藏族传统曲拉制作过程中乳酸菌群变化及曲拉中益生性乳杆菌的筛选和功能性评价

发布时间：2018-06-13 21:34

本文选题：曲拉 + 乳酸杆菌　；参考：《郑州大学》2017年博士论文

【摘要】：本研究以青海省不同地区藏族传统手工制作的牦牛乳曲拉样品为研究对象,对其营养特点、微生物菌群组成、乳杆菌的特征和种类进行了系统的分析,并首次对曲拉在制作过程中各种微生物的动态变化和乳酸菌种类的变化进行了研究,首次对曲拉中的益生性乳酸杆菌进行了体外筛选,并对筛出的优良菌株进行了传代稳定性的分析,同时结合小鼠试验对其免疫调节、抗氧化性进行了较为全面的功能性评价,通过研究发现:(1)从青海省不同地区采集了 43份传统手工制作牦牛乳曲拉样品,根据地区不同,抽取11份代表样品对其pH,水分、灰分、蛋白质、脂肪含量进行了分析,并将分析结果同其他类型的奶酪相比较,发现曲拉蛋白质的含量高于其他奶酪;利用平板计数法对43份曲拉样品的微生物菌群组成进行了分析,通过对曲拉中的乳酸菌、大肠杆菌、霉菌、酵母菌、好氧性细菌、梭菌、芽孢杆菌分析发现:总体来看,乳酸菌、酵母菌是曲拉中数量上的优势菌。(2)根据菌落形态和革兰氏染色的结果,同时结合生理生化特征分析、API碳源发酵试验、16SrRNA基因序列分析和recA试验,43份样品中共分离出69株乳杆菌代表株:Lactobacillus sakei(2株)、Lactobacillus diolivorans(1株)、Lactobacillus casei(28株)、Lactobacillus plantarum(34株)、Lactobacillus kefiri(4 株),Lactobacillus plantarum为优势种,占49%,Lactobacilluscasei 为第二大种,占 41%。(3)通过对曲拉制作过程中微生物的动态变化进行监测,发现:在曲拉制作过程中,均能不同程度地检测到乳酸菌、大肠杆菌、好氧性细菌、丝状真菌以及酵母菌群;乳酸菌和酵母为参与曲拉发酵的优势菌群;参与曲拉发酵的乳酸菌种有:Leuconostoc mesenteriodes、Enterococcus hirae、Enterococcus mundtii、Lactococcus lactis、Lactobacillus plantarum、Lactobacillus sakei、Lactobacillus buchneri、Lactobacillus diolivorans、Lactobacillus casei。在曲拉制作过程中,Leuconostocmesenteriodes 一直为优势种。Leuconostocmesenteriodes、Lactobacillusplantarum、Lactobacillus sakei是原料乳中固生的乳酸菌种类,在曲拉的制作过程中,这三种乳酸菌一直起主导作用。(4)对分离到的69株乳杆菌进行了益生性能的筛选,采用的筛选指标有:耐酸、耐胆盐性能;耐模拟人体胃肠液;抑菌性能;抗生素敏感性;细胞表面疏水性。经过筛选,有7株菌益生性能表现良好:Lactobacillus/splantarum 1197、Lactobacillus pl ant arum 1141、Lactobacillus kefiri1059、Lactobacillus casei1138、Lactobacillus casei1089、Lactobacillus plantarum 1086-1、Lactobacilluscasei 1133。在这 7 株菌中,综合各种指标,选择益生性能较其他5株菌相对优秀的L.plantarum1086-1、L.casei1133进行下一步的评价。(5)对L.plantaruL.086-1、L.casei1133进行了连续传20代遗传稳定性考察,从表型特征看:两株菌的细胞形态、活菌数、产乳酸活力、碳源发酵类型在传代过程中均能稳定遗传;从发酵特性看:L.plantarum1086-1、L.casei1133发酵性能稳定性均较差,L.plantarum1086-1从第15代开始,发酵性能明显下降,L.casei1133从第10代开始,发酵性能明显下降;从益生特性稳定性看:两株菌的耐酸-胆盐性、对模拟人体胃肠液的耐受性、细胞表面疏水性、抗生素抗性在传代过程中均能稳定遗传。(6)通过BALB/c小鼠体内试验对L.plantarum1086-1、L.casei1133进行了免疫调节功能的评价,结果表明:不同剂量的L.plantarum1086-1和L.casei1133均能极显著提高小鼠小肠派伊尔结巨噬细胞的吞噬活性(P0.01),前者无剂量依赖性,后者呈现剂量依赖性;高剂量的L.plantarum1086-1和L.casei1133菌液对小鼠IgA的分泌没有影响,但能显著促进机体IgG、IgM的分泌(P0.05,P0.01);高剂量的L.plantarum1086-1 和 L.casei1133均能极显著提高小鼠血清中IL-6,IK-10,IFN-γ和TNF-α的水平(P≤0.001);不同剂量的L.plantarum1086-1和L.casei1133均能极显著上调小鼠肝脏IL-6,IL-10,IFN-γ 和 TNF-a mRNA 表达水平(P0.01)。(7)通过 BALB/c 小鼠体内试验对 L.plantarum1086-1、L.casei1133 进行了抗氧化功能的评价,结果表明:高剂量的L.plantarum1086-1能极显著提高血清抗氧化能力(P0.01),L.casei1133对血清的抗氧化能力的提高程度不太显著,但有改善的趋势;不同剂量的L.plantarum1086-1和L.casei1133均能显著上调小鼠脾脏SOD、CAT、GSH-Px mRNA表达水平(P0.05,P0.01),各剂量组之间差异显著(P0.05,P0.01),呈现剂量依赖性。
[Abstract]:In this study, the samples of yak lactotra, which were traditionally made by Tibetans in different regions of Qinghai Province, were studied. The nutritional characteristics, the composition of microbial flora, the characteristics and types of lactobacillus were systematically analyzed. The dynamic changes of various microbes and the variety of lactic acid bacteria were studied for the first time in the process of making tra. For the first time, the probiotic Lactobacillus in tra was screened in vitro, and the stability of the screened strains was analyzed. At the same time, a more comprehensive functional evaluation was carried out on the immunoregulation and antioxidation in mice. Through the study, it was found that (1) 43 traditional handcrafts were collected from different regions of Qinghai province. The samples of yak lactotra were selected from 11 representative samples to analyze their pH, moisture, ash, protein, and fat content. Compared with other types of cheese, the content of tra protein was higher than that of other cheeses, and the composition of microbial flora of 43 tra samples was made by flat plate counting method. Analysis, through the analysis of lactic acid bacteria, Escherichia coli, moulds, yeasts, aerobic bacteria, Clostridium, bacillus and bacillus, we found that lactic acid bacteria and Saccharomyces cerevisiae were the dominant bacteria in the number of tra. (2) according to the colony shape and the fruit of Gram stain, and the analysis of physiological and biochemical characteristics, API carbon source fermentation test, 16SrRN A gene sequence analysis and recA test showed that 69 strains of lactobacillus were isolated from 43 samples: Lactobacillus sakei (2 strains), Lactobacillus diolivorans (1 strains), Lactobacillus casei (28 strains), Lactobacillus plantarum (34 strains), Lactobacillus kefiri (4), accounting for 49% and second Large species, accounting for 41%. (3) monitoring the dynamic changes of microbes during the production of TRA, found that lactic acid bacteria, Escherichia coli, aerobic bacteria, filamentous fungi and yeast groups can be detected to varying degrees in the production process of TRA, and lactic acid bacteria and yeast are the dominant groups involved in tra fermentation, and lactic acid fermentation is involved in lactic acid. Leuconostoc mesenteriodes, Enterococcus hirae, Enterococcus mundtii, Lactococcus lactis, Lactobacillus plantarum, Lactobacillus sakei. Es, Lactobacillusplantarum, Lactobacillus sakei are the species of Lactobacillus in raw milk. In the process of making Tra, the three kinds of lactic acid bacteria have been playing the leading role. (4) screening for the probiotic properties of 69 isolated Lactobacillus strains, the screening indexes are acid resistance, bile salt resistance, simulated human gastrointestinal fluid and bacteriostasis. Lactobacillus/splantarum 1197, Lactobacillus pl ant Arum 1141, Lactobacillus kefiri1059, Lactobacillus casei1138, Lactobacillus casei1089, Lactobacillus 1086-1, 1133. in the 7 strains. In order to synthesize various indexes, L.plantarum1086-1, L.casei1133, which was better than the other 5 strains, was selected for the next step. (5) the genetic stability of L.plantaruL.086-1 and L.casei1133 was examined for 20 generations of continuous transmission. From the phenotypic characteristics, the morphology of the two strains, the number of living bacteria, the activity of lactic acid, and the type of carbon source fermentation were found. The fermenting performance of L.plantarum1086-1, L.casei1133 was poor, and the fermentation performance decreased obviously from the fifteenth generation. The fermentation performance of L.casei1133 decreased obviously from the tenth generation. From the probiotic stability, the acid resistance of two strains of bacteria and the simulated human body were in the simulated human body. The tolerance, surface hydrophobicity and antibiotic resistance of the gastrointestinal tract were stable in the process of passage. (6) the immunoregulation function of L.plantarum1086-1 and L.casei1133 was evaluated by BALB/c mice in vivo. The results showed that the different doses of L.plantarum1086-1 and L.casei1133 could significantly improve the small intestinal pary of mice. The phagocytic activity of macrophages (P0.01), the former has no dose dependence, the latter is dose-dependent, and the high dose of L.plantarum1086-1 and L.casei1133 bacteria have no effect on the secretion of IgA in mice, but can significantly promote the secretion of IgG, IgM (P0.05, P0.01), and the high dose of L.plantarum1086-1 and L.casei1133 can greatly improve the small amount of L.plantarum1086-1 and L.casei1133. The levels of IL-6, IK-10, IFN- gamma and TNF- alpha in rat serum (P < 0.001), and L.plantarum1086-1 and L.casei1133 in different doses all significantly increased the level of IL-6, IL-10, IFN- gamma and TNF-a mRNA in mice. (7) the evaluation of antioxidative functions was carried out through the experiment in vivo. The results showed that high dose of L.plantarum1086-1 could significantly increase serum antioxidant capacity (P0.01), and the increase of L.casei1133 on serum antioxidant capacity was not significant, but there was a tendency to improve; L.plantarum1086-1 and L.casei1133 at different doses could significantly increase the mRNA expression of SOD, CAT, GSH-Px (P0.05, P0.01) in the spleen of rats. The difference between the dose groups was significant (P0.05, P0.01), showing a dose-dependent manner.
【学位授予单位】：郑州大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：TS252.54

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