圈养大熊猫肠道乳酸菌分离及益生特性

【背景】大熊猫数量稀少、繁殖困难,在其生长过程中极易感染消化系统疾病甚至死亡。【目的】分析圈养大熊猫肠道可培养乳酸菌的群落结构与功能,筛选具有益生特性的乳酸菌菌株,为大熊猫消化系统疾病的预防和肠道微生物研究提供理论参考及菌种资源。【方法】采用3种培养基分离大熊猫粪便中的乳酸菌,通过革兰氏染色镜检和过氧化氢试验对分离的菌株进行初步鉴定,基于BOXA1R-PCR图谱遗传多样性选取代表菌株进行16S rRNA基因测序分析并进行主成分分析(principal component analysis, PCA),同时分析乳酸菌菌株安全性和益生特性。【结果】通过初步鉴定共分离获得58株乳酸菌,根据BOXA1R-PCR结果挑选20株菌进行测序,结果显示20株菌分属于明串珠菌属(Leuconostoc)、肠球菌属(Enterococcus)、魏斯氏菌属(Weissella)和链球菌属(Streptococcus)这4个属,主成分分析结果表明不同年龄段的大熊猫肠道乳酸菌群落结构存在差异。20株乳酸菌均不溶血,17株乳酸菌对11种抗生素均敏感;11株菌耐pH值2.0酸性条件,14株菌对0.3%的胆盐具有良好...     

生殖道中具有抑菌活性乳酸菌的分离筛选及其抑菌物质分析

目的 从阴道炎患者的棉拭子样本中分离筛选具有抗菌活性乳酸菌,为研究对细菌性阴道炎具有确实疗效的微生态制剂提供理论与菌种保证.方法 应用纯培养技术及双层平板法从阴道样品中分离筛选具有较好抑菌活性的乳酸菌.通过有机酸排除试验、过氧化氢排除试验和蛋白类抑菌物质排除试验对具有较好抑菌活性菌株的主要抑菌物质分析,并通过PCR反应对是否具有细菌素相关基因进行鉴定.结果 从7个棉拭子样本中共分离得到26株乳酸菌,其中有6株呈现抑菌活性.K4M2、K4M5、K4M6和K4M8菌株的主要抗菌物质为有机酸.而K4M9、K4M10两株植物乳杆菌的主要抗菌物质除有机酸外,尚可以产生细菌素.病原菌拮抗试验结果表明,这两株菌株的混合培养上清对具有较好的抑菌效果.结论 K4M9、K4M10菌株有望作为微生态制剂用于细菌性阴道炎的临床防治.     

商品鸡盲肠内容物乳酸菌的分离与鉴定

实验以健康商品鸡盲肠为菌源,对附着其上的乳酸菌进行分离与鉴定,就所获得的菌株的耐酸耐胆汁、黏附特性、抑菌性进行初步研究。实验结果表明,从商品鸡盲肠中分离出6株乳酸菌,根据耐酸耐胆汁试验,病原菌生长抑制试验以及肠道黏膜粘附试验,筛选出2株优势菌株CX001和CX005,经乳杆菌属的生化鉴定,初步确定为短乳杆菌。     

ICR小鼠肠道中乳酸菌组成的克隆文库研究

目的对健康雄性ICR小鼠肠道内乳酸菌的组成结构进行研究。方法收集10只健康雄性ICR小鼠的新鲜粪便样品,提取粪便样品中微生物的总DNA,采用乳酸菌类群特异性引物（Lac1）和细菌通用性引物（1391r）的组合扩增16SrRNA基因并构建乳酸菌特异性克隆文库,研究小鼠肠道内各种乳酸菌的组成和比例。结果克隆文库的分析结果表明罗伊乳杆菌（Lactobacillus reuteri）和约氏乳杆菌（Lactobacillus johnsonii）为ICR小鼠肠道内的优势种,其他还包括鼠乳杆菌（Lactobacillus murinus）、阴道乳杆菌（Lactobacillus vaginalis）、肠乳杆菌（Lacto-bacillus intestinalis）等乳酸菌以及一个潜在的乳酸菌新种。结论健康ICR小鼠肠道内乳酸菌的多样性较高;L.re-uteri种可能具有较高的菌株水平多样性。     

耐受微酸性次氯酸水乳酸菌的分离鉴定、耐受特性及益生特性

【背景】次氯酸水具有杀菌性，当其作为饮用水可能会对畜禽肠道内有益菌造成负面影响。【目的】筛选出具有耐受微酸性次氯酸水(slightly acidic hypochlorous acid water, SAHW)能力的乳酸菌并研究其益生特性。【方法】从内蒙古地区传统自然发酵制品中分离筛选出8株乳酸菌菌株，经16SrRNA基因测序进行鉴定。进一步通过耐受SAHW、耐酸、耐胆盐、疏水性及自凝聚能力试验进行耐受特性和益生特性研究。【结果】菌株MBH3-2为副干酪乳杆菌，其经过质量浓度为20mg/L的SAHW处理2h，菌落对数值仅下降0.3lg(CFU/mL)，存活率为50.10%；而且在酸性(pH 2.0、2.5和3.0)、胆盐环境下均可存活；疏水率达62.12%，属于高度疏水性；自凝聚能力在2 h达到97.29%。【结论】最终确定一株具有优良耐受SAHW及基本满足作为微生态制剂要求的菌株，MBH3-2具有作为新型微生态制剂菌株的潜力。     

奶牛源乳酸菌的分离、鉴定及其微生态制剂的研究

为筛选出能用于防治奶牛子宫内膜炎的益生性乳酸菌菌株并以其为原材料制作微生态制剂,采集产后30～50 d健康奶牛的子宫颈口处分泌物,利用MRS选择性培养基厌氧培养方式进行乳酸菌初步筛选。纯化得到的单株菌株进行染色和镜检,将典型的乳酸菌进行生化鉴定。提取其DNA,扩增16S rDNA全序列并测序,将所得序列与NCBI核酸数据库进行同源性比对,明确各分离菌株种属归属。将鉴定得到的单株乳酸菌过滤以及离心获取上清,得到的上清进行抑菌实验。采用琼脂扩散法检测对大肠埃希菌的抑菌活性。以得到的良好抑菌能力的菌株为原材料进行混合抑菌,进行微生态制剂最优比例的探究,并研究这些菌株的生长曲线和产酸能力。结果显示,经染色镜检、生化鉴定和16S rDNA测序分析和单株抑菌实验得到6株较好的抑菌能力的乳酸菌,其中鼠李糖乳杆菌、魏斯式细菌和粪肠球菌的混合上清抑菌效果最好,具有较强的生长和产酸能力。研究得到了可用于防治奶牛子宫内膜炎益生菌株,可进一步用于临床试验探究。     

功能性乳酸菌系列产品的研制开发试验Ⅰ活性酸乳发酵工艺的研究

乳酸菌很早就成为人们利用的有益微生物,是人体肠道内极为重要的生理菌群。由乳酸菌发酵制成的食品称为功能性保健食品。本试验是1993年由朝阳市科委下达的科研项目,是运用现代生物技术,研究以乳酸菌为主要对象,以含乳酸菌量最高的酸奶为基础原料,进行了乳酸菌奶粉、乳酸菌奶糖、乳酸菌冷饮、乳酸菌片、乳酸菌饮料、乳酸菌奶酪等系列保健食品的深加工技术研究。现将活性酸乳发酵工艺的研究报告如下：l材料与方法1．1材料1．1．1主要原料鲜牛奶、奶粉、优质白糖、乳酸菌、稳定剂等。1．1．2供试菌株共9株（引自山东、天津、北京等地）。…     

高效降解草酸乳酸菌的筛选

尿路结石70%~80%主要由草酸钙结晶构成。人体内的草酸一般通过肠道内微生物降解,经由粪便排出或在泌尿道吸收由尿液排出。本研究对市场上商品化的发酵乳制品、饮料和药品中的乳酸菌进行分离,得到37株菌,包括嗜热链球菌、保加利亚乳杆菌、干酪乳杆菌、鼠李糖乳杆菌、植物乳杆菌、嗜酸乳杆菌、动物双歧杆菌、长双歧杆菌、粪肠球菌、屎肠球菌和乳球菌,并检测这些菌株降解草酸的能力。结果提示,乳酸菌在体外能够有效的降低培养物中的草酸浓度,并筛选出了具有高效降解草酸能力的乳酸菌菌株。有望成为尿石症预防的新措施。     

抑制嗜水气单胞菌乳酸菌的筛选、鉴定及抑菌作用

从健康成年尼罗罗非鱼（Oreochromis niloticus）肠道中分离纯化得到乳酸菌,采用牛津杯双层平板法筛选具有抑制嗜水气单胞菌(Aeromonas hydrophila)活性的乳酸菌菌株,借助16S rRNA分子鉴定乳酸菌种类。通过排除有机酸和过氧化氢干扰及蛋白酶敏感性等试验分析抑菌活性最佳乳酸菌的抑菌活性物质成分,并进行热、酸碱稳定性和扫描电镜观察等确定抑菌活性物质对嗜水气单胞菌的抑菌作用。结果显示,尼罗罗非鱼肠道中有3株乳酸菌对嗜水气单胞菌表现出良好的抑菌效果,经鉴定后分别为植物乳酸菌（Lactobacillus plantarum）和唾液乳杆菌（Lactobacillus salivarius）。其中,菌株LX3的抑菌活性最佳,抑菌圈直径可达到（25.25±0.23） mm且对多种蛋白酶敏感,经不同温度和酸碱性处理后抑菌活性物质仍保持较高的抑菌活性,特别是在pH 12和100 ℃处理后,抑菌圈直径仍达到（13.87±0.12）和（16.65±0.26） mm。此外,扫描电镜观察发现菌株LX3抑菌活性物质主要破坏了嗜水气单胞菌的细胞结构,使细胞内容物流出,致其死亡。上述结果表明,尼罗罗非鱼肠道中拥有较为丰富的抑制嗜水气单胞菌乳酸菌资源,尤其是菌株LX3显示出良好的抑菌活性与抑菌作用,这对于替代抗生素防治嗜水气单胞菌引发的相关鱼类疾病具有重要价值。     

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。     

新疆传统发酵乳品中乳酸菌与 酵母菌的益生特性

目的观察新疆传统发酵乳品中分离的14种菌株的生长特点及产酸能力,筛选出具有较强耐胆盐能力,并能在人工胃肠液中存活的菌株。方法对10株乳酸菌和4株酵母菌进行生长曲线、pH、耐胆盐能力和耐人工胃肠液检测。结果 10株乳酸菌和4株酵母菌具有良好的生长曲线和产酸能力;马乳酒样乳杆菌具有较强的耐胆盐能力;希氏乳杆菌、马乳酒样乳杆菌、乙醇假丝酵母和东方伊萨酵母具有较强的耐人工胃液能力;乳酸乳球菌、哈尔滨乳杆菌、瑞士乳杆菌、马乳酒样乳杆菌、乙醇假丝酵母和东方伊萨酵母具有较强的耐人工肠液能力。结论 10株乳酸菌和4株酵母菌具有优良的益生特性,有望成为益生菌制剂的备用菌株。     

北疆传统发酵生奶酪中乳酸菌的耐受性及益生特性测定

【背景】北疆乳制品中生奶酪作为发酵乳制品,其中有多种微生物的参与,是优质食品级微生物的宝贵来源,然而其中蕴含的丰富的微生物资源也面临着流失。【目的】研究已筛选出的41株乳酸菌的生长曲线变化规律,并对其在低pH环境的耐酸特性及益生特性进行分析。【方法】通过人工模拟胃肠液和含0.3%、0.5%、1.0%浓度牛胆盐溶液培养,对从生奶酪中分离鉴定的41株乳酸菌菌株进行人工胃肠液、牛胆盐耐受性试验。【结果】41株乳酸菌中有6株乳酸菌(编号为QM-5、QM-27、UM-12、UM-18、NM-11和NM-14)均能在pH值分别为2.0、2.5、3.0、3.5、4.0时生长较好,在pH 2.0的环境下也保持一定程度的生长。菌株存活率大于50%,乳酸菌含量为108 CFU/mL以上。从生奶酪分离的乳酸菌有极强的耐酸、耐胆盐特性,可以预测在胃肠道环境生存。41株菌对大肠杆菌(Escherichia coli)、金黄色葡萄球菌(Staphylococcus aureus)和枯草芽孢杆菌(Bacillus subtilis)均有抑制作用。【结论】通过比较耐酸性、胃肠道模拟、胆盐耐受性和抑菌特性等实验结果,初...     

Characterization of enterocin- and salivaricin-producing lactic acid bacteria from the mammalian gastrointestinal tract

Bacteriocin production may be a factor contributing to bacterial dominance within complex microbial populations and may therefore be a common trait within the gut microbiota. However, of 278 antimicrobial-producing culturable lactic acid bacteria (LAB) isolated from a range of mammalian intestinal sources in this study, characterization revealed just 23 distinct strains producing bacteriocin-like inhibitory substances and one Streptococcus hyointestinalis strain producing a potentially novel protease-insensitive antimicrobial. Three class II bacteriocins previously isolated from intestinal-derived LAB were identified as enterocin A and two salivaricin P-like bacteriocins. Moreover, this is the first report of intestinal-derived Streptococcus salivarius producing variants of the lantibiotic salivaricin A.     

Evidence for Lignin Oxidation by the Giant Panda Fecal Microbiome

The digestion of lignin and lignin-related phenolic compounds from bamboo by giant pandas has puzzled scientists because of the lack of lignin-degrading genes in the genome of the bamboo-feeding animals. We constructed a 16S rRNA gene library from the microorganisms derived from the giant panda feces to identify the possibility for the presence of potential lignin-degrading bacteria. Phylogenetic analysis showed that the phylotypes of the intestinal bacteria were affiliated with the phyla Proteobacteria (53%) and Firmicutes (47%). Two phylotypes were affiliated with the known lignin-degrading bacterium Pseudomonas putida and the mangrove forest bacteria. To test the hypothesis that microbes in the giant panda gut help degrade lignin, a metagenomic library of the intestinal bacteria was constructed and screened for clones that contained genes encoding laccase, a lignin-degrading related enzyme. A multicopper oxidase gene, designated as lac51, was identified from a metagenomic clone. Sequence analysis and copper content determination indicated that Lac51 is a laccase rather than a metallo-oxidase and may work outside its original host cell because it has a TAT-type signal peptide and a transmembrane segment at its N-terminus. Lac51 oxidizes a variety of lignin-related phenolic compounds, including syringaldazine, 2,6-dimethoxyphenol, ferulic acid, veratryl alcohol, guaiacol, and sinapinic acid at conditions that simulate the physiologic environment in giant panda intestines. Furthermore, in the presence of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), syringic acid, or ferulic acid as mediators, the oxidative ability of Lac51 on lignin was promoted. The absorbance of lignin at 445 nm decreased to 36% for ABTS, 51% for syringic acid, and 51% for ferulic acid after incubation for 10 h. Our findings demonstrate that the intestinal bacteria of giant pandas may facilitate the oxidation of lignin moieties, thereby clarifying the digestion of bamboo lignin by the animal.     

大熊猫胃肠道中消化酶活力的分析

为了研究大熊猫对食物的化学性消化特点和机制,测定了9只大熊猫唾液和3只大熊猫胃肠道中主要消化酶的活力,并与其他动物进行了比较.结果显示,大熊猫唾液呈碱性,蛋白酶和淀粉酶等消化酶活力低;肠道中淀粉酶活力高,而脂肪酶活力明显低于棕熊.大熊猫小肠粘膜中存在显著量的蔗糖酶、乳糖酶和麦芽糖酶活力.另外,在1只大熊猫胃和直肠液中检测到了少量纤维素酶活力.研究结果提示,大熊猫唾液直接参与食物消化的作用可能很弱;大熊猫对淀粉类食物有很好的消化能力,但对脂肪类食物消化能力相对不高.大熊猫胃肠道消化酶的活力特点适应其消化天然食物中的营养物质.     

葡萄藤叶青贮中乳酸菌的分离与鉴定

为了筛选出性状优良的乳酸菌菌株,本试验以实验室纯培养方式从葡萄藤叶自然发酵的青贮中分离鉴定乳酸菌。经过菌落形态、细胞形态、生理生化鉴定和16S r DNA基因序列以及系统发育树的分析,从葡萄藤叶青贮中分离出5株乳酸菌,其中菌株P-1. 2和P-2. 2为戊糖乳杆菌(Lactobacillus pentosus),菌株P-1. 7为戊糖片球菌(Pediococcus pentosaceus),菌株P-2. 1为粪肠球菌(Enterococcus faecium),菌株P-2. 3为短乳杆菌(Lactobacillus brevis)。测定产酸速率和生长速率,发现菌株P-2. 1的产酸能力和生长性能最好。     

The effect of prebiotics on production of antimicrobial compounds, resistance to growth at low pH and in the presence of bile, and adhesion of probiotic cells to intestinal mucus

AIMS: Screening of five bile salt-resistant and low pH-tolerant lactic acid bacteria for inhibitory activity against lactic acid bacteria and bacterial strains isolated from the faeces of children with HIV/AIDS. Determining the effect of prebiotics and soy milk-base on cell viability and adhesion of cells to intestinal mucus. METHODS AND RESULTS: Lactobacillus plantarum 423, Lactobacillus casei LHS, Lactobacillus salivarius 241, Lactobacillus curvatus DF 38 and Pediococcus pentosaceus 34 produced the highest level of antimicrobial activity (12,800 AU ml(-1)) when grown in MRS broth supplemented with 2% (m/v) dextrose. Growth in the presence of Raftilose Synergy1, Raftilose L95 and Raftiline GR did not lead to increased levels of antimicrobial activity. Cells grown in the presence of Raftilose Synergy1 took longer to adhere to intestinal mucus, whilst cells grown in the absence of prebiotics showed a linear rate of binding. CONCLUSIONS: A broad range of gram-positive and gram-negative bacteria were inhibited. Dextrose stimulated the production of antimicrobial compounds. Adhesion to intestinal mucus did not increase with the addition of prebiotics. SIGNIFICANCE AND IMPACT OF THE STUDY: The strains may be incorporated in food supplements for HIV/AIDS patients suffering from gastro-intestinal disorders.     

乳酸菌在肠道内黏附定殖的研究进展

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。由于其具有安全高效,易获取的优势,在食品、医疗及动物生产方面得到广泛运用。在动物肠道内,它利用糖类物质,生成酸性代谢物,降低肠道pH,改善肠道微生物环境。要实现乳酸菌在动物体内的良好定殖,首先要实现黏附,而黏附过程主要分为特异性结合与非特异性结合。黏附机制的相关研究中黏附素理论被普遍认可,包含了粘附蛋白学说、磷璧酸学说及细菌表面分子学说。本文就乳酸菌的黏附相关因子,体外黏附模型及黏附的影响因素做了阐述。     

Microbial production of lactic acid: the latest development

Lactic acid is an important platform chemical for producing polylactic acid (PLA) and other value-added products. It is naturally produced by a wide spectrum of microbes including bacteria, yeast and filamentous fungi. In general, bacteria ferment C5 and C6 sugars to lactic acid by either homo- or hetero-fermentative mode. Xylose isomerase, phosphoketolase, transaldolase, l- and d-lactate dehydrogenases are the key enzymes that affect the ways of lactic acid production. Metabolic engineering of microbial strains are usually needed to produce lactic acid from unconventional carbon sources. Production of d-LA has attracted much attention due to the demand for producing thermostable PLA, but large scale production of d-LA has not yet been commercialized. Thermophilic Bacillus coagulans strains are able to produce l-lactic acid from lignocellulose sugars homo-fermentatively under non-sterilized conditions, but the lack of genetic tools for metabolically engineering them severely affects their development for industrial applications. Pre-treatment of agriculture biomass to obtain fermentable sugars is a pre-requisite for utilization of the huge amounts of agricultural biomass to produce lactic acid. The major challenge is to obtain quality sugars of high concentrations in a cost effective-way. To avoid or minimize the use of neutralizing agents during fermentation, genetically engineering the strains to make them resist acidic environment and produce lactic acid at low pH would be very helpful for reducing the production cost of lactic acid.