| Steviol glycoside(SG)is a natural sweetener which has a wide range of applications in food,nutrition,medicine and other fields.The output of SGs in China was about 10,000 tons per year,of which 90%was used for export,and the global annual demand increased by10%?20%.However,the purification process of SGs was complicated.First,the aqueous extract of SGs was obtained by percolation,which was then purified by flocculating agent,membrane separation,adsorption resin,ion exchange resin and finally recrystallized to obtain commercial steviol glycosides.This process was not environmentally friendly and the cost was relatively high,which could not satisfy the requirements of green production and consumption of food additives.To solve this problem,this thesis tried to explore a SGs purification method with simpler process,better purification effect,more economic and environmental benefits according to the differences of structures and properties between impurities and glycosides based on the previous research of our group.Firstly,the SGs were extracted from dried Stevia rebaudiana Bertoni leaves(the solid-to-liquid ratio was 1:20,w/v,room temperature,24 h)and then the components of the extract were analyzed.The impurities-to-stevia ratio was 5:1?6:1,glycoside content was7.31±0.26 mg/m L,protein content was 18.71±0.80 mg/m L,polyphenol content was0.98±0.01 mg/m L.Therefore,the main impurities in extract were proteins,and the main pigments were polyphenols.Based on the preliminary results of our research group,the purification effect of reversed-phase chromatography(RPC)was tested.NDR-1 non-polar macroporous resin was used as the adsorption matrix of SGs.The static adsorption and desorption of the extract showed that almost all SGs were adsorped by the resin when the amount of resin was 12 g/g solids,the temperature was 25~oC,and 15%(v/v)ethanol solution was used as the adsorption solvent.And the SGs can be desorbed by absolute ethanol.The purity of purified SGs was46.34%,the SGs loss rate was 1.58%,and the impurity removal rate was 77.62%.Furthermore,dynamic adsorption test was carried out.As the ethanol content in the impurity elution solvent increased from 15%(v/v)to 20%(v/v),the purity of glycosides in the obtained desorption solution increased from 56.13%to 57.42%.However,when the ethanol content increased to 25%(v/v),SGs would be eluted out together with impurities,which made seperation difficult.Therefore,it was hard to obtain SGs of higher purity by one-step RPC.Thirdly,since the ideal impurity removal rate could not be achieved by one-step RPC,other methods were used to preliminarily remove impurities.According to composition result,protein was the main impurity,so D280 anion exchange resin was tested as the adsorbent to remove the protein.The optimized condition test showed that at 25~oC,p H=7,and resin addition of 0.5 g/g solids,the SGs loss rate was 12.56%,and the protein removal rate was57.66%.This result proved that protein can be effectively removed by electrostatic adsorption,however,since the SGs loss rate was too high,and the method was not environmentally friendly,the positively charged chitosan was used to replace the D280 resin for preliminary purification.The flocculation conditions of chitosan were optimized.Under the condition of0.04 g/g solid content,30~oC,p H=5,2.5 h,the protein removal rate was 41.9±0.02%,and the SGs loss rate was 0.9±0.01%.As a result,a two-step purification procedure was performed using chitosan precipitation combined with RPC.At optimized chitosan flocculation conditions,using 25%(v/v)ethanol as the impurity eluent in RPC,the purity of SGs was 82.75%,the SGs recovery rate was98.57%,and the total impurity removal rate was 95.60%.Compared with the industrial production process,this new method had the advantages of shorter process,lower cost and was more environmentally friendly. |
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