Improvement in the yield of lipophilized lysozyme by the combination with Maillard‐type glycosylation

Hen egg white lysozyme was modified using the Maillard‐type glycosylation method prior to the lipophilization with palmitic acid. The yield of lipophilized lysozyme significantly increased by the pre‐glycosylation of the protein. The lipophilized lysozyme derivative was separated into two main fractions with different level of glycosylation. All fractions showed a strong antimicrobial activity against Gram‐negative bacteria,Escherichia coli. The lipophilization of the lysozyme combined with glycosylation is a promising method for potential industrial applications of the lysozyme due to the enhanced antimicrobial activity and the improved yield.     

Functional and biological activities of casein glycomacropeptide as influenced by lipophilization with medium and long chain fatty acid

This study determined the functional and different biological activities of casein glycomacropeptide (GMP) after conjugation with fatty acids. Medium (i.e. caproic, lauric and myristic acid) and long (i.e palmitic and stearic acid) fatty acids were conjugated to GMP at the available amino group. Functionalities of lipophilized GMP conjugates included foaming and emulsifying activities, and biological activities for bacterial growth inhibition, cell damage and anti-invasion. Greater lipophilization of GMP was achieved with medium chain fatty acids (p < 0.05), which resulted in reduced GMP solubility regardless of fatty acid conjugate. Foaming activity of GMP was lost after lipophilization, but emulsification activity of GMP was enhanced (p < 0.05). A parallel increase in growth inhibition of Salmonella spp. coupled with anti-invasion of Salmonella enteritidis (Inv A) into mammalian cells was induced by lipophilization of GMP with long chain fatty acid. Our results show that GMP modified by lipophilization with specific fatty acids provides improved functionality as a surfactant with enhanced antibacterial activity towards gram negative bacteria.     

Multi-functional biopolymer prepared by covalent attachment of galactomannan to egg-white proteins through naturally occurring Maillard reaction

Protein-polysaccharide conjugate was prepared as a functional biopolymer using protein and polysaccharide via a Maillard-type reaction. Ovalbumin and lysozyme were conjugated with galactomannan under controlled heating and humidity conditions. The antioxidant effect of ovalbumin and the antimicrobial activity of lysozyme were enhanced by the glycosylation. The emulsifying properties of the egg protein were also significantly improved by the modification. The increase in lipid affinity due to the conjugation resulted in the enhancement of the radical scavenging ability of ovalbumin. The effectiveness of lysozyme and its glycosylated derivative in restricting the activity of a Gram-negative pathogen, Edwardsiella tarda in fish was also investigated.     

Effect of Maillard‐based conjugation with dextran on the functional properties of lysozyme and casein

The purpose of this research was to find the best experimental conditions for glycosylation of lysozyme and casein with dextran, and to investigate the effect of glycosylation on the functional properties of these proteins. Glycosylation was performed by allowing proteins to react with dextran under Maillard reaction conditions. The extent of glycosylation was determined by sugar analysis, SDS‐PAGE, gel filtration and cation‐exchange chromatography. Glycosylation of lysozyme with a 1:5 weight ratio of protein to dextran, held at 60 °C for one week under a relative humidity of 79% resulted in coupling of 3.0 mole dextran to one mole lysozyme. In the case of casein, at 60 °C, 48 h and 79% relative humidity, 0.1 and 0.13 mole dextran was attached to one mole of casein when a weight ratio of protein to dextran of 1:5 and 1:7.5 was used, respectively. A decrease in the degree of glycosylation occurred when samples were incubated for 72 h. Enzymatic activity of glycosylated lysozyme was reduced by 20% compared with unmodified lysozyme (P < 0.05). Both proteins exhibited improved solubility at different pH values (3, 7 and 9), different temperatures (25, 40 and 60 °C) and increased heat stability, with a better emulsion activity and emulsion stability than with un‐modified proteins (P < 0.05). These changes might increase the applicability of lysozyme as a natural antimicrobial and casein as a protein ingredient in different food systems. Copyright © 2005 Society of Chemical Industry     

Antibacterial activity of dextran-conjugated lysozyme against Escherichia coli and Staphylococcus aureus in cheese curd

The purposes of this research were to glycosylate lysozyme with dextran under Maillard reaction conditions and assess the antimicrobial characteristics of the lysozyme-dextran conjugate in a culture medium and cheese curd. Solutions containing lysozyme and dextran were incubated at 60 degrees C and at 79% relative humidity. Gel permeation chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis were used to follow the glycosylation process. Under optimum conditions 3.7 mol of dextran were coupled to 1 mol of lysozyme. Lytic activity of the conjugate against the cell wall of Micrococcus luteus was about 62% of that of native lysozyme. Evaluation of the lysozyme-dextran conjugate against test microorganisms (Staphylococcus aureus and Escherichia coli) in culture media indicated a progressive increase in antimicrobial activity, with an increase in enzyme-conjugate concentration. The lysozyme-dextran conjugate was also effective against E. coli in a natural food system, as it reduced the bacterial count by 3 log in cheese curd after 40 days of storage. Unlike E. coli, the antimicrobial action of lysozyme against S. aureus was not improved by conjugation with dextran in both in vitro and in vivo tests. Antimicrobial activity of the lysozyme-dextran conjugate against gram-negative bacteria is probably related to the remaining lytic activity as well as the excellent surfactant properties of the lysozyme-dextran conjugate. These results might increase the applicability of lysozyme as a natural antimicrobial ingredient in different food products.     

Multi‐functional biopolymer prepared by covalent attachment of galactomannan to egg‐white proteins through naturally occurring Maillard reaction

Protein‐polysaccharide conjugate was prepared as a functional biopolymer using protein and polysaccharide via a Maillard‐type reaction. Ovalbumin and lysozyme were conjugated with galactomannan under controlled heating and humidity conditions. The antioxidant effect of ovalbumin and the antimicrobial activity of lysozyme were enhanced by the glycosylation. The emulsifying properties of the egg protein were also significantly improved by the modification. The increase in lipid affinity due to the conjugation resulted in the enhancement of the radical scavenging ability of ovalbumin. The effectiveness of lysozyme and its glycosylated derivative in restricting the activity of a Gram‐negative pathogen,Edwardsiella tarda in fish was also investigated.     

Antimicrobial Activity of Monoacyl Hexose Coexistent with Lysozyme against Gram-Positive Bacilli

The antimicrobial activities of myristoyl, palmitoyl, or stearoyl hexoses, which were glucose, mannose, and galactose, coexistent with lysozyme against three Gram-positive bacteria, Bacillus coagulans, Bacillus subtilis, and Bacillus licheniformis, were measured in order to investigate the availability of monoacyl hexose as an antimicrobial co-agent. The lysozyme exhibited an antimicrobial activity against Bacillus subtilis and Bacillus licheniformis, but there was no significant difference between the dependencies of the antimicrobial activity against the two bacteria on the lysozyme concentration. However, the antimicrobial activities of the monoacyl hexoses coexistent with the lysozyme were different between those against the two bacteria. The stearoyl hexose coexistent with the lysozyme exhibited the highest antimicrobial activity against two bacteria. It was indicated that the antimicrobial action of the monoacyl hexose would be exerted parallel with the bacterial lysis of lysozyme. Stearoyl hexose, which has a high hydrophobicity, coexistent with the lysozyme could exhibit a higher antimicrobial activity than only the lysozyme.     

Impedance measurement of growth of lactic acid bacteria in the presence of nisin and lysozyme

Effect of nisin (0.500 g/l), lysozyme (0.035 g/l) and the mix of nisin (0.500 g/l) and lysozyme (0.035 g/l) against two nisin-producing lactococci strains, three non-nisin-producing lactococci strains and four lactobacilli strains with antimicrobial activity was determined by the impedimetric method. None of the tested lactococci and lactobacilli strains were inhibited by lysozyme at concentration of 0.035 g/l. Only the nisin-producing lactococci strains were resistant to nisin at 0.500 g/l, the other lactococci and lactobacilli were inhibited by nisin at 0.500 g/l intensively. The mix of lysozyme and nisin displayed a comparable effect to the nisin addition against the used lactococci strains and was very variable depending on the strain of tested lactobacilli. The impedimetric method was shown to be convenient for screening the growth abilities of lactococci and lactobacilli in the presence of antimicrobial components.     

Characterization of Fish-Skin Gelatin Gels and Films Containing the Antimicrobial Enzyme Lysozyme

ABSTRACT:  Fish skins are rich in collagen and can be used to produce food-grade gelatin. Films cast from fish-skin gelatins are stable at room temperature and can act as a barrier when applied to foods. Lysozyme is a food-safe, antimicrobial enzyme that can also produce gels and films. When cold-water, fish-skin gelatin is enhanced with lysozyme, the resulting film has antimicrobial properties. The objective of this study was to characterize the effect on strength and barrier properties of lysozyme-enhanced fish-skin gelatin gels and films, and evaluate their activity against potential spoilage bacteria. Solutions containing 6.67% fish-skin gelatin were formulated to contain varying levels of hen-egg-white lysozyme. Gels were evaluated for strength, clarity, and viscoelastic properties. Films were evaluated for water activity, water vapor permeability, and antimicrobial barrier capabilities. Fish-skin gels containing 0.1% and 0.01% lysozyme had pH (4.8) and gelling-temperatures (2.1 °C) similar to lysozyme-free fish-skin gelatin controls. However, gel strength decreased (up to 20%). Turbidities of gels, with or without lysozyme, were comparable at all concentrations. Films cast with gelatin containing lysozyme demonstrated similar water vapor permeabilities and water activities. Lysozyme was still detectable in most fish gelatin films. More antimicrobial activity was retained in films cast with higher lysozyme concentrations and in films where lysozyme was added after the gelatin had been initially heated. These results suggest that fish-skin gelatin gels and films, when formulated with lysozyme, may provide a unique, functional barrier to increase the shelf life of food products.     

Antimicrobial activity of immobilized lysozyme on plasma-treated polyethylene films

In this study we tested the antimicrobial activity of polyethylene films modified by means of plasma processes that were followed by the chemical immobilization of lysozyme, an antimicrobial enzyme. To chemically immobilize the enzyme in its active form at the surface of polyethylene, substrates that had been plasma treated under different experimental conditions were soaked in lysozyme solutions at different concentrations. The immobilization of the enzyme was checked, and the antimicrobial activity of the films was investigated by observing the death rate of Micrococcus lysodeikticus cells suspended in phosphate buffer in contact with the films. The results clearly indicate that plasma-treated films loaded with lysozyme are active against the selected microorganism. A modified version of the Gompertz equation was used to quantitatively valuate the dependence of the antimicrobial activity of the films under both plasma treatment conditions and lysozyme concentrations.     

老蒜提取物萃取部位的抑菌活性及成分分析

本实验将大蒜切片后于室温下浸泡于10%乙醇水溶液或者蒸馏水一段时间,得到老蒜提取物样品一和样品二;依次用石油醚、乙酸乙酯、正丁醇对样品一和样品二进行萃取,得到不同极性的萃取部位,以多种细菌、霉菌、放线菌为供试菌进行抑菌圈实验,研究各个萃取部位的抑菌活性。实验结果表明石油醚层和乙酸乙酯层表现出了较强的抑菌活性,正丁醇层抑菌活性较弱,水层没有抑菌活性。选取抑菌活性较强的石油醚层和乙酸乙酯层进行了气质联用分析,样品一石油醚层中含有的多种具有二硫键的化合物可能使其具有较强的抑菌活性,而且样品一和样品二的石油醚层和乙酸乙酯层均鉴定出多种酚类物质和有机酸,对其抑菌活性可能有一定的贡献。老蒜提取物样品一、样品二的功能成分存在的差异导致了其抑菌活性的不同。     

AB-8大孔树脂固定化溶菌酶及酶学性质研究

利用AB-8大孔树脂为载体,戊二醛为交联剂对溶菌酶进行固定化,研究固定化酶的制备条件、酶学性质、微观结构及抑菌效果。结果表明：固定化时间4h、固定化温度25℃、戊二醛质量浓度0.3g/100mL、m酶:m载体＝1:200时固定化溶菌酶的相对酶活力最高;与游离酶相比,溶菌酶经过固定化后耐热性提高、耐酸性增强,米氏方程分析表明,溶菌酶经过固定化后与底物壳聚糖的亲和力下降,固定化酶重复使用5次时,酶活力残留率为57.6%,抑菌实验结果表明,固定化溶菌酶对纯牛奶具有较好的抑菌效果。     

Modified sodium caseinate films as releasing carriers of lysozyme

Films made of sodium caseinate containing lysozyme where modified by chemical or biochemical crosslinkers to achieve a controlled release of the antimicrobial lysozyme. Kinetic studies in buffered solutions at pH 3.02–5.80 were undertaken. Both the pH and the use of crosslinkers had a strong effect on the protein network, and consequently, a significant influence on the releasing of lysozyme activity. Sodium caseinate (NaCas) films became insoluble networks at pHs close to the isoelectric point of caseinate (pH 4.6), therefore decelerating remarkably the diffusion of lysozyme without the addition of crosslinkers. Additionally a slow release of lysozyme was achieved after mixing with glyoxal, achieving a modulation in the antimicrobial activity against Micrococcus lysodeikticus and Staphylococcus aureus. Other crosslinkers, as calcium chloride or transglutaminase, almost blocked enzyme release and were not found adequate to achieve enough antimicrobial activity. Results showed that active caseinate films modified by pH and glyoxal efficiently retarded the release of lysozyme, being a promising way to extend antimicrobial effects during food storage and to enhance food safety.     

Antimicrobial synergism of partially-denatured lysozyme with glycine: effect of sucrose and sodium chloride

Previously we reported that heat-treatment of lysozyme at 80°C for 20 min in pH 6.0 (HL80/6) greatly promoted its antimicrobial action to Gram-negative bacteria without detrimental effects on its inherent action to Gram-positive ones. In this study the effects of sucrose and NaCl on the promoted antimicrobial activity of HL80/6 and synergy with glycine were investigated. This potent antimicrobial lysozyme (HL80/6) retained 54% of the native enzymatic activity. The enhanced bactericidal action of HL80/6 against Escherichia coli K12 was gradually decreased with increasing sucrose and NaCl concentration in the medium, where complete suppression was observed at 2% sucrose and 0.1% NaCl. On the other hand, 1% NaCl concentration was required to suppress the antimicrobial activity against Staphylococcus aureus of either native lysozyme (NLz) or HL80/6, while sucrose up to 8% had no detectable inhibitory effect on this strain. However, similar doses of sucrose or NaCl had no effect on the enzymatic activity of both NLz and HL80/6, indicating that the antimicrobial action of HL80/6 is independent of its muramidase activity. HL80/6 lysozyme, but not NLz, exhibited good synergy with glycine against Gram-negative bacteria even in the presence of the inhibitory doses of sucrose or NaCl, suggesting a possible application in food industry as a food preservative. Thus, the results introduce an interesting finding that partial denaturation of lysozyme can induce its antimicrobial specificity to include the food-borne Gram-negative pathogens and heralded fascinating opportunities for application of HL80/6 with glycine in formulated food systems.     

Mono- and multilayer active films containing lysozyme as antimicrobial agent

Active packaging materials able to release antimicrobial compounds into foodstuff can be used in order to avoid or slow down the bacterial growth during storage. In this work the use of two techniques to control the release of the chosen active compound (lysozyme) from a polymeric material into the foodstuff is proposed: a monolayer cross-linked PVOH film and a multilayer structure made of cross-linked PVOH layers are developed and studied. Lysozyme release tests into water were performed in order to compare the release kinetics from the investigated films. Results suggest that by means of both structures it is possible to control the rate at which lysozyme is released from the PVOH film. The antimicrobial activity of lysozyme released from the investigated films was tested against a suspension of Micrococcus lysodeikticus. Results show that the incorporation of lysozyme into PVOH does not lead to a loss of activity of the enzyme.

Industrial relevance

The increased use of gently processed foods requires packaging to be an integral part of the preservation concept. Consequently additional antimicrobial activity from the packaging material can aid in shelf life extension.This paper concentrates on the release rate of lysozyme, a naturally occuring antimicrobial agent (eg. salvia, mothers milk, raw milk), from multilayer films. A comperision of mono- and multilayer films containing lysozyme regarding their effectiveness on M. lysodeikticus as target organism was also performed. Both aims were met leading to a controlled release of lysozyme with no loss of activity.     

Anticancer,antioxidant, and antimicrobial activities of anemone (Anemone cathayensis)

This study was performed to evaluate the anticancer, antioxidant, and antimicrobial activities of fractions and isolated compounds from anemone (Anemone cathayensis). Fourteen compounds were isolated from extracts. Anticancer activities of fractions and compounds were determined by MTT assay, and all tested fractions showed inhibition activity on human breast cancer cells (MDA-MB-231). The fraction 6 displayed the strongest anticancer activity, and inhibition percent was 50.32%. The antioxidant effect of fractions was evaluated by using DPPH scavenging assays. Fraction 5 had a higher DPPH radical scavenging activity with low IC₅₀ value of 30.578 μg/mL. The antimicrobial activity of the fractions was evaluated against 3 microorganisms using the agar well diffusion method. The fractions also showed moderate antimicrobial activity. These results suggest that anemone could hold a good potential source for human health.     

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.     

Immobilization of lysozyme on polyvinylalcohol films for active packaging applications

A new technique for the immobilization of lysozyme onto the surface of polyvinylalcohol films is presented. The active compound was sprayed along with a suitable bonding agent onto the surface of the cross-linked polymeric matrix. Active compound release tests determined the amount of lysozyme immobilized on the film surface. With the use of Micrococcus lysodeikticus, the antimicrobial activity of the films was determined and the results correlated with the amount of immobilized lysozyme. This new technique was effective for immobilizing the enzyme, and the developed films were active against the test microorganism. Results were compared with those obtained with a different immobilizing technique, in which the active compound was bound into the bulk of the polymeric film. As expected, the surface-immobilized lysozyme films have a higher antimicrobial activity than bulk-bound films.     

乳清蛋白可食用膜抑菌性、溶解性的研究

主要研究了Nisin、纳塔霉素和溶菌酶不同组合添加下乳清蛋白可食用膜的抑菌性和溶解性,结果表明:随着抑菌剂浓度的增加,膜的抑菌效果增强,不同抑菌剂组合下膜的抑菌效果不同,0.35%Nisin和0.08%溶菌酶组合下抑菌效果更好。在酸性环境中,当添加0.03%溶菌酶和0.25%纳塔霉素时,可有效降低膜的溶解性;在中性环境中,各组膜的溶解性几乎相当,在碱性环境中,大部分膜的溶解性要低于其在酸性环境中。     

Inactivation of Lactobacillus brevis in Beer Utilizing a Combination of High‐Pressure Homogenization and Lysozyme Treatment

Inactivation of Lactobacillus brevis in beer by high‐pressure homogenization (HPH) and lysozyme addition was evaluated. The minimum inhibitory concentration of lysozyme against L. brevis was determined and found to be 100 mg.L^?1. The effects of the homogenization process on lysozyme antimicrobial activity and muramidase activity, and the microbial reduction promoted by HPH, and by HPH associated with lysozyme, were evaluated. A significant reduction in lysozyme muramidase activity was observed under 250 MPa, however, no reduction in antimicrobial activity was observed after homogenization up to 300 MPa. The HPH at 100, 140 and 150 MPa promoted 1, 3 and 6 decimal reductions in L. brevis microbial counts, respectively. The HPH and lysozyme association had an additive effect on microbial inactivation, immediately after homogenization, and the lysozyme remained active during 10 days of storage, increasing the inactivation of L. brevis up to a 6 decimal reduction. Therefore, the application of lysozyme with HPH has the potential to reduce the level of pressure required for beer processing, improving the economic costs when utilizing HPH.