甜菊糖苷的提取纯化工艺研究
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摘要
甜菊糖苷(steviol glycosides)是一类从菊科植物甜叶菊(Stevia rebaudiana Bertoni)的叶子中提取得到的高甜度、低热量的天然非营养型甜味剂。甜菊糖苷在体内具有多种药理活性且无毒副作用,已作为天然蔗糖的替代品,引起了广大学者的注意。目前,甜菊糖苷广泛应用于化妆品、食品和医药等工业中。
本论文以甜菊糖苷为研究对象,以甜菊糖苷的提取率、甜菊糖苷的损失率和甜菊糖苷的纯度为评价指标,围绕甜菊糖苷的提取、除杂、纯化、精制和脱色工艺条件的优化为研究目标开展了以下几个方面的工作:
1.甜菊糖苷的提取工艺研究以甜菊糖苷提取率为评价指标,分别考察回流提取法,浸提法,逆流提取法,酶法和回流套提法5种方法提取甜叶菊叶中甜菊糖苷的提取工艺。结果表明:回流法、浸提法、酶法、逆流法和回流套提法在最佳提取条件下的提取率分别为98.08、80.17、86.98、79.98和94.54 %。相比较回流套提法的提取率高、时间短、用水量少。因此,回流套提法用于提取甜菊糖苷具有独特的优越性。
2.甜菊糖苷的除杂工艺研究以甜菊糖苷的损失率和除杂率为评价指标,考察了几种除杂方法对甜叶菊水提取液的除杂效果,实验结果表明,醇沉法对甜叶菊水提取液具有较好的的除杂效果,醇沉的除醇沉最佳条件为:以10 mL/min的速度向水提液中加入95 %的乙醇至体系乙醇浓度达到80 %,静置1 h,离心分离。在此条件下,其最佳工艺除杂率为16.47 %,甜苷损失率为6.13 %。
3.甜菊糖苷纯化精制工艺的研究
(1)通过动态吸附试验,系统的研究了L型大孔吸附树脂对甜菊糖苷的最佳吸附/解吸工艺条件为:使用串联双柱,上样体积为4200mL;上样浓度0.025g/ml;吸附速率为40 BV/h;水洗脱量为800 mL(10BV),洗脱速率为7.2 BV/h;吸附和洗脱温度为40℃;洗脱液为混合溶液;洗脱速率为20 BV/h。
(2)选择4种大孔吸附树脂,通过比较其对甜菊糖苷的静态吸附结果,筛选出较适合精制甜菊糖苷的大孔吸附树脂,且对甜菊糖苷进行了动态吸附,其工艺条件为:上浓度为0.1 g/mL;吸附速率为12 BV/h;上样体积40 mL;洗脱剂为70 %乙醇,收集100 mL。
4.甜菊糖苷的脱色工艺研究
采用活性氧化铝-中压柱对甜菊糖苷进行吸附脱色,发现活性氧化铝对甜菊糖苷有一定的脱色作用,对甜菊糖苷的纯度与质量也产生一定的影响。其优化条件为:以水为溶剂;吸附温度为35℃,吸附时间为30 min;甜菊糖苷与氧化铝的质量比为1: 0.8;脱色后的甜菊糖苷用连续中压柱层析法对甜菊糖苷进行纯化制备,用蒸馏水洗脱,得到的甜菊糖苷的纯度为91.76%。
Steviol glycosides isolated from the leaves of Stevia rebaudiana Bertoni have been regarded as a promising natural sweetener because of their high sweetness and lower calorific value. Steviol glycosides have many biological effects without side effects, they have caused great interest of some researchers and have been regarded as the substitute of sucrose. Nowadays, Steviol glycosides have been widely used in many fields including cosmetic, food and medicine industry.
In this dissertation, the Steviol glycosides were chosen as the research object. The methods of extraction, impurity removal, purification, fine purification and decolorization of Steviol glycosides from Stevia rebaudiana Bertoni were studied. extraction rate, loss rate and purity of Steviol glycosides chosen as the evaluation indexes. The contents of this dissertation are the following:
1.The study on extraction technic of Steviol glycosides in Stevia rebaudiana Bertoni.
the extractrd technology of Steviol glycosides from Stevia rebaudiana Bertoni by reflux extraction, immersion extraction, enzymatic extraction, countercurrent extraction and reflux set extraction were studied. The five methods were compared by their results of extraction rate of Steviol glycosides. The extraction yields of the optimum process for above five methods were 98.08 %, 80.17 %, 86.01 %, 79.98 % and 94.54 %, respectively. The result indicated that reflux set extraction had the advantages of higher extraction rate of Steviol glycosides, shorter extraction time and saving - water. Therefore, the reflux set extraction have their unique superiority over other method.
2.The study on impurity removal of Steviol glycosides in Stevia rebaudiana Bertoni
The impurity removing rate and loss rate of Stevioside were choosen as the assessment index and then three impurity removal process of water extracts of Stevia rebaudiana Bertoni were compared. The results showed that ethanol subsiding method was better than flocculation and modified attapulgite adsorption technology. The best conditions of ethanol subsiding method were as follows: adding 95% ethanol(speed is 10 mL/min)to obtain 80% ethanol concentration in solution, and standing for 1 h, then centrifuging to obtain the supernant, in this optimum process, the loss rate of Stevioside is 6.13 % and impurity removal rate is 16.47 %.
3. The study on purification and fine purification of Steviol glycosides in Stevia rebaudiana Bertoni.
(1)Dynamic adsorption and characteristic of L macroporous resin to Steviol glycosides in Stevia rebaudiana Bertoni were researched. The results showed its best dynamic adsorption and desorption conditions as follow: sample volume is 1400 mL; sample concentration is 0.025 g/ml; adsorption velocity is 40 BV/h; the number of water is 800 mL(10BV), desorption velocity is 7.2 BV/h; temperature of dynamic adsorption and desorption is 40℃; desorption agent is mixed solvent; desorption velocity is 40 BV/h the number of collected tubes is 600 mL(8BV).
(2)choice of four kinds of macroporous resin by comparing the results of static adsorption of Steviol glycosides in Stevia rebaudiana Bertoni, and selected a suitable macroporous resin to Steviol glycosides in Stevia rebaudiana Bertoni, and the study of adsorption of Steviol glycosides in Stevia rebaudiana Bertoni by dynamic adsorption. the results showed that the better adsorption performance to Steviol glycosides in Stevia rebaudiana Bertoni is D-1macroporous resin, and the best adsorption conditions is that: sample concentration is 0.1 g/mL; adsorption velocity is12 BV/h; sample volume is 40 mL; desorption agent is 70 % ethanol; the number of collected tubes is 100 mL. the puity of Steviol glycosides in Stevia rebaudiana Bertoni by purification and fine purification is 75.30 %.
4.The study on decoloration of Steviol glycosides in Stevia rebaudiana Bertoni.
The result of active aluminum oxide -column chromatography-app lied in decolorization of Steviol glycosides revealed that it had some influences not only on decolorization, but also on the purity and quality. The optimal conditions were as follows: desorption agent is water; temperature of decolorization 35℃, time 30 min; the mass ratio of Steviol glycosides to active aluminum oxide 1:0.8. then, Used column chromatography to purify Steviol glycosides, desorption agent is 70 % ethanol. the puity of Steviol glycosides in Stevia rebaudiana Bertoni by Decolorization is 91.76 %.
本论文以甜菊糖苷为研究对象,以甜菊糖苷的提取率、甜菊糖苷的损失率和甜菊糖苷的纯度为评价指标,围绕甜菊糖苷的提取、除杂、纯化、精制和脱色工艺条件的优化为研究目标开展了以下几个方面的工作:
1.甜菊糖苷的提取工艺研究以甜菊糖苷提取率为评价指标,分别考察回流提取法,浸提法,逆流提取法,酶法和回流套提法5种方法提取甜叶菊叶中甜菊糖苷的提取工艺。结果表明:回流法、浸提法、酶法、逆流法和回流套提法在最佳提取条件下的提取率分别为98.08、80.17、86.98、79.98和94.54 %。相比较回流套提法的提取率高、时间短、用水量少。因此,回流套提法用于提取甜菊糖苷具有独特的优越性。
2.甜菊糖苷的除杂工艺研究以甜菊糖苷的损失率和除杂率为评价指标,考察了几种除杂方法对甜叶菊水提取液的除杂效果,实验结果表明,醇沉法对甜叶菊水提取液具有较好的的除杂效果,醇沉的除醇沉最佳条件为:以10 mL/min的速度向水提液中加入95 %的乙醇至体系乙醇浓度达到80 %,静置1 h,离心分离。在此条件下,其最佳工艺除杂率为16.47 %,甜苷损失率为6.13 %。
3.甜菊糖苷纯化精制工艺的研究
(1)通过动态吸附试验,系统的研究了L型大孔吸附树脂对甜菊糖苷的最佳吸附/解吸工艺条件为:使用串联双柱,上样体积为4200mL;上样浓度0.025g/ml;吸附速率为40 BV/h;水洗脱量为800 mL(10BV),洗脱速率为7.2 BV/h;吸附和洗脱温度为40℃;洗脱液为混合溶液;洗脱速率为20 BV/h。
(2)选择4种大孔吸附树脂,通过比较其对甜菊糖苷的静态吸附结果,筛选出较适合精制甜菊糖苷的大孔吸附树脂,且对甜菊糖苷进行了动态吸附,其工艺条件为:上浓度为0.1 g/mL;吸附速率为12 BV/h;上样体积40 mL;洗脱剂为70 %乙醇,收集100 mL。
4.甜菊糖苷的脱色工艺研究
采用活性氧化铝-中压柱对甜菊糖苷进行吸附脱色,发现活性氧化铝对甜菊糖苷有一定的脱色作用,对甜菊糖苷的纯度与质量也产生一定的影响。其优化条件为:以水为溶剂;吸附温度为35℃,吸附时间为30 min;甜菊糖苷与氧化铝的质量比为1: 0.8;脱色后的甜菊糖苷用连续中压柱层析法对甜菊糖苷进行纯化制备,用蒸馏水洗脱,得到的甜菊糖苷的纯度为91.76%。
Steviol glycosides isolated from the leaves of Stevia rebaudiana Bertoni have been regarded as a promising natural sweetener because of their high sweetness and lower calorific value. Steviol glycosides have many biological effects without side effects, they have caused great interest of some researchers and have been regarded as the substitute of sucrose. Nowadays, Steviol glycosides have been widely used in many fields including cosmetic, food and medicine industry.
In this dissertation, the Steviol glycosides were chosen as the research object. The methods of extraction, impurity removal, purification, fine purification and decolorization of Steviol glycosides from Stevia rebaudiana Bertoni were studied. extraction rate, loss rate and purity of Steviol glycosides chosen as the evaluation indexes. The contents of this dissertation are the following:
1.The study on extraction technic of Steviol glycosides in Stevia rebaudiana Bertoni.
the extractrd technology of Steviol glycosides from Stevia rebaudiana Bertoni by reflux extraction, immersion extraction, enzymatic extraction, countercurrent extraction and reflux set extraction were studied. The five methods were compared by their results of extraction rate of Steviol glycosides. The extraction yields of the optimum process for above five methods were 98.08 %, 80.17 %, 86.01 %, 79.98 % and 94.54 %, respectively. The result indicated that reflux set extraction had the advantages of higher extraction rate of Steviol glycosides, shorter extraction time and saving - water. Therefore, the reflux set extraction have their unique superiority over other method.
2.The study on impurity removal of Steviol glycosides in Stevia rebaudiana Bertoni
The impurity removing rate and loss rate of Stevioside were choosen as the assessment index and then three impurity removal process of water extracts of Stevia rebaudiana Bertoni were compared. The results showed that ethanol subsiding method was better than flocculation and modified attapulgite adsorption technology. The best conditions of ethanol subsiding method were as follows: adding 95% ethanol(speed is 10 mL/min)to obtain 80% ethanol concentration in solution, and standing for 1 h, then centrifuging to obtain the supernant, in this optimum process, the loss rate of Stevioside is 6.13 % and impurity removal rate is 16.47 %.
3. The study on purification and fine purification of Steviol glycosides in Stevia rebaudiana Bertoni.
(1)Dynamic adsorption and characteristic of L macroporous resin to Steviol glycosides in Stevia rebaudiana Bertoni were researched. The results showed its best dynamic adsorption and desorption conditions as follow: sample volume is 1400 mL; sample concentration is 0.025 g/ml; adsorption velocity is 40 BV/h; the number of water is 800 mL(10BV), desorption velocity is 7.2 BV/h; temperature of dynamic adsorption and desorption is 40℃; desorption agent is mixed solvent; desorption velocity is 40 BV/h the number of collected tubes is 600 mL(8BV).
(2)choice of four kinds of macroporous resin by comparing the results of static adsorption of Steviol glycosides in Stevia rebaudiana Bertoni, and selected a suitable macroporous resin to Steviol glycosides in Stevia rebaudiana Bertoni, and the study of adsorption of Steviol glycosides in Stevia rebaudiana Bertoni by dynamic adsorption. the results showed that the better adsorption performance to Steviol glycosides in Stevia rebaudiana Bertoni is D-1macroporous resin, and the best adsorption conditions is that: sample concentration is 0.1 g/mL; adsorption velocity is12 BV/h; sample volume is 40 mL; desorption agent is 70 % ethanol; the number of collected tubes is 100 mL. the puity of Steviol glycosides in Stevia rebaudiana Bertoni by purification and fine purification is 75.30 %.
4.The study on decoloration of Steviol glycosides in Stevia rebaudiana Bertoni.
The result of active aluminum oxide -column chromatography-app lied in decolorization of Steviol glycosides revealed that it had some influences not only on decolorization, but also on the purity and quality. The optimal conditions were as follows: desorption agent is water; temperature of decolorization 35℃, time 30 min; the mass ratio of Steviol glycosides to active aluminum oxide 1:0.8. then, Used column chromatography to purify Steviol glycosides, desorption agent is 70 % ethanol. the puity of Steviol glycosides in Stevia rebaudiana Bertoni by Decolorization is 91.76 %.
引文
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