| In this study,the biological characteristics and safety evaluation of three strains of bamboo rat-derived cellulase-producing bacillus subtilis(GL-4,GL-5 and GL-8)were studied.Meanwhile,the cellulase related genes from GL-4 strain were cloned and expressed by genetic engineering technology,and the expressed products had high enzyme activity.And 60-day-old piglets from the inbred line of Guangxi Bama mini-pig were used to evaluate the in vivo feeding effects of diets supplemented with these bacteria strains.All the experimental results are showed as follows:In this experiment,three strains of cellulase-producing bacillus subtilis isolated from the intestinal contents of bamboo rats were subjected to growth curve determination,antibiotic sensitivity test,drug resistance gene detection,biological safety test in mouse,and acid and bile salt resistance,self-aggregation,hydrophobicity,and other properties tests.The results showed that the three tested strains,GL-4,GL-5 and GL-8,all showed the four characteristics as:(1)High proliferation rate: the logarithmic phase could be reached within 1.5 h and the maximum growth could be reached within 5 h;(2)Strong stress resistance: the survival rates of the three tested bacteria reached more than 65% under the condition of p H2.5,and they could continue to survive and proliferate in the media with bile salt concentration of 0.3%and 0.5%;(3)Good self-aggregation and hydrophobicity: after incubation for 24 h,the self-aggregation rates of the three strains reached above 85%,and the hydrophobicity of GL-5 and GL-8 was higher than 40%;(4)all strains were sensitive to most antibiotics and showed no toxic or side effects on mice.According to the cellulase gene sequences of endoglucanase(CP092631)and β-glucosidase(CP026039)from Bacillus subtilis strain disclosed in the NCBI database,primers were designed to clone two cellulase genes,endoglucanase gene Cel31 and β-glucosidase gene Cel39,from Bacillus subtilis GL-4.Both Cel31 and Cel39 genes were expressed in Escherichia coli with p ET32a(+)as a fusion protein.The recombinant proteins of Cel31 and Cel39 were soluble and had a molecular weight of about 73 k Da and 45 k Da,respectively.The highest expression level was obtained after inducing with 0.5 mmol/L IPTG for 6 hours.Enzyme activity assays showed that the expression product of BL21/p ET32a-Cel31 had a specific endoglucanase activity of 209.07 U/m L,which was 4.2 times higher than that expressed by GL-4,and the expression product of BL21/p ET32a-Cel39 had a specific β-glucosidase activity of 52.16 U/m L,which was 1.5 times higher than that expressed by GL-4.The results indicate that the activities of endoglucanase and β-glucosidase expressed at a high level by BL21/p ET32a-Cel31 and BL21/p ET32a-Cel39 respectively in this study.Thirty-two inbred piglets(60-day-old)of Bama mini-pigs were randomly divided into four treatment groups,with eight replicates in each group: 1)basic diet(CK group);2)basic diet+Bacillus subtilis GL-4(Treat1 group);3)basic diet + Bacillus subtilis GL-4,GL-5 and GL-8(Treat2 group);4)basic diet + Bacillus subtilis GL-4 +Lactobacillus casei HJD(Treat3 groups).The experimental results showed that the feed-weight ratio of piglets tended to be improved by adding different combinations of bacteria strains in the diet,but only the feed-weight ratio of Treat3 group was significantly improved(P<0.05),and the difference in feed-weight ratio among the CK group,Treat1 group and Treat2 group was not significant(P>0.05).The results of apparent digestibility of nutrients showed that compared with CK group,apparent digestibility of crude fiber,acidic washing fiber and phosphorus of piglets in Treat3 group were significantly increased(P<0.05),while those in Treat1 group and Treat2 group were not significantly increased(P>0.05).The apparent digestibility of digestive energy in Treat2 group and Treat3 group were significantly higher than those in CK group and Treat1 group(P<0.01),and the difference in apparent digestibility of digestive energy between CK group and Treat1 group was not significant(P>0.05).The addition of different combinations of bacteria strains in the feed tended to increase the apparent digestibility of dry matter,crude protein and neutral detergent fiber,but did not reach a significant level(P>0.05).The supplementation of different combinations of bacteria strains in the diet had no significant effects on serum GLU,TP and ALB(P>0.05),but significantly increased the contents of serum ALP,AST and BUN(P<0.05),and significantly reduced the content of serum T-CHO(P<0.05).Compared with the CK group,the serum contents of various immune factors(IL-6,TNF-α,CRP,Ig G,Ig M and Ig A)in the Treat1 group and the Treat2 group were significantly increased(P<0.01).In addition to the serum contents of IL-6,Ig M and Ig A,the serum contents of TNF-α,CRP and Ig G in the Treat3 group were also significantly increased(P<0.01).The results of microbial diversity detected in piglet feces showed that the ratios of Firmicutes to Bacteroides were increased in the Treat1,Treat2 and Treat3 groups,and the ratio was the largest in the Treat3 group,followed by the Treat1 and Treat2 groups.The fecal short-chain fatty acid content including butyric acid,acetic acid,propionic acid,caproic acid,valeric acid,isobutyric acid,and isovaleric acid in the feces of piglets from Treat 1,Treat 2 and Treat 3group were increased.In conclusion,the addition of Bacillus subtilis or Lactobacillus casei to the diet could improve the growth performance,immune reaction and apparent digestibility of nutrients of piglets.For different bacteria strain combinations,the feeding effect was different.Treat3(Bacillus subtilis GL-4 + Lactobacillus casei HJD)had the best feeding effect,followed by Treat1 group and Treat2 group.In summary,the three tested strains GL-4,GL-5 and GL-8 in the present study have excellent probiotic characteristics,and can be good candidate strains used as probiotics in livestock and poultry production.The feeding experiment showed that adding different combinations of GL-4,GL-5,GL-8 and Lactobacillus casei HJD in the feed could improve the feed-to-weight ratio and apparent digestibility of nutrients,increase the immune reaction,the ratio of Firmicutes to Bacteroides of gastrointestinal microflora and the content of short-chain fatty acids in the feces.These results provide a scientific basis for the application of GL-4,GL-5 and GL-8 as probiotics in pig production. |
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