Induced Breeding And Enzyme Properties Of Bacillus As Probiotics

In this paper,we researched on isolation and identification, mutation breeding, enzyme properties and effect of probiotics of Bacillus as probiotics. The main results were as follows:(1) Strain B1 with cellulase activity and JC12 strain with Proteinase activity were screened from soil and healthy broiler's intestines respectively, according to enzyme activeties and tolerance of bile salts and acid. Cellulase activity of CMC and FPA of B1 strain were 0.256 U/mL and 0.228 U/mL respectively, which with high tolerance of bile salts and acid. B1 strain was identified as Bacillus cereus according to the physiology and biochemistry tests and the comparison of 16S rDNA sequence. JC12 strain survived well in the gastrointestinal tract, and its Proteinase activity reached 519 U/mL. JC12 strain was identified as Bacillus cereus according to the physiology and biochemistry tests.(2) Strain B1-1 with high cellulase activity was screened by microwave and DES mutagenesis, and cellulase activity of CMC and FPA reached 0.373 U/ml and 0.348 U/mL, increased by 45.70% and 52.63% respectively. High Proteinase activity JC128 strain was selected after composite mutagenized by microwave and DES, and its proteinase activity reached 1207 U/mL, enhanced 1.33 times higher. B1-1 and JC128 were genetically stable after five subcultures, and well survived in the gastrointestinal tract.(3) The optimum conditions for cellulase production were 20 g/L maltose, 2.5 g/L ammonium sulphate, pH 5.0 and 37℃culture for 36 h. The optimum fermentation condition for JC128 strain producing Proteinase were: 6 % maltose, 3 % soybean cake, initial pH of 7.0, at 37℃. The proteinase had excellent stability at high temperature and pH, which presented the highest activity at 65℃and pH7.0. The Proteinase activity was stimulated observably by 1 mmol/L Fe²⁺. B1-1 and JC128 strains both can inhibit the growth of Escherichia coli and Staphyloccocus aureus.(4) Strains B1-1 and JC128 were proved to be safety by toxicity test. The two strains both had effect on growth performance, and compound probiotics highly increased the growth performances. Determine the enzyme activeties in intestines after mice were fed with B1-1 and JC128 strains for 14 days. The results indicate that B1-1 strain had Proteinase activity, and JC128 strain had cellulase activity, and compound probiotics had better effect. Cellulase activity of CMC and FPA and protease activity were increased by 48.70% and 30.15% and 30.23% respectively in the compound probiotics preparations compared with the control. B1-1 and JC128 strains increased spleen index and thymus index, and compound probiotics significantly higher than the control (P<0.01).