Study On ?-polyglutamic Acid Production Based On Microbial Fermentation Using Bacillus Subtilis And Its Separation And Purification

?-PGA(poly-?-glutamicacid,y-PGA)is a water-soluble polymer produced by microorganisms.It having a few advantages including water absorption,water retention,biocompatibility and natural degradation.As a novel polymer and its derivatives,?-PGA can be extensively applied in food,medicine and environmental protection.However,its high commercial price is preventing it from being applied because of low production efficiency and high cost of separation and purification.To this end,this paper systematically reveals the effects of submerged fermentation(SMF)and solid state fermentation(SSF)on y-PGA production by comparatively studying SMF and SSF.Based on this,fermentation conditions and the processes of separation and purification were optimized to improve the efficiency in production and separation.As a result,the production cost of ?-PGA was reduced Hopefully,this finding will promote y-PGA and its products to be further developed and applied.The details were shown as following:(1)Study on SMF:The results from a few single factor studies showed that sucrose and tryptone were the optimal sources of carbon and nitrogen,respectively.The substrate L-glutamic acid plays a significant role in y-PGA synthesis.Furthermore,the optimal medium was obtained by response surface design,which is composed of sucrose 28.87 g/L,tryptone 17.56 g/L and L-glutamic acid 15.26 g/L.Also,the fermentation conditions was optimized,and the optimal one is shown as following:inoculums size 7.5%,loaded volume 20%,pH 7,and incubation temperature 37? for 36 h.Finally,a top yield reached 17.42g/L,4.98-fold higher than the one before optimization.(2)Study on SSF:Independent effects of solid matrix type,grinding degree of nutrient matrix,solid matrix/nutrient substrate,inoculum size and related fermentation conditions on y-PGA productionwere studied comprehensively.The results showed that perlite is the optimum solid support,the grinding degree of soybean one-eighth,the optimal ratio of ground soybean to perlite 1:1,the initial water addition into 100 g of solid substance6 mL,the inoculum size 8%,and the optimum pH of the medium7.After incubation for 36 h at 37 ?,the maximum yield of y-PGA,79.12 g/Kg was achieved,which is 1.86 times of the one before optimization.(3)Study of NK effect on y-PGA production:It was found from comparing time courses of nattokinase(NK)and y-PGA in production that the rapid production period of y-PGA isalways later than NK.Furthermore,the molecular weight(MW)distributions of y-PGA produced by SMF and SSF was studied,and the results indicated that the MW of y-PGA produced by SMF is larger than that under SSF,and the former is more concentrated than the latter in the MW distribution.Therefore,it is preliminarily concluded that NK couldaffect y-PGA production.Based on SMF,the effect of exogenous NK on y-PGA production was investigated,and the results indicated that NK can really inhibit the production of y-PGA by substrate competition and degradation of y-PGA.(4)Study on separation and purification of y-PGA:The protocol of separation and purification of y-PGA was improved based on ethanol extraction,acetone treatment,dialysis treatment and phenol treatment,and the results showed that 3-fold volumes of acetone can effectively improve the extraction rate and purity of y-PGA,dialysis treatment can remove small proteins and impurities,and phenol treatment can further improve the purity of y-PGA.Ninhydrin colorimetric detection and HPLC were applied to determine the purity of y-PGA,and the result indicated the purified ?-PGA sample owns a higher purity relative to the one before purification.The highest purity reached 89.12%.It was found from paper chromatography that the purified y-PGA mainly consisted of a single glutamic acid without other free amino acids.SDS-PAGE gel electrophoresis showed that y-PGA MW distributes in the range of 690-700 KD.It indicates the polymerization degree is concentrated.The results of the thesis will provide a crucial reference for the research on y-PGA production us:ing microbial fermentation and its separation and purification.This work is helpful for reducing the production cost of y-PGA to a certain extent.Finally,it will promote further development and application of y-PGA.