The Optimization Of Fermentation Conditions For Feeding Bacillus Licheniformis BL-5

ObjectiveFirstly,the fermentation conditions of Bacillus licheniformis BL-5 were optimized by the viable bacteria count.Then,the optimized fermentation conditions were obtained on the basis of the resulting fermentation conditions by the number and rate of spores.The high rate of spores is conducive to extend shelf life,is conducive to add it as a probiotic preparation in the feed applications.MethodThe growth curve of Bacillus licheniformis BL-5 was measured by spectrophotometry with the indexe of OD value to determine the best culture time of seed liquid.The strain with high rate of spores was selected by the method of gradient dilution plate counting and plate streaking with indexes of number and rate of spores.The optimum number and rate of spores were obtained by response surface methodology with the main indicator of the rate of spores and reference indicator of the number of spores.Result1.Firstly,the growth curve of Bacillus licheniformis BL-5 was measured,slow phase was 0-6 h,logarithmic phase was 7-12 h,stable phase was 13-18 h,decline phase was reached after 19 h.The culture time of seed bacteria wasselected at 14 h according to the growth curve.Secondly,the essential ingredients of medium were determined via single factor experiment.The result showed that the optimal carbon source was sucrose,optimal nitrogen source was ammonium sulfate,and the best ratio of carbon/nitrogen was 2/1.Thirdly,at Plackett-Burman(PB)experimental design,nine main factors were obtained from 12 factors about the culture conditions and medium compositions as follows: sucrose,magnesium sulfate,dipotassium phosphate,yeast extract,p H,fermentation temperature and rotate speed were positive effect,and ammonium sulfate and liquid volume were negative effect.Fourthly,the factorial design experiment was carried out according to the resulting PB experimental design.And the ultimately optimum fermentation conditions of Bacillus licheniformis BL-5 was determined as follows: sucrose 29.85g/L,ammonium sulfate 10.00g/L,dipotassium phosphate 1.80g/L,yeast extract3.00g/L,p H 8,30% liquid volume,fermentation temperature 30℃,rotate speed 150 r/min.2.Firstly,according to the single factor experiment of the rate of spores,the best result of eight factors was determined as follows: the lethal condition was at 75 ℃ with 15 min,fermentation temperature was 30 ℃,fermentation time was 48 h,the seed culture medium was beef extract and peptone,seed strain age was 24 h,Mg2+ concentration was 1.5 g/L,K+ concentration was 0.015mol/L,the amount of inoculum was 5%.Secondly,the three main factors and their effect trends acquired from 6 factors in PB experimental design were as follows: fermentation time and Mg2+ concentration had a positive effect,while K+ concentration had a negative effect.The center point of steepest ascent result in PB experimental design was the best experimental group 3,namely:fermentation time 42 h,K+ concentration 0.004mol/L,Mg2+ concentration 1.5g/L.The number and rate of spores were optimized by response surface design according to this center point.The final optimization of fermentation conditions were: K+ concentration 0.002mol/L,Mg2+ concentration 1g/L,fermentation time36 h.Conclusion1.The culture time of seed Bacillus licheniformis BL-5 was selected at 14 h according to the growth curve by the index of viable bacteria count.The viable bacteria count of Bacillus licheniformis BL-5 were optimized via single factor experiment,PB experimental design and factorial design experiments,successively.The optimum fermentation conditions of the viable bacteria count were as follows: sucrose 29.85g/L,ammonium sulfate 10.00g/L,dipotassium phosphate 1.80g/L,yeast extract 3.00g/L,p H 8,30% liquid volume,fermentation temperature 30℃,rotate speed 150 r/min.The optimum amount of viable Bacillus licheniformis BL-5 cells was 7.17×1010cfu/ml via verification experiment.2.The optimum conditions were obtained by the single factor experiment,PB experimental design,steepest ascent experiment and response surface methodology with the index of the number and rate of spores as follows: the lethal condition was at 75 ℃ with 15 min,the seed culture medium was beef extract and peptone,the optimum fermentation conditions were K+concentration 0.002mol/L,magnesium2+ 1g/L,fermentation time 36 h.The optimum fermentation conditions were verified with three replicates: the average rate of Bacillus licheniformis BL-5 spore was 94.67%,the number of spores was 3.32 × 109cfu/ml.