【摘要】：目的分離鑒定糞便乳桿菌FZB1菌株,并研究其益生特性。材料與方法1.分離鑒定菌株利用16S r DNA、rpo A和rec A保守基因克隆測(cè)序和序列分析,并使用細(xì)菌微量生化管和常規(guī)生化實(shí)驗(yàn)測(cè)定糖發(fā)酵產(chǎn)酸等生化特征。2.探究糞便乳桿菌FZB1菌株耐酸、耐膽鹽、碳源底物的利用能力和溫度生長(zhǎng)情況及糞便乳桿菌FZB1菌株對(duì)腸上皮細(xì)胞HT-29黏附性能和對(duì)病原菌大腸桿菌E57黏附抑制影響。3.我們利用聚合酶鏈?zhǔn)椒磻?yīng)(Polymerase Chain Reaction,PCR)技術(shù)來(lái)追蹤喂飼和不喂飼糞便乳桿菌FZB1的Balb/c J小鼠糞便樣品,研究糞便乳桿菌FZB1菌株在Balb/c J小鼠腸道內(nèi)的黏附定植情況。4.開(kāi)展糞便乳桿菌FZB1菌株對(duì)宿主焦慮癥狀影響的相關(guān)研究。使用RU24969藥物誘導(dǎo)Balb/c J小鼠的焦慮癥狀出現(xiàn),然后運(yùn)用不同行為學(xué)實(shí)驗(yàn)儀器:Morris圓形水迷宮、高架十字迷宮和社交行為實(shí)驗(yàn)對(duì)其進(jìn)行行為學(xué)評(píng)估。結(jié)果1.分離鑒定實(shí)驗(yàn)發(fā)現(xiàn),糞便乳桿菌FZB1的16S r DNA基因和糞便乳桿菌AFL13-2的16S r DNA基因最相似,相似性為99.2%。rpo A的氨基酸殘基序列比對(duì)結(jié)果表明:該菌株和姬鼠乳桿菌、動(dòng)物乳桿菌、鼠乳桿菌的親緣關(guān)系最近,其相似性分別達(dá)到了97.1%、97.1%和96.7%。該菌株的rec A氨基酸殘基序列和糞便乳桿菌完全相同,和姬鼠乳桿菌、動(dòng)物乳桿菌、鼠乳桿菌rec A氨基酸殘基序列有較高的相似性,分別為91.9%、91.5%和91.5%。生化鑒定結(jié)果表明:該菌株符合乳桿菌屬和糞便乳桿菌的生化特征,被鑒定為:糞便乳桿菌FZB1(=CGMCC 10515)。2.在碳源利用和耐酸、耐膽鹽實(shí)驗(yàn)中發(fā)現(xiàn),該菌株可利用乳糖、可耐受p H 3的酸度,可在0.4%的�；鞘懰徕c、0.08%的膽酸鈉、0.15%的禽膽鹽、0.15%的脫氧膽酸鈉、0.15%的�；蛆Z脫氧膽酸鈉和0.06%的牛膽鹽環(huán)境中生長(zhǎng)。溫度生長(zhǎng)實(shí)驗(yàn)結(jié)果表明,該菌在37℃條件下生長(zhǎng)更好。3.糞便乳桿菌FZB1菌株對(duì)腸上皮細(xì)胞HT-29具有很強(qiáng)的黏附能力,黏附率可達(dá)88.7%,比對(duì)照組福菜乳桿菌標(biāo)準(zhǔn)株YM0097高11.8%,阻止大腸桿菌E57的黏附主要通過(guò)置換作用,相對(duì)置換率達(dá)9.9%,競(jìng)爭(zhēng)或排斥作用總體上能促進(jìn)大腸桿菌E57對(duì)腸上皮細(xì)胞的黏附作用。4.糞便乳桿菌FZB1菌株可在Balb/c J小鼠腸道中黏附定植,黏附定植時(shí)間為13天。使用RU24969藥物誘導(dǎo)Balb/c J小鼠的焦慮癥狀出現(xiàn),然后運(yùn)用不同行為學(xué)實(shí)驗(yàn)儀器:Morris圓形水迷宮、高架十字迷宮和社交行為實(shí)驗(yàn)對(duì)其進(jìn)行行為學(xué)評(píng)估。實(shí)驗(yàn)結(jié)果證明,糞便乳桿菌FZB1菌株可緩解Balb/c J小鼠焦慮癥狀。結(jié)論通過(guò)以上研究,我們發(fā)現(xiàn)自白鷺糞便中分離得到的乳桿菌,確定為糞便乳桿菌。這株糞便乳桿菌能夠降解乳糖、耐酸、耐不同來(lái)源不同濃度膽鹽、對(duì)腸上皮細(xì)胞HT-29具有很強(qiáng)的黏附能力及能黏附定植于Balb/c J小鼠腸道,黏附定植時(shí)間為13天,并能緩解Balb/c J小鼠焦慮癥狀,糞便乳桿菌FZB1菌株可進(jìn)一步開(kāi)發(fā)為新型益生菌制劑。
[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，

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