酸马奶源乳酸乳球菌代谢物和大黄联合作用对小鼠盲肠微生物以及营养代谢的影响

发布时间：2018-04-17 13:28

本文选题：乳酸乳球菌代谢物 + 牛源致病性大肠杆菌O_(78)　；参考：《内蒙古农业大学》2016年硕士论文

【摘要】：内蒙古锡林郭勒牧区酸马奶是古老的的低酒精乳饮品之一。现代医学证明酸马奶能够辅助性治疗结核病、降三高、改善消化系统等。近年来食品安全问题已经引起全球人民广泛关注,由Escherichia coli O157:H7、Listeria monocytogenes等致病菌引发的疾病导致了巨大经济损失。解决由Escherichia coli引起的感染,减少畜牧业损失,已经成为当务之急。微生物代谢物作为一种安全且无毒副作用的天然防腐剂,已经引起科研工作者的重视。因此,本试验从酸马奶中分离筛选出和大黄联合用药对E. coli O78有较强抑菌作用的菌株。并通过体内试验,研究对感染E. coli O78小鼠营养代谢和盲肠微生物以及相互关系的影响。试验一：产生抑菌物质乳酸乳球菌的分离筛选和鉴定酸马奶划线接种分离纯化后,观察菌落形态、进行革兰氏染色、显微镜观察以及接触酶试验,其中革兰氏染色为球菌,接触酶试验阴性,牛津杯法筛选有抑菌物质的菌株进一步纯化后,进行生理生化鉴定,16S rRNA扩增鉴定。结合生理生化鉴定,对E.coli O78抑菌效果较好的菌株,进一步进行16S rRNA鉴定,确定该菌株为Lactococcus lactis subsp. LactiS。试验二：乳酸乳球菌代谢物的提取以及体外抑菌测定利用酸碱法提取乳酸乳球菌代谢物,牛津杯法测定抑菌直径,微量平板和TTC法测定最小抑菌浓度(MIC)。乳酸乳球菌代谢物与EDTA联合用药MIC为0.063mg/mL+0.2mg/mL, FIC=0.41。乳酸乳球菌代谢物和大黄联合用药MIC为0.033mg/mL+10.417mg/mL, FIC=0.20.结果表明,乳酸乳球菌代谢物和大黄联合作用效果优于乳酸乳球菌代谢物与EDTA联合作用,更适用于后续试验。试验三：乳酸乳球菌代谢物和大黄联合作用对致病性E. coli O78抑菌机理的测定酶标比浊法测定乳酸乳球菌代谢物与大黄联合作用对致病性E. coli O78抑菌生长曲线的影响。利用AKP和BCA试剂盒,分别检测联合作用对致病性E. coli O78细胞壁和细胞膜通透性的影响。结果表明,联合用药对致病性E. coli O78的抑菌生长曲线呈现“S“型,破坏致病性E. coli O78的细胞壁和细胞膜完整性,使通透性增强,细胞质外渗,增加菌液中蛋白质和碱性磷酸酶含量。试验四：乳酸乳球菌与大黄联合作用对E. coli O78体内抑菌作用(1)致病性E.coli O78对小鼠80%致死率的确定选取昆明系健康小鼠60只,随机分为6组,每组10只。E.coli O78菌液10倍稀释,小鼠腹腔注菌后开始观察小鼠死亡情况,统计小鼠死亡率,选择致小鼠80%死亡的菌浓度,进行后续试验。(2)联合用药最优灌胃剂量的确定选取健康昆明系小鼠110只,公母各半,随机分为11组,每组10只,分别为空白对照组、阳性对照组(盐酸环丙沙星)、阴性对照组(生理盐水)、乳酸乳球菌代谢物组中(0.0150g/kg·bw)、低(0.0075g/kg·bw)剂量组、大黄中(4g/kg·bw)、低(2g/kg·bw)剂量组以及联合用药L1(乳酸乳球菌代谢物0.0150g/kg·bw+大黄2g/kg·bw)、L2(乳酸乳球菌代谢物0.0075g/kg·bw+大黄2g/kg·bw)、L3(乳酸乳球菌代谢物0.0150g/kg·bw+大黄4g/kg·bw)、L4(乳酸乳球菌代谢物0.0075g/kg·bw+大黄4g/kg·bw)。试验期7d,第4d分别灌胃后1h腹腔注射80%致死量E.coli O78。小鼠腹腔注菌后,观察小鼠采食量、饮水次数、被毛粗乱情况、死亡率等指标。结果表明,小鼠灌胃盐酸环丙沙星最优剂量为0.13g/kg·bw,乳酸乳球菌代谢物最优剂量为0.0150g/kg·bw,大黄最优剂量为2g/kg·bw,联合用药最优剂量为L1(乳酸乳球菌代谢物0.0150g/kg·bw+大黄2g/kg·bw)。(3)联合用药对E.coli O78感染小鼠营养代谢和盲肠微生物及其相互作用关系的影响选取健康昆明系小鼠120只,随机分为6组,每组20只,分别为空白对照组、阳性对照组、阴性对照组、乳酸乳球菌代谢物组、大黄组、联合用药组,按照(2)中最优剂量进行灌胃,第4d腹腔注菌后0h、24h、48h、72h眼球采血收集血液,同时无菌采集盲肠内容物,进行盲肠微生物试验。结果表明,联合用药能增加小鼠盲肠内乳酸杆菌和双歧杆菌数量,增强乳酸脱氢酶活性,降低盲肠内氨氮含量,降低血糖和血脂,增加胰岛素和胰高血糖素水平。
[Abstract]:Inner Mongolia is the ancient Xilinguole pastoral koumiss low alcohol milk drinks. One of the modern medical certificate koumiss can tuberculosis, adjuvant therapy for reducing blood, improve the digestive system. In recent years, food safety problem has already caused widespread concern by the global people, Escherichia coli O157:H7, Listeria monocytogenes and other pathogens caused by the disease causes huge economic losses solve. Coli infection caused by Escherichia, reduce the loss of animal husbandry, has become a pressing matter of the moment. Microbial metabolites as a safe and non-toxic side effects of natural preservatives, has attracted the attention of researchers. Therefore, this test from Koumiss in drug screening and separation combined with rhubarb had a strong inhibitory effect on the strain E. coli O78. And through in vivo experiments, research on the infection of E. coli mice of O78 nutrition metabolism and caecum microorganism and their relationship The effect of the test: the separation and purification. Screening and identification of Koumiss isolated streak inoculation produce antibacterial substances of Lactococcus lactis after observation of colony morphology, Gram staining, microscopy and contact enzyme test, including gram negative cocci, catalase test, Oxford cup method screening for further purification of antibacterial substances of strain physiological and biochemical identification, 16S, rRNA amplification. Physiological and biochemical identification of inhibitory effect on E.coli O78 strains, further 16S rRNA identification, this strain was identified as Lactococcus lactis subsp. LactiS. two test: extraction and determination of lactic acid galactococcus metabolites by acid-base method of Lactococcus lactis metabolites extraction and in vitro antibacterial, determination of antibacterial diameter of Oxford cup method of determination of the minimum inhibitory concentration of micro plate and TTC method (MIC). The Lactococcus lactis metabolites and drug combination was 0. MIC EDTA 063mg/mL+0.2mg/mL FIC=0.41., Lactococcus lactis metabolites and Rhubarb Combination of MIC 0.033mg/mL+10.417mg/mL and FIC=0.20.. The results show that the combined effects of lactic acid galactococcus metabolites and rhubarb combined effect is better than the Lactococcus lactis metabolites and EDTA, more suitable for subsequent experiments. Experiment three: Determination of the combined effects of lactic acid galactococcus metabolites and rhubarb on the growth curve of coli O78 bacteria pathogenic E. turbidimetric determination of enzyme combined effect of Lactococcus lactis and metabolites of rhubarb on pathogenicity of E. coli O78. The antibacterial mechanism by using AKP and BCA kit were detected the effect on pathogenic E. coli O78 cell wall and cell membrane permeability. The results showed that the antibacterial drug combination on pathogenic E. coli O78 growth curve show "S" type, cell wall and cell membrane integrity damage of pathogenic E. coli O78, to enhance the permeability, Cytoplasm extravasation, increase the protein content of alkaline phosphatase and bacteria. Experiment four: the combined effect of Lactococcus and rhubarb on E. coli O78 in vivo antibacterial effect (1) pathogenic E.coli O78 on mice 80% death rate of the selected Kunming Department of health 60 mice were randomly divided into 6 groups, 10 rats in each group.E.coli O78 broth 10 times dilution, observation of the death of mice began to mice after inoculation, the death rate of mice, 80% mice induced by selection of bacteria and the concentration of death, for further test. (2) to determine the optimal combination of intragastric dose of 110 healthy Kunming mice were selected, male and female, were randomly divided into 11 groups, 10 rats in each group respectively, the blank control group, positive control group (ciprofloxacin hydrochloride), negative control group (saline), Lactococcus lactis group (0.0150g/kg metabolite BW), low dose group (0.0075g/kg - BW), big yellow (4g/kg - BW), low dose group (2g/kg - BW) And the combination of L1 (0.0150g/kg - bw+ metabolites of Lactococcus lactis rhubarb 2g/kg - BW), L2 (0.0075g/kg - bw+ metabolites of Lactococcus lactis rhubarb 2g/kg - BW), L3 (0.0150g/kg - bw+ metabolites of Lactococcus lactis rhubarb 4g/kg - BW), L4 (0.0075g/kg - bw+ metabolites of Lactococcus lactis rhubarb 4g/kg - BW). The test period 7d. 4D respectively after intragastric administration of intraperitoneal injection of 1H 80% lethal injection in abdominal cavity of O78. mice after E.coli, mice intake, drinking times, by wool thick chaos, mortality and other indicators. The results show that the optimal dose of ciprofloxacin hydrochloride orally for 0.13g/kg BW, the optimal dose of 0.0150g/kg metabolites of Lactococcus lactis BW, rhubarb optimal the dose was 2g/kg / BW, the combination optimal dose of L1 (0.0150g/kg - bw+ metabolites of Lactococcus lactis rhubarb 2g/kg - BW). (3) the combination of E.coli mice infected with O78 nutrition metabolism and caecum microorganism and their interactions The relationship between health and 120 healthy Kunming mice were randomly divided into 6 groups, 20 rats in each group, respectively. The blank control group, positive control group, negative control group, Lactococcus lactis metabolite group, rhubarb group, combination group (2), according to the optimal dose by intragastric administration by intraperitoneal injection of bacteria after 4D 0h, 24h, 48h, 72h eye blood collected blood, and cecal contents were collected aseptically, cecal microbial test. The results show that the combination can increase the number of mice in cecal Lactobacillus and Bifidobacterium, enhanced the activity of lactate dehydrogenase, reduced ammonia nitrogen content in the cecum, lowering blood glucose and blood lipids, increase insulin and glucagon level.

【学位授予单位】：内蒙古农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S816

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