Diversity Analysis Of Lactobacillus Delbrueckii Subsp.bulgaricus Based On Genomics And Whey Proteolysis Characteristics

Lactobacillus delbrueckii subsp.bulgaricus is one of the lactic acid bacteria widely used in the production of fermented dairy products,with an efficient protein hydrolysis system.Studies have shown that there are diversities in the proteolytic properties of L.delbrueckii subsp.bulgaricus,but the genomic differences and the whey protein utilization capacity need to be further investigated.In the present study,24 strains of L.delbrueckii from the fermented yak milk and Qura collected from Qinghai,Sichuan and Xinjiang province were studied,and he gene sketches were sequenced.The 24 strains isolated with 39 L.delbrueckii strains published in NCBI were subjusted to comparative genomics,including homologous evolution analysis,core gene analysis,pan-gene analysis,and the distribution of key genes in the proteolytic system,etc.The result showed that the genome of L.delbrueckii varied among the strains.Based on the results of homologous evolution,it was found that there are multiple evolutionary branches of L.delbrueckii subsp.bulgaricus,with intraspecies diversity.In addition,the pan-genome of the strains were “open”,and evolution were still in progress.The COG function annotation results indicated that the accessory & specific genome of the strains had more functional genes than the core genome.The analysis of key genes in the proteolytic system exhibited that the OppA gene of the oligopeptide transport system with the most diversity,and the peptidase genes PepD,PepQ,PepO and PepI also had diversity,while the genes encoding cell wall proteases were highly conservative and had no difference among strains.Based on the analysis results of genomics,8 strains of L.delbrueckii subsp.bulgaricus with different evolutionary branches and large differences in proteolysis-related genes were selected to determine proteolytic enzyme activities and the utilization of the main components of whey protein.The results showed that during the cultivation process(12 h),the activities of the three peptidases all showed a trend of increasing first and then decreasing,but there were significant differences among strains.The 8 strains had a strong hydrolytic capacity for ?-lactalbumin(26.93%~31.33%),but the ?-lactoglobulin hydrolytic capacity varied among strains.The hydrolysis rates of variants A and B of ?-lactoglobulin were 10.56%~22.82% and 9.04%~23.83%,respectively.There was a certain correlation between the aminopeptidase activities and the hydrolysis ability of ?-lactoglobulin of the 8 strains.DQHXNS8L6 and DQHXNS15M2 reached the highest aminopeptidase activities and hydrolytic capacities of ?-lactoglobulin within 3 hours.The gene numbers of PepD,OppA and OppB were also correlated with the activity of three peptidases and the hydrolytic capacity of ?-lactoglobulin.The 8 strains were fermented in whey protein-lactose system.The results showed that the peptides production capacity(relative molecular mass < 2000)were different among L.delbrueckii subsp.bulgaricus.2 strains with strong peptides production capacity were selected to measure if different initial pH conditions(7.0,6.5,6.0 and 5.5)affected peptides production capacity and enzyme activities.The results showed that the 2 strains had the highest enzyme activities under the initial pH value of 6.0.When the initial pH was 6.0 or 6.5,the activities of cysteine peptidase and proline-specific peptidase was higher and with strain difference,while cell wall proteases and metal peptidase aciticities were basically unaffected in the selected pH range.The peptides in the hydrolysates of the 2 strains were identified,it was found that DQHXNS8L6 had more cleavage sites than DQHXNS1L2.Five kinds of bioactive peptides derived from ?-lactoglobulin were identified in the hydrolysate of DQHXNS8L6,LDAQSAPLR,DAQSAPLRVY,ILAEKTKIPAVF,IDALNENK and LSFNPTQ,respectively.DQHXNS8L6 released four kinds of bioactive peptides containing Pro.In summary,L.delbrueckii subsp.bulgaricus strains exhibited significant differences between genotypic and phenotypic in the proteolytic metabolic system.The peptide-producing ability has a certain correlation with the cysteine peptidase and proline-specific peptidase activities,and they are affected by the system pH.