Screening And Preliminary Application Of Bacillus Subtilis From Pig

The unscientific abuse of antibiotics makes the problems of bacterial resistance and drug residue increasingly prominent,which seriously endangers the healthy development of aquaculture industry and human food safety.It is urgent to develop safe and effective alternatives to antibiotics.Bacillus subtilis is widely found in water,soil and gastrointestinal tract of animals.It can promote the digestion and absorption of animal nutrients,inhibit bacteria,regulate intestinal flora and improve animal immunity,and is easy to separate and culture,so it has become one of the best alternatives feed antibiotics.Therefore,the aim of this study was to screen the dominant Bacillus subtilis from pig manure and apply this dominant strain to animal experiments to explore its prebiotic effect and provide technical support for the development of Bacillus subtilis preparations.The main findings are as follows:1.Screening of Bacillus subtilis :50 strains of Bacillus subtilis were obtained from 100 pig manure samples by heat treatment.25 strains of Bacillus subtilis were identified by microscope,physiological and biochemical tests,PCR identification and 16 S r RNA sequence analysis,respectively named BS-1?BS-25..The results showed that all strains except strain BS-12?BS-13?BS-14 could secrete protease,all strains except strain BS-11 and BS-18 could secrete amylase,and all strains except strain BS-10 could secrete amylase.The results of bacteriostasis test showed that the strain BS-3?BS-15?BS-19 had better bacteriostatic effect on Escherichia coli K99,the strain BS-3?BS-4?BS-5?BS-6?BS-8?BS-15?BS-19?BS-24 had better bacteriostatic effect on Staphylococcus aureus,and the strain BS-3?BS-19 had better bacteriostatic effect on both Escherichia coli K99 and Staphylococcus aureus.As a result,strain 19 was selected as the target strain for the next study and named BS-19.2.The biological characteristics of Bacillus subtilis BS-19.Through analyzing the growth curve,stress resistance and drug sensitivity BS-19 Bacillus subtilis,the results showed that the strain BS-19 entered logarithmic growth period after 4 h and stable growth state after 10 h.After treatment at 90? high temperature for 5 min,the survival rate was 87.94%.The survival rate was 108.29% and 79.68% after culture in 0.1% and 0.3% bile salt solution for 24 h,indicating that the BS-19 had strong resistance to acid,high temperature and bile salt.BS-19 is sensitive to cefotaxime,cefoperazone,streptomycin,ofloxacin,kanamycin,less sensitive to doxycycline and less sensitive to tetracycline,indicating that it has better biosafety.3.Preliminary application of Bacillus subtilis BS-19: using Salmonella typhimurium attack mice as an animal model of diarrhea to study the application effect of strain BS-19.The experiment was divided into blank control group,probiotic group,diarrhea model group,prevention group and treatment group.The average daily gain results of mice showed that the average weight gain of diarrhea group was lower than that of other groups,and the difference was very significant(P<0.01)compared with probiotic group,but not significant(P>0.05)compared with other groups.The diarrhea rate statistics showed that the diarrhea rate of model group,prevention group and treatment group was 87%?53% and 40%,respectively.The effect of BS-19 on immune organs of mice showed that the spleen index of probiotics group was higher than that of blank group(P<0.01),the spleen index of treatment group was higher than that of diarrhea group(P<0.01),the liver index of probiotics group was higher than that of prevention group and blank group(P<0.05),the liver index of prevention group and treatment group was higher than diarrhea group(P<0.01);indicating that Bacillus Subtilis BS-19 has the effect of promoting growth in the spleen and liver of mice,and has protective effect on spleen and liver injury caused by S.Typhimurium.Intestinal section staining results show that the probiotic group has the most complete small intestine structure,intestinal villi row is the most neat,while diarrhea group small intestine structure is incomplete,intestinal villi row disorder and incomplete,and inflammatory cell infiltration,indicating that BS-19 has a maintenance effect on normal intestinal structure,repair effect on intestinal injury of Salmonella infection,and the treatment effect is better than prevention effect.The effect of BS-19 on inflammatory factors in mouse serum showed that the TNF-??IL-17 of prevention group,treatment group and probiotics group were lower than that of blank group(P<0.05),while the IFN-??IL-4 was higher than that of blank group(0.05).The IL-2?IL-6 of probiotics group was lower than that of blank group,and the IL-4 of probiotics group was significantly higher than that of other groups(P<0.05),which indicated that BS-19 could effectively inhibit inflammatory reaction,and the therapeutic effect was better than that of prevention.To sum up,the BS-19 of Bacillus subtilis selected in this study have good growth performance,enzyme production characteristics and strong resistance to stress,and have bacteriostatic effect on Escherichia coli K99 and Staphylococcus aureus;It can effectively improve and maintain the structure of intestinal barrier in animals and inhibit the occurrence of inflammation.