| Lactococcus lactis subsp.lactis is one of the commonly used starters in the dairy industry,and recent studies have shown its potential for application in a variety of fermentation systems.Genomic analysis has been able to predict the potential functional properties of L.lactis subsp.lactis strains,but it still lacks direct guidance due to the lack of corresponding studies in practical application systems.Therefore,the investigation of evolutionary patterns and functional properties of L.lactis subsp.lactis by the whole genome analysis and analysis of the fermentation characteristics of the strains in the fermentation system would provide support for comprehensively understanding of the subgroups of L.lactis subsp.lactis and the metabolism and accumulation of substances during the fermentation of starter cultures.In this study,the average nucleotide identity analysis,homologous gene analysis and pan-/core-genome analysis were performed on the genomes of 75 L.lactis strains,and the subgroups identification was performed by phylogenetic analysis based on all homologous gene sequences.The functional characteristics of the genomes among subgroups were predicted,including genes encoding for glycoside hydrolase(GH),cellenvelope proteinase and bacteriocin.Based on the distribution of GH family genes,we analyzed the ability of 34 L.lactis subsp.lactis strains to utilize 22 carbohydrates under single carbon source conditions,and then studied the lactose and fructoside metabolism characteristics from growth and acid production characteristics,enzymatic characteristics,gene expression level,and correlation between genotypes and phenotypes.Finally,the basic fermentation and metabolic properties of the strains were studied in milk and soymilk systems.The genomic analysis revealed that L.lactis subsp.lactis strains could be divided into two subgroups(environmental and dairy-domesticated groups)in the phylogenetic analysis,and environmental strains were isolated from plants,human feces and raw milk samples,while dairy-domesticated strains were isolated from dairy niches.The two subgroups showed significant difference in carbohydrate utilization,protein hydrolysis and bacteriocin synthesis,as environmental strains had more types of GH family genes and bacteriocin synthesis genes than dairy-domesticated strains,while genes encoding cellenvolope proteases were mainly distributed in dairy-domesticated strains.The analysis of carbohydrate utilization ability of the strains under single carbon source conditions revealed that L.lactis subsp.lactis strains generally had the ability to utilize glucose,fructose,mannose,trehalose,galactose and maltose,and 8 environmental strains had the ability to utilize xylooligosaccharides,fructooligosaccharides,galactooligosaccharide and xylan,and5 of them also had the ability to utilize arabinose.In addition,13 L.lactis subsp.lactis strains had the ability to utilize lactose rapidly,among which 3 strains belonged to the environmental group,while 9 strains belonged to the dairy-domesticated group.The two lactose metabolism pathways were found to have four distributions in L.lactis subsp.lactis by genomic functional prediction,among which the strains containing phosphate transport system(PTS)and tagatose6-phosphate(T6P)pathway had excellent growth and acid production characteristics,fast lactose metabolism rate and significant 6-phosphate-?-galactosidase activity,while the strains only containing permease and Leloir pathway had poor growth and acid production characteristic with a slower lactose metabolism rate,and the strains lacking both pathways could barely metabolize lactose.In addition,three fructoside metabolic pathways distributed in L.lactis subsp.lactis,and pathways with the PTS and major facilitator super family transporter were found to be important for sucrose and fructooligosaccharides metabolism,while the pathway with ABC transporter play a key role in inulin metabolism based on the expression levels of related genes under different fructoside conditions.The metabolomics method combined with basic fermentation characteristics and sensory evaluation was used to comprehensively evaluate the milk fermentation characteristics of the strains.The results showed that strains DSCAB11M15 and DYNDL21-2 had excellent growth and acid production characteristics in milk fermentation,which were able to completely curd after cultured for 8 h.The differential metabolites among the samples were mainly related to the pyruvate metabolism,citric acid cycle and amino acid metabolism pathways of microorganisms,and the important non-volatile and volatile compounds such as lactic acid,acetaldehyde,acetoin,2,3-butanedione and ?-aminobutyric acid were significantly up-regulated,indicating that they could give excellent flavor characteristics and additional functional values to the fermented milk.In addition,the abundance of 3-methylbutanal was significantly upregulated in the fermented milk of strain DYNDL21-2,which give a special caramel flavor in the sensory evaluation.The fermentation characteristics of L.lactis subsp.lactis in soymilk were further investigated,and the results showed that strains DYNDL21-2,DYNDL1-2 and FJNDD18M8 had excellent growth and acid production characteristics,and could completely curd after 6 h of fermentation in soymilk.The further metabolomics analysis revealed that the abundance of hexanal and various furan heterocyclic compounds were significantly downregulated,which could effectively reduce the intensity of beany flavor.In addition,the significant up-regulation of acetaldehyde,acetoin and 2,3-butanedione in the fermented soymilk of strain FJNDD18M8 could improve the cream and buttery flavor.In total,the distribution of genes related to carbohydrate metabolism and proteolysis was associated with the evolution L.lactis subsp.lactis species,which suggested that PTS-T6 P pathway was mainly responsible for metabolism.The strains DSCAB11M15 and DYNDL21-2 with genes encoding cellenvolope proteases promoted the abundances of amino acids after fermentation,while the abundances of metabolites from amino acids also increased as the latter contained the genes encoding ?-keto acid decarboxylase. |
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