猪肠道产细菌素菌株的筛选和鉴定

发布时间：2018-02-08 22:25

  本文关键词： 肠道抗病菌 细菌素 菌株分离 发酵条件优化　出处：《江西农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：本研究通过对健康成年猪肠道内生菌群的分离,利用点种法筛选出对临床分离的致病性大肠杆菌有明显抑制作用的肠道活性菌株作为研究对象,通过排除酸、过氧化氢试验、酶解试验来初步确定活性菌株所产抑菌活性产物为多肽或蛋白类物质。通过对活性菌株盲肠2号菌菌落形态、代谢试验及16S r DNA的检测,鉴定其为枯草芽孢杆菌。通过对活性菌株盲肠2号菌生产活性产物时的接种量、初始p H、培养温度、发酵时间、培养基组成的研究,确定了该菌株生产活性物质的最佳条件,为今后进一步开发和利用该活性菌株奠定了基础。主要内容包括以下三个部分:1.猪肠道内生菌群活性菌株的分离。从健康成年猪肠道不同区段(十二指肠、空肠、回肠、盲肠、结肠、直肠)的菌群中进行肠道内生菌株的分离和培养,分离得到形态特征尽可能不同的菌落共计2317株,以临床分离的致病性大肠杆菌(Escherichia coli)作为指示菌,点种法对峙培养,将对指示菌具有明显抑菌作用的肠道内生菌株进行分离纯化,作为候选活性菌株。利用牛津杯琼脂扩散法对这些候选活性菌株的发酵上清液的抑菌活性进行检测,排除产酸、过氧化氢的可能后,再进行胰蛋白酶、木瓜蛋白酶的酶解处理,将酶解后发酵上清抑菌活性明显降低或完全丧失的菌株推定为产蛋白质类抑菌物质的活性菌株,留作进行后续研究。2.活性菌株的鉴定。以活性菌株盲肠2号菌的菌落特征、革兰氏染色,以及各种代谢反应试验结果为依据,初步确定盲肠2号菌株为芽孢杆菌(Bacillus)。进一步通过对盲肠2号菌的16S r DNA测序结果:盲肠2号菌株与枯草芽孢杆菌(Bacillus subtilis)同源性达到99%以上,鉴定本试验筛选所得盲肠2号菌为枯草芽孢杆菌。3.盲肠2号菌株生产活性产物时的最佳生产条件研究。通过研究不同接种量、初始p H、培养温度、发酵时间、培养基组成对活性菌株盲肠2号菌生产活性产物的影响,结果表明该菌株在利用PCA肉汤,接种量为3%、初始p H为7.0、培养温度为37℃、发酵时间为6h时生产活性物质的抑菌活性最强。
[Abstract]:Through the isolation of intestinal endophytic bacteria from healthy adult pigs, the enteric active strains with obvious inhibitory effect on clinical pathogenic Escherichia coli were screened by point breeding method. The bacteriostatic products of the active strains were identified as polypeptide or protein by enzymatic hydrolysis test. The colony morphology, metabolic test and 16s r DNA of the active strain cecum 2 were detected. Bacillus subtilis was identified as Bacillus subtilis. The optimum conditions for the production of active substances were determined by studying the inoculation amount, initial pH, culture temperature, fermentation time and composition of culture medium of the active strain cecum 2. It lays a foundation for the further development and utilization of this active strain. The main contents include the following three parts: 1. Isolation of the active strains of endophytic bacteria from the intestinal tract of healthy adult pigs (duodenum, jejunum, ileum, cecum, intestinal tract, ileum, cecum, duodenum, jejunum, ileum, cecum). The intestinal endophytic strains were isolated and cultured in colon and rectum. A total of 2317 strains with different morphologic characteristics were isolated. Escherichia coli, which is clinically isolated, was used as an indicator. The enteric endophytic strains with obvious bacteriostatic effect were isolated and purified as candidate active strains. The inhibitory activity of the fermentation supernatants of these candidate active strains was detected by Oxford cup Agar diffusion method, and the acid production was excluded. After the possibility of hydrogen peroxide, trypsin and papain were treated by enzymatic hydrolysis. The strains that had obviously decreased or completely lost the bacteriostatic activity of the supernatant of fermentation after enzymatic hydrolysis were presumed to be active strains producing protein-producing antimicrobial substances. 2. Identification of the active strains. Based on the colony characteristics of the active strain cecum 2, Gram staining, and the results of various metabolic reactions. The cecum 2 strain was identified as Bacillus subtilis. The result of 16s r DNA sequencing showed that the homology of cecum 2 strain and Bacillus subtilis was more than 99%. Cecum 2 was identified as Bacillus subtilis. 3. The optimum production conditions of cecum 2 were studied. Different inoculation amount, initial pH, culture temperature and fermentation time were studied. The effect of the composition of culture medium on the production of active product of Cecum 2 strain showed that the strain was using PCA broth, the inoculation amount was 3%, the initial pH was 7.0, and the culture temperature was 37 鈩，

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