2种乳酸菌对牙鲆体表、消化道粘液粘附及对4种致病菌的粘附抑制性

目的 研究2株乳酸菌（L1来自牙鲆肠道,L2为干酪乳杆菌）在牙鲆体表和消化道粘液的粘附及其对4种致病菌的粘附抑制情况。方法 异硫氰酸荧光素（FITC）标记法。结果 2种乳酸菌在牙鲆体表和消化道粘液中均有粘附性。在胃的粘附效果最好,粘附百分率分别是48．18％和63．0％,L2在牙鲆体表、盲囊和肠的粘附能力强于L1。L1对白色念珠菌（Candida albicans）在各部位均无粘附抑制作用,对荧光假单胞菌（P．Fluorescens）粘附抑制作用最强．使其在牙鲆消化道粘液的粘附百分率下降了19．01％,其余依次为非01型霍乱弧菌（V．cholerae non-Оl strains）、嗜水气单胞菌（Aeromonas hydrophila）,分别降低了5．22％、3．22％;L2对非Оl型霍乱弧菌粘附抑制效果最强,使其粘附百分率降低了22．31％。使荧光假单胞菌、嗜水气单胞菌和白色念珠菌分别下降了17．50％、12．69％、5．15％。结论 对4种常见致病菌的粘附抑制在消化道粘液中好于体表。     

乳杆菌表面粘附蛋白的提取、鉴定及粘附特异性的初步研究

以从健康牙鲆肠道中分离筛选的乳杆菌L15(Lactobacillussp.L15)和嗜酸乳杆菌ATCC4356为实验材料,应用5mol/L LiCl提取其表面蛋白,利用蛋白印迹法鉴定出在L15表面蛋白中分子量为61.8kDa和54.6kDa的蛋白质分别参与对牙鲆和鲤鱼粘液的粘附过程,为新发现的粘附蛋白种类,将其命名为MAPPpo1和MAPPcc。ATCC4356中分子量分别为43.0kDa和63.3kDa的两个表面蛋白参与对牙鲆粘液的粘附,而分子量为43.0kDa的蛋白参与对鲤鱼粘液的粘附。同时,蛋白质印迹法显示,L15和ATCC4356在牙鲆和鲤鱼肠粘液中均具有相同的粘附受体,在牙鲆肠粘液中是分子量为29.7kDa和30.3kDa的两种蛋白质,而在鲤鱼肠粘液中只有分子量为26.2kDa的蛋白作为受体参与L15和ATCC4356的粘附过程。结果显示,乳杆菌对肠粘液的粘附不但具有菌种的特异性,而且也有宿主的特异性。     

牙鲆肠道乳酸菌的分离和鉴定

目的：根据微生态学原理,从健康牙鲆的肠道固有菌群中分离乳酸菌。方法：需氧与厌氧培养法。结果：两种方法获得了不同的结果,用LBS（pH6.5）直接分离,从19株分离菌中只获得6株乳酸菌,其中1株P15为乳杆菌;而用LBS（pH5.4）和SL(pH5.4）先富集后分离得到了许多单一的乳杆菌菌落。对所分离的乳杆菌进行生化鉴定,均符合该菌的生化特征。牛津杯抑菌试验显示,乳酸菌对弧菌均有抑制作用,其中乳杆菌P15在pH6.8和pH7.5生长良好,并对弧菌有强力的抑制作用;而在pH8.0和pH8.5时该菌生长不良且无抑菌活性。     

中国微生态学杂志2007年第19卷总目次

论著两株乳杆菌对牙鲆体表及消化道粘液的粘附特性……………………………………………………………刘倩,邵占涛,陈营(1)新生儿肠道双歧杆菌数量影响因素与其婴儿期腹泻关系………………………………………………………刘泉波,刘作义(4)四君子汤及其纳米制剂对微生态失调小鼠的调整作用………………………………………………鞠宝玲,陆叶,唐小云,等(6)艰难梭菌细胞毒素B功能区的克隆及序列分析…………………………………………………………刘红升,张清华,蒋知新(9)PBP2a多克隆抗体制备及其胶乳凝集检测法的建立……………………………………     

环丙沙星预先处理牙鲆消化道对干酪乳杆菌定植的影响

利用1株干酪乳杆菌,通过实验研究用环丙沙星预先处理牙鲆消化道后乳杆菌的定植和演替规律。在投喂含有1.2×10^9CFU/g乳杆菌的饲料5 d后,消化道定植的乳杆菌超过106CFU/g,其后维持在10^6～10^8CFU/g动态平衡中。同时随着乳杆菌的投喂,不经环丙沙星预先处理牙鲆消化道的正常组,鱼消化道的弧菌数从10^7～8CFU/g降低到10^6CFU/g左右;而经环丙沙星预先处理牙鲆消化道的药饵组,鱼胃、小肠和盲囊的弧菌数则是先增加,然后显著下降。停喂乳杆菌7 d后,2个实验组鱼消化道的乳杆菌均从10^5～6CFU/g下降到10^4CFU/g,干酪乳杆菌正常组鱼盲囊中弧菌从10^5CFU/g回升到10^6CFU/g,胃和小肠中弧菌数量基本不变。干酪乳杆菌药饵组则有所不同：除胃中弧菌数量则基本不变外,盲囊和小肠中弧菌数量继续下降,其原因有待进一步研究。实验结果表明,干酪乳杆菌能在牙鲆消化道内定植,而用预先处理牙鲆消化道后,更有利于乳杆菌的定植;乳杆菌的投喂和定植,使牙鲆消化道中的弧菌数量明显下降。     

棒状腐败乳杆菌Lc7的生物学特性及益生作用

【目的】对分离自健康成人粪便样本的棒状腐败乳杆菌(Loigolactobacillus coryniformis)Lc7进行分类学鉴定和益生潜力评估。【方法】基于16SrRNA基因和基因组核心基因构建系统发育树,对Lc7进行分类学鉴定;通过耐酸和胆汁酸盐、粘附、抗氧化和抑菌实验,以及溶血、明胶酶活性和抗菌药物敏感性实验,评估Lc7的益生特性。同时,构建小鼠溃疡性结肠炎模型,评估Lc7的体内抗炎潜力。【结果】Lc7鉴定为L. coryniformis,在酸和胆汁酸盐的连续作用下,Lc7的存活率为70.17%。Lc7对HT-29细胞的粘附指数为56.33 CFU/cell,其自聚集和疏水性分别为80%和40%;Lc7对福氏志贺菌和鼠伤寒沙门菌等7个常见致病菌均有较强的抑制能力;对1,1-二苯基-2-苦基肼(1,1-diphenyl-2-picryl-hydrazyl,DPPH)和羟自由基(hydroxyl radicals,·OH)的清除率分别为91.70%和48.53%;Lc7无溶血现象和明胶酶活性,对选取的大多数抗生素均敏感。在小鼠结肠炎实验中,Lc7干预组小鼠结肠长度明显长于模型组(...     

三株益生菌粘附性质及机制的初步研究

目的研究3株益生菌株的粘附性质,初步探讨细菌与肠细胞的粘附机制。方法选取已被深入研究的LGG作为阳性对照,应用显微观察和平板计数的方法测定3株乳杆菌和肠上皮细胞Caco-2的粘附,选择疏水性、表面电荷和自聚合能力3项指标来描述细菌的表面性质,应用粘附抑制试验和酵母凝集试验来测定糖类专一性粘附。结果无论是用显微观察还是平板计数,ST-Ⅲ均是所测试的4株菌中粘附能力最强的,当加入细菌和细胞比约为60∶1时,ST-Ⅲ在Caco-2上的粘附数为(7.43±0.65)CFU/细胞,强于对照菌LGG[(3.99±0.57)CFU/细胞]。在所测试的4株菌中,粘附能力和疏水性具有很好的相关性,同时自聚合能力对粘附也表现出一定的促进作用。除LGG外,D-甘露糖和甲基-α-D-甘露糖苷均能抑制另外3株乳杆菌的粘附,同时这3株乳杆菌也能不同程度地凝集酵母。结论3株益生菌的粘附均涉及到非特异性的疏水结合和甘露糖特异性粘附两个过程;ST-Ⅲ是1株具有良好粘附能力的益生菌,有待进一步的研究。     

高密度共生发酵和强黏附性优势菌株的筛选CSCD

目的 比较10株乳酸菌的活菌数,筛选高密度共生发酵和高黏附性的共培养菌株,为后期开发应用奠定基础。方法 对单个菌株及共培养菌株进行发酵活菌数的筛选,筛选出对Caco-2细胞黏附性强的优势菌株,并比较其耐酸、耐胆盐和抑制肠道致病菌能力。结果 10株益生菌菌株中,鼠李糖乳酪杆菌AI-11与发酵粘液乳杆菌AI-25、两歧双歧杆菌AI-91与动物双歧杆菌乳亚种AI-01的组合菌株较单个菌株具有协同效应,活菌数显著高于单个菌株(均t=8.878 0,P<0.001),且具有较好的抑制肠道致病菌大肠埃希菌和奇异变形杆菌的效果,较好的耐受pH 2.5的酸性环境和胆汁盐环境的特性,较鼠李糖乳酪杆菌GG和发酵粘液乳杆菌CECT 5716具有较好的Caco-2细胞黏附性(t=9.004 1, P<0.001;t=11.301 2,P<0.001)。结论 筛选出的2个共培养组合菌株在发酵能力和细胞黏附性方面具有活菌数高、黏附性强的优势,可应用于食品、功能食品及膳食补充剂中,以增强改善肠道菌群的功效性。     

乳杆菌阴道分离株16S rDNA鉴定及优势菌株产H_2O_2和自凝集能力分析

目的从阴道分泌物标本分离鉴定乳杆菌,分析与健康相关优势菌种产过氧化氢(H2O2)和自凝集能力,揭示菌株特异性的潜在益生特性。方法利用MRS固体培养基从41例阴道分泌物标本中分离单菌落,纯培养后提取细菌基因组DNA,PCR扩增16S rDNA序列,依据序列一致性确定细菌种属。利用TMB-HRP-MRS培养基测定优势乳杆菌菌株产H2O2能力,并测定其自凝集能力。结果获得155个细菌分离株,分属乳杆菌属104株(67.1%)、肠球菌属21株(13.5%)、链球菌属28株(18.1%)、葡萄球菌属1株(0.6%)和双歧杆菌属1株(0.6%)。各菌属在无或有妇科临床症状患者标本中的构成比例差异有统计学意义(χ2=7.4918,P=0.0236),乳杆菌在前者检出率为83.3%,显著高于其在后者47.1%的检出率(χ2=4.4879,P=0.0341)。乳杆菌中卷曲乳杆菌分离株所占比例最高(67.3%,70/104)。来自不同标本的22株卷曲乳杆菌中77.3%菌株产H2O2,且这些菌株具有强弱不同的自凝集能力(27.5%~97.3%)。结论乳杆菌属特别是卷曲乳杆菌种是健康阴道环境中优势菌属,其产H2O2和自凝集能力存在明显菌株特异性,有2株卷曲乳杆菌此两特性均很强可供进一步研发。     

1株植物乳杆菌生物学特性的研究

研究1株植物乳杆菌(N3)的生物学特性,实验结果表明该菌能耐受80~85℃的高温和0.20kg/cm2蒸汽压力;直接分解玉米淀粉的乳酸产率为7.05%(36 h)和8.19%(48 h);能耐受pH为4.5的酸性环境;在人工胃液中的活菌数为4.1×106CFU/g;对金霉素、土霉素、痢特灵和氟哌酸等抗生素的敏感性强,而对防霉剂和脱霉素不敏感。植物乳杆菌(N3)是1株优良的益生素生产菌。     

Adhesion of probiotic lactobacilli to chick intestinal mucus

In the present work, interactions between three Lactobacillus strains (Lactobacillus fermentum CRL1015, Lactobacillus animalis CRL1014, and Lactobacillus fermentum CRL1016) and chicken small intestinal mucus were determined. Three lactobacilli isolated from chicken and selected by their potentially probiotic properties were able to grow in mucus preparations. Three peaks from gel filtration chromatography of intestinal mucus were obtained. The adhesion to three mucus fractions (I, II, and III), especially fraction III, was higher (P < 0.01) in L. fermentum CRL1015 than L. animalis CRL1014. Pretreatment of this fraction with proteases and metaperiodate showed lower (P < 0.01) adhesion values than that of the control, suggesting that a glycoprotein from the mucus acts as a receptor for L. fermentum CRL1015. Highest adhesion values were obtained at pH 7 and 42 degrees C, and neither the removal of divalent cations with ethylenediaminetetraacetic acid (EDTA) nor the addition of calcium produced significant variation from the adhesion values of the control (P > 0.01). This adhesion was only inhibited by N-acetyl-glucosamine. Salmonella pullorum and Salmonella gallinarum showed high (P < 0.01) values of adhesion to chick intestinal mucus. The results obtained from assays of the inhibition of adherence of Salmonella spp. to mucus, immobilized in polystyrene tissue culture wells, indicated that the pathogen adhesion was not reduced by lactobacilli (P > 0.05) or their spent culture supernatants (P > 0.05), suggesting that these strains did not interfere with the binding sites for Salmonella spp. adhesion to the small intestinal mucus.     

Characterization of the properties of human- and dairy-derived probiotics for prevention of infectious diseases in fish

The present study aimed to investigate the potential probiotic properties of six lactic acid bacteria (LAB) intended for human use, Lactobacillus rhamnosus ATCC 53103, Lactobacillus casei Shirota, Lactobacillus bulgaricus, L. rhamnosus LC 705, Bifidobacterium lactis Bb12, and Lactobacillus johnsonii La1, and one for animal use, Enterococcus faecium Tehobak, for use as a fish probiotic. The strains for human use were specifically chosen since they are known to be safe for human use, which is of major importance because the fish are meant for human consumption. The selection was carried out by five different methods: mucosal adhesion, mucosal penetration, inhibition of pathogen growth and adhesion, and resistance to fish bile. The adhesion abilities of the seven LAB and three fish pathogens, Vibrio anguillarum, Aeromonas salmonicida, and Flavobacterium psychrophilum, were determined to mucus from five different sites on the surface or in the gut of rainbow trout. Five of the tested LAB strains showed considerable adhesion to different fish mucus types (14 to 26% of the added bacteria). Despite their adhesive character, the LAB strains were not able to inhibit the mucus binding of A. salmonicida. Coculture experiments showed significant inhibition of growth of A. salmonicida, which was mediated by competition for nutrients rather than secretion of inhibitory substances by the probiotic bacteria as measured in spent culture liquid. All LAB except L. casei Shirota showed tolerance against fish bile. L. rhamnosus ATCC 53103 and L. bulgaricus were found to penetrate fish mucus better than other probiotic bacteria. Based on bile resistance, mucus adhesion, mucus penetration, and suppression of fish pathogen growth, L. rhamnosus ATCC 53103 and L. bulgaricus can be considered for future in vivo challenge studies in fish as a novel and safe treatment in aquaculture.     

Quantitative evaluation of adhesion of lactobacilli isolated from human intestinal tissues to human colonic mucin using surface plasmon resonance (BIACORE assay)

AIMS: To isolate lactobacilli from the mucus layer of the human intestine and evaluate their adhesion abilities using a BIACORE assay. METHODS AND RESULTS: Thirty strains of lactobacilli were isolated from the mucus layer of normal human intestinal tissues using conventional plate culture. The strains were identified using homology comparisons of the 16S rDNA sequence to databases as Lactobacillus salivarius (26%), Lactobacillus fermentum (13%), Lactobacillus gasseri (10%), Lactobacillus paracasei (7%), Lactobacillus casei (3%), Lactobacillus mucosae (3%) and Lactobacillus plantarum (3%). Lactobacillus plantarum LA 318 shows the highest adhesion to human colonic mucin (HCM) using the BIACORE assay at 115.30 +/- 12.37 resonance unit (RU). The adhesion of cell wall surface proteins from strain LA 318 was significantly higher to HCM than to bovine serum albumin (BSA; P < 0.05). CONCLUSIONS: We isolated 30 strains of lactobacilli. Lactobacillus salivarius was the predominant species of lactobacilli isolated in this study. The adhesion of strain LA 318 isolated from human transverse colon to its mucin was shown. The adhesion could be mediated by lectin-like components on the bacterial cell surface. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study where lactobacilli were isolated from human intestinal tissues and shown to adhere to HCM.     

Kinetics of adhesion of selected fish-pathogenic Vibrio strains of skin mucus of gilt-head sea bream (Sparus aurata L.).

The kinetics of adhesion of Vibrio strains isolated from diseased fish to skin mucus of gilt-head sea bream was studied. A modified Langmuir adsorption isotherm was calculated, and the results obtained indicate that the strains tested (Vibrio alginolyticus DP1HE4 and Vibrio anguillarum-like DC12R8 and DC12R9) showed a saturation kinetics except for V. alginolyticus (CAN), which showed a proportional adsorption kinetics. The adhesive capability for skin mucus does not seem to be an essential virulence factor of pathogenic strains of Vibrio, since this specific interaction depended on several environmental factors, temperature and salinity being the most important. However, the absence of an inhibitory effect of mucus on the pathogenic microorganisms, and the capability of the Vibrio strains to utilize mucus as a carbon source, could favor their settlement on the skin with a potential for infection of cultured, stressed fish.     

Good adhesion properties of probiotics: a potential risk for bacteremia?

The ability to adhere to human intestinal mucus was tested for lactic acid bacteria of clinical blood culture, human fecal and dairy origin. The blood culture isolates were found to adhere better than the dairy strains. Of the Lactobacillus rhamnosus strains (nine clinical, 10 fecal and three dairy), blood culture isolates adhered better than the fecal strains. Although these results indicate a trend for blood culture isolates to bind to intestinal mucus in higher numbers than strains of dairy and human fecal origin, other factors are also likely to be involved in the etiology of lactobacillemia since some of the clinical Lactobacillus isolates exhibited a relatively low level of adhesion.     

Adhesion of Vancomycin-Resistant Enterococcus to Human Intestinal Mucus

The intestinal mucus layer provides a potential niche for colonization by vancomycin-resistant Enterococcus faecium (VREF). We therefore examined the ability of six VREF strains to adhere to human intestinal mucus and determined binding kinetics. Four of six (67%) VREF strains demonstrated significant adhesion to immobilized intestinal mucus compared with a Salmonella typhimurium–negative control strain, but the level of adherence was low compared with Lactobacillus rhamnosus GG. Binding kinetics studies demonstrated that the maximum number of these four VREF strains that could adhere to a unit surface area of immobilized mucus was similar to or higher than the maximum number of L. rhamnosus GG that could adhere; however, L. rhamnosus GG demonstrated 20- to 130-times higher affinity than the VREF strains. These results demonstrate that VREF strains may adhere to human intestinal mucus and suggest that L. rhamnosus GG might be able to displace VREF strains.     

Assessment of the probiotic potential of lactic acid bacteria isolated from kefir grains: evaluation of adhesion and antiproliferative properties in in vitro experimental systems

The main objective of this study was to isolate lactic acid bacteria from kefir grains and investigate their probiotic potential. In this study, 48 bacterial strains were isolated from kefir grains, whereas 39 strains were categorized to the genus Lactobacillus. Evaluation of the probiotic potential of the isolated stains was performed, including resistance to low pH, tolerance to pepsin, pancreatin and bile salts, and antibiotic resistance. In addition, evaluation of adhesion and antiproliferative properties in in vitro experimental systems was also conducted. Strains SP2 and SP5 that displayed the best performance in the conducted in vitro tests were selected for further studies. Firstly, genotypic identification of the two strains was performed by partial 16S rRNA gene sequencing, BLAST analysis, and species-specific multiplex PCR assay. The two strains were confirmed to be Pediococcus pentosaceus SP2 and Lactobacillus paracasei SP5. Then, the adhesion properties of the two strains were examined in vitro. Both strains displayed substantial adherence capacity to HT-29 human colon cancer cells. Moreover, a significant decrease of HT-29 cell growth after treatment with viable P. pentosaceus SP2 or L. paracasei SP5 was recorded. In addition, downregulation of anti-apoptotic genes and over-expression of cell cycle–related genes was recorded by real-time PCR analysis. Treatment with conditioned media of the two strains also caused significant reduction of cancer cell proliferation in a time- and concentration-dependent manner. P. pentosaceus SP2 and L. paracasei SP5 displayed the best probiotic properties that exerted substantial adherence on human colon cancer cells as well as significant anti-proliferative properties.     

两株乳酸杆菌益生菌特性的研究

对两株乳酸杆菌作为益生菌的特性进行分析。采用人工胃液、人工肠液,及体外培养人结肠癌细胞株HT-29,模拟 上消化道环境,检测候选菌株L.acidophilus1.1878和L.rhamnosus1.120对上消化道环境的耐受性和粘附性;以琼脂扩散打孔 法,检测候选菌株SCS对4种常见肠道致病菌的抑菌能力。结果表明,两株候选菌株可耐受pH>3.0的人工胃液,及0.2%的 胆盐浓度;光镜观察L.acidophilus1.1878、L.rhamnosus1.120粘附于HT-29细胞边缘,平均4.5～7个/细胞,扫描电镜观察 L.acidophilus1.1878、L.rhamnosus1.120粘附于HT-29细胞表面的刷状缘,且细胞表面结构完整;对4种肠道致病菌的抑菌 圈直径均在12mm以上。证明实验观察的两株乳酸杆菌具有较好的耐酸、耐胆盐及粘附、抑菌特性,符合益生菌的标准。