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Separation and purification of stevioside directional conversion and the product of sweet tea glycosides

Author: JiangZhongYu
Tutor: ChenYuRu
School: Nanjing Normal University
Course: Microbiology
Keywords: stevioside Chryseobacterium sp. rubusoside biotransformation intracellular enzymes
CLC: S566.9
Type: Master's thesis
Year: 2011
Downloads: 25
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Abstract


Steviol glycosides, the main sweet ingredients in the leaves of Stevia rebaudiana (Bertoni), have two sweet components stevioside (SS) and rebaudioside A (RA). Rubusoside, the main sweet ingredient in the leaves of Rubus suavissimus which is alpine plant, has low yield because of the limitation of geographical conditions. This work could provide a new method to obtain rubusoside(RS) using stevioside biotransfomation, therefore reducing the planting of Rubus suavissimus.The bacillus that could convert stevioside specifically was identified on the basis of morphology features and 16S rDNA sequence analysis, which was ultimately identified as Chryseobacterium sp.(CCTCC NO:M209202). The bacillus could not convert any other glycosides in the steviol glycosides.The conditions of culture and biotransformation were studied. The results showed that the culture medium was 1% cassava starch,1% peptone,0.5% K2HPO4,0.5% KH2PO4,0.2% NaCl and 0.04% MgSO4.The conversion rate could reach 100% after 48 h when the concentration of stevia glycosides was 10 mg/ml, including 7.2 mg/ml stevioside under the condition of pH7.0,37℃, bacteria media volum 50 mL/250 mL and 10% inoculum.The enzyme that could convert stevioside into rubusoside was intracellular enzyme. The bacteria cells were broken by high pressure method. When stevia glycosides was incubated with the enzyme for 12 h under the condition of pH 7.0,45℃, stevia glycosides concentration 10 mg/ml, the conversion rate of stevioside reached 95.0%. It could reach 100% after 15 h. Different surfactants had used for broking the cells. The optimum addition of SDS was 0.2% with 90% conversion rate after 12 h, and CTAB was 1% with 74% conversion rate. The optimum addition of Triton-100 was 2%, which could reach 100% conversion rate after 12 h.The methods of immobilized enzymes and conditions of conversing stevioside were investigated with the sodium alginate as embedding material. The optimum preparation conditions were as follows:the concentration of sodium alginate and CaCl2 solution were 4% and 2% respectively, immobilization time 2 h. The results showed that 10 mg/ml stevioside could be converted to rubusoside by 80% for 2 days under the condition of 45℃, pH7.0,15 g/lOmL immobilized enzyme. The conversion rate maintained more than 80% when the immobilized enzyme recycled 3 times. Several macroporous resins were used to separate and purify the biotransformation product. CN-308 was selected. The results showed that 1BV resins could adsorpt all glycosides included in 10BV biotransformation fluid. RS and RA were adsorpted saturated when the sample fluid was 27.5BV. Desorption solvent was 70% ethanol. When desorption solvent was 1.5BV, maximum concentration of RS was obtained.

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