盾叶薯蓣的乙醇发酵与皂苷双水相萃取的集成
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摘要
盾叶薯蓣是用来生产甾体激素类药物前体薯蓣皂苷元的重要植物资源,其根茎中不仅含有1.1～16.5%的甾体皂苷,还含有40-50%的淀粉和40-50%的木质纤维素,传统酸水解法生产薯蓣皂苷元是直接将盾叶薯蓣根茎与酸混合进行水解反应,其中的淀粉和纤维素不仅没有得到利用而且其产物对环境造成了极大的污染。本论文针对传统酸水解法浪费资源、污染环境等问题,先将盾叶薯蓣中的淀粉和纤维素进行糖化和酒精发酵,再采用新型双水相体系对发酵液中的皂苷进行提取,整个工艺操作简单易行,达到了资源综合利用和清洁生产的目的。
首先对盾叶薯蓣根茎中的淀粉和木质纤维素的含量进行了测定,测定结果：淀粉含量38.7%,木质纤维素含量40.9%,其中纤维素含量15.9%。
其次对盾叶薯蓣淀粉的两种水解方式分别进行了考察,酸水解的最优条件为：盾叶薯蓣与酸液的料液比1：4,硫酸浓度1.2%,浸泡20h左右,高温蒸汽灭菌锅中130℃,处理50min。在此条件下葡萄糖浓度达到了89g/L,而且此过程没有对皂苷产生明显影响。采用文献报道的双酶法处理盾叶薯蓣后得到的葡萄糖浓度也为89g/L,与酸解淀粉法得到的葡萄糖浓度相同。
再次,采用摇瓶实验对同步糖化发酵工艺进行了初步优化,结果表明盾叶薯蓣经淀粉水解后可直接作为酿酒酵母的发酵培养基,无需额外添加N源；发酵培养基的最佳初始pH为5.0；同步糖化发酵中纤维素酶的最佳加入时间为12h。采用酸解法和双酶法水解盾叶薯蓣淀粉后,在同步糖化发酵中纤维素酶的最佳用量分别为大于或等于120和100U/g盾叶薯蓣,发酵液中乙醇浓度分别为39.2和39.5g/L。所以在以后的实验中我们选择采用双酶法水解淀粉。
在发酵罐实验中,进一步优化纤维素酶的加入时间为16h,纤维素酶的最佳用量为100U/g盾叶薯蓣,此时发酵液的乙醇浓度达到了50.6g/L,比对照提高了36.4%。纤维素到乙醇的转化率为33.9%,为理论转化率的59.8%。
最后考察了微波辅助对乙醇-硫酸铵双水相萃取盾叶薯蓣乙醇发酵液中皂苷成分的影响,实验证明单纯使用双水相即能达到同微波辅助同样的效果；而对于盾叶薯蓣药材中皂苷成分,微波辅助双水相则比单纯双水相提取率提高了25.9%。发酵液的皂苷得率比盾叶薯蓣药材的皂苷得率高17.6%,而且在淀粉、纤维素的水解和发酵过程中,高级皂苷实现了逐步向低级皂苷的转化。
Dioscorea zingiberensis C. H. Wright (DZW) is an important plant resource to produce diosgenin which is the precursor of steroidal hormone drugs. Its tuber contains not only steroidal saponins of 1.1-16.5%but also starch of 40-50%and lignocelluse of 40-50%. Acid hydrolysis is traditionally used to produce diosgenin by directly mixing the DZW tuber and acid, in which the starch and cellulose are wasted and a great pollution exists to environment. Aiming at solving the problem of recource waste and environmental pollution, this dissertation focuses on the research of the saccharification and ethanol fermentation using the starch and cellulose in DZW and extraction of saponins using a novel aqueous two-phase system. The integrated process is simple and can achieve the goal of integrated utilization and clean production.
First of all, the contents of starch and lignocellulose in the tuber of DZW were determined as follows:starch 38.7%, lignocellulose 40.9%, and cellulose 15.9%.
The two hydrolysis methods (acid hydrolysis and two enzymes hydrolysis) were then inspected. The optimum conditions of acid hydrolysis are determined:the ratio of DZW and water of 1:4, the concentration of H2SO4 of 1.2%, the soak condition about 20h, hydrolyzing at 130℃for 50min. Under these conditions, the concentration of glucose achieves 89g/L and the content of saponins doesn't change much meanwhile. On the other hand, two kinds of enzymes were used to hydrolyze DZW and get the same concentration of glucose as the acid hydrolysis.
The simultaneous saccharification and fermentation (SSF) was performed and optimized in the shake bottles. The experimental result showed that the hydrolyzed DZW can directly be used as the fermentation medium without addition of N resource. The optimal initial pH is 5.0 in culture medium. The optimal time for adding cellulase is 12h. The optimal adding amount of cellulase is more than or equal to 120 and 100 U/g DZW for acid hydrolysis and two enzymes hydrolysis, respectively. The concentration of ethanol is 39.2 and 39.5 g/L, respectively, in the fermentation broths. So the two-kind enzymes method is chosen for the starch hydrolisis.
The SSF experiment was studied further in a fermentator. The optimal conditions of the adding time and amount of cellulase are 16h and 100 U/g DZW respectively. The transformation rate from cellulose to ethanol is 33.9%, which is 59.8%of the theoretical value.
Finally, the microwave-assisted extraction of saponin from the fermentation broth was investigated using an aqueous two-phase system of ethanol-(NH4)2SO4, which shares the same result as one without microwave-assisted. While using DZW as material to extract saponins, the microwave-assisted extraction ratio is 25.9%higher than one without microwave-assisted. And the extraction rate of saponins from fermentation broth is 17.6%higher than that from DZW and there are more saponins with less sugar ligand in fermentation broths than that in DZW.
首先对盾叶薯蓣根茎中的淀粉和木质纤维素的含量进行了测定,测定结果：淀粉含量38.7%,木质纤维素含量40.9%,其中纤维素含量15.9%。
其次对盾叶薯蓣淀粉的两种水解方式分别进行了考察,酸水解的最优条件为：盾叶薯蓣与酸液的料液比1：4,硫酸浓度1.2%,浸泡20h左右,高温蒸汽灭菌锅中130℃,处理50min。在此条件下葡萄糖浓度达到了89g/L,而且此过程没有对皂苷产生明显影响。采用文献报道的双酶法处理盾叶薯蓣后得到的葡萄糖浓度也为89g/L,与酸解淀粉法得到的葡萄糖浓度相同。
再次,采用摇瓶实验对同步糖化发酵工艺进行了初步优化,结果表明盾叶薯蓣经淀粉水解后可直接作为酿酒酵母的发酵培养基,无需额外添加N源；发酵培养基的最佳初始pH为5.0；同步糖化发酵中纤维素酶的最佳加入时间为12h。采用酸解法和双酶法水解盾叶薯蓣淀粉后,在同步糖化发酵中纤维素酶的最佳用量分别为大于或等于120和100U/g盾叶薯蓣,发酵液中乙醇浓度分别为39.2和39.5g/L。所以在以后的实验中我们选择采用双酶法水解淀粉。
在发酵罐实验中,进一步优化纤维素酶的加入时间为16h,纤维素酶的最佳用量为100U/g盾叶薯蓣,此时发酵液的乙醇浓度达到了50.6g/L,比对照提高了36.4%。纤维素到乙醇的转化率为33.9%,为理论转化率的59.8%。
最后考察了微波辅助对乙醇-硫酸铵双水相萃取盾叶薯蓣乙醇发酵液中皂苷成分的影响,实验证明单纯使用双水相即能达到同微波辅助同样的效果；而对于盾叶薯蓣药材中皂苷成分,微波辅助双水相则比单纯双水相提取率提高了25.9%。发酵液的皂苷得率比盾叶薯蓣药材的皂苷得率高17.6%,而且在淀粉、纤维素的水解和发酵过程中,高级皂苷实现了逐步向低级皂苷的转化。
Dioscorea zingiberensis C. H. Wright (DZW) is an important plant resource to produce diosgenin which is the precursor of steroidal hormone drugs. Its tuber contains not only steroidal saponins of 1.1-16.5%but also starch of 40-50%and lignocelluse of 40-50%. Acid hydrolysis is traditionally used to produce diosgenin by directly mixing the DZW tuber and acid, in which the starch and cellulose are wasted and a great pollution exists to environment. Aiming at solving the problem of recource waste and environmental pollution, this dissertation focuses on the research of the saccharification and ethanol fermentation using the starch and cellulose in DZW and extraction of saponins using a novel aqueous two-phase system. The integrated process is simple and can achieve the goal of integrated utilization and clean production.
First of all, the contents of starch and lignocellulose in the tuber of DZW were determined as follows:starch 38.7%, lignocellulose 40.9%, and cellulose 15.9%.
The two hydrolysis methods (acid hydrolysis and two enzymes hydrolysis) were then inspected. The optimum conditions of acid hydrolysis are determined:the ratio of DZW and water of 1:4, the concentration of H2SO4 of 1.2%, the soak condition about 20h, hydrolyzing at 130℃for 50min. Under these conditions, the concentration of glucose achieves 89g/L and the content of saponins doesn't change much meanwhile. On the other hand, two kinds of enzymes were used to hydrolyze DZW and get the same concentration of glucose as the acid hydrolysis.
The simultaneous saccharification and fermentation (SSF) was performed and optimized in the shake bottles. The experimental result showed that the hydrolyzed DZW can directly be used as the fermentation medium without addition of N resource. The optimal initial pH is 5.0 in culture medium. The optimal time for adding cellulase is 12h. The optimal adding amount of cellulase is more than or equal to 120 and 100 U/g DZW for acid hydrolysis and two enzymes hydrolysis, respectively. The concentration of ethanol is 39.2 and 39.5 g/L, respectively, in the fermentation broths. So the two-kind enzymes method is chosen for the starch hydrolisis.
The SSF experiment was studied further in a fermentator. The optimal conditions of the adding time and amount of cellulase are 16h and 100 U/g DZW respectively. The transformation rate from cellulose to ethanol is 33.9%, which is 59.8%of the theoretical value.
Finally, the microwave-assisted extraction of saponin from the fermentation broth was investigated using an aqueous two-phase system of ethanol-(NH4)2SO4, which shares the same result as one without microwave-assisted. While using DZW as material to extract saponins, the microwave-assisted extraction ratio is 25.9%higher than one without microwave-assisted. And the extraction rate of saponins from fermentation broth is 17.6%higher than that from DZW and there are more saponins with less sugar ligand in fermentation broths than that in DZW.
引文
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