and subsequent heating revealed that the interactions between gelatin and chitosan were stronger in the blends made with tuna-skin gelatin than in the blends made with bovine-hide gelatin. As a result, the fish gelatin–chitosan films were more water resistant (∼18% water solubility for tuna vs 30% for bovine) and more deformable (∼68% breaking deformation for tuna vs 11% for bovine) than the bovine gelatin–chitosan films. The breaking strength of gelatin–chitosan films, whatever the gelatin origin, was higher than that of plain gelatin films. Bovine gelatin–chitosan films showed a significant lower water vapour permeability (WVP) than the corresponding plain films, whereas tuna gelatin–chitosan ones were only significantly less permeable than plain chitosan film. Complex gelatin–chitosan films behaved at room temperature as rubbery semicrystalline materials. In spite of gelatin–chitosan interactions, all the chitosan-containing films exhibited antimicrobial activity against Staphylococcus aureus, a relevant food poisoning. Mixing gelatin and chitosan may be a means to improve the physico-chemical performance of gelatin and chitosan plain films, especially when using fish gelatin, without altering the antimicrobial properties.     

Effect of Molecular Weight,Acid, and Plasticizer on the Physicochemical and Antibacterial Properties of β‐Chitosan Based Films

Abstract: Effects of chitosan molecular weight (1815 and 366 kDa), type of acid (1% acetic, formic, and propionic acid, or 0.5% lactic acid) and plasticizer (0, 25% glycerol or sorbital w/w chitosan) on the mechanical, water barrier, and antibacterial properties of β‐chitosan films were investigated. Tensile strength (TS) of high molecular weight (Hw) films was 53% higher than that of low molecular weight (Lw) ones, acetate, and propionate films had the highest TS (43 and 40 MPa) among tested acids, and plasticizer‐reduced film TS 34%. Film elongation at break (EL) was higher in Hw films than in Lw ones, in which formate and acetate films were the highest (9% and 8%, respectively), and plasticizer increased the film EL 128%. Molecular weight of chitosan did not influence water vapor permeability (WVP) of the films. Acetate and propionate films had lower WVP than other acid types of films, and plasticizer increased film WVP about 35%. No difference was found between glycerol and sorbitol films in terms of film mechanical and water barrier properties. Lw β‐chitosan films showed significant antibacterial activity against E. coli and L. innocua. This study demonstrated that β‐chitosan films are compatible to α‐chitosan films in physicochemical properties and antibacterial activity, yet with simple sample preparation. Practical Application: β‐chitosan based edible films at molecular weight of about 300 kDa showed great antibacterial activity against Gram‐positive and Gram‐negative bacteria. The films have similar mechanical and water barrier properties to α‐chitosan based films at the similar molecular weight, but simple sample preparation procedures and more attractive color. The release of active chitosan fragment from the film matrix acts as an antibacterial agent, making β‐chitosan films suitable as intelligent food wraps or coatings for a wide range of food products to control moisture loss and prevent surface bacterial growth.     

The antimicrobial,mechanical, physical and structural properties of chitosan–gallic acid films

Chitosan films incorporated with various concentrations of gallic acid were prepared and investigated for antimicrobial, mechanical, physical and structural properties. Four bacterial strains that commonly contaminate food products were chosen as target bacteria to evaluate the antimicrobial activity of the prepared gallic acid–chitosan films. The incorporation of gallic acid significantly increased the antimicrobial activities of the films against Escherichia coli, Salmonella typhimurium, Listeria innocua and Bacillus subtilis. Chitosan films incorporated with 1.5 g/100 g gallic acid showed the strongest antimicrobial activity. It was also found that tensile strength (TS) of chitosan film was significantly increased when incorporating 0.5 g/100 g gallic acid. Inclusion of 0.5 g/100 g gallic acid also significantly decreased water vapor permeability (WVP) and oxygen permeability (OP). Microstructure of the films was investigated by Fourier transform infrared spectroscopy (FT-IR) and scanning electron microscopy (SEM) and it was found that gallic acid was dispersed homogenously into the chitosan matrix.     

Physico-mechanical and antimicrobial properties of gelatin film from the skin of unicorn leatherjacket incorporated with essential oils

Gelatin films incorporated with bergamot (BO) and lemongrass oil (LO) at various concentrations as glycerol substitute were prepared and characterised. Incorporation of BO and LO at 5–25% (w/w protein) resulted in the decreases in both tensile strength (TS) and elongation at break (EAB) of the films. Water vapour permeability (WVP) were decreased in LO incorporated films, while it was increased in film added with BO at level higher than 5% (P < 0.05). Film solubility and transparency values decreased, and the films had the lowered light transmission in the visible range when BO and LO were incorporated. Films incorporated with LO showed inhibitory effect in a concentration dependent manner against Escherichia coli, Listeria monocytogenes, Staphylococcus aureus and Salmonella typhimurium, but BO added film inhibited only L. monocytogenes and S. aureus. Films containing both BO and LO did not inhibit Pseudomonas aeruginosa. Significant change of molecular organisation and higher intermolecular interactions among gelatin molecules were found in the film structure as determined by FTIR. Thermo-gravimetric analysis (TGA) demonstrated that films added with BO and LO exhibited enhanced heat stability with higher degradation temperature, compared with control film. Scanning electron microscopic (SEM) images revealed the presence of micro-pores in the essential oil incorporated films, which contributed to physical properties of the resulting films. Thus, gelatin films incorporated with BO and LO can be used as active packaging, but the properties could be modified, depending on essential oil added.     

Polypropylene/Montmorillonite Nanocomposites Containing Nisin as Antimicrobial Food Packaging

Antimicrobial nanocomposites prepared with polypropylene, montmorillonite, and nisin were developed as food packaging material. Nisin was incorporated at 1, 2.5, and 5 % (w/w) and the characterization included antimicrobial, mechanical, thermal, barrier, and structural properties. Composite films inhibited the Gram-positive bacteria Listeria monocytogenes, Staphylococcus aureus, and Clostridium perfringens when tested on skimmed milk agar plates. Antimicrobial activity was released in food simulants after contact with the nanocomposites, increasing until 48 h in solutions containing the surfactant Tween 20 or acetic acid. The addition of nisin caused no significant modification in deformation at break values as compared with control films. However, results of tensile strength and Young modulus differed significantly among samples. The higher value for Young modulus was observed for films with 5 % nisin. Water vapor barrier properties were not significantly different among control and antimicrobial films, whereas oxygen permeability was higher for nanocomposites containing nisin. The nanocomposites tested had no significant differences in the melting temperature (165 to 167 °C), and the crystallization temperature ranged from 121 to 129 °C, with lower values for films containing 5 % nisin. Scanning electron microscopy showed that nanocomposites containing 1 and 2.5 % nisin present similar homogeneity to that of control films. Some film properties were affected after nisin incorporation in polypropylene/montmorillonite matrix but active antimicrobial films were obtained, showing suitable behavior as a food packaging material.     

Biocomposite Films Based on κ-Carrageenan/Locust Bean Gum Blends and Clays: Physical and Antimicrobial Properties

The aims of this work were to evaluate the physical and antimicrobial properties of biodegradable films composed of mixtures of κ-carrageenan (κ-car) and locust bean gum (LBG) when organically modified clay Cloisite 30B (C30B) was dispersed in the biopolymer matrix. Film-forming solutions were prepared by adding C30B (ranging from 0 to 16 wt.%) into the κ-car/LBG solution (40/60 wt.%) with 0.3 % (w/v) of glycerol. Barrier properties (water vapour permeability, P _vapour; CO₂ and O₂ permeabilities), mechanical properties (tensile strength, TS, and elongation-at-break, EB) and thermal stability of the resulting films were determined and related with the incorporation of C30B. Also, X-ray diffraction (XRD) was done in order to investigate the effect of C30B in film structure. Antimicrobial effects of these films against Listeria monocytogenes, Escherichia coli and Salmonella enterica were also evaluated. The increase of clay concentration causes a decrease of P _vapour (from 5.34?×?10^?11 to 3.19?×?10^?11 g (m s?Pa)^?1) and an increase of the CO₂ permeability (from 2.26?×?10^?14 to 2.91?×?10^?14 g (m s?Pa)^?1) and did not changed significantly the O₂ permeability for films with 0 and 16 wt.% C30B, respectively. Films with 16 wt.% clay exhibited the highest values of TS (33.82 MPa) and EB (29.82 %). XRD patterns of the films indicated that a degree of exfoliation is attained depending on clay concentration. κ-car/LBG–C30B films exhibited an inhibitory effect only against L. monocytogenes. κ-car/LBG–C30B composite films are a promising alternative to synthetic films in order to improve the shelf life and safety of food products.     

Preparation and characterization of agar/clay nanocomposite films: the effect of clay type

Abstract: Agar‐based nanocomposite films with different types of nanoclays, such as Cloisite Na⁺, Cloisite 30B, and Cloisite 20A, were prepared using a solvent casting method, and their tensile, water vapor barrier, and antimicrobial properties were tested. Tensile strength (TS), elongation at break (E), and water vapor permeability (WVP) of control agar film were 29.7 ± 1.7 MPa, 45.3 ± 9.6%, and (2.22 ± 0.19) × 10^?9 g·m/m²·s·Pa, respectively. All the film properties tested, including transmittance, tensile properties, WVP, and X‐ray diffraction patterns, indicated that Cloisite Na⁺ was the most compatible with agar matrix. TS of the nanocomposite films prepared with 5% Cloisite Na⁺ increased by 18%, while WVP of the nanocomposite films decreased by 24% through nanoclay compounding. Among the agar/clay nanocomposite films tested, only agar/Cloisite 30B nanocomposite film showed a bacteriostatic function against Listeria monocytogenes.     

Storability of Antimicrobial Chitosan-Lysozyme Composite Coating and Film-Forming Solutions

ABSTRACT:  Chitosan-lysozyme (CL) film and coating solutions were prepared aseptically by incorporating 60% lysozyme (w/w chitosan) into 3% chitosan solution. The solutions were stored at 10, 21, and 37 °C up to 6 mo for monthly evaluation of physicochemical and antimicrobial properties. Solutions were made into films at each sampling time to investigate lysozyme release, water solubility, water vapor permeability (WVP), tensile strength (TS), and elongation (EL) of the films. During the 6-mo storage, the pH and translucency of CL solutions did not change ( P > 0.05) and microorganisms were not detected with total aerobic count media. CL solutions became darker, with a more saturated yellow hue developing with increased storage time and temperature. Storage enhanced the antimicrobial activities of the solutions against E. coli and L. monocytogenes , and films made of solutions stored at 37 °C exhibited higher antimicrobial activities against these 2 pathogens than those stored at 10 and 21 °C. Increased water solubility and lysozyme release and decreased TS and EL were observed in films made from solutions stored at 37 °C. WVP was not significantly affected by storage temperature and time. These changes might be attributed to increased chitosan degradation by lysozyme hydrolysis at a higher storage temperature and longer storage time. Results indicated that with storage at 10 to 20 °C, premade CL solutions are stable and may be distributed as a commercial product for coating or film applications or both in different foods for at least 6 mo.