婴儿肠道双歧杆菌种群多样性分析

使用分子生物学方法,对4例出生时间为17～120h婴儿的肠道双歧杆菌种群多样性进行分析。肠道中剖腹产婴儿在出生17h后,双歧杆菌就已定植;另外,婴儿肠道中双歧杆菌群有短双歧杆（Bifidobacterium breve）和长双歧杆菌（Bifidobacterium longum）,分别占双歧杆菌种群的65％和35％。     

Adhesion of four Bifidobacterium strains to human intestinal mucus from subjects in different age groups

The number of bifidobacteria in faeces and intestinal contents has been shown to be reduced with increasing age of the subject. The adhesion of four Bifidobacterium strains was tested to mucus isolated from subjects of different age. All strains bound significantly less to mucus isolated from elderly subjects, compared to mucus from the other age groups. Two of the tested strains also showed decreased adhesion to mucus isolated from 6-month-old and adult subjects compared to the adhesion to mucus from 2-month-old subjects. The results suggest that reduced adhesion may be a factor involved in the decreasing colonisation of elderly subjects by bifidobacteria.     

两歧双歧杆菌胞外多糖对胃癌细胞及hTERT的影响

【目的】研究从双歧杆菌属两歧双歧杆菌(Bifidobacterium bifidum)提取的细胞表面成分胞外多糖(Exopolysaccharide, B.EPS)对人胃癌细胞BGC-823的生长抑制作用及对端粒酶限速因子hTERT活性的影响。【方法】将三种不同浓度B.EPS体外作用于胃癌细胞BGC823,MTT法检测细胞生长抑制率并辅以形态学观察;异硫氰酸盐(FITC)联合PI染色,通过流式细胞术检测肿瘤细胞初期调亡情况;肿瘤细胞端粒酶限速因子hTERT mRNA经RT-PCR检测B.EPS对端粒酶活性抑制作用;通过荧光分光光度计显示B.EPS 对胃癌细胞作用后胞内Ca2+含量变化。【结果】经过检测发现,B.EPS对人胃癌细胞BGC823的生长显著抑制(P＜0.05)呈剂量时间反应关系;细胞中hTERT mRNA在B.EPS的作用下表达降低(P＜0.05),有一定剂量效应关系;随着B.EPS对肿瘤细胞的抑制,细胞内Ca2+含量显著增加(P＜0.05)。【结论】B.EPS诱导人胃癌细胞BGC823调亡的机制可能与改变肿瘤细胞端粒酶限速因子hTERT mRNA表达量和细胞内钙离子浓度有关。     

Substrate specificity of three recombinant α-L-arabinofuranosidases from Bifidobacterium adolescentis and their divergent action on arabinoxylan and arabinoxylan oligosaccharides

Bifidobacterium adolescentis possesses several arabinofuranosidases able to hydrolyze arabinoxylans (AX) and AX oligosaccharides (AXOS), the latter being bifidogenic carbohydrates with potential prebiotic properties. We characterized two new recombinant arabinofuranosidases, AbfA and AbfB, and AXH-d3, a previously studied arabinofuranosidase from B. adolescentis. AbfA belongs to glycoside hydrolase family (GH) 43 and removed arabinose from the C(O)2 and C(O)3 position of monosubstituted xylose residues. Furthermore, hydrolytic activity of AbfA was much larger towards substrates with a low amount of arabinose substitutions. AbfB from GH 51 only cleaved arabinoses on position C(O)3 of disubstituted xyloses, similar to GH 43 AXH-d3, making it to our knowledge, the first reported enzyme with this specificity in GH 51. AbfA acted synergistically with AbfB and AXH-d3. In combination with AXH-d3, it released 60% of arabinose from wheat AX. Together with recent studies on other AXOS degrading enzymes from B. adolescentis, these findings allowed us to postulate a mechanism for the uptake and hydrolysis of bifidogenic AXOS by this organism.     

Development of a high-performance affinity chromatography-based method to study the biological interaction between whole micro-organisms and target proteins

Aims: The bacteria–host molecular cross‐talk is the matter of primary importance both in pathogenesis and in commensalism. Principally based on immunological methods, the methodologies commonly utilized for these studies are laborious and require specific antibodies. Here, we developed a new high‐performance affinity chromatography (HPAC)‐based approach that allows a direct measure of the interaction between whole bacterial cells and host molecules. Methods and Results: Bifidobacterium lactis BI07 cells immobilized on amino‐derivatized silica beads were utilized as stationary phase in a high‐performance affinity chromatography approach. The analytes plasminogen, collagen I and collagen IV were injected, and interactions were evaluated by the insertion in an HPLC system with UV detection. According to our data, Bif. lactis BI07 is capable of interacting with plasminogen, while it does not exhibit any binding activity to collagen I and IV. Conclusions: In this study, we implemented a high‐performance affinity chromatography‐based method to characterize the biological interaction between whole micro‐organisms and target proteins. Significance and Impact of the Study: With respect to the approaches commonly utilized to study the interaction between bacteria and host proteins, this HPAC‐based approach is fast and cheaper than other methods and allows a direct measure of the interaction between bacterial cells and target molecules.     

双歧杆菌发酵果蔬汁对小鼠免疫功能的影响

目的研究双歧杆菌发酵果蔬汁对小鼠免疫功能的影响。方法将小鼠随机分成纯净水对照组与发酵果蔬汁低、中和高3个剂量组,饮水法喂饲小鼠,测定小鼠胸腺和脾脏指数、腹腔巨噬细胞吞噬功能、血清溶血素测定、皮肤迟发型超敏反应(DTH)程度。结果与对照组比较,3种果蔬汁能显著增强巨噬细胞吞噬功能,增加血清溶血素抗体水平和DTH程度。结论双歧杆菌发酵果蔬汁能提高机体的免疫功能。     

双歧杆菌脂磷壁酸对结肠癌细胞CD44v6和MMP-2表达的影响

目的研究双歧杆菌脂磷壁酸(LTA)对结肠癌细胞中CD44v6与基质金属蛋白酶2(MMP-2)表达的影响,探讨其在抑制结肠癌转移中的作用。方法结肠癌LoVo细胞及HT-29细胞用含50 mg/L双歧杆菌LTA的培养液培养24 h后,RT-PCR和免疫细胞化学染色检测CD44v6和MMP-2在结肠癌细胞中的表达变化。结果结肠癌LoVo细胞及HT-29细胞中CD44v6和MMP-2的mRNA和蛋白质均呈高表达,经双歧杆菌LTA处理后,其表达均明显下降,与对照组比较,差异有非常显著性(P0.01)。结论双歧杆菌LTA可能通过下调CD44v6和MMP-2的表达来抑制结肠癌的转移。     

长双歧杆菌完整肽聚糖对小鼠免疫功能的影响

目的比较长双歧杆菌及其完整肽聚糖的免疫调节作用。方法通过研究长双歧杆菌完整肽聚糖对植瘤小鼠淋巴细胞的转化、抑瘤率、生命延长期以及对细胞Bcl-2和Bax表达的影响来探讨它们对小鼠细胞免疫的调节作用。结果与对照组相比,完整肽聚糖使淋巴细胞转化增加、抑瘤率提高、延长生命期增加、Bcl-2阳性表达率减小而Bax基因表达增加。结论长双歧杆菌与其完整肽聚糖均有抑制S180肿瘤的作用,但完整肽聚糖的效果优于长双歧杆菌。     

用生料制备功能性双歧醋生产工艺的实验室研究

目的将双歧杆菌、醋酸菌、酵母菌和粉碎的制醋原料及麸曲共同发酵,通过生料制醋的方法来制备功能性双歧醋。方法将粉碎的玉米与麸曲、酵母液、麸皮和水搅拌均匀,使其经过液态糖化和酒精发酵后,接入醋酸菌和双歧杆菌(二者比例为1∶1),同时加入辅料,进行醋酸发酵,当检测到醋酸酸度为5.0%~7.5%时,加入食盐终止发酵,经过过滤,除菌澄清得到功能性双歧醋。结果双歧醋的最终醋酸度为3.2%,外观红棕色,光泽度好,清澈透明,无沉淀和悬浮物。总菌数:醋酸菌为3.3×1011/m l,双歧杆菌为1.9×107/m l;活菌数:醋酸菌为1.7×1011/m l,双歧杆菌为6.8×106/m l;大肠菌群数3个/100 m l;致病菌:不得检出。结论双歧杆菌及其代谢物可以在双歧醋中存活,生料固态发酵制备双歧醋的方法可行。     

一种含双歧杆菌种属特异性复制子的新型大肠杆菌-双歧杆菌穿梭质粒的构建

目前,双歧杆菌的转化是一个技术难题,与大肠杆菌等宿主菌的高转化效率不同,采用普通的原核质粒无法转化双歧杆菌.为此,本文提出双歧杆菌转化对质粒复制子具有"种属特异性"要求,并通过构建含有双歧杆菌特异复制子的新型穿梭质粒,以求解决这一难题.首先从GenBank获取长双歧杆菌隐性质粒pMB1的序列信息,采用Overlap-PCR方法获得其全长DNA,作为拟构建质粒的复制子;继而采用重组技术,将其与pMK4质粒片段(含大肠杆菌复制子pUC和抗氯霉素基因Cat)重组,构建大肠杆菌-双歧杆菌穿梭质粒;用电穿孔法将重组质粒转化双歧杆菌,通过观察不同电转参数下的转化效率,选择双歧杆菌转化的最佳条件.结果,成功获得全长1899bp的pMB1复制子并构建成功含有pMB1和pUC双复制子的原核重组质粒,经酶切和测序鉴定正确,命名为pCMB1.以重组质粒成功转化了长双歧杆菌NCC2705和NQ1501,而其它3种野生型双歧杆菌(包括1株长双歧杆菌)未能转化成功.结论:质粒中含有双歧杆菌种属特异的复制子是实现双歧杆菌转化的必要条件;即使是含有特异复制子的质粒也只能转化有限数量种型甚至有限数量种株的双歧杆菌;选择最佳电转化条件能显著提高转化效率.