优良乳酸菌的选育及在保育猪生产中的应用研究
发布时间：2018-09-08 11:40
【摘要】：对猪样品及东部地区苜蓿样品中的乳酸菌进行系统的分类鉴定及理化性能研究;筛选具有较强抑菌活性、耐受人工胃肠液环境并快速产酸的优良乳酸菌应用于保育猪生产中,评价其对保育猪生长过程中排泄物及肠道微生物多样性,以及对保育猪生长性能的影响。为抗生素低减型生猪生产找到了新的途径,为乳酸菌添加剂的制作提供优良的菌种。根据乳酸菌的生理生化特征、16S r RNA基因序列分析,可以从猪样品中一共分离到52株乳酸菌,这些菌株经过鉴定,提示一共包括了Lactobacillus reuteri,Lactobacillus acidophilus,Lactobacillus amylovorus,Lactobacillus johnsonii,Lactobacillus fermentum,Lactobacillus,Bifidobacterium,Lactobacillus salivarius,Weissella,Morganella morganii,Streptococcus alactolyticus,Enterococcus faecalis,Lactococcus lactis和Enterococcus在内的14种乳酸菌菌种。通过双层牛津杯平板法从这些菌株中筛选到2株对大肠杆菌、沙门氏菌及李斯特氏菌均有较强抑菌活性的乳酸菌P83和P84。对其抑菌活性物质做初步分析的时候可以发现,两株菌株的发酵上清液在排除酸和过氧化氢的作用后依然保持较强的抑菌活性;以上的结果表明P83和P84的发酵上清液中除了酸和过氧化氢外,还可能存在着细菌素等抑菌活性物质。从我国东部河北,上海,辽宁,江苏、山东和北京6个地区共收集到苜蓿样品125份,并且从其中分离出242株的乳酸菌,采用双层牛津杯法评定了这242株乳酸菌的抑菌活性,并且从中筛选出来了62株抑菌活性比较强的优良的乳酸菌;对这62株乳酸菌进行16S r RNA基因序列分析发现,分离到的杆菌较少共13株,其中包括魏斯氏菌属5株,共4个种:Weissella,Weissella mino,Weissella confusa和Weissella viridescens;乳酸杆菌属一共8株,一共有3个种:Lactobacillus curvatus,Lactobacillus sakei和Lactobacillus plantarum。分离到的球菌数量比较多,一共有49株球菌,其中包括了肠球菌属9株,一共有4个种:Enterococcus faecalis,Enterococcus mundtii,Enterococcus和Enterococcus hirae;明串珠菌属一共17株,一共有3个种:Leuconostoc,Leuconostoc citreum和Leuconostoc mesenteroides;戊糖片球菌属一共9株,共1个种:Pediococcus pentosaceus;乳酸乳球菌属一共14株,共3个种:Lactococcus lactis,Lactobacillus sakei和Lactococcus garvieae;通过生理生化实验以及人工模拟胃肠液实验对这些乳酸菌的一些生长的特性和对胃肠液耐受的能力进行进一步研究,其中菌株ZZU A190在p H3.0~10.0,盐浓度0~6.5%,人工肠液和人工胃液等条件下都表现出很强的耐受能力。通过生理生化实验、抑菌活性检测实验以及人工胃肠液耐受实验,对从猪样品和苜蓿样品中分离到的及实验室保存的乳酸菌进行筛选,筛选到抑菌活性较强和对人工胃肠液有较好耐受性能的菌株ZZU A190和ZZU A203作为制备乳酸菌添加剂的菌种。将它们作为添加剂进行保育猪饲喂研究的结果显示,抗生素或抗生素与乳酸菌发酵饲料联合使用对保育猪粪便中乳酸菌的数量无显著影响。单独使用乳酸菌发酵饲料不仅能够显著增加保育猪粪便中乳酸菌的含量,而且使得粪便中大肠杆菌的数量显著减少。这可能与抗生素的长期使用导致产生了致病菌的抗性菌株,致使肠道微生物菌群失调有关。与抗生素阴性对照相比,日粮中只添加乳酸菌发酵饲料或抗生素与乳酸菌发酵饲料联合添加均可以显著提高保育猪的日增重;与抗生素阳性对照相比,仅单独添加乳酸菌发酵饲料能够显著提高保育猪的日增重,降低腹泻率与死亡率。
[Abstract]:This * is a systematic identification and identification of Lactobacillus in pig samples and alfalfa samples in the eastern region, and screening of excellent lactic acid bacteria with strong antibacterial activity, tolerance to the artificial gastrointestinal fluid environment and rapid acid production. The * * is used to evaluate the diversity of feces and intestinal microflora in the growing process of conservation pigs. And the effect on the growth performance of the * * pigs. It has found a new way for the production of low production of antibiotics, and provided excellent strains for the production of lactic acid bacteria additives. According to the physiological and biochemical characteristics of lactic acid bacteria, 16S * r RNA gene sequence analysis, 52 strains of lactic acid bacteria can be isolated from pig samples. Including Lactobacillus reuteri, Lactobacillus acidophilus, Lactobacillus amylovorus, Lactobacillus johnsonii, Lactobacillus fermentum, Lactobacillus, Bifidobacterium, Lactobacillus salivarius, Weissella, Morganella morganii, Streptococcus alactolyticus, Enterococcalis, Lactobacillus lactius and Enterococcus Two strains of lactic acid bacteria P83 and P84 with strong antibacterial activity against Escherichia coli, Salmonella and Listeria were screened from these strains by double-layer Oxford cup plate method. The results showed that there might be bacteriostatic substances such as bacteriocins in the supernatant of P83 and P84 besides acid and hydrogen peroxide. 125 alfalfa samples were collected from 6 areas of Hebei, Shanghai, Liaoning, Jiangsu, Shandong and Beijing in eastern China and were separated from them. A total of 242 strains of lactic acid bacteria were isolated and evaluated by double-layer Oxford cup method, and 62 strains of lactic acid bacteria with strong antibacterial activity were screened out. Weissella, Weissella mino, Weissella confusa and Weissella viridescens; Lactobacillus, Lactobacillus curvatus, Lactobacillus sakei and Lactobacillus plantarum. A total of 49 strains of cocci were isolated, including 9 strains of Enterococcus, and 4 species: Enterococcus FA FA fa. Eccalis, Enterococcus mundtii, Enterococcus and Enterococcus hirae; Leuconostoc, Leuconostoc citreum and Leuconostoc mesenteroides; Pediococcus pentosaceus; Lactococcus lactis, Lactococcus lactis, Lactococci, Lactococcus lactis, Lactobacillus enteroides; Pediococcus pentosaceus Lus sakei and Lactococcus garvieae. Some growth characteristics of these lactic acid bacteria and their ability to tolerate gastrointestinal juice were further studied by physiological and biochemical experiments and artificial gastrointestinal juice simulation experiments. Strain ZZU A190 showed strong tolerance under the conditions of pH 3.0-10.0, salt concentration 0-6.5%, artificial intestinal juice and artificial gastrointestinal juice. Through physiological and biochemical experiments, bacteriostatic activity test and artificial gastrointestinal juice tolerance test, * lactic acid bacteria isolated from pig and alfalfa samples and lab preserved were screened. Strain ZZU A190 and ZZU A203, which had strong antibacterial activity and good tolerance to artificial gastrointestinal fluid, were screened for Lactobacillus preparation. The results of the study showed that antibiotics * or antibiotics combined with Lactobacillus fermented feed had no significant effect on the number of lactic acid bacteria * in the pig feces. Lactobacillus fermented feed alone could significantly increase the content of lactic acid bacteria in pig feces, and * This may be related to the long-term use of antibiotics resulting in the production of pathogenic bacteria resistant strains, resulting in the imbalance of intestinal microbial flora. * the daily gain of raising pigs is higher than that of the positive antibiotics. Only adding lactic acid bacteria fermented feed alone can significantly increase the daily gain * and reduce the diarrhoea rate and mortality of the pigs.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S828
[Abstract]:This * is a systematic identification and identification of Lactobacillus in pig samples and alfalfa samples in the eastern region, and screening of excellent lactic acid bacteria with strong antibacterial activity, tolerance to the artificial gastrointestinal fluid environment and rapid acid production. The * * is used to evaluate the diversity of feces and intestinal microflora in the growing process of conservation pigs. And the effect on the growth performance of the * * pigs. It has found a new way for the production of low production of antibiotics, and provided excellent strains for the production of lactic acid bacteria additives. According to the physiological and biochemical characteristics of lactic acid bacteria, 16S * r RNA gene sequence analysis, 52 strains of lactic acid bacteria can be isolated from pig samples. Including Lactobacillus reuteri, Lactobacillus acidophilus, Lactobacillus amylovorus, Lactobacillus johnsonii, Lactobacillus fermentum, Lactobacillus, Bifidobacterium, Lactobacillus salivarius, Weissella, Morganella morganii, Streptococcus alactolyticus, Enterococcalis, Lactobacillus lactius and Enterococcus Two strains of lactic acid bacteria P83 and P84 with strong antibacterial activity against Escherichia coli, Salmonella and Listeria were screened from these strains by double-layer Oxford cup plate method. The results showed that there might be bacteriostatic substances such as bacteriocins in the supernatant of P83 and P84 besides acid and hydrogen peroxide. 125 alfalfa samples were collected from 6 areas of Hebei, Shanghai, Liaoning, Jiangsu, Shandong and Beijing in eastern China and were separated from them. A total of 242 strains of lactic acid bacteria were isolated and evaluated by double-layer Oxford cup method, and 62 strains of lactic acid bacteria with strong antibacterial activity were screened out. Weissella, Weissella mino, Weissella confusa and Weissella viridescens; Lactobacillus, Lactobacillus curvatus, Lactobacillus sakei and Lactobacillus plantarum. A total of 49 strains of cocci were isolated, including 9 strains of Enterococcus, and 4 species: Enterococcus FA FA fa. Eccalis, Enterococcus mundtii, Enterococcus and Enterococcus hirae; Leuconostoc, Leuconostoc citreum and Leuconostoc mesenteroides; Pediococcus pentosaceus; Lactococcus lactis, Lactococcus lactis, Lactococci, Lactococcus lactis, Lactobacillus enteroides; Pediococcus pentosaceus Lus sakei and Lactococcus garvieae. Some growth characteristics of these lactic acid bacteria and their ability to tolerate gastrointestinal juice were further studied by physiological and biochemical experiments and artificial gastrointestinal juice simulation experiments. Strain ZZU A190 showed strong tolerance under the conditions of pH 3.0-10.0, salt concentration 0-6.5%, artificial intestinal juice and artificial gastrointestinal juice. Through physiological and biochemical experiments, bacteriostatic activity test and artificial gastrointestinal juice tolerance test, * lactic acid bacteria isolated from pig and alfalfa samples and lab preserved were screened. Strain ZZU A190 and ZZU A203, which had strong antibacterial activity and good tolerance to artificial gastrointestinal fluid, were screened for Lactobacillus preparation. The results of the study showed that antibiotics * or antibiotics combined with Lactobacillus fermented feed had no significant effect on the number of lactic acid bacteria * in the pig feces. Lactobacillus fermented feed alone could significantly increase the content of lactic acid bacteria in pig feces, and * This may be related to the long-term use of antibiotics resulting in the production of pathogenic bacteria resistant strains, resulting in the imbalance of intestinal microbial flora. * the daily gain of raising pigs is higher than that of the positive antibiotics. Only adding lactic acid bacteria fermented feed alone can significantly increase the daily gain * and reduce the diarrhoea rate and mortality of the pigs.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S828
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