乳酸菌理化特性及其对鸡白痢沙门氏菌的拮抗作用研究

发布时间：2018-05-14 00:16

  本文选题：发酵乳酸菌1.2133 + 理化特性　； 参考：《安徽农业大学》2017年硕士论文

【摘要】：畜禽抗生素的滥用对畜禽产品造成了药物残留和耐药性菌株的污染等问题,严重危害了动物性食品的质量与安全,造成了严重的社会公共卫生和食品安全。动物微生态制剂是绿色抗生素替代品之一,具有广泛的应用价值。乳酸菌是研制动物微生态制剂的典型菌种,对动物肠道内环境微生态的稳定具有调节作用,对宿主肠道疾病具有防治作用。鸡白痢沙门氏菌(Salmonella pullorum)是危害养鸡业最严重的肠道细菌病之一,是重要的公共卫生问题。防治S.pullorum对鸡的感染,对保障家禽健康养殖和动物性食品质量与安全具有重要的意义。在乳酸菌属中,乳酸杆菌是最主要的一类,其中包括干酪乳杆菌(Lactobacillus casei)、发酵乳杆菌(Lactobacillus fermentum)、嗜酸乳杆菌(Lactobacillus acidophilus)等。L.fermentum天然存在肠道具有很强的胃肠道环境耐受力等优点,有关其在预防和治疗S.pullorum感染的报道较少。因此,本研究旨在体外筛选出能抑制S.pullorum生长的乳酸菌株,以此为基础,研究其体内外对S.pullorum的抗感染作用,进一步阐述乳酸菌对鸡抗病原菌感染的保护性作用研究。具体研究内容和结果如下:1.乳酸菌理化特性及抑菌活性比较在本实验室乳酸菌筛选的基础上,选取L.casei1.2435、L.fermentum1.2133、L.acidophilus1.1854、S.thermophillus 1.2471,4株乳酸菌进行抑菌特性比较分析。通过耐酸性和胆盐耐受实验、细胞黏附实验、疏水性实验、抑菌实验进行乳酸菌理化特性比较。实验结果表明,4株乳酸菌在pH值2.5的培养基中培养2h后,L.fermentum1.2133的存活率达到68%,显著高于其他3株菌。在胆盐浓度为0.3%的培养基中培养2h后,L.fermentum1.2133存活率大于60%。表面疏水性实验结果表明,各菌株间比较差异显著,L.fermentum 1.2133具有较高的表面疏水性;各菌株对目标菌株均具有凝聚能力,其中L.fermentum1.2133自聚合能力最强。抑菌实验结果表明,4株乳酸杆菌对病原菌E.coli AE17、E.coli O78以及S.pullorum CVCC533均具有一定的抑菌能力,且存在菌株差异性。L.fermentum1.2133对S.pullorum CVCC533有显著的抑菌能力,L.acidophilus1.1854对E.coli AE17抑菌活性显著,L.casei1.2435对E.coli O78抑菌能力显著,S.thermophillus1.2471抑菌活性较弱。2.L.fermentum1.2133拮抗S.pullorum CVCC533体外抑菌作用观察采用荧光显微镜对L.fermentum1.2133发酵上清作用于S.pullorum CVCC533生物被膜中细菌的生存能力进行观察,在L.fermentum1.2133发酵上清液与S.pullorum CVCC533共培养24h后,显微镜中观察到实验组中S.pullorum CVCC533的生物被膜中S.pullorum CVCC533菌体大部分呈现红色荧光,只有少量点状的绿色荧光;对照组视野中基本为绿色荧光,仅有少量红色荧光。在体外对L.fermentum1.2133与S.pullorum CVCC533进行竞争性共培实验的结果表明,在0-18h,L.fermentum1.2133对S.pullorum CVCC533的生长抑制随着时间的推移逐渐增强,S.pullorum的数量逐渐减少,到18h后,趋于稳定。L.fermentum1.2133对S.pullorum CVCC533生长具有拮抗作用,且不同的初始菌液浓度形成的竞争性效果存在差异,菌液浓度10:1的竞争性优于1:1。3.L.fermentum1.2133对感染S.pullorum CVCC533的鸡肠道菌群和组织病理学观察将120只1日龄的罗曼肉鸡随机分为4个组,分别为对照组(第I组)、1日龄开始灌喂L.fermentum1.2133(2.5×108 cfu/chick)组(第II组)、1日龄开始灌喂L.fermentum1.2133至14日龄攻毒S.pullorum CVCC533(5×108 cfu/chick)的预处理组(第III组)以及第14日龄直接感染S.pullorum CVCC533组(第IV组),分别进行肠道细菌计数和病理切片观察。结果表明,鸡感染后24h,回肠和盲肠细菌数均发生变化,第II组乳酸菌数量显著高于第III组、第IV组,而沙门氏菌数量明显有所减少,好氧菌总数增多。第II组鸡的十二指肠、空肠、回肠肠状态正常,肠绒毛完整,无明显病理变化较第I组鸡小肠形态发育更完整;第III组鸡十二指可见肠绒毛固有层出现轻微肠炎性细胞侵润;空肠绒毛中心轴有部分红细胞,回肠绒毛表现局部损伤。第IV组十二指肠绒毛断裂,绒毛自溶现象明显,空肠绒毛中心轴红细胞累积,回肠肠绒毛上皮的杯状细胞被溶解,绒毛自溶现象明显,第III组与第IV组相比较,鸡小肠各肠段的炎症和出血点等症状减轻。4.L.fermentum1.2133对感染S.pullorum CVCC533的鸡肠道AvBD2 mRNA转录水平的影响本实验采用RT-PCR方法,测定了L.fermentum1.2133对感染S.pullorum CVCC533的鸡肠道AvBD2 mRNA的转录水平。在鸡感染72 h内(0 h,12 h,24 h,36、48h和72h)采鸡小肠各段组织。结果表明,AvBD2在小肠各段中均有表达。在整个时间段内,第II组AvBD2 mRNA的转录水平在鸡小肠内的转录水平基本呈稳定趋势且转录水平显著高于第I组。第III组和第IV组小肠段AvBD2 mRNA的转录水平与第II组差异显著;第III组在24h后,AvBD2 mRNA的转录水平基本呈波动性变化趋势降低,第IV组呈显著性下降,都显著低于第I组转录水平,实验结果表明,L.fermentum1.2133对鸡肠道防御素AvBD2的表达具有调控作用。上述研究表明,L.fermentum1.2133对鸡肠道内环境具有很好耐受能力,且其发酵上清液对S.pullorum CVCC533生物被膜的中S.pullorum CVCC533有显著的抑菌作用;能稳定定植于肠道,对鸡肠道菌群有调节作用,同时还能促进鸡小肠内β-防御素2的表达量,在抗S.pullorum CVCC533感染过程中发挥着重要的作用。
[Abstract]:The abuse of livestock and poultry antibiotics caused the drug residues and the contamination of drug resistant strains, seriously endangering the quality and safety of animal food, causing serious social public health and food safety. Animal microecological preparation is one of the substitute of green antibiotics and has extensive application value. Lactic acid bacteria is the development of the animal products. The typical strains of animal microecological agents regulate the stability of the microecological environment in the intestinal tract of animals and have the control effect on the intestinal diseases of the host. Salmonella pullorum is one of the most serious intestinal bacterial diseases endangering the poultry industry, and it is an important public health problem. The prevention and control of S.pullorum infection to chickens. It is of great significance to protect the health of poultry and the quality and safety of animal food. Lactobacillus is the most important category in the genus Lactobacillus, including Lactobacillus casei (Lactobacillus casei), Lactobacillus fermentum, Lactobacillus acidophilus (Lactobacillus acidophilus) and other natural intestinal tract. With the advantages of strong tolerance to the gastrointestinal environment and so on, there are few reports on the prevention and treatment of S.pullorum infection. Therefore, this study aims to screen out the lactic acid strains that can inhibit the growth of S.pullorum in vitro. On the basis of this study, the anti infection effect of S.pullorum in vitro and in vitro and in vivo is studied, and the resistance of lactic acid bacteria to the chicken is further elaborated. Study on the protective effect of dyeing. The specific contents and results are as follows: 1. comparison of physical and chemical properties and bacteriostasis activity of lactic acid bacteria on the basis of lactic acid bacteria screening in our laboratory, the bacteriostasis characteristics of L.casei1.2435, L.fermentum1.2133, L.acidophilus1.1854 and S.thermophillus 1.2471,4 strains were compared and analyzed. Acid resistance and bile salt were used. Tolerance test, cell adhesion experiment, hydrophobic experiment and bacteriostasis experiment were used to compare physical and chemical properties of lactic acid bacteria. The experimental results showed that after 4 strains of lactic acid bacteria were cultured in the medium of pH value 2.5, the survival rate of L.fermentum1.2133 reached 68%, which was significantly higher than that of other 3 strains. After the culture of 2h in the medium with the concentration of bile salt concentration 0.3%, L.fermentum1.2133 survived. The results of the hydrophobicity greater than the 60%. surface showed that there was a significant difference among the strains, and the L.fermentum 1.2133 had a high surface hydrophobicity, and all the strains had the ability to condense to the target strains, and the self polymerization ability of L.fermentum1.2133 was the strongest. The results of bacteriostasis experiment showed that 4 strains of lactobacillus were E.coli AE17, E.coli O78. And S.pullorum CVCC533 all had certain bacteriostasis ability, and the existence of strain differential.L.fermentum1.2133 had significant inhibitory ability to S.pullorum CVCC533, L.acidophilus1.1854 had significant antibacterial activity to E.coli AE17, L.casei1.2435 was significant to E.coli O78, and the inhibitory activity of S.thermophillus1.2471 was weak. The bacteriostasis effect of anti S.pullorum CVCC533 in vitro was observed by fluorescence microscopy on the viability of bacteria in S.pullorum CVCC533 biofilm by L.fermentum1.2133 fermentation supernatant. After L.fermentum1.2133 fermentation supernatant and S.pullorum CVCC533 were co cultured 24h, the S.pullorum CVCC533 in the experimental group was observed in the micromirror. Most of the S.pullorum CVCC533 mycelium in the biofilm showed red fluorescence, only a small number of spot green fluorescence; the control group was basically green fluorescence and only a small amount of red fluorescence. The results of competitive co culture experiment on L.fermentum1.2133 and S.pullorum CVCC533 in vitro showed that in 0-18h, L.fermentum1.2133 to S.pullor. The growth inhibition of UM CVCC533 gradually increased with time, and the number of S.pullorum gradually decreased. After 18h, a stable.L.fermentum1.2133 had an antagonistic effect on S.pullorum CVCC533 growth, and the competitive effects of different initial bacteria concentration were different, and the competitiveness of the liquid concentration 10:1 was better than 1:1.3.L.fermentum1.21. 33 S.pullorum CVCC533 infected chicken intestinal tract flora and histopathological observation of 120 1 day old Roman broilers were randomly divided into 4 groups, respectively, the control group (group I), 1 days of age to start feeding L.fermentum1.2133 (2.5 x 108 cfu/chick) group (group II), 1 days of age to start feeding L.fermentum1.2133 to 14 days old S.pullorum CVCC533 (5 * 108. Cfu/chick) pretreated group (group III) and fourteenth day old direct infection S.pullorum CVCC533 group (group IV), the intestinal bacterial count and pathological section were observed respectively. The results showed that the number of bacteria in the ileum and the cecum were all changed after the chicken infection, and the number of lactic acid bacteria in group II was significantly higher than that of the group III, group IV, and the number of Salmonella was obvious. The number of aerobic bacteria increased. The duodenum, jejunum, ileum and intestinal villi in group II were normal and the intestinal villi were intact. No obvious pathological changes were found in group I. The twelve fingers of group III showed slight enteritis invasion of the lamina propria; the central axis of the jejunum had some red cells and ileum. In group IV, the duodenal villi was broken, the villus autolysis was obvious, the central axis of the jejunum villi was accumulated, the goblet cells in the intestinal villi epithelium were dissolved and the villus autolysis was obvious. Group III was compared with the group IV, and the symptoms of the intestinal segments of the small intestine of the chicken reduced the sense of.4.L.fermentum1.2133 to the feeling. The transcriptional level of AvBD2 mRNA in chicken intestinal tract infected with S.pullorum CVCC533 was influenced by RT-PCR method, and the transcriptional level of AvBD2 mRNA of chicken intestinal tract infected with S.pullorum CVCC533 was measured by L.fermentum1.2133. In chicken infection 72 h (0 h, 12, 24, and so), it was found that all segments of the small intestine of chicken were found in all segments of the small intestine. In the whole time period, the transcriptional level of AvBD2 mRNA in group II was basically stable in the small intestine of the chicken and the transcriptional level was significantly higher than that in the I group. The transcriptional level of AvBD2 mRNA in group III and IV group was significantly different from that in the group II; in the III group, the transcriptional level of AvBD2 AvBD2 was basically fluctuant after 24h. The potential of group IV decreased significantly and was significantly lower than that of group I. The results showed that L.fermentum1.2133 had a regulatory effect on the expression of chicken intestinal defensin AvBD2. The above study showed that L.fermentum1.2133 was well tolerated in the environment of chicken intestinal tract, and the fermentation supernatant had a biofilm of S.pullorum CVCC533 biofilm. S.pullorum CVCC533 has significant bacteriostasis, stable colonization in the intestinal tract, regulating the intestinal flora of Chicken Intestines, and also promoting the expression of beta defensin 2 in the small intestine of chicken, and plays an important role in the anti S.pullorum CVCC533 infection process.

【学位授予单位】：安徽农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.31

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