Mechanism On Companion Strain Relieving The Oxidative Stress From Acid-producing Strain In Vitamin C Co-culture Fermentation System

Vitamin C（Vc）is an important water-soluble vitamin required by human body and has important application value in the food industry as well.Its derivatives are usually added as antioxidants and nutritional supplements in food.Precursor of Vitamin C,2-keto-L-gulonic acid（2-KLG）can be obtained by microbial fermentation.This method is called two-step fermentation,and the second step of two-step fermentation is co-culture fermentation,which includes Bacillus as a companion strain and Ketogulonicigenium vulgare as an acid-producing strain.The relationship between the two bacteria is mutually beneficial,but the interaction mechanism is not clear.It is known that a variety of Bacillus genus can be used as companion bacteria,but the companying abilities are different.Analysis of the mechanism of companion strain relieve oxidative stress from K.vulgare 25B-1 in co-culture fermentation system,and the mechanism of different ability from variety companion strains,which not only can establish a theoretical and methodological foundation for further use of traditional mutagenesis technique,genetic engineering strategy or metabolic engineering method to enhance Vitamin C production,but also can give significant clues for the study of other complex microbial consortia.The main results of the research were as follows:The physiological properties of B.endophyticus ST-1 and K.vulgare 25B-1 in mono-and co-culture fermentation systems before and after B.endophyticus ST-1 sporulation.Results showed that B.endophyticus ST-1 could stimulate the growth and 2-KLG yield of K.vulgare 25B-1,while K.vulgare25B-1 promoted the sporulation of B.endophyticus ST-1.At the later stage of fermentation,the yield of2-KLG increased gradually,affecting on the growth of B.endophyticus ST-1.The 2,6-Pyridinedicarboxylic acid（DPA）content of B.endophyticus ST-1 in co-culture was higher than that in mono-culture.The pH value of co-culture fermentation was more stable than that of mono-culture fermentation systems,which was beneficial to the growth and 2-KLG yield of K.vulgare 25B-1.The oxidation-reduction potential（ORP）decreased gradually in co-culture fermentation system,and ORP was negatively correlated with2-KLG production.Four sorbose dehydrogenase genes,Kvsdh1,Kvsdh2,Kvsdh3 and Kvsdh4 and one sorbitone dehydrogenase gene Kvsndh1 were acquired from K.vulgare 25B-1.Bioinformatics analysis showed that these water-soluble proteins were more stable,and only KvSDH1 was membrane-bound protein.Tertiary structure of four sorbose dehydrogenase were similar,a calcium binding site and a pyrroloquinoline quinone（PQQ）binding site were predicted in the tertiary structure of KvSDH1,there was also a PQQ binding site in KvSDH4.The expression of sorbose dehydrogenase gene and the enzyme activity in co-culture fermentation were increased highest at 40 h,and then decreased.The sorbitone dehydrogenase gene and enzyme activity increased gradually and peaked at 66 h in co-culture fermentation system.Subsequent results showed that the reactive oxygen species（ROS）of the two strains was significantly increased at 18 h in co-culture fermentation system,and then decreased gradually.The extracellular activity of total superoxide dismutase（T-SOD）,catalase（CAT）,total antioxidant capacity（T-AOC）increased gradually.The expression of oxidative stress-related genes in two strains of different fermentation systems was analyzed.The results showed that,for B.endophyticus ST-1,the gene expression of sodA and katE for direct scaving ROS was increased and the expression of gp and zwf for redox balance was up-regulated.For K.vulgare 25B-1,the expression of grxC,gor,sodB and zwf were increased.B.endophyticus ST-1accelerated growth and caused excessive accumulation of reactive oxygen species in K.vulgare 25B-1,resulting in oxidative stress.Oxidative stress signals could be transmitted to B.endophyticus ST-1.The release of spores and lysing of B.endophyticus ST-1 cells provided a favorable oxidation-reduction state for K.vulgare 25B-1,leading to increase antioxidant capacity.B.endophyticus ST-1 regulates the oxidation-reduction state in Vc fermentation system,thereby enhancing the antioxidant capacities of K.vulgare 25B-1.The proteomics of the companion strain in different co-culture fermentation systems showed that,both B.pumilus A9 and B.endophyticus ST-1 could promote the growth of K.vulgare 25B-1,and companion ability of B.pumilus A9 was better.Pearson correlation coefficient analysis showed that ORP of two fermentation systems was negatively correlated with 2-KLG yield at 0.01 level.Proteomic analysis showed that B.pumilus A9 and B.endophyticus ST-1 in co-culture fermentation system was more potent in terms of glucose metabolism,thus providing more energy and reducing power for cells to provide more adequate intermediate metabolites.In the amino acid biosynthesis pathway,B.pumilus A9 promoted the conversion of glycine and serine,and more synthesis of threonine compared with those of B.endophyticus ST-1,which enhanced the synthesis of glutamic acid and converted it into glutamine.Bioinformatics analysis of the consensus differential proteins of Bacillus showed that B.pumilus A9 was more capable of cooperating with K.vulgare 25B-1,and could be used to carry out glucose metabolism and metabolic ability in both ways.In ribosomal protein pathway,the expression of large subunit ribosomal proteins,L6,L22 and L23of the companion B.pumilus A9 was up-regulated.At the same time,it was found that B.pumilus A9 had better antioxidant ability and better ability to regulate the redox balance of fermentation system.Studies on proteomics of K.vulgare 25B-1 in different fermentation systems showed that the companion ability of B.pumilus A9,whose was strong,lead to the TCA cycle,the metabolism of pyruvate and the synthesis of various amino acids in K.vulgare25B-1 were promoted.B.pumilus A9 enhanced the growth and 2-KLG yield of K.vulgare 25B-1.In the vitamin and cofactor metabolic pathways,the up-regulation of the expression of the 5’phosphate pyridoxamine oxidase,which was associated with vitamin B₆ metabolism,promoted the formation of pyridoxine,facilitated the conversion of amino acids to ammonia,decarboxylation and sulfur-containing amino acids.In one carbon pool by folate pathway,the up-regulation of the expression of related enzymes enhanced the flux of carbon units and provided the necessary material basis for cell growth.Up-regulation of enzymes associated with peptidoglycan synthesis indicated that B.pumilus A9 was more conducive to the synthesis of biogenic peptidoglycan and cell wall formation.Only two proteins related to oxidative stress in K.vulgare 25B-1 were found,which were iron superoxide dismutase and Spx-regulated thioredoxin reductase,both of which were up-regulated.It can be seen that the ability of K.vulgare 25B-1 in clearance of reactive oxygen species（ROS）companioning with bacteria,B.pumilus A9,has been improved,and the ability to regulate the balance of thiols has also been enhanced to eliminate the adverse effects of cell growth factors and metabolism of K.vulgare 25B-1.