Application Of Magnetically Immobilized Edible Fungus For The Biotransformation Of Panax Notoginseng Saponin Rb1 To Rd And Rg3

Panax notoginseng(Burk.)F.H.Chen,a well-known Traditional Chinese Medicine and healthy herb additive,is the dried roots of the member of-the genus Panax,family Araliaceae.The ginseng saponins(ginsenoside)are the main active ingredients of Panax notoginseng,Which contain wide pharmacological effects.And it was composed of ginsenoside Rbl,Rb2,Rd and Rg3 et al.Among them,Rbl(>20%)is the most abundant compound than other ones but its activity is poor.Although the contents of Rd(<6%)and Rg3(<0.4%)of the total saponins are low,but they showed various bioactivities including the effects on the cardiovascular and the immune system,as well as anti-atherosclerotic activity,anticancer activities,hepatoprotective properties,hypoglycemic or anti-diabetic activities,immunomodulatory properties and so on.The natural saponins of Panax Notoginseng carrry out their important pharmacological activities only when the ginsenosides aer transformed into minor saponins.As many factors affect the transformation in vivo,the transformation efficiency is very low.Recently,microbial transformation has attracted more and more researcher's attention because it possesses many advantages including good selectivity,high-level production,more economically and environmental compatibility.However,there still existed low conversion yields,low reusability and high costs in conventional microbial transformation process.In this work,we firstly used magnetically immobilized microorganism for the biotransformation of Rbl to Rd and Rg3.The higher activity strains among seven kinds of edible fungus were selected for the preparation of magnetically immobilized microorganism.And the abilities of biotransformation were evaluated.In order to increase conversion yields,central composite design(CCD)was used for the optimization of biotransformation conditions.Additionally,the morphological changes of samples were examined by scanning electron microscopy(SEM).Meanwhile,the reusability of the magnetically immobilized microorganism and the enzyme activity were tested.1.The method of screening microorganism for biotransformation of Rd and Rg3 from Panax Notoginseng was established.In this work,we firstly used magnetically immobilized microorganism for the biotransformation of Rb1 to Rd and Rg3.The higher activity strains among seven kinds of edible fungus were selected for the preparation of magnetically immobilized microorganism.And the evaluated by the biotransformation abilities with the content of Rd and Rg3 as an index.Finally,an effective method has been developed and applied for the biotransformation of Rb1 to Rd and Rg3by magnetically immobilized Asp.niger and Asp.oryzae.The yields of Rd and Rg3 reached 41.40 mg/g and 6.29 mg/g which increased 6.81-fold and 3.27-fold to that of untreated control one,respectively.2.The immobilized conditions were optimized.The sodium alginate embedding method of immobilized microorganisms was applied in biotransformation of Rbl.The immobilized conditions were optimized and applied in the production of the target compounds.The effects of Fe3O4 particles concentration,alginate concentration,CaCl2 concentration,concentration of bacterium,diameter of alginate bead on immobilized conditions were evaluated.The optimal immobilization conditions for biotransformation of Rbl were as follows:Fe3O4 particles 0.25%,alginate concentration 7.0%,CaCl2 concentration 2%,concentration of bacterium 7.4 log cfu/mL and alginate bead diameter 5.0 mm.In the stability experiment,the residual activity of magnetically immobilized microorganism(83.8%)was higher than that of non-magnetic immobilized microorganism(78%)after 15 runs.The recycling experiments demonstrated that magnetically immobilized microorganism can provide a promising technique for improving not only the productivity of Rd and Rg3,but also pharmacologically active substances.Therefore,the magnetically immobilized microorganism could be applied easily in industrial production and food industry with higher reusability.3.The fermentation conditions of biotransformation with magnetically immobilized microorganisms were optimized.In this study,the innovation method was found that magnetically immobilized microorganisms technology was combined with biotransformation technology.The fermentation conditions of biotransformation with magnetically immobilized microorganisms were optimized.After that,the response surface analysis was applied to obtain the optimum fermentation conditions.The optimized fermentation conditions achieved with RSM were as follows:pH 6.5,temperature 32 °C,time 48 h,wet weight of microorganism beads 0.8 g/g material and ratio of liquid to solid 20:1 mL/g.The yields of Rd and Rg3 reached 41.40 mg/g and 6.29 mg/g which increased 6.81-fold and 3.27-fold to that of untreated control one,respectively.This study demonstrated a promising simple and environmental-friendly alternative technology that can be applied for the production of valuable natural production in industrial and healthy industry.SEM was used to examine the morphological changes of P.notoginseng samples before and after treated with magnetically co-immobilized Asp.niger and Asp.oryzae.The destruction degree of cell wall by magnetically co-immobilized Asp.niger and Asp.oryzae was obvious than that of non-magnetic immobilized microorganism.Magnetically immobilized have a large specific surface and superparamagnetic properties,which can enhance the activity of microorganism and stimulate it continuously produce more hydrolytic enzymes.SEM demonstrated that vestured pits and cell walls of samples were destroyed obviously which was beneficial to the Rb1 biotransformation and target Rd and Rg3 release.Under the optimal fermentation process,the combination of magnetic nanoparticles and biotransformation technology can more effectively obtain the target products Rd and Rg3.It possesses many advantages including low cost,high production efficiency and environmental protection.Moreover,the immobilized microorganism can be facilely separated from the reaction mixture and can be reused for reducing cost from the perspective of industrial applications.These results proved that this new magnetically immobilized microorganism could be applied for industrial production and pharmaceutical industry with good efficiency.