Study On Mechanism For2-Keto-L-Gulonic Acid Production Of New Strains In Two-Step Fermentation Of Vitamin C

Two-step fermentation is the method of vitamin C（Vc）production industrily. It isindependent and used widespread by our country. The conversion process from L-sorbose to2-keto-L-gulonic acid（2-KGA）, in vitamin C two-step fermentation is mixed culturefermentation by two kinds of strains including acid-producing strain commonly known assmall strain which grow slow and the productin of acid is low, whereas companion bacteriumcalled big strain which could promote the growth and the acid production of small strain.Many strains could be as companion bacteria, but the mechanism of companion bacteria isstill not clear. The search for cohelper bacterium with high companion ability and theresearch of its bio-effects is significance to vitamin C industry production. The characteristicsof new companion bacterium and the optimization for fermentation conditions are exploredas well as the bio-effects of the companion bacterium. The main results are as follows:（1）An efficient companion strain of Bacillus subtilis A9（B.sA9）, which facilitatingthe growth of Gluconobacter oxydans（G.o）and2-KGA production is screened. Based on thecharacteristic of growth and the bio-effects of B.s A9, the results obtained are as follows: B.sA9without the ability to transform L-sorbose into2-KGA but can promotes the cellproliferation and2-KGA production of G.o. The ability of2-KGA production by new strainsof G.o and B.s A9is obviously higher than that of G.o and B.m2980used in the industrialvitamin C fermentation. The alkaline components released by B.s A9could promot thegrowth and2-KGA production of G.o and help to maintain the pH stability in the mixedfermentation of2-KGA.（2）In order to better promote the coordination between the two strains and make itreach to the best state, the nutrient condition and the culture condition are researched byresponse surface methodology （RSM） and by single factor experiment. The optimalfermentation media are as follows（g/L）: L-sorbose of90, urea of12.2, corn steep liquor of14.2, CaCO3of4.1, MgSO4of0.2. The optimal condition is10mL of liquid culture with15%of inoculum in100mL flask cultivating at temperature29℃. The ability of2-KGA production by new strains G.o and B.s A9is obviously higher than that of G.o and B.m2980used in the industrial vitamin C fermentation. The yield of2-KGA increased by2.45%underthe optimized culture medium conditions comparing with the original medium. Thefermentation time is60hours which is16hours shorter than original condition optimizatingbefore.（3）The bioactive components from the supernatant of Bacillus subtilis A9is isolatedand purified by ammonium sulfate fractionation, DEAE Sepharose Fast Flow ion-exchangecolumn chromatography and Sephadex G-100column chromatography respectively.Themolecular weights of active components are between35kD and66.4kD by ammonium sulfatefractionation. The bioactive elution fraction of the peak A4which promot the activity of SDHis purified from the supernatant of Bacillus subtilis A9by DEAE Sepharose Fast Flowion-exchange column chromatography. The molecular weights of active component of peakA4are between41kD and66.4kD. In addition, the bioactive elution fraction of the peak B3ispurified from the peak A4by Sephadex G-100column chromatography. Two protein stripsare presented with molecular weight45kD and61kD by SDS-PAGE electrophoresis.