Study On The Improvement Of 2-KLG Production In Mixed Culture Fermentation Of Vitamin C

Two-step fermentation method of Vitamin C(Vc)is mixed fermentation composed of acid-producing strain and companion stra:in.The acid-producing strain have the ability to synthesize Vc precursor——2-keto-L-gulonic acid(2-KLG),but the growth was weak and the yield of 2-KLG was low under mono-culture of Ketogulonigenium vulgare(K.v).The companion strain could not synthesize 2-KLG,however,it could significantly promote the growth and 2-KLG production of K.v under mixed culture fermentation of Vc.In order to take the advantages of Vc mixed culture fermentation,domestic and foreign researchers conducted many research and made breakthrough progress on analysis of physiological relationship between two strains,the regulation of fermentation conditions,addition of exogenous substances,breeding and construction of good strains,construction of gene metabolic network model.The physiological characteristics and the complex interaction between the two strains from different Genus restrict the potential of Vc mixed culture fermentation.In this paper,we selected a strain of Bacillus pumilus as a companion strain and based on the understanding of the physiological and the fermentation characteristics of two strains,we conducted the optimization of the addition of L-sorbose and GSH in the Vc fermentation system respectively.The main results are as follows:1.In period of decline,B.subtilis was lytic death,and a large number of alkaline substances were released,which led to the pH increase in fermentation environment.On the other hand,2-KLG lead to the pH decrease produced by K.valgure.Eventually,the pH was stabilized in the neutral with a little base range in the Vc mixed culture fermentation system,which ensuring the normal growth and 2-KLG production of K.valgure.2.The yield of 2-KLG was increased by 30.59%,8.34%in Vc mixed culture fermentation when with B.pumilus as companion strain compared with B.cereus and B.endophyticus as companion strain respectively.This result confirmed that the B.pumilus had stronger promoting ability for 2-KLG production.3.When the concentration of L-sorbose was 6%in the fermentation medium,the average 2-KLG production rate was up to 0.737 mg/(mL·h),and the sugar-acid conversion rate was 81.75%.However,when the concentration of L-sorbose was 8%,the average 2-KLG production rate was decreased,2-KLG accumulation significantly slowed down,the sugar-acid conversion rate decreased by 13.13%,the fermentation cycle extended to 84 h.The results showed that the high concentration of L-sorbose in the culture medium inhibited the 2-KLG production of Vc mixed culture fermentation.4.The staging L-sorbose addition strategy was put forwarded.The initial L-sorbose concentration in the fermentation medium was adjusted to 6%,and the L-sorbose was added to the total sugar concentration of 7%and 8%at 24 h and 48 h respectively.The average 2-KLG production rate,the 2-KLG yield and sugar-acid conversion rate increased by 22.91%,5.36%,5.35%respectively compared with initial L-sorbose of 8%at the end of fermentation point,and the fermentation cycle shortened by 14.29%.The inhibitory effect of the initial high concentration of L-sorbose on Vc mixed culture fermentation was eliminated,and the mixed culture fermentation system showed more strong metabolic activity.5.Cell growth of K.valgure and 2-KLG production were promoted significantly by adding exogenous GSH with the optimal concentration of 0.5 mg/mL to the mono-culture of K.valgure.The concentration of the cells was 1.7 times,4.1 times and 7.6 times higher than that of mono-culture of K.valgure without GSH(the control group)at 24 h,48 h and 72 h respectively.The yield of 2-KLG was 22.23 mg/mL in the mono-culture of K.valgure at 72 h,which increased by 6.6 times compared with the control group.6.Cell growth of B.pumilus was promoted by adding exogenous GSH with the optimal concentration of 1.0 mg/mL to the mono-culture of B.pumilus.The concentration of the cells was 1.14 times,1.58 times and 2.29 times higher than that of mono-culture of B.pumilus without GSH(the control group)at 18 h,42 h and 60 h respectively.7.Cell growth and 2-KLG production were promoted significantly by adding exogenous GSH with the optimal concentration of 1.0 mg/mL to the mixed culture fermentation.the yield 2-KLG was 69.92 mg/mL in the mixed culture fermentation at 72 h,which increased by 10.46%compared with mixed culture fermentation without GSH(the control group).The concentration of B.pumilus was 1.09 times,1.12 times and 1.41 times higher than that of the control group,and the concentration of K.valgure was 1.07 times,1.14 times and 1.05 times of the control group at the 24 h,48 h and 72 h respectively.8.The total antioxidant capacity analysis showed that GSH increased the total antioxidant capacity in the fermentation system.It is speculated that as an antioxidant,the GSH removed a large amount of reactive oxygen free radicals that impede the normal propagation and released the oxidative stress response of K.valgure.