Study On The Fermentation Conditions Of Pyrazine By Bacillus Subtil Is

2,3,5,6-tetramethylpyrazine, also called as ligustrazine, was proved to be active constituent of Rhizoma Chuanxiong. It is widely used in clinical application to cure disease such ascerebral vascular, pulmonary edema, gastric ulcers, nephrosis, and so on. Found in cocoa beans, soy products, guava, tea, mushrooms, shrimp, meat, peanuts, nuts, cheese, liquor, it is considered as an important aroma substance with barbecue aroma. National standard of GB 2760-2007 stipulates that 2,3,5,6-tetramethylpyrazine is allowd to be used of the edible spice.At present,2,3,5,6-tetramethylpyrazine is mainly produced by chemical synthesis method, but this method has always been faced with serious environmental protection issues and low transformation efficiency. These factors undoubtedly hiders the industri-alization of 2,3,5,6-tetramethylpyrazine. By contrast, microbiological fermentation is getting more and more attention with unique advantages such as environment friendly type, mild reaction condition and good controll ability. The key was to find a 2,3,5,6-tetramethylpyrazine-producing strain with prominent fermentation level. In order to screen the excellent strain efficiently, a suitable detection method should be established. A high performance liquid chromatographic method on a column of Hypersil NH2 (300mm×4.6mm,5μm) with an mobile phase containing acetonitrile and water(90:10,V/V) was used for the analysis of ligustrazine in fermentation broth, the flow rate was 1ml/min and the column temperature was 35℃, the UV detection wavelength was 295nm. The chromatography was completed in just 10 min.The method was proved to be a rapid, effective technique to determine the 2,3,5,6-tetramethylpyraz-ine in fermentation broth.In this study, taking Jingzhi sesame flavor liquor medium temperature Daqu as separation materials, VP qualitative tests were combined with metabolite quantitaty. Ultimately, a 2,3,5,6-tetramethylpyrazine-producing strain was isolated. According to the morphology, physiological and biochemical characteristics and 16S rDNA gene sequence, the strain was identified as Bacillus subtilis. Using the one-factor experim-ental design and orthogonal experiment, the optimum culture medium and culture cond-ition were ascertained(glucose 80g/L, peptone 30 g/L, yeast extract 10 g/L, ammonium dibasic phosphate 30g/L, dipotassium hydrogen phosphate 4 g/L, initial pH6.5, liquid volume 50mL/500mL, inoculation amount 5%, shaking speed 200r/min, culture temp- erature 37℃). When incubation time was 60h,40g/L ammonium dibasic phosphate was added into the broth. Under the optimal culture condition, the maximum 2,3,5,6-tetram-ethylpyrazine accumulation achieved was up to 20.168g/L in the end of fermentation.This study take the flocculation-cooling crystallization and recrystallization pro-cess to extract 2,3,5,6-tetramethylpyrazine from fermentation broth, then crude product was obtained by cold air drying. The procedure has the advantages of simple operation, high yield, unsophisticated process, low cost and environmentally friendly. The best dosage of natural flocculant determined was 0.8g/L, but the loss rate of 2,3,5,6-tetramet-hylpyrazine is simply 0.104%.So the loss rate of 2,3,5,6-tetramethylpyrazine can be considered as nary, but 92.9% cell and 78.4% protein were removed, edulcoration is fairly excellent. Crystals purified by recrystallization are pure and transparent, white needle. Yield of 2,3,5,6- tetramethylpyrazine throughout the whole separation process was 87.20%. The purity of the product was up to 99.94% and had totally met the commercial grade.