| Diosgenin is an important precursor in the synthesis of steroidal hormones and steroidal contraceptives, and about 60% steroidal hormone medicines are produced from it. Dioscorea zingiberensis C. H. Wright (DZW) is one of the important resources for diosgenin production. Diosgenin exists as a form of glycosidal steroidal saponin in DZW. In industry, acids are usually used to hydrolyze DZW tubers to produce diosgenin. However, the structure of diosgenin is easily damaged and numerous byproducts are then generated in the traditional acid hydrolysis, which results in lower diosgenin yield and serious pollution. It is well known that biological hydrolysis for natural product has many advantages, such as high specificity, mild reaction conditions and clean production as well as low cost. In this paper, a microbial transformation method was developed to convert the saponins in DZW to diosgenin, and the transformation kinetics and the extraction and separation of biotransformed products were studied.Firstly, a specific strain was selected from a wide range of strains offered according to its recognized ability to produce diosgenin. Morphological characters indicated the strain belongs to Trichoderma. GenBank BLAST showed that its ITS sequence shared 100% homology with that of Trichoderma harzianum. Comparing the post-biotransformed sample with the pre-biotransformed one, no other new saponins and no byproducts were found. Biotransformation of DZW by T. harzianum with a high yield of diosgenin was deserved to be studied further.Secondly, fermentation of DZW by T. harzianum was investigated based on the above results. The transformation conditions were optimized for maximum diosgenin production. The optimum fermentation temperature was 30℃, the optimum concentration of DZW in the medium was 33.33 g/L and the optimum inoculation amount was 6%. Diosgenin yield was increased by 50.28% in phosphate buffer at pH 6.0 over that of control sample. The yield of diosgenin was enhanced by 22.07% and 33.35%, respectively, if adding 2 mmol/L Fe2+ or 0.03% (w/v) Tween-85 into medium. The optimum medium obtained by response surface methodology was composed of 0.06 mol/L phosphate buffer,0.07% (w/v) Tween-85 and 0.93 mmol/L Fe2+. Under these conditions, the maximum diosgenin yield of 30.05±0.59 mg/g was achieved, which was slightly higher than that obtained from traditional acid hydrolysis, and the conversion percentage was 73.63±0.54%. Thirdly, the main biotransformation pathway and kinetic feature were determined by kinetics modelling and analysis. The results showed that the coefficients of determination of total steroid, diosgenin, zingibernsis newsaponin, diosgenin-triglucoside, deltonin, trillin, diosgenin.-diglucoside and prosapogenin were 0.97,0.99,0.98,0.94,0.85,0.82,0.79 and 0.66, respectively. During the biotransformation the hydrolysis of rhamnosyl residue and glucosyl residue occurred simultaneously. The hydrolysis of glucosyl residue included the hydrolysis of mono-glucosyl residue and diglucosyl residue. The hydrolysis of glycoside bond linked with glucosyl and aglycone was different from that of glycoside bond linked with two glucosyls. For mono-glucosyl, the hydrolysis rate of glycoside bond linked with glucosyl and aglycone was higher than that of other glycoside bond in sugar chain. On the contrary, for diglucosyl, the hydrolysis rate of glycoside bond linked with glucosyl and aglycone was lower than that of other glycoside bond. Kinetic modelling suggested that the main pathway to produce diosgenin was as follows:the terminal rhamnosyl residue of zingibernsis newsaponin was firstly hydrolyzed to produce diosgenin-triglucoside, the terminal diglucosyl residue of diosgenin-triglucoside was then hydrolyzed to produce trillin, and the terminal glucosyl residue of trillin was eventually hydrolyzed to yield diosgenin.Fourthly, saponins and diosgenin were extracted and separated by aqueous two-phase and three-liqud-phase extraction. An improved microwave-assisted aqueous two-phase extraction with 30% ethanol and 15% (NH4)2SO4 could thoroughly extract the steroids from the fermentation broth of DZW by T. harzianum, and the total saponin yield and diosgenin yield were 96.8% and 97.1%, respectively. Subsequent application of three-liquid-phase system composed of 30% ethanol,17% (NH4)2SO4 and 40% petroleum ether resulted in the separation of diosgenin, untransformed saponins from other impurities such as raw herb residuals, microbial cells and glucose. In the three-liquid-phase extraction, the recovery of diosgenin was 97.2% in the top phase (petroleum ether phase) and after the recovery of petroleum ether high purity (> 98%) diosgenin crystals was obtained by crystallization separation with methanol, the recoveries of zingibernsis newsaponin, deltonin and diosgenin-diglucoside in the middle phase (ethanol phase) were almost 100%, the recoveries of diosgenin-triglucoside and trillin in the middle phase (ethanol phase) were 98.8% and 96.0%, respectively, and 72.0% of glucose was extracted into the bottom phase (salt phase). Moreover, the microbial cells and residuals of the raw herb were concentrated at the interface between the middle phase and bottom phase.Lastly, an integrated process of biotransformation and acid hydrolysis following the three-liquid-phase extraction was applied to produce diosgenin. Diosgenin yield increased by 38.5% compared to traditional acid hydrolysis. Moreover, COD and reduced sugar in wastewater produced by this integrated process were only 3.3% and 0.3% of that from the traditional method, respectively. |
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