国内5种医用益生菌产品的菌种分析

发布时间：2018-03-13 18:37

  本文选题：益生菌　切入点：16S　出处：《第三军医大学》2006年硕士论文　论文类型：学位论文

【摘要】： 益生菌制剂产品菌种与活菌数含量是其产品质量的关键。国外益生菌产品的微生物分析表明,部分产品的微生物种属和活菌数含量与产品标签上标注的种属和数量不符。然而,国内还没有这方面的研究报道。本课题对国内主要的五种医用益生菌制剂产品进行菌种分析。考察了五种医用益生菌制剂多个批次产品的活菌数含量,有统计意义的反映产品在有效期内的活菌数含量情况。通过结合表型特征分析法与16S rDNA序列分析法对从产品中分离的10株菌进行种属鉴定。同时将PCR-DGGE分析方法应用到鉴定益生菌制剂产品菌株组成,为益生菌制剂产品的质量控制提供快速、可靠、重复性好的分析方法。 方法与结果:(1)通过筛选获得到适合的选择培养基,并用于5种医用益生菌制剂25批产品的活菌计数,计数结果显示:产品Ⅰ中的三种菌的活菌数变化范围分别为10~5～10~(10)CFU/粒、10~5～10~8CFU/粒、10~6～10~8 CFU/粒,产品Ⅱ的活菌数变化范围为10~8～10~9 CFU/粒,产品Ⅲ的活菌数变化范围为10~8～10~9 CFU/粒,产品Ⅳ中的两种菌的活菌数变化范围分别为10~7～10~8 CFU/粒和10~8～10~(10) CFU/粒,产品Ⅴ中的三种菌的活菌数分别为10~7 CFU/粒、10~6 CFU/粒、10~6 CFU/粒。(2)分离纯化10株产品菌株,提取单菌株基因组DNA,PCR扩增16S rRNA基因,纯化并测定PCR产物序列,将测得序列与GenBank数据库的序列进行同源性分析,并建立系统进化树。在GenBank数据库中登陆其10株菌的16S rDNA序列,获得如下序列号:PFK1(DQ295034),PFK2(DQ295035),PFK3(DQ295036),LZCL(DQ295040),ZCS1(DQ295037),MMA1(DQ295041),MMA2(DQ295038),JSQ1(DQ295042),JSQ2(DQ295039),JSQ3(DQ295043)。基于菌株16S rDNA序列的Blast和系统进化树分析结果表明产品Ⅰ中含有的三种菌分别为长双歧杆菌、植物乳杆菌和粪肠球菌;产品Ⅱ中含有的菌是动物双歧杆菌;产品Ⅲ中含有的菌是地衣芽孢杆菌;产品Ⅳ中含有的两种菌是屎肠球菌和枯草芽孢杆菌;产品Ⅴ中含有的三种菌分别为动物双歧杆菌、保加利亚乳杆菌、嗜热链球菌。(3)将分离菌株进行形态、生理和糖发酵实验,结果表明这些表型鉴定的结论与16 S rRNA基因序列同源性分析结论一致,分离纯化5种产品的10株菌中仍有相同的3株菌与标签标注的不一致,即其中标签上分别标注为青春双歧杆菌和长双歧杆菌的两株菌鉴定结果均为动物双歧杆菌,
[Abstract]:The microbiological analysis of probiotic products in foreign countries shows that the microbial species and the number of live bacteria in some products are not consistent with the species and quantity labeled on the product label. There is no such research report in China. In this paper, the main five kinds of medical probiotics products in China were analyzed, and the number of live bacteria in five kinds of medical probiotics products were investigated. It is statistically significant to reflect the content of live bacteria in the valid period of product. By combining phenotypic characteristic analysis and 16s rDNA sequence analysis, the species of 10 strains isolated from the product were identified. Meanwhile, the method of PCR-DGGE analysis was applied to identify the species of 10 strains isolated from the product. Used to identify the composition of probiotic product strains, To provide a rapid, reliable and reproducible analytical method for the quality control of probiotics. Methods and results: a suitable medium was obtained by screening and used to count live bacteria in 25 batches of 5 kinds of medical probiotics. The counting results showed that the number of live bacteria in product I ranged from 10 ~ 5 ~ 10 ~ (10) to 10 ~ (10) ~ 10 ~ (10) ~ (10) ~ (10) ~ (10) ~ (10) C ~ (10) C ~ (8) ~ (10) C ~ (10) C ~ (10) ~ (8) CFU/, to 10 ~ (8) ~ 10 ~ (9) CFU/ for product 鈪，

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