The Purification And Biotransformation Of Steviol Glycosides From Leaves Of Stevia Rebaudiana Bertoni And Rubus Suavissimus S. Lee

Stevia rebaudiana Bertoni and Rubus suavissimus S. Lee are sweetplants that contain large quantities of steviol glycosides, e.g. stevioside,rebaudioside C, rebaudioside A and rubusoside. The steviol glycosidesare of high sweetness and low calorie, which can be used in the diary ofthe diabetic, phenylketonuria patients and obese persons. Hence, thesteviol glycosides became the highlight of the additive industry. For now,the steviol glycosides are mainly used as the sweetener.In this paper, the extraction and purification of single steviolglycoside was studied, including the following aspects: The first was theextraction and separation of stevioside、rebaudioside A and rebaudiosideC form the leaves of Stevia rebaudiana Bertoni. The deionized water wasused as the solvent for extraction. The macroporous resin and anionexchange resin were used for purification. As for the extraction andseparation of rubusoside from leaves of Rubus suavissimus S. Lee, waterwas also used. The purification of rubusoside was also resin-based. Thesecond was the purification of stevioside、 rebaudioside C and rebaudioside A from the crude extract using preparative HPLC underHILIC mode, and the purification of rubusoside from the crude extract ofRubus suavissimus S. Lee using preparative HPLC under RP-HPLC mode.The third was the biotransformation of stevioside. The detailed technicalparameters were as follows:1. First，Use HILIC analytical column to analyze steviol glycosidesfrom the leaves of Stevia rebaudiana Bertoni. Use C18column to analyzerubusoside from the leaves of Rubus suavissimus S. Lee.2. The extraction, separation and purification of steviol glycosidefrom the leaves of Stevia rebaudiana Bertoni were as follows: solid-liquidration was1:10, extraction temperature was100℃, extraction time was60min and extracted for three times, the extraction rate was97.23%. Themacroporous resin was used to separate stevioside, rebaudioside C andrebaudioside A from the extraction solution: HP-20resin was selected,sample concentration was6mg/mL,4BV of the solution at4BV/h wasloaded on to the resin, and washed with3BV deionized water, the elutionsolvent was70%ethanol in water and the elute flow rate was3BV/h.Followed was decoloration of the product using D-941anion-exchangeresin. The content of the steviol glycosides in the leaves of Steviarebaudiana Bertoni was8.21%(w/w),100g leaves were extractedfollowed by the procedure mentioned above, and after the treatment ofHP-20, and14.25g crude extract can be obtained at the purity of48.9%. Then the extract was submitted to the D-941resin,10.39g crude extractcan be obtained at the purity of63.7%. The recovery of the resin-basedprocedure was83%. After separated by resins, the crude extract of Steviarebaudiana Bertoni was submitted to preparative HPLC under HILICmode, the condition was as follows: the mobile phase was83%acetonitrile in water; flow rate was10mL/min, detection wavelength was213nm; the loading amount can reached at200mg and79.2mg ofstevioside,7.4mg of rebaudioside C and33.7mg of rebaudioside A can beobtained, at the purity of97.5%,96.8%and97.2%, respectively.3. The extraction of rubusoside from leaves of Rubus suavissimus S.Lee was the same as the Stevia rebaudiana Bertoni and the extraction ratewas97.44%. As for the separation and purification of rubusoside fromRubus suavissimus S. Lee, the HP-20macroporous resin and D941anion-exchange resin were also used, but with different condition. Theextraction was submitted to the low-pressure glass chromatographiccolumn wet-packed with HP-20macroporous resin at the flowingcondition: the concentration of the extract was2mg/mL, the flow rate was3BV/h, the deionized water and30%ethanol were then used to wash theimpurity at the flow rate of4BV/h; then90%ethanol in water was usedas eluent at the flow rate of3BV/h. Then the extract was decolored byD-941anion-exchange resin. The content of the steviol glycosides in theleaves of Rubus suavissimus S. Lee is4.5%(w/w),100g leaves was used for extraction, after the treatment of HP-20,7.31g crude extract can beobtained at the purity of52.1%, after the treatment of the D-941resin,5.22g crude extract can be obtained at the purity of68.6%, Theresin-based procedure recovery is82%. The crude extract of Rubussuavissimus S. Lee was purified by RP-HPLC, the condition was asfollows: C18column (19×300mm,7μm, Waters), flow rate was10mL/min, the mobile phase was75%methanol in water, the detectionwavelength was213nm. The loading amount can reached200mg, and123.4mg of rubusoside was yielded at the purity of97.9%.4. As stevioside has the after-taste bitterness, which restricts its uses.Rubusoside have better flavor but low yield. So, in this article thebiotransformation of stevioside was also studied. A fungus was selectedto hydrolyze stevioside into rubusoside. After the pretreatment of thefermentation medium using HP-20resin, the purity reached to75.44%,and the relatively content of rubusoside, stevioside, rebaudioside C andrebaudioside A were43.8%,20.4%,7.9%,27.9%respectively. On thecontrary, the relatively content of rubusoside, stevioside, rebaudioside Cand rebaudioside A of the substrate were0%,66.72%,11.28%,22%.Hence the relative content of stevioside was decreased, the relativecontent of the rubusoside and rebaudioside A was magnified which leadto the improvement of the taste.