粪便乳杆菌FZB1的分离鉴定与益生特性研究
发布时间：2018-11-07 18:54
【摘要】：目的分离鉴定粪便乳杆菌FZB1菌株,并研究其益生特性。材料与方法1.分离鉴定菌株利用16S r DNA、rpo A和rec A保守基因克隆测序和序列分析,并使用细菌微量生化管和常规生化实验测定糖发酵产酸等生化特征。2.探究粪便乳杆菌FZB1菌株耐酸、耐胆盐、碳源底物的利用能力和温度生长情况及粪便乳杆菌FZB1菌株对肠上皮细胞HT-29黏附性能和对病原菌大肠杆菌E57黏附抑制影响。3.我们利用聚合酶链式反应(Polymerase Chain Reaction,PCR)技术来追踪喂饲和不喂饲粪便乳杆菌FZB1的Balb/c J小鼠粪便样品,研究粪便乳杆菌FZB1菌株在Balb/c J小鼠肠道内的黏附定植情况。4.开展粪便乳杆菌FZB1菌株对宿主焦虑症状影响的相关研究。使用RU24969药物诱导Balb/c J小鼠的焦虑症状出现,然后运用不同行为学实验仪器:Morris圆形水迷宫、高架十字迷宫和社交行为实验对其进行行为学评估。结果1.分离鉴定实验发现,粪便乳杆菌FZB1的16S r DNA基因和粪便乳杆菌AFL13-2的16S r DNA基因最相似,相似性为99.2%。rpo A的氨基酸残基序列比对结果表明:该菌株和姬鼠乳杆菌、动物乳杆菌、鼠乳杆菌的亲缘关系最近,其相似性分别达到了97.1%、97.1%和96.7%。该菌株的rec A氨基酸残基序列和粪便乳杆菌完全相同,和姬鼠乳杆菌、动物乳杆菌、鼠乳杆菌rec A氨基酸残基序列有较高的相似性,分别为91.9%、91.5%和91.5%。生化鉴定结果表明:该菌株符合乳杆菌属和粪便乳杆菌的生化特征,被鉴定为:粪便乳杆菌FZB1(=CGMCC 10515)。2.在碳源利用和耐酸、耐胆盐实验中发现,该菌株可利用乳糖、可耐受p H 3的酸度,可在0.4%的牛磺石胆酸钠、0.08%的胆酸钠、0.15%的禽胆盐、0.15%的脱氧胆酸钠、0.15%的牛磺鹅脱氧胆酸钠和0.06%的牛胆盐环境中生长。温度生长实验结果表明,该菌在37℃条件下生长更好。3.粪便乳杆菌FZB1菌株对肠上皮细胞HT-29具有很强的黏附能力,黏附率可达88.7%,比对照组福菜乳杆菌标准株YM0097高11.8%,阻止大肠杆菌E57的黏附主要通过置换作用,相对置换率达9.9%,竞争或排斥作用总体上能促进大肠杆菌E57对肠上皮细胞的黏附作用。4.粪便乳杆菌FZB1菌株可在Balb/c J小鼠肠道中黏附定植,黏附定植时间为13天。使用RU24969药物诱导Balb/c J小鼠的焦虑症状出现,然后运用不同行为学实验仪器:Morris圆形水迷宫、高架十字迷宫和社交行为实验对其进行行为学评估。实验结果证明,粪便乳杆菌FZB1菌株可缓解Balb/c J小鼠焦虑症状。结论通过以上研究,我们发现自白鹭粪便中分离得到的乳杆菌,确定为粪便乳杆菌。这株粪便乳杆菌能够降解乳糖、耐酸、耐不同来源不同浓度胆盐、对肠上皮细胞HT-29具有很强的黏附能力及能黏附定植于Balb/c J小鼠肠道,黏附定植时间为13天,并能缓解Balb/c J小鼠焦虑症状,粪便乳杆菌FZB1菌株可进一步开发为新型益生菌制剂。
[Abstract]:Objective to isolate and identify Lactobacillus faecalis FZB1 strain and study its probiotic characteristics. Materials and methods 1. The 16s r DNA,rpo A and rec A conserved genes were cloned, sequenced and sequenced. The biochemical characteristics of sucrose fermentation and acid production were determined by microtubes and routine biochemical experiments. 2. To investigate the acid resistance, bile salt tolerance, carbon substrate utilization and temperature growth of Lactobacillus faecalis FZB1 strain, and the effect of Lactobacillus faecalis FZB1 strain on the adhesion to HT-29 of intestinal epithelial cells and the inhibition of E57 adhesion to pathogenic bacteria E. coli. 3. Polymerase chain reaction (Polymerase Chain Reaction,PCR) technique was used to trace fecal samples of Balb/c J mice fed or not fed with Lactobacillus faecalis FZB1, and to study the adhesion and colonization of Lactobacillus faecalis FZB1 strain in the intestine of Balb/c J mice. 4. To study the effect of Lactobacillus faecalis FZB1 strain on host anxiety symptoms. The anxiety symptoms of Balb/c J mice were induced by RU24969 drugs, and then were evaluated by using different behavioral experimental instruments: Morris circular water maze, elevated cross maze and social behavior experiment. Result 1. The results of isolation and identification showed that the 16s r DNA gene of Lactobacillus faecalis FZB1 and the 16s r DNA gene of Lactobacillus faecalis AFL13-2 were the most similar, and the amino acid residues of 99.2%.rpo A were similar to that of Lactobacillus agrarius. The genetic relationship of Lactobacillus animalis and Lactobacillus muris was the most recent, and the similarity reached 97.1% and 96.7.1%, respectively. The amino acid residues of rec A were identical to those of Lactobacillus faecalis, and were similar to those of Lactobacillus agrarius, Lactobacillus animalis and Lactobacillus muris rec A amino acid residues, which were 91.9% and 91.5%, respectively. The results of biochemical identification showed that the strain accorded with the biochemical characteristics of Lactobacillus and Lactobacillus faecalis and was identified as Lactobacillus faecalis FZB1 (= CGMCC 10515). 2. In the experiments of carbon source utilization, acid resistance and bile salt resistance, it was found that the strain could use lactose, tolerate pH 3 acidity, and be able to use 0.4% sodium taurocholate, 0.08% sodium cholate and 0.15% poultry bile salt. 0.15% sodium deoxycholate, 0.15% sodium taurocholate and 0.06% sodium oxalate grew in the environment. The results of temperature growth test showed that the strain grew better at 37 鈩，
本文编号：2317278
[Abstract]:Objective to isolate and identify Lactobacillus faecalis FZB1 strain and study its probiotic characteristics. Materials and methods 1. The 16s r DNA,rpo A and rec A conserved genes were cloned, sequenced and sequenced. The biochemical characteristics of sucrose fermentation and acid production were determined by microtubes and routine biochemical experiments. 2. To investigate the acid resistance, bile salt tolerance, carbon substrate utilization and temperature growth of Lactobacillus faecalis FZB1 strain, and the effect of Lactobacillus faecalis FZB1 strain on the adhesion to HT-29 of intestinal epithelial cells and the inhibition of E57 adhesion to pathogenic bacteria E. coli. 3. Polymerase chain reaction (Polymerase Chain Reaction,PCR) technique was used to trace fecal samples of Balb/c J mice fed or not fed with Lactobacillus faecalis FZB1, and to study the adhesion and colonization of Lactobacillus faecalis FZB1 strain in the intestine of Balb/c J mice. 4. To study the effect of Lactobacillus faecalis FZB1 strain on host anxiety symptoms. The anxiety symptoms of Balb/c J mice were induced by RU24969 drugs, and then were evaluated by using different behavioral experimental instruments: Morris circular water maze, elevated cross maze and social behavior experiment. Result 1. The results of isolation and identification showed that the 16s r DNA gene of Lactobacillus faecalis FZB1 and the 16s r DNA gene of Lactobacillus faecalis AFL13-2 were the most similar, and the amino acid residues of 99.2%.rpo A were similar to that of Lactobacillus agrarius. The genetic relationship of Lactobacillus animalis and Lactobacillus muris was the most recent, and the similarity reached 97.1% and 96.7.1%, respectively. The amino acid residues of rec A were identical to those of Lactobacillus faecalis, and were similar to those of Lactobacillus agrarius, Lactobacillus animalis and Lactobacillus muris rec A amino acid residues, which were 91.9% and 91.5%, respectively. The results of biochemical identification showed that the strain accorded with the biochemical characteristics of Lactobacillus and Lactobacillus faecalis and was identified as Lactobacillus faecalis FZB1 (= CGMCC 10515). 2. In the experiments of carbon source utilization, acid resistance and bile salt resistance, it was found that the strain could use lactose, tolerate pH 3 acidity, and be able to use 0.4% sodium taurocholate, 0.08% sodium cholate and 0.15% poultry bile salt. 0.15% sodium deoxycholate, 0.15% sodium taurocholate and 0.06% sodium oxalate grew in the environment. The results of temperature growth test showed that the strain grew better at 37 鈩，
本文编号：2317278
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