Screening Of The Probiotic Bacillus From Piglets And Stuty Of Antimicrobial Activity

Probiotics has non-toxic,no residue and remarkable antibacterial effect,it is green substitute for antibiotics.Bacillus is a focus on the study of probiotics, because it has good resistance against high temperature, acid-base,and the processing is convenient.The study includes screening a strain which can in inhibit growth of indicator from fresh feces of healthy piglets,identification strain,optimization culture medium and culture conditions of strain,and preliminary researched antimicrobial activity.The main consequences are as follows:Thirty seven strains of Bacillus were isolated form feces of piglets by Gram staining and spore staining. Out of them,11 strains showed strong inhibition ability in Escherichia coli and Staphylococcus aureus. The tolerance test of these strains for porcine bile salt medium, artificial gastric juice, artificial intestinal juice and copper sulfate, and the secretion activity of enzyme include amylase, protease, cellulose were studied. The results showed that strains of pig enterogenous Bacillus possess fortis tolerance to the bile and artificial digestive juice and copper ions inhibited the proliferation of Bacillus. All Bacillus strains can produce amylase, but the cellulose and protease hydrolysis activity was not obvious. The strain YB5 which had good tolerance and enzyme production capacity was identified as Bacillus subtilis according to physiological and biochemical characteristics and the analysis of 16S rDNA.The fermentation conditions of Bacillus subtilis were optimized,to increase biomass spore forming rate.By single factor experiment,the optimal ingredients of culture medium were confirmed:glucose 2%,peptone 1%,magnesium sulfate 0.02%,calcium chloride 0.02%,dipotassium phosphate:monopotassium phosphate 0.2:0.05(%) and the optimal culture conditions were confirmed by orthogonal experiment:initial pH=7.0,inoculum size 7%,temperature 37℃,shaking speed 150r/min.Under the optimized conditions,viable count of YB5 from 2.03×109cfu/ml increased to 3.83×109cfu/ml, increased by 89% and spore forming rate of YB5 from 77% increased to 96%,increased by 25%.Staphylococcus aureus as the indicator bacteria,preliminary researched character of antimicrobial substance.Putting liquid supernatant in different temperature to research its antibacterial activity fond that 40℃ to 70℃ processing,the antibacterial activity did not change significantly;80℃ processing,the antibacterial activity were gradually decreased with the rise of temperature;after 121℃ processing,the antibacterial activity were almost disappeared. The antibacterial activity declined after acid base and organic solvent processing.Liquid supernatant was sensitive about protease when using pepsin, trypsin and papain processed 1h,the antibacterial activity were almost disappeared.The above results estimated that antimicrobial substance produced by Bacillus subtilis YB5 belongs to protein.