【作者】 张燕；

【导师】 敖日格乐；

【作者基本信息】 内蒙古农业大学 ， 动物生产学， 2016， 硕士

【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。更多还原

【Abstract】 Inner Mongolia Xilinguole pastoral koumiss become old low alcohol milk drinks. Modern medicine can prove koumiss adjuvant treatment of tuberculosis, reducing blood and improve the digestive system. In recent years, food safety issues have aroused widespread concern around the world, caused by Escherichia.coli O157:H7、 monocytogenes Listeria and other diseases caused by a huge economic losses caused by the disease. To solve the infection caused by Escherichia.coli, reduce the loss of animal husbandry, has become a top priority. Microbial metabolites as a safe and non-toxic side effects of natural preservatives, has attracted the attention of researchers. Therefore, this test from Koumiss separation screened antibacterial effect of strong strain and rhubarb combination, together to suppress the E.coli O78. Through in vivo experiment, the research of the E.coli O78 on the nutrition metabolism and the microorganism in the appendix of the infected mice.Experiment 1:The isolation and identification of antibacterial substance produced by Lactococcus lactisKoumiss crossed inoculated with the isolated and purified to observe colony morphology, Gram staining, microscopy and catalase test, Gram staining for cocci, catalase test was negative, Oxford cup method for screening the antibacterial substances of strains for further purification, physiological and biochemical identification,16S rDNA amplification assay. Combined with physiological and biochemical identification, the antibacterial pathogenicity E.coli O78 effect of better strains, further 16S rRNA identification, to determine the strain for Lactococcus lactis subsp. lactis.Experiment 2:Lactococcus lactis crude extracts and in vitro antibacterial metabolite determinationThe acid-base extraction of Lactococcus lactis metabolites and Oxford cup method was used to measure the bacteriostasis diameter and microtiter plate and TTC method determination of minimal inhibitory concentration (MIC). Lactococcus lactis metabolites with EDTA combination of MIC 0.063mg/mL+0.2mg/mL, FIC=0.41. Lactococcus lactis metabolites and Rhubarb Combination of MIC 0.033mg/mL+10.417mg/mL, FIC=0.20. The results show that the combined effect of Lactococcus lactis metabolites and rhubarb combined action effect is better than that of Lactococcus lactis metabolites with EDTA, more suitable for in subsequent trials.Experiment three:Determination of the combined effect of lactic acid galactococcus metabolites and antibacterial mechanism of rhubarb on pathogenicity E.coli O78Enzyme labeled turbidimetric method determination of Lactococcus lactis metabolites with rhubarb combined effects on pathogenicity E.coli O78 inhibitory growth curve and by alkaline phosphatase and BCA kit combined effects were detected on pathogenicity E.coli O78 cell wall and cell membrane permeability of the impact.The results show that the combination of pathogenicity E.coli O78 antibacterial growth curve showed "s" type, damage the integrity of cell wall and cell membrane of pathogenicity E.coli O78, enhance the permeability and extravasation of cytoplasm, increase in the bacteria protein and alkaline phosphatase (AKP).Experiment four:the combined effect of Lactococcus lactis and rhubarb in vivo antibacterial effect of on pathogenicity E.coli O78(1) The determination of the 80% lethal rate of pathogenicity E.coli O78 in mice60 Kunming healthy mice were selected and randomly divided into 6 groups,10 rats in each group.10 times pathogenicity E.coli O78 broth 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, follow-up test.(2)The determination of optimal dosage of combined administration of Medicine110 healthy Kunming mice were selected, male and female, were randomly divided into 11 groups,10 rats in each group were control group, positive control group (ciprofloxacin hydrochloride), negative control group (saline), Lactococcus lactis group (0.0150g/kg·bw), metabolite low dose group (0.0075g/kgbw),rhubarb (4g/kg-bw), low dose group (2g/kg·bw) and the combination of Li (lactic acid galactococcus metabolites 0.0150g/kg·bw+rhubarb 2g/kg·bw), L2 (lactic acid galactococcus metabolites 0.0075g/kg·bw+rhubarb 2g/kg·bw), L3 (Lactococcus lactis0.0150g/kg·bw+rhubarb metabolite 4g/kg·bw), L4 (lactic acid galactococcus metabolites 0.00.75g/kg·bw+ rhubarb 4g/kg·bw). Test period 7d, in 4d after filling the stomach with 1h the stomach injected into intraperitoneal injection of 80% lethal dose of pathogenicity E.coli O78. After intraperitoneal injection of mice, the feed intake, the number of drinking water, the gross disorder, mortality and other indicators were observed. The results show that the intragastric administration of ciprofloxacin hydrochloride in the optimal dose for 0.13g/kg·bw, Lactococcus lactis optimal dose for 0.0150g/kg·bw, the optimal dose of rhubarb 2g/kg·bw, combination optimal dose for L1 (Lactococcus lactis metabolites 0.0150g/kg·bw+rhubarb 2g/kg·bw).(3) The effects of combined administration of drugs on the nutritional metabolism and the relationship between the pathogenicity E.coli O78 and the micro flora and their interaction in mice120 healthy Kunming mice were selected and divided into 6 groups, with 20 rats in each group, compared with blank control group, positive control group, negative control group, Lactococcus lactis metabolite group, rhubarb group, combined treatment group, according to the (2) the optimal dose of intragastric administration,4d after intraperitoneal injection of bacteria after Oh,24h,48h,72h eyeball blood collected blood, while the sterile acquisition cecal contents, microecology of the caecum test.The results showed that the combination therapy can increase the number of lactobacilli and bifidobacteria in the caecum of mice, enhanced the activity of lactate dehydrogenase and reduce the content of ammonia nitrogen in the cecum, lowering blood glucose and blood lipids, insulin and glucagon levels increased.更多还原