Screening Of Probiotics Bacillus Coagulans And Optimization Of Its High Density Cultivation Conditions

In this study,B.coagulans were isolated from cecal and feces of healthy piglets,and their probabilities and safety were studied.A strain of B.coagulans BC303 with good prebiotics and no antibiotic resistance was obtained.The high-density culture and rapid spore of the cell were realized by the stage control strategy,and the key points of BC303 sporulationprocess were studied by transcriptome sequencing.The sporulation initiation pathways and related regulatory models of BC303 were preliminarily constructed.The main findings are as follows:1.A total of 40 strains of Bacillus were isolated and screened from cecal and feces of healthy piglets.Four strains of B.coagulans were identified by morphological,physiological and biochemical analysis and 16 S rDNA.They were named BC147,BC235,BC310 and BC303 respectively.All four strains had good adhesion ability and showed strong tolerance to bile salt and low acid environment.The survival rate of BC303 on piglets intestinal mucus was as high as 41.67%,the survival rate of vegetative cells was 91.0% at pH 4,and the survival rate was above 40% when exposed to 0.3% bile salt for 2 hours.Pathogen antagonism test showed that four strains showed good antibacterial activity against common intestinal pathogens,including for Salmonella typhimurium ATCC 13311 have the highest antagonism ability;At the same time,four strains have certain protease,amylase and cellulase production ability,and they are sensitive to antibiotics in different degree,and BC303 is highly sensitive to antibiotics.The results showed that BC303 was a good candidate for probiotics in mice,and the strain had good safety and could increase the daily gain,feed conversion and immunity.2.B.coagulansBC303 was selected as the research strain,the fermentation conditions and fermentation medium were studied by single factor test and response surface methodology(RSM),and ultimately determine the optimum culture conditions of BC303 for the time 20 h,initial pH8.0,culture temperature is 37℃,speed 230 rpm,inoculum 3%;the optimum ratio of fermentation medium: glucose 15.80g/L,peptone 15.90g/L,yeast powder 11g/L,MgSO4 4.60g/L.The vegetative cells density of BC303 was increased to 2.13×1010CFU/mL by adding 15g/L glucose as the best feeding regimen at 8h.On this basis,the addition of 1.8g of calcium carbonate,1g of bran and 0.1mg of isoleucine can make the spore density reached1.3×108CFU/mL.It was found that the optimum sporulation conditions were 40℃and280rpm,respectively.Through the screening,addition and optimization of fermentation conditions,the BC303 spores were increased to 8.4×108CFU/mL.3.The key points of B.coagulans BC303 spore formation were studied by sequencing the prokaryotic chain specific transcriptome,the sporulation initiation pathway and related regulatory models were constructed.The sporulation pathway was initiated by the self-phosphorylation of KinC and KinE.Then theypass the phosphate to Spo0 F and Spo0 B,and finally the phosphorylation of Spo0 A was produced.At the same time,KinC self phosphorylation may also bypass Spo0 F and Spo0 B,and directly transfer the phosphateto Spo0 Aand start the sporulation process.In this process,the activity of Spo0 A was affected by cell density,DNA damage,redox status,nutrient condition and dephosphorylation.Among them,Opp,AIP,dnaA and LexA can indirectly regulate the level of self-phosphorylation of histidine protein kinase;sda and codY-GTP directly inhibit the self-phosphorylation of histidine protein kinase;Spo0A-PO4 is susceptible to dephosphorylation of Spo0 E.The above regulation will eventually lead to the expression of Spo0 A and the level of phosphorylation,which may regulate the initiation and formation of B.coagulans.