Incorporation of the Antilisterial Bacteriocin-like Inhibitory Substance from Pediococcus parvulus VKMX133 into Film-forming Protein Matrices with Different Hydrophobicity

ABSTRACT: The antilisterial bacteriocin-like inhibitory substance (BLIS) produced by Pediococcus parvulus VKMX133 was incorporated into protein film matrices of ethanol-soluble corn zein (CZ), and water-soluble whey protein isolate (WPI). Various BLIS concentrations were added to film-forming solutions (FFS), cast, dried, and cut in circular sections (28.27 mm²). Antimicrobial activity of films was evaluated by measuring inhibition zones against Listeria innocua ATCC 33090 on tryptic soy broth (TSB) agar. BLIS released from films into water at 10 °C was determined. Film effectiveness was evaluated by measuring the reduction of L. innocua population (10⁸ colony-forming units [CFU]/mL) in peptone water where film sections were immersed. Film topography was analyzed by scanning electron microscopy (SEM). The minimum BLIS concentration in FFS to generate films with antimicrobial activity was 833 and 3333 arbitrary units per milliliter (AU/mL) for CZ and WPI, respectively. BLIS released into water was detected only for CZ films. Antimicrobial CZ films were more effective in reducing L. innocua population than WPI films at the same BLIS concentrations. SEM showed that surface topography was porous for CZ and more closed and compact for WPI films. BLIS can be entrapped into film protein matrices to produce edible antimicrobial packaging. However, BLIS inhibitory action against L. innocua and release were dependent on film nature and topography, and probably on hydrophobic and electrostatic interactions arising between the protein matrices and BLIS. High concentration of bacteriocin in films does not necessarily improve their effectiveness against L. innocua.     

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.     

Innovative multilayer antimicrobial films made with Nisaplin® or nisin and cellulosic ethers: Physico-chemical characterization,bioactivity and nisin desorption kinetics

In this study, ethylcellulose/hydroxypropylmethylcellulose/ethylcellulose (EC/HPMC/EC) three-layer films including Nisaplin® or nisin and lecithins were formulated. Lecithins were used as plasticizers to ensure cohesion between hydrophobic ethylcellulose and hydrophilic HPMC layers. It was observed that the introduction of pure nisin or its non-pure commercial form Nisaplin® into films didn't significantly alter their mechanical and optical properties. Additionally, these nisin or Nisaplin-loaded multilayer films showed significant antimicrobial activity. The comparison of inhibition diameters obtained with EC/HPMC film used as control and EC/HPMC/EC films demonstrated that the three-layer films delayed nisin desorption. This was confirmed by the kinetics of nisin release in a (0.8% w/v) NaCl solution at 28 °C: nisin from two-layer EC/HPMC films totally desorbed within 0.5 h, while the three-layer films allowed to expand nisin release time over 20 h. The ratio of nisin desorption coefficients (k_d): k_{d (EC/HPMC)}/k_{d (EC/HPMC/EC)} was determined after desorption modelling, and was found to be up to 118, proving that multilayer films with hydrophobic layers could be a potential way to control nisin release from antimicrobial bio-packagings.Industrial relevanceThis paper concerns active packaging, considered as a new approach to preserve food shelf life. Active packaging is a real gain for plastic and food industrials. Coating was used to obtain antimicrobial packaging. The impact of incorporating the antimicrobial agent in multilayer films on the release kinetics is investigated.     

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 effects of corn zein films impregnated with nisin, lauric acid, and EDTA

Bacterial growth during food transport and storage is a problem that may be addressed with packaging materials that release antimicrobials during food contact. In a series of five experiments, EDTA, lauric acid (LA), nisin, and combinations of the three antimicrobial agents were incorporated into a corn zein film and exposed to broth cultures of Listeria monocytogenes and Salmonella Enteritidis for 48 h (sampled at 2, 4, 8, 12, 24, and 48 h). Four experiments used starting cultures of 10(8) CFU/ml in separate experiments tested against each bacterium; the fifth experiment examined the inhibitory effect of selected antimicrobial agents on Salmonella Enteritidis with an initial inoculum of 10(4) CFU/ml. L. monocytogenes cell numbers decreased by greater than 4 logs after 48 h of exposure to films containing LA and nisin alone. No cells were detected for L. monocytogenes (8-log reduction) after 24-h exposure to any film combination that included LA. Of all film agent combinations tested, none had greater than a 1-log reduction of Salmonella Enteritidis when a 10(8)-CFU/ml broth culture was used. When a 10(4) CFU/ml of Salmonella Enteritidis initial inoculum was used, the films with EDTA and LA and EDTA, LA, and nisin were bacteriostatic. However, there was a 5-log increase in cells exposed to control within 24 h. The results demonstrate bacteriocidal and bacteriostatic activity of films containing antimicrobial agents.     

乳酸链球菌素与植酸大豆蛋白复合膜的功能特性

为了开发新型大豆蛋白可食膜,将天然减菌剂乳酸链球菌素（Nisin）和植酸加入到大豆蛋白膜中,研究它们对膜的遮光、隔氧、阻湿功能及蛋白质交联度和膜结构的影响。结果表明：添加Nisin会引起大豆分离蛋白膜阻湿性和蛋白质交联度下降,但遮光性和隔氧性增强。植酸对膜的遮光性、阻湿性、隔氧性和蛋白质的交联度的影响与其添加量有关。电镜扫描分析结果表明,大豆分离蛋白膜表面平整光滑,添加了Nisin后,膜中出现复杂纹路,但仍然呈大理石状;继续加入植酸,膜的大理石状结构遭到了破坏。     

Physical properties and antioxidant activity of an active film from chitosan incorporated with green tea extract

An active film from chitosan incorporated with aqueous green tea extract (GTE) was developed. The effects of GTE concentrations including 2, 5, 10 and 20% (w/v) of green tea in the film-forming solution on the film properties were determined by measuring physical properties, total polyphenolic content and antioxidant activity of the active films. Fourier Transform Infrared (FTIR) spectrometry was carried out to observe the potential modifications of the chitosan films when incorporated with GTE. The results suggested that incorporation of GTE into chitosan films improved mechanical and water vapor barrier properties and enhanced polyphenolic content and antioxidant activity of the films. Changes in the FTIR spectra of the chitosan films were observed when GTE was incorporated, suggesting some interactions occurred between chitosan and the polyphenols from GTE. This study showed the benefits of incorporation of GTE into chitosan films and the potential for using the developed film as an active packaging.     

ANTIMICROBIAL PROPERTIES OF NISIN-COATED POLYMERIC FILMS AS INFLUENCED BY FILM TYPE AND COATING CONDITIONS

Polymeric films were coated with nisin, an antimicrobial peptide, for potential application in shelf-life extension of food. Five commercial packaging films with varying surface energies were compared. The highest antimicrobial activity was observed on the most hydrophobic nisin-coated films (surface energy ± 32 dyne/cm). The binding affinity of nisin to the surface of ethylene acrylic acid copolymer (surface energy = 32 dyne/cm) was investigated. Films were coated in nisin solutions under varying conditions including nisin concentration, contact time, solution's volume to film area ratio, pH of coating solution and temperature during coating. Stronger nisin activity was detected on films as the coating time increased from 30 s to 2 h. Higher nisin coating concentration also promoted the binding of nisin to films with saturation level observed at 250 μg/mL. Films coated with nisin using a higher solution to film area ratio (0.84 compared to 0.14 and 0.28 mL/cm²) also exhibited higher nisin activity.     

Physical and Chemical Properties of Edible Films Containing Nisin and Their Action Against Listeria Monocytogenes

ABSTRACT: The effects of hydrophobicity/hydrophilicity of edible films against Listeria monocytogenes strain V7 by various nisin concentrations (4.0 - 160 IU/film disk) and pH values ranging from 2.0 to 8.0 were determined and the mechanical properties and water vapor permeability of films prepared with or without nisin were compared. Surface hydrophobicities (446, 282, 232 and 142, respectively) of whey protein isolates, soy protein isolates, egg albumen and wheat gluten were determined. As the nisin concentration increased, the amount of inhibition progressively increased in all tested films. Using nisin, edible films with higher hydrophobicity values of 280 to 450 units under an acidic environment exerted a greater inhibitory effect against L. monocytogenes. Different interactions of nisin with the proteins of the different films resulted in variation in the mechanical properties and water vapor permeabilities of the films tested.     

Functional Properties of Antimicrobial Lysozyme-Chitosan Composite Films

ABSTRACT: Lysozyme-chitosan composite films were developed for enhancing the antimicrobial properties of chitosan films. A 10% lysozyme solution was incorporated into 2% chitosan film-forming solution (FFS) at a ratio of 0%, 20%, 60%, and 100% (w lysozyme/w chitosan). Films were prepared by solvent evaporation. Lysozyme release from the film matrix, the antimicrobial activity of films against Escherichia coli and Streptococcus faecalis , and basic film properties were investigated. The lysozyme release proportionally increased with increasing initial concentration of lysozyme in the film matrix, and the amount of released lysozyme was in natural log relationship with time. The films with 60% lysozyme incorporation enhanced the inhibition efficacy of chitosan films against both S. faecalis and E. coli , where 3.8 log cycles reduction in S. faecalis and 2.7 log cycles reduction in E. coli were achieved. Water vapor permeability of the chitosan films was not affected by lysozyme incorporation, whereas the tensile strength and percent elongation values decreased with increased lysozyme concentration. Scanning electron microscopy images revealed that lysozyme was homogeneously distributed throughout the film matrix. This study demonstrated that enhanced antimicrobial activity of lysozyme-chitosan composite films can be achieved by incorporating lysozyme into chitosan, thus broadening their applications in ensuring food quality and safety.     

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.     

Development and characterization of an antimicrobial packaging film coating containing nisin for inhibition of Listeria monocytogenes

The purpose of this study was to develop and characterize a packaging film coating containing nisin. A spot-on-lawn assay was used to determine the effect of acid type (ascorbic, acetic, hydrochloric, lactic) and nisin level (equal increments from 10,000 IU to 9 IU) to be used in the formulation of the film coating. Zones of inhibition were measured after incubation on tryptic soy agar (37 degrees C, 48 h). Low-density polyethylene films coated with differing levels of nisin were characterized by field emission scanning electron microscopy, tensile strength, elongation, and water vapor transmission rate. The MIC of nisin in solution was 157 mg/ml. All acids were equally inhibitory (P > 0.05), but acetic acid produced the largest zone of inhibition (21 mm). Field emission scanning electron microscopy confirmed that the cloudy appearance of the films was due to sodium chloride found in the commercially prepared nisin. Tensile strength increased as nisin concentration increased, which also corresponded to increasing film thickness. The nisin coatings (10,000 and 2,500 IU/ml) did not have a significant effect (P > 0.05) on the water vapor transmission rate of the low-density polyethylene film.     

Listeria monocytogenes inhibition by defatted mustard meal-based edible films

An antimicrobial edible film was developed from defatted mustard meal (Sinapis alba) (DMM), a byproduct from the bio-fuel industry, without incorporating external antimicrobials and its antimicrobial activity against Listeria monocytogenes and physical properties were investigated. The DMM colloidal solution consisting of 184 g water, 14 g DMM, and 2g glycerol was homogenized and incubated at 37°C for 0.2, 0.5, 24 or 48 h to prepare a film-forming solution. The pH of a portion of the film-forming solution (pH 5.5) was adjusted to 2.0 or 4.0. Films were formed by drying the film-forming solutions at 23°C for 48 h. The film-forming solution incubated for 48 h inhibited L. monocytogenes in broth and on agar media. Antimicrobial effects of the film prepared from the 48 h-incubated solution increased with decrease in pH of the solution from 5.5 to 2.0. The film from the film forming solution incubated for 48 h (pH 2.0) initially inhibited more than 4.0 log CFU/g of L. monocytogenes inoculated on film-coated salmon. The film-coating retarded the growth of L. monocytogenes in smoked salmon at 5, 10, and 15°C and the antimicrobial effect during storage was more noticeable when the coating was applied before inoculation than when it was applied after inoculation. The tensile strength, percentage elongation, solubility in watercxu, and water vapor permeability of the anti microbial film were 2.44 ± 0.19 MPa, 6.40 ± 1.13%, 3.19 ± 0.90%, and 3.18 ± 0.63 gmm/kPa hm(2), respectively. The antimicrobial DMM films have demonstrated a potential to be applied to foods as wraps or coatings to control the growth of L. monocytogenes.     

Effectiveness and stability of plastic films coated with nisin for inhibition of Listeria monocytogenes

Plastic films were coated with a cellulose-based carrier solution containing nisin, a natural antimicrobial peptide with the potential to inhibit growth of food spoilage and pathogenic microorganisms such as Listeria monocytogenes. Five commercial plastic films with different chemical compositions and surface properties were compared in this study: low-density polyethylene, ethylene-vinyl acetate copolymer, and three types of ethylene-methacrylic acid copolymers: Surlyn 1601, Nucrel 0403, and Nucrel 0903. The films were coated with nisin at a concentration of 1000 IU/cm2. Nisin-coated films were stored at room temperature (21 degrees C) and at 4 degrees C and analyzed weekly for 12 weeks. Antimicrobial activity of the different nisin-coated films against a nisin indicator strain, Lactococcus lactis subsp. cremoris ATCC 14365, and against L. monocytogenes ATCC 19115 was assessed using an inhibition zone assay. Nisin incorporated into the films was recovered by a boiling and extraction procedure, and its activity was quantified using an agar well diffusion assay. Film type did not have any significant effect on the antimicrobial activity of the nisin-coated films (P < 0.05); all five film types had comparable inhibition zones on both assays. The films maintained stable activity for the duration of the study, both at room temperature and refrigeration. The results of this study demonstrate that commercially available packaging films can be coated with nisin and the resulting antimicrobial films can be conveniently stored at room temperature with no adverse effect on nisin activity.     

Antimicrobial activity of lactoferrin against foodborne pathogenic bacteria incorporated into edible chitosan film

The objectives of this research were to develop and characterize edible chitosan film containing lactoferrin as a natural antimicrobial agent, and to investigate the combination effects of lactoferrin with lysozyme in chitosan film against the growth of Escherichia coli O157:H7 and Listeria monocytogenes. Chitosan films containing lactoferrin, lysozyme, or nisin were fabricated, and the antimicrobial concentrations were 0.5, 1, or 2 mg in a circular disc of chitosan film. Three concentrations of lactoferrin or EDTA (0.28, 0.56, or 1.12 mg per disc) were also incorporated into the chitosan film containing lysozyme to investigate the combination effects of lactoferrin. The water barrier properties of the chitosan films containing lactoferrin were characterized. The antimicrobial activities against E. coli O157:H7 and L. monocytogenes were determined using the agar diffusion assay and cell count assay. The chitosan films containing lactoferrin less than 1 mg per disc did not alter the water vapor permeability of the chitosan film. Although the film containing lysozyme exhibited significant antimicrobial activity, the incorporation of lactoferrin alone into chitosan film did not exhibit significant antimicrobial activity against both E. coli O157:H7 and L. monocytogenes. However, the combination of lactoferrin with lysozyme-containing chitosan film significantly decreased the growth of E. coli O157:H7, exhibiting a comparable effect to that of the combination of EDTA with lysozyme (P < 0.05). Furthermore, the combination of lactoferrin with lysozyme in chitosan film exhibited greater reduction in the growth of L. monocytogenes than did the combination EDTA with lysozyme, resulting in an approximate 3-log reduction.     

Influence of the homogenization conditions and lipid self-association on properties of sodium caseinate based films containing oleic and stearic acids

The effect of the homogenization conditions of the film-forming emulsions and lipid self-association on the physical properties of sodium caseinate films containing oleic and stearic acids was studied. For this purpose, different film-forming emulsions were prepared by using different homogenization methods and were characterized as to particle size distribution and rheological properties. Likewise, mechanical, structural and optical properties and water vapour permeability (WVP) of the obtained films were also determined. While films containing stearic acid showed a laminar-like structure, oleic acid was more homogeneously dispersed in the film matrix. These differences in structure make the stearic acid films less flexible, showing more surface roughness and less gloss and transparency than films containing oleic acid. The film microstructure also affects the WVP. In this sense, for oleic acid films, water barrier efficiency increased when homogenization conditions were more intense, whereas for films containing stearic acid, the opposite effect was observed. This different behavior was attributed to the different kind of lipid self-association in the aqueous media, protein interactions and their impact on the final film microstructure.     

Enhanced antimicrobial activity of nisin in combination with allyl isothiocyanate against Listeria monocytogenes,Staphylococcus aureus,Salmonella Typhimurium and Shigella boydii

This study was designed to evaluate the synergistic antimicrobial effect of nisin and allyl isothiocyanate (AITC) against Listeria monocytogenes, Staphylococcus aureus, Salmonella Typhimurium and Shigella boydii. The synergistic interactions between nisin and AITC were observed against all foodborne pathogens, showing the fractional inhibitory concentrations <1. The populations of L. monocytogenes and S. aureus at the combined treatment of nisin and AITC were decreased to below 1 log CFU mL^?1 after 10‐h incubation at 37 °C. The changes in fatty acid profiles of all strains were substantially influenced by nisin alone and the combined treatment of nisin and AITC. A good agreement was observed among cell viability, membrane permeability and depolarisation activity in response to nisin and AITC. The results suggest that nisin and AITC as synergistic inhibitors could be an effective approach to achieve satisfactory antimicrobial activity against a wide range of foodborne pathogens.     

复合抗菌膜对腊肉品质的影响

采用羧甲基纤维素(CMC)与明胶制作复合型抗菌膜用于腊肉的防腐保鲜。以膜的水蒸气透过率、膜的厚度和透光度为主要指标,对膜液的组成进行优化,以此为基础加入适量的山梨酸钾,制备出具有良好性能的复合型抗菌膜。结果表明,将CMC与明胶以质量比1:2的比例配制成质量浓度10g/L的成膜液,添加质量分数0.4%的甘油和0.08%的山梨酸钾制成复合抗菌膜,对腊肉进行涂膜保鲜够延缓腊肉的腐败速度,延长贮藏时间。     

Characterisation of gelatin–fatty acid emulsion films based on blue shark (Prionace glauca) skin gelatin

Incorporation of fatty acids (stearic and oleic) into edible films based on blue shark (Prionace glauca) skin gelatin was investigated to modify properties such as water vapour barrier and flexibility due to their hydrophobicity and plasticizing effect, respectively. Addition of stearic acid from 0% to 100% of protein concentration in the film-forming solution considerably decreased water vapour permeability of gelatin–fatty acid emulsion films compared to addition of oleic acid at the same fatty acid concentration. Increasing concentrations of both fatty acids decreased tensile strength, but increased elongation at break due to their plasticizing effect. At the same concentration, oleic acid gave a greater plasticizing effect than did stearic acid. On the other hand, transparency of the gelatin–stearic acid emulsion film was lower than that of the gelatin–oleic acid emulsion film. Faster stirring speed of homogenisation improved properties of only the gelatin–stearic acid emulsion film.     

Enhancing Physical Properties and Antimicrobial Activity of Konjac Glucomannan Edible Films by Incorporating Chitosan and Nisin

ABSTRACT: The antimicrobial effect of konjac glucomannan (KGM) edible ûlm incorporating chitosan (CHI) and nisin at various ratios or concentrations was studied. This activity was tested against pathogenic bacteria, namely, Escherichia coli, Staphylococcus aureus, Listeria monocytogenes , and Bacillus cereus. Mechanical and physical properties were determined, and the results indicated that the blend film KC2 (mixing ratio KGM 80/ CHI 20) showed the maximum tensile strength (102.8 ± 3.8 MPa) and good transparency, water solubility, and water vapor transmission ratio. Differential scanning calorimetry (DSC) and Fourier transform infrared (FTIR) spectroscopy were used to characterize the structural change of the blend films; and the results showed that strong intermolecular hydrogen bonds occurred between CHI and KGM. Incorporation of nisin at 463IU per disk of film for the selected KC2 was found to have antimicrobial activity against S. aureus, L. monocytogenes , and B. cereus. The mean value of inhibition zone diameter of the CHI-N series and the KC2-N series were higher than the KGM-N series at each corresponding concentration and with significant difference ( P < 0.05), however, there was no significant difference in the antimicrobial effect between CHI and KC2 incorporating nisin. At all these levels, the blend ûlm KC2-nisin had a satisfactory appearance, mechanical and physical properties, and antimicrobial activity. Therefore, it could be considered as a potential "active" packaging material.