Study On Mechanism Of Different Companion Strains In Vitamin C Two-Step Fermentation

The mixed fermentation process for producing 2-keto-L-gulonic acid (2-KGA), the precursor of Vitamin C (Vc) transformed from L-sorbose, is the major method of Vc industrial production in China. The mixed fermentation is realized by the cooperation of the two different strains of acid-producing strain and companion strain. Ketogulonigenium vulgare is the acid-producing strain and producing 2-KGA is slowly in culture alone, and the companion strain which could promote the growth of acid-producing strain finish the conversion. There are many candidate strains could be companion strain. However, the mechanism of this phenomenon is not well known. This paper deals with the mechanism for B, megaterium 2980、B.subtilis A9 promoting the growth and the 2-KGA production of K.vulgare in "Two-Step fermentation" of Vc.(1) An efficient companion strain of Bacillus subtilis A9 can be used to facilitate the growth of K.vulgare and the production of 2-KGA. Two optimization models, namely response surface methodology and artificial neural network are built to optimize the medium components for mixed cultures fermentation of 2-KGA. The results indicate that the fitness and prediction accuracy of the ANN model are higher than those for the RSM model. Furthermore, using genetic algorithm, the input space of the ANN model is optimized, predicting that the maximum 2-KGA production of 72.54 g/L would be obtained at the GA-optimized concentrations of the medium components (L-sorbose,92.5 g/L; urea,10.2 g/L; corn steep liquor,16 g/L; CaCO3,3.96 g/L; MgSO4,0.28 g/L). The 2-KGA production experimentally obtained using the ANN-GA designed medium is 71.21±1.53 g/L, which is in good agreement with the predicted value.(2) The bioactive components from the supernatant of B. megaterium 2980 and B. subtilis A9 by ammonium sulfate fractionation, DEAE Sepharose Fast Flow ion-exchange column chromatography and Sephadex G-100 column chromatography. The bioactive components can promote the cell proliferation and 2-KGA production of K. vulgare, and also enhances the activity of SDH. Two protein bands of B. megaterium 2980 are presented with molecular weight 36kDa and 43kDa; At the same time, two protein bands of B.subtilis A9 are presented with molecular weight 45kD and 61kD both by SDS-PAGE electrophoresis.(3) Additions of small exogenous nutrition molecules produce different effects on promoting 2-KGA production. Appropriate amount of glutathione, adenosine 5’-triphosphate, nitrogenous bases and dihydrofolic acid can promote 2-KGA production of K.vulgare. They are mainly to improve the yield of 2-KGA by increasing the nutritional materials of microorganism. But folic acid, coenzyme I and glycerol cannot promote 2-KGA production of K.vulgare. Moreover, the mixed strains of stronger acid-producing ability are particularly sensitive to small exogenous nutrients molecules, higher 2-KGA production.(4) The changes of extracellular amino acids, Vitamin B, oxidation-reduction potential and bacteria concentrations by different companion strains associated with different fermentation time, the results are basically same.The abundant of growth factors are released to the medium from the companion strain during sporulation, including glycine, phenylalanine, leucine, threonine, aspartic acid, isoleucine and pyridoxine hydrochloride. These nutritional components are important to promote the growth and production of acid producing strain. The changes of oxidation-reduction potential in mixed culture system are consistent with the changes of bacteria concentrations during the fermentation process. The change of oxidation-reduction potential in the fermentation system is a comprehensive embodiment of the fermentation process, and it can be used as a parameter for optimizing the fermentation process.(5) Through the comparative study on total antioxidant capacity, total superoxide dismutase, glutathione reductase, the level of reactive oxygen species, mercapto group content, malonaldehyde content, H2O2 content, inhibition and producing superoxide anion free radical content between mono-cultures system and co-cultures system (K.v 25-B-1+2980 system and K.v 25-B-1+A9 system), the results show that a good deal of reactive oxygen species is produced by K.vulgare in two-step fermentation of vitamin C. But it does not have enough ability to clean up the ROS itself. However, the companion strain removes the ROS in the co-cultures system, it means that the companion strain can not only provide the growth factor but also provid a beneficial environment for K.vulgare growth and 2-KGA production. Through the comparative study on T-AOC, T-SOD, GR, ROS,-SH, MDA, H2O2 of two mixed culture system, the results show that the stronger companying ability of companion strain, the stronger ability of resistance to ROS.(6) Based on qualitative analysises of phospholipids in the acid-producing strain, companion strain and mixed cultures strains show that the mixed cultures system of phosphatidyl glycerol, phosphatidyl ethanolamines, phosphatidyl serines and phosphatidyl inositols have been associated with moleculars don’t appear alone in acid-producing strain culture system and the companion strain culture system. At the same time, the cell membrane permeability and ATP activity of the mixed cultures system are stronger than that of alone in acid-producing strain culture system and the companion strain culture system. The mixed strains of 2-KGA production ability is stronger, the more specific phospholipid molecules and the relative contents are also more, the cell membrane permeability and ATP activity are stronger, too. In the process of mixed cultures, with the participation of the companion strain, which makes the cell membrane of the acid-producing strain and the companion strain change and the active transport capacity is enhanced which is beneficial to the material exchange.