| Wine fermentation is a complex ecological and biological process that involves a sequential development of various yeast genera and species.The interactions among different yeasts during the fermentation can affect wine quality.It is well known that nitrogen-containing compounds play a necessary role during yeast growth and metabolism.Therefore,in order to understand the patterns of yeast assimilable nitrogen(YAN)consumption,fermentation characteristics of Chinese indigenous yeasts and commercial strains were firstly investigated under four YAN concentrations.Then,we monitored the population dynamics of selected Saccharomyces cerevisiae NX11424 and commercial yeast UCD522 HO::Kanmx and UCD2610 HO::Kanmx in mixed fermentation(inoculation ratio 1:1),which provided the basis for the study of yeast metabolism interaction during mixed fermentation.Finally,the endo-and exo-metabolites during pure and mixed fermentations were investigated to understand the metabolic characteristics of Chinese indigenous S.cerevisiae strain NX11424 and interactions with commercial yeast UCD522 HO::Kanmx.This study contributed new insights for the control of the alcoholic fermentation and the improvement of wine quality.The main findings are as follows:1.Fermentation characteristics of 8 S.cerevisiae strains and 2 non-Saccharomyces(Hanseniaspora uvarum and Issatchenkia orientalis)were studied under four different YAN levels(433 mg/L,208 mg/L,123 mg/L and 55 mg/L).S.cerevisiae under the four YAN concentrations achieved successful completion of fermentaions.S.cerevisiae showed the hightest fermentation rate,followed by I.orientalis.In addition,the fermentation curve expressed as the accumulated amount of released CO2 during the fermentation differed among the studied S.cerevisiae strains at the same level of YAN.The fermentation rates showed the maximum difference at a YAN concentration of 55 mg/L.The fermentation rate of UCD522(Montrachet)was the fastest,followed by Chinese indigenous yeasts LEF1225 and NX11424,while UCD2610(EC1118)showed the slowest fermentation rate.Fermentation rates showed the minimum difference at a YAN level of 433 mg/L.2.The construction of recombinant strains of UCD522 HO::Kanmx,UCD2610 HO::Kanmx and UCD2610 HO::Hyg,and their fermentation characteristics were studied.Fermentations under YAN concentrations of 433 and 55 mg/L showed that the fermentation rate and yeast population dynamics did not change between the recombinant strains and the corresponding parental strains.During the mixed fermentations of NX11424 with UCD522 HO::Kanmx,or with UCD2610 HO::Kanmx at high YAN level,NX11424 dominated the entire time course of fermentation.The contribution of NX11424 to the mixed fermentation was more pronounced in comparing fermentation profiles.The population ratio of NX11424 to UCD522 HO::Kanmx,or to UCD2610::Kanmx showed a ratio of 1:1 throughout the fermentations under low YAN level,with the exception of the end stage which showed a minor increased ratio.3.Characteristics of NX11424 metabolism.(1)The changes of key biomarkers during the fermentation of NX11424 under high and low YAN concentrations showed a similar trend.The uptake of glucose,myo-inositol and amino acids resulted in the accumulation of glycyl-proline,histidine,glutamin,glycine,glutamic acid,alanine,1-deoxyerythritol and 2-deoxyerythritol inside yeast cells,and the production and secretion of glycerol,trehalose,succinic acid and butane-2,3-diol.(2)The levels of the metabolites during NX11424 fermentation were affected by different YAN concentrations,which resulted in the significant difference in the levels of pyruvic acid,?-oxoglutarate,palmitoleic acid,proline,butane-2,3-diol,citrulline,ornithine,galactinol,citramalic acid,tryptophan,alanine,phosphate and phenylethanol.The central carbon metabolism,amino acids metabolism,fatty acid metabolism,purine metabolism and energy metabolism were significantly affected.NX11424 could utilize proline to produce protein under a low YAN level.The intracellular level of citrulline and ornithine under high YAN concentration was higher than that under low YAN level.NX11424 is more suitable for the fermentation under lower YAN level.4.The difference of intracellular metabolism of NX11424 and UCD522 HO::Kanmx was studied at low YAN level.(1)The levels of endo-metabolites were significantly different between the fermentation of NX11424 and UCD522 HO::Kanmx,and 14 metabolites were identified as key endogenous biomarkers.The levels of palmitoleic acid,glycerol-3-galactoside,glycerol-alpha-phosphate,diglycerol,inosine,glycyl-proline and phenylalanine were higher in NX11424 fermentation,while the concentrations of ethanol phosphate,erythritol,galactinol,cytidine-5-monophosphate,succinic acid,malic acidand 2-isopropylmalic acid were higher during the fermentation of UCD522 HO::Kanmx.(2)Metabolic pathway analysis showed that nine pathways including Glycerolipid metabolism;Citrate cycle(TCA cycle);Nitrogen metabolism;Arginine and proline metabolism;Alanine,aspartate and glutamate metabolism;Glycine,serine and threonine metabolism;Pyrimidine metabolism;Pentose and glucuronate interconversions;and Butanoate metabolism were of significant difference(P < 0.05).The difference of the metabolic pathways during fermenations was transformed from carbon metabolism pathway to amino acid and nitrogen metabolic pathways.5.The metabolites and metabolic pathway analysis during the pure(NX11424 and UCD522 HO::Kanmx)and mixed fermentations at days 2,4 and 6 under YAN levels of 433 mg/L and 55 mg/L were performed.(1)Generally,there was a significant difference between the metabolites at day 2 and at day 4 as well as day 6 during the fermentation,and there was no significant difference between the latter two.(2)In terms of different strains,the metabolites at day 2 showed the most significant difference.Compared with the pure fermentation,the levels of intracellular amino acids and TCA related metabolites decreased during the mixed fermentation under low YAN concentration,while the levels of fatty acids increased.In terms of mixed fermentation under high YAN level,intracellular concentrations of TCA related metabolites decreased,while the concentration of amino acid and EMP related metabolites increased.The comparison of pure and mixed fermentation showed that the metabolic pathways were significantly changed during the mixed fermentation(P < 0.05).S.cerevisiae changed various types of metabolic pathways to cope with the competition between strains at low YAN concentration.Metabolic pathways affected by the interaction between NX11424 and UCD522 HO::Kanmx under low YAN level were determined by nine significant pathways,with the top three of Glycerolipid metabolism;Citrate cycle(TCA cycle);and Alanine,aspartate and glutamate metabolism.However,eight pathways were significantly changed under high YAN level,with Arginine and proline metabolism;Alanine,aspartate and glutamate metabolism;and Nitrogen metabolism showing the most significant differences.(3)The composition of the metabolites during the mixed fermentation at day 6 depended mainly on the contribution of NX11424 under both high and low YAN levels.The levels of tyrosol,phenylethanol,glycerol,butane-2,3-diol and succinic acid under low YAN level were significantly higher than those under high YAN concentration. |
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