| Ginsenoside compound K (C-K) is a kind of protopanaxadiol ginsenosides with one glucosemolecule at the C-20position of aglucone and its content is extremely low in ginseng. It has previouslybeen shown that ginsenoside C-K is the main derivative of protopanaxadiol ginsenosides metabolizedby human intestinal flora. This metabolite is considered as an active substance that is absorbed into thebloodstream and plays medicinal activity.In this paper, we transform the total saponins extracted in stems and leaves of Panax ginseng with10anaerobic bacteria, from which we screened a specific strain that can metabolize protopanaxadiolginsenosides to compound K. The results were detected by TLC, HPLC and MS. We also optimizedfermentation medium and conditions to raise ginsenoside C-K yield through single factor experiments,Plackett-Burman design and response surface methodology. The main results are shown as follows:1. Selected10anaerobic bacteria (7of them have strong ability to degrade cellulose and the otherswere enriched from different ginseng soil) to transform total saponins via liquid state fermentation.Based on the size and color of spots of TLC analysis, strain2#and6#were considered having strongesttransformation ability. Based on HPLC-MS analysis, stain2#and6#can metabolize total saponins toproduce ginsenoside C-K.2. Considering cell growth, ginsenoside C-K yield and medium costs comprehensively, we usedsingle factor experiments and found that the best carbon source of strain2#is glucose and its optimumconcentration is2.0g/L to8.0g/L; the best nitrogen source is yeast extract and its optimumconcentration is3.0g/L to5.0g/L; the best C/N is2:1and improving C/N is favourable to cell growthand to reducing C/N is favourable to ginsenoside C-K yield; added vitamin into fermentation medium isfavourable to cell growth and ginsenoside C-K yield, but there is no obvious impact to adding traceelements.3. Plackett-Burman experiment was used to screen the9fermentation medium components.Though ANOVA and regression analysis, for2#strain, magnesium chloride, ferrous sulfate and yeastextract were the main factors that affect the production of ginsenoside C-K. Box-Behnken experimentwas used to optimize the concentration of the3factors. Though quadratic regression analysis andANOVA, we built a model fitting equation. Computing first-order partial derivatives of the equation, weachieved an optimal fermentation medium program: magnesium chloride0.63g/L, ferrous sulfate1.53mg/L, yeast extract3.84g/L. Under the optimal program, ginsenoside C-K yield could inrease to(163.2261.141) μg, and more50.26%than before. Though plotting response surfacce and contourplots, we found the interaction effect between ferrous sulfate and yeast extract is significant, and theinteraction effect between magnesium chloride and yeast extract took the second place. Therefore, theconclusion is that yeast extract is the most significant factor effecting ginsenoside C-K yield. 4. The best inoculation amount of2#strain was7%(v/v), and the optimal total saponinsconcentration was1.2mg/mL to2.0mg/mL. Based on the further studies of cell growth law, glucosemetabolism and ginsenoside glycosidase synthesis law, we optimized the fermentation conditions ofstain2#and presented a strategy about controlling the temperature and pH of the fermentation process,which is in the pre-fermentation (0to18h) control pH6.5, temperature40℃, and during18h to24hcontrol pH7.0, temperature30℃, after24h, control pH7.5, temperature30℃. This strategy canshorten the fermentation period of stagnation, implement higher cell specific growth rate andgensenoside glycosidase synthesis rate, finally improve ginsenoside C-K conversion rate and shortenthe fermentation cycle.5. Though UPLC analys is, we studied the conversion process of ginsenoside Rb1by strain2#andverified that its main metabolic pathway was Rb1â†'Rdâ†'F2â†'C-K. Meanwhile, the molar conversionrate of ginsenoside C-K was60.90%. |
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