Citric Acid-degradation And Tolerance Of High Concentration Of Citric Acid By Issatchenkia Terricola

Issatchenkia terricola WJL-G4 was used as the research object,to investigate the optimal conditions for degradation of citric acid and the cell physiological characteristics of citric acid tolerance,and to provide more theoretical basis for the biological acid reduction of juice and its processed products.In the medium with citric acid as the sole carbon source,the growth characteristics of the strain,the best conditions for degrading citric acid and its tolerance were investigated,and the best acid-reducing medium and conditions were determined.Different kinds of carbon sources were added as the second carbon source of the culture medium.The carbon source and the changes of organic acid types and contents of the bacteria under the dual-carbon source mixing condition were studied,and the way to degrade citric acid was preliminarily determined.Finally,with the help of different concentrations of citric acid fermentation and culture simulation system,the stress of high concentration of citric acid on the terrestrial Isa yeast WJL-G4 and its stress phenotype change mechanism were studied.The main conclusions obtained are as follows:Using Issatchenkia terricola WJL-G4 isolated and obtained in our laboratory as the experimental strain,in the medium with citric acid as the only carbon source,the acid reduction rate and growth were used as the judgment indicators to select the citric acid concentration,nitrogen source type and concentration,inorganic salt type and concentration,and used single factor test to screen temperature,liquid volume,inoculation volume,shaker speed,culture time,and then used orthogonal design experiments to further optimize the deacidification conditions of Issatchenkia terricola WJL-G4.The results showed that:the best formula for deacidification was:citric acid concentration 10 g/L;the best nitrogen source was yeast extract powder,and the addition amount was 5 g/L;the best inorganic salt was magnesium sulfate,and the optimal concentration is 0.001 mol/L;the deacidification conditions were screened at a temperature of 28?,a filling volume of 40 mL/250 mL conical flask,an inoculation volume of 1%(V/V),a rotation speed of 160 r/min,and time at 12 h,the deacidification rate was 91.82%,which was four times higher than that before optimization,and reduced energy consumption and cost savings.In addition,the maximum mass concentration of the strain that can tolerate SO2 is 8 mg/L,and the tolerable volume fraction of alcohol is 5%(V/V).Using glucose,sucrose,fructose,succinic acid,and malic acid as other carbon sources,they were added to the medium with citric acid concentration of 10 and 20 g/L,respectively.The utilization of various carbon sources and the changes in the types and contents of organic acids of Issatchenkia terricola WJL-G4 under different dual-carbon source conditions.The results showed that:under the mixed culture conditions of glucose,sucrose and fructose and citric acid,when the culture time was 0-24 h,the utilization rate of citric acid by Issatchenkia terricola WJL-G4 was significantly different from that under pure citric acid conditions.The utilization rate of sugars by Issatchenkia terricola WJL-G4 was greater than 70%,which was also greater than the utilization rate of citric acid by Issatchenkia terricola WJL-G4.When the culture time was 48 h,the sugar and acid were all consumed,indicating that the bacteria preferentially used glucose,sucrose and fructose.However,citric acid was partially consumed at the same time;when the carbon source of added sugars was exhausted,citric acid would continue to be metabolized as the carbon source,and the degree of acid reduction could be controlled by controlling the incubation time.For succinic acid and malic acid as additional carbon sources in dual-carbon source fermentation,the addition of succinic acid and malic acid carbon sources affected the utilization of citric acid by Issatchenkia terricola WJL-G4 in the early stage of fermentation during 0-24 h.However,when the culture time was 48 h,Issatchenkia terricola WJL-G4 also consumed citric acid and reach the purpose of acid reduction.This provided theoretical guidance for the biological acid reduction process of citric acid-type fruits and their processed products,but the bacteria degrade lemons.The mechanism of acid remains to be further explored.With the help of scanning electron microscope,transmission electron microscope,fluorescence microscope and other instruments,observed the morphological structure and cell membrane permeability changes of Issatchenkia terricola WJL-G4 under different citric acid concentrations,and then used cell membrane fatty acids,intracellular and extracellular pH,and antioxidant properties.Sex and other indicators were used to explore the stress characteristics of strains tolerating high concentrations of citric acid.The results showed that under the culture condition of pH=3.0,when the control group and the citric acid concentration were 20 g/L,Issatchenkia terricola WJL-G4 was ellipsoidal,the cell structure was complete,the vacuoles were clearly clear,and there were no obvious wrinkles and impurities on the surface.In addition,strains had already experienced stress changes when the citric acid concentration is 20 g/L.The intracellular pH,oil,malondialdehyde,superoxide anion,and superoxide dismutase content have all increased,and the types of cell membrane fatty acids have increased,C15:2,?-2,C18:2,?-6,C20:1,?-9 and C18:1,?-9 these four unsaturated fatty acids content increased.At this time,the strain can tolerate the stress of citric acid through its own regulation;When the concentration of citric acid is?80 g/L,the growth of Issatchenkia terricola WJL-G4 was significantly reduced,the cell morphology changed significantly,the vacuole was no longer full and clear,the cell wall became thinner,no longer complete and clear,and the cell structure began to degrade.At this time,the permeability of the cell membrane becomes larger,and the content of oil,malondialdehyde and superoxide anion increases,but the intracellular pH and superoxide dismutase decrease,and the substantial damage caused to the strain exceeds the cell's own stress ability.This provided a theoretical basis for the mechanism of strains tolerating high concentrations of citric acid,and also provided a data reference for biological acid reduction.