抗汞益生乳酸菌筛选及其缓解汞毒性机制研究

发布时间：2018-06-06 00:34

本文选题：汞 + 乳酸菌　；参考：《东北农业大学》2017年硕士论文

【摘要】：重金属污染由于其不可生物降解性、危险性和毒害性不仅使环境受到破坏,更威胁到了人类和动物的公共健康。汞是机体内一种非必需的重金属元素,可以通过食物链积累,大大增加机体受到损害的风险。本研究旨在分离、筛选耐汞益生乳酸菌,完善乳酸菌解重金属中毒的机理,同时为解汞中毒提供一个新的思路及理论基础,为将来乳酸菌作为生物制剂或者动物饲料中的添加剂打下基础。本试验从健康动物肠道内容物中分离出有耐汞特性的乳酸菌,对分离出的菌株进行鉴定及益生试验进行筛选,挑选出具有优良耐汞性能的益生菌株,筛选抗性基因,随后进行动物试验,通过抗氧化应激、细胞因子的检测及p38 MAPK和NF-κB通路的表达来探究筛选的益生乳酸菌解重金属汞中毒的机理。主要研究结果如下:(1)从健康的猪、马、牛、羊、犬和鸡肠道内容物中共分离出83株耐汞(25mg/L)菌株,经触酶试验、革兰氏染色、耐酸、耐胆盐试验共筛选出21株触酶试验阴性、耐强酸(pH=2)、耐高浓度胆盐(0.8%)的革兰氏阳性菌。(2)经抑菌试验、药敏试验、体外抗氧化试验及汞离子吸附能力测定筛选出4株能够对五种致病菌标准菌株有抑制作用、对抗生素普遍敏感、有较优良体外抗氧化能力且在体外对汞离子吸附能力在90%以上的优势菌株。(3)经分子生物学鉴定,四株菌株分别为两株短乳杆菌(Lactobacillus brevis)、一株嗜淀粉乳杆菌(Lactobacillus amylovorus)、一株约氏乳杆菌(Lactobacillus johnsonii)。(4)对4株乳酸菌进行汞及其他重金属MIC测定,结果显示菌株耐汞能力在50~58 mg/L,同时对硒、镉和镍都有不同程度的耐受能力。(5)综合体外各项指标的结果,选出最优菌株23017进行抗性基因的筛选,结果显示抗性基因开放阅读框区域与乳酸菌水通道蛋白Aqy1基因相似度为96%~100%,证实为水通道蛋白Aqy1基因。(6)测定小鼠的半数致死量为52.69mg/kg,建立急性汞中毒小鼠模型,采用筛选出的最优势菌株短乳杆菌23017进行益生菌缓解金属汞中毒的动物试验,有如下结果:a急性汞中毒可使小鼠体重大幅度减轻,短乳杆菌23017可以有效减缓体重下降;b通过病理切片显示短乳杆菌23017可有效缓解急性汞中毒引起的机体肝脏、肾脏以及肠道的损伤;c急性汞中毒后可以在肝脏、肾脏部位形成蓄积,部分可通过粪便排出体外,短乳杆菌23017可加速汞的排出,减少肝脏和肾脏的负担;d短乳杆菌23017可加速急性汞中毒后引起的体内氧化-抗氧化系统平衡的恢复;e短乳杆菌23017可使急性汞中毒引起的促炎、抑炎因子以及紧密结合蛋白的调控失衡得到缓解。f急性汞中毒可以使p38 MAPK通路和NF-κB通路激活,短乳杆菌23017可以调节相关细胞因子及蛋白表达以缓解炎性通路带来的损伤。
[Abstract]:Heavy metal pollution not only destroys the environment, but also threatens the public health of human and animals because of its non-biodegradability. Mercury is a non-essential heavy metal element in the body, which can accumulate through the food chain and greatly increase the risk of damage to the body. The purpose of this study was to isolate and screen mercury-tolerant probiotic lactic acid bacteria, improve the mechanism of heavy metal poisoning by lactic acid bacteria, and provide a new way of thinking and theoretical basis for mercury poisoning. To lay a foundation for the future of lactic acid bacteria as biological agents or animal feed additives. In this study, mercury tolerant lactic acid bacteria were isolated from intestinal contents of healthy animals. The strains were identified and prebiotic tests were carried out to select probiotic strains with excellent mercury tolerance and to screen resistant genes. Then animal experiments were conducted to explore the mechanism of heavy metal mercury poisoning by screening probiotic lactic acid bacteria through antioxidant stress cytokine detection and the expression of p38 MAPK and NF- 魏 B pathways. The main results were as follows: (1) A total of 83 strains of mercury-resistant 25 mg / L mercury-resistant strains were isolated from healthy pig, horse, cattle, sheep, dog and chicken intestinal contents. A total of 21 strains were screened negative by thixozyme test, Gram staining, acid tolerance and bile salt tolerance test. Through bacteriostatic test, drug sensitivity test, antioxidant test in vitro and mercury ion adsorption ability test, 4 strains were selected to inhibit five pathogenic bacteria standard strains, which were resistant to strong acid and high concentration of bile salt (0.8% gallbladder salt), and 4 strains were screened out by antimicrobial test, drug sensitivity test, in vitro antioxidant test and mercury ion adsorption ability test. The dominant strain, which is generally sensitive to antibiotics, has excellent antioxidant capacity in vitro and adsorbs more than 90% mercury ions in vitro, has been identified by molecular biology. Four strains of Lactobacillus brevisvisus, Lactobacillus amylovorusus and Lactobacillus johnsoniii.f.) were tested for mercury and other heavy metals MIC. The results showed that the mercury tolerance of the strain was 5058 mg / L, and that of selenium. Cadmium and nickel have different degrees of tolerance. 5) combined with the results of in vitro indicators, the optimal strain 23017 was selected for screening resistance genes. The results showed that the similarity between the open reading frame region of resistance gene and the Aqy1 gene of Lactobacillus aquaporin (Aqy1) gene was 96,100, and the median lethal dose was 52.69 mg / kg. The animal experiment of alleviating mercury poisoning by probiotics was carried out with the most dominant strain of Lactobacillus brevis 23017. The results showed that the weight of mice could be greatly reduced by the acute mercury poisoning of 1: a. Lactobacillus brevis 23017 can effectively slow down weight loss. Pathological sections show that Lactobacillus brevis 23017 can effectively relieve acute mercury poisoning in the liver, kidney and intestine after acute mercury poisoning. The kidney accumulates, partly through feces, and Lactobacillus breast-23017 accelerates the release of mercury. Reducing the burden on the liver and kidney by Lactobacillus brevis 23017 can accelerate the recovery of the balance of oxidation-antioxidant system in vivo caused by acute mercury poisoning. Lactobacillus brevis 23017 can promote inflammation caused by acute mercury poisoning, The imbalance of anti-inflammatory factor and tight binding protein was alleviated. F acute mercury poisoning could activate p38 MAPK pathway and NF- 魏 B pathway. Lactobacillus brevis 23017 could regulate the expression of related cytokines and proteins to alleviate the injury caused by inflammatory pathway.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S816.7

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