母乳源乳双歧杆菌Probio-M8的遗传特征及基因组差异比较分析

【目的】母乳源乳双歧杆菌（Bifidobacterium animalis subsp. lactis） Probio-M8具有优良的益生特性,本文拟从全基因组水平解析Probio-M8的遗传特征,并与已有益生功效的乳双歧杆菌的基因组进行比较分析。【方法】本研究基于NCBI已公开的21株乳双歧杆菌和1株模式菌株DSM10140^T的基因组数据,构建了核心基因集与泛基因集,解析该群体的系统发育关系,比较分析Probio-M8的遗传特征及功能基因组。【结果】22株乳双歧杆菌的泛基因集包含1 618个基因,其中核心基因1 514个,占泛基因集的93.57%,表明乳双歧杆菌核心基因集高度保守。以1 514个核心基因构建系统发育树,发现22株乳双歧杆菌分为两个分支,AD011单独为一个分支,Probio-M8和其他菌株与模式菌株DSM10140^T聚在同一分支,且Probio-M8与V9、BB-12、Bi-07、HN019的遗传距离极为接近。进一步分析耐药基因和毒力基因,在Probio-M8与V9、BB-12、Bi-07、HN019基因组上均检测到DfrA...     

牦牛源产细菌素屎肠球菌的分离鉴定和益生特性

【背景】抗生素的滥用导致牦牛肠道常见病原菌耐药性增加,益生菌作为对抗耐药性细菌的新型武器,应用前景广阔。【目的】获取益生特性优良的牦牛源益生菌。【方法】将20份牦牛粪便样本在含0.5%CaCO3的MRS培养基上分离纯化,以大肠杆菌和金黄色葡萄球菌为指示菌,用牛津杯法筛选有抑菌活性的菌株;排除酸和过氧化氢后,经耐酸耐热试验和蛋白酶敏感试验筛选产细菌素菌株,用形态学和16S rRNA基因序列分析鉴定;通过对大肠杆菌、沙门氏菌等腹泻病原菌体外抑菌试验、耐模拟胃肠液、测定自聚集能力和疏水性及抗生素敏感试验分析益生特性。【结果】从20份牦牛粪便样本中共分离出11株产生溶钙圈的菌株,其中6株对大肠杆菌和金黄色葡萄球菌抑菌效果显著,经复筛得到2株产细菌素的乳酸菌SC6和SC9,经鉴定均为屎肠球菌(Enterococcus faecium)。其中SC9对腹泻病原菌抑菌效果明显,有良好的耐受性和肠道黏附能力,对5种常用抗生素均敏感。【结论】屎肠球菌SC9有一定的抗逆性和潜在的益生能力,具备作为益生菌的潜力。     

甲基营养型芽孢杆菌拮抗玉蜀黍尾孢菌、链格菌和灰葡萄孢菌及环脂肽分析

【目的】探究甲基营养型芽孢杆菌(Bacillus methylotrophicus)对植物病原菌玉蜀黍尾孢菌(Cercospora zeae-maydis Tehon et Daniels)、链格菌(Alternaria alternate)和灰葡萄孢菌(Botrytis cinerea)的拮抗作用并鉴定抗菌物质,为其病害防治提供优良生防菌。【方法】平板对峙法初筛和杯碟法筛选拮抗菌株;微生物形态学和16S rRNA基因鉴定拮抗菌株;薄层色谱(TLC)和编码基因分析鉴定抗菌物质;玉米田间生防试验评估拮抗菌对3种病原菌的防治效果。【结果】筛选到一株能够明显拮抗玉蜀黍尾孢菌、链格菌和灰葡萄孢菌的甲基营养型芽孢杆菌B-1841,抑制率分别为65.95%、71.04%和46.69%,抑菌物质为伊枯草菌素类脂肽。玉米田间生防试验表明,菌株B-1841对玉蜀黍尾孢菌、链格菌和灰葡萄孢菌感染的玉米病害均有防治效果,相对防效分别为60.25%、69.89%和45.21%。【结论】甲基营养型芽孢杆菌B-1841对玉蜀黍尾孢菌、链格菌和灰葡萄孢菌引起的病害有防治作用,在农作物真菌病害防治方面具有潜在应用价值。     

Evaluation of in vitro antagonism and of in vivo immune modulation and protection against pathogenic experimental challenge of two probiotic strains of Bifidobacterium animalis var. lactis

The aim of the present study was to compare the effect of intragastric administration with two strains of Bifidobacterium animalis subsp. lactis (Bifido A and Bifido B), in gnotobiotic and conventional mice, challenged with Salmonella Typhimurium. In vitro antagonism test showed that the two strains were able to produce antagonistic substances against various pathogenic microorganisms. In an ex vivo antagonism test the production of antagonistic substances was observed only against three out ten pathogens tested. Both Bifidobacterium strains were able to colonize and to maintain high population levels in the digestive tract of gnotobiotic mice. In addition, the two strains had low and limited translocation ability and did not cause any histological lesion in any of the organs analyzed. Both strains were able to reduce the fecal number of Salmonella in gnotobiotic mice challenged with the pathogen, but only Bifido B was able to confer a protection as demonstrated by a lower mortality. Higher levels of sIgA and IL-10 were observed only in Bifido B mono-associated mice when compared to germ free group. We could conclude that, among the parameters analyzed, the strain Bifido B exhibited the more desirable characteristics to be used as a probiotic.     

Probiotic Lactobacillus casei Expressing Human Lactoferrin Elevates Antibacterial Activity in the Gastrointestinal Tract

In this study, Lactobacillus casei was used to deliver and express human lactoferrin (hLF) to protect the host against bacterial infection. Full-length hLF cDNA was cloned into a Lactobacillus-specific plasmid to produce the L. casei transformants (rhLF/L. casei). Antimicrobial activity of recombinant hLF was examined in inhibition of bacteria growth in vitro. A mouse model was established to test in vivo antibacterial activity and protective effect of orally-administered probiotic L. casei transformant in the gastrointestinal tract. Trials were conducted in which animals were challenged with E. coli ATCC25922. E. coli colony numbers in duodenal fluid from the group fed with rhLF/L. casei were significantly lower than those of the group fed with wild-type L. casei or placebo (P < 0.01). Histopathological analyses of the small intestine, showed both decreased intestinal injury and increased villi length were observed in the mice fed with rhLF/L. casei as compared with the control groups (P < 0.01). Our results demonstrate that L. casei expressing hLF exhibited antibacterial activity both in in vitro and in vivo. It also provides a potentially large-scale production of hLF as applications for treatment of infections caused by clinically relevant pathogens.     

肠毒素大肠杆菌定居因子CS6抗原基因在痢疾杆菌中的表达

通过体外重组的方法,将asd基因插入重组表达质粒,使抗生素抗性失活,并与弗氏志贺氏菌FWL01构成宿主-载体平衡致死系统. 通过蛋白质印迹结果表明,在没有抗生素条件选择的情况下,可稳定表达肠毒素大肠杆菌定居因子抗原CS6. 重组菌通过口服和鼻饲免疫小鼠后,可以诱生CS6血清IgG抗体;同时可以检测到分泌型IgA产生,表明重组菌可以诱导相应的黏膜免疫反应.     

Experiences with the on-line measurement of culture fluorescence during cultivation of Bacillus subtilis, Escherichia coli and Sporotrichum thermophile

Bacillus subtilis and Escherichia coli K12 (both transformed for human leukocyte interferon production) and Escherichia coli B/r and Sporotrichum thermophile (a deuteromycete) were cultivated in submersed culture and the culture fluorescence recorded on-line using a fluorometer. During the cultivation of B. subtilis the signal from the fluorometer correlated with cell density and interferon production and thus could be used for process control (interferon production). However, the culture fluorescence of the other organisms did not increase (S. thermophile), was too weak to be measured with the fluorometer used (E. coli transformed for interferon production), or the signal from the fluorometer was not an accurate measure of the culture fluorescence because of the accumulation of a fluorophor in the culture medium (E. coli B/r).     

Antagonistic activity of Lactobacillus acidophilus RY2 isolated from healthy infancy feces on the growth and adhesion characteristics of enteroaggregative Escherichia coli

Enteroaggregative Escherichia coli (EAggEC) infection is an important cause of acute diarrhea, affecting children in developing countries and travelers visiting tropical or subtropical areas. Three probiotics can exert bacteriostatic or bactericidal effects on human and animal intestinal pathogens, the efficiency of probiotics on EAggEC infection remains unclear. In this study, the antagonistic activity of probiotic bacteria isolated from infant faeces was examined against several EAggEC stains. While three isolates, Lactobacillus acidophilus RY2, Lactobacillus salivarius MM1 and Lactobacillus paracasei En4 were shown to significantly inhibit the growth of EAggEC. In addition, the antagonistic activity of the Lactobacillus species was maintained despite heating (100 °C, 15 min) of cell free culture supernatant (CFCS). The antagonistic activity of the CFCS however, could be reduced following lactate dehydrogenase treatment and at pH 7.2. Furthermore, in an adhesion–inhibition assay, L. acidophilus RY2 was shown to be more effective than L. salivarius MM1 and L. paracasei EN4. This study suggests that L. acidophilus RY2 could be used as a probiotic organism against EAggEC.     

Novel probiotic Enterococcus faecium IS-27526 supplementation increased total salivary sIgA level and bodyweight of pre-school children: A pilot study

Enterococcus faecium IS-27526 is a novel probiotic isolated from dadih, an Indonesian traditional fermented buffalo milk. A 90 days randomized double-blind placebo-controlled study ofpre-post trial was conducted in pre-school children with two groups, placebo and probiotic group. Ultra High Temperature low fat milk was used as a carrier in each group. The aims of this study were to investigate the effect of E. faecium IS-27526 in milk on humoral immune response and on bodyweight of pre-school children. Total serum IgA and total salivary sIgA were measured by sandwich ELISA. The bodyweight of young children was measured. The results showed that total serum IgA did not significantly increase in the probiotic group compared with the placebo group. Total salivary sIgA level and the bodyweight significantly increased (p < 0.05) in probiotic groups compared to the placebo. Changes of total salivary sIgA level were significantly higher in underweight children supplemented with probiotic. Weight gain was observed significantly in children with normal bodyweight supplemented with probiotic. Neither mortality nor weight loss was recorded throughout the study. Taken together, novel probiotic E. faecium IS-27526 has significant positive effects on humoral immune response, salivary sIgA, in underweight pre-school children, and on weight gain of pre-school children.     

Saccharomyces boulardii stimulates sIgA production and the phagocytic system of gnotobiotic mice

The effect of Saccharomyces boulardii on the immune system was evaluated, comparing germ-free Swiss/NIH mice monoassociated with the probiotic with germ-free mice. Saccharomyces boulardii colonized the gut of germ-free mice and survived the gastrointestinal conditions. An increase in sIgA production, both total and anti-S. boulardii, was observed in the intestinal contents of monoassociated mice when compared with germ-free controls. The number of Kupffer cells was significantly higher in monoassociated mice than in germ-free controls. In S. boulardii-monoassociated mice, clearance of Escherichia coli B41 was higher than in germ-free controls. TNF-alpha, IFN-gamma and IL-12 serum levels were higher at earlier time points in monoassociated mice when compared with germ-free mice. These results show that the yeast S. boulardii modulates the host immune responses. This effect may be of interest for improving the resistance to enteropathogenic bacterial infections.     

香坊肠球菌和罗伊氏乳杆菌在无菌猪肠道中的定殖

【背景】已有研究表明,微生物在宿主肠道中的定殖受宿主、肠道环境、微生物物种特性和菌株来源等多个因素的影响。一般认为,来源于同类宿主的微生物菌株,在该类宿主肠道中具有定殖优势,但缺乏在物种和菌株水平上研究微生物自身特性在宿主肠道中定殖的研究报道。【目的】将不同来源(同类宿主肠道、非同类宿主肠道和非肠道环境)、具有不同生物学特性的3株香坊肠球菌(Enterococcus xiangfangensis)和4株罗伊氏乳杆菌(Lactobacillus reuteri)对无菌猪肠道进行定殖,在物种和菌株2个水平上探究物种特性和菌株来源对宿主肠道定殖的偏好性,揭示影响微生物定殖效率的关键因素。【方法】在本项研究中,将从藏猪(Tibetan pigs)、小鼠(ob/ob mice)、食蟹猴(Macaca fascicularis)和发酵食品中分离得到的多株香坊肠球菌和罗伊氏乳杆菌,制成混合菌剂对无菌巴马香猪(Bama miniature pig)进行为期4周的饲喂,并通过实时荧光定量PCR方法检测这7株菌在无菌猪肠道中的定殖情况。【结果】在物种水平上,香坊肠球菌和罗伊氏乳杆菌在无菌猪体内具有相近的定殖细胞数目。但是在菌株水平上,小鼠来源的香坊肠球菌(EX-MS)和罗伊氏乳杆菌(LR-MS)定殖的细胞数目显著高于其他5株菌,表明小鼠来源的菌株具有定殖优势。在体外混合培养中,将3株香坊肠球菌和4株罗伊氏乳杆菌分别等比例混合培养72 h后,发现香坊肠球菌EX-MS菌株的细胞数目高于香坊肠球菌EX-MK和EX-PG菌株;在罗伊氏乳杆菌中,4株菌的细胞数目相近。7株菌相互作用表明,EX-MS菌株对EX-MK和EX-PG菌株均有明显的抑制作用,但对4株罗伊氏乳杆菌无抑制作用。【结论】本次定殖实验的结果表明,在物种水平上,香坊肠球菌和罗伊氏乳杆菌均具有在无菌猪肠道中良好的定殖能力;但在菌株水平上,小鼠来源的香坊肠球菌EX-MS菌株和罗伊氏乳杆菌LR-MS菌株具有定殖优势;同时发现,小鼠来源的香坊肠球菌EX-MS菌株通过抑制同物种的EX-PG和EX-MK菌株,获得了在宿主肠道中的定殖优势。     

Probiotic Potential,Antioxidant Activity,and Phytase Production of Indigenous Yeasts Isolated from Indigenous Fermented Foods

While many bacteria have been used as probiotics by industries, only two yeasts, Saccharomyces cerevisiae var. boulardii and Kluyveromyces fragilis (B0399), have been used for this purpose. In the present work, a total of 116 yeasts isolated from Brazilian indigenous fermented food, cocoa fermentation, and kefir were in vitro characterized for probiotic attributes. From 116 isolates, 36 were tolerant to gastrointestinal conditions evaluated by tolerance to pH 2.0, bile salts (0.3% w/v), and 37 °C temperature. From those, 15 isolates showed a similar or higher percentage (P < 0.05) of hydrophobicity, autoaggregation, and coaggregation with E. coli than the reference strain S. boulardii. All these strains showed a high percentage of adhesion to Caco-2 cells (> 63%) and antioxidant activity (ranging from 18 to 62%). Phytate hydrolysis was evaluated for these yeasts and 13 strains showed positive results, which is important for nutrient availability in plant-based foods. These results are important insights for characterization of novel probiotic yeast strains as well as to aggregate functional value to these food products.

     

Selective cloning of genes encoding RNase H from Salmonella typhimurium, Saccharomyces cerevisiae and Escherichia coli rnh mutant

Summary We have cloned genes encoding RNase H from Escherichia coli rnh mutants, Salmonella typhimurium and Saccharomyces cerevisiae. Selection was accomplished by suppression of the temperature-sensitive growth phenotype of Escherichia coli strains containing the rnh-339::cat and either recB270 (Ts) or recC271 (Ts) mutations. RNases H from E. coli and S. typhimurium contained 155 amino acid residues and differed at only 11 positions. The S. cerevisiae and E. coli RNases H were about 30% homologous. A comparison of the amino acid sequences of several RNases H from cellular and retroviral sources revealed some strongly conserved regions as well as variable regions; the carboxyl-terminus was particularly variable. The overlapping, divergent promoter gene organization found in E. coli was observed to be present in S. typhimurium.     

Bioconversion of isoflavone glycosides to aglycones,mineral bioavailability and vitamin B complex in fermented soymilk by probiotic bacteria and yeast

Aim: To study the role of β‐glucosidase producing probiotic bacteria and yeast in the biotransformation of isoflavone glycosides to aglycones, mineral bioavailability and vitamin B complex in fermented soymilk. Methods and Results: Five isolates of probiotic lactic acid bacteria (LAB), Lactobacillus acidophilus B4496, Lactobacillus bulgaricus CFR2028, Lactobacillus casei B1922, Lactobacillus plantarum B4495 and Lactobacillus fermentum B4655 with yeast Saccharomyces boulardii were used to ferment soymilk to obtain the bioactive isoflavones, genistein and daidzein. High‐performance liquid chromatography was used to analyse the concentration of isoflavones. Bioactive aglycones genistein and daidzein after 24 and 48 h of fermentation ranged from 97·49 to 98·49% and 62·71 to 92·31% respectively with different combinations of LAB with yeast. Increase in bioavailability of minerals and vitamin B complex were also observed in fermented soymilk. Conclusions: LAB in combination with yeast S. boulardii has great potential for the enrichment of bioactive isoflavones, enhancing the viability of LAB strains, decreasing the antinutrient phytic acid and increasing the mineral bioavailability in soymilk fermentation. Significance and Impact of the Study: Fermentation of soymilk with probiotic organisms improves the bioavailability of isoflavones, assists in digestion of protein, provides more soluble calcium, enhances intestinal health and supports immune system. Increased isoflavone aglycone content in fermented soymilk improves the biological functionality of soymilk.     

一株链霉菌21-1的分离鉴定及其对禾谷镰刀菌的抑菌活性

【背景】由禾谷镰刀菌(Fusarium graminearum)引起的小麦赤霉病严重威胁我国的小麦生产。【目的】筛选对禾谷镰刀菌具有拮抗能力的链霉菌菌株,为生防菌剂开发提供理论基础。【方法】利用平板对峙法筛选对禾谷镰刀菌具有拮抗能力的链霉菌;通过形态特征、生理生化特征和16S rRNA基因序列分析对其进行鉴定;通过病原菌菌丝生长、孢子产生及萌发抑制试验分析其发酵液的抑菌活性;利用人工接种试验测定该菌株发酵液的防病效果。【结果】筛选到一株对禾谷镰刀菌具有较强拮抗活性的链霉菌21-1,抑菌率为59.5%。依据形态特征、生理生化特性和16S rRNA基因序列分析,将该菌株鉴定为黄三素链霉菌(Streptomycesflavotricini)。菌株21-1发酵液能够抑制禾谷镰刀菌的菌丝生长、孢子产生及萌发过程,而且可以降低禾谷镰刀菌菌丝中可溶性蛋白质的含量,并增加丙二醛的含量。菌株21-1可以产生蛋白酶及纤维素酶。菌株21-1菌液10倍稀释液对小麦赤霉病的防效最佳,为70.1%。此外,菌株21-1发酵液对其他8种植物病原菌均有较好的抑制作用。【结论】菌株21-1对禾谷镰刀菌有较好的抑菌活性,具...     

Functional Heterologous Protein Expression by Genetically Engineered Probiotic Yeast Saccharomyces boulardii

Recent studies have suggested the potential of probiotic organisms to be adapted for the synthesis and delivery of oral therapeutics. The probiotic yeast Saccharomyces boulardii would be especially well suited for this purpose due to its ability, in contrast to probiotic prokaryotes, to perform eukaryotic post translational modifications. This probiotic yeast thus has the potential to express a broad array of therapeutic proteins. Currently, however, use of wild type (WT) S. boulardii relies on antibiotic resistance for the selection of transformed yeast. Here we report the creation of auxotrophic mutant strains of S. boulardii that can be selected without antibiotics and demonstrate that these yeast can express functional recombinant protein even when recovered from gastrointestinal immune tissues in mice. A UV mutagenesis approach was employed to generate three uracil auxotrophic S. boulardii mutants that show a low rate of reversion to wild type growth. These mutants can express recombinant protein and are resistant in vitro to low pH, bile acid salts, and anaerobic conditions. Critically, oral gavage experiments using C57BL/6 mice demonstrate that mutant S. boulardii survive and are taken up into gastrointestinal immune tissues on a similar level as WT S. boulardii. Mutant yeast recovered from gastrointestinal immune tissues furthermore retain expression of functional recombinant protein. These data show that auxotrophic mutant S. boulardii can safely express recombinant protein without antibiotic selection and can deliver recombinant protein to gastrointestinal immune tissues. These auxotrophic mutants of S. boulardii pave the way for future experiments to test the ability of S. boulardii to deliver therapeutics and mediate protection against gastrointestinal disorders.     

Survival of bifidobacteria administered to calves

Twenty-five bifidobacteria were isolated from feces of calves. Isolates were identified, and their functional properties and antimicrobial activity were determined. From 10 strains with suitable properties rifampicin-resistant mutants (RRBs) were prepared and mixture of RRBs was administered to 2-d-old calves. These strains were identified by sequencing as Bifidobacterium animalis ssp. animalis (6 strains), B. thermophilum (2 strains), B. choerinum (1 strain) and B. longum ssp. suis (1 strain). The control group was without probiotic treatment. Survival ability of administered bifidobacteria was monitored in fecal samples by cultivation on modified TPY agar supplemented with mupirocin, acetic acid, and rifampicin. Administered bifidobacteria survived in gastrointestinal tract of calves for at least 60 d. Other bacteria were also determined after cultivation using fluorescence in situ hybridization (FISH). Bifidobacteria and lactobacilli dominated in fecal microflora. Significantly lower amounts of E. coli and higher amounts of bifidobacteria and total anaerobes were found in the treated group relative to the control group.     

High efficiency transformation of Salmonella typhimurium and Salmonella typhi by electroporation

Summary Salmonella typhimurium and S. typhi were transformd with high efficiency by electroporation. Transformation efficiencies of up to 10¹⁰ transformants per g of pBR322 were obtained. In contrast to chemical transformation methods, neither the smooth lipopolysaccharide of S. typhimurium nor the Vi capsular polysaccharide of S. typhi greatly affected transformation efficiency. The introduction of a galE mutation slightly improved transformation efficiency in S. typhimurium (< tenfold) while the Vi antigen of S. typhi had no detectable effect. The transformation efficiency of S. typhimurium with DNA derived from Escherichia coli was increased greatly by the removal of the hsd restriction system (100-fold). Under these conditions electroporation can be used for the routine and direct transformation of Salmonella strains with partially purified (alkaline lysis) plasmid DNA from E. coli.     

一株产低温蛋白酶地衣芽胞杆菌NWMCC0046全基因组测序及分析

地衣芽胞杆菌（Bacillus licheniformis）NWMCC0046是从西藏日喀则地区屠宰场废弃血污放置土壤中分离得到的一株益生菌，产生的碱性蛋白酶在低温下有作为洗涤剂添加酶的潜力。深入分析菌株NWMCC0046的基因组序列信息，并挖掘该菌株功能特性基因及潜在应用价值。使用PacBio RS II平台和Illumina HiSeq 4000平台对菌株NWMCC0046的基因组进行测序，并对测序数据进行基因组组装、基因预测与功能注释、共线性分析、进化分析及次级代谢产物合成基因簇预测。菌株NWMCC0046全基因组大小为4 321 565 bp，平均GC含量为46.78%，共编码4 504个基因。基因注释揭示了其益生菌特性，如胃肠道内独特的适应性、抗氧化活性和抗菌活性。此外，菌株NWMCC0046还编码工业上许多重要的酶。进化树及共线性结果表明，菌株NWMCC0046属地衣芽胞杆菌且与地衣芽胞杆菌ATCC 14580具有较好的共线性。同时，预测到菌株NWMCC0046中有11个次级代谢产物合成基因簇，编码地衣素、丰原素、杆菌肽和丁酰苷菌素等生物活性物质。基因组信息存储于GenBank，登录号为CP090312。菌株NWMCC0046全基因组数据为其益生特性提供基础数据，对菌株NWMCC0046后续相关研究具有重要意义。     

Prevention of Pathogenic Escherichia coli Infection in Mice and Stimulation of Macrophage Activation in Rats by an Oral Administration of Probiotic Lactobacillus casei I-5

Lactobacillus casei I-5 isolated from an alcohol fermentation broth enhanced immunity and prevented pathogenic infection as a probiotic. Mice fed with I-5 cells for 11 days prior to an intraperitoneal challenge with pathogenic Escherichia coli Juhl exhibited a high survival rate compared with the control group. Rats fed with I-5 cells for 10 days significantly increased the phagocytosis of peritoneal macrophages. In a cell culture system employing peritoneal macrophages from rats, the I-5 administration activated NF-κB stimulated by LPS. It also enhanced LPS-stimulated IL-12 and TNF-α production, but not IL-6 production. These results show that L. casei I-5 effectively prevented infection by pathogenic E. coli possibly through the activation of peritoneal macrophages. The strain would be useful to prevent pathogenic microbial infections in humans and farm animals.