低pH处理对两歧双歧杆菌KLDS2.0603黏附能力的影响

【目的】确定低pH处理对两歧双歧杆菌KLDS2.0603黏附能力及其表面物理化学性质的影响。【方法】将两歧双歧杆菌KLDS2.0603菌体在不同低pH的PBS溶液中处理一定时间后,采用平板菌落计数法和直接镜检法,测定其经历不同pH的酸性环境后的黏附能力,及其表面疏水性和自动聚集能力。【结果】不同pH的PBS溶液处理后的双歧杆菌菌体,其黏附能力均不同程度下降,除pH 5.0的处理组外,其余处理组均显著低于空白组。此外,经不同pH的PBS溶液处理后,仅pH 3.0和3.5的两处理组,双歧杆菌表面疏水性显著提高。除pH 1.0、1.5和5.0的处理组外,其余处理组的自动聚集能力均显著下降。【结论】低pH的酸性环境会降低两歧双歧杆菌KLDS2.0603的黏附能力,并且双歧杆菌的自动聚集能力和表面疏水性也发生相应变化。除pH 3.0和3.5的处理组外,三者之间呈现一定的正相关性。     

两种生物状态肠道益生菌的粘附和粘附拮抗效应的对比研究

目的:研究活菌和灭活菌两种生物状态的肠道主要益生菌--德氏乳杆菌、双歧杆菌和肠球菌对肠黏膜上皮细胞粘附性及其对肠道几种常见病原菌的粘附拮抗效应.方法:用光镜和电镜技术分析了两种生物状态的三种益生菌对肠黏膜上皮细胞的粘附指数,通过排除实验、竞争实验和替代实验研究了两种生物状态益生菌对侵袭性大肠埃希菌、产毒性大肠埃希菌和痢疾志贺菌的粘附拮抗效应,应用平板扩散法观察了三种益生菌的代谢乏液对上述肠道病原菌的抑制能力.结果:德氏乳杆菌和肠球菌的灭活状态较活菌状态对肠黏膜上皮细胞的粘附性显著增高,双歧杆菌经灭活后对细胞的粘附性与活菌相比差异无显著性,两种生物状态的三种益生菌对肠道致病菌均具有粘附拮抗作用.滤过后的德氏乳杆菌、双歧杆菌和肠球菌的代谢乏液对侵袭性大肠埃希菌、产毒性大肠埃希菌和痢疾志贺菌均具有较明显的抑制作用,经42℃、65℃和100℃加热不影响德氏乳杆菌和双歧杆菌代谢乏液的抑菌作用.结论:灭活状态的德氏乳杆菌、双歧杆菌和肠球菌是具有潜在开发价值的微生态制剂.     

乳酸杆菌对感染大肠杆菌O157:H7小鼠肠道黏膜免疫的影响

【目的】分析小鼠在感染Escherichia coli O157:H7及补充嗜酸乳杆菌KLDS AD1和瑞士乳杆菌KLDS1.8701期间小肠黏膜中SIgA和细胞因子的变化规律,结合小鼠表象特征,探讨2株乳酸杆菌对小鼠腹泻的治疗效果。【方法】将小鼠分成4组,空白组、致病对照组、嗜酸乳杆菌组和瑞士乳杆菌组,对实验组小鼠连续7 d灌胃大肠杆菌致病后,再连续7 d分别灌胃2株乳酸杆菌,采集小鼠小肠利用ELISA法测得各组小鼠肠道组织中SIgA和4种细胞因子IL-2、IFN-γ、IL-4和IL-6的含量。【结果】瑞士乳杆菌可极显著提高感染大肠杆菌O157:H7小鼠的体重,嗜酸乳杆菌的效果较小;感染E.coli O157:H7后,SIgA、IL-2和IFN-γ的含量在第3天达到最大值,第5天开始下降,而IL-4和IL-6在第5天达到最大值,第7天开始下降。补充嗜酸乳杆菌和瑞士乳杆菌后,SIgA和4种细胞因子的含量都迅速增加,并保持较高水平,与其他两组差异显著。【结论】嗜酸乳杆菌KLDS AD1和瑞士乳杆菌KLDS 1.8701都可通过增加细胞因子和SIgA的分泌增强肠道黏膜免疫,对小鼠腹泻有一定的缓解作用。     

嗜酸乳杆菌LA85对小鼠免疫机能的研究

目的：研究嗜酸乳杆菌LA85对小鼠免疫机能的影响。方法：将160只雄性小鼠随机分为4组,即对照组、低、中、高三个剂量组。采用灌胃的方式给予实验小鼠不同剂量的嗜酸乳杆菌LA85。通过对小鼠平均体重、脾脏和胸腺免疫指数、细胞免疫、体液免疫、单核-吞噬性细胞和自然杀伤细胞（NK细胞）活性的评价,探讨嗜酸乳杆菌LA85对小鼠免疫机能的影响机制。结果：与对照组相比,嗜酸乳杆菌LA85低、中、高剂量组能够显著提升小鼠的胸腺指数,嗜酸乳杆菌LA85高剂量组能够显著提升小鼠的脾脏指数。同时能够显著提高小鼠的淋巴细胞增殖分化能力、半数溶血值、巨噬细胞的吞噬能力、NK细胞的细胞活性;嗜酸乳杆菌对小鼠的体重没有影响。结论：给予小鼠口服嗜酸乳杆菌LA85可以通过改善细胞免疫和体液免疫功能,从而提高免疫力。     

嗜酸乳杆菌产细菌素生物学特性的研究

嗜酸乳杆菌在MRS培养基中培养得到的细菌素经30、60、90、121℃处理20min后,活性几乎不变,对以金黄色葡萄球菌为代表的革兰阳性菌、大肠埃希菌为代表的革兰阴性菌有明显的抑制作用,Tricine—SDS—PAGE和不同pH值抑菌实验测定结果证明,嗜酸乳杆菌细菌素是一组低分子量,并在pH2—4时有抑菌作用的活性物质。     

灵芝子实体-益生菌发酵液对环磷酰胺诱发的肠黏膜损伤及菌群紊乱的调节作用

目的建立灵芝子实体-益生菌发酵体系,并研究该发酵液对于环磷酰胺诱导的肠黏膜损伤及菌群紊乱的防治作用。方法通过吸光度(A值)、多糖含量检测等方法评价嗜酸乳杆菌及短双歧杆菌在灵芝子实体提取液中的生长情况。将混合益生菌菌种与灵芝培养基进行共同发酵,发酵液用于对环磷酰胺诱导的肠黏膜损伤模型小鼠的治疗。结果嗜酸乳杆菌、短双歧杆菌均能在灵芝子实体的培养基上生长,且A值及总糖含量均高于对照组。该发酵液对环磷酰胺诱导的肠黏膜损伤模型动物有一定的治疗作用,表现为发酵液干预后由环磷酰胺导致的小鼠体质量降低、结肠组织损伤、炎细胞浸润均有所缓解;并恢复肠道菌群结构,显著增加乳杆菌属丰度,降低肠球菌属和鞘脂单胞菌属丰度。结论灵芝子实体-益生菌发酵液可以有效改善环磷酰胺诱导的肠黏膜损伤的症状,其机制可能与调节肠道微生态平衡密切相关。     

益生菌联合柳氮磺胺吡啶治疗溃疡性结肠炎的疗效观察

【摘 要】 目的 通过建立葡聚糖硫酸钠(DSS)诱导的急性期溃疡性结肠炎(UC)小鼠模型,观察嗜酸乳杆菌以及联合柳氮磺胺吡啶对小鼠溃疡性结肠炎(UC)的治疗作用,并检测Hsp70、Hsp27在肠黏膜的表达,探讨其可能的作用机制。方法　5% DSS 7 d建立急性UC动物模型。将60只BALB/c小鼠随机分为6组：正常对照组、模型组、阴性对照(生理盐水,NS)组、嗜酸乳杆菌组、柳氮磺胺吡啶组和嗜酸乳杆菌联合柳氮磺胺吡啶组,观察指标包括：疾病活动指数(DAI)、结肠黏膜肉眼改变及病理组织学积分;采用免疫组化SABC 法检测热休克蛋白(HSP70)和(HSP27)的表达量。结果 嗜酸乳杆菌可降低实验小鼠DAI积分和改善结肠组织损伤;与模型组、阴性对照组相比,嗜酸乳杆菌联合柳氮磺胺吡啶组的HSP70表达增加(P＜0.05),其中以嗜酸乳杆菌联合柳氮磺胺吡啶组效果最佳。结论 嗜酸乳杆菌和柳氮磺胺吡啶对小鼠溃疡性结肠炎都有治疗作用,且二者疗效相当;两药联合应用效果最佳。其机制可能与增加结肠黏膜HSP70的表达有关。     

细菌对肉鸡肠粘液的粘附作用

研究两歧双歧杆菌、嗜酸乳杆菌、禽大肠杆菌O78、大肠杆菌 ATCC 25922、鸡白痢沙门氏菌和鼠伤寒沙门氏菌与肉鸡不同部位肠粘液糖蛋白的粘附性能,探讨两歧双歧杆菌和嗜酸乳杆菌对所试病原菌的抗粘附作用。结果表明：在不同的肠道部位,两歧双歧杆菌、嗜酸乳杆菌、鸡白痢沙门氏菌和鼠伤寒沙门氏菌与肠粘液糖蛋白均有不同的粘附作用,而禽大肠杆菌O78、大肠杆菌 ATCC 25922在各肠段粘液上的粘附性能则相近;在相同的肠道部位,所试益生菌的粘附能力大于病原菌;两歧双歧杆菌和嗜酸乳杆菌对所试病原菌的粘附有不同的阻断作用,同时二者有时还存在互补抗粘附作用。     

复方嗜酸乳杆菌片联合美沙拉嗪对 溃疡性结肠炎患者肠黏膜的修复作用

目的探讨复方嗜酸乳杆菌片联合美沙拉嗪缓释颗粒对溃疡性结肠炎(UC)患者肠黏膜的修复作用。方法选取活动期UC患者74例,随机分为观察组和对照组各37例。两组患者均给予相同的饮食指导及营养支持治疗。对照组患者给予美沙拉嗪缓释颗粒1.0g/次,4次/d,口服。观察组在对照组基础上加用复方嗜酸乳杆菌片1.0g/次,3次d/。两组患者均连用12周。观察并记录两组患者治疗前和治疗12周后血清D-乳酸和PCT水平及肠镜下肠黏膜评分的变化,并比较其临床效果。结果治疗12周后,两组患者血清D-乳酸和PCT水平及及肠镜下肠黏膜评分均较治疗前有不同程度下降(P0.05),且观察组下降幅度较对照组更大(P0.05);同时观察组临床总有效率高于对照组(94.59%vs78.38%;χ~2=4.16,P0.05)。结论复方嗜酸乳杆菌片联合美沙拉嗪缓释颗粒对UC的治疗效果较肯定,可明显减轻患者肠黏膜病变程度,保护肠黏膜屏障,加快其修复。     

双歧杆菌四联活菌片中嗜酸乳杆菌的分离及生长动力学研究

目的以双歧杆菌四联活菌片为实验材料,利用酸化的MRS培养基筛选分离得到嗜酸乳杆菌,对其进行进一步的生长动力学研究,确定嗜酸乳杆菌的生长数学模型。方法通过浓度梯度稀释法利用改良MRS培养基对双歧杆菌四联活菌片中的嗜酸乳杆菌进行分离,利用分光光度仪和平板菌落计数两种不同的方法测定嗜酸乳杆菌在发酵过程中不同发酵时间的细胞浓度的动态变化,经软件处理后拟合出嗜酸乳杆菌细胞生长的Logistic数学模型。结果Logistic方程能很好地拟合嗜酸乳杆菌细胞生长的动态变化,并得到嗜酸乳杆菌在本实验条件下的数学模型,为进一步研究、利用嗜酸乳杆菌生长能力、产酸能力和产香能力等具有重要的理论指导意义。     

嗜酸乳杆菌NCFM粘附宿主后对其粘附相关基因表达的影响

摘要：【目的】益生菌粘附于肠道上皮细胞上是它的一种益生作用。本研究通过体内外实验,分析嗜酸乳杆菌NCFM对粘附相关基因的影响。【方法】利用GO (Gene Ontolog) 分类筛选Human Genome U133 Plus 2.0 Array基因表达谱芯片中的粘附相关基因,通过体外Caco-2细胞培养模型和体内小鼠粘附模型,采用Real-time PCR方法对粘附相关基因进行验证分析。【结果】经NCFM作用后,12个粘附相关基因呈上调表达。利用Real-time PCR验证,12个基因在体内和体外经嗜酸乳杆菌NCFM作用后亦均同样为上调表达,其中CCL2基因上调表达最为明显。【结论】经体内外研究表明,嗜酸乳杆菌NCFM粘附肠上皮细胞后能够引起宿主粘附相关基因出现特定表达变化,为今后深入揭示其粘附作用提供必要基础。     

嗜酸乳杆菌NCFM对Caco-2细胞中PTX3基因表达的影响

【目的】研究嗜酸乳杆菌NCFM对肠道上皮细胞中免疫与炎症介质因子PTX3表达的影响,并进一步揭示其调节机制。【方法】嗜酸乳杆菌NCFM与Caco-2细胞共培养0、2、4、8和12 h,提取细胞RNA,采用RealTime RT-PCR方法检测PTX3基因的表达。嗜酸乳杆菌NCFM与Caco-2细胞共培养0、0.5、1、2和4 h,提取细胞蛋白质,采用Western blot方法检测NF-κB的磷酸化水平;用NF-κB的特异性抑制剂PDTC预处理Caco-2细胞30 min,然后加入嗜酸乳杆菌NCFM作用2 h,提取细胞RNA,采用Real Time RT-PCR方法检测PTX3基因的表达。【结果】嗜酸乳杆菌NCFM与Caco-2细胞共培养后能诱导PTX3的表达,并且在共培养4 h的时候PTX3的表达量达到最大,然后逐渐下降;嗜酸乳杆菌NCFM能快速的诱导NF-κB的磷酸化,并且在加入其特异性抑制剂PDTC后,PTX3的表达显著下降。【结论】嗜酸乳杆菌NCFM作用于肠道上皮细胞后能够通过迅速激活NF-κB途径暂时性的调控PTX3的表达。     

Inhibition of Escherichia coli O157:H7 attachment by interactions between lactic acid bacteria and intestinal epithelial cells

The intestinal epithelial cell (IEC) layer of the intestinal tract makes direct contact with a number of microbiota communities, including bacteria known to have deleterious health effects. IECs possess innate protective strategies against pathogenic challenge, which primarily involve the formation of a physicochemical barrier. Intestinal tract mucins are principal components of the mucus layer on epithelial surfaces, and perform a protective function against microbial damage. However, little is currently known regarding the interactions between probiotics/pathogens and epithelial cell mucins. The principal objective of this study was to determine the effects of Lactobacillus on the upregulation of MUC2 mucin and the subsequent inhibition of E. coli O157:H7 attachment to epithelial cells. In the current study, the attachment of E. coli O157:H7 to HT-29 intestinal epithelial cells was inhibited significantly by L. acidophilus A4 and its cell extracts. It is also important to note that the expression of MUC2 mucin was increased as the result of the addition of L. acidophilus A4 cell extracts (10.0 mg/ml), which also induced a significant reduction in the degree to which E. coli O157:H7 attached to epithelial cells. In addition, the mRNA levels of IL-8, IL-1beta, and TNF-alpha in HT-29 cells were significantly induced by treatment with L. acidophilus A4 extracts. These results indicate that MUC2 mucin and cytokines are important regulatory factors in the immune systems of the gut, and that selected lactobacilli may be able to induce the upregulation of MUC2 mucin and specific cytokines, thereby inhibiting the attachment of E. coli O157:H7.     

Importance of S-layer proteins in probiotic activity of Lactobacillus acidophilus M92

AIMS: To investigate the functional role of surface layer proteins (S-layer) in probiotic strain Lactobacillus acidophilus M92, especially its influence on adhesiveness to mouse ileal epithelial cells. METHODS AND RESULTS: Sodium dodecyl sulphate polyacrylamide gel electrophoresis of cell surface proteins revealed the presence of potential surface layer (S-layer) proteins, ca at 45 kDa in L. acidophilus M92. Southern blot with pBK1 plasmid, containing slpA gene, gave a positive signal, suggesting that L. acidophilus M92 has a slpA gene coding for the S-layer proteins. S-layer proteins of this strain are present during all phases of growth. The S-layer proteins appeared when cells treated with 5 mol l(-1) LiCl were allowed to grow again. Removal of the S-layer proteins reduced adhesion of L. acidophilus M92 to mouse ileal epithelial cells. Furthermore, the viability of cells without S-layer were reduced in simulated gastric juice at low pH range (2, 2.5, 3) and simulated pancreatic juice with bile salts (1.5 and 3 g l(-1)). S-layer proteins of L. acidophilus M92 were resistant to pepsin and pancreatin, in contrast, the treatment with proteinase K led to a significant proteolysis of the S-layer proteins. CONCLUSIONS: These results demonstrated functional role of S-layer; it is responsible for adhesiveness of Lactobacillus acidophilus M92 to mouse ileal epithelial cells and has a protective role for this strain. SIGNIFICANCE AND IMPACT OF THE STUDY: S-layer proteins have an important role in the establishment of probiotic strain Lactobacillus acidophilus M92 in the gastrointestinal tract.     

Factors involved in adherence of lactobacilli to human Caco-2 cells.

A quantitative assay performed with bacterial cells labelled with [3H]thymidine was used to investigate factors involved in the adherence of human isolates Lactobacillus acidophilus BG2FO4 and NCFM/N2 and Lactobacillus gasseri ADH to human Caco-2 intestinal cells. For all three strains, adherence was concentration dependent, greater at acidic pH values, and significantly greater than adherence of a control dairy isolate, Lactobacillus delbrueckii subsp. bulgaricus 1489. Adherence of L. acidophilus BG2FO4 and NCFM/N2 was decreased by protease treatment of the bacterial cells, whereas adherence of L. gasseri ADH either was not affected or was enhanced by protease treatment. Putative surface layer proteins were identified on L. acidophilus BG2FO4 and NCFM/N2 cells but were not involved in adherence. Periodate oxidation of bacterial cell surface carbohydrates significantly reduced adherence of L. gasseri ADH, moderately reduced adherence of L. acidophilus BG2FO4, and had no effect on adherence of L. acidophilus NCFM/N2. These results indicate that Lactobacillus species adhere to human intestinal cells via mechanisms which involve different combinations of carbohydrate and protein factors on the bacterial cell surface. The involvement of a secreted bridging protein, which has been proposed as the primary mediator of adherence of L. acidophilus BG2FO4 in spent culture supernatant (M.-H. Coconnier, T. R. Klaenhammer, S. Kernéis, M.-F. Bernet, and A. L. Servin, Appl. Environ. Microbiol. 58:2034-2039, 1992), was not confirmed in this study. Rather, a pH effect on Caco-2 cells contributed significantly to the adherence of this strain in spent culture supernatant.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of autoinducer-2 on the adhesion ability of Lactobacillus acidophilus

Aims:  Lactobacilli adhere to the intestinal epithelium and this intimate association likely promotes retention in the gastrointestinal tract and communication with the immune system. The aim of this study was to investigate whether or not the quorum-sensing signalling molecule, autoinducer (AI)-2, was produced by Lactobacillus acidophilus and affected adherence to intestinal epithelial cells.
Methods:  Microarray analysis of concentrated cells of L. acidophilus NCFM revealed several genes involved in a classic stress response and potentially adhesion. Putative genes linked to the synthesis of the interspecies signalling molecule, AI-2, were overexpressed. Examination of the NCFM genome revealed the complete pathway for AI-2 synthesis. AI-2 activity from NCFM was detected using the Vibrio harveyi BB170 assay system. Using site-specific integration, an isogenic mutation was created in luxS and the resulting mutant did not produce AI-2. In addition to some minor metabolic effects, the luxS mutation resulted in 58% decrease in adherence to Caco-2 cells.
Conclusion:  L. acidophilus NCFM encodes the genes for synthesis of the quorum-sensing signal, AI-2, and produces this molecule during planktonic growth.
Significance and Impact of the Study:  The ability to produce AI-2 affects the ability of L. acidophilus to attach to intestinal epithelial cells.     

Competitive exclusion of diarrheagenic Escherichia coli (ETEC) from human enterocyte-like Caco-2 cells by heat-killed Lactobacillus

Diarrheagenic Escherichia coli (ETEC) bearing CFA/I or CFA/II adhesive factors specifically adhere onto the brush border of the polarized epithelial human intestinal Caco-2 cells in culture. Heat-killed Lactobacillus acidophilus strain LB, that adheres onto Caco-2 cells, inhibits diarrheagenic Escherichia coli adhesion in a concentration-dependent manner. Since the L. acidophilus does not express ETEC-CFA adhesive factors, it can be postulated that the heat-killed L. acidophilus LB cells inhibit diarrheagenic E. coli attachment by steric hindrance of the human enterocytic ETEC receptors.     

嗜酸乳杆菌SR-1产类细菌素发酵条件及生物学特性的研究

本研究通过正交试验获得嗜酸乳杆菌sR-1分泌类细菌素的最适培养条件,即葡萄搪2％,大豆蛋白胨2．5％,酵母粉0．4％,血浆蛋白2．5％。并首次利用流加培养的方式,改进了类细菌素产生菌的发酵条件,抑菌直径从19mm提高到25mm,同时对类细菌素生物学特性进行了初步的探索。结果表明：嗜酸乳杆菌产生的类细菌素对多种蛋白酶不敏感,在低pH下能抑制革兰阴性、阳性的致病菌,在pH6．5下吸附菌体作用最强。     

Buffering capacity and membrane H+ conductance of lactic acid bacteria

Abstract Buffering capacity and membrane conductance to H⁺ were measured in Enterococcus faecalis and Lactobacillus acidophilus by a pulse technique. The magnitude of these parameters varied between one species and another. Over the pH range studied, from pH 3.72 to 7.74, the acidophile Lactobacillus acidophilus showed higher values of buffering capacity and membrane H⁺ conductance than the neutrophile Enterococcus faecalis . These results support the idea that acidophiles have high cytoplasmic buffering capacity, which may allow them to resist changes in cytoplasmic pH.     

嗜酸乳杆菌及其制剂对大鼠血脂代谢的影响

本实验表明,口服嗜酸乳杆茵及其制剂,能升高大鼠ADL—C和HDL—C/T比值及降低[VLDL LDL]—C的浓度。这一作用对于防止动粥发生显然是十分有利的。若将嗜酸乳杆菌及其制剂,与其它降血脂药物配伍,用于高血脂症的辅助治疗,将会产生良好的效果。