Screening And Mouse Test Of Facultative Anaerobic Bacillus

The abuse of antibiotics has brought many negative effects on human and animal health.The state and society pay more and more attention to food safety,and comprehensively prohibit the addition of antibiotics in feed as growth promoting agents.According to research,microbial preparations prepared by probiotics have become one of the products that replace antibiotics.Probiotics can not only effectively replace antibiotics to promote growth,but also stimulate the development of immune organs and improve the body’s resistance to pathogenic microorganisms.It can secrete a variety of enzymes to help absorb nutrients that are difficult to digest and utilize in feed.When the body intake enough probiotics can promote the development of gastrointestinal epithelial cells and promote barrier function.Probiotics can secrete a variety of vitamins,antibacterial proteins;competition for intestinal oxygen is conducive to the growth of intestinal resident anaerobic bacteria,limiting the survival of other aerobic and facultative anaerobic pathogenic bacteria,and playing a health performance conducive to livestock and poultry health.In this study,121 strains of Bacillus with protease-producing activity were screened from samples collected from different sources after 2h treatment with 0.3%bile salt and10min heat treatment at 80℃.Four facultative anaerobic Bacillus strains capable of growing in thin oxygen environment and anaerobic environment were selected by continuous passage in micro-oxygen environment.The antibacterial test showed that four Bacillus strains had broad-spectrum antibacterial effect on Escherichia coli,Salmonella,Staphylococcus haemolyticus and Staphylococcus aureus.Four Bacillus strains were identified as Bacillus subtilis using 16sr DNA universal primers and named as A1,C1,C3 and Y19-6.The resistance test of four Bacillus strains showed that Bacillus subtilis Y19-6 had better tolerance than A1,C1 and C3.The survival rate of Bacillus subtilis Y19-6 was 79%after being treated for 3 h in p H=2 environment,and 97%after being treated for 3 h in 0.3%bile salt environment.The survival rate of bacillus subtilis Y19-6 was 71%,66%and 54%after 10min of heat treatment at 60℃,80℃and 100℃respectively.The growth curve of bacteria showed that Y19-6 had more advantages.Y19-6 was identified as Bacillus subtilis by gyr A gene and MLST multilocus sequence typing.Seven pairs of housekeeping genes were amplified and sequenced and compared with Pub MLST.The new alleles ilv D 115 and pta 115 were uploaded and the new ST type255 was obtained.In the acute toxicity test and bacterial translocation test of mice,intragastric administration of high concentration bacterial liquid had no effect on the mental state,appetite and excretion of mice,and there was no lesion in the internal organs of mice after dissection,indicating that Y19-6 did not transfer to other organs,and was sensitive to most clinical medications with high safety.The results of Bacillus subtilis isolation on the 8th and12th days after intragastric administration of Y19-6 in mice showed that the Bacillus subtilis content was 5.0~5×10~4 cfu/g on the 8th day after intragastric administration,and 6.2~8×10~3cfu/g remained on the 12th day after intragastric administration,indicating that Y19-6 with facultative anaerobic characteristics could remain in the intestinal tract of mice for a long time.The isolation of Lactobacillus after intragastric administration for 30 days showed that Y19-6 could promote the number of intestinal Lactobacillus.However,it was found that Y19-6 had good protective performance and effectively reduced the mortality of mice after challenge with Streptococcus suis type 9 ss9-P10.In summary,one facultative anaerobic Bacillus subtilis Y19-6 was successfully screened out for the first time from 121 Bacillus strains with protease-producing activity isolated from samples from different sources in this experiment.In addition to the broad-spectrum antibacterial effect,good acid resistance,bile salt resistance and heat resistance,and the ability to secrete a variety of enzymes,this strain can be colonized in the gastrointestinal tract for a long time due to its facultative anaerobic characteristics,and the number of Lactobacillus in the intestinal tract can be effectively improved by gavage once.The safety of Y19-6 was verified by the mouse test.The ss9-P10 challenge test of Streptococcus suis type 9 showed that feeding mice could improve their nonspecific immunity and enhance their disease resistance.It indicates that Y19-6 has a certain research value.