Analysis The Differences Of Vitro Feed Protein Degradation And Protease Genes Encoding Of Three Bacillus Subtilis Strains From Different Sources

Bacillus subtilis is an important microbial feed fermentation agent. However, Bacillus subtilis isolated from intestines of different animal species have the differences of characteristics in ability of dietary protein and protease genes encoding degradation, and that is a subject worthy of further study. This will help us deeply understand the activity and structure stability of protease of Bacillus subtilis strains under the different environments,and it has an important significance on the development and application of microbial preparations in swine, poultry and aquaculture industry.This study was conducted on the three excellent Bacillus subtilis strains isolated from different animals (Bacillus subtilis A7 from aquaculture、Bacillus subtilis H6 from swine、 Bacillus subtilis S1 from poultry). The optimal culture conditions of Bacillus subtilis A7、H6 and S1 were determined by using the single factor experiment, which were composed respectively of culture temperature 28℃、37℃、39℃,original pH 7.0、7.0、6.8.Then, an orthogonal test was made on design of the best solid fermentation conditions, which containing forced filling rate 40g/250ml、inoculation amount 5% and solid fermentation time 48h. Then, the bacteria powder of Bacillus subtilis were made on the basis of the solid fermentation results,and the viable bacteria powder counts of Bacillus subtilis A7、H6、S1 can respectively reach 8.90×109 CFU/g,5.60×1010 CFU/g and 6.80×1010 CFU/g, moreover, the ratio of spore forming was about 90%. Investigation into the reactivation of bacteria powder in preservation period showed that,Bacillus subtilis A7 had the best stability during the conservative period test, till the trimester, the revival rate can still keep around 70% and spore rate was above 80%.beside. Bacillus subtilis H6 took second place and with Bacillus subtilis S1 the last. In addition,the duration of the preservation period at 4℃ was better than under room temperature.The results in bacterial degradation of protein demonstrated that:Bacillus subtilis A7 showed strong enzymatic hydrolysis ability to protein while Bacillus subtilis H6 and S1 had the similarity degradation, however, Bacillus subtilis S1 had a poor influence on the degradation of casein. Generally speaking, enzymatic capacity of the three were; A7> H6> S1.The feed degradability rates of soybean and corn in test group of Bacillus subtilis A7 were respectively 53.13%、11.17%,which increased by 17.64%、7.49% compared with control group; The protein degradability rates were respectively 28.06%、17.31%,improving 16.42%、 8.44% vs control group.Bacillus subtilis protease genes were successfully cloned and the fragment size is 1579 bp。Homology to reported Bacillus nprE were above 99%, besides, the similarity of nucleotide ORF sequence and amino acid similarity among these three strains was respectively 99.30%、 99.47%. Enzyme structure prediction showed that, the neutral protease was a stable alpha/beta protein with molecular weight of medium size.Results showed that Bacillus subtilis strains isolated from different animals had a distinguished difference of feed protein degradation and culture conditions. Although these Bacillus subtilis protease had high homology of genes and identical spatial structure, the primary structure were far apart with 8 AA discrepancy. We suggested that it is the 8 AA discrepancy which were the key points leading to differences in the ability of feed protein degradation.