Screening And Fermentation Optimization Of Ba Cillus Co A Gulans And Enterococcus Faecalis

Antibiotic has been widely used as feed additive.However,the abuse of antibiotic has resulted in negative effect on human and environment.Antibiotic was forbidden as feed addive in many countries.Thus,it is urgent to develop an alternative feed additive to replace the antibiotic.Probiotics is beneficial to the growth and reproduction of animal,and helps preserve animals' good health.Therefore,probiotics is an ideal replacement of antibiotic as feed additive.In this study,three probiotics strains were isolated.The stability in stimulated internal environment and the antibacterial ability of these strains were measured as well.The fermentation process was also optimized,and the encapsulation of probiotics was performed.The results were as follows:Acorrding to the general screening methods for probiotics,three strainss are isolated from animal gut contenes as well as animal wastes and they are calassified as Enterococcus faecium RS3?Enterococcus faecalis J and Bacillus coagulans 202 by the analysis of 16S rDNA sequences,which are all safe micro-organisms approved by MOP P.R.China.B.coagulans 202 and E.faecalis J were stable in stimulated internal environment.All the strains possess good antibacterial ability to Escherichia coli and Salmonella enteritidis which caused the diarrhea to animal.The optimum medium component of B.coagulans 202 were as follows(g/L):glucose 6,soya bean meal 30,K2HPO4·3H2O 2,MgSO4·7H2O 1,MnSO4·H2O 0.005,CaCl2 0.3,with the optimum cultivation conditions were as follows:inoculation quantity was 2%(v/v),the initial pH was 6.5,inoculum age was 7,the optimal temperature was 37? and the shaker speed was 240 rpm.The biomass reached 15×108 CFU/mL at 48 h,spore forming rate was 90%.The biomass was increased 50%compared with the initial experiment.By feeding ammonia and glucose,the biomass in a 5L bioreactor was 20×108 CFU/mL,and spore forming rate was 90%.The optimum medium component of E.faecalis J were as follows(g/L):sucrose 10,soya bean meal 60,sodium acetate 5,MnSO4·H2O 0.15,MgSO4·7H2O 0.4,K2HPO4·3H2O 2.The optimum cultivation conditions were as follows:inoculation quantity was 1%(v/v),the initial pH was 6.6 and inoculum age was 7.By feeding ammonia and sucrose,the biomass of E.faecalis J in a 5-L bioreactor reached 1.8×1010 CFU/mL,which was 10 times than that of the initial experiment.The encapsulation of E.faecium RS3 was performed by using calcium alginate.The capsule wall material,cross-linking agent concentration,reaction temperature and time were also optimized.After the optimization,the encapsulation rate reached 95%.Furthermore,poly-y-glutamic acid(y-PGA)was added in alginate solution,it showed the survival rate of E.faecium RS3 in stimulated gastric fluid was significantly increased by 70 times,the number of viable bacteria improved from 9×1 05 CFU/mL to 5.30×107 CFU/mL.