【作者】 潘康成；

【导师】 崔恒敏；

【作者基本信息】 四川农业大学 ， 预防兽医学， 2009， 博士

【摘要】 本文对实验室分离保存的芽孢杆菌进行产酶活性筛选,并对筛选得到的一株产酶活性高的芽孢杆菌进行序列分析鉴定,研究其对雄性小鼠的生殖系统安全16S rDNA性、采用实时荧光定量检测在肉鸡肠道中消长规律、PCR (RT-qPCR)和ERIC-PCRPCR-DGGE检测肉鸡口服该菌悬液后肠道菌群多样性,然后制成活菌制剂观察其对肉鸡生长性能、肌肉品质、肠道菌群、消化酶活性和免疫功能的影响。结果如下：1.采用平板培养法对10株芽孢杆菌产蛋白酶、纤维素酶、淀粉酶活性进行初选,然后采用比色法测定芽孢杆菌发酵培养液酶活性进行复选。筛选出产酶活性较高的芽孢杆菌Pab02菌株。根据细菌的16SrDNA序列两端的保守性设计通用引物,提取菌株的基因组DNA,对菌株的16SrDNA的进行PCR扩增,并对扩增得到的目标片段进行测序,将测序结果与NCBI上已知菌种的16SrDNA序列进行同源性比较和构建系统发育进化树进行分析,结合细菌形态特征、生理生化特性,最终确定芽孢杆菌Pab02为枯草芽孢杆菌(Bacillus subtilis)。2.为了评价该菌的安全性,选用72只昆明系雄性小鼠,随机分成6组。药物对照组,每只小鼠按20 mg/kg体重经口灌喂环磷酰胺,1次/d,连续5d,诱导小鼠产生生殖毒性；药物对照组和空白对照组小鼠饲喂不含任何益生菌和抗生素的日粮,芽孢杆菌Ⅰ组、Ⅱ组、Ⅲ组和Ⅳ组分别饲喂含有105,106,107CFU/g和108CFU/g的饲料。饲喂至30d时,每组随机剖杀6只,检测小鼠睾丸、附睾重,精子畸形、精子活力,睾丸组织中MDA含量和SOD活性,精子顶体酶活力,血清睾酮(T)含量,并通过组织切片观察雄性小鼠睾丸的组织结构。结果显示,芽孢杆菌Pab02对小鼠体重、小鼠睾丸和附睾重均有明显的增重效果；芽孢杆菌Pab02可提高小鼠精子活率,对精子畸形的减少没有明显的作用；芽孢杆菌Pab02可降低小鼠翠丸组织中MDA含量,提高睾丸组织中SOD活性、精子顶体酶活力和血清睾酮的含量,芽孢杆菌Pab02对小鼠的睾丸组织结构有一定的改善作用。结果表明,芽孢杆菌Pab02对雄性小鼠是安全的,没有造成生殖毒性,对雄性小鼠生殖功能有一定的促进作用。3.芽孢杆菌Pab02菌悬液按2ml/kg体重(109CFU/ml)灌喂28日龄的肉鸡,2次/d,连续3d后,收集肠道内容物,采用RT-qPCR检测肠道内容物中芽孢杆菌的数量。结果,对照组(灌喂等量的PBS液)肉鸡肠道内芽孢杆菌数在5.37-6.10logCopies/g之间,当连续口服芽孢杆菌Pab02菌悬液后,十二指肠内的芽孢杆菌数量从12h到第4d显著高于对照组,第5d后恢复到对照组水平；空肠、回肠和直肠从12h到第5d显著高于对照组,第6d后恢复到对照组水平。盲肠内的芽孢杆菌数量在36h和60h时显著高于对照组。结果表明,芽孢杆菌在对照组肉鸡肠道中的数量较少,芽孢杆菌Pab02作为微生态制剂饲喂动物,须长期投服或间断时间不超过3d。4.芽孢杆菌Pab02菌悬液连续灌喂28日龄肉仔鸡3d后(2次/d),收集停喂3d的肠道内容物,采用ERIC-PCR／PCR-DGGE方法分析电泳指纹图谱条带数及相似性指数,以及对DGGE特异性条带回收、测序分析其肠道菌群。结果显示,两种方法都证明肉鸡口服芽孢杆菌Pab02后各肠段的条带数及特征性条带均明显多于对照组,说明芽孢杆菌Pab02具有提高肉鸡肠道菌群多样性和种群密度；通过对电泳指纹图谱条带的聚类分析,两种检测方法显示肉鸡肠道菌群结构的多样性变化规律相似,两组肉鸡肠道细菌之间存在着种群的差异性,但两组之间肠道总菌群的相似性系数为53.2%,存在着较高的相似性,说明该年龄段的肉鸡肠道微生物种群存在着一定的稳定性；尽管两种检测方法检测结果存在着一定的相似性,但从电泳指纹图谱和统计条带数量分析,PCR-DGGE明显优于ERIC-PCR;通过刘DGGE图谱上出现的7条共同条带和8条试验组条带进行回收鉴定,结果表明,肉鸡在4周龄到5周龄的肉鸡肠道中主要以乳酸菌为主要菌群,饲喂芽孢杆菌Pab02后能提高肉鸡肠道菌群的丰度和种群密度。5.利用芽孢杆菌Pab02制成活菌制剂后饲喂肉鸡,观察其对肉鸡生长性能、肌肉品质、肠道菌群、消化酶活性及免疫功能的影响。空白对照组饲喂基础日粮,抗生素对照组在日粮中添加0.05%的硫酸新霉素制剂,Pab02组在日粮中添加0.1%的芽孢杆菌Pab02制剂。饲喂至6周龄时,与空白对照组相比,芽孢杆菌Pab02提高了肉鸡的末期体重、净增重和日增重(P<0.05),但比抗生素组低(P>0.05)；对采食量和饲料转化率没有影响(P>0.05);Pab02组肉鸡胴体品质的各项指标与两对照组相比,显著提高了腿肌率(P<0.05),其余各项指标无明显差异(P>0.05);Pab02组鸡肌肉中的蛋白含量显著高于两对照组(P<0.05)；肌肉中的氨基酸总量和鲜味氨基酸含量极显著高于两对照组(P<0.01),必须氨基酸量极显著高于抗生素组(P<0.01)；在6周龄时,芽孢杆菌Pab02可以提高肠道中乳酸菌的数量,减少大肠杆菌和总需氧菌数量,在盲肠段中表现差异显著(P<0.05)；在4周龄和6周龄时,芽孢杆菌Pab02组肠道淀粉酶和蛋白酶活性均显著或极显著高于两对照组(P<0.05或P<0.01),芽孢杆菌Pab02可促进免疫器官的发育,提高血清对新城疫病毒的抗体效价,提高6周龄时的血清IgG含量(P<0.01)。结果表明该菌制成的微生态制剂可以在肉鸡饲养整个期间替代抗生素添加剂。更多还原

【Abstract】 The paper was focused on the screening the production enzyme from Bacillus spp. which isolated and maintained by the laboratory of fermentation engineering of Sichuan Agricultural University; one strain of production high enzyme activity of Bacillus spp. was identified by 16S rDNA sequence analysis; designing to evaluate toxic effects of probiotic Bacillus spp. on reproductive system of male mice; a fluorescent quantitative real-time PCR (RT-qPCR) had been developed to detect and quantitate the Bacillus spp. in the broiler alimentary tract after the broilers were administered it as probiotics by oral gavage; ERIC-PCR and PCR DGGE were used to detect intestinal flora diversity after the broilers were administered it as probiotics by oral gavage; the effects of it as probiotics on the growth performance, carcass and chicken quality, intestinal micro flora and digestive enzyme activity and immune function of broilers were investigated in the trial. The results were as follows.1. Secretion of extracellular enzyme of cellulase, amylase and protease from 10 strains of Bacillus spp. were screened by agar medium at the first and mensurated the enzyme activity in medium of Bacillus spp. fermentation by spectrophotometric method at the final. The enzyme activity of Bacillus Pab02 was higher than other strains. The conserved primers were designed according to conservative sequence of 16 S rDNA of Bacillus spp. The genomic DNAs were extracted from Bacillus Pab02, the target 16 S rDNA fragments were amplified by PCR, and the sequence analysis was performed using the NCBI with BLAST data base. According to the morphological and physiochemical properties, and based on their positions in the phylogenetic tree, Pab02 was finally identified to be Bacillus subtilis.2. In order to evaluate safety of B. subtitlis Pab02,72 Kunming healthy male mice were divided into six groups. Mice of the drug group were administered cyclophosphamide (CP) (20mg/kg body weight one time a day for five consecutive days by oral gavage) to induce the reproductive toxicity. Mice of a drug group and a control group accepted the diets without antibiotics and other probiotics. Mice of Bacillus Pab02 I group,Ⅱgroup, III group and IV group were fed the diets with 105 CFU/g,106 CFU/g,107CFU/g and 108 CFU/g of B. subtilis Pab02, respectively. The experimental period was lasted for 30 days. At the end of experimental period, six mice of per group were sacrificed and analyzed for the reproductive toxicity by measured the body weight, testes and epididymes weight, epididymal sperm motility and abnormal, histology of testes, serum testosterone, testis SOD and malondialdehyde value and sperm acrosin activity assay. The results showed that B. subtilis Pab02 treatment significantly improved the body weight, the testicular and epididymal weights, SOD and sperm acrosin activity, serum testosterone level as compared to the control group; the number of abnormal sperms in epididymidis and histomorphology of testes did not show significant difference in comparison to the control group. The present results indicated that B. subtilis Pab02 was safety for male mice; it did not induce reproductive toxicity and had a slightly positive effect on the reproductive function of male mice.3. A fluorescent quantitative real-time PCR (RT-qPCR) had been developed to detect and quantitate the Bacillus spp. in the broiler alimentary tract after 28 days-broilers were administered the suspension of B. subtilis Pab02 (2x109CFU/kg body weight a twice a day for 3 consecutive days by oral gavage). The results showed that the numbers of Bacillus spp. in different alimentary tract of the control broiler (administered PBS by oral gavage) ranged from 5.37 to 6.10 log copies/g fecal. After chicken were orally administered the B. subtilis Pab02 suspension for 3 days, the numbers of Bacillus sp. in duodenum were significant higher than the control group from 12h to 4th day, and returned to the control group level at the 5th day. In jejunum, ileum and rectum, the treatment group was significant higher than the control group from 12h to 5th day, and returned to the control group level at the 6th day. In cecum, the treatment group was significant higher than the control group at 36h and 60h. The results indicated that the numbers of Bacillus spp. were lower in the intestine of chicken in the natural state; B. subtilis Pab02 as probiotics for animal must be fed for a long-term or intermittent phase no more than 3 days.4.28 days-broilers were administered the suspension of B. subtilis Pab02 (2×10 CFU /kg body weight a twice a day for 3 consecutive days by oral gavage), the contents of duodenum, jejunum, ileum, caecum and rectum were collected respectively after ceased feeding the probiotic Bacillus for 3 days. ERIC-PCR and PCR-DGGE assayed the electrophoretic bands and the similarity of correlation coefficients, as well as specific-DGGE bands recovered and sequences assayed their intestinal flora. The results showed that both methods had proved that a number of characteristic bands of Bacillus Pab02 group chickens were significantly more than the control group. It was suggested that B. subtilis Pab02 could improve intestinal flora diversity and population density of the broiler. Assaying the cluster of the electrophoresis fingerprint bands, the structures of intestinal flora of broiler were similar to the diversity of changes. Although there was the existence of differences between the Pab02 group and the control group of broiler intestinal bacterial population, the similarity coefficient of the total intestinal flora of two groups was 53.2%. It indicated that there was a high similarity of intestinal flora and a certain degree of stability of intestinal flora at the age of 4 and 5 weeks in broiler. Despite there was a certain amount of similarity, through statistical analysis of electrophoresis fingerprints of the number of bands, PCR-DGGE was superior to ERIC-PCR.7 bands of common and 8 bands of the Pab02 group in DGGE were recovered and identified by sequencing, the results indicated that the gut microflora were mainly lactic acid bacteria at the 4 week age of and 5 week age of broiler chickens; Bacillus Pab02 could increase the abundance and population density of microflora in broiler intestine.5. The effects of B. subtilis Pab02 as probiotics on the growth performance, carcass and chicken quality, intestinal micro flora and digestive enzyme activity and immune function of broilers were investigated in the trial. The control group was fed with the basic diets. The antibiotics group was fed with the basic diets supplemented with 0.05% sulfuric acid neomycin. The Pab02 group was fed with the basic diets supplemented with 0.1% B. subtilis Pab02 additive. The broilers were fed for 35 days. At the age of 6 weeks, the average body weight, weight gain and daily weight gain of the Pab02 group broiler were significant higher than those of the control group (P<0.05), but lower than that of the antibiotics group (P>0.05). The average feed intake of the pab02 group was slightly higher than those of the control groups (P>0.05). Feed conversion rate of the Pab02 group was no significance among the three groups (P>0.05). There were no significant diferences among the Pab02 group and two matched control groups in various indexs of carcass quality (P>0.05), in addition to the leg muscles and the leg muscle rate (P<0.05). Protein content of chicken meat in the Pab02 group was significant higher than those of two control groups (P>0.05). The contents of total amino acids, essential amino acids and taste amino acids of chicken in the Pab02 group were all higher than those in tow control groups (P<0.05 or P<0.01). B. subtilis Pab02 could improve the number of Lactic acid bacteria and decrease the number of E. coli and aerobe bacterial, but only in caecum show a significant difference (P<0.05). At 4 and 6 weeks, intestinal amylase and protease activity of Pab02 group were significantly or strongly significant higher than those of two control groups (P<0.05 or P<0.01). B. subtilis Pab02 could promote the development of immune organs and improve on the Newcastle disease virus serum antibody titer at 4 and 6 weeks, and could increase serum IgG level at 6 weeks (P<0.01). The results indicated that B. subtilis Pab02 as probiotics in broiler could replace the antibiotics additive for livestock throughout the period.更多还原