The Study Of H6-3 Bacillus Subtilis On Separating, Biological Characteristics, Optimization Of Culture Conditions And Clinical Effectiveness

The study separated 15 strains of Spore-Forming from young pig fresh excreta which gathered from many scale pig farms.There were three strains of Bacillus subtilis that had been bacteriological identified,and then through the experments of acid resistance and bile salt tolerance,production of protease and amylase test,bacteriostasis test in vitro,and animal safety test,finally filted out No.H6-3 strain,which could adapt the pig intestinal, degrade the protein and starch in feed and be safe to the pig.Through the optimization experiments of liquid and solid-state fermentation conditions,we determined the laboratory conditions to the best culture conditions of H6-3. The best liquid culture conditions included:pH7.0,temperature39℃,shaker speed 150r/min.we used the above-mentioned method to culture the H6-3,when the concentration of seed solution was 10⁷cfu/ml and inoculation was 1%(v/v),the viable number of 6.5×10⁸cfu/ml after cultured 48h.The best Solid-state fermentation included: temperature 39℃,charge 40g/250ml,material:water ratio 1:1.1,when the seed liquid 10⁷cfu/ml concentration,the inoculum size 5%(v/w),a few solid bacterial culture of 1.33×10¹¹cfu/g.after cultured 48h.H6-3 possessed the capacity of producing protease,when the temperature was 40℃, pH 6 The highest protease activity got to 753.187IU after cultured 72h.Clinical study results showed that adding the H6-3 in pig feed could significantly promote the growth of piglets and weight gain,and the effect of the weight gain was better than the effect,when feeding with the yeast(J44) or with lactic acid bacteria(19 #).After feeding for 20 days,the average daily gain of H6-3 group,H6-3 and J44 group,the H6-3 and 19 # group were 0.61 kg,0.56 kg and 0.46 kg respectively.The average daily gain of control group was 0.37g.Through analysis of variance,the differences between the daily gain of test group and the control group were significant(P＜0.05),and the the differences between the daily gain of H6-3 group and the othe testing groups were highly significant(P＜0.05).We respectively fed piglets with the solid dosage forms and liquid preparations of H6-3.20 days later we found that the effect of solid dosage forms group is superior to liquid formulations group and the average daily gain of piglets were 0.25 kg,0.21kg respectively,while the average daily gain of the control group was 0.16 kg.Through analysis of variance,the daily gain of piglets by fed solid dosage forms group was significantly higher than the liquid formulations fed group and the control group(P＜0.05). The further study found that the ratio of iarrhea of the experimental group piglets was only 4.7%and the survival rate was 100%after the piglets was fed by H6-3 solid dosage forms, while the ratio of iarrhea of the control group piglets was 10.85%and the survival rate was 89%.Compared with control group,Test group improved the average daily gain weight by 11.44%,feed conversion ratio reduced 8.9 I%.By the T lymphocyte proliferation test found feeding H6-3 20 days later,the peripheral blood T lymphocyte proliferation rate of experimental group piglets was 47.7%, compared with the control group increased 8.4%.