溶菌酶及其在食品工业中的应用

溶菌酶是一种对人安全且具有保健作用的蛋白酶,是国际公认的绿色天然酶制剂。它在食品工业中被广泛用作食品添加剂和防腐剂。溶菌酶广泛存在于人、动植物及微生物体内,尤以鸡蛋清中含量较高,具有巨大的开发应用价值。     

溶菌酶的性质及其在食品防腐中的应用

溶菌酶对细菌、真菌生长具有显著的抑制作用,可以作为一种安全的食品防腐剂应用于食品的保鲜。本文介绍了溶菌酶的物理性质、抑菌作用及其机理、免疫学功能等性质,并综述了溶菌酶在食品防腐中的应用进展。     

溶菌酶在酱油中的抑菌效果研究

溶菌酶为多肽链组成的蛋白质分子,酶活在酸性条件下具有良好的热稳定性,且对革兰氏阳性细菌具有专一的抑制杀灭作用,是成品酱油的一种良好的天然抑菌防腐剂。本文研究了溶菌酶在酱油中的抑菌防腐效果。结果表明:(1)在酱油中,溶菌酶的抑菌防腐效果理想,且具有缓慢杀菌的功能,其最佳添加量为150 mg/kg,抑菌防腐效果比0.1%苯甲酸钠要好;(2)在高盐分含量酱油中的抑菌防腐效果比低盐分含量酱油要好,添加溶菌酶后菌落总数下降趋势更加明显;(3)酱油的巴氏灭菌操作对溶菌酶的活性无显著影响;(4)在起始菌落总数为10000 cfu/ml或以下的酱油中使用溶菌酶能使酱油具有较好的抑菌防腐效果,在货架期内菌落总数增长缓慢,产品质量稳定。     

生物防腐剂在肉品保鲜中的应用(二)——溶菌酶和乳铁蛋白

溶菌酶和乳铁蛋白(LF)属于生物防腐保鲜剂的范畴。溶菌酶由于其来源不通其结构也不同,其具有抗菌消炎、抗病毒、增强免疫力、促进双歧杆菌增殖等作用,乳铁蛋白则是一种结合糖蛋白,具有抑制细菌生长的特性。本文分别介绍了溶菌酶和乳铁蛋白的结构及其生物学特性,并介绍了他们的抑菌特性。并详细阐述了溶菌酶和乳铁蛋白在肉及肉制品中防腐保鲜方面的研究进展。     

溶菌酶及其在肉制品保鲜中的应用

溶菌酶是一类安全无毒的碱性蛋白质,作为天然防腐剂广泛用于食品工业中。肉类食品因其含有丰富的营养物质,极易受到微生物的侵害。综述了溶菌酶的发现、种类、理化特性、抑菌机理及其在肉制品贮藏保鲜方面的应用,证明溶菌酶是一种有效的肉类防腐保鲜剂。     

Detection of Hen Lysozyme on Proteic Profiles of Grana Padano Cheese through SELDI-TOF MS High-Throughput Technology during the Ripening Process

Lysozyme is used in cheese manufacture in order to prevent blowing in cheeses caused by Clostridium tyrobutyricum. Being an egg derivative, the presence of lysozyme must be included on the label for residual allergenic risk (2003/89/CE). The aim of this study was to evaluate the presence of lysozyme on proteic profiles of typical Italian cheeses such as Grana Padano through surface-enhanced laser desorption/ionization time-of-flight mass spectrometry. The proteolytic activity of ripening (from 0 to 24 months), confirmed by a decrease in casein, did not influence the intensity of lysozyme peaks. Furthermore, ripened Grana Padano cheese could be differentiated on mass profiling from immature Grana Padano by the presence of particular signals that are probably related to casein proteolysis.     

Prevalence and stability of lysozyme in cheese

The use of the preservative and potential allergen hen egg white lysozyme in cheese production has to be declared. In the present study, an HPLC method with fluorescence detection (HPLC-FLD) was optimised and validated for the analysis of lysozyme in cheese. Lysozyme was detected in concentrations between 30.8 and 386.2 mg/kg cheese in 30 out of 46 analysed commercial cheese samples. During cheese production and storage for 0–54 weeks a lysozyme satellite peak (LSP) was detected, which totals up to 18% of the lysozyme content. Mass spectrometry and peptide mass fingerprint revealed that LSP possesses the same primary structure as lysozyme. Since disulphide scrambling could not be detected, LSP was assigned to a conformational isomer of lysozyme. As a consequence, LSP was included in the HPLC-FLD analysis of lysozymes in cheese.     

Effect of starch on the thermal kinetics and transmittance properties of lysozyme

Lysozyme is being tested for use as a preservative in the food industry. The interaction between starch and lysozyme will help in recommending starch or starch fractions as carriers for lysozyme. The effect of starch fractions on the folding and unfolding of lysozyme was estimated by differential scanning calorimetry (DSC), kinetics and transmittance turbidometry. Lysozyme was unfolded (heated to 90 °C) and folded (cooled to 20 °C) five times in the presence of starch fractions. Starch was added at 1 and 2%. Overall, a trend of higher onset temperature (T_o) values occurred at 2% addition of all starch fractions except amylose. The increase in the number of cycles influenced the effect of starch on lysozyme denaturation. The percentage of lysozyme's ΔH values decreased as a new heating and cooling cycle was performed (ie 74.4% of the ΔH remained from the first cycle). The effect of amylose (AM) and amylopectin (AP) on the kinetics of lysozyme unfolding and folding was found to be different based on the assumption that the peak DSC temperature is the fastest step of the reaction. The unfolding showed higher activation energy (E_a) in the presence of both AM and AP, while the folding was not significantly changed. The turbidity of the solution containing lysozyme and potato starch showed transmittance in between that of lysozyme and starch. Stirring of the blend kept the transmittance unchanged while an increase in the transmittance was noticed when stirring ceased. Copyright © 2004 Society of Chemical Industry     

A sensitive HPLC method to detect hen's egg white lysozyme in milk and dairy products

Because of the lytic activity on the cell wall of bacteria like Clostridium tyrobutyricum, hen's egg white lysozyme is used in cheese manufacturing to prevent late blowing. A HPLC method capable to quantify as low as 0.8 ppm lysozyme in milk and cheese is proposed. Lysozyme was extracted with 1 m NaCl at pH 6.0 and the extract was deproteinized at low pH values, reaching a recovery up to 90%. Reversed-phase HPLC was performed on a polymeric column and monitoring lysozyme under fluorescence detection (excitation at 280 nm and emission at 340 nm). The repeatability of this determination tested on a 15-month-aged hard cheese and expressed as relative standard deviation was 1.47 (n=6) and no interference of peptides formed during ripening was observed. Four commercial preparations of egg white lysozyme gave a similar fluorescence response and the partitioning over cheese and whey was studied with one of them. About 80% of the lysozyme added to the cheesemilk at concentrations up to 80 ppm was retained in the cheese and the concentration factor of lysozyme from the cheesemilk to the cheese proved to be 8.2 on average. This HPLC method and the microbiological assay using Micrococcus luteus were compared in cheese analysis, proving the former to be more accurate and reliable than the latter. Eighteen commercial samples including both generic cheeses and cheeses having protected designation of origin, all of them not declared to contain lysozyme, showed concentrations of this enzyme ranging from 0 to 111 ppm.     

THE EFFECT OF LYSOZYME ON THE APPARENT HEAT RESISTANCE OF NONPROTEOLYTIC TYPE B CLOSTRIDIUM BOTULINUM

Lysozyme has been shown to increase the recovery of heated spores of type E Clostridium botulinum, thereby increasing the measured heat resistance. This study evaluated the effect of lysozyme on the apparent heat resistance of spores of nonproteolytic type B strains of C. botulinum, which had heat resistances up to 30 times greater than type E strains. Adding lysozyme to the recovery medium increased the apparent heat resistance 16 to 160 times that of values previously established. This increase was comparable to that obtained with type E strain Saratoga (73 times). Lysozyme had no effect on the recovery of heated spores of a proteolytic B strain.     

Lysozyme in Wine: An Overview of Current and Future Applications

Lysozyme, a muramidase enzyme from egg whites (EC 3.2.1.17), is widely used in soluble form to control lactic acid bacteria in different foods. Moreover, hen egg white lysozyme is a hydrolytic enzyme that can be used to the control malolactic fermentation (MLF) during winemaking. MLF is only desirable in red and in some white wine, this suggest that MLF is at fault and needs to be controlled in all other types of wine. Lysozyme exhibits selective antimicrobial activity based on the hydrolysis of peptidoglycan cell wall constituents in lactic acid bacteria. In the last decade, several studies identified allergic reactions due to the presence of lysozyme in food. Given this relatively high incidence of lysozyme sensitization, and in accordance with the recently changed EC food legislation (1266/2010/CE), the use of lysozyme as an additive has to be declared on the ingredient label. To overcome this problem, the immobilization of the enzyme on insoluble supports, which allows the enzyme to be removed, has been the preferred strategy. In this context, this article offers a review of reports on lysozyme enological use over the last decade. It surveys the immobilization techniques and support materials used for lysozyme food preservation. This study attempts to provide useful guidance from the wealth of available immobilization data in the literature and, more importantly, to develop an integrated perspective on how to customize lysozyme for future enological uses.     

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.     

Effect of pH,temperature and sodium bisulfite or cysteine on the level of Maillard-based conjugation of lysozyme with dextran,galactomannan and mannan

The antimicrobial activity of lysozyme against Gram positive bacteria is well known. Application of this enzyme as a natural antimicrobial or preservative agent in food and pharmaceutical industry is under consideration in many laboratories. The antimicrobial effect of lysozyme can be extended towards Gram negative bacteria by chemical modification including conjugation with carbohydrates. The purpose of this investigation was to find the optimum experimental conditions for glycation of lysozyme with polysaccharides dextran, galactomannan and mannan and to evaluate some functional properties of the modified enzyme. Lysozyme was allowed to react with dextran under Maillard reaction condition at different pH and temperatures and in the presence of 50 mmol/L sodium bisulfite or cysteine and the extent of glycation was determined by ion exchange chromatography and SDS-PAGE. The optimum condition for glycation was pH 8.5 and 60 °C with a protein to derxtran molar ratio of 1:5. Same results were obtained for galactomannan but mannan did not react. Under these condition three moles dextran was attached to one mole lysozyme. Sodium bisulfite inhibited glycation at pH 8.5 and temperatures above 40 °C while cysteine prevented glycation at all pH and temperatures. Dextran-conjugated lysozyme exhibited improved heat stability, pH and heat solubility and better emulsifying property as compared with the unmodified lysozyme. These results suggest that it is possible to improve the properties of this enzyme in order to make it more suitable for application in foods and pharmaceuticals.     

Effects of reduced levels of sulfite in wine production using mixtures with lysozyme and dimethyl dicarbonate on levels of volatile and biogenic amines

Sulphur dioxide (SO₂) is an important preservative for wine, but its presence in foods can cause allergies and this has given impetus to the research for alternatives. The aim of this study was to reduce levels of sulfite in wine production using mixtures with lysozyme and dimethyl dicarbonate and examine the influence on levels of volatile and biogenic amines. To do so, vinifications were carried out using lysozyme, dimethyl dicarbonate (DMDC) and mixtures of these with SO₂ in different concentrations (25 and 50 mg l^?1). Results were compared with a control vinification with only SO₂ (50 mg l^?1). Mixing low concentrations of SO₂ with lysozyme and DMDC reduced the concentration of biogenic amines (histamine, tyramine, putrescine, cadaverine, phenylethylamine + spermidine and spermine). In general, the total concentration of volatile amines (dimethylamine, isopropylamine, isobutylamine, pyrrolidine, ethylamine, diethylamine, amylamine and hexylamine) was higher in the sample fermented only with SO₂. The concentrations of amines with secondary amino groups (dimethylamine, diethylamine, pyrrolidine) were higher in the sample only fermented with SO₂ than those fermented with DMDC and lysozyme or with a mixture of preservatives. When SO₂ was the only preservative in wine, total amine concentration (biogenic and volatile amines) was higher than for the rest of the treatments. Lysozyme by itself, and lysozyme mixed with SO₂, both reduced the formation of biogenic amines but given the antioxidant activity of SO₂ the use of the preservative mixture seems more advisable.     

Effects of Lysozyme on the Microbiological Stability and Organoleptic Properties of Unpasteurized Beer

Lysozyme is an antimicrobial enzyme that could be applied to counteract those bacterial species which, due to their own metabolic activity, possess notable beer spoilage ability and lead to loss of beer quality. Experiments were carried out to assess lysozyme potential to prevent the growth of beer spoilage bacteria, and to verify the effect of lysozyme on the microbiological stability and sensory characteristics of unpasteurized beer. Eight replicates, all from the same lot of Italian beer, were treated with 0 and 100 ppm lysozyme. Microbiological analyses were conducted bimonthly to investigate the presence of spoilage bacteria. Sensory analyses were performed to determine whether there were any significant differences in sensory impressions between beers produced with and without lysozyme. Lysozyme exerted a strong inhibitory action on the lactic acid bacteria (LAB) present in the beer and was very stable throughout the shelf life. Sensory tests revealed no unfavourable influence on beer flavour when using lysozyme. Indeed, the shelf life of beer with added lysozyme proved to be extended. Even as late as 1 month after the expiry date it still met with the panellists' approval. Lysozyme may be regarded as an effective agent for preventing microbiological contamination and prolonging the stability of unpasteurized beer.     

Lysozyme: just an additive or a technological aid as well?

The effects of lysozyme on coagulation of milk and cheese making were studied by means of the gelograph, tristimulus colorimetry, ANS-fluorescence (hydrophobicity) and SDS-PAGE. Lysozyme binding to caseins caused structural differences during coagulation and it is proposed that, if the products have similar qualitative properties, lysozyme might be used as a technological aid giving shorter clotting times and higher yields.     

Isolation and Characterization of Buffalo Milk Lysozyme

Lysozyme is a commercially valuable enzyme, and is applied in many fields, concerning products such as foods, drugs, and the like. In this work, lysozyme was isolated and purified from buffalo milk using sephadex G-50 and cation exchanger carboxymethyl cellulose. Lysozyme active fractions from buffalo milk were assayed against Gram positive substrate Micrococcus luteus at 450 nm and a decline in absorbance of 0.001 per min was observed. The optimum activity of lysozyme (158.3 ± 1.7 units/mL) was at 7.5 pH and 37°C temperature. Lysozyme activity at pasteurization temperatures 62.5°C, 30 min and 75°C, 15 s were (156.08 ± 1.03 and 156 ± 2 units/mL) not affected significantly; however, 47% activity of lysozyme was reduced at 100°C for 5 min. Antibacterial susceptibility testing of lysozyme (chicken egg white lysozyme and buffalo milk lysozyme) was performed on Micrococcus luteus (ATCC 4698) and Escherichia coli (ATCC 25235). Both lysozymes showed no inhibition effect against Escherichia coli.     

Use of active compounds for prolonging the shelf life of mozzarella cheese

The effectiveness of lysozyme and ethylenediaminetetraacetic disodium salt (Na₂-EDTA) against the spoilage microorganisms of mozzarella cheese was studied. Mozzarella cheeses were packaged in a conditioning solution (diluted brine), which contained lysozyme (0.25 mg mL⁻¹) and different amounts of Na₂-EDTA (10, 20 and 50 mmol L⁻¹), and stored at 4 °C for 8 days. The population of spoilage microorganisms (total coliforms and Pseudomonadaceae), along with the functional microbiota of mozzarella cheese (lactic acid bacteria) was enumerated. Lysozyme and Na₂-EDTA significantly inhibited the growth of coliforms and Pseudomonadaceae during the first 7 days of storage, whereas the functional microbiota (or lactic acid bacteria) were not affected. The results of this study showed that it is possible to extend the shelf life of mozzarella cheese through the use of lysozyme and Na₂-EDTA in the conditioning brine.     

Modified Lysozymes as Novel Broad Spectrum Natural Antimicrobial Agents in Foods

In recent years much attention and interest have been directed toward application of natural antimicrobial agents in foods. Some naturally occurring proteins such as lactoperoxidase, lactoferrin, and lysozyme have received considerable attention and are being considered as potential antimicrobial agents in foods. Lysozyme kills bacteria by hydrolyzing the peptidoglycan layer of the cell wall of certain bacterial species, hence its application as a natural antimicrobial agent has been suggested. However, limitations in the action of lysozyme against only Gram‐positive bacteria have prompted scientists to extend the antimicrobial effects of lysozyme by several types of chemical modifications. During the last 2 decades extensive research has been directed toward modification of lysozyme in order to improve its antimicrobial properties. This review will report on the latest information available on lysozyme modifications and examine the applicability of the modified lysozymes in controlling growth of Gram‐positive and Gram‐negative bacteria in foods. The results of modifications of lysozyme using its conjugation with different small molecule, polysaccharides, as well as modifications using proteolytic enzymes will be reviewed. These types of modifications have not only increased the functional properties of lysozyme (such as solubility and heat stability) but also extended the antimicrobial activity of lysozyme. Many examples will be given to show that modification can decrease the count of Gram‐negative bacteria in bacterial culture and in foods by as much as 5 log CFU/mL and in some cases essentially eliminated Escherichia coli. In conclusion this review demonstrates that modified lysozymes are excellent natural food preservatives, which can be used in food industry.     

Simultaneous Isolation of Avidin and Lysozyme from Egg Albumen

A single column cation exchange method was developed which allowed simultaneous recovery of lysozyme and avidin from undiluted egg white. A unique application-elution sequence was developed, involving accumulation of avidin on the column through several cycles of egg white application and lysozyme elution. Lysozyme was recovered with higher yields than reported for the isoelectric precipitation methods often used in the industry (86% vs 60–80%). Lysozyme peaks appeared homogeneous on SDS-PAGE. Avidin recovery was also as good or better than that of previously reported ion exchange methods (74%–80%). The purity of the avidin fraction (up to 40.9%) was superior to that of other reported primary avidin fractions.