Study On The Effect Of Sporulation Of Companion Bacteria On The Growth And 2-KLG Production Of Ketogulonigenium Vulgare In Vc Mixed Bacteria Fermentation

Vitamin C is an indispensable vitamin for human life activities,and is widely used in many industries such as food,cosmetics,medicine and feed.At present,the main method for industrial production of vitamin C(Vc)is the "two-step fermentation method",in which the second step uses L-sorbose as raw material,and uses acid-producing bacteria and companion bacteria to compose mixed fermentation system for producing Vc precursor of 2-L-Gulonic acid(2-KLG).The acid-producing bacterium in the fermentation process is Ketogulonigenium vulgare(K.vulgare,K.v),which have weak growth and low 2-KLG production when cultured alone,but it can convert L-sorbose to 2-KLG;Most of the companion bacteria are Bacillus spp.,which do not have the ability to produce 2-KLG,but can produce spores at the end of growth and under the condition of lack of nutrients.Studies have shown that the sporulation of companion bacteria is essential for the growth and 2-KLG production of K.vulgare.On this basis,this research selects two strains of companion bacteria with certain differences in companion ability.By analyzing the changes of the sporulation process of the two companion bacteria in the mixed bacterial fermentation relative to the single bacterial culture and the effect of this process on the growth and 2-KLG production of K.vulgare,to further study on the effect of the sporulation process of the companion bacteria on K.vulgare in order to provide a new direction for the study of the interaction mechanism between the two bacteria.The main findings are as follows:(1)When K.vulgare 25B-1 was cultured alone,its growth was weak and 2-KLG production ability was very low,the yields of 2-KLG in three different mixed bacterial fermentation systems were: B.s+K.v: 78.43±0.79 mg/m L;B.e+K.v: 69.71±1.26 mg/m L;B.p+K.v: 71.88±1.12 mg/m L,two strains of B.subtilis A9 and B.endophyticus ST-1 with certain differences in companion ability were selected.In the mixed fermentation system,K.vulgare promoted the growth and sporulation of B.subtilis A9 and B.endophyticus ST-1.When K.vulgare 25B-1 was fermented alone,the p H of the fermentation broth was acidic,while in mixed fermentation,B.subtilis A9 and B.endophyticus ST-1 could adjust the p H value of and avoid of p H decrease of the fermentation broth,which promote the large production of 2-KLG.In the single-strain fermentation of K.vulgare 25B-1,the ORP of the fermentation broth was maintained between-57 m V and 26 m V.With the increase of fermentation time,the ORP gradually increased,and the fermentation broth was oxidative.B.subtilis A9 and B.endophyticus ST-1 can reduce the ORP value of the fermentation broth,making the fermentation broth less reductive,which is more conducive to the growth of K.vulgare 25B-1.(2)In the mixed fermentation system,K.vulgare 25B-1 promoted the production of spores of B.subtilis A9 and B.endophyticus ST-1,and the sporulation time of the companion bacteria was shortened and the number of spores increased.The extracellular fluid of B.subtilis A9 and B.endophyticus ST-1 cultured for 12 h,24 h,36 h and 48 h could promote 2-KLG production of K.vulgare 25B-1,the promotion effect is much better at 48 h with a large number of spores production.In the mixed-fermentation system,the activities of 2-KLG production related enzymes SDH and SNDH of K.vulgare 25B-1 were higher than those in K.v fermentation system.With the massive spores production of the companion bacteria,the enzyme activities increased and reached the highest value at 48 h,at the same time,with the spores production of the companion bacteria,the number of viable cells of K.vulgare 25B-1 gradually increased,and the production rate of 2-KLG in the fermentation system accelerated(3)In the mixed fermentation,the expression levels of B.subtilis A9 quorum sensing gene lux S and signal peptide genes phr C and phr A were significantly increased at 12 h,it is speculated that the signal molecule AI-2 and the signal peptides Phr A and CSF may be produced in large quantities at this time,thereby promoting the phosphorylation of the sporulation-related protein Spo0 A.At the same time,the early expression of the sporulation-related genes spo0 A,spo0F,and spo VFA may lead to the increase of Spo0 A content and phosphorylation level,thus making the sporulation time of B.subtilis A9 earlier.The sporulation of the companion bacteria induced the expression of DNA replication initiation-related genes dna G and dna B and the 2-KLG production genes sdh and sndh in K.vulgare 25B-1,which promotes the growth and reproduction of K.vulgare 25B-1 and increases enzyme activities of SDH and SNDH.Therefore,a large amount of 2-KLG accumulated in the system.