哈茨木霉生物转化盾叶薯蓣中的皂苷及其产物提取分离
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摘要
薯蓣皂苷元(Diosgenin)是合成多种甾体激素类药物和甾体避孕药的理想前体,占甾体激素类药物原料的60%。盾叶薯蓣(Dioscorea zingiberensis C.H. Wright)是我国特有的多年生藤本植物,也是我国生产薯蓣皂苷元的最主要原料之一。薯蓣皂苷元在植物中以甾体皂苷的形式存在,通常用酸将皂苷水解为苷元。传统的酸水解生产方法不仅破坏了薯蓣皂苷元的结构,造成苷元提取率低,而且对环境造成极其严重的污染。生物转化法条件温和、操作简单、成本低廉、环境友好。本文利用生物转化法直接将盾叶薯蓣药材中的皂苷转化为薯蓣皂苷元,并对其转化途径及产物的提取分离进行了研究。
首先,筛选出一株能够直接利用盾叶薯蓣药材发酵生产薯蓣皂苷元的微生物菌种,经形态学、分子生物学鉴定为哈茨木霉(Trichoderma harzianum)。哈茨木霉转化盾叶薯蓣过程中甾体成分种类没有变化,也没有其它副产物形成,薯蓣皂苷元得率高,该菌是一株高效的、具有应用潜力的菌种。
其次,对哈茨木霉转化盾叶薯蓣中皂苷的发酵培养条件进行了优化。以薯蓣皂苷元产量为优化目标,最适的摇瓶培养温度为30℃,培养基最佳药材载量为33.33g/L,发酵培养基的最适接种量为6%。当培养基以pH 6.0的Na2HPO4-KH2PO4缓冲液为基质、添加2 mmol/L的Fe2+或0.03%(w／v)的吐温-85时,薯蓣皂苷元产量分别比空白对照提高50.28%、22.07%和33.35%。响应面法优化得到的盾叶薯蓣发酵培养基组成为0.06mol/L的Na2HPO4-KH2PO4、0.07%(w/v)的吐温-85和0.93 mmol/L的Fe2+,优化条件下哈茨木霉发酵盾叶薯蓣120 h,薯蓣皂苷元产量为30.05±0.59 mg/g,转化率为73.63±0.54%。
再次,建立了描述哈茨木霉转化皂苷的动力学模型,用数学软件Matlab对皂苷转化过程进行模拟。结果表明：总甾体浓度实际值与动力学模拟计算数值拟合较好,可决系数(coefficient of determination)为0.97；薯蓣皂苷元、盾叶新苷、葡萄糖三糖苷、三角叶薯蓣皂苷、延龄草次苷、葡萄糖二糖苷以及prosapogenin的浓度变化符合M-M酶催化动力学,其可决系数(coefficient of determination)分别为0.99、0.98、0.94、0.85、0.82、0.79和0.66；皂苷转化过程中鼠李糖和葡萄糖的水解同时存在,葡萄糖的水解包括末端葡萄二糖和单糖的水解,甾体母核C3位相连的糖苷键的水解与糖链上其它葡萄糖苷键的水解不同,对于单葡萄糖的水解甾体母核C3位相连的糖苷键水解活性高于其它糖苷键,对于葡萄二糖的水解甾体母核C3位相连的糖苷键水解活性低于其它糖苷键；生成薯蓣皂苷元的主要途径为盾叶新苷先水解鼠李糖,再水解葡萄二糖,最后水解末端单葡萄糖生成薯蓣皂苷元。
最后,应用双水相和三液相对皂苷及薯蓣皂苷元进行了提取分离研究。以乙醇／硫酸铵双水相体系为溶剂,利用微波辅助双水相提取发酵液中的皂苷及薯蓣皂苷元,总皂苷提取率为96.8%,薯蓣皂苷元提取率为97.1%。在双水相体系中加入石油醚,形成三液相。由30%(w／w)乙醇、17%(w／w)硫酸铵、40%石油醚组成的三液相体系实现了发酵液中薯蓣皂苷元、皂苷与葡萄糖、微生物细胞和药渣的有效分离。上相(石油醚相)中薯蓣皂苷元的回收率为97.2%,回收上相石油醚后,薯蓣皂苷元甲醇结晶样品色谱检测纯度大于98%；中相(醇相)中盾叶新苷、三角叶薯蓣皂昔、葡萄糖二糖苷的回收率接近100%,葡萄糖三糖苷和延龄草次苷的回收率分别为98.8%和96.0%；72.0%的葡萄糖分配在下相(盐相)；微生物细胞和药渣形成沉淀层悬浮于中相与下相之间。
生物转化结合三液相萃取后的酸水解,获得的薯蓣皂苷元产量比传统酸水解增加38.5%,而产生的COD、还原糖仅为酸水解的3.3%和0.3%。
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.
首先,筛选出一株能够直接利用盾叶薯蓣药材发酵生产薯蓣皂苷元的微生物菌种,经形态学、分子生物学鉴定为哈茨木霉(Trichoderma harzianum)。哈茨木霉转化盾叶薯蓣过程中甾体成分种类没有变化,也没有其它副产物形成,薯蓣皂苷元得率高,该菌是一株高效的、具有应用潜力的菌种。
其次,对哈茨木霉转化盾叶薯蓣中皂苷的发酵培养条件进行了优化。以薯蓣皂苷元产量为优化目标,最适的摇瓶培养温度为30℃,培养基最佳药材载量为33.33g/L,发酵培养基的最适接种量为6%。当培养基以pH 6.0的Na2HPO4-KH2PO4缓冲液为基质、添加2 mmol/L的Fe2+或0.03%(w／v)的吐温-85时,薯蓣皂苷元产量分别比空白对照提高50.28%、22.07%和33.35%。响应面法优化得到的盾叶薯蓣发酵培养基组成为0.06mol/L的Na2HPO4-KH2PO4、0.07%(w/v)的吐温-85和0.93 mmol/L的Fe2+,优化条件下哈茨木霉发酵盾叶薯蓣120 h,薯蓣皂苷元产量为30.05±0.59 mg/g,转化率为73.63±0.54%。
再次,建立了描述哈茨木霉转化皂苷的动力学模型,用数学软件Matlab对皂苷转化过程进行模拟。结果表明：总甾体浓度实际值与动力学模拟计算数值拟合较好,可决系数(coefficient of determination)为0.97；薯蓣皂苷元、盾叶新苷、葡萄糖三糖苷、三角叶薯蓣皂苷、延龄草次苷、葡萄糖二糖苷以及prosapogenin的浓度变化符合M-M酶催化动力学,其可决系数(coefficient of determination)分别为0.99、0.98、0.94、0.85、0.82、0.79和0.66；皂苷转化过程中鼠李糖和葡萄糖的水解同时存在,葡萄糖的水解包括末端葡萄二糖和单糖的水解,甾体母核C3位相连的糖苷键的水解与糖链上其它葡萄糖苷键的水解不同,对于单葡萄糖的水解甾体母核C3位相连的糖苷键水解活性高于其它糖苷键,对于葡萄二糖的水解甾体母核C3位相连的糖苷键水解活性低于其它糖苷键；生成薯蓣皂苷元的主要途径为盾叶新苷先水解鼠李糖,再水解葡萄二糖,最后水解末端单葡萄糖生成薯蓣皂苷元。
最后,应用双水相和三液相对皂苷及薯蓣皂苷元进行了提取分离研究。以乙醇／硫酸铵双水相体系为溶剂,利用微波辅助双水相提取发酵液中的皂苷及薯蓣皂苷元,总皂苷提取率为96.8%,薯蓣皂苷元提取率为97.1%。在双水相体系中加入石油醚,形成三液相。由30%(w／w)乙醇、17%(w／w)硫酸铵、40%石油醚组成的三液相体系实现了发酵液中薯蓣皂苷元、皂苷与葡萄糖、微生物细胞和药渣的有效分离。上相(石油醚相)中薯蓣皂苷元的回收率为97.2%,回收上相石油醚后,薯蓣皂苷元甲醇结晶样品色谱检测纯度大于98%；中相(醇相)中盾叶新苷、三角叶薯蓣皂昔、葡萄糖二糖苷的回收率接近100%,葡萄糖三糖苷和延龄草次苷的回收率分别为98.8%和96.0%；72.0%的葡萄糖分配在下相(盐相)；微生物细胞和药渣形成沉淀层悬浮于中相与下相之间。
生物转化结合三液相萃取后的酸水解,获得的薯蓣皂苷元产量比传统酸水解增加38.5%,而产生的COD、还原糖仅为酸水解的3.3%和0.3%。
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.
引文
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