Physical properties and antilisterial activity of bioactive edible films containing Lactobacillus plantarum

Bioactive polymeric films were developed from the incorporation of Lactobacillus plantarum into polysaccharide or protein edible films. Lactic acid bacteria (LAB) were added directly to the film forming solution and films were obtained by casting. Four different biopolymers were tested: sodium caseinate, pea protein, methylcellulose and hydroxypropylmethylcellulose. In order to study the impact of the incorporation of the protective culture into the biopolymer matrix, the water vapour permeability (WVP), optical and mechanical properties of the dry films were evaluated. Furthermore, survival of L. plantarum and the antimicrobial potential of bioactive films against Listeria innocua were studied. Whereas optical and mechanical properties of the films were not notably altered by the LAB incorporation, WVP values increased for all biopolymers. Viability of the strain was lower in cellulose derivative matrices than in protein films, although bacteriocin production in the newly prepared films was greater in these matrices. As a result, a significant antimicrobial activity against L. innocua was observed for polysaccharide matrices, which was not observed for protein films, where the bacteriocin production is delayed. In pea protein films the maximum bacteriocin production occurs at 15 storage days and afterwards decreases. So, the nature of the biopolymer and the knowledge of time when an adequate bacteriocin concentration is produced are key to use bioactive films in an effective way.     

Development of Tapioca Starch/Decolorized Hsian-Tsao Leaf Gum-Based Antimicrobial Films: Physical Characterization and Evaluation Against Listeria monocytogenes

The physical properties and antimicrobial activities against Listeria monocytogenes of the tapioca starch/decolorized hsian-tsao leaf gum (the polysaccharide gum extracted from hsian-tsao herb followed by removing the color matters, abbreviated as dHG hereafter)-based films incorporated with potassium sorbate (KS) or the ethanolic extract of thyme (TH) were investigated. It was found that the cross-sectional microstructure of starch/dHG films with KS showed some rough texture, and films with TH showed a relatively smooth microstructure with dispersed microparticles. The equilibrium moisture content and water vapor permeability of starch/dHG films with 20 % KS (based on the weight of starch/dHG) was higher than the others tested, possibly due to the plasticizing effect of KS. In general, the tensile strength and tensile modulus decreased with increasing antibacterial concentration. As compared with the KS solutions, the TH solutions showed a pronounced antimicrobial activity against L. monocytogenes as determined by the inhibition zone test. However, the antimicrobial migration of both KS and TH in the starch/dHG matrix was limited to a certain extent, as evidenced by the significantly lower antimicrobial activity in the film system. When applying the starch/dHG films with antimicrobials to the fresh beef slices, all samples showed detectable improvement against the growth of L. monocytogenes, implying that starch/dHG films incorporated with TH or KS were effective against L. monocytogenes in conjunction with some modification of the physical properties due to the interactions between the antimicrobials and the components of film matrix.     

Effects of ingredient composition on optical and mechanical properties of pullulan film for food-packaging applications

In the present research, the effects of pullulan (Pul), glycerin (Gly), xanthan gum (Xa) and locust bean (Lb) concentrations on pullulan film properties were investigated using a Central Composite Rotatable Design. Optimal ingredient combination was determined and antimicrobial activity of films combined with sakacin A was confirmed against Listeria monocytogenes. Using predictive models, contour plots and the characteristics of commercial LDPE films as constraints, the following combination within the optimal region was selected: Pul 100 g/l, Gly 10 g/l, Xa 1 g/l and Lb 1 g/l. Statistical analysis demonstrated that Pul and Gly significantly influenced the properties of pullulan films. Strong interactions were observed between Pul-Gly and Xa-Gly. When sakacin A was added to the film mixture, no significant influence on films optical properties was reported, while an increase in flexibility was observed. Results obtained indicate the potential application of pullulan film developed in this study as an effective antimicrobial biopolymer in food-packaging systems.     

Effects of drying methods and conditions on antimicrobial activity of edible chitosan films enriched with galangal extract

The aim of this work was to study the effects of drying methods and conditions (i.e., ambient drying, hot air drying at 40 °C, vacuum drying and low-pressure superheated steam drying within the temperature range of 70–90 °C at an absolute pressure of 10 kPa) as well as the concentration of galangal extract on the antimicrobial activity of edible chitosan films against Staphylococcus aureus. Galangal extract was added to the film forming solution as a natural antimicrobial agent in the concentration range of 0.3–0.9 g/100 g. Fourier transform infrared (FTIR) spectra and swelling of the films were also evaluated to investigate interaction between chitosan and the galangal extract. The antimicrobial activity of the films was evaluated by the disc diffusion and viable cell count method, while the morphology of bacteria treated with the antimicrobial films was observed via transmission electron microscopy (TEM). The antimicrobial activity, swelling and functional group interaction of the antimicrobial films were found to be affected by the drying methods and conditions as well as the concentration of the galangal extract. The electron microscopic observations revealed that cell wall and cell membrane of S. aureus treated by the antimicrobial films were significantly damaged.     

Evaluation of antimicrobial and physical properties of edible film based on carboxymethyl cellulose containing potassium sorbate on some mycotoxigenic Aspergillus species in fresh pistachios

Active packaging is a relatively novel concept of packaging that changes the conditions of the packaged food to extend its shelf-life and improve its safety. In this study, antimicrobial effects of carboxymethyl cellulose based-edible film containing potassium sorbate as an antimicrobial agent were studied against Aspergillus flavus (PTCC-5004), Aspergillus parasiticus (PTCC-5286) and A. parasiticus (PTCC-5018) by using agar diffusion assay. Results showed suitable inhibition effects against A. parasiticus (PTCC-5286) and A. flavus (PTCC-5004) in comparison with A. parasiticus (PTCC-5018). Pistachios were coated with this edible antimicrobial film containing three concentrations of sorbate (1, 0.5 and 0.25 g/100 mL film solution); all concentrations showed no growth of molds. Tensile strength values of films with potassium sorbate, decreased when compared to control, and film’s flexibility, was 28.82 percent for 3 g/100 mL sorbate, while higher concentration of sorbate, decreased the flexibility. The water vapor permeability values (WVP) of films were determined to be 1.18 (g mm/m² day kPa) for films plasticized with glycerol, without sorbate while WVP values for the films containing 1 and 2 g/100 mL sorbate increased to 3.77 and 15.5 (g mm/m² day kPa), respectively. The observed glass transition temperature (T_G) depression for these polymer blends was related to the plasticizer content (water, polyethylene, and glycerol), especially water.     

Antimicrobial compression-moulded wheat gluten films containing potassium sorbate

Antimicrobial glycerol-plasticized wheat gluten (WG) films containing potassium sorbate (PS) were successfully produced by compression moulding; a thermoplastic process involving high temperature and high pressure. Antifungal properties of the films were tested against Aspergillus niger and Fusarium incarnatum by the agar diffusion assay. The results indicated that films containing more than 10 wt.% PS showed antimicrobial activity against A. niger while films containing 2.5 wt.% or more of PS showed antimicrobial activity against F. incarnatum. It was also found that when the film was exposed to an absorbing medium (the agar solution), most of the PS was released, an interesting feature for edible active packaging. Despite the loss, a very promising result was that, without seeding of spores, the films resisted microbial growth for at least one week when the films were left in the agar solution. X-ray diffraction and field emission scanning electron microscopy revealed that the PS crystals were dissolved in the wheat gluten material. In addition to the antimicrobial properties, dynamic mechanical, tensile, PS loss, water vapour transmission rate and oxygen permeability data also indicated that PS acted as a plasticiser in the wheat gluten film.     

Modelling natamycin release from alginate/chitosan active films

In antimicrobial active films, the active agent is incorporated in the polymeric matrix and is released from the film to the product surface where most deterioration processes take place. In this study, films structured solely by alginate and by blends of alginate:chitosan (82.5:17.5 and 65:35) were obtained by casting. The film formulations received natamycin (4 and 8 g/100 g biopolymer) as antimicrobial agent. Films were characterised according to their thickness and morphology. Diffusion experiments in water indicated very slow release kinetics of the antimicrobial, being markedly hindered in the alginate and chitosan composite films, probably due to electrostatic interactions between chitosan and natamycin. Fickian diffusion prevailed and the diffusion coefficients found for the films varied from 2.6 × 10^?11 to 2.5 × 10^?12 cm² s^?1.     

Characterization of salmon gelatin based film on antimicrobial properties of chitosan against E. coli

Salmon gelatin and chitosan are high potential biopolymer to obtain edible films with antimicrobial effect for fresh meat. Therefore, it is important to characterize the structural (glassy or rubbery) state of the gelatin film on antimicrobial properties of chitosan. Extracted salmon (Salmo Salar) gelatin (acid-basic extraction) and low molecular weight chitosan solutions (0; 0.25; 0.5 and 1% w/w) at pH 5.5 were prepared to obtain a final concentration to 7% w/w (gelatin + chitosan). Films were obtained by casting at 5 °C and equilibrated at 33% and 85% of relative humidity (RH) in order to obtain a glassy and rubbery state, respectively. Water content, specific volume and glass transition temperature (Tg) were measured and antimicrobial properties against Escherichia coli (E.c.) (105 ufc/ml) were obtained by agar plate diffusion and kinetic measurements at 37 °C. Chitosan diffusion in agar plate was determined using liquid solutions and films over agar plate. The results showed diminution of Tg (maintaining structural state) as increase chitosan concentration, increasing also specific volume and water content due their high capacity to adsorb water. Although inhibition by chitosan of bacteria growth kinetic was 100%, solutions and films samples could not diffuse in the agar. In conclusion, the chitosan could not diffuse on the films matrix and also in agar plate and therefore in food when is combined with salmon gelatin. The implication of this work have been investigate the use of edible films from marine sources with antimicrobial effects in an effort to growing demand from consumers for safer and better quality foods.     

Preparation of active antimicrobial methyl cellulose/carvacrol/montmorillonite nanocomposite films and investigation of carvacrol release

Methyl cellulose (MC)/carvacrol (CRV)/montmorillonite (MMT) nanocomposite films were prepared to obtain active antimicrobial packaging materials. The characterization of film samples by X-ray diffraction and transmission electron microscopy showed that composite films were of nano structures. CRV addition to the MC film and MC/MMT nanocomposite films led to a decrease in the thickness and opacity values of them, whereas MMT addition to the film matrix caused an increase in these values. Thermal stability of films slightly increased with increasing MMT concentration in film matrix. CRV release from films was investigated at different temperatures for 30 days. An increase in the MMT concentration matrix caused a decrease in CRV release at 25.0 ± 0.5 °C and in 60 ± 4% relative humidity (RH). CRV release increased with temperature at a constant RH. The antimicrobial activities of films were tested against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) by the microatmosphere method and these organisms were completely inhibited on the nutrient broth/bacteriological agar medium when film samples containing 11.1 ± 0.2 mg CRV were present. MC/CRV film and MC/CRV/MMT-60% nanocomposite films on sausage reduced E. coli and S. aureus counts by 0.9 and 0.7 log cfu/mL, respectively, compared to the control film. The amount of CRV release from developed antimicrobial films can be controlled by MMT concentration within the film matrix and by the storage temperature of film.     

Characterization of antimicrobial polylactic acid based films

Olive leaf extract (OLE) (Olea europaea L.), which has antimicrobial effect on many food pathogens, was incorporated as antimicrobial agent into polylactic acid (PLA) films. Antimicrobial activities of films were tested against Staphylococcus aureus. Increasing amount of the OLE in the film discs from 0.9 mg to 5.4 mg caused a significant increase in inhibitory zones from 9.10 mm to 16.20 mm, respectively. Moreover, incorporation of OLE and/or increasing the amount in the film formulation significantly enhanced the water vapor permeability (WVP). The water solubility and the degradation rates of films increased up to 19.3% and 22.4%, respectively. Thus, OLE incorporated PLA films have a prospectively potential in antimicrobial food packaging to reduce post-process growth of S. aureus with improved properties.     

Antimicrobial assays of natural extracts and their inhibitory effect against Listeria innocua and fish spoilage bacteria, after incorporation into biopolymer edible films

The antimicrobial activity of twelve natural extracts was tested against two fish spoilage bacteria (Pseudomonas fluorescens and Aeromonas hydrophila/caviae) and Listeria innocua, in order to assess their potential utilization in the preservation and safety of minimally processed fish products. After a screening of the active extracts by agar diffusion and vapour diffusion methods, oregano and thyme essential oils and citrus extract were selected. The minimum inhibitory concentration (MIC) of the selected extracts was determined by disc diffusion method against target bacteria and at two temperatures: bacteria's optimal growth temperature (30°C or 37°C) and refrigeration temperature (4°C). Due to its better solubility, lack of odour and greater inhibitory effect obtained against L. innocua at refrigerated temperature, citrus extract was selected and incorporated at 1% (v/v) into different biopolymer film forming solutions (gelatin, methyl cellulose and their blend 50:50 w/w). The antimicrobial activity of the developed films was then evaluated, just after preparation of the films and after one month of storage at 43±3% relative humidity and 24±3°C. Regardless of the biopolymer matrix, all the developed films showed antimicrobial activity against the target bacteria. The most sensitive bacterium towards active films was L. innocua while P. fluorescens appeared as the most resistant one, in accordance with the previously performed antimicrobial tests for pure extracts. The differences in activity of the films between the tested two temperatures were not significant except for L. innocua, for which three times higher inhibition diameters were observed at refrigerated temperature. The inhibitory effectiveness of the films against the tested strains was maintained regardless of the biopolymer matrix for at least one month. Therefore, these edible films show potential for their future use in fresh fish fillets preservation.     

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.     

Properties and antimicrobial activity of edible films incorporated with kiam wood (Cotyleobium lanceotatum) extract

The antimicrobial properties of wood extracts are well known; however their application to edible films is limited. In this study, the minimum bactericidal concentration (MBC) of kiam wood extract was established as 300 mg/L at which bacterial growth was completely inhibited. The antimicrobial properties of hydroxypropyl methylcellulose (HPMC) films containing 1-5 fold of MBC of kiam wood extract were tested against Escherichia coli O175:H7, Staphylococcus aureus and Listeria monocytogenes. The edible films containing kiam wood extract exhibited more effective impact on the growth reduction of L. monocytogenes than S. aureus and E. coli (p < 0.05). The use of kiam wood extract at 1 and 2 fold of MBC incorporated into edible HPMC films did not exhibit any antimicrobial activity. However, the inhibitory effect of edible HPMC films containing kiam wood extract was observed at 3, 4 and 5 fold of MBC. The greatest zone of inhibition was observed at 5 fold of MBC incorporated in edible HPMC films. Tensile strength and elongation at break significantly decreased with the incorporation of kiam wood extract, whereas water vapor permeability and film solubility increased. The color of edible films became darker and more reddish-yellowish as well as having a lower transparency as the level of kiam wood extract was increased. Kiam wood extract incorporated in edible film provided the films with a rougher surface than pure edible film. Our results pointed out that the incorporation of kiam wood extract as a natural antibacterial agent has potential for use in extending the shelf life of food products.     

Antimicrobial activity of whey protein based edible films incorporated with oregano,rosemary and garlic essential oils

The use of edible films to release antimicrobial constituents in food packaging is a form of active packaging. Antimicrobial properties of spice extracts are well known, however their application to edible films is limited. In this study, antimicrobial properties of whey protein isolate (WPI) films containing 1.0–4.0% (wt/vol) ratios of oregano, rosemary and garlic essential oils were tested against Escherichia coli O157:H7 (ATCC 35218), Staphylococcus aureus (ATCC 43300), Salmonella enteritidis (ATCC 13076), Listeria monocytogenes (NCTC 2167) and Lactobacillus plantarum (DSM 20174). Ten millilitres of molten hard agar was inoculated by 200 μl of bacterial cultures (colony count of 1 × 10⁸ CFU/ml) grown overnight in appropriate medium. Circular discs of WPI films containing spice extracts, prepared by casting method, were placed on a bacterial lawn. Zones of inhibition were measured after an incubation period. The film containing oregano essential oil was the most effective against these bacteria at 2% level than those containing garlic and rosemary extracts (P < 0.05). The use of rosemary essential oil incorporated into WPI films did not exhibit any antimicrobial activity whereas inhibitory effect of WPI film containing garlic essential oil was observed only at 3% and 4% level (P < 0.05). The results of this study suggested that the antimicrobial activity of some spice extracts were expressed in a WPI based edible film.     

Gelatine based films added with bacteriocins and a flavonoid ester active against food-borne pathogens

Active films, based on food grade gelatine added with bacteriocins and/or the flavonoid ester prunin laurate as antimicrobial compounds, were prepared. The films were characterized by water vapor permeability, mechanical property measurements and scanning electron microscopy. Film antibacterial activity was determined by the agar diffusion and direct contact microplate techniques against Listeria monocytogenes 01/155, Staphylococcus aureus ATCC29213 and Bacillus cereus 1. Physical properties of gelatine films were not significantly affected by active compound incorporation. Antibacterial effect on each strain was dependent on the active compound incorporated into the film, but in all cases a synergistic inhibitory action was observed when both antimicrobial compounds were added. Thus, the results of this study suggest that gelatine based active films here presented could provide an alternative strategy for food biopreservation.Industrial relevanceThis work proposes the elaboration of active films based on food grade gelatine and the addition of enterocins A, B and P synthesized by Enterococcus faecium SM21 and the flavonoid ester, prunin laurate, as antimicrobial active compounds. Films were prepared using a simple and low cost methodology and their application can be adapted to different kinds of food systems. The inclusion of the antimicrobial compounds on the film matrix did not alter their functional properties. The obtained films were active against L. monocytogenes, S. aureus and B. cereus, providing an alternative tool for food preservation, especially in the case of foods susceptible to contamination by these pathogens. Although the inhibitory effect of active films was different for each indicator strain, in all cases a synergistic inhibition effect was observed when both active compounds, ES and PL, were added to gelatine films. This synergistic effect between both antimicrobial compounds offers a novel potential hurdle technology for food preservation.     

Study of the performance of nisin supported in edible films

The antimicrobial activity of nisin supported in edible films prepared with suspensions of tapioca starch containing glycerol, was studied. Films were prepared by casting the systems after gelatinization. The effect of the edible film as antimicrobial barrier to external hazard as well as the diffusional characteristics of the nisin and its release characteristics were studied in parallel to antimicrobial inactivation. Studies were performed with L. innocua, after equilibration of edible films at a relative humidity (RH) of 57.5% and at 25 °C. Results obtained showed that nisin supported in starch-based films is active and that the film is a useful barrier to further product contamination. Gradual release of the antimicrobial from the edible film can also help to preclude microorganism proliferation better than nisin directly added because it seems to counterbalance, at least partially, the inactivation of nisin.     

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.     

Characterization of a starfish gelatin film containing vanillin and its application in the packaging of crab stick

To explore the use of starfish gelatin (SFG) films as a biodegradable material, SFG from starfish was extracted and used as a film material. In addition, to provide antimicrobial activity and enhanced flavor of SFG films, vanillin was incorporated. As the concentration of vanillin increased, the tensile strength of the films increased and water vapor permeability decreased. With regard to the structural characteristics of SFG films containing vanillin, the microstructure of the SFG films was not affected by the addition of vanillin. In addition, the SFG films containing vanillin exhibited antimicrobial activity against Listeria monocytogenes. As the application of the SFG films, crab sticks were packed with SFG films containing 0.05% vanillin. During storage, the populations of L. monocytogenes inoculated on crab sticks wrapped with SFG films containing vanillin were lower than those on the control sample, suggesting that SFG films containing vanillin can be useful in active food packaging.     

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.     

Effects of nisin concentration on properties of gelatin film-forming solutions and their films

The addition of nisin into a gelatin matrix can change properties of the film. The aim of this work was to develop gelatin-based films containing different nisin concentrations in order to study their influence on the film's antimicrobial and physical properties and their rheological properties as a film-forming solution (FFS). The FFS was characterised by rheological assays, and the gelatin-based active films were characterised and assessed by the effects of nisin concentrations on their various properties, including antimicrobial activity. Nisin's concentration affected not only its viscoelastic properties of FFS but also its film solubility in water, film surface roughness and light barrier. The addition of nisin also slightly modified the water contact angle and the mechanical properties of the gelatin films. Finally, the films demonstrated activity against Staphylococcus aureus and Listeria monocytogenes at concentrations above 56 mg of nisin g⁻¹ of gelatin.