不同嗜热链球菌菌株的胞外多糖合成能力及eps基因簇分析

发布时间：2018-06-11 21:43

本文选题：嗜热链球菌 + 胞外多糖　；参考：《哈尔滨工业大学》2017年硕士论文

【摘要】：嗜热链球菌(Streptococcus thermophilus,S.thermophilus)是世界上公认的最具经济价值的同型发酵乳酸菌之一,广泛用于乳制品的生产。嗜热链球菌胞外多糖(Exopolysaccharides,EPS)由于其具有改善乳制品的品质,并已被证实具有抗氧化、抑菌等生物活性,而具有广泛的应用前景。本研究首先采用苯酚硫酸法和3,5-二硝基水杨酸法对从本实验室前期分离获得的22株S.thermophilus CS1~CS22产EPS能力进行评价,比较不同菌株所产EPS含量的差异,从中筛选确定高产EPS的优良菌株CS5、CS6、CS9和CS18。本研究以产EPS含量较高的S.thermophilus CS6为研究对象,对该菌EPS合成条件进行优化。采用单因素实验确定传代次数、接种量、发酵温度、后熟时间和碳源(乳糖、半乳糖、果糖、葡萄糖和蔗糖)等影响因素的范围。利用Plackett-Burman设计来筛选单因素中关键因素;利用响应面的中心复合实验CCD法确定最佳培养条件如下:接种量4.34%,发酵温度44°C,脱脂乳含量10%,后熟时间24 h,后熟温度4°C,传代次数3代,乳糖含量为1.7%,并进行验证,得到EPS粗提取物的含量为343.75 mg/L,与预测值接近,且比优化前提高了11.64%。本研究通过对S.thermophilus CS6进行补充碳源(麦芽糖、甘露糖、甘露醇、木糖和核糖)和氮源(蛋白胨、大豆蛋白胨、胰蛋白胨、牛肉膏和乳清浓缩蛋白)优化实验,同时对最大影响EPS产量的碳源(麦芽糖)和氮源(大豆蛋白胨)进行梯度实验设计,得到它们的最佳浓度范围,为碳源和氮源的进一步优化做准备,最终为后续即将完成eps基因簇序列分析的全部22株S.thermophilus产EPS的培养条件优化奠定基础。本研究对22株S.thermophilus eps基因簇的测定:首先对已公布的eps基因簇进行分析、归类,并根据其5’端deoD和3’端orf14.9之间基因的保守序列设计引物(共计8对);分别以22株S.thermophilus的基因组DNA为模板,扩增eps基因簇,对PCR产物鉴定、纯化及测序,序列进行拼接,获得完整的eps基因簇序列。本研究最终得到3株S.thermophilus(CS2、CS6和CS10)完整的eps基因簇序列信息,以及其它菌株大部分eps基因簇片段的序列信息。序列分析发现,菌株CS2、CS6和CS10均与S.thermophilus ASCC 1275菌株(ID:CP006819.1)的eps基因簇具有高度同源性,含有额外的与EPS长度相关的eps2C和eps2D基因,推测有利于EPS的合成;同时含有丰富的糖基转移酶基因,负责葡萄糖、半乳糖、鼠李糖、UDP-N-乙酰葡萄糖胺和UDP-呋喃半乳糖的转运,有利于形成独特单体组成的EPS。以上研究,为进一步研究eps基因簇在菌株产EPS差异中的作用奠定基础。
[Abstract]:Streptococcus thermophilus S. thermophilus (S. thermophilus) is recognized as one of the most economically valuable homozygous lactic acid bacteria in the world and is widely used in dairy production. Streptococcus thermophilus Exopolysaccharide EPSs (EPSs) have a wide application prospect due to its improved quality of dairy products, and has been proved to have antioxidant, bacteriostatic and other biological activities. In this study, the EPS production ability of 22 S.thermophilus CS1 CS22 strains isolated from our laboratory was evaluated by phenol sulfuric acid method and 3zhudinitrosalicylic acid method, and the EPS content of different strains was compared. The high EPS producing strains CS5, CS6, CS9 and CS18 were screened. In this study, S. thermophilus CS6 with high EPS content was used as the research object to optimize the EPS synthesis conditions of S. thermophilus CS6. Single factor experiments were used to determine the range of factors affecting the passage, inoculation, fermentation temperature, post-ripening time and carbon sources (lactose, galactose, fructose, glucose and sucrose). Plackett-Burman design was used to screen the key factors in the single factor, and the optimum culture conditions were determined by the CCD method of central composite experiment of response surface as follows: inoculation amount 4.34, fermentation temperature 44 掳C, degreasing milk content 10, ripening time 24 h, post-ripening temperature 4 掳C, passage times 3 generations. The content of lactose was 1.7mg / L, and the content of crude extract of EPS was 343.75 mg / L, which was close to the predicted value and increased 11.64% than that before optimization. In this study, S. thermophilus CS6 was optimized for carbon sources (maltose, mannitol, xylose and ribose) and nitrogen sources (peptone, soybean peptone, tryptone, beef extract and whey concentrate protein). At the same time, the gradient experimental design of carbon source (maltose) and nitrogen source (soybean peptone), which has the greatest influence on EPS yield, was carried out, and the optimum concentration range was obtained, so as to prepare for the further optimization of carbon source and nitrogen source. The results laid a foundation for the optimization of the culture conditions of all 22 strains of S. thermophilus which were about to complete the sequence analysis of eps gene cluster. In this study, 22 strains of S. thermophilus eps gene cluster were determined. Firstly, the published eps gene cluster was analyzed and classified. According to the conserved sequence of 5 'end deoD and 3' terminal orf14.9, primers were designed (8 pairs in total), 22 S. thermophilus genomic DNA was used as template to amplify the eps gene cluster, and the PCR products were identified, purified, sequenced and sequenced. The complete eps gene cluster sequence was obtained. In this study, the complete sequence information of eps gene cluster and the sequence information of most eps gene cluster fragments of other strains were obtained. Sequence analysis showed that the eps gene cluster of S.thermophilus ASCC 1275 had high homology and contained additional EPS length related eps2C and eps2D genes, which suggested that CS2 + CS6 and CS10 were beneficial to the synthesis of eps, and there were abundant glycosyltransferase genes. The transport of glucose, galactose, rhamnose UDP-N-acetylglucosamine and UDP-furan galactose is beneficial to the formation of unique monomer EPSs. The above studies laid a foundation for further study on the role of eps gene cluster in the differences of EPS production.
【学位授予单位】：哈尔滨工业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TS252.1

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