High-density Cultivation Of Bacillus Coagulans And Preparation Of Its Viable Tablets

With the abuse of antibiotics,the drug resistance of some pathogenic bacteria is becoming more and more strong,and the problems are becoming more and more serious,even endangering human health.It’s important to be able to find alternatives to antibiotics.Two candidate Bacillus coagulans strains were isolated from fermented soya bean by high temperature treatment,acid production test,Gram staining and spore staining microscopic examination,and contact enzyme test.The two strains were named GBC-1 and GBC-2 respectively for acid production,spore production,Gram staining and contact enzyme positive.They were identified as Bacillus coagulans by 16s r DNA identification.The probiotic characteristics of two strains of Bacillus coagulans were analyzed,including stomach,intestinal juice,bile salt,heat resistance and bacteriostasis,antibiotic sensitivity and extracellular enzyme hydrolysis activity.The survival rate of GBC-1 was higher than that of GBC-2 at 0.3%concentration for 2 h.GBC-1 was higher than that of GBC-2 at 80℃for 30 h GBC-1 was not resistant to common antibiotics,but GBC-2 was resistant to penicillin,chloramphenicol and rifampicin,and GBC-1 was more safe than GBC-2;both strains produced amylase,protease and cellulase,and the two strains had little difference in hydrolysis ability of starch,protein and cellulose.After comparing the probiotics of the two strains,Bacillus coagulans GBC-1with better probiotic characteristics was selected for follow-up test.The optimal culture medium and culture conditions of Bacillus coagulans were obtained as follows:fermentation time 22.56 h,fermentation temperature39.20℃,microelement concentration 0.62 g/L,initial p H 6.5,inoculation amount 5%,carbon source sucrose concentration 20 g/L,nitrogen source yeast extract concentration 10 g/L,the theoretical maximum number of viable bacteria is 4.14×10⁹cfu/m L。The number of viable bacteria was（4.27±0.06）×10⁹cfu/m L,which was 5.93-folds as much as that before optimization,and the number of spores was 2.82-folds of that before optimization.After that,5 L fermentor was used for high density culture.The dissolved oxygen content was controlled at about 30%,and the p H value of the medium was kept at 6.5 with20%Na OH.The glucose content was controlled between 1%and 2%,cultured for 48 hours,the number of viable bacteria reached（9.13±0.72）×10¹⁰cfu/m L,which was 21.38-folds that before high-density culture,and the number of spores was（10.37±0.23）×10⁹cfu/m L,which was 24-folds before high-density culture.The bacteria were collected by centrifugation at the speed of 7000 r/min for 10 min.the porous starch was used as the carrier,the material thickness was2 cm,the moisture content was about 3%,and 5%sorbitol was used as the protective agent to prepare the bacterial powder.The tablets were prepared by adding disintegrating agent crosslinking povidone 2%,lubricant magnesium stearate 1%,hardness about 60 N;the best storage temperature is 4℃.In this experiment,the isolated Bacillus coagulans were cultured in high density,and the powder was prepared and pressed directly.It has certain guiding significance for the industrial scale production of Bacillus coagulans solid bacteria preparation.