【作者】 任雁；

【导师】 张惟广；

【作者基本信息】 西南大学 ， 食品科学， 2007， 硕士

【摘要】 盾叶薯蓣（英文名Chinese Yam，拉丁名：Dioscorea Zingiberensis C．H．Wright）是我国特有种，又名火头（藤）根，苦良姜。薯蓣皂苷元（diosgenin）为白色晶体或粉末，有刺激性气味，难溶于水，易溶于苯、氯仿等有机溶剂，化学式及分子量：C₂₇H₄₂O₃，414.61，化学名称：（25^R）^Z-异螺旋甾烯-3β-醇，属异螺旋甾烯烷的衍生物，是薯蓣科薯蓣属植物根茎中薯蓣皂苷（dioscin）的水解产物。皂苷元具有多种生理功能，生物界称其为“生命的钥匙”，医药界称其为“药物黄金，激素之母”。本试验以四川遵生药用植化有限公司出售的盾叶薯蓣为原料，对盾叶薯蓣的组成成分，常用盾叶薯蓣保藏方法进行了研究，对薯蓣皂苷元理化性质进行了定性判断，比较了传统皂苷元提取方法与酶解法，确定了盾叶薯蓣中皂苷元酶解法的最佳工艺条件。另外，考虑到超声波对薯蓣结构的影响，对比了超声波辅助提取法与超声-酶解法。最后，采用大孔树脂对皂苷元粗品进行精制，发现树脂ADS-7对皂苷元的吸附及解析较好，在一定程度上能有效的起到精制效果。主要研究结果如下：1．测定盾叶薯蓣中各种成份，发现其中淀粉（26.99％）和粗纤维（57.7％）含量最大，皂苷含量约5.24％。因此选择淀粉酶，纤维素酶，复合酶进行酶解发酵，更具有理论依据。研究鲜盾叶薯蓣的保存方法，得出冷藏是一种可取的方法。2．通过两种方法对皂苷元标准品及样品的扫描，可知硫酸-甲醇法较优，在414nm处出现单一峰。通过稳定性试验（RSD=4.49‰），精密度试验（RSD=6.34‰）及回收率试验（RSD=9.53‰），可知相对标准偏差较小，结果重现性好，精密度较高，所得数据精确。直接酸水解法最佳工艺：酸浓度为3mol／L，酸解时间为4h，发酵时间为24h，加水量为60mL（即料液比1∶3）。影响因素大小：酸的浓度＞酸解时间＞发酵时间＞加水的量，所得皂苷元量为0.4108g3．选择影响耐温淀粉酶四个主要因素，确定三个水平，得出最佳工艺参数：A₂B_3C3D₂，即pH为6，酶用量为0.1mL，酶作用温度为50℃，酶作用时间为36h。影响因素大小：酶作用温度＞pH＞酶用量＞酶作用时间，所得皂苷元量为0.4935g，比直接酸解法提高了20.13％。4．选择影响耐高温淀粉酶的四个主要因素，得出最佳工艺参数：A₃B₃C₃D₂，即pH为6，酶用量为0.1mL，温度为50℃，酶作用时间为24h，影响因素大小为：酶解温度＞酶解时间＞pH＞酶用量，可得0.4999g皂苷元，比直接酸解法提高了21.69％。通过加酶与不加酶提取过程中几个参数的对比，可以看出，加酶后所得皂苷元量远大于不加酶组，且黄色废液中所得糖含量高，为绿色无污染糖，经酸解后两次得糖相差不大。分析酸用量与皂苷元，渣重，糖的关系，可知酶作用提取皂苷元，可以减少酸的用量（皂苷元得率达到直接发酵法提取得率时，可节约酸33.33％），降低污染。5．将原材料经超声波处理后，索氏提取，未能明显得到皂苷元。超声后不加酶法与超声后加酶法所得皂苷元量基本相当，分别为0.4977g／20g粉末和0.4994 g／20g粉末，比直接发酵法分别提高了21.15％和21.56％。可见，超声能够一定程度上取代酶作用，但其发酵后糖量略有减少，绿色糖减少。考虑到超声波对人体的负作用，以及超声波法成本要远大于生物酶法，认为酶解发酵法较优。6．从熔点及纯度来看，酶法所得皂苷元样品质量均要高于直接发酵法所得皂苷元。酶法所得皂苷元样品熔点大致在202-210℃，纯度均在88％以上，而直接水解法所得皂苷元样品熔点一般在198-205℃，纯度均低2个百分点。7．树脂ADS-7对皂苷元的吸附作用很好，吸附率高达94.35％，用无水乙醇作为解析剂，可将其全部析出，说明它是一种很好的树脂。乙醇的解析作用很强，95％乙醇基本上能将所吸附的皂苷元全部解析下来。最终得出：8g预处理过的树脂ADS-7，加入200mL浓度为0.094mg／mL样品溶液，用数显恒流泵控制样液以1.0mL／min的速度流入装有树脂ADS-7的层析柱，待吸附完成后，收集吸附后的流出液，取1mL进行检测皂苷元含量，算出吸附量。将层析柱内树脂凉干（避免层析柱破裂），然后加入250mL解析剂，解析率可达到87.76％。收集解析液，检测皂苷元含量，算出皂苷元纯度为94.12％，比纯化前提高4.5％。更多还原

【Abstract】 Dioscorea Zingiberensis C. H. Wright(Chinese yam is a special kind of plant, which also named HuoTou geng or KuLiang jiang. The diosgenin is white crystal with stimulating smell. It’s hard to dissolve in water, but easy to dissolve in some organic solvent like benzene, chloroform and so on. The structural formula of diosgenin is C₂₇H₄₂O₃, and the molecular weight is 414.61.The diosgenin is the hydrolysate of Dioscorea Zingiberensis C. H. Wright, which has tremendous medical value and marketable potential. The diosgenin has many physiological functions which plays an important role in human health, the world of biology calls it as "the key of life", the field of medication calls it as "the gold in medication, the mother of hormone". This paper studied the component of Dioscorea Zingiberensis C. H. Wright and the different methods of conservation. We judged the diosgenin’s properties of physical chemistry, compared the extractation methods, including the tradition method and enzymatic zymotechnics. Finally, confrimed the best technics conditions. Ultrasonic may influence the structure of Dioscorea Zingiberensis C. H. Wright, so we contrasted ultrasonical extractation and ultrasonic with enzyme. The optimized conditions for the extraction and purification of diosgenin were obtained. The resultsindicated:1. Starch （26.99%） and coarse fiber （57.7%） content were the most among all the components of Diascorea Zingiberensis C. H. Wright which also had 5.24% dioscin. So we chose amylase, cellulose enzyme and compound enzyme to use in the experiment. Refrigeration was a good way to preserve the fresh Dioscorea Zingiberensis C. H. Wright and had no adverse effect on the extraction of diosgenin. 2. In the diosgenin determination experiment, we used two methods. One was Colorimetry of vanillin-acetic acid, another was Colorimetry of vitriol-methanol.The latter one was better with single apex appears at 414nm, which had small relatively standard deviation. We can find the best technics from orthogonal experiment by acid hydrolyzing directly: acid concentration is 3 mol/L, the time of acid hydrolyzing was 4 hours and the fermentation time with 60ml water was 24 hours. Acid concentration leads the first factor in the method and the water amount was the last one.3. Monofactorial experiment helped select out 4 factors and 3 levels which influenced thermoduric amylase and gave the best processing parameters as A₂B₃C₃D₂ which can accept 0.4935g diosgenin, 20.13% higher than traditional method, namely the Parameters just like the pH was 6, the dosage of emzyme was 0.1mL, the temperature was 50℃, and the time of fermenting was 36 hours. Of the 4 factors, temperature affected most while action time of amylase was the least. In other words, pH affected greater than amylase dosage while smaller than temperature.4. Slected four factors which effected more on thermostable amylase, we got the best technics parameters as A₃B₃C₃D₂, which can accept 0.4999g diosgenin, 21.69% higher than traditional method, the Parameters just like the pH was 6, the dosage of emzyme was 0.1mL, the temperature was 50℃, and the time of fermenting was 24 hours. Of these four factors, we found that the tempreture effected most on amylase, and the dosage of emzyme was the least. Comparing the two experiments with amylase or not, we found the output of diosgenin adding amylase was more than without amylase, also we can get more green sugar by adding amylase athough the total sugar was almost same. Analysing the relationship among the output of diosgenin, the weight of residue and sugar, we came to a conclusion that using little acid can decrease pollute by adding amylase which saving 33.33% acid.5. Considering the influence of ultrasonic on the structure of Dioscorea Zingiberensis C. H. Wright, we have compared the diosgenin extraction processing.We cannot get diosgenin only using ultrasonic extraction without fermentation. If we used ultrasonic to assist amylase extraction, the output of diosgenin were not higher obviously than amylase extraction, it would decrease if the ultrasonic power was too high or too low. If using ultrasonic instead of adding amylase with fermentation, we can get 0.4977g diosgenin which was 21.15% higher than taditional method while get 0.4994g diosgenin which was 21.56% higher by using ultrasonic with amylase fermentation. That was to say, ultrasonic can instead of amylase in a way, but the weight of sugar we got has decreased but the total sugar was invariability. Finally, considering the side effect to people and the higher cost of ultrasonic, we tought the amylase method was better.6. The diosgenin sample got from enzymolysis extraction was better than that got from direct fermentation in terms of sample melting point and purity. The melting point of the sample from enzymolysis extraction was 202-210℃and purity was over 88% while the latter from 198℃to 205℃while purity was 86%.7. Resin ADS-7 can adsorb and desorb diosgenin well with 94.35% absorption capability in a short adsorption and a short desorption. Ethanol had a strong ability both in adsorption and desorption, 95% of ethanol could almost desorb all the absorption. Add 8g ADS-7 into 200mL diosgenin solution with a concentration of 0.094mg/mL, then pump into ADS-7 with a constant flow of 1.0mL/min while collect effusion. After completed adsorption, taking 1 mL effusion to determine the content of diosgenin,drying the resin, adding 250mL desorption solvent into it, desorption ratio can come to 87.76%. Considering the cost, to use 250mL desorption solvent to determine the content of diosgenin, we got diosgenin purity was 94.12% which increased by 4.5% than before.更多还原