Study On The Probiotic Characteristics Of Bacillus And Its Rapid Optimization And Preparation In Spore Fermentation

Commercial Bacillus probiotics are used in the form of spores.In this dissertation,a strain of Bacillus as probiotics was available base on a comprehensive evaluation of commercial Bacillus strains.The strain was then used as an example to study on the method of specific detection of spore concentration based on DPA contents.The fast and accuate method of spore detection was used study the feasibility of rapid optimization on spore production in liquid and solid-state fermentation,respectively.The research contents and related results are as follows:?1?In this study,15 samples from commercial Bacillus probiotics from medicine and feed sources were included for screening their probiotic characteristics.The enzyme tests including amylase,cellulase and protease,antimicrobial activities,tolerance tests to acid and bile salts,oxygen-consumptions rate and growth rate were compared in all the 15 Bacillus strains.?2?Based on previous studies in the laboratory,2,6-pyridine dicarboxylic acid?DPA?of spores can be fully released after treatment by high temperature and pressure.DPA mixed with Eu²⁺and chelating agent CyDTA can produce fluorescent complexes,and the fluorescence intensity of complexes is linearly correlated with the spore concentrions.?3?This study optimized the culture medium for the spore production of Bacillus spp and investigated the feasibility of using a dipicolonic acid?DPA?fluorimetry assay as a simpler alternative to plate counting for evaluating spore yields.Through the single-factor experiment and response surface methodology?RSM?,the metal ions and agro-industrial raw materials that significantly enhanced spore production were determined.The flocculation techniqure was used for fast recovery of spores from liquid broth.?4?The feasibility of DPA fluorimetry assay as a simpler alternative to plate counting in optimization design for spore yields in solid state fermentation was further studied.Based on the optimized culture medium and culture conditions,the fermentation process and metabolic activity were studied.?5?The experiment further discusses the feasibility of compound solid-state fermentation process of Bacillus strain in non-sterilized fermentation substrates.?1?The results showed strain S9 was the highest compared with other strains at exerted enzyme activities and antimicrobial activity against all the three indicator bacteria.The vegetative cells of three strains of S9,Y2 and S8 were tolerant to low acid environment and the survival rates in 0.3%bile salt environment were over 90%in S9and Y2.The strain S9 grew faster with more rapid consumption of oxygen.The strain S9,identified as B.amyloliquefaciens and named B.amylolyquefaciens BS-20.?2?The results showed that the fluorescence intensity of fluorescence complexes was within linearity with spore concentrations?R²>0.999?varying from 10⁴ CFU/mL to 10⁷CFU/mL.The limit of detection?LOD?reached 8×10³ CFU/mL.The spore yield detected by fluorimetry assay were consistent to that by plate counting assay based on the results of spore concentrations from fermented broth at different sampling time analyzed by both methods.?3?After conducting a response surface methodology?RSM?analysis of several metal ions and supplementing soybean meal and corn meal,the optimized media produced an 8.8-fold increase in spore yields.The culture conditions in flask fermentation was further optimized through the single-factor experiment and the finally spore yields could reach 8.35×10⁹ CFU/mL.The flocculation techniqure was used for fast recovery of spores from liquid broth.The flocculating efficiency was optimized by mixing the complex of chitosan and ZnSO₄?the proportion of 3:1?with fermentation broth at pH 4.5.The concentrating rate was reached 90%after 30 min settlement and the recovery rate of viable spores was 93%.?4?Through the single-factor experiment,the solid substrates ratio with corn flour:soybean meal flour:wheat bran=1:1:3 produced higher spore yields.Carbon sources,nitrogen sources,inorganic metal ions and their concentrations were screened by a single factor experiment and central composite design was determined.When the liquid substrate consisted of glucose 8 g/L,peptone 2 g/L,Mn²⁺1.0 mM,spore concentration reached 6.6×10¹⁰ CFU/g.The cultivation conditions including inoculation size,initial moisture content,moisture content,matrix loading and culture time was studied by single factor experiment step by step.The results showed that inoculum 6%,initial pH 7.0 moisture content 50%,substrate loading 30 g/250mL and culture time 48 h were optimum culture conditions.The spore concentration finally reached 7.1×10¹⁰ CFU/g after optimization.The spore yield detected by fluorimetry assay were consistent to that by plate counting assay in each optimization steps during solid state fermentation.The maximum yield of spore concentration and other active substances were obtained at 48 h of ferementation with protease 573.0 mg/g,amylase 188.8 mg/g,cellulase 186.8 mg/g,acid soluble protein3.45 mg/g,respectively.?5?Lactobacillus reuteri C5 and Saccharomyces boulardii SC18 were introduced as single or compound starter cultures to prevent contamination of during culture process.The pH value and concentrations of viable substances as different sampling time were analyzed among different starters.The results showed the combination of L.reuteri C5,S.boulardii SC18 and B.amylolyquefaciens BS-20benefited the fermentation process with the faster decreased pH,reducing sugar concentrations and more TCA leves and higher concentraions of lactaobacillli,yeast and spores and none pollution.In conclusion,the whole study screened probiotic Bacillus strains by comparing biological characteristics of various kinds of commercial Bacillus probiotic strains.A rapid quantitative method for detecting Bacillus spores was established and applied successfully in rapid optimization production of liquid and solid-state fermentation.The paper attempts to establish a whole application system for Bacillus probiotics including strain evaluation,fermentation optimization and preparation,which provide methodological basis for research and industrialization production of Bacillus probiotics preparations.