Study On Synthesis Of Genipin By Microbial Fermentation

Genipin is a kind of non-toxic ring ene ether terpenoids substances, easily soluble in methanol, ethanol, ethyl acetate, acetone, etc. It has obvious effects used for the treatment of inflammation, jaundice, hepatic disorders, lipid peroxidation, hypertension, defaecation, et al. Genipin also is a kind of excellent natural biological crosslinking agent, has the advantages at biocompatibility, cell low toxicity, resistance to degradation ability and application field widely. The concentration of genipin is very low in Gardenia fruits, only about 0.005%～0.01%. It is difficult to extract genipin from gardenia fruits directly. And it could be obtained through the method of enzymolysis, namely using theβ-glucosidase hydrolyze geniposide into genipin. But it is failed to realize industrial production because of higher cost.A great deal of genipin could be obtained through the method of microbial transformation at the conditions of normal temperature and atmospheric. It can protect the effective components from being destroyed, but it also can simplify the production process, reduce the cost and improve the yield. So the microbial transformation is one of the best ways to transform geniposide. And it will change to genipin, improve effect, reduce the toxicity and enlarge the adaptability of the drug.This paper studies the whole process of producing genipin by the method of microbial fermentation, including strain breeding, fermentation and converting conditions optimization and product purification.There are those works and main results of this study include:1. By preliminary and secondary screening, a strain with high activity ofβ-glucosidase named FYS-9 was selected. It was identified as Aspergillus awamori according to its morphology characteristics and 18S ribosomal DNA gene sequence analysis.2. Through mutating the spore and protoplasts with UV, a mutant 70C-3 was screened. The enzyme activity of 70C-3 was 36.15 IU/mL, with an improvement of 39.4% comparing to the FYS-9. In order to enhance the enzyme activity of the strain 70C-3, the fermentation medium were optimized by single factor and orthogonal experiments. That is, bran+corncob (1:1) 3%, (NH4) 2SO40.5%, KH2PO4 0.2%, tween80 0.1%, MgSO4·7H2O 0.05%, trace ionic liquid 1.5 mL/L. The enzyme activity of 70C-3 was reached 45.16 IU/mL under the optimized fermentation medium.β-glucosidase enzymatic properties of Aspergillus awamori 70C-3 were also studied. The optimum temperature for the enzyme is 60℃, and the optimum pH is 5.0.3. The fermentation conditions were optimized. That is,30℃,150 r/min, convert for 24 h with 1.5% geniposide, the final genipin yield was 8.5 g/L and the molar conversion rate of geniposide reached 97.7%.70C-3 was cultured at 30℃,170 r/min, with 6% Gardenia powder, for 96 h. The maximum conversion of geniposide reached 116.36%.4. H-103 macroporous resin was used to separate genipin and the flow rate was 2 mL/min. The genipin was eluted by 60% alcohol solution. The purity of genipin was 98.2%, and the yield of genipin in the whole separation process reached 77.8%. The combinative method of HPLC, MS, NMR and IR was used to identify the genipin.