Development Of Two Probiotic Formulations Through Solid And Liquid Fermentation

Probiotics owns numerous physiological functions,of which the most important is theypromote the proliferation of beneficial microorganisms in the gut and reduce the number of pathogens.Because of great advantages including non-toxic,no residue,no side effects and no pollution,probiotic formulations become effective alternatives to antibiotics.With a stain of Bacillus subtilis which has strong resistance and Lactobacillus acidophilus which had been domesticated with acid and bile,this paper is mainly for the following aspects of research:?1?we got a strain of Bacillus subtilis?5?,which had good antibacterial activities against E.coli,Salmonella,Staphylococcus aureus and Penicillium.Then we performed bile domestication and acid domestication on Lactobacillus acidophilus to enable it a strong acid and bile resistant ability.All of these laid the solid foundation for the two strains used in feed or feed additives.?2?Through single factor and orthogonal experiments on optimization for liquid fermentation medium of Bacillus subtilis and its conditions,we cultured Bacillus subtilis for 12 hours and measured the size of its OD_{600 nm} using UV spectrophotometry.Then we chose OD_{600 nm}00 nm as an index to select the medium.After that,optimized medium was as follows:6%for broken rice hydrolyzate,0.7%for yeast extract,0.5%for potassium dihydrogen phosphate,and OD_600nm as high as 7.18.On the other hand,we determined the optimum cultured conditions of the medium by single factor experiment,namely,5 for initial pH,37?for cultured temperature,6%for inoculum size,200 r/min for trained speed.Broth should be harvested after cultured for 19 hours.Under the optimum conditions,OD_{600 nm}00 nm can reach as high as 7.38,and the number of viable is 5.2×10¹⁰.?3?We selected out the hydrolyzed sugar solution of 4%broken rice which can be carbon source for liquid fermentation of Lactobacillus acidophilus instead of glucose.Then we added growth factors into the basis medium,performed single factor and orthogonal experiments,and determined the best combination of growth factors as A₂B₁C₃,that is,0.8%for lactose,5%for malt extract and 7%for white radish juice.According to the above formula,OD?diluted 20 times?of liquid fermentation of Lactobacillus acidophilus can reach as high as 0.463,6.20×10⁹ cfu/mL for bacteria concentration.Compared with basis medium?5.31×10⁸cfu/mL?,it had a substantial growth in the number of bacteria.?4?After determining the materials and their ratios of solid state fermentation medium,we selected the best additional carbon and nitrogen one by one,as well as their concentrations.At the moment,the number of Bacillus subtilis can reach as high as 14.3×10⁸ cfu/g,the number of Lactobacillus acidophilus viable can reach as high as 8.5×10⁸ cfu/g.Then we recognized the optimum fermentation temperature is 37?,and the optimum fermentation time is:24 h for Bacillus subtilis and 48 h for Lactobacillus acidophilus.Bacillus subtilis takes halfway flip in its fermentation,while Lactobacillus acidophilus was allowed to just stand.?5?Probiotic granulation got the best results when the proportion of rice flour,wheat flour and soluble starch is 1:1:2.There is not only no adhesion in granulation,but also intact particle shape and tight structure after drying.On the basis of univariate multilevel experiment,we conducted orthogonal experiments,and obtained the optimum combination of protective agent Bacillus subtilis.It was A₃B₁C₃,namely 15%for sucrose solution,5%for skim milk powder,15%for glycerol concentration and 75.05%for survival rate.The best combination of Lactobacillus acidophilus was A₂B₃C₁:15%for trehalose solution concentration,15%for skim milk powder,10%for glycerol concentration and 45.46%for survival rate.Storage properties of Bacillus subtilis formulation were superior to Lactobacillus acidophilus as survival rate of the former after 100 days decreased by only1.39%,which the latter declined by 25.26%.