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Study on Separating and Purifying Process of Rebaudioside-A

Author: FuJuanJuan
Tutor: WenHuiLiang
School: Nanchang University
Course: Of Food Science
Keywords: rebaudioside-A steviol glycosides silica gel column chromatography macroporous adsorption resin
CLC: TS201.2
Type: Master's thesis
Year: 2013
Downloads: 37
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Abstract

Rebaudioside-A is a glycoside which is part of steviol glycosides, and compared to the other containing glycosides, its sweetness is about350～450times of sucrose and no bitterness. Nowadays it has found that the content of rebaudioside-A is higher, the sweet taste is purer, and steviol glycosides has widely used in the field of medicine, food and beverage, therefore, it has a very important significance to research how to improve the content of rebaudioside-A in steviol glycosides products.This paper took steviol glycosides as the research object. Firstly steviol glycosides was extracted from Stevia rebaudiana Bertoni by using ultrasonic auxiliary enzymatic method, and the extraction conditions were optimized by the orthogonal experiment. Then rebaudioside-A was separated from steviol glycosides by using silica gel column chromatography method, and the orthogonal experiment was used to find the optimal separation conditions. Lastly the paper studied to use macroporous adsorption resin to separate and purify stevioside and rebaudioside-A from the steviol glycosides solution which was crystallized. The relevant conclusions were as follows:(1)The study optimized extraction conditions of the single factor experiment and the orthogonal experiment. The results showed that:in the liquid-solid ratio (V/W) was12, the extraction solution of pH for4.5～5.5, the enzyme quality solubility of0.15%, the ultrasonic time of80min, the extraction temperature of60℃, and the ultrasonic power of50W, then merged four times extraction from the filtrate. Under these conditions, the extraction rate of steviol glycosides was12.9%. Compared with traditional extraction method, the method of the ultrasonic assisted cellulase extraction of steviol glycosides not only cut short the required extraction time, but also reduced the required extraction temperature, thereby saved energy.(2)In the process of removing impurity of extracting solution, the decoloring rate and the loss rate of steviol glycosides were used to determine the dosage of composite flocculant which were potassium aluminium sulfate and calcium oxide. The results showed that:when the dosage ratio of potassium aluminium sulfate and calcium oxide was1.4:1, the decolorizing rate was maximum and the loss rate of steviol glycosides was smaller.(3)In the experiment of separated rebaudioside-A by the silica gel column chromatography, the sample forms, pressurized or not of the elution process and eluant type were first considered. Results showed that:when the sample was the steviol glycosides mixed with silica gel, the elution process was pressurized, and the eluant was dichloromethane and methanol which were mixed according to a certain proportion, these impacted greater on the separation of rebaudioside-A. Then in the experiment process, the study considered single factors like as the dosage of the steviol glycosides, the dosage of the silica gel, the ratio of eluent and elution velocity and did the orthogonal test. According to the analysis of experiments, when the ratio of steviol glycosides’ dosage and silica gel’ dosage was1:17.5, the eluent ratio of dichloromethane and methanol was3:2, and the elution velocity was60drops/min, the purity of rebaudioside-A could reach93.4%and the yield could reach51.3%. Lastly, amplification experiments were done under these conditions, the results were close and good reliability, and the purity of rebaudioside-A was up to95.1%in these results.(4)The steviol glycosides is composed of a variety of glycosides, including rebaudioside-A and stevioside, after crystallized the steviol glycosides, the mother solution contains rebaudioside-A and a lot of stevioside. In this experiment, first let steviol glycosides to crystallize, it could be obtained with rebaudioside-A that the purity was91.9%and the yield was51.0%, then the mother solution was be adsorbed with3-4h by ADS-8macroporous adsorption resin. After this, it used85%methanol solution and under elution speed of60drops/min to elute, it could be obtained with stevioside that the purity was93.8%and the yield was78.5%. In addition, the purity of rebaudioside-A was90.5%that could be obtained, but the yield of rebaudioside-A was very low.

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