Study On Key Technology In Production Of Saponins By Microbial Method From Dioscorea Zingiberensis C. H. Wright Rhizomes

Dioscorea zingiberensis C.H.Wright(DZW),is a unique medicinal plant resource in China.DZW rhizomes are generally used to produce diosgenin,and diosgenin can be used in the synthesis of hundreds of steroidal hormones,known as the"medicinal gold".Diosgenin is existed in DZW rhizomes in the form of saponin,which is an amphiphilic compound with glycosylation at C3 and C26 positions of diosgenin.Diosgenin is usually produced via acid hydrolysis of all components of DZW together in industry.There are bottlenecks such as high pollution,low yield and high cost.To break the bottleneck and produce diosgenin cleanly,a method is needed to separate saponins from other components of DZW to enrich the saponins from solid phase to the water phase,and then convert saponins into diosgenin by adding microbial strains to the water phase.Therefore,how to ensure the stable content of aqueous saponins in DZW rhizomes and how to efficiently promote the release of saponins in solid phase of WSDP into aqueous phase are the key issues that need to be studied.In this study,systematic research on the above key issues were conducted and the following research results were obtained:(1)Factors influencing stability of aqueous saponins content in DZW rhizomes were found and the corresponding control technology was established.It was found in this study that endogenous enzymes and microbial strains of four genera could affect the stability of aqueous saponins in DZW,that is,endogenous enzymes could only degrade aqueous saponins in a small amount,and Myroides,Enterococcus,Alcaligenes and Stenotrophomonas together degraded a large amount of aqueous saponins probably;For this reason,an optimal control method was established.In other words,in the process of harvesting and storage of DZW rhizomes,the tissue structure of DZW rhizomes should be kept intact,so as to avoid that the endogenous enzymes in DZW rhizomes and the miscellaneous bacteria of the above species contacted with saponins in DZW rhizomes.Measures(such as high temperature treatment)should be taken immediately after homogenating of DZW rhizomes to inhibit the activity of endogenous enzymes and the metabolism of various bacteria in the homogenate.The above control methods were tested and can effectively avoid the hydrolysis of aqueous saponins in fresh DZW rhizomes in the process of harvesting,storage and diosgenin production,and ensure that a large amount of saponins are concentrated in aqueous phase.(2)The key factors promoting the release of saponins from DZW rhizomes into aqueous phase and its control technology were studied,it was found that starch hydrolase,lignocellulose degradation enzyme and surfactants could promote the release of saponins from solid pahse of water suspension of DZW powder(WSDP)into aqueous phase.It was found in this study that starch hydrolase,lignocellulose degradation enzyme and surfactants were the key factors in promoting the release of saponins from DZW rhizomes into aqueous phase;the control technology of these key factors was carried out with the WSDP,the utilization of starch hydrolase and lignocellulose degradation enzyme(mainly cellulase,xylanase,mannase,pectinase and laccase)in specific conditions can promote the release of saponins from solid pahse of WSDP into aqueous phase;while adding 50 g/L sodium dodecyl sulfate to WSDP can effectively promote the saponins from the solid phase of WSDP to dissolve or disperse in aqueous phase,so as to realize the enrichment of saponins from solid phase of WSDP to aqueous phase.(3)Microbial strain promoting release of saponins from DZW rhizomes to aqueous phase was screened,the effect was evaluated and the reason promoting the release of saponins from solid pahse of WSDP into aqueous phase was revealed.Bacillus sp.HG224 was screened and selected directionally for its ability in promoting release of saponins from solid phase of WSDP to aqueous phase.The strain treated WSDP in the fermentation condition of tow-stage temperature could promote release of saponins from solid phase of WSDP to aqueous phase and effectively increase the saponins content in aqueous phase of WSDP.And its effect was evaluated and analyzed.It was found that the enzymes that degraded lignocellulose and starch secreted by this strain effectively promote release of saponins from solid phase of WSDP.Meanwhile,the surface active substances secreted by this strain further promoted the release of saponins from the solid phase of WSDP into the aqueous phase,thereby significantly increasing the saponins content in aqueous phase of WSDP.Compared with untreated group,the saponins content in aqueous phase of WSDP treated by this strain increased from 50.85%(ratio in total saponins content)to 66.23%by 30.25%,resulting that a large amount of saponins were enriched in aqueous phase.(4)The production process of DZW saponins by microbial method was established,industrial experiment research on this process was carried out,enriching the saponins from the solid phase of WSDP into aqueous phase in industrial scale,and large amount of nutrient-rich sugar liquid and solid residue were obtained.A new technology and process for producing DZW saponins by microbial method was established.That is,WSDP or fresh DZW homogenate was treated with Bacillus sp.HG224,the starch in the water suspension was hydrolyzed with double enzyme method,then the saccharification liquid containing saponins was obtained by solid-liquid separation;after that,the saponins concentrate was obtained by membrane filtration,so as to achieve enriching saponins from solid phase into aqueous phase.40 m~3 fermenter level industrial scale tests of the new process were subsequently carried out in the cooperative enterprise.The results showed that the production time cycle could be greatly shortened by the new process,and 91.7%of saponins were released to saponins concentrate from the solid phase of WSDP;the diosgenin yield from saponins concentrate was comparable to that of the enterprise natural fermentation treatment process;Large amount of nutrient-rich sugar liquid and solid residue were produced in the new process,which could be high-valued utilized to produce other by-products.This study laid a solid foundation for the industrialization application of the microbial method production process of DZW saponins,provided a guiding ideology and important reference basis for the storage and processing methods of medicinal plants rich in starch,and provided a brand-new research idea for the extraction of DZW saponins by microbial fermentation.