双肽（IIb类）细菌素的结构特点及其与功能的关系

双肽（IIb类）细菌素通常是由革兰氏阳性菌产生的热稳定小分子（＜10 kDa）双组分抗菌肽,该类肽能够依靠特定模式基序介导肽-肽互作,形成具有跨膜能力的活性二聚体蛋白。大量研究表明,双肽细菌素具有可靠的安全性和理想的抑菌效果,在耐药菌控制方面有着巨大的应用潜力。因此,双肽细菌素发挥功能的结构特征与作用机制受到研究者们的广泛关注。本文按照双肽细菌素结构形成、肽-肽互作及肽-膜互作的逻辑梳理该类抗菌肽发挥抑菌作用的机制,同时综合现有研究阐述双肽细菌素的结构规律及影响其活性的结构要点,以期为双肽细菌素的相关研究提供新的思路。     

乳酸菌素的研究现状和发展趋势

乳酸菌素是由乳酸菌产生的一种具有活性的多肽或蛋白质物质，大部分细菌素对近缘关系的细菌有抑制作用，少数细菌素抑菌谱很广。主要可分为热稳定的小分子肽，热敏感的大分子蛋白和羊毛硫抗生素。乳酸菌素是一种纯乳酸菌产生的制剂，是将脱脂乳经杀菌、真空浓缩．再经乳酸菌发酵后干燥而制成的乳酸菌体及其代谢产物混价粉状物，可作医药用或食品添加剂，属疗效型乳制品。本文对乳酸菌素的分类、抑菌机理及在食品和医药方面的应用作了全面系统的综述，并指出了今后的研究方向和应用前景。     

食物蛋白质来源的水解物或多肽的现状及进展(Ⅱ)活性多肽概况

食品蛋白水解物中含有大量具有生理活性的多肽,包括有类吗啡活性多肽、抑制血管收缩素转化酶(ACE)活性的多肽类(即降血压多肽类)、免疫调节肽、促进矿物质吸收的多肽、抗菌活性肽、抗血栓活性的多肽等.不同食物蛋白中所含有的活性多肽会有所不同,其中乳蛋白(如酪蛋白和乳清蛋白)是含有活性多肽最为丰富的蛋白质.本文概述了乳蛋白、植物蛋白(主要为大豆蛋白)、畜产物以及水产蛋白等来源的活性多肽的研究进展以及开发现状.特别对乳蛋白来源的几类新型活性肽(酪蛋白糖巨肽和乳铁蛋白活性肽)作了较为详细的论述.     

利用米糠蛋白制备类阿片拮抗肽和降血压肽的研究

采用离体豚鼠回肠(GPI)检定法测定不同米糠蛋白胰蛋白酶水解物的类阿片拮抗活性,结果发现跟新鲜米糠一样,低温脱脂米糠蛋白的酶解物具有较高的类阿片拮抗活性,高温脱脂米糠蛋白的酶解物无类阿片拮抗活性。采用低温脱脂米糠蛋白为原料,研究其制备类阿片拮抗肽后的残留蛋白酶解物的ACE抑制活性,结果发现在6种酶解物中,水解度(DH)为15.4%的碱性蛋白酶水解物的ACE抑制活性最高。     

唾液乳杆菌LH1F所产细菌素的纯化及其生物学特性研究

本文对唾液乳杆菌(Lactobacillus Salivarius)LH1F发酵液中的抑菌物质的特性进行了研究,在排除菌体细胞、有机酸、过氧化氢干扰及经胰蛋白酶、胃蛋白酶处理后,确定抑菌成分为肽类细菌素,然后经过60%硫酸按盐析及超滤初步纯化后,对其生物学特性进行了研究。结果表明,唾液乳杆菌LH1F所产的细菌素具有良好的热稳定性,121℃处理30 min仍保留75.5%的抑菌活性;在p H2.0~p H5.0条件下稳定;对胃蛋白酶、胰蛋白酶和蛋白酶K敏感;该细菌素具有较广的抑菌谱,对供试的多种革兰氏阳性菌(G+)及革兰氏阴性菌(G-)具有明显的抑制作用,为作为天然食品防腐剂的进一步研究奠定基础。     

几种常见植物蛋白生物活性肽的研究概述

对大豆、大米、小麦、玉米四种植物蛋白的生物活性肽进行了介绍，主要包括：降血压肽、阿片样活性肽、类阿片样拮抗肽、免疫调节肽等。这些活性肽的发现为今后进一步利用植物蛋白资源，生产功能性食品基料或药品提供了一条新途径。     

拮抗黄瓜花叶病毒(CMV)细菌粗提蛋白对CMV粒体形态的影响

为研究拮抗细菌活性蛋白对CMV的影响,从烟田耕作层土样中分离到对CMV有拮抗作用的细菌菌株B6,通过离子交换层析和凝胶过滤层析初步确定其活性物质是分子量大小为40.6kD的蛋白质。粗提病毒经活性蛋白处理后,检测活性蛋白对病毒的抑制率,体外混合对CMV粒体形态的影响及利用叶圆片法,检测活性物质处理后寄主体内病毒复制特点,结果表明:1)拮抗细菌B6发酵产生的活性蛋白对CMV的体外钝化作用为86.97%;2)电镜下观察到活性蛋白能使CMV粒体变形,从而降低侵染力;3)寄主体内的病毒复制作用受到了拮抗活性物质的抑制,并且随着拮抗活性物质浓度的增加病毒的复制作用减弱。     

仿刺参相关微生物对致病灿烂弧菌的拮抗及机理研究

从健康的仿刺参体表以及肠道中分离得到37株细菌,以仿刺参"腐皮综合症"致病菌灿烂弧菌(Vibriosplendidus)为指示菌,采用十字交叉划线法和纸片法进行体外拮抗试验,两种方法筛选出对灿烂弧菌(Vibriosplendidus)有拮抗作用的菌株分别为20种和8种。选择对灿烂弧菌有较强拮抗作用的7种菌株进行拮抗机理研究。研究表明:7株菌均是通过营养物质和生存空间竞争及产生有抑菌活性的胞外产物两种方式协同抑菌。其中,编号为CG-6-1的Pseudoalteromonas sp.主要分泌蛋白类等和比较大的极性分子,其胞外产物经过硫酸铵沉淀,在80%饱和度时抑菌活性最高,因此推断其产生的抑菌物质是蛋白类物质。     

一株后生元菌株的抑菌特性研究及其细菌素基因簇的挖掘

本文旨在筛选一株可拮抗中国大鲵源嗜水气单胞菌的后生元菌株,并对其进行抑菌特性的研究和细菌素基因簇的挖掘。以中国大鲵病原性嗜水气单胞菌（Aeromonas hydrophila, Ah2）为指示菌,筛选一株产细菌素乳酸菌株并评估其药敏特性;采用有机溶剂萃取法初步纯化菌株产细菌素;通过pH、温度和消化酶耐受性、贮藏稳定性、抑菌谱及最小抑菌（MIC）和杀菌浓度（MBC）共六类指标评价细菌素的抑菌特性;溶血反应与细胞毒性实验测试细菌素对Ah2的抑菌效果,经扫描电子显微镜初步探究细菌素的抑菌机制;经由紫外全波长扫描定性细菌素以及Tricine-SDS-PAGE电泳测定细菌素的分子量范围;最后根据菌株的全基因组序列挖掘其潜在的细菌素基因簇（RiPPs）。结果显示,从青岛市售腐乳中筛出一株产细菌素植物乳植物杆菌M4L1（Lactiplantibacillus plantarum M4L1）。初步纯化M4L1产细菌素并命名为LP01。细菌素LP01具备良好的消化酶耐受性,且在pH2~10、?20~121 ℃和9个月贮藏期内均表现出稳定的抑菌活性,并对单增李斯特菌、弗氏柠檬酸杆菌等14株革兰氏阳性和阴性菌均有不同程度的抑制作用;另外,LP01对Ah2的MIC和MBC分别为12.94和25.88 μg/mL。经MBC浓度的LP01处理后的Ah2,溶血活性和细胞毒性均得到明显缓解;SEM观察其通过破坏Ah2的细胞壁具有抑制或杀伤作用。LP01在波长200~220 nm处的肽类特征吸收峰显著,电泳后条带显示其分子量在3.3~4.0 kDa,LP01为小分子肽类细菌素。此外,基因注释到M4L1有2个细菌素基因簇（RiPPs）,对应产物分别为Plantaricin K和Plantaricin E,均属于植物乳杆菌II类细菌素。菌株M4L1不仅含有多种抗菌物质相关基因簇,而且其细菌素LP01具备了优良的抑菌性能,能有效抑制多种水产病原菌、食物腐败菌及食源性致病菌等。产细菌素植物乳植物杆菌M4L1作为一株潜在的后生元菌株具有较好的应用前景。     

一株安徽本地泡菜中产细菌素乳酸菌的筛选与鉴定

从安徽本地泡菜中筛选出一株具有抑菌活性的乳酸菌(编号为PC-3)。通过菌落形态观察、染色镜检、生理生化和分子生物学实验鉴定分析,鉴定为植物乳杆菌(Lactobacillus plantarum),并命名为Lactobacillus plantarum PC-3。通过实验排除乳酸、乙酸和过氧化氢干扰后,L. plantarum PC-3的无细胞发酵上清液(cell-free fermentation supernatant,CFS)对大肠杆菌(ATCC 8099)和金黄色葡萄球菌(ATCC 6538)仍具有抑制作用,且经胰蛋白酶、胃蛋白酶和胃蛋白酶K处理后抑菌作用下降明显,说明L. plantarum PC-3所产主要抑菌物质为蛋白类物质,初步分析该抑菌活性物质为细菌素类。L. plantarum PC-3可作为食品运输和贮藏过程中控制大肠杆菌和金黄色葡萄球菌的拮抗制剂出发菌株,能有效地防止杂菌污染,延长食品保质期。     

Antimicrobial proteinaceous compounds obtained from bifidobacteria: from production to their application

Antimicrobial proteinaceous compounds such as bacteriocins or bacteriocin-like compounds produced by bifidobacteria are largely unknown but have been found to have potent antimicrobial activities toward closely related bacteria and undesirable harmful microorganisms. They are useful in the fields of food preservation or safety, health care, and pharmaceutical applications. The inhibition activity of these substances has been reported to be strain-dependent. Binding to the epithelial cell on the gastrointestinal surfaces is one of the important factors of resident microflora to colonize the intestine. Certain Bifidobacterium strains are able to produce substances that compete and prevent pathogenic bacteria from adhering to the receptors on epithelial cells of intestinal surfaces. The potential probiotic effects of bifidobacteria is well known in the human ecosystem and their production of antimicrobial peptides can contribute to elucidate the precise mechanisms by which bifidobacteria can dominate the intestinal microbiota and achieve their probiotic function. This paper presents a review of the antimicrobial proteinaceous compounds produced by various Bifidobacterium strains, the attempts made to purify them, their characterization, identification and useful applications.     

Characterisation of Antimicrobial Producing Lactic Acid Bacteria from Malted Barley

Thirty‐three putative inhibitor‐producing lactic acid bacteria (LAB) were isolated from malted barley based on their ability to inhibit growth of two indicator strains. Eleven of the inhibitor‐producing LAB produced an antimicrobial activity which was active across a wide pH range, relatively insensitive to heat treatment while sensitive to treatment with proteolytic enzymes indicating that the inhibitory compounds are proteinaceous in nature and therefore bacteriocin‐like inhibitory substances. Ten of these eleven malt isolates were observed to secrete the inhibitory compounds into the cell‐free supernatant with optimal production occurring in the late exponential growth phase. The inhibitory spectra of these isolates included various Gram‐positive bacteria among which a variety of beer‐spoiling bacteria.     

Antimicrobial‐Producing Lactic Acid Bacteria Isolated from Raw Barley and Sorghum

From a total of four thousand presumed lactic acid bacteria, obtained from raw, unmalted sorghum and barley, 308 isolates were shown to exhibit inhibitory activity against the indicator strain Listeria innocua 4202. Six of these inhibitor‐producing isolates were selected for further study on the basis of their relatively wide antimicrobial spectrum, which showed that these producers inhibited several Gram‐positive bacteria, including a range of beer spoiling bacteria. The proteinaceous nature, anti‐microbial activity against closely related species, heat resistance and pH stability of the inhibitory substances produced by these six bacteria identified these compounds as bacteriocins. All six isolates were shown to secrete the inhibitory compounds into the cell free supernatants. Bacteriocins produced by five of the six producers were purified to homogeneity. Further analytic data was obtained for three of the inhibitory compounds by means of mass spectroscopy and/or N‐terminal amino acid sequencing.     

酱油渣中具有抑菌活性的乳酸菌的筛选及其抑菌特性

本研究以实验室前期从酱油渣中分离的16株乳酸菌ZW1~ZW16为出发菌株,拟筛选对大肠杆菌、金黄色葡萄球菌抑制效果较强的菌株,并对菌株产生的抑菌物质、生长特性及其影响因素进行分析。结果显示,乳酸菌ZW2、ZW9、ZW14对大肠杆菌、金黄色葡萄球菌均有较高的抑菌活性,其中ZW9抑菌效果最好,排除有机酸和H₂O₂影响后,胰蛋白酶、胃蛋白酶、蛋白酶K、α-蛋白酶、碱性蛋白酶、中性蛋白酶处理后抑菌活性均显著性降低（P<0.05）,初步推断抑菌物质是一种具有蛋白质属性的细菌素。抑菌动力学曲线显示,菌株培养至20 h抑菌活性趋于稳定。抑菌稳定性显示,抑菌物质在40~100 ℃具有较好的热稳定性,在紫外线照射、表面活性剂处理下均保持较高的抑菌活性;在pH2.0~6.0抑菌活性无明显变化,pH10.0抑菌能力完全丧失。酱油渣中筛选的3株乳酸菌具有抑菌功能且产生的抑菌物质稳定,对开发成新型生物抑菌剂具有重要参考价值。     

Bacteriocins of lactic acid bacteria: Their potentials as food biopreservative

Abstract

Numerous strains of lactic acid bacteria used in the fermentation of foods are known to produce bacteriocins. In general, bacteriocins are a group of proteinaceous antimicrobial substances that inhibit the growth of closely related bacteria. However, some bacteriocins produced by lactic acid bacteria (LAB) exhibit a relatively broad antimicrobial spectrum and are active against several food‐spoilage and health‐threatening microorganisms. Many investigators have reported on the use of bacteriocins as food preservative to extend the shelflife of various foods. This review decribes the research that has been conducted on bacteriocinogenic lactic acid bacteria— isolated from a wide variety of foods and in some instances of animal origin—and the characteristics of bacteriocins. Special emphasis is placed on their potentials for use as food preservative and on their physicochemical nature, antibacterial spectrum, and genetic behavior.     

Antimicrobial spectrum activity of bacteriocinogenic Staphylococcus strains isolated from goat and sheep milk

Bacteriocins have attracted great attention as potential alternatives to antibiotics and chemical food additives. In the present study, 243 Staphylococcus isolates from milk samples (n = 110) of goat and sheep herds located in Fars province, Iran, were screened for antimicrobial substance production. Twenty-eight isolates showed an antagonistic activity against the indicator strain Micrococcus luteus ATCC 4698. The susceptibility of all antimicrobial substances to proteolytic enzymes allowed us to consider them as bacteriocin-like inhibitory substances (BLIS). The term BLIS is applied to uncharacterized proteinaceous antimicrobials produced by gram-positive bacteria. Based on molecular identi?cation methods, the isolates belonged to the species Staphylococcus chromogenes, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus pseudintermedius, Staphylococcus aureus, and Staphylococcus agnetis. Pulsed-?eld gel electrophoresis revealed a high level of genotype diversity among the Staph. chromogenes isolates. All of the isolates harbored nukA or bsaA2 genes, suggesting that their BLIS were related to nukacin or Bsa. The antimicrobial compounds from test strains were not effective against gram-negative pathogens, including Salmonella enterica serovar Typhimurium, Escherichia coli O157:H7, and Klebsiella pneumonia as well as the indicator mold Aspergillus fumigatus. All the gram-positive targets, including Bacillus cereus, Listeria monocytogenes, Enterococcus faecalis Ef37 (a tyramine-producer strain), Lactobacillus saerimneri 30a (a histamine-producer strain), and methicillin-resistant Staph. epidermidis, were inhibited by the Staph. chromogenes isolates. Staphylococcus haemolyticus 4S12 was able to inhibit the majority of gram-positive bacteria. Listeria monocytogenes strains were the only indicators sensitive to the antimicrobial agents produced by Staph. agnetis 4S97B. The other Staphylococcus strains were ineffective on all the organisms tested. Based on their inhibitory capacities, the BLIS produced by the Staph. chromogenes isolates seem to be interesting candidates for developing novel antimicrobial agents.     

Preliminary characterization of bacteriocin-like substances from lactic acid bacteria isolated from organic leafy vegetables

This study presents the characterization of new strains of lactic acid bacteria (LAB) from organic vegetables. Forty-five strains of LAB isolated from vegetables were investigated by its antimicrobial activity against taxonomically related microorganisms. Genetic identification of selected LAB was performed by means of PCR method. These strains were Enterococcus faecium, Lactococcus lactis, Enterococcus hirae and Enterococcus canis. Bacteriocin-like substances were active against Gram-positive bacteria and Gram-negative foodborne pathogens (Listeria monocytogenes and Escherichia coli, respectively). The antimicrobial activity of LAB strains was inactivated by the addition of proteases, thus confirming the proteinaceous nature of the inhibition. In all four strains the bacteriocin activity was stable after extended refrigerated storage and freezing-thawing cycles. This fact suggests that bacteriocin produced by the four LAB strains may find application as biopreservatives in minimally processed vegetables.     

Chemical and genetic characterization of bacteriocins: antimicrobial peptides for food safety

Antimicrobial peptides are produced across all domains of life. Among these diverse compounds, those produced by bacteria have been most successfully applied as agents of biocontrol in food and agriculture. Bacteriocins are ribosomally synthesized, proteinaceous compounds that inhibit the growth of closely related bacteria. Even within the subcategory of bacteriocins, the peptides vary significantly in terms of the gene cluster responsible for expression, and chemical and structural composition. The polycistronic gene cluster generally includes a structural gene and various combinations of immunity, secretion, and regulatory genes and modifying enzymes. Chemical variation can exist in amino acid identity, chain length, secondary and tertiary structural features, as well as specificity of active sites. This diversity posits bacteriocins as potential antimicrobial agents with a range of functions and applications. Those produced by food‐grade bacteria and applied in normally occurring concentrations can be used as GRAS‐status food additives. However, successful application requires thorough characterization. © 2013 Society of Chemical Industry     

Molecular identification of naturally occurring bacteriocinogenic and bacteriocinogenic-like lactic acid bacteria in raw milk and soft cheese

Lactic acid bacteria (LAB) are currently used by food industries because of their ability to produce metabolites with antimicrobial activity against gram-positive pathogens and spoilage microorganisms. The objectives of this study were to identify naturally occurring bacteriocinogenic or bacteriocinogenic-like LAB in raw milk and soft cheese and to detect the presence of nisin-coding genes in cultures identified as Lactococcus lactis. Lactic acid bacteria cultures were isolated from 389 raw milk and soft cheese samples and were later characterized for the production of antimicrobial substances against Listeria monocytogenes. Of these, 58 (14.9%) LAB cultures were identified as antagonistic; the nature of this antagonistic activity was then characterized via enzymatic tests to confirm the proteinaceous nature of the antimicrobial substances. In addition, 20 of these antagonistic cultures were selected and submitted to genetic sequencing; they were identified as Lactobacillus plantarum (n = 2) and Lactococcus lactis ssp. lactis (n = 18). Nisin genes were identified by polymerase chain reaction in 7 of these cultures. The identified bacteriocinogenic and bacteriocinogenic-like cultures were highly variable concerning the production and activity of antimicrobial substances, even when they were genetically similar. The obtained results indicated the need for molecular and phenotypic methodologies to properly characterize bacteriocinogenic LAB, as well as the potential use of these cultures as tools to provide food safety.     

Purification and characterization of pedioxanthin (carotenoid pigment) produced by Pediococcus pentosaceus N33 strain isolated from pickles

Forty-two lactic acid bacteria were isolated from 28 pickles and yoghurt samples, and screened for their antimicrobial efficacy against certain multidrug resistant (MDR) bacteria using well diffusion method. A strain Pediococcus pentosaceus N33 (MF062087) showed the best inhibitory effects against both Brevibacillus brevis H18 (MG637284) and Lysinibacillus fusiformis R29 (MG637285). Pediococcus pentosaceus N33 strain produced high thermostable antimicrobial compound as indicated by heat treatment between 60 and 100°C for 20 min as well as at 121°C for 15 min. It is a proteinaceous compound highly active at acidic pH (2–6). The antimicrobial compound was extracted and precipitated by 70% ammonium sulfate and finally fractionated using Sephadex G₅₀ column. The molecular weight of pure compound was 10 kDa using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The spectroscopic analysis based on UV, IR, NMR and MS analyses were used to characterize and identify the pure antimicrobial compound. From the characterization and identification of this inhibitory compound, it was observed that the target compound contains carotenoid pigment in addition to protein. This carotenoid pigment was identified as pedioxanthin which is the first observed from this strain. Our results evaluated the potential activity of Pediococcus pentosaceus N33 strain as a probiotic agent and its potential contribution for food industry and pharmaceutical applications.