| Panax quinquefolius is a perennial medicine of Araliaceae,which has the effects of tonifying vital energ y,protecting liver and kidney,anti-inflammation,analgesia,prevention of cardiovascular and cerebrovascular diseases and so on P.quinquefolium is similar to ginseng in appearance and is used in medicine because of its roots.Modern medical and pharmacological studies have shown that ginsenosides are the main active components,among which rare ginsenosides play an irreplaceable role.It has become a hot research direction to convert ginsenoside Rb1,which is rich in American ginseng,into rare ginsenosides with higher medicinal value.Microbial transformation of ginsenosides is the hydrolysis of glycosyl groups on the side chain of ginsenosides byβ-glucosidase secreted by microorganisms.It mainly acts on C-3 and C-20 sites of diol ginsenoside type and C-6 and C-20 sites of triol ginsenoside type.The main purpose of this study is to screen the microorganisms that can convert panaxadiol-type ginsenoside Rb1 into rare ginsenoside C-K,and analyze its transformation path and conditions,so as to provide a theoretical basis for the production of high value-added American ginseng products.The soil samples used in this stud y came from the rhizosphere soil of P.quinquefolium collected from the American ginseng plantation in Wendeng District of Weihai.The strains capable of transforming ginsenoside Rb1 were screened by microbial screening and saponins transformation experiments.After the DNA of the strains was being extracted,the strains were identified by 16sr DNA and phylogenetic tree.After that,the single factor experiment was carried out to determine the best transformation interval of each condition,and then through the response surface experiment,the best transformation condition was determined.The results are as follows:1.26 strains were isolated from soil,of which 13 strains could secreteβ-glucosidase.Ginsenoside Rb1 could be transformed into rare ginsenoside Compound K by two strains S’4 and S’6.2.Through analysing of 16s r DNA and phylogenetic tree analysis of these strains,it was determined that strain S’4 was Dyella jiangningensis SBZ3-12 and strain S’6 was Cohnella thermotolerans CCUG 47242.3.Through the single factor experiment on the transformation of saponins,the results showed that the transformation efficiency of the two strains was the best at 37℃,the optimal transformation p H range of strain S’4 was 5～6,and the optimal transformation p H range of strain S’6 was 6～7.The optimal solution concentration range of strain S’4 was 1×10~9 CFU/m L～1.5×10~9CFU/m L,and the optimal solution concentration range of strain S’6 was 6×10~8CFU/m L～9×10~8CFU/m L.The optimal transformation time range of strains S’4and S’6 was 6～8 days.4.Through the regression equation obtained from the response surface experiment,the best transformation conditions of strain S’4 were determined as follows:the transformation temperature was 37℃,the strain concentration was 1.5×10~9CFU/m L,the p H value was 5.6,and the time was 8 days.The theoretical conversion rate of ginsenoside C-K was 69.1%,the actual conversion rate of ginsenosid C-K was 66.25%.The optimal transformation conditions of strain S’6 were as follows:the transformation temperature was37℃,the strain concentration was 9.0×10~8CFU/m L,the p H was 6.4,and the time was 7.5 days.The theoretical conversion rate of ginsenoside C-K was 82.3%,and the actual conversion rate of ginsenosid C-K was 78.36%.5.Through thin layer chromatography and high performance liquid chromatography,the transformation pathways of strain S’4 and strain S’6were determined to be Rb1→Rd→F2→CK.The two strains with transformation ability obtained in this study can efficiently convert ginsenoside Rb1 into ginsenoside C-K,which has a good application prospect and provides a more feasible method and theoretical basis for the industrial production of ginsenoside C-K. |
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