饲用益生菌的筛选及其菌剂制备技术研究
发布时间：2018-10-15 19:23
【摘要】：罗伊氏乳杆菌作为一种重要的动物微生态制剂中的益生菌,其菌体本身及代谢产物能够抑制仔猪肠道过路菌和病原菌的粘附及生长,因此能够提高仔猪的免疫力和降低仔猪死亡率,从而大大的改善了养殖行业的质量。本课题是从畜禽粪便中有针对性、目的性地筛选出产酸菌株,且对其进行体外性能评价。主要研究如下:(1)饲用益生菌的筛选。经过初筛及耐酸耐胆盐复筛,从仔猪粪便中筛选出三株产酸菌株,通过菌体特征、糖发酵等生理生化实验以及分子生物学鉴定,最后确定其为罗伊氏乳杆菌,且其中耐受性最强的为NCU801。(2)NCU801生长曲线绘制。罗伊氏乳杆菌NCU801属于异型发酵,能够利用葡萄糖代谢产生乳酸、乙酸,同时还能够产生乙醇、二氧化碳等,0-4 h、4-16 h、16-24 h及24 h后分别为延滞期、对数期、稳定期和衰亡期。(3)NCU801的动物胃肠道耐受性能研究。L.reuteri NCU801在pH 2.5动物胃液时,菌株D值大于60 min;通过模拟动物肠液耐受性实验,菌体在肠液作用4 h后,存活率仍有38.6%。在胆盐浓度为0.3%的改良MRS培养基中作用24 h后,活菌数下降一个多数量级,但活菌数仍有9.7×105 CFU/m L。可见,L.reuteri NCU801具有较强抵抗动物胃肠道逆环境的能力。(4)NCU801的抑菌性能研究。通过对肠道中常见的9种致病菌的抑菌实验,发现罗伊氏乳杆菌NCU801的菌体、发酵液和无菌上清液均具有抑菌性,且发酵液和发酵液的无菌上清液的抑菌效果较菌体更强,其中发酵液和无菌上清液对大肠埃希氏菌抑制作用最强,抑菌圈直径达18 mm以上,由此可以判断出罗伊氏乳杆菌NCU801的抑菌作用主要依赖于本身的代谢产物。(5)NCU801肠道粘附性能研究。NCU801对Caco-2细胞的粘附,其粘附指数同菌体浓度、菌体发酵时间、孵化时间及pH值都有对应的关系;而NCU801对Caco-2细胞粘附指数最大值达(5.08±0.15)CFU/cell。NCU801活菌、死菌均能在置换、竞争和排斥性的抑制病原菌对Caco-2细胞粘附;其中抑制粘附作用最大的为活菌悬液对大肠杆菌排斥粘附抑制,抑制粘附率达86.8±4.5(%)。(6)NCU801高密度培养条件研究。通过单因素试验,确定了L.reuteri NCU801最佳碳源、氮源、缓冲盐、无机盐离子分别为葡萄糖、牛肉膏和酵母膏、NaAc、Mg2+;而正交试验优化的培养基配方为:牛肉膏1.5%,酵母膏1.5%,葡萄糖5%,MgSO4 0.06%,MnSO4 0.01%,Na Cl 0.001%,K2HPO4 0.2%,柠檬三铵0.2%,CH3COONa 0.3%。菌液浓度达1.3×109 CFU/mL。
[Abstract]:Lactobacillus Roy is one of the most important probiotics in animal microecology. Its bacteria and metabolites can inhibit the adhesion and growth of intestinal tract bacteria and pathogens in piglets. Therefore, it can improve the immunity of piglets and reduce the mortality of piglets, thus greatly improving the quality of the breeding industry. The purpose of this study was to screen acid-producing strains from animal feces and evaluate their performance in vitro. The main results are as follows: (1) screening of probiotics for feeding. Three strains of acid-producing strains were screened from piglets' feces after primary screening and repeated screening of acid-tolerant bile salt. The strains were identified as Lactobacillus Roy by physiological and biochemical experiments, such as sucrose fermentation and molecular biological identification. Among them, NCU801. (2) NCU801 growth curve was the most tolerant. Lactobacillus Roy NCU801 is a kind of abnormal fermentation, which can produce lactic acid, acetic acid, ethanol, carbon dioxide and so on by glucose metabolism. After 0-4 h, 4-16 h, 16-24 h and 24 h, the delay period is logarithmic phase, respectively. (3) the animal gastrointestinal tolerance of NCU801. When pH 2.5 animal gastric juice, the strain D value was greater than 60 min;. The bacteria survived 38.6% after the intestinal fluid was exposed for 4 h. After 24 h exposure to the modified MRS medium with 0.3% bile salt concentration, the number of viable bacteria decreased by one order of magnitude, but the number of live bacteria was still 9.7 脳 10 ~ 5 CFU/m 路L ~ (-1). It can be seen that L.reuteri NCU801 has a strong ability to resist the adverse environment of gastrointestinal tract. (4) the bacteriostasis of NCU801. The bacteriostatic experiments of 9 common pathogenic bacteria in intestinal tract showed that the bacteria of Lactobacillus Roy NCU801, fermentation broth and aseptic supernatant were all bacteriostasis, and the bacteriostasis effect of aseptic supernatant of fermentation broth and fermentation broth was stronger than that of bacteria. Among them, fermentation broth and aseptic supernatant had the strongest inhibitory effect on Escherichia coli, and the diameter of inhibition circle was more than 18 mm. It can be concluded that the bacteriostatic effect of Lactobacillus Roy NCU801 mainly depends on its metabolites. (5) the adhesion of NCU801 to Caco-2 cells is studied. The adhesion index of NCU801 to Caco-2 cells is the same as the cell concentration and fermentation time. The maximum adhesion index of NCU801 to Caco-2 cells was (5.08 卤0.15) CFU/cell.NCU801, and the dead bacteria could inhibit the adhesion of pathogenic bacteria to Caco-2 cells by replacement, competition and rejection. Among them, the most effective adhesion inhibition was the rejection of adhesion by live bacteria suspensions to Escherichia coli, and the inhibition rate of adhesion was 86.8 卤4.5 (%). (6) NCU801 high density culture conditions. The optimum carbon source, nitrogen source, buffer salt and inorganic salt ion of L.reuteri NCU801 were determined as glucose, beef paste and yeast extract, NaAc,Mg2 respectively. The optimum medium formula of orthogonal experiment was: beef paste 1.5, yeast extract 1.5, glucose 5 and MgSO4 0.06, MnSO4 0.0110, Na Cl 0.001, K2HPO4 0.2cm, lemon triammonium 0.2, CH3COONa 0.3. The concentration of bacteria solution was 1.3 脳 10 ~ 9 CFU/mL..
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S816.3
[Abstract]:Lactobacillus Roy is one of the most important probiotics in animal microecology. Its bacteria and metabolites can inhibit the adhesion and growth of intestinal tract bacteria and pathogens in piglets. Therefore, it can improve the immunity of piglets and reduce the mortality of piglets, thus greatly improving the quality of the breeding industry. The purpose of this study was to screen acid-producing strains from animal feces and evaluate their performance in vitro. The main results are as follows: (1) screening of probiotics for feeding. Three strains of acid-producing strains were screened from piglets' feces after primary screening and repeated screening of acid-tolerant bile salt. The strains were identified as Lactobacillus Roy by physiological and biochemical experiments, such as sucrose fermentation and molecular biological identification. Among them, NCU801. (2) NCU801 growth curve was the most tolerant. Lactobacillus Roy NCU801 is a kind of abnormal fermentation, which can produce lactic acid, acetic acid, ethanol, carbon dioxide and so on by glucose metabolism. After 0-4 h, 4-16 h, 16-24 h and 24 h, the delay period is logarithmic phase, respectively. (3) the animal gastrointestinal tolerance of NCU801. When pH 2.5 animal gastric juice, the strain D value was greater than 60 min;. The bacteria survived 38.6% after the intestinal fluid was exposed for 4 h. After 24 h exposure to the modified MRS medium with 0.3% bile salt concentration, the number of viable bacteria decreased by one order of magnitude, but the number of live bacteria was still 9.7 脳 10 ~ 5 CFU/m 路L ~ (-1). It can be seen that L.reuteri NCU801 has a strong ability to resist the adverse environment of gastrointestinal tract. (4) the bacteriostasis of NCU801. The bacteriostatic experiments of 9 common pathogenic bacteria in intestinal tract showed that the bacteria of Lactobacillus Roy NCU801, fermentation broth and aseptic supernatant were all bacteriostasis, and the bacteriostasis effect of aseptic supernatant of fermentation broth and fermentation broth was stronger than that of bacteria. Among them, fermentation broth and aseptic supernatant had the strongest inhibitory effect on Escherichia coli, and the diameter of inhibition circle was more than 18 mm. It can be concluded that the bacteriostatic effect of Lactobacillus Roy NCU801 mainly depends on its metabolites. (5) the adhesion of NCU801 to Caco-2 cells is studied. The adhesion index of NCU801 to Caco-2 cells is the same as the cell concentration and fermentation time. The maximum adhesion index of NCU801 to Caco-2 cells was (5.08 卤0.15) CFU/cell.NCU801, and the dead bacteria could inhibit the adhesion of pathogenic bacteria to Caco-2 cells by replacement, competition and rejection. Among them, the most effective adhesion inhibition was the rejection of adhesion by live bacteria suspensions to Escherichia coli, and the inhibition rate of adhesion was 86.8 卤4.5 (%). (6) NCU801 high density culture conditions. The optimum carbon source, nitrogen source, buffer salt and inorganic salt ion of L.reuteri NCU801 were determined as glucose, beef paste and yeast extract, NaAc,Mg2 respectively. The optimum medium formula of orthogonal experiment was: beef paste 1.5, yeast extract 1.5, glucose 5 and MgSO4 0.06, MnSO4 0.0110, Na Cl 0.001, K2HPO4 0.2cm, lemon triammonium 0.2, CH3COONa 0.3. The concentration of bacteria solution was 1.3 脳 10 ~ 9 CFU/mL..
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S816.3
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