银杏叶黄酮和多糖综合分离技术的研究
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摘要
银杏叶是一类蕴藏量相等大的潜在资源，主要包括黄酮类、萜类、多糖类、生物碱、酚类、氨基酸、微量元素等有多种有效成分，具有广阔的开发前景。我国银杏叶产量占世界产量的70％左右，本文着眼于银杏叶活性成分的综合利用，研究了银杏叶黄酮的酶法提取工艺，优化了工艺参数；研究了多糖的提取工艺和超滤浓缩分离工艺；研究了银杏叶黄酮的超临界CO_2萃取工艺和技术，优化了工艺参数。主要研究结果如下：
1．常规的乙醇提取法之前，对银杏叶进行酶预处理，具有较好的破壁作用，可提高银杏叶黄酮的得率。在单因子实验结果的基础上，采用响应曲面法研究了酶浓度、pH值、酶解温度和酶解时间对银杏叶黄酮提取得率的影响。结果表明，纤维素酶具有较好的酶解能力，当酶浓度为100U／mL，酶解时间为116min，温度为40℃，pH值为4.5时，总黄酮得率达到2.63％，相对单一醇提法得率提高了约30％。
2．选用水提取法浸提银杏叶多糖，通过正交实验优化水提取法工艺，提高多糖得率；并将超滤膜技术引入对银杏叶多糖提取液的浓缩分离中，可去除杂质，纯化多糖。在单因子实验的基础上，采用正交实验得到较优的银杏叶多糖提取工艺参数为料液比为1∶30，提取时间3.5h，提取温度为85℃，提取次数2次，多糖得率达到5.98％。通过超滤，初步判断银杏叶多糖的分子量范围，确定银杏叶多糖主要分布在100Kd以上和30-100Kd。初步建立了银杏叶多糖提取液的超滤工艺参数为膜截留分子量30Kd，压力0.050MPa，温度为30℃，浓缩液中多糖的截留率大约为90％，平均膜通量为0.980L／(min·m~2)。
3．采用超临界CO_2萃取技术萃取银杏叶黄酮，实现了萃取和分离过程的一体化，操作方便，萃取效率高，耗能较低。通过单因素实验，初步确定银杏叶黄酮萃取最佳工艺参数：CO_2流速为20L／h时，乙醇浓度为80％，乙醇夹带剂用量为100mL／100g银杏叶，萃取时间为2h，萃取温度为45℃，萃取压力为30MPa，黄酮的得率为1.30％。
Ginkgo biloba leaves is a large potential resources that reserves largely and has broad prospects, including mainly flavonoids, terpene, polysaccharides, alkaloids, phenols, amino acids, trace elements and other multiple active ingredients. The output of Ginkgo biloba leaves in China is 70% of world output, this thesis focused on the comprehensive use of active components from Ginkgo biloba leaves. In this thesis, the research work focused on the following three aspects: (1) optimizing the extraction techniques of flavonoids from Ginkgo biloba leaves by enzymatic hydrolysis; (2) extracting polysaccharides from Ginkgo biloba leaves and isolating by ultrafiltration; (3) extracting parameters of flavonoids from Ginkgo biloba leaves by supercritical carbon dioxide. The main research findings are as follows:
1. The hydrolysis pretreatment with enzymatic before extraction with ethanol is helpful to get rid of cell wall of Ginkgo biloba, the yield of flavonoids from Ginkgo biloba can be raised. At the base of single factor experiments, the effect of the concentration of enzyme, pH value, temperature and time of enzymatic hydrolysis on the yield of the extraction of total flavonoids from Ginkgo biloba leaves was studied by response surface methodology (RSM). The results indicated that cellulase showed the better ability of enzymatic hydrolysis. The optimum conditions of enzymatic hydrolysis were: 100U/ml concentration, 116mins time, 40℃temperature and 4.5 pH value, the highest yield of total flavonoids was 2.63%. Comparing with single extraction with alcohol, the yield of total flavonoids was increased by 30%.
2. The yield of polysaccharides from Ginkgo biloba leaves was increased by optimizing water extraction technology through orthogonal test. The ultrafiltration technology was introduced into the concentration and separation of polysaccharides from Ginkgo biloba leaves, which can be used to get rid of the impurities. At the base of single factor experiment, the effect of ratio of material water, time of extraction, temperature of extraction and times of extraction on the yield of the extraction of polysaccharides from Ginkgo biloba leaves was studied by orthogonal test. The result indicated that the optimal technical conditions of extraction were ratio of material water 1:30, time of extraction 3.5h, temperature of extraction 85℃and times of extraction 2 times, the highest yield of polysaccharides was 5.98%. It was judged roughly by ultrafiltration that molecular mass distribution of polysaccharides from Ginkgo biloba leaves contain two parts, one more than 100Kd and the other between 30 and 100Kd. The ultrafiltration technology of polysaccharides from Ginkgo biloba leaves was established primarily, which were molecular weight cut-off (MWCO) of membrane 30Kd, pressure 0.050MPa and temperature 30℃.The highest retention rate of polysaccharides was 90%; meanwhile the average membrane flux was 0.980 L/(min·m2).The optimal technology of extraction and ultrafiltration that operates steadily is viable and effective.
3. Supercritical carbon dioxide extraction technique was used in the flavonoids from Ginkgo biloba leaves, which achieve to integrate with the extraction and separation processes. Supercritical carbon dioxide extraction that operates easily is of higher efficiency and lower energy consumption. At the base of single factor experiment, the yield of the extraction of flavonoids from Ginkgo biloba leaves was studied. The result indicated that the optimal technical conditions of supercritical carbon dioxide extraction were velocity of CO_2 20L/h, concentration of alcohol 80%, time 2h, temperature 45℃and pressure 30MPa. The highest yield of flavonoids was 1.30%.
1．常规的乙醇提取法之前，对银杏叶进行酶预处理，具有较好的破壁作用，可提高银杏叶黄酮的得率。在单因子实验结果的基础上，采用响应曲面法研究了酶浓度、pH值、酶解温度和酶解时间对银杏叶黄酮提取得率的影响。结果表明，纤维素酶具有较好的酶解能力，当酶浓度为100U／mL，酶解时间为116min，温度为40℃，pH值为4.5时，总黄酮得率达到2.63％，相对单一醇提法得率提高了约30％。
2．选用水提取法浸提银杏叶多糖，通过正交实验优化水提取法工艺，提高多糖得率；并将超滤膜技术引入对银杏叶多糖提取液的浓缩分离中，可去除杂质，纯化多糖。在单因子实验的基础上，采用正交实验得到较优的银杏叶多糖提取工艺参数为料液比为1∶30，提取时间3.5h，提取温度为85℃，提取次数2次，多糖得率达到5.98％。通过超滤，初步判断银杏叶多糖的分子量范围，确定银杏叶多糖主要分布在100Kd以上和30-100Kd。初步建立了银杏叶多糖提取液的超滤工艺参数为膜截留分子量30Kd，压力0.050MPa，温度为30℃，浓缩液中多糖的截留率大约为90％，平均膜通量为0.980L／(min·m~2)。
3．采用超临界CO_2萃取技术萃取银杏叶黄酮，实现了萃取和分离过程的一体化，操作方便，萃取效率高，耗能较低。通过单因素实验，初步确定银杏叶黄酮萃取最佳工艺参数：CO_2流速为20L／h时，乙醇浓度为80％，乙醇夹带剂用量为100mL／100g银杏叶，萃取时间为2h，萃取温度为45℃，萃取压力为30MPa，黄酮的得率为1.30％。
Ginkgo biloba leaves is a large potential resources that reserves largely and has broad prospects, including mainly flavonoids, terpene, polysaccharides, alkaloids, phenols, amino acids, trace elements and other multiple active ingredients. The output of Ginkgo biloba leaves in China is 70% of world output, this thesis focused on the comprehensive use of active components from Ginkgo biloba leaves. In this thesis, the research work focused on the following three aspects: (1) optimizing the extraction techniques of flavonoids from Ginkgo biloba leaves by enzymatic hydrolysis; (2) extracting polysaccharides from Ginkgo biloba leaves and isolating by ultrafiltration; (3) extracting parameters of flavonoids from Ginkgo biloba leaves by supercritical carbon dioxide. The main research findings are as follows:
1. The hydrolysis pretreatment with enzymatic before extraction with ethanol is helpful to get rid of cell wall of Ginkgo biloba, the yield of flavonoids from Ginkgo biloba can be raised. At the base of single factor experiments, the effect of the concentration of enzyme, pH value, temperature and time of enzymatic hydrolysis on the yield of the extraction of total flavonoids from Ginkgo biloba leaves was studied by response surface methodology (RSM). The results indicated that cellulase showed the better ability of enzymatic hydrolysis. The optimum conditions of enzymatic hydrolysis were: 100U/ml concentration, 116mins time, 40℃temperature and 4.5 pH value, the highest yield of total flavonoids was 2.63%. Comparing with single extraction with alcohol, the yield of total flavonoids was increased by 30%.
2. The yield of polysaccharides from Ginkgo biloba leaves was increased by optimizing water extraction technology through orthogonal test. The ultrafiltration technology was introduced into the concentration and separation of polysaccharides from Ginkgo biloba leaves, which can be used to get rid of the impurities. At the base of single factor experiment, the effect of ratio of material water, time of extraction, temperature of extraction and times of extraction on the yield of the extraction of polysaccharides from Ginkgo biloba leaves was studied by orthogonal test. The result indicated that the optimal technical conditions of extraction were ratio of material water 1:30, time of extraction 3.5h, temperature of extraction 85℃and times of extraction 2 times, the highest yield of polysaccharides was 5.98%. It was judged roughly by ultrafiltration that molecular mass distribution of polysaccharides from Ginkgo biloba leaves contain two parts, one more than 100Kd and the other between 30 and 100Kd. The ultrafiltration technology of polysaccharides from Ginkgo biloba leaves was established primarily, which were molecular weight cut-off (MWCO) of membrane 30Kd, pressure 0.050MPa and temperature 30℃.The highest retention rate of polysaccharides was 90%; meanwhile the average membrane flux was 0.980 L/(min·m2).The optimal technology of extraction and ultrafiltration that operates steadily is viable and effective.
3. Supercritical carbon dioxide extraction technique was used in the flavonoids from Ginkgo biloba leaves, which achieve to integrate with the extraction and separation processes. Supercritical carbon dioxide extraction that operates easily is of higher efficiency and lower energy consumption. At the base of single factor experiment, the yield of the extraction of flavonoids from Ginkgo biloba leaves was studied. The result indicated that the optimal technical conditions of supercritical carbon dioxide extraction were velocity of CO_2 20L/h, concentration of alcohol 80%, time 2h, temperature 45℃and pressure 30MPa. The highest yield of flavonoids was 1.30%.
引文
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