Studies On The Mixture Of Mechanisms And New Associated Strains In The Fermentation Of Vitamin C

Vitamin C, as known L-ascorbic acid, is one of the most important water-soluble vitamins. It has been widely used due to its important physiological functions. At present, the huge production of the vitamin C in China has taken up more than 90% of the global yield and more than 80% output is exported. "Two-step fermentation method" is the unique method producing the vitamin C in China: in the first-step fermentation process, D-sorbitol is converted into L-sorbose by the oxidation of Acetobacter melanogenuim; in the second-step mix fermentation process, L-sorbose is transformed into 2-keto-L-gulonic acid (2-KGA). At the last step, the 2-KGA is changed into vitamin C through chemical method. In the second step, the mix fermentation is realized by the cooperation of the two different strains: Bacillus megaterium and Ketogulinigenium vulgare which are commonly called as mega- and micro-bacteria respectively. Now, more than 98% sorbose can be converted into KGA through mix fermentation, but the basic physiological relationship between the two strains is still unclear. In this paper, the intrinsic relationship between the mixed bacterium is studied.The main results are shown as follows:(1)Either B. megaterium or K. valgare can be cultured individually, and each shows the ability of developing colonies and the stability of heredity.(2)It is not B. megaterium but K. valgare shows the ability of conversion from L-sorbose to 2-KGA. And the conversion rate is about 20% after 72 h fermentation.(3)The rate of conversion from L-sorbose to 2-KGA can be increased evidently through the optimization of the mix fermentation conditions: the conversion rate is more than 95% after 48 h fermentation.(4)The effects of the extracellular and intracellular fluid from B. megaterium on the growth and acid production of K. valgare are also studied. The results show that the extracellular fluid, instead of intracellar fluid, from B. megaterium cultured for 24 h has the best effect on the growth and acid production of K. valgare. And the conversion rate is about 23%. These results are not consistent with previous reports.(5)The growth of B. megaterium can be guided by K. valgare. In other words, the growth of B. megaterium tends towards following the track of that of K. valgare, instead of E. coli which is the control in this experiment. This result indicates the complexity of the relationship between the two strains. (6)A substance named as AP1 by us has firstly been selected and has also been verified the promotion on the growth and acid production of K. valgare. Under the addition of 0.1% AP1 which is the optimum content in the experiment, the content of 2-KGA produced by individual K. valgare fermentation for 48 h could reach to 55.05 mg/mL, three times as that under without AP1. Moreover, the conversion rate is about 70% and the viability of K. valgare is more than 1.3 times higher than that of without AP1. These results suggest that the mechanism of the acid production is closely related to the redox.(7)B601, in possession of a bigger promotion to K. valgare than B. megaterium, has been screened from soil. Under the same mix fermentation conditions, the number of its viable bacterium is less than that of B. megaterium, but the number of its spores is more than that of B. megaterium. In the early fermentation, B601 also contributes much to the growth of K. valgare. The B601 fermentation is ended in 44 h, 4 h ahead of B. megaterium, L-sorbose is increased to 10%.(8)The culture conditions and medium of the new culture system of mixed B601 and K. valgare are optimized and the results are as follows: 10% L-sorbose, 0.6% urea, 1.0% corn steep liquor, pH 6.8, temperature of culture 30℃, with 10% of volume, inoculum 8%, age of seed 20 hours. Compared with the previous fermentation, L-sorbose is enhanced by 25%, urea and corn steep liquor are decreased by 50% and 33% respectively, culture temperature is increased by 2, and the fermentation cycle is shortened by 4 h while keeping the conversion rate of 98%.In this paper, the innovation points are as follows:(1)Some new results about the physiological relationship between the two associated strains in mix fermentation have been received, especially the result that extracellular and intracellar fluid from B. megaterium show hardly effect on the acid production of K. valgare.(2)That K. vulgare guide growth of B. megaterium is firstly found.(3)it is firstly found that AP1 can promote the growth and the production of the acid of K.vulgare;(4)B601 is selected from the soil that associated as K. vulgare in the mixed culture, and it can better contribute to the growth and the production of the acid of K. vulgare.