Studies On The Processing Technology Of The Probiotic With Bacillus Subtillis

Probiotics are perfect substitutes of antibiotic, which are effective in preventing and treating animal diseases and promoting the growth and development of the animal. There are some other advantages, such as non-toxic, no side effects,no residue and no drug-resistant.Probiotics have a good prospect of application in aquaculture. Bacillus subtilis is allowed to be added in feed by both China’s Ministry of Agriculture and the U.S.FDA. Bacillus subtilis BSl is a strain preserved in our laboratory. Preliminary studies indicate that the add of this strain’s culture in the diets could effectively improve the growth performance of weaned pigs, and improve the animal’s disease resistance.In the practical application of probiotic, there are some shortcomings such as a low number of viable cells, poor product stability, short shelf life which limit its industrial production and large-scale application. Bacillus subtillis forms spore in its life cycle, which has a very strong resistance to adversity. Therefore B. subtilis has an inherent advantage in terms of stability. The fermentation medium, fermentation process and spray-drying process of B.subtilis BS1were optimized in this study. By these methods, we got an effective industrialized production process of BS1.The main conclusions are as follows:1. Through the single factor experiment, we found that the best carbon source, nitrogen source and inorganic salt were malt extract, soybean powder and calcium carbonate. Meanwhile, the result showed that solo quick nitrogen source was not conducive to the Bacillus subtilis BS1spores formation. By orthogonal experiment and regression orthogonal combination test, we determined the primary and secondary factors that influence the spore formation of BS1, they were:calcium carbonate, malt extract powder and soybean powder. The best liquid fermentation medium for the spore production of Bacillus subtilis BS1was:malt extract lg/L, soybean powder15g/L, CaCO36.68g/L, MnSO40.4g/L, MgCl22g/L. Using this formula, we cultured BS1for48h under the condition of180r/min,37℃.At last, the spore yield could be up to8.55×1010cfu/mL.2.50L fermenter experiment showed that under the condition of liquid volume60%(V/V),37±1℃, stirring speed200rpm, aeration rate1.5m3/h, tank pressure0.03～0.05Mpa, natural pH, the BS1entered its logarithmic phase8h later, entered its stable phase16h later, began to form spores20h later,28h later a large number of spores were formed and at46h the number of spores reached the highest value.3. Through500L fermenter test, we got an appropriately fermentation process control strategy:8h, stirring speed100rpm, aeration rate12m3/h;8to20h, stirring speed150rpm, aeration rate18m3/h;20～48h, stirring speed100rpm, aeration rate10m3/h.During the whole process, the temperature was37±1℃, the tank pressure was0.03-0.05Mpa, natural pH.5000L fermenter trial production showed the fermentation average fermentation time was35.8h; the average spore yield was1.02×1010cfu/mL.4.Using corn starch as drying aid, under this spray drying conditions:inlet air temperature210℃, feeding frequency13Hz, We sprayed dry the fermentation broth of BS1. The product yield was48.8%, the moisture content was5.27%. If being stored at room temperature, the shelf life of the spray drying powder was6months at least, the survival rate of bacteria was around84%.