Screening And Genome Analysis Of Lactobacillus Helveticus Strain GY-3 With High ACE Inhibitory Activity From The Sediment In Bohai Sea

Lactobacillus helveticus is intestinal probiotics,with the frequent observation of high angiotensin-I-convertingen enzyme(ACE)inhibitory activity in its fermented milk.The marine is the optimal source for biological flora,including abundant types Lactobacillus.In order to exploit the sources for ACE inhibitory peptide,this study screened a Lactobacillus helveticus with high ACE inhibitory activity detected in its fermented milk.Furthermore,the optimization of the fermentation condition and genomic analysis was done to lay the foundation for developing high-yield ACE inhibitory peptide dairy products.The main contents of this study are as following:1.The composition of the bacterial communities in the surficial sediments from Bohai Sea indicated that the genus Lactobacillus with relative abundance of 0.24%was abundant.Based on the bromocresol green method and calcium carbonate method,conducting a series of studies such as microscope observation,catalase measurement,gelatin liquefaction,H2S production,indole test,litmus milk test and other physiological and biochemical experiments,5 strains were eventually obtained and identified as Lactobacillus helveticus via 16S rDNA sequencing.2.The screened strains were inoculated to skim milk medium with a inoculation quantity of 3%.After 24h incubation at constant temperature of 37 ℃,the ACE inhibitory activity of 5 strains were 38.57%、5.25%、58.56%、26.60%、45.27%.Choosen 3 of them which had high ACE3.inhibitory activity to re-fermented,the ACE inhibitory activity were 27.27%、52.10%、13.27%.Comprehensive two experimental results,the strain with highest inhibition on fermented milk was chosen,and named as Lactobacillus helveticus strain GY-3.4.Fermentation characteristics(temperature,inoculum and the amount of carbon,nitrogen and Inorganic salts added)of Lactobacillus helveticus strain GY-3 was investigated in skim milk through the ACE inhibitory activity.The results showed that the optimal inoculation quantity for Lactobacillus helveticus strain GY-3 was 3%,and the optimal fermentation temperature was 37℃.Through optimization in Lactobacillus helveticus strain GY-3 medium,the results indicated that respective addition of 1.5%glucose,1.0%yeast extract powder,0.6%soy peptone,0.04%MnSO4·4H2O to skim milk could elevated the ACE inhibition rate of Lactobacillus helveticus strain GY-3 fermented milk.By orthogonal test analysis,the highest inhibition rate of 79.52%was achieved by simultaneous addition of 1.0%glucose,0.6%soy peptone,1.0%yeast extract powder,0.04%MnSO4·4H2O to skim milk medium.5.Whole genome DNA of Lactobacillus helveticus strain GY-3 was extracted for the analysis and prediction of encoding gene,non-coding RNA,repetitive sequence,and the functions of encoding gene sequences were annotated against different database.The results showed that the whole genome length of Lactobacillus helveticus strain GY-3 was 2,022,469 bp,and the number of encoding gene was 2,159,and it contained Cell-envelope Proteinase(PrtH4),Polypeptide Transport system(OppB1)and Peptidase system(PepT、PepD、PepO3、PepE、PepO、PepF、PepI、PepCE et al.).Lactobacillus helveticus strain GY-3 has a high6.level of protein hydrolysis capacity,and it has less genes for hydrolysis ACE inhibits peptide.In this study,the screening of Lactobacillus helveticus strain GY-3 provides a broader source for the development of high-yield ACE inhibitory peptide lactic acid bacteria.Genomics analysis also made it possible to improve the ACE inhibition rate with metabolic regulation method.It is of great significance for the development of antihypertensive function products.