Study On Biotransformation Of Steroidal Saponins In Dioscorea Zingiberensis By Aspergillus Oryzae

Diosgenin is an important precursor in the synthesis of steroidal hormones and steroidal contraceptives,it also has biological activities,for example,inducing apoptosis.Diosgenin exists in plant tissues in the form of steroidal saponins,whose sugar chains connect with the cell wall of the plant cells.As a result,the traditional method for diosgenin production is acidic hydrolysis,which hydrolyzes the sugar chains of steroidal saponins to obtain diosgenin. However,this method results in serious wastewater pollution,which is a formidable challenge to deal with.In this paper,a microbial transformation method was developed to convert the steroidal saponins from Dioscorea zingiberensis directly to produce diosgenin by Aspergillus oryzae.The steroidal saponins in the herb and the transformed products were identified and analyzed both qualitatively and quantitatively.The transformation process was optimized. Furthermore,the transformation pathway and mechanism were preliminarily explored.Firstly,the steroidal compounds in the herb and the transformed products were separated and purified,and their chemical structures were identified.Four steroidal saponins were separated by solvent extraction,silica gel column chromatography and crystallization,and their structures were identified as zingiberensis newsaponin,deltonin,3-O-[β-D-(1→3)-glucopyranosyl-β-D-(1→4)-glucopyranosyl]-β-D-glucopyranosyl-diosgenin (diosgenintriglucoside) and diosgenin-diglucoside by MS,IR ¹³C NMR and ¹H NMR.Other two steroidal compounds were identified as trillin and diosgenin by comparing their HPLC retention times with standards.Secondly,the qualitative and quantitative analytical method using HPLC-UV-ELSD was established.Based on this,the transformation conditions were investigated,including the buffer in culture media,culture and transformation temperature,amount of microbe inoculation,and loading capacity of herb.The optimized results were as follows:pH 5.0/6.0 Na₂HPO₄-KH₂PO₄ buffer,3.33%raw herb,inoculating 8%microorganism,culturing at 30 or 37℃for 84h,then 50℃for 8h.After conversion by A.oryzae,17.06 mg diosgenin can be produced from one gram raw herb.Thirdly,the transformation pathway and mechanism were preliminarily explored by experiments,including direct microbial transformation of raw herb and monomeric compounds,direct enzymatic transformation of raw herb by crude enzymes,and reaction kinetics simulation by computer.The results showed that this process was a complicated course,composed of parallel reactions and consecutive reactions,involving multi-substrate and multi-enzyme.The whole process began with the cleavage of rhamnoside linkages,and then moved onto the hydrolysis of glycosidic linkages,which consisted of one-by-one mode and two-at-a-time mode.The cleavage of O-glycosidic bond was different from other glycosidic bonds in the sugar chain.