| Steviol glycosides exist in blade of Stevia rebaudiana Bertoni,which is one kind of perennial herb originating in Paraguay. Steviol glycosides is similar to sucrose in sweet.There are10different kinds of sweet substances in steviol glycosides. Rebaudioside A,(RA) is one main component in steviol glycosides and the sweetness is450times more than sugar, which has the nearest sweetness for sweet sucrose. Steviol glycosides are widely applied in medicine, beverage, flavoring agent and other fields because of its low-calorie, high sweetness, the characteristics of non-toxic and no side effects.This paper firstly focused on steviol glycosides and studied and compared systematically on two separation methods of Stevia rebaudiana Bertoni leaves, that is, hot-water treatment and enzyme extraction. This paper also studied some rebaudioside A(RA)separation methods of steviol glycosides and optimized the best recrystallization conditions to separate RA by orthogonal test. Main research contents and conclusions were as follows:1.The study optimized hot-water treatment and cellulase assited technique on the process of steviol glycosides extraction by orthogonal test. Anthrone sulfuric acid method was used to detect the amount of steviol glycosides in extract. The results showed that:in leaf-water ratio of1:10,2hours of extraction time conditions, the process of hot-water treatment, the best conditions were extraction temperature of80℃,2times of extraction. Under these conditions, the extraction rate of steviol glycosides was11.2%. In the cellulase assited process, the best treatment conditions were0.2%dosage of cellulase,extraction temperature of50℃,1.5hours of extraction time, pH5.0. The extraction rate of steviol glycosides reached14.0%under these process conditions. Combined with these various factors, the paper got the conclusion that enzymatic extraction was better than hot-water treatment.2. In order to detecte the content of stevioside in extract, this research made use of gravimetric method. Compared with the result of anthrone sulfuric acid and the Standards, the result of gravimetric method was slightly lower. However, taking into account of the experimental error, gravimetric method still could be used to detect the steviol glycosides content.3. UV and hydrogen peroxide can be used as sterilization, as well as odium benzoate and potassium sorbate have antimicrobial effect. According to this principle, this study utilized UV sterilization on stevia leaves. Furthermore, hydrogen peroxide, odium benzoate and potassium sorbate were added into the extract to remove the microorganism. The results showed that UV sterilization had no effect on microbiological content in extract. However, hydrogen peroxide with0.15%dosage could kill all microorganism in extract under the conditions of25℃,time of1h or50℃,time of0.5h. Sodium benzoate and potassium sorbate could inhibit the growth of microorganisms in the extract under the antibacterial conditions of25℃,time of1h,pH4.4. In this study, steviol glycosides was recrystallized by mixed solution of ethanol and acetone. And orthogonal test was applied to optimize and get the best crystallization conditions. At last, the crystalline product was dissolved in the mixture of ethanol and water, then we can got high purity product of RA. The results showed that:When the solid-liquid ratio of ethanol in sample was1:4and the amount of acetone was15%that of ethanol, and the product was firstly dissolved in the constant temperature water bath at70℃,then the sample was recrystallized in water bath at40℃.The crystallization rate of the product was45.6%. Moreover the crystallization rate of RA was70.33%and the content of RA reached74.8%. After the seed was added into the sample under these best crystallization conditions, there were no significant changes in the purity of RA and crystallization rate. But when the sample was stirred under the best crystallization conditions, the crystallization rate of the product, the crystallization rate and the content of RA were respectively increased to54.9%,81.5%and72.2%. The crystalline product was dissolved in aqueous ethanol solution with the content of5%water and1:10of the solid-liquid ratio, then stirring1h at90℃. At last, the sample was filtrated and RA was got as the filtration product with the purity of93.8%. The sample yield was54.5%and crystallization rate of RA was57.7%calculated from the initial product. 5. Thin layer chromatography was used separation stevioside and rebaudioside A, which the main components of steviol glycosides. The ST and RA was separated for the solvent when N-Butanol:water=20:3. When the solvent was N-Butanol:water=5:1, the experiment could enrichment ST and DA, which in the steviol glycoside. |
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