| Steviol glycoside(SG),a kind of non-nutritive,non-fermentable and high-intensity sweetener extracted from Stevia rebaudiana Bertoni,has the advantages of non-toxicity,low calerie,high sweetness and stabibility.It is an ideal sweetener with broad development prospects in the food industry.Currently,solvent extraction has been the main process of commercial SGs extraction.The residual mother liquor after crystallization is generally spray-dried as powder,named mother liquor sugar(MLS)and sold as a low-price sweetner in China.MLS contains about 60%SGs(dry weight),while the residual impurities are polyphenols and flavonoids.The impurities existed significantly aggravate the bitter aftertaste of MLS,hindering the further recovery of SGs.About 2000 t of MLS was produced every year in China.If the impurities can be removed from SGs,the price of the product can be increased by 2~3times.Aimed at abovementioned problems,this paper focused on developing a simple and industrialized method of SGs recovery.Additionally,the enzymatic modification of SG has attracted wider attention after the approval of the enzymatic modification product of SG,especially rebaudioside D(RD)and rebaudioside M(RM),which have one or two more?-glycosyls than RA have more excellent properties in sweetness.The drawback of current enzymatic modification of RA lied in the unability of achieving industrialization.This paper tried to use commercial enzyme to modify RA.Firstly,the non-polar macroporous resin NDR-1 was wisely selected as the adsorption matrix and used for the purification of MLS.Results showed that the adsorption ratio was92.24%,the desorption ratio was 86.61%,and the impurity content was reduced from 34.13%to 12.88%after the static adsorption and desorption of MLS under the optimal conditions:adsorption solvent(25%(v/v)ethanol),pH 7.00,10~oC;desrption solvent(absolute ethanol),30~oC,with pH unadjusted.Based on the optimized ethanol content of static adsorption and desorption,the dynamic chromatography of NDR-1 resin(column column:1.1×100 cm)was conducted,after which the impurity content of MLS was reduced to 4.78%,and the SGs recovery rate was 89.88%.300 g(dry weight)of MLS was treated with a 10×100 cm glass column.After the resin chromatography,the impurity content was reduced to 13.79%and the SGs recovery rate was 90.00%.The results abovementioned verified the industrial feasibility of this procedure.Secondly,the impurities of MLS were removed by methanol recrystallization combined with resin column chromatography.Absolute methanol working as the solvent can recrystallize the MLS and effectively remove the impurities.Crystals with different SG composition can be obtained under different crystallization conditions.Results showed that the lowest impurity content(3.11%)and the crystallization ratio(42.45%)in the crystal was attained at the ratio of solid to liquid(1:5),temperature of 25~oC and stirring time of 24 h.The remaining mother liquor was again treated with macroporous resin NDR-1.Dynamic chromatography(column column size:1.1×100 cm)was performed under optimized conditions(adsorption solvent of 35%ethanol,adsorbent to resin mass ratio of 1:10),thereafter SGs(purity of 98.10%)were obtained,with recovery rate of 95.20%.A pilot-scale experiment of the recrystallization combined with column chromatography method was conducted in purification of MLS.The recovery rate of SGs from MLS was 94.18%,and the impurity content of the obtained SGs reached 2.83%after treating 300 g of MLS(The impurity content of sample after column chromatography was1.92%).Finally,cellulase(Trichoderma reesei)and cellobiose served as the transglycosylase and substrate donors(using RA as a receptor)hold the higest efficiency.More specifically,the highest conversion yield of RA(36.05%)was obtained under the optimized reaction conditions:RA 10 g/L,cellobiose 100 g/L,enzyme concentration of 2000 U/mL,temperature 70~oC,pH3.97,while the taste of modified product was improved to certain extent.Using cellobiose as the donor substrate,the analysis of RA-modified products by cellulase showed that the major five transglycosylation products were isomers of monoglycosyl-rebaudioside A. |
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