Antimicrobial and physical properties of edible chitosan films enhanced by lactoperoxidase system

Effects of lactoperoxidase system (LPOS) incorporated directly into chitosan films at different concentrations (0.5, 1 and 1.5%) were studied. Films obtained were tested on the inhibition of phytopathogenic strains such as Xanthomonas campestris pv. Mangifera indica, Colletotrichum gloeosporioides (C. 64, C. 4612 and C. 62) and Lasiodiplodia theobromae ngr 05A. Water vapor permeability and mechanical properties of films with LPOS and/or not iodine were also studied. Antibacterial effect obtained by disc diameter technique indicated that chitosan concentration at 1 and 1.5% (w/w) incorporated with LPOS and/or not iodine inhibited higher X. campestris pv. M. indica than chitosan film alone or at low concentrated of 0.5% incorporated by LPOS. The antimicrobial technique using puncture gave very good information on the antifungal effect and on the variability in susceptibility of strains of fungi. C. gloeosporioides C64 and L. theobromae were inhibited completely by 1 and 1.5% chitosan incorporated by LPOS contained or not iodine, while C. gloeosporioides C4612 was sensitive to the presence of iodine and C62 were resistant strains. The properties of chitosan films were not significantly changed by the incorporation of the enzyme system.     

Antimicrobial Action of Chitosan against Spoilage Organisms in Precooked Pizza

ABSTRACT: Chitosan antifungal capacity when the biopolymer is used as an edible film and as a dough ingredient in precooked pizza was studied. This action was evaluated against the fungi that produce major spoilage in that product. It was observed that the use of chitosan in acetic acid as edible coating (0.079 g/100 g pizza) delayed Alternaria sp, Penicillium sp, and Cladosporium sp (Deuteromycetes) growth. This behavior was similar compared with the action of calcium propionate (0.103 g/100 g pizza) and potassium sorbate (0.034 g/100 g pizza) preservatives. On the other hand, chitosan showed a little sensibility on Aspergillus sp (Zygomycetes). It was demonstrated that the use of chitosan into the dough was not effective.     

Antifungal activity of films and solutions based on chitosan against typical seed fungi

The use of eco-freindly polymers as antimicrobial materials is in growth due to the need to reduce the negative impact of conventional treatments on the environment and the human health. The purpose of the present study was to assess the antifungal properties of films and solutions based on chitosan with different molecular weight at different concentrations. Surfactants were added to the formulation to assess their impact on treatment efficiency. The antifungal activity was conducted against tree fungi, Aspergillus niger, Alternaria alternata and Rhizopus oryzae. Results indicated important and significant differences of the antifungal activity between chitosan based solutions and chitosan based films. Furthermore, the antifungal activity of the different treatment depended on the type of fungus treated. Thus, chitosan film treatments were significantly more effective on A. niger than solution treatments. On the other hand, solution treatments resulted in higher radial inhibition when applied against A. alternata or R. oryzae. The highest radial inhibition was observed against A. alternata (97%) using a chitosan solution. The influence of the other parameters (concentration, molecular weight and surfactant type) on treatment efficiency was not as important and their significance depended on treatment type and fungus nature.     

Antimicrobial Effects of Lactoferrin, Lysozyme, and the Lactoperoxidase System and Edible Whey Protein Films Incorporating the Lactoperoxidase System Against Salmonella enterica and Escherichia coli O157:H7

Lactoferrin (LF), lysozyme (LZ), the lactoperoxidase system (LPOS), and edible whey protein isolate (WPI) films incorporating LPOS were studied for inhibition of Salmonella enterica and Escherichia coli O157:H7. Antimicrobial effects of LF (5 to 40 mg/mL), LZ (1 to 20 mg/mL), and LPOS (0.5% to 5.0% [w/v] [0.03–.25 g/g, dry basis]) were examined by measuring turbidity of antimicrobial‐containing media after inoculation and by examining cell inhibition by WPI films incorporating LPOS (LPOS‐WPI films) on an agar recovery medium. Elastic modulus (EM), tensile strength (TS), percent elongation (%E), oxygen permeability (OP), and Hunter L, a and b of WPI films incorporating 0.03 to 0.25 g/g of LPOS were compared with those of plain WPI films without LPOS. The growth of S. enterica and E. coli O157:H7 (4 log colony‐forming units [CFU]/mL) in tryptic soy broth (TSB) was not prevented by LF at ≥20 and ≥40 mg/mL, respectively. S. enterica and E. coli O157:H7 in TSB were not inhibited by LZ at ≥ 6 and ≥ 20 mg/mL, respectively. LPOS at concentrations of 2.75% (w/v) and 1.0% (w/v) reduced S. enterica and E. coli O157:H7 to below the limit of detection (1 CFU/mL) in TSB, respectively. LPOS‐WPI films (0.15 g/g) completely inhibited S. enterica and E. coli O157:H7 (4 log CFU/cm²), inoculated either onto agar before placing the film disc or onto top of the film disc. Incorporation of 0.25 g/g of LPOS decreased EM, TS, and %E. The oxygen barrier property of WPI films was improved with the incorporation of LPOS at 0.15 to 0.25 g/g.     

Listeria monocytogenes Inhibition by Whey Protein Films and Coatings Incorporating the Lactoperoxidase System

Antimicrobial effects of whey protein isolate (WPI) films and coatings incorporating the lactoperoxidase system (LPOS) against Listeria monocytogenes were studied by turbidity, plate counting, disc‐covering, and disc‐surface‐spreading tests using various growth media. Survival of L. monocytogenes applied to smoked salmon before or after the coating was monitored immediately after application and during storage at 4 °C and 10 °C for up to 35 d. Tensile properties (elastic modulus [EM], tensile strength [TS], elongation [E]), oxygen permeability (OP), and color (Hunter L, a, b) of WPI films, with and without LPOS, were also compared. LPOS inhibited L. monocytogenes in broth and on agar media. WPI films incorporating 29 mg of LPOS per gram of film (dry basis) inhibited 4.2 log colony‐forming units (CFU)/cm² of L. monocytogenes inoculated on agar media. WPI coatings prepared with LPOS at 0.7% (w/w) in a coating solution (40 mg LPOS/g coating [dry basis]) initially reduced >3 and 1 log CFU/g of L. monocytogenes and total aerobic microorganisms in smoked salmon, respectively. The WPI coatings incorporating LPOS prevented the growth of L. monocytogenes in smoked salmon at 4 °C and 10 °C for 35 d and 14 d, respectively. The tensile properties, oxygen permeability, and color of WPI films were not significantly changed by incorporation of LPOS (P >0.05).     

Antibacterial Activities of Chitosans and Chitosan Oligomers with Different Molecular Weights on Spoilage Bacteria Isolated from Tofu

Seven bacteria were isolated from spoiled tofu and identified as Bacillus sp. (S08), B. megaterium (S10), B. cereus (S17, S27, S28, S32), and Enterobacter sakazakii (S35). In a paper disc test with 6 chitosans and 6 chitosan oligomers of different molecular weights, chitosans showed higher antimicrobial activity than did chitosan oligomers at a 0.1% concentration. Results of inhibitory effects of 6 chitosans on growth of Bacillus sp. (S08) failed to detect viable cells after incubation for 24 hrs at 37 C, even at 0.02% concentration. With B. megaterium (S10) and B. cereus (S27), a 3 to 4 log cycle reduction was found in the chitosan‐treated group. The growth of Enterobacter sakazakii (S35) was completely suppressed in the presence of 0.04% chitosan except for 1 chitosan product. The minimum inhibitory concentration of chitosan differed with products and isolates, ranging from 0.005% to above 0.1%.     

Inactivation of Gram-negative pathogens in refrigerated milk by reuterin in combination with nisin or the lactoperoxidase system

Inhibitory activity of reuterin (β-hydroxypropionaldehyde) combined with the antimicrobial peptide nisin or the lactoperoxidase system (LPOS) against food-borne Gram-negative pathogens in milk refrigerated at 4 and 8 °C was investigated. At 4 °C, reuterin (8 AU/ml) and LPOS were bactericidal against Salmonella enterica, Campylobacter jejuni, Aeromonas hydrophila and Yersinia enterocolitica, whereas the only effect recorded for nisin was a slight inhibition of Escherichia coli O157:H7. At 8 °C, reuterin was bactericidal against all the Gram-negative pathogens studied. Same results were attained with LPOS, except for its effect on Y. enterocolitica which was only bacteriostatic. The combination of reuterin with nisin did not enhance the antimicrobial effect of reuterin. A strong synergistic bactericidal activity of reuterin in combination with LPOS on E. coli O157:H7 and S. enterica was observed in milk at 4 °C, and against all the Gram-negative bacteria assayed in milk refrigerated at 8 °C. The application of both antimicrobials would be a useful means to inhibit pathogenic microorganisms, which may be present in milk due to postpasteurization contamination.     

Antifungal Coatings on Fresh Strawberries (Fragaria × ananassa) to Control Mold Growth During Cold Storage

ABSTRACT: Chitosan (2%) -based or hydroxypropyl methylcellulose (HPMC) (1%) -based coatings were applied on fresh strawberries to evaluate their antifungal efficacies against Cladosporium sp. and Rhizopus sp. Potassium sorbate (PS) was also incorporated into coating formulas to evaluate any additional inhibitory effects on mold inhibition. Strawberries were inoculated with Cladosporium sp. or Rhizopus sp. at a level of approximately 10³ log colony-forming units (CFU) /g, coated with 2% chitosan, 2% chitosan containing 0.3% PS, or 1% HPMC containing 0.3% PS, and stored at about 5°C and about 50% RH up to 23 d for enumeration of mold, yeast, total aerobes, and coli forms. To assess antifungal activity of the coating materials in vitro, coating solutions were embedded into agar plates and the diameters of radial mold growth were measured after inoculation. In addition, weight loss of coated strawberries and water vapor permeability of the coatings were measured. No significant combined inhibitory effects between chitosan and PS on fungal growth on fresh strawberries were detected. However, significant combined inhibition activity was observed in in vitro testing when PS was formulated into chitosan. Antifungal activity of chitosan against Cladosporium sp. and Rhizopus sp. was not affected by the autoclaving process of coating solutions. Coating treatment also reduced total aerobic count, coliforms, and weight loss of strawberries during storage. Hence, chitosan can be used as a natural antimicrobial coating on fresh strawberries to control the growth of fungi, thus extending shelf-life of the fruits.     

Evidence of cell-associated proteinases from Virgibacillus sp. SK33 isolated from fish sauce fermentation

Abstract: Cell‐associated proteinases from Virgibacillus sp. SK33 isolated from fish sauce fermentation were extracted and characterized. Proteinases were effectively released when washed cells were incubated in 0.3 mg/mL lysozyme in 50 mM Tris‐maleate (pH 7) at 37 °C for 2 h. Major cell‐associated proteinases exhibited molecular mass of 17, 32, and 65 kDa, but only a 32‐kDa proteinase showed strong amidolytic activity toward Suc‐Ala‐Ala‐Pro‐Phe‐AMC. Activity of all cell‐associated proteinases was completely inhibited by phenylmethanesulfonyl fluoride, indicating a characteristic of serine proteinase. In addition, a 65‐kDa serine proteinase was also inhibited by ethylenediaminetetraacetic acid, implying a metal‐dependent characteristic. Optimum activity toward a synthetic peptide substrate was at 50 °C and pH 8 and 11. Proteinases with molecular mass of 17 and 32 kDa exhibited caseinolytic activity at 25% NaCl and activity based on a synthetic peptide substrate increased with NaCl concentrations up to 25%, suggesting their role in hydrolyzing proteins at high salt concentrations. This is the first report of liberated cell‐associated proteinases from a moderate halophile, Virgibacillus sp. Practical Application: The cell‐associated proteinases could be extracted from Virgibacillus sp. SK 33 using lysozyme. The extracted enzyme could be applied to hydrolyze food proteins at NaCl content as high as 25%. In addition, this study demonstrated that not only extracellular but also cell‐associated proteinases are key factors contributing to protein‐degrading ability at high salt environment of Virgibacillus sp. SK 33.     

Influence of temperature and relative humidity on the immobilized lactoperoxidase system in a functional chitosan film

The effect of temperature (10–30 °C), relative humidity (RH, 50–90%), and storage period (2–10 days) were studied on the production and sustainability of thiocyanate (SCN⁻) and hypothiocyanite–hypoiodite (OSCN⁻, OI⁻) components of a lactoperoxidase system (LPOSI) in chitosan film by applying response surface methodology and Doelhert experimental design. The results of analysis of variance indicated that the temperature and storage period exhibited a significant effect on the film properties. Temperature had a positive influence on OSCN⁻, OI⁻, while these products (OSCN⁻, OI⁻) decreased during the storage period, and SCN⁻ disappeared after 2 days. Lactoperoxidase systems with iodine (LPOSI) or without iodine (LPOS), incorporated into chitosan films, showed inhibition of Xanthomonas campestris pv. Mangiferae indicae and did not changed the chitosan film permeability to gas and water vapor.     

2-Hydroxy-4-methoxybenzaldehyde inhibits the growth of Aspergillus flavus via damaging cell wall,cell membrane,manipulating respiration thus creating a promising antifungal effect on corn kernels

This study investigated the antifungal activity and the potential antifungal mechanisms of 2-hydroxy-4-methoxybenzaldehyde (HMB) against Aspergillus flavus (A. flavus). The minimum inhibitory concentration (MIC) of HMB in preventing spore germination was 70 μg mL⁻¹. HMB at MIC disrupted cell wall integrity by reducing the number of septa by 86.66% (P < 0.05) in mycelia and increased cell membrane permeability by about 14-fold (P < 0.05) evidenced by propidium iodide (PI) staining. Furthermore, HMB at MIC inhibited respiration by 33.33%. These results revealed that the antifungal activity of HMB against A. flavus could be attributed to the damaged cell wall integrity, cell membrane permeability and respiration metabolism. What’s more, A. flavus was completely restrained in corn kernels due to HMB. Therefore, HMB could be applied as an effective antifungal agent.     

抗青霉芽孢杆菌优良菌株的分离、筛选及鉴定

为了获得一株对青霉有强烈拮抗作用的芽孢杆菌以应用于食品的防腐保鲜中,从木瓜、柑橘、葡萄、全麦面包、太子参、陈皮等食品及中草药共计30个样品中分离得到132株芽孢杆菌。通过平板对峙法,筛选出18株对青霉有较强拮抗作用的芽孢杆菌。对其中3株抑菌效果显著的芽孢杆菌利用杯碟法测定其发酵液的抑菌效果,最终筛选出一株芽孢杆菌LPL40,其发酵上清液对青霉的抑菌圈直径达到(13.94±1.90)mm。通过形态学、生理生化及16S rDNA鉴定,最终将该菌株命名为解淀粉芽孢杆菌LPL40(Bacillus amyloliquefaciens LPL40)。     

Potential of chitosan coating in delaying the postharvest anthracnose (Colletotrichum gloeosporioides Penz.) of Eksotika II papaya

The in vitro and in vivo fungicidal activity of chitosan was studied against Colletotrichum gloeosporioides, the causal agent of anthracnose in papaya fruits. Chitosan at 1.5% and 2.0% concentrations showed a fungistatic effect with 90–100% inhibition (significant at P ≤ 0.05) of the fungal mycelial growth. Changes in the conidial morphology were also observed with the higher chitosan concentrations after 7‐ h incubation. In vivo studies showed that 1.5% and 2.0% chitosan coatings on papaya not only controlled the fruit decay but also delayed the onset of disease symptoms by 3–4 weeks during 5 weeks storage at 12 ± 1 °C and slowed down the subsequent disease development. However, when leaving the fruits to ripen at ambient temperature (28 ± 2 °C), 2.0% chitosan was less effective than 1.5% in controlling the disease development. Chitosan coatings also delayed the ripening process by maintaining the firmness levels, soluble solids concentration and titratable acidity values during and after storage.     

Antifungal activity of five different essential oils in vapour phase for the control of Colletotrichum gloeosporioides and Lasiodiplodia theobromae in vitro and on mango

Mango fruit has high commercial value; however, major postharvest losses are encountered throughout the supply chain due to postharvest diseases. These results lead to the search for natural fungicide for postharvest diseases control. The antifungal effects of five essential oils (thyme, clove, cinnamon, anise and vitex) were assessed by disc volatilisation method. Thyme oil vapours at 5 μL per Petriplate, and clove and cinnamon oil at 8 μL per Petriplate showed 100% growth inhibition of mango pathogens in vitro. GC/MS analysis of essential oil showed thymol (23.88), o‐cymol (23.88) and terpinolene (23.88) as the major constituents of thyme oil. Clove and cinnamon oils contain 3‐allyl‐2‐methoxyphenol (37.42%) and benzofuran 3‐methyl (17.97%), respectively. Thyme oil as a fumigant at 66.7 μL L^?1 showed a significant (P < 0.05) inhibition on postharvest pathogens of mango fruits stored at 25 °C for 6 days. Results of our study suggest the possibility of using thyme oil as an alternate natural fungicide to manage postharvest diseases in mango.     

Purification and characterization of two types of chitosanase from Aspergillus sp. CJ22-326

Aspergillus CJ22-326, a fungi strain capable of utilizing chitosan as a carbon source, was isolated from soil samples. Two types of chitosanase (ChiA and ChiB) produced from the culture supernatant of Aspergillus CJ22-326 were purified to an apparent homogeneity identified by SDS–PAGE through ammonium sulfate precipitation, CM-Sepharose FF chromatography, and Sephacryl S-200 gel filtration. Molecular weights of the enzymes were 109 kDa (ChiA) and 29 kDa (ChiB). Optimum pH values and temperature of ChiA were 4.0 and 50 °C, respectively, those of ChiB were 6.0 and 65 °C. The enzyme activities of ChiA and ChiB were increased by about 0.5-fold and 1.5-fold, respectively, by the addition of 1 mM Mn²⁺. However, 2.5 mM Ag⁺, Hg²⁺ and Fe³⁺strongly inhibited ChiA and ChiB activities. Viscosimetric assay and analysis of reaction products of these enzymes, using chitosan as a substrate, by TLC indicated endo- and exo-type cleavage of chitosan by ChiB and ChiA, respectively. ChiB catalysed the hydrolysis of glucosamine (GlcN) oligomers larger than pentamer, and chitosan with a low degree of acetylation (0–30%), and formed chitotriose with chitohexaose as the major products. ChiA released a single glucosamine residue from chitosan and glucosamine oligomers. Both of the activities of ChiA and ChiB increased with the degree of deacetylation of chitosan. The enzyme ChiB had a useful reactivity and a high specific activity for producing functional chitooligosaccharides with high degree of polymerization.     

Fabrication of chitosan nanoparticles incorporated with Pistacia atlantica subsp. kurdica hulls’ essential oil as a potential antifungal preservative against strawberry grey mould

Essential oil was extracted from Pistacia atlantica subsp. kurdica hulls and encapsulated in chitosan nanoparticles (NPs) by an emulsion-ionic gelation method. A desirable retention rate (43.3–61.5%) of P. atlantica subsp. kurdica hulls’ essential oil (PAHEO) encapsulated in chitosan NPs was confirmed. In addition, the Fourier transform infrared spectroscopy and X-ray diffraction analysis results revealed the success of PAHEO encapsulation. The formed NPs illustrated a shrunk and spherical shape with a size range of 187.2–632.5 nm as indicated by scanning electron microscopy and dynamic light scattering. The encapsulated PAHEO had a high antifungal activity against Botrytis cinerea under both the in vitro and in vivo conditions. It also significantly decreased the incidence and disease severity of grey mould on strawberries during storage. The spoilage process was postponed by the 8th days of storage at 4 °C in the strawberry fruit treated by PAHEO-incorporated chitosan NPs. These findings imply that NP-encapsulated PAHEO will have promising novel applications in food industries.     

Antibacterial effects of chitosan coating containing Mentha aquatica L. essence against Escherichia coli,Staphylococcus aureus and Listeria monocytogenes in Iranian white cheese

The effect of a chitosan coating and Mentha aquatica L. essence on Iranian white cheese was investigated. Results showed 100% inhibition of Escherichia coli growth using 1.5% essence after 10 days. After 15 days of incubation, the Staphylococcus aureus population was reduced by 44.2%, 70.0%, and 88.5% using 0.5, 1.0 and 1.5% essence, respectively. After 15 days, Listeria monocytogenes growth was inhibited by 63.84%, 70.12%, and 85.9% using 0.5, 1.0, and 1.5% essence, respectively. Inhibition zone diameter studies also confirmed the antibacterial effects of applied coating against all the above‐mentioned bacteria in Iranian white cheese.     

Optimization of Chitosanase Production by <Emphasis Type="Italic">Trichoderma koningii</Emphasis> sp. Under Solid-State Fermentation

This study aimed at the optimization of the production of chitosanase in solid culture. Trichoderma koningii sp., an entomopathogenic fungus, was used to produce chitosanase under solid-state fermentation using a mixture of wheat bran and chitosan. The incubation period; addition of moistening water and culture medium composition were optimized. The protocol to extract the enzyme was also optimized. The optimal conditions for chitosanase production by T. koningii were obtained using a mixture of 3.0 g of wheat bran and 1.5 g of chitosan, with the addition of 2.5 mL of moistening water (pH 5.5) and of 2.5 mL of saline solution (pH 5.5) containing NaNO₃ (1.0 g/L), (NH₄)₂HPO₄ (1.0 g/L), MgSO₄.7H₂O (1.0 g/L), and NaCl (1.0 g/L). Optimal enzyme extraction was carried out adding 20 mL of sodium acetate buffer (200 mM, pH 5.5) at 30 °C under orbital agitation at 150 rpm for 6 min. The optimized production yielded 4.84 IU/gds.     

Natural Control of Corn Postharvest Fungi Aspergillus flavus and Penicillium sp. Using Essential Oils from Plants Grown in Argentina

The objective in this study was to evaluate the antifungal activity of essential oils from native and commercial aromatic plants grown in Argentina against corn postharvest fungi and to link the essential oil bioactivity with lipid oxidation and morphological changes in fungus cell membrane. Essential oil (EO) of oregano variety Mendocino (OMen), Cordobes (OCor), and Compacto (OCom), mint variety Inglesa (Mi), and Pehaujo (Mp), Suico (Sui); rosemary (Ro), and Aguaribay (Ag) were tested in vitro against 4 corn fungi: A. flavus (CCC116–83 and BXC01), P. oxalicum (083296), and P. minioluteum (BXC03). The minimum fungicidal concentration (MFC) and the minimum inhibitory concentration (MIC) were determined. The chemical profiles of the EOs were analyzed by GC‐MS. Lipid oxidation in cell membrane of fungi was determined by hydroperoxides and related with essential oil antifungal activity. The major compounds were Thymol in OCor (18.66%), Omen (12.18%), and OCom (9.44%); menthol in Mi and Mp; verbenone in Sui; dehydroxy‐isocalamendiol in Ag; and eucaliptol in Ro. OCor, Omen, and OCom showed the best antifungal activity. No antifungal activity was observed in Ag and Ro EO. The hydroperoxide value depended on the fungi (P < 0.001) and the antimicrobial agent (P < 0.001).Membrane lipids were oxidized by Sui EO in A. flavus BXC01 and A. flavus CCC116–83 (0.021 and 0.027 meqO₂/kg, respectively). The results suggest that the EOs of OCor, OMen, OCom, Mi, Mp, and Sui grown in Argentina can be used as natural alternatives to control fungi that produce mycotoxin in maize.     

Dephytinization of food stuffs by phytase of Geobacillus sp. TF16 immobilized in chitosan and calcium-alginate

In this work, Geobacillus sp. TF16 phytase was separately immobilized in chitosan and Ca-alginate with the efficiency of 38% and 42%, respectively. These enzymes exhibited broad substrate specificity. Maximal relative phytase activity was measured at pH 5.0 and 95°C and pH 3.0 and 75°C for chitosan and Ca-alginate, respectively. The enzymes were highly stable in a wide pH and temperature range. Values of K_m and V_max were determined as 2.38 mM and 3401.36 U/mg protein for chitosan, and 7.5 mM and 5011.12 U/mg protein for Ca-alginate. The immobilized enzymes showed higher resistance to proteolysis. After 4 h incubation, hydrolysis capacities of chitosan- and Ca-alginate immobilized enzymes for soymilk phytate were calculated as 24% and 33%, respectively. The chitosan- and Ca-alginate immobilized phytases conserved its original activity after 8 and 6 cycles of reuse, respectively. The features of the enzymes were very attractive and they might be useful for some industrial applications.