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。     

桂花黄酮的提取纯化及抑菌活性研究

研究桂花黄酮提取纯化技术,并探讨其抗菌活性.结果表明,解析-热提法提取效率最高,其最优条件为:用样品量1.6倍的90%乙醇解析15 min,40倍80%乙醇提取3 h,所得桂花黄酮纯度、得率分别为45.64%和12.54%.纯化采用HPD400大孔吸附树脂,以浓度为0.46 mg/mL(pH 5.0)样品液上样,吸附流速3 BV/h,上样体积15 Bv,洗脱剂为7 BV的70%的乙醇溶液,洗脱流速2 BV/h,所得桂花黄酮纯度达92.84%.抑菌试验表明,桂花黄酮对金葡球菌、大肠杆菌、枯草杆菌、稻瘟病菌均有较好的抑菌效果.纯化后的桂花黄酮抑菌效果优于对照苯甲酸钠,表明桂花黄酮有较好的应用前景.     

枯草芽孢杆菌HS-A38产抗菌脂肽Bacillomycin D的组分分析及鉴定

对一株枯草芽孢杆菌HS-A38产生的脂肽类物质进行分离鉴定及抑菌活性研究。通过酸沉淀分离和有机溶剂抽提的方法,从枯草芽孢杆菌HS-A38发酵液中得到脂肽粗提物LP,产率为1.956 g/L。利用薄层色谱和茚三酮染色法确定该脂肽粗提物中存在四个组分,分别为LP1、LP2、LP3和LP4;抑菌活性检测显示,组分LP3对两株海洋致病菌副溶血性弧菌和铜绿假单胞杆菌的活性较高。组分LP3经硅胶柱层析纯化分离后,应用反相高效液相色谱(RP-HPLC)和基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)对该组分做进一步纯化和鉴定。分析表明,LP3样品在保留时间20 min~28 min产生单峰团LP3-1,其纯度为85.24%;经MALDI-TOF-MS分析和数据比对,组分LP3-1中的主要成分为杆菌霉素Bacillomycin D。     

抑制猪霍乱沙门氏菌的乳酸菌的分离、鉴定及潜在抑菌物质分析

猪霍乱沙门氏菌(Salmonella choleraesuis)是一种常见的食源性致病菌。为了预防和治疗该菌引起的疾病,饲养者在饲料中大量添加抗生素,致使猪肉存在严重的食品安全问题。从健康成年无量山乌骨鸡肠道内容物中筛选出具有抑菌作用的乳酸菌,测定其对猪霍乱沙门氏菌的抑菌效果,分析乳酸菌抑制猪霍乱沙门氏菌的有效成分,并对筛选的乳酸菌种进行了分子生物学鉴定。采用双层平板法对具有抑制猪霍乱沙门氏菌的乳酸菌进行筛选,采用牛津杯法对抑菌效果进行测定,并采用酶蛋白敏感性测定、热处理、有机酸处理等方法分析抑菌活性物质有效成分,采用16S rDNA分子标记对乳酸菌进行鉴定,并构建系统发育树。结果显示,从健康鸡肠道中筛选出18株乳酸菌,其中2株对猪霍乱沙门氏菌(Salmonella choleraesuis)、大肠埃希菌(Escherichia coli)、金黄色葡萄球菌(Staphylococcus aureus)、肠炎沙门氏菌(Salmonella enteritidis)、肠炎沙门氏菌亚种(Salmonella enteritidis subspecies)、志贺氏菌(Shigella)、无乳链球菌(Streptococcus agatactiae)具有良好的抑菌效果;不同蛋白酶、pH处理对乳酸菌无细胞培养液抑菌效果均有不同程度的影响,但是经80℃处理的乳酸菌无细胞培养液,其抑菌效果未明显改变。经鉴定,2株乳酸菌分别为植物乳杆菌(Lactobacillus plantarum)和短乳杆菌(Lactobacillus brevis)。从健康成年无量山乌骨鸡肠道内容物中分离得到的植物乳杆菌菌株L2和短乳杆菌菌株L4对猪霍乱沙门氏菌等致病菌具有明显地抑制作用,推测其抑菌有效成分可能为小肽类及有机酸,这对减少抗生素使用,提高猪肉食品的品质与安全性具有一定价值。     

猪源乳酸菌产乳酸及其抑菌特性研究*

研究了5株(L1、L2、L3、L5和L7)分离自仔猪肠道的乳酸菌的产乳酸能力及抑菌特性。结果表明:L5菌株产乳酸的速度最快,培养液中乳酸含量最高,L5菌株培养液pH值的下降速度最快,终末pH值最低,而L1菌株产乳酸的速度最慢,培养液乳酸含量最低。5株乳酸菌对大肠杆菌K88、K99、987P、O141和大肠杆菌E1及金黄色葡萄球菌均有不同程度的抑制作用;排除酸的影响后仍有22%～53%抑菌效果;经热处理后保持有92%以上的抑菌效果;蛋白酶处理后保持85%以上的抑菌效果。     

猪源乳酸菌产乳酸及其抑菌特性研究

研究了5株(L1、12、L3、L5和L7)分离自仔猪肠道的乳酸菌的产乳酸能力及抑菌特性。结果表明：L5菌株产乳酸的速度最快,培养液中乳酸含量最高,L5菌株培养液pH值的下降速度最快,终末pH值最低,而L1菌株产乳酸的速度最慢,培养液乳酸含量最低。5株乳酸菌对大肠杆菌K88、K99、987P、O141和大肠杆菌E1及金黄色葡萄球菌均有不同程度的抑制作用;排除酸的影响后仍有22％～53％抑菌效果;经热处理后保持有92％以上的抑菌效果;蛋白酶处理后保持85％以上的抑菌效果。     

鸡卵黄抗体IgY的分离纯化及鉴定

采用水溶稀释法结合硫酸钠二次盐析沉淀法分离纯化鸡卵黄中蛋白IgY,实验中比较了不同pH值的水溶稀释液对卵黄除脂效果的影响;并采用SDS-PAGE及western blotting对提取产物进行鉴定。结果显示,pH值5.2水溶稀释液除脂效果最好,IgY得率最高。实验优化了鸡卵黄抗体IgY分离纯化技术,得到的IgY产量高、纯度高,特异性强;此外,水溶稀释法制备IgY具有利用小体积样品获得大量蛋白及纯化效率高的优点。     

一株乳酸菌胞外多糖产生的影响因素及其提取*

应用苯酚—硫酸法对乳酸菌胞外多糖产生的影响因素进行了研究,表明该菌株在培养温度为30℃,培养时间为40~48h,pH值降到4时,胞外多糖的产量最大。葡萄糖是乳酸菌产生多糖的良好碳源。在对乳酸菌的培养物进行离心、透析、脱蛋白、脱色,最后用乙醇沉淀,得到粗品多糖,粗品多糖至少含有两种分子量和含量相差很大的多糖。经过SephadexG-200凝胶柱得到多糖精品EPS-Ⅱ,薄层层析结果显示其为一纯化的样品。     

凝胶过滤法纯化裂褶多糖检测方法的改进

凝胶过滤洗脱液中多糖含量一般采用硫酸-苯酚等化学显色法测定,然后根据洗脱曲线得到纯化的组分,但化学方法费时且消耗试剂.本研究采用350 nm非特征性吸收波长对2种不同纯度裂褶多糖样品PSG1和PSG2的洗脱液直接测定吸光度,与硫酸-苯酚法490 nm测定得到的洗脱曲线基本一致,即分别可得到4个和2个组分,同时采用HPLC对分离效果进行了检测.研究表明,采用350 nm波长直接检测可实现不同纯度裂褶多糖的分离.利用本法检测其他多糖的效果有待进一步研究.     

两种高效抑菌乳酸菌素BSN4和BCN4的特性研究

对筛选到具有高效抑菌效果的植物乳杆菌SN4和粪肠球菌CN4产生的乳酸菌素进行进一步生物学特性研究。采用牛津杯法测定细菌素对金黄色葡萄球菌的抑菌效果,发酵上清制取乳酸菌素初提液,通过对两株乳酸菌产生的乳酸菌素进行蛋白酶、热和酸碱处理,研究其抑菌效果的稳定性,以及菌株的生长合成曲线和抑菌谱。试验结果表明:两种乳酸菌素对大部分蛋白酶较敏感,胰蛋白酶处理后乳酸菌素BSN4和BCN4分别能保留84%和55%活性;两种乳酸菌素在p H 4~10之间保持80%的抑菌活性(抑菌圈20 mm),在25~100℃处理20 min后能分别保留89.1%和74.4%以上的活性,乳酸菌素BSN4经120℃处理5 min后仍保留69%活性;菌株SN4在发酵10 h就能达到稳定期,并表现出较好地产酸性和抑菌活性。抑菌谱显示乳酸菌素BSN4和BCN4对革兰阴性菌和真菌无明显抑菌效果,但对大部分革兰阳性菌有较好的抑菌活性。结果显示,两种乳酸菌素对大部分革兰阳性菌有较好的抑菌效果,具有较好的环境耐受性,可为乳酸菌素在生产生活中的应用提供参考。     

Antioxidative potential of lactobacilli isolated from the gut of Indian people

Oxidative stress is one of the major causes of degenerative conditions occurring at cellular level with serious health implications. This study was aimed at investigating the antioxidative potentials of probiotic lactobacilli of Indian gut origin and their ability to augment antioxidant defense enzyme systems in the host cells under oxidative stress conditions. A total of 39 Lactobacillus cultures were assessed for their resistance against reactive oxygen species. Most of the cultures were moderately to strongly resistant towards 0.4 mM H(2)O(2). The Lactobacillus isolate CH4 was the most H(2)O(2) resistant culture with only 0.06 log cycle reduction. Majority of the cultures demonstrated high resistance towards hydroxyl ions and Lp21 was the most resistant with log count reduction of 0.20 fold only. Almost all the cultures were also quite resistant to superoxide anions. Lp21 also showed the highest superoxide dismutase content (0.8971 U). Amongst the 39 cultures, Lactobacillus spp. S3 showed the highest total antioxidative activity of 77.85 ± 0.13 % followed by Lp55 (56.1 ± 1.2 %) in terms of per cent inhibition of linolenic acid oxidation. Lp9 up-regulated the expression of superoxide dismutase 2 gene in HT-29 cells both at 0.1 mM (1.997 folds) and 1.0 mM H(2)O(2) (2.058 folds) concentrations. In case of glutathione peroxidase-1, Lp9, Lp91 and Lp55 showed significant (P < 0.001) up-regulation in the gene expression to the level of 5.451, 8.706 and 10.083 folds, respectively when HT-29 was challenged with 0.1 mM H(2)O(2). The expression of catalase gene was also significantly up-regulated by all the cultures at 0.1 mM H(2)O(2) conditions. It can be concluded that the antioxidative efficacy of the putative probiotic lactobacilli varied considerably between species and strains and the potential strains can be explored as prospective antioxidants to manage oxidative stress induced diseases.     

产碱性磷酸酶乳杆菌的筛选鉴定、酶的纯化及特性

【背景】碱性磷酸酶(alkaline phosphatase,ALP)是生物体内参与磷酸代谢的调控酶,不同物种的ALP性质与其生理功能有关,提纯后的ALP常用作工具酶,广泛应用于基因工程中,但目前关于乳酸菌中ALP的相关研究甚少。【目的】筛选出一株产ALP且具有潜在益生作用的乳杆菌,对该酶进行分离纯化,并对其性质进行探究,为今后益生菌的开发利用和ALP的工业化生产提供新的微生物资源。【方法】采集蒙古国4个地区的酸马奶样品,通过显色反应初筛和酶活检测复筛对产酶菌株进行筛选,经形态学观察、生理生化鉴定及16S rRNA基因序列同源性比较分析进行菌种鉴定。采用超声破碎法提取ALP,经硫酸铵沉淀、DEAE-52离子交换层析、Sephadex G-200凝胶过滤层析纯化该酶,SDS-PAGE电泳法检测其纯度。【结果】从78株乳酸菌中分离筛选出一株产ALP酶活性最高的乳杆菌(编号为Z23),16S rRNA基因序列长度为1 473 bp,鉴定结果表明为鼠李糖乳杆菌。纯化后的酶比活力为180.27 U/mg,纯化倍数为48.37,酶活回收率为17.05%,该酶亚基相对分子质量为46.7 kD。菌株所产ALP的最适温度为37℃,4℃时酶活最为稳定;最适pH为9.5,在pH 9.0-10.0之间,酶活稳定性可达90%以上;Mg2+和K+对ALP有明显激活作用,Ba2+和Cu2+在低浓度时对ALP有激活作用,高浓度时有抑制作用,Ca~(2+)、Zn~(2+)和EDTA对ALP有强烈的抑制作用。以不同浓度的p-NPP为底物,测得酶的Km值为3.42 mmol/L,Vmax值为1.24 mmol/(L·min)。【结论】本研究对蒙古国地区酸马奶中的益生菌资源有了更为明确的认知,为今后碱性磷酸酶产生菌的筛选和酶的应用开辟了新途径。     

一株植物乳杆菌Lp3对高脂模型大鼠的益生作用

【目的】本研究通过构建大鼠高脂结构模型来探究一株植物乳杆菌Lp3的益生作用。【方法】植物乳杆菌Lp3筛选自青藏高原地区传统发酵的牦牛酸奶,初步认定Lp3是一株具有良好耐受力的降胆固醇菌株,且体外益生特性突出,本研究通过建立高脂SD大鼠模型,在饲喂试验动物高脂饲料的同时灌胃植物乳杆菌Lp3,来确定该菌株对试验动物血脂的影响效果,并同时研究其对大鼠肠道菌群、粪便水分、粪便中胆固醇和胆汁酸含量的影响,以及对肝脏组织中的胆固醇(TC)和甘油三酯(TG)的影响。【结果】结果表明,植物乳杆菌Lp3对大鼠没有任何明显的毒副作用,对高脂模型大鼠具有良好的降血脂效果。饲喂高脂饲料并灌喂乳酸菌Lp3组大鼠(HL)的血清总胆固醇、甘油三酯和低密度脂蛋白胆固醇含量较饲喂高脂饲料组(HC)显著减少(P0.05),但是高密度脂蛋白胆固醇的含量变化并不明显。HC组大鼠与HL组及饲喂普通日粮组(对照组)大鼠相比较,HC组大鼠粪便中大肠杆菌数量明显增加,双歧杆菌、乳杆菌数量明显减少。但是灌胃乳酸菌的HL组大鼠的粪便中乳杆菌数略高于对照组,大肠杆菌和双歧杆菌数量和对照组大鼠的基本一致。表明植物乳杆菌Lp3具有维持肠道菌群平衡的作用。此外灌胃乳酸菌后HL组大鼠粪便含水量比HC组要高6.44%。HC组大鼠肝脏组织中胆固醇和甘油三酯要显著高于HL组(P0.05),说明Lp3可以减少脂类物质在肝脏组织中的沉积。从肝脏组织切片来看,也可以得出上述结论。【结论】结果表明本研究所筛选的植物乳杆菌Lp3对高脂大鼠具有值得深入研究的益生作用。     

植物乳杆菌黏附大鼠小肠黏液及机制的研究

分析了6种植物乳杆菌黏附大鼠小肠粘液的能力,并分析了介导黏附性的主要因素。结果表明,植物乳酸杆菌向大鼠小肠粘液的黏附具有菌种特异性,其黏附作用是甘露糖特异性的,细胞外表蛋白质、碳水化合物和(脂)磷壁酸可能参与了黏附过程。     

脂蛋白(a)的赖氨酸结合异质性对人动脉SMC增殖的影响

为探讨脂蛋白 ( a) [Lp( a) ]的赖氨酸结合功能在致动脉粥样硬化中的作用 ,利用短时超速离心结合凝胶层析分离纯化 Lp( a) ,以 Lysine- Sepharose4B亲和层析分离出能与柱结合的 Lp( a)Lys+ 和不能与柱结合的 Lp( a) Lys-.采用 MTT比色法、流式细胞仪分析细胞增殖状况 ,同时以ELISA法检测细胞培养液中转化生长因子β( TGF-β)的活化水平 ,观察了两种 Lp( a)对培养的人动脉平滑肌细胞 ( SMC)增殖的影响 .结果表明 :Lp( a)、Lp( a) Lys+ 较 Lp( a) Lys-能有效地促进SMC增殖 ,刺激细胞从 G0 /G1期进入 S和 G2 /M期 ,并显著降低培养液中 TGF- β活化量 .提示 :Lp( a) Lys+ 可能是 Lp( a)促人 SMC增殖的主要组分 ,其机制可能与干扰纤溶酶原活化 ,从而抑制TGF-β活化有关 .     

Characterization and regulation of lens-specific calpain Lp82

Eye tissues contain splice variants of muscle-preferred p94 (calpain 3), such as lens-specific Lp82 and Lp85, retina-specific Rt88, and cornea-specific Cn94. The purpose of the present experiment was to analyze the activation and regulation of the best characterized p94 splice variant, Lp82. Recombinant rat Lp82 (rLp82) was expressed using the baculovirus system, purified with Ni-NTA affinity and DEAE-ion exchange chromatographies, and characterized by SDS-PAGE, casein zymography, and immunoblotting. After incubation with calcium, rLp82 autolyzed into two major fragments at approximately 60 and 22 kDa. Sequencing of the autolytic fragments showed loss of three amino acids from the N terminus and cleavage near the IS2 region. Also, Lp82 and calpain 2 were found to hydrolyze each other. Calpastatin inhibited calpain 2 activity, but not Lp82. Homology modeling suggested that the lack of inhibition of Lp82 by calpastatin was due to molecular clashes at the unique AX1 region of Lp82. Lp82 also hydrolyzed calpastatin. These results suggested that Lp82 might regulate other calpain activities and cause hydrolysis of substrates such as crystallins during lens cataract formation.     

乳酸杆菌细胞裂解物对金黄色葡萄球菌、大肠埃希菌的抑制作用

目的探讨乳杆菌DM8909裂解物在体内外对金黄色葡萄球菌、大肠埃希菌的抑制作用。方法通过对乳杆菌超声波破碎制成裂解物,分别用乳杆菌裂解物原液、裂解物稀释液、发酵上清液、乳杆菌活菌制剂进行体内、体外实验,观察乳杆菌各成分对金黄色葡萄球菌、大肠埃希菌的抑制作用。结果德氏乳酸杆菌裂解物对金黄色葡萄球菌、大肠埃希菌的抑制作用与乳杆菌活菌制剂的抑制作用相近。结论德氏乳酸杆菌裂解物在体内外对金黄色葡萄球菌、大肠埃希菌均有较强的抑制作用。     

Assessment of tolerance to multistresses and in vitro cell adhesion in genetically modified Lactobacillus plantarum 590

Lactobacillus plantarum (Lp) is a lactic acid bacterium that has many excellent traits that meet the needs of industrial production. Genetically modified (GM) Lp590 was obtained from Lp that was modified by the insertion of the gene nisI, which can confer resistance to nisin and play a role as a bio-preservative. Here, explorations were made to assess the safety of GM Lp590 and establish an in vitro evaluation model. The ability of Lp590 to tolerate both environmental stresses (such as temperatures ranging from 52 to 4 °C, or exposure to ethanol, oxygen, and osmotic stresses) and gastrointestinal transit was assessed. Lp590 showed a tolerance to 4 °C and ethanol (20%) within a period of 240 min that was similar to Lp. Notably, Lp590 can tolerate higher temperature (52 °C) and higher levels of H(2)O(2) (2%) and NaCl (4.0 M) than Lp. In contrast, Lp590 has the same gastrointestinal transit tolerance as Lp. In addition, Lp590 can adhere to Caco-2 cells, and it has no adverse effect on the cell membrane in vitro. These results indicate that GM Lp590 has many desirable biological characteristics and has good prospects for industrial applications. A useful and comprehensive exploration has been undertaken to establish a new in vitro evaluation model for genetically modified microorganisms (GMMs).     

Factors involved in binding of Lactobacillus plantarum Lp6 to rat small intestinal mucus

AIM: To investigate the adhesion determinants of Lactobacillus plantarum Lp6, a dairy isolate. METHODS AND RESULTS: Small intestinal mucus extracted from rats was used as a substrate for adhesion. Adhesion determinants were studied by physical, chemical and enzymatic pretreatments of the bacteria, and adhesion inhibition assay. The mannose-specific adhesins were explored by studying the effect of d-mannose on adhesion and the yeast-agglutinating ability of the bacteria. It was found that adhesion decreased after bacteria were treated with sodium metaperiodate, protease K, trypsin, lithium chloride and trichloroacetic acid. However, adhesion did not decrease after trypsin-treated bacteria were incubated with cell surface protein extract. Cell surface bound exopolysaccharides were found to inhibit the adhesion. D-mannose inhibited the adhesion in a dose-dependent manner. The bacteria could significantly agglutinate yeast and lost this ability after protease K treatment. CONCLUSIONS: Adhesion was mainly mediated by the mannose specific adhesins, which might be proteins that reversibly bind to the cell surface components. Cell surface-bound exopolysaccharides were also involved in adhesion. SIGNIFICANCE AND IMPACT OF THE STUDY: The mannose-specific adhesion of Lact. plantarum Lp6 to rat mucus might be important for competing with pathogens-binding sites in gut, which may be used to resist the colonization of the pathogens.     

Identification, characterisation and specificity of a cell wall lytic enzyme from Lactobacillus fermentum BR11

Screening of a genomic library with an antiserum raised against whole Lactobacillus fermentum BR11 cells identified a clone expressing an immunoreactive 37-kDa protein. Analysis of the 3010-bp DNA insert contained within the clone revealed four open reading frames (ORFs). One ORF encodes LysA, a 303 amino acid protein which has up to 35% identity with putative endolysins from prophages Lj928 and Lj965 from Lactobacillus johnsonii and Lp1 and Lp2 from Lactobacillus plantarum as well as with the endolysin of Lactobacillus gasseri bacteriophage Phiadh. The immunoreactive protein was shown to be encoded by a truncated ORF downstream of lysA which has similarity to glutamyl-tRNA synthetases. The N-terminus of LysA has sequence similarity with N-acetylmuramidase catalytic domains while the C-terminus has sequence similarity with putative cell envelope binding bacterial SH3b domains. C-terminal bacterial SH3b domains were identified in the majority of Lactobacillus bacteriophage endolysins. LysA was expressed in Escherichia coli and unusually was found to have a broad bacteriolytic activity range with activity against a number of different Lactobacillus species and against Lactococcus lactis, streptococci and Staphylococcus aureus. It was found that LysA is 2 and 8000 times more active against L. fermentum than L. lactis and Streptococcus pyogenes, respectively.