Fermentation Characteristics Of Fermented Milk By Lactobacillus Delbrueckii Subsp.Bularicus And Subsp.Lactis

Lactobacillus delbrueckii subsp.bulgaricus and subsp.lactis are commonly used in dairy fermentation.However,the variation in the genome,carbohydrate utilization,fermentation characteristics,and functional characteristics between Lactobacillus delbrueckii subsp.bulgaricus and subsp.lactis have not been systematically clarified.This study focused on the genomic characteristics,the fermentation characteristics,and the blood pressure regulation function of fermented milk between the two subspecies,in order to provide theoretical and application guidance for mining the LAB resources and screening the starter culture strains.In this study,the phylogenetic tree was constructed with the homologous genes of 75 genomes of Lactobacillus delbrueckii.The functional characteristics of the genomes among subspecies were predicted,including glycoside hydrolase(GH)family genes and proteolytic system-related genes.Functional characteristics in the genomes between Lactobacillus delbrueckii subsp.bulgaricus and subsp.lactis were analyzed.The utilization capacity of 19 carbohydrates by Lactobacillus delbrueckii subsp.bulgaricus and subsp.lactis was investigated.Then,the representative strains of two subspecies were selected to compare the growth and acidifying characteristics during fermentation and to analyze the volatile flavor compounds,non-volatile compounds,and polypeptide composition of their fermented milk.Finally,the ACE inhibitory activity in vitro and blood pressure regulation function in vivo of whey of their fermented milk were discussed.The subspecies of Lactobacillus delbrueckii were identified according to the phylogenetic tree.The result of the prediction of GH genes showed that variation of GH gene families existed between different subspecies of Lactobacillus delbrueckii.Lactobacillus delbrueckii subsp.lactis had more GH genes for the hydrolysis of plant carbohydrates than Lactobacillus delbrueckii subsp.bulgaricus.Notably,Lactobacillus delbrueckii subsp.lactis ND02、YNF-5、UB0003、D2M49、D10M169 and D11M188 had unique α-galactosidase genes.The result of the prediction of proteolysis system-related genes showed that no obvious distribution pattern existed among Lactobacillus delbrueckii.However,Cell-envelope proteinase(CEP)amino acid sequence showed significant subspecies or subgroup differences.The phylogenetic tree constructed by the amino acid sequence of CEP shows that the phylogenetic position of CEP of Lactobacillus delbrueckii subsp.bulgaricus is independent of the CEP of other subspecies;What’s more,the alignment result of site 222 of the CEP sequence which specifically binds to the substrate showed that Lactobacillus delbrueckii subsp.lactis D11M188 and the other five strains were consistent with Lactobacillus delbrueckii subsp.bulgaricus were threonine,while the other four species and the rest 12 strains of Lactobacillus delbrueckii subsp.lactis were alanine at the site.The CEP and its encoding gene of Lactobacillus delbrueckii subsp.bulgaricus were classifed as Prt B,whereas Lactobacillus delbrueckii subsp.lactis D11M188 and the other five strains were classifed as Prt L-1 with the remaining Lactobacillus delbrueckii subsp.lactis strains were classifed as Prt L-2.The result of functional gene classification annotation revealed that Lactobacillus delbrueckii subsp.lactis possessed notable potential with carbohydrate transport and metabolism,transcription and adaptation to environmental evolution and stress resistance due to their shorter milk fermentation history,whereas Lactobacillus delbrueckii subsp.bulgaricus already have adapted to the evolutionary pressures in the milk.Analysis of fermentation characteristics of Lactobacillus delbrueckii subsp.bulgaricus and Lactobacillus delbrueckii subsp.lactis in skim milk.The results of carbohydrate utilization experiments showed that Lactobacillus delbrueckii subsp.lactis could utilize more carbohydrates than Lactobacillus delbrueckii subsp.bulgaricus.The results of fermentation characteristics of Lactobacillus delbrueckii subsp.bulgaricus and Lactobacillus delbrueckii showed that the volatile flavor compounds of fermented milk by the two subspecies had a distribution pattern,in which 2,3-butanedione mainly existed in Lactobacillus delbrueckii subsp.lactis and 2-pentanone was mainly found in Lactobacillus delbrueckii subsp.bulgaricus.The metabolism difference in the late fermentation period was more significant than that in the early fermentation period,the difference pathway in the late fermentation period mainly focused on carbohydrate and fatty acid metabolism.Peptidomics of fermented milk had a distinguishable distribution pattern between the two subspecies with rare variation was found between peptidomics of fermented milk by Lactobacillus delbrueckii subsp.lactis.For the hydrolysis of αs2-casein,Lactobacillus delbrueckii subsp.bulgaricus had powerful hydrolysis ability to αs2-casein regions(69–86)and(95–115),while Lactobacillus delbrueckii subsp.lactis prefer to hydrolyze regions(142–172)and(186–207).In the hydrolysis of β-casein,Lactobacillus delbrueckii subsp.bulgaricus has more extensive hydrolysis than Lactobacillus delbrueckii subsp.lactis.The peptidomics of fermented milk after digestion showed that the regions(73–97)and(124–163)of β-casein could resist digestion and also were the main source regions of bioactive peptides.The peptidomics characteristics of fermented milk revealed that the 12 h fermented milk by Lactobacillus delbrueckii subsp.bulgaricus DQHXNS8L6 and Lactobacillus delbrueckii subsp.lactis D11M188 had the most number of ACE-inhibitory peptides after digestion.The24 h fermented milk of Lactobacillus delbrueckii subsp.bulgaricus DQHXNS8L6 had notable content of antihypertensive peptide Tyr-Pro(YP),and high ACE inhibitory activity before and after digestion in vitro,while Lactobacillus delbrueckii subsp.lactis D11M188 only had high ACE inhibitory activity in fermented milk.In the in vivo experiment,the fermented milk of Lactobacillus delbrueckii subsp.bulgaricus DQHXNS8L6 had antihypertensive effect on Spontaneous hypertension rat(SHR)in single oral gavage and long-term experiment.The mechanism of blood pressure regulation of fermented milk of Lactobacillus delbrueckii subsp.bulgaricus DQHXNS8L6 may be through reducing the level of oxidative stress in the serum and aorta,inhibiting the level of ACE gene transcription and protein expression,and improving aortic fibrosis and vascular thickening.