猪源益生乳酸菌的筛选及其菌剂制备技术研究
发布时间：2018-11-27 10:01
【摘要】：为研制出高活性饲用益生菌制剂替代仔猪饲料中添加的抗生素。本研究从健壮仔猪粪便中筛选出具有较强产酸能力的乳酸菌,并对菌株生长特性,耐受动物消化道逆环境能力、抑制病原菌、粘附小肠上皮细胞等益生特性及菌剂制备工艺进行研究。主要研究成果如下:(1)猪源乳酸菌的筛选及鉴定。通过添加溴甲酚紫指示剂的MRS进行初次筛选,得到26株疑似目标菌株;经过耐酸耐胆盐复筛,得到7株对低pH和高浓度胆盐具有一定耐受能力的菌株。通过生理生化、分子生物等手段将其中耐酸耐胆盐性能最好的两株菌分别鉴定为植物乳杆菌NCU156和戊糖片球菌NCU301。(2)NCU156生长特性。NCU156能够产生大量的乳酸使培养基pH降低至3.6左右。0-2 h是生长延滞期,2-12 h是对数期,12-32 h是稳定期,32 h以后为衰亡期。(3)NCU156动物胃肠道环境耐受性。NCU156在pH2.5的MRS中4 h存活率为87.31%。在3 g/L的胆盐浓度下4 h存活率仍有41.88%;在模拟胃液中处理4 h菌数稍有增加(P0.05),接入模拟肠液后10 h活菌数在108 CFU/mL以上,说明NCU156能够顺利达到动物肠道。(4)NCU156粘附性能。NCU156粘附能力在一定范围内呈菌浓度和Ca2+浓度依赖性,在作用2 h粘附达到平衡,在稳定期中期(20 h)粘附最稳定;对病原菌粘附的抑制作用中,排斥性的粘附抑制作用效果最强,其次是竞争性的粘附抑制,最后是置换性的粘附抑制。(5)NCU156抑菌性能。NCU156对常见7株致病菌的抑菌性研究表明,对铜绿假单胞菌抑制效果最好,抑菌圈的直径达23 mm以上,菌体没有抑菌性;发酵液抑菌性不受高温、过氧化性酶、蛋白酶等处理方式的影响,故其抑菌性可能主要来源于所产生的乳酸;NCU156药敏结果显示其对四环素类、青霉素类抗生素敏感,对头孢类、糖肽类、喹诺酮类抗生素均耐药。(6)NCU156菌剂制备工艺优化。以活菌数为指标通过单因素实验和BOX-Behnken响应面实验设计优化菌剂制备的工艺,最佳发酵条件为:接种量4.45%、料液比1:0.61、发酵温度30.6℃、发酵时间46.40 h。优化后的烘干条件为热风干燥50℃、4 h。
[Abstract]:In order to develop a high-activity probiotic agent for feeding to replace the antibiotics added in the feed of piglets. In this study, lactic acid bacteria with strong acid-producing ability were screened from the feces of healthy piglets, and the growth characteristics of the strains, tolerance to the adverse environment of the digestive tract of animals, and inhibition of pathogenic bacteria were studied. The probiotics of adhesion to intestinal epithelial cells and the preparation of bacteria were studied. The main results are as follows: (1) screening and identification of porcine lactic acid bacteria. Through the initial screening of MRS with bromocresol violet indicator, 26 suspected target strains were obtained, and 7 strains with certain tolerance to low pH and high concentration bile salt were obtained by repeated screening with acid tolerance and bile salt tolerance. Through physiology and biochemistry, The two strains with the best acid-tolerant bile salt tolerance were identified as Lactobacillus plantarum NCU156 and NCU301. (2) NCU156 by molecular biology. NCU156 can produce a large amount of lactic acid to reduce the pH to 3. The growth delay period was about 0.6 h and 0.2 h, respectively. 2-12 h was logarithmic phase, 12-32 h was stable phase, and 32 h later was decaying stage. (3) NCU156 animal gastrointestinal environment tolerance. The survival rate of NCU156 in MRS of pH2.5 was 87.31 h. The survival rate of 4 h was still 41.88 at the concentration of 3 g / L of bile salt. The number of bacteria in simulated gastric juice increased slightly after 4 h treatment (P0.05), and the number of live bacteria was more than 108 CFU/mL after 10 hours of inoculation. The results showed that NCU156 could reach the animal intestinal tract smoothly. (4) the adhesion ability of NCU156 was in a certain range of concentration and Ca2 concentration dependent, the adhesion of NCU156 reached equilibrium at 2 h, and the most stable in the middle of stable phase (20 h). In the inhibition of the adhesion of pathogenic bacteria, the repellent adhesion inhibition was the strongest, followed by the competitive adhesion inhibition. The results showed that NCU156 had the best inhibitory effect on Pseudomonas aeruginosa, the diameter of inhibition circle was over 23 mm, and the bacteriostasis of NCU156 was not. The bacteriostasis of fermentation broth is not affected by high temperature, superoxide enzyme, protease and so on, so its bacteriostasis may mainly come from the lactic acid produced. The results of NCU156 showed that it was sensitive to tetracyclines penicillin antibiotics and resistant to cephalosporins glycopeptides and quinolones. (6) the preparation process of NCU156 bacteria was optimized. The optimum fermentation conditions were as follows: inoculation amount 4.45, ratio of material to liquid 1: 0.61and fermentation temperature 30.6 鈩，
本文编号：2360317
[Abstract]:In order to develop a high-activity probiotic agent for feeding to replace the antibiotics added in the feed of piglets. In this study, lactic acid bacteria with strong acid-producing ability were screened from the feces of healthy piglets, and the growth characteristics of the strains, tolerance to the adverse environment of the digestive tract of animals, and inhibition of pathogenic bacteria were studied. The probiotics of adhesion to intestinal epithelial cells and the preparation of bacteria were studied. The main results are as follows: (1) screening and identification of porcine lactic acid bacteria. Through the initial screening of MRS with bromocresol violet indicator, 26 suspected target strains were obtained, and 7 strains with certain tolerance to low pH and high concentration bile salt were obtained by repeated screening with acid tolerance and bile salt tolerance. Through physiology and biochemistry, The two strains with the best acid-tolerant bile salt tolerance were identified as Lactobacillus plantarum NCU156 and NCU301. (2) NCU156 by molecular biology. NCU156 can produce a large amount of lactic acid to reduce the pH to 3. The growth delay period was about 0.6 h and 0.2 h, respectively. 2-12 h was logarithmic phase, 12-32 h was stable phase, and 32 h later was decaying stage. (3) NCU156 animal gastrointestinal environment tolerance. The survival rate of NCU156 in MRS of pH2.5 was 87.31 h. The survival rate of 4 h was still 41.88 at the concentration of 3 g / L of bile salt. The number of bacteria in simulated gastric juice increased slightly after 4 h treatment (P0.05), and the number of live bacteria was more than 108 CFU/mL after 10 hours of inoculation. The results showed that NCU156 could reach the animal intestinal tract smoothly. (4) the adhesion ability of NCU156 was in a certain range of concentration and Ca2 concentration dependent, the adhesion of NCU156 reached equilibrium at 2 h, and the most stable in the middle of stable phase (20 h). In the inhibition of the adhesion of pathogenic bacteria, the repellent adhesion inhibition was the strongest, followed by the competitive adhesion inhibition. The results showed that NCU156 had the best inhibitory effect on Pseudomonas aeruginosa, the diameter of inhibition circle was over 23 mm, and the bacteriostasis of NCU156 was not. The bacteriostasis of fermentation broth is not affected by high temperature, superoxide enzyme, protease and so on, so its bacteriostasis may mainly come from the lactic acid produced. The results of NCU156 showed that it was sensitive to tetracyclines penicillin antibiotics and resistant to cephalosporins glycopeptides and quinolones. (6) the preparation process of NCU156 bacteria was optimized. The optimum fermentation conditions were as follows: inoculation amount 4.45, ratio of material to liquid 1: 0.61and fermentation temperature 30.6 鈩，
本文编号：2360317
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