Isolation,identification And Biological Activity Of Lactic Acid Bacteria From Acidified Whey Of Dali Milk Fan

Acidified whey is produced from whey with spontaneous fermentation and is often used as a natural coagulant in traditional dairy products such as milk fans and cakes.Acidified whey is a resource rich in lactic acid bacteria（LAB）.In this study,LAB were isolated and identified from acidified whey collected from Dengchuan Town,Eryuan County,Dali Bai Autonomous Prefecture,Yunnan Province.Antibacterial activity and in vitro antioxidant activity of those LAB strainswere determined.Using non-targeted metabolomics,differential metabolites in culture supernatant of LAB with different antioxidant activity were analyzed.The results would providea theoretical basis for the exploration and utilization of microbial resources in traditional fermented dairy products.The main research findings are as follows:1.Samples of acidified whey of Dali milk fan were subjected to isolate LAB followed by identification.A total of 69 suspected LAB isolates were obtained using MRS selective culture medium.Based on physiological and biochemical characteristics and 16S r RNA sequence analysis,69 strains were all identified as Weissella confusa.2.Antibacterial activity of 69 strains of Weissella confusa against three indicator pathogens including Escherichia coli,Salmonella and Shigella was studied.The results showed that strains WR7-1,WR7-2,WR6-2,WR6-14,WR5-3,and WR5-8 all inhibited the growth of three indicator pathogens.Strain WR7-1 exhibted the greatest antagonistic activity and significantly suppressed the growth of three indicators.3.Antioxidant activity of culturesupernatant,intact cells,and cell-free extracts of 69strains of Weissella confusa was deterimined using DPPH and ABTS⁺free radical scavenging capacity.The results showed that all three fractions of 69 strains scavenaged DPPH and ABTS⁺free radicals.Fermentation supernatant exhibed the greatest ability scavenging DPPH free radical ranging from 88.94%to 96.22%,followed by intact cells(in a range from 1.90%to 36.77%and cell-free extracts ranging from 3.06%to 14.83%.Similarly,the strongest capacity scavenging ABTS⁺free radical was also observed for culture supernant（76.45%～93.28%）,followed by intact cells（12.91%～38.13%）and cell-free extracts（5.81%～15.19%）.According to these previous data fermentation supernatant was chosen for ferric reducing antioxidant power（FRAP）analysis.The results showed that the FRAP of culturesupernatant ranged from 53.06%to 77.63%.There was slight difference in DPPH scavenging ability of culture supernatant between different strains,while supernatant of those strains differed in their ABTS⁺scavenging ability and FRAP differed.4.Differential metabolites in culture supernatant of strains having better ABTS^（10）scavenging ability（group H1 including WR7-15,WR7-16,WR7-17,and WR7-20）VS those with weaker ABTS^（10）scavenging ability（group D1 including WR7-5,WR7-6,WR7-7,and WR7-8）as well as strains with higher FRAP（group H2 including WR5-1,WR5-7,WR5-8,and WR5-12）VS those having lower FRAP（group D2 including WR3-2,WR7-10,WR7-11,and WR7-18）was detected using ultra-high performance liquid chromatography tandem time of flight mass spectrometry（UHPLC-Q-TOF MS）.The results showed that a total of 31 differential metabolites were screened out in the positive and negative ion mode from culture supernatant in srrains from two groups with different ABTS⁺scavenging ability.Compared with lower group D1,Pro His and R-1 formamide phosphate were upregulated in supernant of strains with higher ABTS⁺scavenging rate（H1）.The positive correlation was observed between R-1 formamide phosphate and ABTS⁺scavenging capacity.A total of 32 differential metabolites were screened out in the positive and negative ion mode from culture supernatant in srrains from two groups with different FRAP.When comparing with lower group D2,compounds belonging to benzene and its derivatives,organic acids and their derivatives,heterocyclic compounds,fatty acyls and amino acids and their metabolites were upregulated in supernant of strains with greater FRAP.Several compounds such Lys-Tyr-Gln-Glu-Ala,Leu-Gln-Tyr-Lys-Asn,Cys-Ser-Ile,Pantoic acid and Methyl cis-9-tetradecenoate were positively related with FRAP.Taken together,69 LAB strains isolated from Dali milk fan acidified whey are all Weissella confusa,and 6 strains significantly inhibited the growth of Escherichia coli,Salmonella and Shigella.Among them,WR7-1 had the strongest antibacterial activity.Culture supernatant exhibited strongest DPPH and ABTS⁺free radical scavenging activity as well as FRAP.Non-targeted metabonomics research revealed that metabolites in culture supernatant such as amino acids and their metabolites,organic acids and their derivatives,fatty acyls,heterocyclic compoundwere related to antioxidant activity of Weissella confusa.These findings provide a solid base for application of LAB in acidified whey.