Screening Of Bacillus Coagulans And High-density Fermentation

Probiotics is a general term for a group of active microorganisms that can improve the micro-ecological balance of organisms,regulate the physiological functions of organisms,and improve the immunity of organisms.Bacillus coagulans is a kind of probiotics,its main characteristics are:（1）Ability to survive the gastric transport and tolerate to gastric acid and bile;（2）Ability to adhere and colonize the intestinal epithelium;（3）Ability to maintain intestinal homeostasis;（4）It can inhibit the growth of intestinal pathogens and prevent epithelial colonization or invasion;（5）No pathogenicity and other characteristics.Nowadays the number of viable Bacillus coagulans on the market is relatively small,and the high fermentation cost restricts its development in various fields.This study first screened Bacillus coagulans from the environment;then,using biomass as an index,the fermentation conditions were optimized through single factor experiments,and several biostatistics methods were applied to determine the most suitable fermentation medium components,such as Plackett-Burman design and Central Composite Design.Subsequently,the influence of different carbon and nitrogen sources on the sporulation ability of the strain was investigated,and the cultivation conditions for both biomass and sporulation efficiency were determined.Finally,the high-density fermentation was carried out in a 30 L mechanical agitated aeration fermenters in order to provide a reference for the development of feed industry.In this study,Bacillus coagulans was first screened from the environment,and then the optimal fermentation conditions for the biomass of this strain were optimized by single factor experiments.Through the Plackett-Burman design test,the Central Composite Design center composite test and other biostatistics methods,the optimal fermentation medium composition of the strain’s biomass was determined.Then the ability of spores production of this strain was optimized.Finally,high-density fermentation was conducted on the basis of the optimized process,and the main research results are as follows:（1）Isolation and identification of B.coagulans CNJD from kimchi:using kimchi as a sample,a target strain was isolated and purified by two-step method based on transparenting,then the strain was identified as Bacillus coagulans（Bacillus coagulans）,named CNJD through colony morphology observation,cell morphology observation and strain identification.（2）Optimizing of fermentation conditions of B.coagulans CNJD:the growth curve of the strain was measured by spectro-photometer turbidit,and the optimal seed culture time was selected as 24h according to this growing curve.Using single factor experiment,Plackett-Burman design experiment,Central Composite Design design experiment and other methods,the optimal fermentation conditions for this strain were determined as fermentation time 16 h,fermentation temperature 55 ^oC,and inoculum2%,The shaker speed is 180 rpm/min,the medium volume of shaking flask is 50m L/250 m L,and the initial fermentation p H is 5.The optimal fermentation medium composition is sucrose 48.59 g/L,yeast extract 10.00 g/L,tryptone 10.00 g/L,sodium chloride 1.50 g/L,calcium chloride 1.70 g/L,and manganese sulfate 0.33 g/L.The predicted biomass is 7.99×10¹⁰ CFU/m L,and the measured biomass is 7.86×10¹⁰CFU/m L.The results are similar,indicating that the experimental results obtained by the above optimization method are reasonable and credible.After optimizing the fermentation conditions and medium composition,the biomass of Bacillus coagulans was 5.31 times that before optimization.（3）Optimizing of spore-forming conditions of B.coagulans CNJD:By investigating the effects of different carbon and nitrogen sources on it,the highest sporulation efficiency was obtained when corncob powder was used as a single carbon source.The analysis of the activity of polysaccharide hydrolase and the soluble sugar content of the fermentation medium indicated that after 22 hours of fermentation,the cellulase activity in the broth was 2.323 U/m L,and the xylanase activity was 23 U/m L,the presence of xylo-oligosaccharides in the broth and the digestion and utilization of cellulose and xylan may be one of the reasons for increased sporulation efficiency.The optimal fermentation conditions of spore-forming by this strain were determined by a single factor test as follows:fermentation time 22 h,fermentation temperature 55 ^oC for the first stage（0-16h）,45 ^oC for the second stage（16-22h）,and inoculum 2%,the shaker speed is 180 r/min,the liquid volume is 50 m L/250 m L,and the initial fermentation p H is 5.0.The best fermentation medium composition is corncob powder50.00 g/L,yeast extract powder 6.00 g/L,tryptone 6.00 g/L,sodium chloride 1.50 g/L,calcium chloride 1.70 g/L,and manganese sulfate 0.33 g/L.After optimizing the fermentation conditions and medium composition,the number of spores of Bacillus coagulans reached 1.7×10⁸CFU/m L.（4）High-density fermentation:the biomass and spores production of Bacillus coagulans were increased through high-density fermentation.Based on the fermentation conditions after biomass optimization,high-density culture of Bacillus coagulans was carried out using a fed-batch fermentation method.The number of viable bacteria was3.5×10¹¹CFU/m L,which was 5 times that of the shake flask optimization.After being collected and spray-dried,the number of spores was 6×10⁷ CFU/m L.Based on the fermentation conditions after spore optimization,high-density culture of Bacillus coagulans was carried out by fed-batch fermentation.The number of viable bacteria was3.2×10¹² CFU/m L,the number of spores reached 4.3×10⁹ CFU/m L,and the number of spores was relatively shaken,which was 25 times as much as flask fermention.