Screening, Identification Of A1-Deoxynojirimycin Producing Bacteria Strain And The Optimization Of Fermentation Conditions

1-Deoxynojirimycin (DNJ) is a piperidine alkaloids which can be used toinhibit glucose absorption，reduce postprandial hyperglycemia, and resist to tumorsand anti-virus. At present, DNJ is mainly taken from Mulberry, but the output is low.Besides, the process of chemical synthesis is complex. Based on this problem, wescreened a bacterial strain producing DNJ, determined the optimization offermentation DNJ which including fermentation conditions and medium. Usingdefatted silkworm powder to got silkworm peptone, we replaced market peptone bysilkworm peptone for larger fermentation test.We screened a bacteria strain from281strains which were isolated frommulberry-park by the agar plate method and reversed-phase high performanceliquid chromatography. Its DNJ yield was approximately80.22mg/L, which wasnamed for WB-1.The bacteria strain WB-1was screened by a series of experiments, includingphysiological, biochemical and molecular identification. The strain surface wasbeige, moist, smooth, neat edges and slightly uplift. The strain which belonged tonon-moving aerobic bacteria, could hydrolyze starch, utilize citrate as the solecarbon source, it also could decompose hydrogen peroxide. H2S producing, gelatinliquefaction, phenylalanine deaminase and methyl red test for WB-1were allnegative. The16srDNA fragments were amplified by PCR method, and retrievedthe NCBI database after sequencing. The sequence had nearest genetic relationshipsto Lysinibacillus sphaericus, so we determined that the bacteria strain WB-1wasbelong to Lysinibacillus. The growth curve of the strain was measured, and itsvarious stages of growth were:0~4h in a lag phase,4~14h in the logarithmic phase,14~22h in a stable period, after22h it entering the decline phase.The single-factor experiments were used to determine the basic ingredientsand conditions in the flask fermentation. The conditions of flask fermentation were:fermentation temperature of30°C, fermentation initial pH of6.5, shaking speed of180rpm, liquid amount of30mL, fermentation time of2d. The fermentation mediaand culture conditions of WB-1producing DNJ were conducted a multi-factor optimization by successively PB and Box-Behnken experimental design. Theoptimization fermentation conditions were as follows:0.5%glucose,0.3%sucrose,0.5%beef extract,0.795%peptone,0.255%MgSO4,0.05%NaCl, the shakingspeed of180rpm, liquid amount of38mL. Under this condition, the yield of DNJwas to184.95mg/L, which was1.31times to the production before fermentation.The silkworm peptone was obtained by enzymatic hydrolysis of defattedsilkworm powder. Adding it with market peptone to culture strain WB-1, we foundthat the silkworm peptone could delay the strain into the decline phase. Usingsilkworm peptone which was made by ourselves instead of market peptone for DNJfermentation, we determined the addition content was0.75%. Larger fermentationtest was carried out by strain WB-1in a5L fermenter. Taking appropriatefermentation broth in regular time, detected the OD600value and DNJ production ofthe fermentation broth, respectively. Then rendered the growth curves of the strainand changing yield of DNJ with different fermentation time when we culturedWB-1in fermentor. The result showed it was time to put the tank when fermentingto50h, at this time we could obtain the higher DNJ production.