Mechanisms On The Oxidative Stress Relieving Of Ketogulonicigenium Vulgare By Rhodotorula Mucilaginosa In Vitamin C Mixed Culture Fermentation

The“two-step fermentation method”is the main method for the production of vitamin C?Vc?in China,in which the second step is conducted by acid-producing strain of Ketogulonigenium vulgare?K.vulgare?and some Bacillus spp.as companion strain to produce 2-keto-L-gulonic acid?2-KLG,the precursor of Vc?.Researches have confirmed that the growth of K.vulgare without companion strain is weaker and 2-KLG production is lower.However,with mixed culture fermentation companied by Bacillus spp.K.vulgare grows well and the yield of 2-KLG is higher.Some studies have shown that in the process of Vc mixed culture fermentation,the metabolites released by the companion bacteria promote the growth and 2-KLG of K.vulgare.According to research advances,it is speculated that the problem of weak growth and low 2-KLG production caused by K.vulgare alone is due to the disturbance of ROS metabolism and oxidative stress in K.vulgare,and metabolites released by companion bacteria may relieve K.vulgare oxidative stress resulting in promoting its growth and 2-KLG production.In this study,Vc mixed culture fermentation system consisting of a new companion strain of R.mucilaginosa A8?R.m?and K.vulgare?K.v?was used as the research object.Firstly,the initial fermentation inoculation ratio of R.m and K.v was optimized.Then,the physiological characteristics of R.m and K.v were analyzed,and then the conditions of the mixed culture fermentation were optimized.Secondly,in the mixed culture fermentation system and mono culture of K.vulgare fermentation system,the antioxidant capacity of K.vulgare was studied.Finally,based on the electron flow in K.vulgare cells,the reasons for the weak growth and low 2-KLG of K.vulgare alone were analyzed,and the analysis of how R.mucilaginosa A8 relieved the oxidative stress of K.vulgare was analyzed.The mechanism of interaction between the two strains of R.m and K.v provides a theoretical basis and new ideas.The main findings are as follows:?1?A new companion strain was isolated from mud of Longwei Lake in Dongling,Shenyang.The morphological identification and molecular identification confirmed that the strain was Rhodotorula mucilaginosa,which was named as R.mucilaginosa A8.By the same initial inoculum concentration(OD₆₅₀ value)of the two strains of R.m and K.v and the initial inoculum?2 m L?of K.vulgare,the inoculation amount of R.mucilaginosa A8 was sequentially increased to produce 2-KLG from the mixed culture fermentation,and the optimal initial inoculation ratio of R.m and K.v was 1:3.33?R.m:K.v?.The growth of R.mucilaginosa A8 was in the lag phase within the first 6 h,logarithmic growth phase within6-18 h,stable phase within 18-48 h,and decline after 48 h,repectively.The growth of K.vulgare showed fluctuating change within the first 52 h,stable period within 52-76 h and decline after 76 h.When K.vulgare was fermented without companion of R.m,2-KLG accumulation was extremely slow;while K.vulgare fermented with companion of R.m,2-KLG accumulated slowly within 0-12 h;accumulated rapidly within 12-52 h;accumulated no longer after 56 h.During the whole fermentation period,the ORP value of the single strain of K.vulgare system fluctuates little and stays between-39 m V and 16 mV,while the ORP value of the mixed culture system fluctuates to a large extent and remains between-277 mV and-26 mV.In the rapid accumulation stage of KLG within 12-52 h,the average ORP value of the mixed culture system was 14.6 times lower than that of the single strain of K.vulgare system.In the mixed culture system,the proportion of two strans of R.m and K.v was maintained between 1:4 and 1:63?R.m:K.v?during the rapid accumulation of 2-KLG.?2?The response surface method was used to optimize Vc mixed culture fermentation conditions for producing 2-KLG by K.vulgare companion with R.mucilaginosa A8.The optimum fermentation conditions were as follows:L-sorbose 80 g/L,urea 14 g/L,corn steep liquid?CSL?15 g/L,CaCO₃ 1.58 g/L,MgSO₄ 0.3 g/L,KH₂PO₄ 1.0 g/L,pH 6.2 of unsterilized medium,with the liquid volume of 40/250 mL and the temperature of 23°C.2-KLG production increased from 52.56 g/L to 71.95 g/L after optimizing the fermentation conditions.Conversion rate from L-sorbose to 2-KLG was increased from 60.97%to 83.46%,and fermentation cycle was shortened from 96 h to 56 h.?3?In the culture of K.vulgare,the Fe-S cluster protein gene expression in the electron transport complex I/II/III/VI on the respiratory chain is lower,and the electron carrier CoQ and Cytc gene expression is lower as well.The lower expression of ATP synthase-related genes in energy metabolism coupled with electron transport results in weaker growth of K.vulgare cells.On the other hand,more electrons are leaked from the CoQ substrate at the end of respiratory chain of K.vulgare's,which results in increasing ROS(H₂O₂,O₂^-·,HOO·,and·OH)generation by a series of metabolic pathways and producing autooxidative stress.Because of lower expression levels of antioxidant-related genes?SOD,CAT,and NADPH:quinone reductase gene?in K.vulgare,it cannot synthesize sufficient antioxidants to defense against ROS in K.vulgare resulting in disorder of ROS metabolism and weaker growth of K.vulgare cells.K.vulgare intracellular SDH and SNDH gene expression levels are lower,which leads to K.vulgare lower 2-KLG production.In addition,due to the low expression of intracellular Cytc gene in K.vulgare,there is a selective electronic fluctuation in Cytc?first,Cytc will choose to leak electrons for the removal of intracellular excess H₂O₂;secondly,Cytc will select the normal respiratory chain electron transport,it is used to enhance the energy metabolism coupled with electron transfer;finally,Cytc will choose to transfer the electrons after the dehydrogenation of the substrate by SDH and SNDH in 2-KLG metabolic pathway,respectively,in order to maintain the flow of metabolism?,therefore leads to weaker growth of K.vulgare and lower 2-KLG production.?4?R.mucilaginosa A8 induces the increase of gene expression levels of electron transport-related substances?including NADH dehydrogenase iron-sulfur protein,succinate dehydrogenase flavoprotein subunit,CoQ,cytochrome reductase iron-thixoprotein,Cytc,cells Expression of pigment oxidase,ATP synthase?and?subunits?,antioxidant enzymes?including SOD,CAT and NADPH:quinone reductase?and enzymes?including SDH and SNDH?for 2-KLG production in K.vulgare,to relieve K.vulgare oxidation Stress and promote its growth and 2-KLG production.