| Lactic acid bacteria are widely distributed in nature,and the metabolic products of its fermentation include various organic acids(lactic acid,acetic acid,formic acid)and ethanol.Lactobacillus and its metabolites are widely used in the food industry,such as important flavor substances,fermentation agents of fermented dairy products,meat products,pickles and wine.In this thesis,the metabolic network models of four kinds of lactic acid bacteria were constructed by means of fermentation experiment and bioinformation database query.The metabolic characteristics of different lactic acid bacteria were compared by metabolic flux analysis.The research results are as follows:In this work,Lactobacillus bulgaricus 1.2717?Streptococcus thermophilus 1.2718?Lactobacillus plantarum 1.246 and ctobacillus panis 1.3924 were fermented,their OD(600nm)and pH value were measured,so as to study their growth curve and acid production capacity.The results showed that,after entering the logarithmic growth period in 8-28 h,Lb.1.2717,St.1.2718 and Lp.1.246 were found,Meanwhile,the activity of the strain was good,the reproduction ability was strong,and the acid production performance was good.Lp.1.3924 entered logarithmic growth for a long time at 4-12 h,at this time,the strain had strong reproduction,good activity,and good acid production performance.Under the same culture condition,the OD(600nm)value and pH value of four kinds of lactic acid bacteria were different.Lp.1.3924 had a shorter delay period and enters the logarithmic growth period first,so its pH value first dropped,while after entering the logarithmic period,Lp.1.246 had the fastest increment rate.However,the lowest pH value exerted in Lb.1.2717 and St.1.2718.By UPLC,LC-MS/MS and HPLC,the supernatant of four lactobacillus was determined.The column was Agilent diamonsilc18(2)(250 * 4.6mm,5 ?m),the mobile phase was 0.1% phosphoric acid water: methanol=95:5,and eluted with equal degree.The detection wavelength was 210 nm,the flow rate was 1mL/min,and the column temperature was 30?.Six kinds of organic acids present a good linear relationship in the set concentration range,the correlation coefficient is in the range 0.992220~0.999061.By comparing the retention time of the organic acid peak of the supernatant with that of the organic acid standard,the kinds and contents of organic acids,such as lactate,malate,fumatate,?-ketoglutarate,succinate and citrate,were determined.The central carbon metabolism network information of four kinds of lactic acid bacteria was extracted by the KEGG database and BRENDA integrated enzyme information system.After analyzing the intracellular EMP pathway,TCA cycle,oxidative phosphorylation and other pathways,the central carbon metabolism network models of four kinds of lactic acid bacteria were constructed by MetaFluxNet 1.8 software.The metabolic flux distribution of four kinds of lactic acid bacteria was analyzed and compared by metabolic flux analysis.Assuming that the flux of metabolic end products of the four lactic acid bacteria was consistent,the distribution characteristics of metabolic flux in the glycolytic pathway,tricarboxylic acid cycle and pentose phosphate pathway were discussed respectively.Based on this,it was determined that the key nodes in the metabolic network were g6 p,f6p,pep,pyr,oxa and akg.By establishing a metabolic flux distribution chart for fixing the concentration of all metabolites except lactic acid,it was found that the production of lactic acid had a great change.Lb.1.2717 produced the most lactic acid,which was 118%,128%,and 120% of St.1.2718,Lp.1.246,and Lp.1.3924,respectively.Compared with St.1.2718,Lp.1.246,and Lp.1.3924,the growth rates were 18.9%,27.5%,and 20.3%,respectively.The metabolic characteristics of four lactic acid bacteria were further analyzed and compared,and the result showed that the lactic acid production was inversely proportional to the OD value of the lactic acid bacteria. |
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