模拟人体胃肠道环境筛选益生乳杆菌

【目的】筛选具有益生特性的乳杆菌作为保健型酸奶的候选菌株。【方法】从健康人肠道和奶豆腐中分离筛选出耐受人工胃液的乳杆菌,对其进行体外益生特性(人工胃肠液耐受性、胆盐耐受性、抑菌活性及胆固醇降解能力)研究。【结果】从在乳杆菌分离培养基上有溶钙圈的41株菌株中筛选出5株耐酸、耐人工胃液较强的菌株,经16S rR NA基因测序鉴定,其中3株为乳杆菌,分别命名为植物乳杆菌Lp MT-3、植物乳杆菌Lp MT-5和唾液乳杆菌LsA F-7。在人工胃液中3株菌的耐受力均强于商品化的对照菌株LGG(鼠李糖乳杆菌GG);转入肠液4 h后直至26 h,Lp MT-5存活率基本稳定在45%左右,仅次于LGG。胆盐浓度为0.10%时,3株乳杆菌的耐胆盐能力均强于LGG;胆盐浓度为0.20%时,Lp MT-3和LsA F-7仍能存活。3株乳杆菌均具有抑菌活性,对粪肠球菌的抑制最明显,其次是金黄色葡萄球菌,对大肠杆菌、沙门氏菌的抑制作用较差。3株乳杆菌对胆固醇的清除效力依次为Lp MT-3LpM T-5Ls AF-7;清除率依次为Ls AF-7Lp MT-3LpM T-5。【结论】筛选出3株适应人体胃肠液环境、耐胆盐、抑菌及降胆固醇活力强的乳杆菌,可作为进一步开发新的益生菌产品和保健型酸奶的菌株。     

双歧杆菌试管内生物拮抗作用研究

目的 本实验从正常青少年体内分离出1株双歧杆菌,通过试管内研究双歧杆菌对大肠埃希菌和粪肠球菌的生物拮抗作用.为双歧杆菌临床应用及食品开发提供一定的基础理论依据和广阔的市场前景.方法 本项研究以从佳木斯大学健康学生体内分离出1株双歧杆菌为试材,将大肠埃希菌、粪肠球菌接种于PYG液体培养基中,37℃厌氧培养48 h,将菌液浓度利用分光光度计法调到2麦氏.将大肠埃希菌对照组分为10支,分离的双歧杆菌+大肠埃希菌组10支,分离的双歧杆菌对照组10支,分离的双歧杆菌+粪肠球菌组10支,粪肠球菌对照组10支,将以上50个试管全部置于厌氧培养箱内37℃培养48 h,将培养好的大肠埃希菌和粪肠球菌进行梯度稀释,然后用微量移液器吸取标本于每个培养基上滴3滴,每滴滴上由高稀释度向低稀释度,液体量为10 μL的菌液.晾干滴种好的培养基于适宜条件进行培养并选出适合菌落生长的稀释度,计算同一稀释度平均菌落数(x).结果 双歧杆菌12 h时对大肠埃希菌生长无显著性,在24h时检测不到大肠埃希菌的存在.12和24h时粪肠球菌与双歧杆菌混合组与粪肠球菌对照组相比,差异均无统计学意义(P＞0.05).结论 体外对照实验研究该双歧杆菌对大肠埃希菌和粪肠球菌的生物拮抗作用,此株双歧杆菌只对大肠埃希菌有抑制作用,而对粪肠球菌无抑制作用,机制需要深入研究.     

一株替代粪肠球菌的 生产用芽胞杆菌筛选及其性质

摘要：目的 筛选1株能够产业化、替代粪肠球菌的芽胞杆菌。方法 从健康鸡、鸭、仔猪的粪便与肠道内容物中筛选,采用选择性培养基和耐酸耐胆盐发酵,通过耐人工肠液和人工胃液试验与粪肠球菌比较得到1株产酸能力较好的替代粪肠球菌的芽胞杆菌,并对其性质进行研究。结果 所筛选的芽胞杆菌（GY0520）对人工胃液、人工肠液有很好耐受性,90 ℃水浴15 min存活率为97%,能够产生大量有机酸,有利于提高动物机体的抗病能力及改善其生理性能,但对抗生素有一定的敏感性,不能配伍使用。结论 所筛选的芽胞杆菌能够替代粪肠球菌用于生产,为养殖业的微生态产品提高稳定性提供参考。     

屎肠球菌降胆固醇作用的初步研究

血液中胆固醇含量增加会严重威胁人类健康。利用皂化-比色法检测了22株屎肠菌株体外降胆固醇的能力,结果显示屎肠菌株的胆固醇去除率在85.93%~21.25%之间。选取3株胆盐耐受能力较好的菌进行后续研究,其中,屎肠球菌SP5-6L的耐酸能力较差;JS2和ML13-5菌株具有一定的耐酸和耐胆盐能力;3株菌株的胆盐水解酶活性都很高。在0.3%胆盐培养基中,屎肠球菌的胆固醇去除率不同,其中ML13-5菌株的去除率由52.71%增加到68.01%。此外,屎肠球菌无溶血性,但其抗药性各有不同。本文对屎肠球菌降低胆固醇的机制进行了初步研究,为进一步利用其开发相关功能性食品和药物提供了依据。     

一株具降胆固醇功能乳酸菌的机制研究

目的对动物双歧杆菌SQ-0001的体外环境耐受性及其降胆固醇作用进行研究。方法用邻苯二甲醛法对pH 3.0和0.3%牛胆酸钠有一定耐受性的动物双歧杆菌SQ-0001的体外降胆固醇机制进行了研究。结果动物双歧杆菌SQ-0001对pH 3.0及0.3%胆盐环境具有一定的耐受性;该菌在胆盐浓度为0.2%,培养时间为24 h时,胆固醇脱除率最高,达到91.59%;该菌降胆固醇的作用主要以吸收为主,吸收率达到63.87%,其次是降解率,为23.81%。结论动物双歧杆菌SQ-0001的环境耐受能力良好,具有很好体外降胆固醇作用,该菌株可以作为日后降胆固醇的潜在益生菌。     

降胆固醇乳酸菌的应用及其机理研究进展

乳酸菌除了能够改善肠道微生态环境促进人体健康外,有些乳酸菌还具有能降低人体血清胆固醇的特性。近年来许多研究都已证明某些乳杆菌、双歧杆菌及肠球菌等具有调节血脂代谢、降低人体血清胆固醇的能力。本文综述了降胆固醇乳酸菌的体外筛选模型及其降胆固醇的作用机理,以期为进一步开发相关产品提供技术依据。     

重组干酪乳杆菌在模拟消化环境中生存性能的研究

目的 探讨重组干酪乳杆菌Lactobacillus casei 393在模拟胃肠道环境中的存活能力.方法 人工模拟胃肠道环境,即人工胃液（pH=1.5～4.5）、人工肠液、胆汁（质量浓度0.3～3.0 g/L）和高盐（质量浓度40～90g/L）.结果 重组干酪乳杆菌在pH为2.5～4.5的人工胃液中具有较强的生存能力,3 h活菌数仍达108/ml;在人工肠液中经过不同时间的作用后,重组干酪乳杆菌显出生长趋势;在0.3%的胆汁环境作用8 h仍有存活,且能耐受7%NaCl浓度的高渗环境.结论 实验为干酪乳杆菌Lactobacillus casei 393能否作为益生菌制剂在胃肠道中发挥作用提供了理论基础.     

基于耐酸性与抑菌性效果的益生菌候选菌株体外筛选

目的基于筛选耐酸性强和抑菌效果好的菌株为靶点,为益生菌研发提供优良的候选菌株。考察菌株耐酸性与抑菌性的相关性,为益生菌快速筛选探索新方法。方法选取62株不同乳杆菌、魏斯菌、粪肠球菌,在酸性程度不同的培养基中采用接种培养的方式研究受试菌株耐酸能力。用大肠埃希菌和金黄色葡萄球菌作为指示菌,采用牛津杯法研究受试菌株的抑菌能力。分析同种细菌的耐酸能力和抑菌能力的相关关系。结果在酸性环境下,受试菌株存活率超过50%的有22株,耐酸性较强的菌株有DMLA16017、DMLA16116、DMLA16117、DMLA16118和DMLA16131。受试菌株对病原菌的抑菌力较强的有DMLA16017、DMLA16009、DMLA16118、DMLA16002和DMLA15015。结论鼠李糖乳杆菌DMLA16017和发酵乳杆菌DMLA16118在耐酸性和抑菌性上表现出优良性状。可以通过受试菌株对酸性耐受能力指示受试菌株对病原菌抑制能力,实现对受试菌株的快速筛选。     

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.     

三种益生菌混合培养及共生机理研究

确定酪酸菌、肠膜芽孢杆菌与粪肠球菌三种菌混合工艺.研究了三种菌混合培养的生长曲线,混合批式传49代、混合连续培养考察三种菌的共存稳定性.三种菌无细胞上清液彼此之间均有促进生长作用,混合培养液的菌数较单独培养增加,在稀释率0.042/h,填充床连续培养11d,三种菌可稳定共存,但批式传49代过程中,肠膜芽孢杆菌有消失现象,而酪酸菌、粪肠球菌均较传代前增加了100倍.平板打孔生长圈法、点种法实验分别表明酪酸菌、肠膜芽孢杆菌对粪肠球菌有显著的促进作用,连续培养较批式传代可更好的研究菌际关系.并得到简单易行的复方益生菌剂组方方法.     

In vitro study on bacteriocin production of Enterococci associated with chickens

In recent years, the approach of using innovative strategies such as probiotics or bacteriocins for the prevention or treatment of bacterial infections has come into focus. The present study was undertaken to check in vitro ability of Enterococci-isolated from the gastrointestinal tract of chickens-to produce a bacteriocin-like substance and to describe some further probiotic properties in five selected Enterococcus faecium strains. All strains (n=17) were found to produce bacteriocin-like substances against 14 out of 20 indicator bacteria of animal, food or environmental origin. Selected E. faecium strains expressed sufficient survival by pH 3.0 after 3h, in the presence of 1% bile after 24h and they were sensitive to most of antimicrobials tested. All tested strains adhere to the human, canine and porcine intestinal mucus (between 1.5% and 9.2%). However, better adhesion ability was observed for the canine mucus. PCR detection of enterocin structural genes determined presence of enterocins A and P genes in all selected strains. Characterization of bacteriocin substance in detail was performed in E. faecium EF55. The EF55 strain produced a bacteriocin-like substance (during the late logarithmic and early stationary growth phase) with inhibitory activity mostly against Gram-positive bacteria (100-51,200 AU/mL) including Listeria monocytogenes. Proteinaceous character of the bacteriocin substance was confirmed (its inhibitory activity was lost after its treatment with proteases), it was found to be stable after heating (100 degrees C 10 min) and during 12 months storage at -20 degrees C. The highest inhibitory activity of bacteriocin produced by EF55 strain (growing in MRS) broth was achieved between pH 7.0 and 9.0.     

Virulence factors and<Emphasis Type="Italic">in Vitro</Emphasis> adherence of<Emphasis Type="Italic">Enterococcus</Emphasis> strains to urinary catheters

The ability to adhere in vitro to urinary catheters and the presence of enterococcal virulence factors was determined in 30 Enterococcus urinary isolates (12 E. faecalis, 12 E. faecium, 3 E. casseliflavus, 3 E. gallinarum). Silicone, siliconized latex and polyvinyl chloride (PVC) were examined by sonication quantitative culture technique and scanning electron microscope. As compared to E. faecalis and E. faecium, E. casseliflavus and E. gallinarum displayed lower adhesion to all synthetic materials. All the tests performed showed higher adherence of all tested strains to siliconized latex and silicone than to PVC. Biofilmforming ability was observed in 5 E. faecalis but in none of the remaining strains. The gene coding enterococcal surface protein (Esp) was detected in 7 E. faecalis and 6 E. faecium strains. Gelatinase was found in 1 E. faecalis, 2 E. faecium and hemolysins were found in 6 E. faecalis and 1 E. faecium strains. All E. casseliflavus and E. gallinarum strainswere negative for these traits. Hydrophobic type of cell surface (measured by its affinity for n-hexadecane) was shown in a few isolates. Bacterial adherence was not significantly associated with the above pathogenic factors.     

Interaction of Arcobacter spp. with human and porcine intestinal epithelial cells

Little is known about the pathogenic mechanisms or potential virulence factors of Arcobacter spp. The aim of the study described here was to obtain more insights in the pathogenicity mechanisms of Arcobacter spp. by testing their ability to adhere to, invade and induce interleukin-8 expression in human Caco-2 and porcine IPI-2I cell lines. Eight Arcobacter strains were tested. Four strains were obtained from a culture collection, and represent the four Arcobacter spp. known to be associated with animals and humans. The other four strains were field isolates from the amniotic fluid of sows and from newborn piglets. All eight Arcobacter strains were able to adhere to both cell lines, and induced interleukin-8 production as early as 2 h after a 1h incubation period. This production was still increased 6 h postinfection. Differences in the cell association of the eight strains were obvious, with A. cibarius showing the highest adhesion ability. Invasion of intestinal epithelial cells was only observed for A. cryaerophilus strains. No correlation between invasiveness or strong adhesion of the tested strains and the level of interleukin-8 induction was observed.     

Antibiotic-resistant strains of Enterococcus isolated from Swedish and Danish retailed chicken and pork

Two hundred and seventy-seven Enterococcus isolates representing the predominating enterococcal flora of retailed chicken and pork were identified by phenotypical features, and by random amplified polymorphic DNA. The resistance to nine different antibiotics was determined. Enterococcus faecium was the most frequently occurring species in both Swedish and Danish chicken. Enterococcus faecalis and unidentified groups of Enterococcus dominated in pork. Seventy-three per cent of the Enterococcus isolates from Swedish chicken were resistant to one or more of the tested antibiotics. The corresponding values for Swedish pork, Danish chicken and Danish pork were 9%, 55% and 14%, respectively. Tetracycline resistance was most frequent in isolates from Danish pork and Swedish chicken, while erythromycin resistance was most frequent in Danish chicken. Strains with acquired vancomycin resistance, mainly Ent. faecium , were found only on Danish chicken, except for one isolate from Danish pork. All vancomycin-resistant isolates contained the van A gene. Vancomycin resistance could be transferred to a vancomycin-sensitive Ent. faecium strain from four of nine tested donor strains.     

Probiotic potential of enterococci isolated from canine feed

Enterococci isolated from 28 different commercially available feeds (10-1000 CFU/mL) were identified and their probiotic potential was determined. Species identification of 22 selected strains was performed by intergenic length-polymorphism analysis (tRNA-PCR); PCR products were analyzed using capillary electrophoresis. Six strains were allotted to the species Enterococcus faecium, four to E. faecalis, one to E. hirae; the remaining strains were not classed. The strains were sensitive to vancomycin, ampicillin, tetracycline and rifampicin. They were able to adhere to human as well as canine intestinal mucus. They produced lactic acid (0.99-1.04 mmol/L) and most of them were urease-positive with sufficient survival in 5 % Oxgall-bile. They did not show any inhibitory activity due to antimicrobial substances. Plasmid DNA was detected in 8 strains, the bands responding to small molecular size (10 kbp). Considering all probiotically important properties, E. faecium strain EE3 was suggested as potential feed additive.     

Isolation and molecular characterization of vancomycin-resistant Enterococcus faecium in Malaysia

Nineteen strains of vancomycin-resistant Enterococcus faecium isolated from 10 of 75 (13.3%) tenderloin beef samples were examined for resistance to selected antibiotics, presence of plasmids, and genetic diversity by random amplification of polymorphic DNA analysis. All strains showed multiple resistant to the antibiotics tested. Multiple antibiotic indexing of the vancomycin-resistant E. faecium strains showed that all (100%) originated from high risk contamination environments where antibiotics were often used. Plasmids ranging in size from 1.5 to 36 megadalton were detected in 15 of 19 (79%) strains. Thus, three plasmid profiles and eight antibiotypes were observed among the E. faecium strains. A high degree of polymorphism was obtained by combining the results of the two primers used; with the 19 E. faecium strains being differentiated into 19 RAPD-types. These preliminary results suggest that RAPD-PCR has application for epidemiologic studies and that resistance patterns and plasmid profiling could be used as an adjunct to RAPD for the typing of E. faecium in the study area.     

肝硬化患者肠球菌感染及体外药敏监测

目的研究肝硬化患者肠球菌感染的现状及药敏特点,以加强对肝硬化肠球菌感染的认识,指导用药。方法留取标本进行细菌培养、鉴定及药敏试验。结果所有纳入研究范围的肝硬化患者共检出肠球菌112株,其中粪肠球菌89株,占79.5%,比例最高,屎肠球菌占14.3%,居第2位。腹水中检出肠球菌64株,占57.1%,其次为痰和尿液,分别为38.1%和14.4%。肠球菌对红霉素、奈替米星的耐药率超过80%,对青霉素G、氨苄西林、呋喃妥因的敏感率超过70%,对左氧氟沙星的敏感率为58.9%,对头孢唑啉、头孢派酮的敏感率分别只有25.0%和33.0%,检出高耐氨基糖苷类肠球菌(HLAR)58株,占51.8%,未检出耐万古霉素和替考拉宁肠球菌,但检出万古霉素中介肠球菌4株,占3.6%。结论肠球菌是肝硬化患者医院感染的重要致病菌,尤以粪肠球菌和屎肠球菌为主,屎肠球菌的耐药率高于粪肠球菌,万古霉素和替考拉宁是治疗肠球菌感染的首选药物。     

The correlation between surface hydrophobicity and adherence of Bifidobacterium strains from centenarians' faeces

To develop food-grade bifidobacteria micro-ecologics, screening for Bifidobacteria strains which can adhere to intestinal epithelial cells was finished. Twenty-three bifidobacterial strains tested were isolated from centenarians in Bama country, the fifth long-lived district in the world. Surface hydrophobicity and adherence capability to intestinal epithelial cells in vitro of bifidobacteria were simultaneously investigated for the first time. It has been demonstrated that all the strains exhibited adhesive properties to some extent using intestinal Caco-2 cell line in in vitro model. It could be conclude that the higher hydrophobic strains the stronger adhesive capability. The highest value of hydrophobicity (37.24+/-1.45% and 32.06+/-1.21%) was obtained for strains H-10 and I-6, respectively; correspondingly, the strongest adherence ability (49.47+/-4.88/cell and 47.33+/-2.72/cell) was achieved, respectively. Correlation between surface hydrophobicity and adherence ability of different Bifidobacterium strains including polynomial regression equation (R2=0.78) had been achieved. The present study provided a liable and effective method for screening bifidobacteria with the ability to adhere to intestinal epithelial cells.     

Drug resistance of 100 clinical strains of Enterococcus spp

The aim of this study was to evaluate the drug susceptibility of 100 Enterococcus spp. strains isolated from patients hospitalized in State Clinical Hospital No 1 in Warsaw. All strains were identified (API 20 STREP) and their susceptibility to antibiotics was tested (ATB STREP) in automatic ATB system. Additionally, PYRase activity, beta-lactamase production (in nitrocefin test), MICs for vancomycin and teicoplanin (E test), HLAR--high level aminoglycoside resistance and susceptibility to vancomycin, teicoplanin, piperacillin and piperacillin/tazobactam (disc diffusion method) were determined. E. faecalis ATCC 29212 was used as the control strain. Fifty E. faecalis, 45 E. faecium, 2 E. casseliflavus, 2 E. durans and 1 E. avium strain were cultured. All strains were PYRase-positive and beta-lactamase-negative. Ten isolates demonstrated intermediate susceptibility to vancomycin (6--E. faecalis and 4--E. faecium). One E. faecalis strain was intermediately susceptible to both glycopeptides. One E. casseliflavus strain showed low-level resistance to vancomycin, but this strain was susceptible to teicoplanin--phenotype Van C. HLAR strains were found among 31 E. faecalis and 40 E. faecium strains. 48 E. faecalis strains were susceptible to piperacillin and 49 to piperacillin/tazobactam. Whereas, 41 E. faecium were resistant to both these drugs. Thirty six per cent of isolates were resistant to penicillin and ampicillin, 73% to erythromycin, 87% to tetracycline, 89% to lincomycin and 56% to nitrofurantoin. Some discrepancies were noticed between the results of different methods applied for susceptibility testing--ATB system, E test and disc diffusion. These discrepancies concerned HLAR detection and susceptibility to glycopeptides determination. The best methods were: disc-diffusion for HLAR detection and E test for determination of resistance to vancomycin and teicoplanin. Increasing resistance to antimicrobial agents is observed in clinical Enterococcus spp. isolates cultured in our laboratory, especially in E. faecium strains. It is necessary to control the dissemination of multiresistant Enterococcus spp. strains in hospital wards.     

Genetic transformation of various species of Enterococcus by electroporation

A transformation system for Enterococcus faecalis was developed which uses untreated (i.e., non-protoplasted) cells and the electroporation technique. The optimized protocol resulted in transformation efficiencies of up to 4 x 10(6) transformants per microgram of plasmid DNA. All strains of E. faecalis tested could be transformed by this method, albeit with differing transformation efficiencies. Using the protocol optimized for E. faecalis we successfully transformed Enterococcus faecium, E. hirae, E. malodoratus and E. mundtii.