Sreening And Identification Of Production Enzyme Activity From Bacillus Spp., As Potential Probiotics In Broilers

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.