| "Two-step fermentation" was developed by the Chinese scientists for Vitamin C industrial producing.There were two strains involved in the fermentation-an companion strain and an acid-producing strain.They each plays an important role in the fermentation process.The interaction between the two bacteria has been studied as a hot spot.In this paper,a new companion strain of ST-1 and Ketogulonicigenium vulgare 25-B-1(K.vulgare 25-B-1)were used as research objects.The physiological characteristics of the companion strain ST-1 and the K.vulgare 25-B-1 were analyzed.The spore formation and germination regularities were explored,and their effects on the 2-keto-L-gulonic acid(2-KGA)yield.The effects of companion strain of ST-1 on the K.vulgare 25-B-1 antioxidant capacity were analyzed by studying the intracellular and extracellular reactive oxygen species(ROS)level in K.vulgare 25-B-1.The expression differences of related genes in K.vulgare were studied.This research provides some theoretical basis and new ideas for analysising the interaction mechanism between the companion strain and the acid-producting strain.The main findings are as follows:(1)The phylogenetic analysis of the companion strain ST-1 was conducted.The phylogenetic tree showed that the strain of ST-1 is homologous to several other Bacillus endophyticus strains,therefore the strain of ST-1 was identified as Bacillus endophyticus and named Bacillus endophyticus ST-1(B.endophyticus ST-1).The homologous relationship between B.endophyticus ST-1 and B.endophyticus Hbe603 is the closest.B.endophyticus ST-1 had a stronger ability to promote K.vulgare producting 2-KGA comparing with the other two companion bacteria of Bacillus subtilis HJ-04 and Bacillus megaterium 25-B.(2)K.vulgare 25-B-1 can promote the growth of B.endophyticus ST-1 and shorten its growth period.The biomass of co-culture B.endophyticus ST-1 at each time point was higher than that of mono-culture B.endophyticus ST-1.The growth of the former peaked at 20 h,48 h and 64 h,which were about 12.7 times,11.9 times and 11.1 times as much as that of the initial biomass,the growth of the latter peaked at 36 h and 64 h,which were about 9.6 times and 11.3 times as much as that of the initial biomass.During the 72 h fermentation,B.endophyticus ST-1 in co-culture system had twice growth periods,the first period lasted for 28 h.the second period lasted for 24h,while the B.endophyticus ST-1 itself had only one complete grow-th period.(3)B.endophyticus ST-1 can promote the 2-KGA production and the growth of K.vulgare 25-B-1.The 2-KGA yield in the fermentation broth of mono-cuture K.vulgare 25-B-1 was 2.7 mg/mL.and in the co-culture fermentation broth was 66.5 mg/mL,which was about 16 times as much as that of the_former.In co-culture system K.vulgare 25-B-1's biomass is more than that in mono-culture system at each time point.The peaking time points of the K.vulgare 25-B-1 acid-producing rate were consistent with that of sporulation of B.endophyticus ST-1.Therefore,it was presumed that the spore-formation of B.endophyticus ST-1 could be a key factor for increasing the 2-KGA production.(4)A comprehensive analysis of the physiological characteristics of K.vulgare 25-B-1 and B.endophytius ST-1 showed that:?B.encdophytius ST-1 experienced 8 h to generate spores when co-cultured with K.vulgare 25-B-1;?In co-culture system,the spore of B.endophytius ST-1 took 4 h to germinate,In mono-culture system,the spore of B.endophytius ST-1 took 8 h during 28 h to 60 h,and 4 h during 60 h to 68 h,it is speculated that B.endophytius ST-1 secreted some substances to promote spore germination,and K.vulgare 25-B-1 can stimulate the secretion of these substance in advance.The results showed that the decrease of pH in culture broth was an important condition for spore activitation,and B.endophytius ST-1 was able to secrete alkaline substance to neutralize the acid partially in co-culture.(5)K.vulgare 25-B-1 in co-culture system has a stronger ability to produce and remove ROS.The ROS level in cells of co-cuture K.vulgare was about 4.6,4.1 and 3.4 times higher than that in mono-culture K.vulgare 25-B-1 at 18 h,24 h and 40 h respectively,and the extracellular ROS of the co-cuture K.vulgare 25-B-1 was about 1.1,1.1,1.2 times than that of mono-culture K.vulgare 25-B-1.The intracellular total antioxidant capacity(T-AOC),superoxide dismutase(SOD)and catalase(CAT)activities of co-culture K.vulgare 25-B-1 were 1.33-36.17,0.93-1.56 and 1.14-1.77 times than that of mono-culture K.vulgare 25-B-1;The extracellular T-AOC,SOD and CAT activities of co-culture K.vulgare 25-B-1 were 2.54-23.5,7.50-23.3,1.56-6.97 than that of mono-culture K.vulgare 25-B-1.At 40 h of fermentation,the extracellular antioxidant capacity of co-culture K.vulgare 25-B-1 was rapidly increased,suggesting that it was related to the release of intracellular nutrients and antioxidant substances of the companion strain ST-1.(6)Studies on the expression differences of the related genes in K.vulgare showed that,the electrons transfer from NADH dehydrogenase to coenzyme Q was inhibited by adding 10?M rotenone to the fermentation medium.which decreased the 2-KGA yield by 10.9%after fermenting 18 h.Meanwhile,the expressions of genes related to 2-KGA metabolic pathway(Sorbose dehydrogenase and sorbitan dehydrogenase),cytochrome c551 and cytochrome oxidase genes were significantly down-regulated,which indirectly indicated that there might be a coupling relationship between the 2-KGA metabolic pathway and the NADH dehydrogenase downstream of respiratory chain;The expressions of genes which coding antioxidant enzymes(superoxide dismutase and catalase)in mono-culture K.vulgare 25-B-1 were higher than that of co-culture K.vulgare 25-B-1,suggesting that the mono-culture K.vulgare 25-B-1 was subjected to oxidative stress,and induced the up-regulation of antioxidant-related gene expression. |
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