Studies On Fermentation Of Micrococcus Luteus-Y2 Producing 1-deoxynojimycin In Mulberry

1-deoxynojirimycin(DNJ) is anα-glucosidase inhibitor and could lower blood glucose efficiently. In ancient China, the root of mulberry tree was thought to be an excellent treatment for Diabetes. The modern science now proved that DNJ is the main active constituent of the mulberry tree lowering the blood glucose. View of that, former studies has determined the DNJ concentration from these materials as follows: different mulberry varieties, different mulberry positions, different leaf positions, different seasons, different metropolis as well as insects feeding on mulberry. However, there is no conclusion of the exact origin of DNJ in mulberry.Now it becomes an important direction of researche works that obtained to active compound was extracted frome Chinese crude drug and plant endophyte. In our former studies endophytes producing DNJ stably, Y1 Stenotrophomonas maltophilia and Y2 Micrococcus luteus, have been isolated. Further researches were as follows: the determination Physiology and biochemistry indicator and bionomics of Y2, the optimization of Y2 fermenting conditions, the initial identification and characterization of DNJ produced by the endophytes, theα-glucosidase inhibition activity of fermentation crude extract. At the same time, We determined the DNJ content of germ free mulberry. The main results are as follows:1 The determination Physiology and biochemistry indicator and bionomics of Y2. Contrast with Micrococcus luteus, the characteristic of Y2 is similar to it. Strain Y2, inoculating it, enter logarithmic growth phase in 36 h, stationary phase is 36 h~60 h, enter senescence phase after 60 h. The suitable temperature is 25℃~35℃, and minimum 4℃. maximum 40℃. The suitable pH is 6.5~8.0, and the most suitable one is 7.0. At neutral and alkaline conditions, it could grow well, and strong salt tolerance.2 The effects of environmental conditions and nutrition of strain Y2 producing DNJ were studied through the cultivation in shake-flask fermentation.The optimal shaking flask fermentation conditions were confirmed by one-factor-at-a-time experiment.The optimized fermentation conditions for DNJ production were: inoculum volume 2%(v/v), solution amount 50 mL/250 mL, fermentation temperature 30℃, initial pH 7.0, fermentation time 30 h.Based on the results above, the effects of nutrition on production of DNJ were studied involving the optimal carbon, the optimal nitrogen, inorganic salt combination by one-factor-at-a-time experiment. In the optimization of medium, the influences of peptone, maltose, MgSO4, KH2PO4 and NaCl on DNJ production were first evaluated using Plackett Burman design. Among of the test components, the effect of peptone reach extremely significant level and the KH2PO4 shows significant effect on the DNJ production. However, maltose, MgSO4 and NaCl have no significant effects on DNJ production. The path of steepest ascent was used to approach the optimal region of the medium composition. In the last step, the optimal concentrations of test components were determined by central composite design and response surface analysis.The components of optimum medium for production of DNJ were: maltose 20.0g/L, peptone 36.53 g/L, KH2PO4 2.22 g, MgSO4 0.5 g/L, NaCl 4 g/L, demineralized water 1000 mL.Based on the optimal fermentation condition and medium, the DNJ concentration of fermentation broth after RP-HPLC-UV reached 213.18μg/ml, 184.63% higher than that of basal medium.3 The initial extraction, purification of DNJ produced by strain Y2 was studied by Rep-HPLC-UV analysis. Strain Y2 fermentation broth was concentrated, precipitated by ethanol, separated by NKA-9 and duolite, concentrated again, and freezed to dehydration. Determined by RP-HPLC-UV analysis after derivatization, the DNJ concentration was up to 0.337%, enhanced by 5.3 times, the sample product was purified effective.4 Determination of theα-glucosidase inhibition activity of fermentation crude extract byα-glucosidase inhibition model in vitro. The results are as follows: The sample products both better than acarbose have effectα-glucosidase inhibition activity. The IC50 of strain Y2 is 111.58 mg/L, and Y1 is 2307.56 mg/L. Its activity is positively relative with concentration in certain range, and exhibits dose-dependent inhibition. 5 Determination of the germ free mulberry DNJ concentration. The germ free mulberry were detected with bacteria medium and fungus medium, and were proved no parachorium endophyte. Compared with mulberry growing in field, the DNJ concentration of germ free mulberry are equivalent, 0.197%. The DNJ production depended on not only endophyte but also mulberry.