几种天然保鲜剂抑菌能力的研究

通过抑止菌圈实验研究荼多酚、溶菌酶、丁香和桂皮提取液的最小抑菌浓度(MIC)和抑菌强度.结果表明:茶多酚对冷却肉中常见腐败菌和致病菌都具有很强的抑菌作用,最低抑菌浓度为0.5%;溶菌酶主要对革兰氏阳性菌有较强的抑制作用,但对革兰氏阴性菌作用弱或无作用,最低抑菌浓度为0.015%;丁香和桂皮提取液具有广谱抑菌,最低抑菌浓度为5%.     

溶菌酶功能化金纳米颗粒抑菌性能研究

为了提高溶菌酶的稳定性及对革兰氏阴性菌的抑菌性能,以金纳米颗粒为核,通过表面定向修饰溶菌酶,制备了绿色、高效的溶菌酶功能化金纳米颗粒抑菌材料,研究溶菌酶与金纳米的比例、纳米颗粒质量浓度等对抑菌效果的影响,研究溶菌酶功能化纳米材料对大肠杆菌的抑菌机理及对人神经母细胞瘤(SH-SY5Y)的细胞毒性。结果表明:与未修饰的金纳米及单纯的溶菌酶相比,溶菌酶功能化金纳米颗粒对大肠杆菌和金黄色葡萄球菌的抑菌效果均显著增强,表现出协同抑菌作用,在最优条件下,0.1 g/L溶菌酶功能化金纳米颗粒可以完全杀死2种细菌,并且该溶菌酶功能化金纳米颗粒具有持久抑菌性及良好的生物相容性,对哺乳细胞没有毒性。透射电子显微镜和活菌/死菌荧光染色结果表明:溶菌酶功能化金纳米颗粒主要通过破坏菌体细胞壁和细胞膜结构,从而杀死细菌。     

2株中国红豆杉内生细菌代谢产抑菌活性物质的研究

自秦巴山区野生中国红豆杉(Taxus chinensis)中分离的25株内生细菌中筛选出2株具有较广抑菌活性的菌株TC-3和TC-2,通过生物学特征研究和16S rDNA序列分析,初步鉴定为Bacillus sp.和Bacillus subtilis.在排除酸性产物和过氧化氢的干扰后,TC-3和TC-2菌株发酵上清液对金黄色葡萄球菌(Staphyloccocus aureus)、枯草芽孢杆菌(Bacillus subtilis)等革兰氏阳性菌和大肠杆菌(Escherichia coli)等革兰氏阴性菌具有较好的抑菌作用,而且对部分真菌也有抑菌作用;经胰蛋白酶、胃蛋白酶和蛋白酶K处理后发酵液抑菌活性略有下降,表明该活性物质为非蛋白成分,或起抑菌作用的非单一物质.该活性物质不同于一般细菌素,属于类细菌素.它具有很好的热稳定性,在酸性条件下稳定且活性高,对蛋白酶敏感性低,且具有较广的抗菌谱.     

化学法改性溶菌酶抑菌性及其结构研究

采用化学法改性溶菌酶,以EDAC作为缩合剂,肉桂酸、咖啡酸和对香豆酸作为改性剂,形成一定程度的改性酶,并对改性酶的抑菌性及结构进行研究。结果表明:与天然酶相比,改性酶的活力有所下降,但是对革兰氏阴性菌的抑制作用增强,咖啡酸改性酶和对香豆酸改性酶对大肠杆菌和铜绿假单胞菌的最小抑菌浓度均为0.5 mg/mL,肉桂酸改性酶为0.75 mg/mL,但对革兰氏阳性菌的抑菌能力有所降低,咖啡酸改性酶和对香豆酸改性酶对金黄色葡萄球菌和溶壁微球菌的最小抑菌浓度均为1.25 mg/mL,肉桂酸改性酶对二者的最小抑菌浓度分别为1.25 mg/mL和1.50 mg/mL,3种改性酶的各二级结构含量均发生一定的变化。     

3种取代度的羧甲基纤维素对溶菌酶抑菌活性影响的研究

溶菌酶活性易受食品组分和环境因素影响。研究了3种取代度的羧甲基纤维素与溶菌酶之间的静电相互作用,考察在不同温度、pH值和离子强度条件下,羧甲基纤维素对溶菌酶抑菌活性的影响。结果表明:取代度为0.7和0.9的羧甲基纤维素可提高溶菌酶活性,取代度为1.2的可降低溶菌酶活性。由于取代度越高,与溶菌酶的静电相互作用越强,溶菌酶的抑菌性随着羧甲基纤维素取代度的增加而逐渐降低。由于溶菌酶与羧甲基纤维素相互作用形成较大粒子,复合物中溶菌酶的热稳定性、pH稳定性均有所降低。Na~+会抑制溶菌酶的酶活且会使溶菌酶/羧甲基纤维素复合物解离,酶活逐渐恢复至单独溶菌酶酶活。并且进一步通过对2种革兰氏阳性菌(金黄色葡萄球菌、枯草芽孢杆菌)和2种革兰氏阴性菌(大肠杆菌、阴沟肠杆菌)的抑菌圈实验证明,羧甲基纤维素与溶菌酶复合后,低取代度的羧甲基纤维素能提高对革兰氏阳性菌的抑制作用;而且,和游离溶菌酶比较,羧甲基纤维素/溶菌酶复合物对大肠杆菌抑制作用增强。     

植物乳酸菌类细菌素特性研究及其产生条件的优化

通过单因素实验和正交实验优化了乳酸菌产乳酸菌素的培养条件并分析了其特性。结果表明：植物性乳酸杆菌生长的最佳培养条件组合是温度30℃,培养基初始pH为6.0,接种量4%,种龄12h,厌氧条件下,培养时间40h;乳酸菌菌素对革兰氏阴性菌和酵母菌的抑菌作用最大的最佳培养条件组合是温度28℃,培养基初始pH为7.0,接种量2%,种龄10h,厌氧条件下,培养时间24h。乳酸菌菌素革兰氏阳性菌的抑菌作用最大的最佳培养条件组合是温度28℃,培养基初始pH为6.5,接种量1%,种龄6h,厌氧条件下,培养时间40h。在排除酸性产物和过氧化氢的干扰后,该乳酸菌发酵液对金黄色葡萄球菌、枯草芽孢杆菌等革兰氏阳性菌和大肠杆菌、鸡白痢沙门氏菌等革兰氏阴性菌具有较好的抑制作用,而且对部分真菌也有抑制作用;经胰蛋白酶、胃蛋白酶和木瓜蛋白酶处理后发酵液抑菌活性略有下降,表明乳酸菌素为非蛋白成分或起抑菌作用的非单一物质。它具有很好的热稳定性,在酸性条件下稳定且活性高,对蛋白酶敏感性低,且具有较广的抑菌谱。     

溶菌酶的修饰、功能特性及其在食品保鲜中的应用研究进展

溶菌酶是一种自然界中广泛存在的天然防腐剂,能选择性溶解目标微生物的细胞壁而使其失去生理活性,已被广泛应用于食品工业。天然溶菌酶主要抑制革兰氏阳性菌的生长,而对革兰氏阴性菌几乎不起作用或作用较弱;因此,对溶菌酶实施分子结构调整,扩展其抑菌谱具有十分重要的意义。本文重点将近年来国内外已报道的各类溶菌酶增效技术（物理、化学与生物改性技术等,特别是辅以纳米制备技术）、改性后溶菌酶对有害微生物的抑制效果及作用机制和改性后溶菌酶的其他功能特性变化（活性、稳定性、乳化性等）等做了详细综述;同时介绍了改性溶菌酶在食品贮藏与保鲜等领域中的应用情况,并对溶菌酶的分子改性策略、溶菌酶与纳米及材料科学结合应用的潜力进行了展望。     

碘乙酰胺改性溶菌酶抑菌效果研究

在单因素实验的基础上,应用正交分析,优化碘乙酰胺改性溶菌酶的条件,并测定此改性条件下溶菌酶对革兰氏阴性菌的抑菌活性。结果表明,0.06 mol/L碘乙酰胺与5 mg/mL溶菌酶改性时对大肠杆菌和沙门氏菌的抑菌效果最好,在45℃,反应时间30 min,pH=5的条件下改性后的溶菌酶,其对大肠杆菌的抑菌圈直径为25.7 mm,是改性前抑菌圈的2.7倍。在55℃,反应时间70 min,pH=6的条件下改性后的溶菌酶对大肠杆菌的抑菌圈直径为24.1 mm,是改性前抑菌圈的2.6倍。这充分说明蛋清溶菌酶经过碘乙酰胺改性后,可以用于对革兰氏阴性菌(大肠杆菌K12、沙门氏菌)抑菌,而且效果良好。     

乳酸菌混合发酵对抑菌作用的影响研究

通过测定6株乳酸菌及其混合发酵对3种革兰氏阳性菌和3种革兰氏阴性菌抑制作用,探究单一发酵及混合发酵对抑菌作用的影响。结果显示,6株不同乳酸菌及其混合发酵对3种革兰氏阳性菌和3种革兰氏阴性菌的抑制效果均不同。建立主成分分析模型,根据模型中累计贡献率达75.284%两个主成分,进行综合评分,可得乳酸菌混合发酵抑菌效果高于任一单菌株发酵,乳酸菌混合发酵表现出广谱抑菌性。通过排除乳酸及过氧化氢干扰,以及经p H 6.0处理、121℃,20 min热处理、蛋白酶K处理,进一步对乳酸菌及其混合发酵抑菌物质分析,可知除乳酸外,不同乳酸菌及其混合发酵的发酵产物中存在其他抑菌物质,且抑菌产物中过氧化氢浓度不足以产生抑菌效果。乳酸菌发酵抑菌物质在酸性条件下活性高,对热和蛋白质酶K敏感。     

不同蜂产品抑菌效果的研究

采用滤纸片法,测定和比较了不同浓度的蜂蜜、蜂王浆、蜂胶、蜂花粉溶液对革兰氏阳性菌(金黄色葡萄球菌)和革兰氏阴性菌(大肠杆菌)的抑菌效果.实验结果表明:蜂蜜、蜂王浆、蜂胶、蜂花粉溶液对革兰氏阳性菌和革兰氏阴性菌均具有抑菌效果,而且蜂胶在抑制革兰氏阳性菌,蜂花粉在抑制革兰氏阴性菌方面效果比其它蜂产品更好.蜂蜜、蜂王浆、蜂胶、蜂花粉具有很好的抑菌效果,是优良的天然保健品.     

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.     

Antibacterial activity of hen egg white lysozyme modified by thermochemical technique

Lysozyme is an important component of egg white with antibacterial properties especially against Gram-positive bacteria. Research indicates that the range of lysozyme activity may be extended thanks to chemical or physical modifications. The purpose of the study was to determine the effectiveness of antibacterial action against selected strains of bacteria of lysozyme modified by the thermochemical technique. Our investigations indicated a possibility of extending the range of lysozyme activity using modification. We found that under the adopted conditions modification promotes an increased polymerisation of lysozyme. It was observed that the temperature and time of modification have an effect on the content of the forming polymers. It was found that the application of lysozyme modification leads to an increase in its antibacterial activity in comparison to the activity of lysozyme monomer. The experiments have shown that the used lysozyme preparations demonstrate different activity in relation to different types of bacteria. The observed increase in antibacterial activity of modified lysozyme preparations against Gram-negative bacteria were not associated with a decrease of that activity against Gram-positive bacteria. The greatest reduction of growth of the examined Gram-negative bacteria species was recorded for the preparation which contained the highest quantity of dimer (34–37.6%). A more effective action against bacteria Staphylococcus epidermidis was observed for preparations containing 25–27% of dimer.     

Enhancement of nisin, lysozyme, and monolaurin antimicrobial activities by ethylenediaminetetraacetic acid and lactoferrin

A microtiter plate assay was employed to systematically assess the interaction between ethylenediaminetetraacetic acid (EDTA) or lactoferrin and nisin, lysozyme, or monolaurin against strains of Listeria monocytogenes, Escherichia coli, Salmonella enteritidis, and Pseudomonas fluorescens. Low levels of EDTA acted synergistically with nisin and lysozyme against L. monocytogenes but EDTA and monolaurin interacted additively against this microorganism. EDTA synergistically enhanced the activity of nisin, monolaurin, and lysozyme in tryptic soy broth (TSB) against two enterohemorrhagic E. coli strains. In addition, various combinations of nisin, lysozyme, and monolaurin with EDTA were bactericidal to some gram-negative bacteria whereas none of the antimicrobials alone were bactericidal. Lactoferrin alone (2000 microg ml(-1)) did not inhibit any of the bacterial strains, but did enhance nisin activity against both L. monocytogenes strains. Lactoferrin in combination with monolaurin inhibited growth of E. coli O157:H7 but not E. coli O104:H21. While lactoferrin combined with nisin or monolaurin did not completely inhibit growth of the gram-negative bacteria, there was some growth inhibition. All combinations of EDTA or lactoferrin with antimicrobials were less effective in 2% fat UHT milk than in TSB. S. enteritidis and P. fluorescens strains were consistently more resistant to antimicrobial combinations. Resistance may be due to differences in the outer membrane and/or LPS structure.     

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.     

Ultrafiltration-modified chicken egg white lysozyme and its antibacterial action

The aim of the study was to produce a lysozyme preparation with an increased content of enzyme polymeric forms using the ultrafiltration (UF) technique. The effect of selected parameters of the membrane process on the extent of enzyme modification was examined. The study showed that the membrane technique, developed for the production of lysozyme monomer, could also be used in the direct production of a preparation containing enzyme polymers. Under optimal conditions of the modification procedure, the obtained preparation contained 53.3% of lysozyme polymeric forms. The effectiveness of the antibacterial action of UF-modified lysozyme against selected strains of bacteria was determined. Its bacteriostatic activity depended on the applied modification conditions. Among lysozyme preparations modified by UF, the highest bacteriostatic activity against selected strains of bacteria was recorded in the preparation containing 53.3% polymeric forms. The modification procedure facilitates the extension of the antibacterial spectrum of lysozyme against Gram-negative bacteria ( Pseudomonas fluorescens ) .     

Inhibition of Bacterial Growth by Two Chlorine Sources in a Model System

The bactericidal effectiveness of two sources of aqueous chlorine, hypochlorous acid and chlorine dioxide, against 18 pure and two mixed cultures of spoilage and potentially pathogenic bacteria normally associated with meat and poultry products was evaluated. Hypochlorous acid was significantly more effective than chlorine dioxide against Gram-negative bacteria. Chlorine dioxide was more effective against Gram-positive than Gram-negative bacteria. The type and concentration of chlorine compound used and the type of microorganisms present determine the effectiveness of aqueous chlorine treatments.     

INHIBITION OF SPOILAGE AND PATHOGENIC BACTERIA ASSOCIATED WITH FOODS BY COMBINATIONS OF ANTIMICROBIAL AGENTS

The antibacterial activity of combinations of lysozyme, monolaurin (ML), triglycerol 1,2 laurate (TGL) and butylated hydroxyanizole (BHA) against 7 Gram-positive and 8 Gram-negative bacteria was studied at different pH, NaCl and EDTA concentrations by the spiral gradient end point test. The inhibitory effect of lysozyme in combination with ML was slightly greater for Gram-positive than for Gram-negative bacteria, but their combined effect was not markedly more inhibitory than ML alone. Lysozyme and TGL together were only inhibitory at low pH and high NaCl concentrations in the presence of EDTA. There was an increase in inhibition when lysozyme and BHA were combined. For Gram-positive bacteria, inhibition by ML and BHA together was more marked than when either was present singly. However, ML decreased the antibacterial activity of BHA against Gram-negative bacteria. Similarly, TGL was antagonistic to BHA action against both Gram-positive and Gram-negative bacteria. In general, the inhibition produced by all combinations was greater as the pH decreased and the NaCl concentration increased, especially in the presence of EDTA .     

Purification,characterization, antibacterial activity and N-terminal sequencing of buffalo-milk lysozyme

Lysozyme from buffalo milk was purified to homogeneity and its N-terminal amino acid sequence, biochemical properties and antibacterial spectrum were determined. The purification procedure, comprising ion-exchange chromatography using CM-cellulose and size-exclusion chromatography using Sephadex G-50, conferred 8622-fold purification and 39.3% recovery of lysozyme. The purified enzyme migrated as a single band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and native PAGE. Immunological purity of lysozyme preparation was confirmed by immuno-electrophoresis. Molecular weight of buffalo-milk lysozyme as determined by SDS-PAGE was 16 kDa and its amino acid composition was determined by reverse phase high performance liquid chromatography (HPLC). The sequence of 23 amino acid residues at the N-terminal end showed 56.5% homology with bovine milk lysozyme and 30.4% with equine milk lysozyme. The specific activity of buffalo milk lysozyme was ten-times that of bovine milk lysozyme. Buffalo-milk lysozyme was active over a wide range of pH and its activity was strongly influenced by molarity of the medium. Antibacterial activity of buffalo-milk lysozyme was determined against 11 species of bacteria; out of seven Gram-positive bacteria tested, four were inhibited, while Gram-negative bacteria were resistant.     

THE EFFECT OF LYSOZYME AND BUTYLATED HYDROXYANIZOLE ON SPOILAGE AND PATHOGENIC BACTERIA ASSOCIATED WITH FOODS

The effect of pH (5 to 7), NaCl concentration (0.5 to 5%) and chelating agents on the antibacterial activity of lysozyme and butylated hydroxyanizole (BHA) was investigated by the Spiral Gradient End Point test. Of all the bacteria tested (including 7 Gram-positive and 8 Gram-negative organisms), lysozyme was active only against Lactococcus lactis NCK 400. When EDTA was added, lysozyme was inhibitory for all of the Gram-positive and 4 of the Gram-negative species. Butylated hydroxyanizole was active against all the organisms tested, except for 2 pseudomonads, but these were inhibited when EDTA was added. The inhibition effected by lysozyme and BHA, both in the presence and absence of EDTA, was greatest at low pH and high NaCl concentrations. The inhibition by lysozyme was unaffected by other chelators (sodium citrate and monoglycerol citrate). However, sodium citrate promoted the inhibitory effect of BHA towards Gram-negative bacteria and monoglycerol citrate lowered the minimum inhibitory concentration required by BHA to inhibit 5 of the 7 Gram-positive bacteria by 10%.