微生物转化虎杖中白藜芦醇苷及其产物的分离纯化
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摘要
中药虎杖是蓼科植物虎杖(Polygonum cuspidatum Sieb.et Zucc.)的根和茎,具有祛风利湿,散瘀定痛,止咳化痰等功效。白藜芦醇是虎杖中最有价值的成分,具有抗菌、抗炎、抗病毒、抗氧化和抗癌等功效,现已广泛应用在食品添加剂、保健品、化妆品等行业。但是白藜芦醇的含量偏低,加大了其生产成本。白藜芦醇的糖苷化合物白藜芦醇苷和白藜芦醇具有相似的药理作用,其含量远高于白藜芦醇,但其生物利用度远低于白藜芦醇。将糖苷转化为其苷元通常由酸水解或碱水解来实现。但是反应需要高温高压等剧烈条件,不仅对设备要求高,而且对环境造成很大的污染。相比之下,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虎杖粗药材中的白藜芦醇苷转化为白藜芦醇,并对其分离纯化工艺进行了研究。
首先,筛选β-葡萄糖苷酶的高效产生菌食品级微生物米曲霉,对其所产β-葡萄糖苷酶的性质进行了研究,并用此酶液对虎杖粗药材中的白藜芦醇苷进行转化。结果表明,该β-葡萄糖苷酶的最适pH值为4.8,最适温度为50℃。用β-葡萄糖苷酶粗酶液对虎杖粗药材中的白藜芦醇苷转化12小时,体系中白藜芦醇苷的转化率达100%,白藜芦醇的含量提高到原来的2.9倍。
在此基础上,用米曲霉直接将虎杖粗药材中的白藜芦醇苷转化为白藜芦醇,并对其转化机理进行了研究。摇瓶发酵46小时使白藜芦醇苷完全转化为白藜芦醇,白藜芦醇的提取百分率提高到1.36%,是微波辅助提取的3.6倍。商品纤维素酶水解粗药材12小时后达到了同样高的提取百分率,但是酶的昂贵价格限制了其工业应用。酸水解粗药材20分钟可以将79.0%的白藜芦醇苷水解为白藜芦醇,但同时46.7%的白藜芦醇在此过程中被破坏,因此酸水解不是一种可行的策略。相比较而言,微生物转化法条件温和,操作简单,产物收率高,具有很大的工业化潜力。
因此,在摇瓶发酵的基础上对菌种进行驯化,并用5L发酵罐进行发酵工艺的放大与优化。结果表明,在温度为37℃,通气量为1.5vvm下发酵20小时后,将温度升高到50℃继续反应2小时,白藜芦醇苷的转化率达105%,白藜芦醇的浓度提高到原来的3.4倍,达到了文献报道的自然发酵72小时的水平。转化机理研究表明,白藜芦醇和白藜芦醇苷以游离状态存在于药材细胞中,发酵过程中白藜芦醇苷被转化为白藜芦醇,同时药材细胞壁上的纤维素被部分水解,从而有利于产物的提取。
其次,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分别从虎杖粗药材和虎杖发酵液中提取了白藜芦醇苷、白藜芦醇和大黄素,并对其分相行为进行了研究。选择25%(w/w)的乙醇和21%(w/w)的硫酸铵为提取剂从虎杖粗药材中提取所得的白藜芦醇苷和加热回流与微波辅助提取的提取率相等,而白藜芦醇和大黄素的提取率分别为微波辅助提取和加热回流提取的1.1倍和1.9倍。选择此双水相体系对虎杖发酵液进行提取,所得的白藜芦醇和大黄素的提取率分别是微波辅助提取的1.2倍和3.3倍,分别是常温搅拌提取的1.6倍和4.2倍。发酵液提取物中白藜芦醇和大黄素的含量分别为微波辅助提取的1.3倍和2.3倍,分别为常温搅拌提取的1.4倍和2.6倍,而葡萄糖和多糖的含量分别为后两者的27%和79%。微波辅助双水相提取同时具备微波辅助提取和双水相萃取的优点,并把两个操作步骤合为一步,提高了收率,降低了成本。因此,是一种很有前景的提取和分离目标产物的方法。
最后,利用大孔树脂结合聚酰胺柱层析从发酵液提取物中纯化白藜芦醇,并测定了大孔树脂和聚酰胺对白藜芦醇的吸附动力学参数。结果表明,大孔树脂和聚酰胺对白藜芦醇的表观静态吸附量分别为11.70mg/g和57.90mg/g,表观动态吸附量分别为12.47mg/g和58.98mg/g,吸附均符合Langmuir吸附特征。利用大孔树脂结合聚酰胺柱层析两步纯化发酵液中的白藜芦醇,使其纯度达到95.8%,总收率达69.8%。同时,提取液中的大黄素得到回收,其纯度为18.6%,收率为84.2%。此方法操作简单,成本低廉,白藜芦醇的纯度和收率高,有良好的工业化应用前景。
Dry caudex of Polygonum cuspidatum Sieb.et Zucc.(Huzhang in Chinese) is a Chinese traditional herbal medicine,which has been used for dispelling wind and dampness, eliminating stasis to activate blood circulation,relieving cough and reducing sputum. Resveratrol,the most valuable component in Huzhang,is widely used in food additive,health products and cosmetic industries due to its various pharmaceutical properties such as antimicrobial,anti-inflammatory,anti-virus,antioxidant and anticancer activities.However, its content is very low,which increases its production cost.Piceid,the glucoside of resveratrol, has similar pharmaceutical functions,and its content is much higher,however,its bioavailability is much lower than resveratrol.In general,aglycone is acquired by hydrolysis of its glucoside,and acid or alkaline is often used as the hydrolytic reagent.However,the hydrolytic reaction is always carried out under extreme conditions such as high temperature and high pressure,which not only requires high equipment characteristic but also causes pollution.In contrast,biotransformation only requires mild conditions,simple procedures, lower cost and results in less pollution.In this paper,biotransformation of piceid in P. cuspidatum to resveratrol was performed by Aspergillus oryzae and its separation and purification was studied.
A food-grade microorganism A.oryzae with highβ-glucosidase activity was firstly selected.Properties of the enzyme were studied and biotransformation of piceid in raw herb of P.cuspidatum by the crude enzyme was investigated.The results indicated that the optimum pH of theβ-glucosidase is 4.8 and the optimum temperature is 50℃.Concentration of resveratrol increased to 2.9 times higher after 12h transformation of raw herb by the crudeβ-glucosidase,with the percent conversion of piceid 100%.
Based on the above results,biotransformation of piceid in raw herb of P.cuspidatum to resveratrol by A.oryzae was investigated,and mechanism of the bioprocess was discussed. Percent extraction of resveratrol reached 1.36%after 46h shake flask cultivation,3.6 times higher than that by microwave-assisted extraction of raw herb.The same results were obtained by enzymatic hydrolysis of raw herb for 12h with commercial cellulase,but the high cost of the enzyme limits its industrial application.Seventy nine percent of piceid was hydrolyzed to resveratrol after acid hydrolysis of raw herb for 20min,while 46.7%of resveratrol was destroyed during the process,so acid hydrolysis is not a feasible strategy. Compared with enzymatic and acid hydrolysis,biotransformation only requires mild conditions as well as simple procedures and results in high yield,so it has great potential for industrial application.
Therefore,fermentation in a 5L bioreactor was carried out by the acclimated strain based on the shake flask cultivation,and the operation conditions were optimized.After 20h fermentation at 37℃and air flow rate of 1.5vvm,temperature was increased to 50℃and maintained for 2h.The concentration of resveratrol was 3.4 times higher,reaching the level of the reported spontaneously fermentation for 72h,with the percent conversion of piceid 105%. The investigation about the mechanism of transformation and release of resveratrol and piceid indicates that they exist in herb cells in a free state.Biotransformation of piceid to resveratrol and hydrolysis of cellulose in the cell walls to release the two effective constituents are simultaneously conducted during fermentation.
Microwave-assisted aqueous two-phase extraction(MAATPE) was then investigated with ethanol/ammonium sulphate to obtain effective constituents,including piceid,resveratrol and emodin from both raw herb and fermentation broths,and the partition behavior of the products was studied.The aqueous two-phase system consisting of 25%(w/w) ethanol and 21%(w/w) ammonium sulphate was selected to obtain equal yield of piceid,and 1.1 and 1.9 times higher yields of resveratrol and emodin,respectively,from raw herb of P.cuspidatum than that by microwave-assisted extraction(MAE) and heat reflux extraction.Yields of resveratrol and emodin from fermentation broths were 1.2 and 3.3 times higher,respectively, than that by MAE,and 1.6 and 4.2 times higher,respectively,than that by stirring extraction at room temperature.Contents of resveratrol and emodin in the extract of fermentation broths were 1.3 and 2.3 times higher,respectively,than that by MAE,and 1.4 and 2.6 times higher, respectively,than that by stirring extraction at room temperature,while contents of glucose and polysaccharides in the extract were 27%and 79%,respectively,of that by MAE and stirring extraction at room temperature.MAATPE possesses advantages over both MAE and aqueous two-phase extraction,and integrates the two processes into a single step to get higher yields at lower cost.Therefore,it is a potential method for the extraction and purification of target products.
Macroporous resin and polyamide column chromatography were finally combined to purify resveratrol from the fermentation broths,and the adsorption dynamics of resveratrol was evaluated.The results show that apparent static adsorptive capacities of resveratrol on macroporous resin and polyamide are 11.70mg/g and 57.9mg/g,respectively,and apparent dynamic adsorptive capacities are 12.47mg/g and 58.98mg/g,respectively.Both of the adsorption isotherms correlate well with the Langmuir type.After macroporous resin and polyamide two-step column chromatography,the purity of resveratrol reached 95.8%,with the yield of 69.8%.Emodin in the culture was also recovered,with the purity of 18.6%and the yield of 84.2%.This method is proved potential for industrial application due to its simple procedure,lower cost as well as high purity and yield of resveratrol.
首先,筛选β-葡萄糖苷酶的高效产生菌食品级微生物米曲霉,对其所产β-葡萄糖苷酶的性质进行了研究,并用此酶液对虎杖粗药材中的白藜芦醇苷进行转化。结果表明,该β-葡萄糖苷酶的最适pH值为4.8,最适温度为50℃。用β-葡萄糖苷酶粗酶液对虎杖粗药材中的白藜芦醇苷转化12小时,体系中白藜芦醇苷的转化率达100%,白藜芦醇的含量提高到原来的2.9倍。
在此基础上,用米曲霉直接将虎杖粗药材中的白藜芦醇苷转化为白藜芦醇,并对其转化机理进行了研究。摇瓶发酵46小时使白藜芦醇苷完全转化为白藜芦醇,白藜芦醇的提取百分率提高到1.36%,是微波辅助提取的3.6倍。商品纤维素酶水解粗药材12小时后达到了同样高的提取百分率,但是酶的昂贵价格限制了其工业应用。酸水解粗药材20分钟可以将79.0%的白藜芦醇苷水解为白藜芦醇,但同时46.7%的白藜芦醇在此过程中被破坏,因此酸水解不是一种可行的策略。相比较而言,微生物转化法条件温和,操作简单,产物收率高,具有很大的工业化潜力。
因此,在摇瓶发酵的基础上对菌种进行驯化,并用5L发酵罐进行发酵工艺的放大与优化。结果表明,在温度为37℃,通气量为1.5vvm下发酵20小时后,将温度升高到50℃继续反应2小时,白藜芦醇苷的转化率达105%,白藜芦醇的浓度提高到原来的3.4倍,达到了文献报道的自然发酵72小时的水平。转化机理研究表明,白藜芦醇和白藜芦醇苷以游离状态存在于药材细胞中,发酵过程中白藜芦醇苷被转化为白藜芦醇,同时药材细胞壁上的纤维素被部分水解,从而有利于产物的提取。
其次,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分别从虎杖粗药材和虎杖发酵液中提取了白藜芦醇苷、白藜芦醇和大黄素,并对其分相行为进行了研究。选择25%(w/w)的乙醇和21%(w/w)的硫酸铵为提取剂从虎杖粗药材中提取所得的白藜芦醇苷和加热回流与微波辅助提取的提取率相等,而白藜芦醇和大黄素的提取率分别为微波辅助提取和加热回流提取的1.1倍和1.9倍。选择此双水相体系对虎杖发酵液进行提取,所得的白藜芦醇和大黄素的提取率分别是微波辅助提取的1.2倍和3.3倍,分别是常温搅拌提取的1.6倍和4.2倍。发酵液提取物中白藜芦醇和大黄素的含量分别为微波辅助提取的1.3倍和2.3倍,分别为常温搅拌提取的1.4倍和2.6倍,而葡萄糖和多糖的含量分别为后两者的27%和79%。微波辅助双水相提取同时具备微波辅助提取和双水相萃取的优点,并把两个操作步骤合为一步,提高了收率,降低了成本。因此,是一种很有前景的提取和分离目标产物的方法。
最后,利用大孔树脂结合聚酰胺柱层析从发酵液提取物中纯化白藜芦醇,并测定了大孔树脂和聚酰胺对白藜芦醇的吸附动力学参数。结果表明,大孔树脂和聚酰胺对白藜芦醇的表观静态吸附量分别为11.70mg/g和57.90mg/g,表观动态吸附量分别为12.47mg/g和58.98mg/g,吸附均符合Langmuir吸附特征。利用大孔树脂结合聚酰胺柱层析两步纯化发酵液中的白藜芦醇,使其纯度达到95.8%,总收率达69.8%。同时,提取液中的大黄素得到回收,其纯度为18.6%,收率为84.2%。此方法操作简单,成本低廉,白藜芦醇的纯度和收率高,有良好的工业化应用前景。
Dry caudex of Polygonum cuspidatum Sieb.et Zucc.(Huzhang in Chinese) is a Chinese traditional herbal medicine,which has been used for dispelling wind and dampness, eliminating stasis to activate blood circulation,relieving cough and reducing sputum. Resveratrol,the most valuable component in Huzhang,is widely used in food additive,health products and cosmetic industries due to its various pharmaceutical properties such as antimicrobial,anti-inflammatory,anti-virus,antioxidant and anticancer activities.However, its content is very low,which increases its production cost.Piceid,the glucoside of resveratrol, has similar pharmaceutical functions,and its content is much higher,however,its bioavailability is much lower than resveratrol.In general,aglycone is acquired by hydrolysis of its glucoside,and acid or alkaline is often used as the hydrolytic reagent.However,the hydrolytic reaction is always carried out under extreme conditions such as high temperature and high pressure,which not only requires high equipment characteristic but also causes pollution.In contrast,biotransformation only requires mild conditions,simple procedures, lower cost and results in less pollution.In this paper,biotransformation of piceid in P. cuspidatum to resveratrol was performed by Aspergillus oryzae and its separation and purification was studied.
A food-grade microorganism A.oryzae with highβ-glucosidase activity was firstly selected.Properties of the enzyme were studied and biotransformation of piceid in raw herb of P.cuspidatum by the crude enzyme was investigated.The results indicated that the optimum pH of theβ-glucosidase is 4.8 and the optimum temperature is 50℃.Concentration of resveratrol increased to 2.9 times higher after 12h transformation of raw herb by the crudeβ-glucosidase,with the percent conversion of piceid 100%.
Based on the above results,biotransformation of piceid in raw herb of P.cuspidatum to resveratrol by A.oryzae was investigated,and mechanism of the bioprocess was discussed. Percent extraction of resveratrol reached 1.36%after 46h shake flask cultivation,3.6 times higher than that by microwave-assisted extraction of raw herb.The same results were obtained by enzymatic hydrolysis of raw herb for 12h with commercial cellulase,but the high cost of the enzyme limits its industrial application.Seventy nine percent of piceid was hydrolyzed to resveratrol after acid hydrolysis of raw herb for 20min,while 46.7%of resveratrol was destroyed during the process,so acid hydrolysis is not a feasible strategy. Compared with enzymatic and acid hydrolysis,biotransformation only requires mild conditions as well as simple procedures and results in high yield,so it has great potential for industrial application.
Therefore,fermentation in a 5L bioreactor was carried out by the acclimated strain based on the shake flask cultivation,and the operation conditions were optimized.After 20h fermentation at 37℃and air flow rate of 1.5vvm,temperature was increased to 50℃and maintained for 2h.The concentration of resveratrol was 3.4 times higher,reaching the level of the reported spontaneously fermentation for 72h,with the percent conversion of piceid 105%. The investigation about the mechanism of transformation and release of resveratrol and piceid indicates that they exist in herb cells in a free state.Biotransformation of piceid to resveratrol and hydrolysis of cellulose in the cell walls to release the two effective constituents are simultaneously conducted during fermentation.
Microwave-assisted aqueous two-phase extraction(MAATPE) was then investigated with ethanol/ammonium sulphate to obtain effective constituents,including piceid,resveratrol and emodin from both raw herb and fermentation broths,and the partition behavior of the products was studied.The aqueous two-phase system consisting of 25%(w/w) ethanol and 21%(w/w) ammonium sulphate was selected to obtain equal yield of piceid,and 1.1 and 1.9 times higher yields of resveratrol and emodin,respectively,from raw herb of P.cuspidatum than that by microwave-assisted extraction(MAE) and heat reflux extraction.Yields of resveratrol and emodin from fermentation broths were 1.2 and 3.3 times higher,respectively, than that by MAE,and 1.6 and 4.2 times higher,respectively,than that by stirring extraction at room temperature.Contents of resveratrol and emodin in the extract of fermentation broths were 1.3 and 2.3 times higher,respectively,than that by MAE,and 1.4 and 2.6 times higher, respectively,than that by stirring extraction at room temperature,while contents of glucose and polysaccharides in the extract were 27%and 79%,respectively,of that by MAE and stirring extraction at room temperature.MAATPE possesses advantages over both MAE and aqueous two-phase extraction,and integrates the two processes into a single step to get higher yields at lower cost.Therefore,it is a potential method for the extraction and purification of target products.
Macroporous resin and polyamide column chromatography were finally combined to purify resveratrol from the fermentation broths,and the adsorption dynamics of resveratrol was evaluated.The results show that apparent static adsorptive capacities of resveratrol on macroporous resin and polyamide are 11.70mg/g and 57.9mg/g,respectively,and apparent dynamic adsorptive capacities are 12.47mg/g and 58.98mg/g,respectively.Both of the adsorption isotherms correlate well with the Langmuir type.After macroporous resin and polyamide two-step column chromatography,the purity of resveratrol reached 95.8%,with the yield of 69.8%.Emodin in the culture was also recovered,with the purity of 18.6%and the yield of 84.2%.This method is proved potential for industrial application due to its simple procedure,lower cost as well as high purity and yield of resveratrol.
引文
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