乳酸菌粘附肠道上皮细胞的研究进展

近些年来,乳酸菌因对人体健康功能的促进作用而成为研究热点。目前普遍将乳酸菌的粘附能力作为评价乳酸菌益生性的首要指标。乳酸菌对肠道上皮细胞或细胞外基质组分的粘附会引起机体产生有益的生物学效应。本文从乳酸菌的黏附素和宿主受体的相互作用阐述乳酸菌的粘附机制以及影响其粘附能力的内、外因。归纳目前国内外有关评价乳酸菌粘附特性的方法,以期为乳酸菌粘附特性的研究和应用奠定理论基础。     

乳酸菌黏附特性及其免疫调节作用研究进展

乳酸杆菌一般被认为是人体胃肠道中的常驻微生物群。摄入"益生菌乳酸菌"可与致病微生物竞争平衡肠道微生物群,同时对调节免疫也具有一定影响。乳酸菌借助其表面成分进行黏附,黏附过程通过化学键共价结合,此过程中黏附成分与黏附受体为特异性的,发挥生理功能的基础即为黏附并定殖,因此乳酸菌的黏附能力通常作为评价乳酸菌益生性的首要指标同时也是当前研究的热点。文章综述了乳酸菌黏附的黏附机制、生物学效应以及其与免疫调节的关系。     

双歧杆菌黏附能力的检测方法

双歧杆菌是人肠道的重要菌群,会为人体带来很多益生作用,然而双歧杆菌只有黏附于肠道,定居形成稳定的菌群,才能发挥其益生作用.因此双歧杆菌黏附能力的检测具有重要的意义.本文综合论述了体外模拟实验、表面疏水性分析和自身凝集能力分析等检测双歧杆菌黏附能力的方法,并分析总结了各方法的优缺点.     

益生乳酸菌黏附特性评价体系的研究进展

从益生性乳酸菌黏附特性模型和黏附能力方法入手探究其评价系统,并总结体外细胞模型、黏液黏附模型、组织器官模型、间接评价体系以及细菌黏附能力评价方法在乳酸菌黏附特性研究中的优劣,以期为乳酸菌粘附特性的研究和应用奠定理论基础。     

乳酸菌S-层蛋白的多样性及其研究方法

乳酸菌是人体和许多动物肠道的正常菌群,在预防肠道感染、促进人体健康方面,具有不可或缺的作用.乳酸茵通过竞争性黏附来抑制致病茵的繁殖.研究表明,乳酸菌的黏附作用与细胞表层蛋白密切相关.表层蛋白又名S-层蛋白(surface layer protein,Sip),是许多细菌及古生菌细胞壁表面所包被的生物活性大分子,在一些乳酸茵中也发现了S-层蛋白的存在.S-层蛋白的相对分子质量在40 000～200 000间,而在已发现的乳酸菌中,S-层蛋白相对分子质量在25 000～70 000间.作者讨论了乳酸菌中S-层蛋白的多样性及主要特性,对乳酸菌S-层蛋白的研究方法进行了初步探索.     

luxS基因介导乳酸菌益生特性研究进展

一些乳酸菌具有高肠道耐受性、高粘附肠上皮细胞和产具有抑菌活性的细菌素等益生特性。某些乳酸菌的细菌素合成量、耐受性及黏附特性可以被诱导物-2（autoinduction-2,AI-2）提高,AI-2是通过甲基循环合成的一种信号分子。luxS基因可以编码合成LuxS蛋白,而LuxS蛋白是AI-2合成的关键酶,因此展开对luxS在细菌素合成量和耐受性、黏附特性方面的作用研究具有重要意义。该文通过讨论luxS基因在乳酸菌益生特性的研究现状,提出该研究领域中存在的问题以及发展趋势,从而为提高乳酸菌的益生特性提供理论依据。     

乳酸菌表层蛋白功能研究进展

乳酸菌作为益生菌的主要类群,不仅常用于食品发酵,还具有维持肠道稳态、调节宿主免疫、疾病的预防和治疗等功能。乳酸菌对肠道的黏附能力是其发挥功能的重要前提,也是其在肠道中定植的必要条件。表层蛋白(SLP)是乳酸菌表面主要的黏附因子,在菌相之间、菌相与环境之间的相互关系中发挥重要作用。本文概述乳酸菌SLP的结构特征及功能,重点论述SLP对菌体的支撑保护作用,在菌相识别中的黏附作用,抑制病原菌入侵作用,免疫调节方面的作用等,并阐述SLP在生物技术、纳米技术、疫苗研发中的应用,以期为乳酸菌SLP的进一步研究与开发利用提供参考。     

15株乳酸菌的表面性质及其黏附能力

为了初步探讨乳酸菌的黏附机制,对15株乳酸菌的表面疏水性、自凝聚能力以及体外黏附Caco-2细胞的能力进行了测定,筛选出高黏附性乳酸菌菌株。采用化学试剂和酶处理嗜酸乳杆菌KLDS 1.0901细胞壁表面成分,再经过cFDA-SE荧光标记后,测定其对Caco-2细胞黏附能力的变化,分析影响黏附的主要黏附素。结果表明:嗜酸乳杆菌KLDS 1.0901对Caco-2细胞的黏附率最高,为12.73%;不同乳酸菌之间的表面疏水性、自凝聚能力以及对Caco-2细胞的黏附能力存在差异;经高碘酸钠、苯酚、胃蛋白酶、胰蛋白酶,尤其是氯化锂和热处理,能显著降低KLDS 1.0901的黏附性(p<0.05)。表明KLDS 1.0901对Caco-2细胞的黏附可能是以表层蛋白为主的多种黏附素共同作用的结果。     

乳酸菌生物学特性与其拮抗空肠弯曲杆菌在小鼠肠道定植能力的分析

采用3周龄C57BL/6小鼠建立了空肠弯曲杆菌感染模型,评价了8株在体外具有抑制空肠弯曲杆菌生长能力的乳酸菌在体内拮抗空肠弯曲杆菌定植的能力,同时对该8株菌生长速率、产酸能力、生物膜形成能力、黏附能力等生物学特性进行了测定,通过主成分分析对8株乳酸菌生物学特性的评价以及皮尔森相关性系数对乳酸菌生物学特性及其在体内拮抗空肠弯曲杆菌定植能力相关性的分析,发现8株乳酸菌种间生物学特性差异明显大于种内生物学特性差异,且乳酸菌在小鼠体内拮抗空肠弯曲杆菌定植的能力与其细胞黏附能力呈正相关。具有最高黏附指数的植物乳杆菌N8、N9可有效清除模型小鼠肠道内的空肠弯曲杆菌,对保护人体免受空肠弯曲杆菌感染具有潜在的应用价值,同时也为具有拮抗致病菌能力的益生菌的筛选提供了理论依据。     

乳酸菌在肠道定植的影响因素及研究方法

乳酸菌是一类无芽孢的革兰氏阳性菌,能利用碳水化合物发酵产酸,其作为益生菌家族中重要的一员,具有调节肠道菌群平衡、参与免疫应答和抑制肠道病原菌的生长繁殖等多种益生功能。这些益生功能持续发挥作用的先决条件是乳酸菌能定植于肠道中,但到目前为止,乳酸菌在肠道定植的机制还并不清楚。本综述介绍了乳酸菌在肠道定植中的干扰因素（黏附能力、运动性、乳酸的分泌、与肠道菌群的作用、宿主基因与体征因素、胃酸与胆盐的耐受性以及饮食）,概述了乳酸菌肠道定植的研究方法（平板计数法、荧光标记、聚合酶链式反应以及新型纳米材料）,旨在加深对乳酸菌在肠道定植的认识,为进一步揭示乳酸菌定植肠道的机理提供参考。     

具有潜在改善炎症反应益生菌的筛选

目的:探讨供试菌株对脂多糖(LPS)诱导THP-1细胞分泌炎性细胞因子的调节作用,筛选出具有潜在改善炎症反应的益生菌。方法:通过佛波酯(PMA)诱导THP-1成为成熟巨噬细胞,脂多糖(LPS)刺激成熟巨噬细胞产生炎症反应,评价益生菌对这种炎症反应的改善作用,并对具有改善炎症反应潜力的益生菌株进行胃肠道耐受能力、粘附能力、抑菌能力及抗生素敏感性评价。结果:植物乳杆菌SS-9、SD-H9、鼠李糖乳杆菌SD-L8及罗伊氏乳杆菌WX-94具有改善机体炎症反应的潜力,其中SS-9和SD-H9能显著降低炎症模型细胞IL-1β的分泌(P<0.05),WX-94和SS-9能显著降低炎症模型细胞的IL-6的分泌(P<0.05);同时四株菌均具有较高的胃肠道耐受能力、粘附能力,具有安全通过肠道的潜力;并可以不同程度地抑制致病菌大肠杆菌、金黄色葡萄球菌、铜绿假单胞菌及阴沟肠杆菌的生长,具有抑制肠道感染的潜力;抗生素敏感性研究表明,四株菌均未发现抗生素耐受的风险。结论:植物乳杆菌SS-9、SD-H9、鼠李糖乳杆菌SD-L8及罗伊氏乳杆菌WX-94均可作为具有潜在炎症调节能力的益生菌进行开发和应用。     

Exopolysaccharides produced by probiotic strains modify the adhesion of probiotics and enteropathogens to human intestinal mucus

Exopolysaccharides (EPSs) are exocellular polymers present in the surface of many bacteria, including Lactobacillus and Bifidobacterium. The genome sequence of several strains revealed the presence of EPS-encoding genes. However, the physiological role that EPSs play in the bacterial ecology still remains uncertain. In this study, we have assessed the effect of EPSs produced by Lactobacillus rhamnosus GG, Bifidobacterium longum NB667, and Bifidobacterium animalis IPLA-R1 on the adhesion of probiotic and enteropathogen strains to human intestinal mucus. The EPS fraction GG had no significant effect on the adhesion of L. rhamnosus GG and B. animalis IPLA-R1. However, the EPS fractions NB667 and IPLA-R1 significantly reduced the adherence of both probiotic strains. In contrast, the three EPS fractions increased the adhesion of Enterobacter sakazakii ATCC 29544 and Escherichia coli NCTC 8603. Higher adherence of Salmonella enterica serovar Typhimurium ATCC 29631 and Clostridium difficile ATCC 9689 was detected in the presence of the EPS fractions GG and NB667. In general, these effects were obtained at EPS concentrations of up to 5 mg/ml, and they were EPS dose dependent. The competitive exclusion of probiotics in the presence of EPS could suggest the involvement of these biopolymers in the adhesion to mucus. The increase in the adherence of enteropathogens could be explained if components of the pathogen surface are able to bind to specific EPSs and the bound EPSs are able to adhere to mucus. To the best of our knowledge, this is the first work reporting the effect of EPSs from probiotics on bacterial adhesion properties.     

Binding of aflatoxin B1 alters the adhesion properties of Lactobacillus rhamnosus strain GG in a Caco-2 model

Lactic acid bacteria have been previously reported to possess antimycotoxigenic activities both in vitro and in vivo. The objective of this study was to investigate the effect of aflatoxin B1 on adhesion capability of Lactobacillus rhamnosus strain GG using a Caco-2 adhesion model. Removal of aflatoxin B1 by L. rhamnosus strain GG reduced the adhesion capability of this strain from 30% to 5%. It is therefore concluded that aflatoxins may influence the adhesion properties of probiotics able to sequester them, and subsequently these bacteria may reduce the accumulation of aflatoxins in the intestine via increased excretion of an aflatoxin-bacteria complex.     

益生菌与益生元对人体肠道正常菌群的调节作用

为了解益生菌与益生元对人体肠道菌群的调节作用，选择符合试验要求的成年志愿者，分别食用益生菌和益生元，其中益生菌组150人，益生元组200人，男女各半。结果表明：益生菌与益生元均可增加人体肠道内的双歧杆菌和乳杆菌：均可使肠道内肠杆菌数量下降。但益生菌组双歧杆菌和乳杆菌增加的数量及肠杆菌和拟杆菌降低的数量均高于益生元组，说明在该试验条件下，益生菌组的效果要好于益生元组。     

六株人源益生菌的表面性质与黏附性能研究

通过测定六株人源益生菌的自凝集及疏水性能力,并以人结肠腺癌细胞系Caco-2和HT-29作为体外细胞黏附模型评价其黏附能力,最后探究生长阶段、菌体浓度和孵育时间对发酵乳杆菌CECT5716黏附能力的影响。结果表明嗜酸乳杆菌F-1的自凝集率最高,为69.92%;疏水性最强的是鼠李糖乳杆菌MP-108,达到90.10%;乳双歧杆菌AD011对Caco-2和HT-29的黏附性均为最强,分别为(42.80±0.68)、(47.01±0.20) CFU/cell;不同益生菌的自凝集能力、疏水性及黏附能力均存在菌株特异性,其自凝集能力、疏水性及黏附能力间无明显相关性。此外,发酵乳杆菌CECT5716黏附能力随菌体浓度增大而显著(p<0.05)增加,当菌体浓度达108 CFU/mL以上时黏附能力不再显著提高;随着共孵育的时间延长,其黏附能力也增强,在共孵育2 h后不再提升;且菌体生长稳定期时表现出的黏附能力最强,达到(3.61±0.20) CFU/cell。所考察的益生菌大部分都具有较强的潜在黏附能力,其中以乳双歧杆菌AD011最强。这些益生菌具有可在人体肠道定殖生长的潜在益生功能,在食品和药品等行业中具有良好的应用前景。     

传统乳制品中潜在益生植物乳杆菌的体外筛选

对从内蒙古牧区乳制品中分离到的29株植物乳杆菌的耐酸性（pH=3．0）、耐胆汁性（质量浓度3g／L牛胆酸钠）、抗菌活性进行了系统性研究。结果表明，有10株菌具有较好的耐酸耐胆盐特性，结合抗菌特性及黏附性试验结果，菌株WZ47-1，WZ32-2-1和WH13-2有可能成为潜在益生菌菌株。     

Study of adhesion of Lactobacillus casei CRL 431 to ileal intestinal cells of mice

It is well known that the cell wall of Lactobacillus casei CRL 431, a strain present in probiotics, presents lectinlike surface molecules. Presence of these molecules stimulates the immune system. Given the role that lectins and lectinlike substances play in the adhesion phenomenon, it is probable that this is an initial stage in the immunostimulation produced by this bacterium. To confirm this, adhesion of this microorganism to exfoliated mouse ileal epithelial cells was studied in vitro. Other L. casei strains isolated from adult human intestines and one of dairy origin were also examined for their ability to adhere to ileal epithelial cells. Another strain, which was included in the present study, was Lactobacillus acidophilus CRL 730. L. casei strains isolated from humans showed good ability to adhere to ileal epithelial cells, whereas L. casei isolated from dairy origin did not. Adhesion was only observed at 37 degrees C and at a pH between 6 and 7.5. The exposure time needed for highest adhesion was 30 min. Presence of lectinlike substances on the surface of L. casei CRL 431 is important to this adhesion phenomenon, since adherence capacity was lost after removal of these substances.     

Functionality of the S-layer proteins from <Emphasis Type="Italic">Lactobacillus</Emphasis> in the competitive against enteropathogens infection

The interest of probiotics as remedy for a broad number of infectious diseases has gained wide interest over the last few years, but little is known about their underlying mechanism of action. In this study, five selected Lactobacillus isolated from human intestinal and ferment milk were preliminarily identified by 16S rDNA gene sequencing and assessed the ability to inhibit the adhesion of enteropathogens using HT-29 cells model through a process which may be related to specific components of the bacterial surface. Surface layer proteins are located in a paracrystalline layer outside the bacterial cell wall and are thought to play a role in tissue adherence. Removal of S-layer proteins from the Lactobacillus (treated with 5 M LiCl) reduced inhibition activity as revealed in exclusion, competition and displacement assays, which suggested that S-layer proteins had involved in the adhesion of probiotics. SDS-PAGE analysis confirmed the presence of S-layer proteins with dominant band which was approximately 60 kDa. Further analysis of S-layer proteins revealed that the hydrophobic amino acids accounted for 43.2 % of the total amino acid for the Lactobacillus paracasei M7. So, these probiotics could be used in the health-promoting food products, which could prevent the diarrhea caused by pathogens.     

Short communication: effect of exopolysaccharide isolated from "viili" on the adhesion of probiotics and pathogens to intestinal mucus

The strong ropy character of the Scandinavian fermented milk viili is conferred by the exopolysaccharides (EPS) produced by lactococcal strains. These biopolymers can be responsible for some health benefits. We have assessed the influence of the EPS fraction isolated from commercial viili on the adhesion of some probiotics and pathogens to human intestinal mucus. Concentrations of viili EPS greater than 0.1 mg/mL promoted a decrease in adherence of Bifidobacterium lactis Bb12 and Lactobacillus rhamnosus GG and this effect was dose-dependent. However, no modifications were detected on the adhesion levels of the pathogenic strains tested at a concentration of 1 mg/mL of EPS. Results obtained in the present work should be considered in the design of new probiotic products.     

Use of Caenorhabditis elegans for preselecting Lactobacillus isolates to control Salmonella Typhimurium

Host-specific probiotics have been used to control enteric pathogens, including foodborne pathogens, in food animal production. However, evaluation of the efficacy of these probiotics requires costly in vivo assays in the target animal. The nematode Caenorhabditis elegans has been used for prescreening of antimicrobial agents and for studies of host-pathogen interactions. In the present study, 17 Lactobacillus isolates from chicken and pig intestines were tested with C. elegans, and the ability of these isolates to prevent death from Salmonella infection was variable. Two Lactobacillus isolates (S64, which gave full protection, and CL11, which gave no protection) were further studied. Both isolates exhibited a similar colonization profile in the C. elegans intestine. Although different culture fractions of CL11 were not protective, both live and heat-killed S64 cells provided full or partial protection of C. elegans from death caused by Salmonella infection. In contrast, different culture fractions from both isolates had similar effects on the colonization of the nematode intestine by Salmonella Typhimurium DT104. Our preliminary results from a pig performance trial revealed a correlation between the degree of protection in the C. elegans survival assay and the performance of 35-day-old weaned piglets that were treated with the same Lactobacillus isolates, suggesting that C. elegans can be used as a laboratory animal model for preselecting probiotics for control of Salmonella infections.