Screening,Probiotic Properties Of β-Glucosidase Producing Lactic Acid Bacteria And Biotransformation Of Flax Lignans

Lignans were reported to possess antioxidative,estrogenic and antiestrogenic activities,therefore have preventive effects on breast cancer and atherosclerosis.Flaxseed is a rich source of lignans.Flax lignans existed mainly in the form of secoisolariciresinol diglucoside(SDG).SDG needs to be metabolized by intestinal microflora to produce metabolites such as secoisolariciresinol(SECO),demethyl-SECO,demethyl-dehydroxy-SECO,enterodiol,and enterolactone before it can be effectively absorbed in the colon.Therefore,SDG metabolites normally have higher bioavailability than SDG.In this study,8 LAB strains with high β-glucosidase activity were screened and used to transform SDG by resting cells or liquid fermentation.Their growth and probiotics characteristics were studied.Ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)was applied to analyze the metabolic pathways of 4 of the LAB strains in fermentation of flax lignans.The main results were as follows:(1)Eight LAB strains with high β-glucosidase activity were obtained by MRS-cellobiose plate and p-NPG method screening.Among them,5 strains(C1,C2,C5,C8,D2)were isolated from fermented food which were identified as Lactiplantibacillus plantarum by 16S r DNA sequencing and physiological tests.Results indicated that β-glucosidase activities of the 8 LAB strains were significantly increased via the induction of cellobiose as a sole carbon source(P<0.05).The maximum β-glucosidase activity(16.02 U/mL)was observed with Enterococcus durans GW18275.The β-glucosidase produced by the strains are mainly intracellular enzymes or membrane enzymes.The resting cells of the 8 LAB strains were able to hydrolyze SDG to SECO.SECO generation rate was used as the optimization index to optimize SDG transformation efficiency of Lactiplantibacillus plantarum C5 and Lactiplantibacillus plantarum SCB0151.The optimum substrate concentration was 10 mM for Lactiplantibacillus plantarum C5 and 5 mM for Lactiplantibacillus plantarum SCB0151.The optimum reaction temperature was 37℃.The optimum initial pH was both pH 6.While the maximum SDG conversion rate were 54.48%and 98.56%for Lactiplantibacillus plantarum C5 and Lactiplantibacillus plantarum SCB0151 respectivley,and the SECO generation rate were 17.06%and 68.39%,respectively.(2)The growth and probiotics characteristic of 8 LAB strains were studied.Eight LAB strains could grow at 22-37℃,which Lactiplantibacillus plantarum SCB0151,SCB0667 and Enterococcus durans GW18275 could also grow in 45℃.The growth pH of strains range from pH5-pH10.Lactiplantibacillus plantarum C1,C2,SCB0667 and Enterococcus durans GW18275 can also grow at 9%wt.%NaCl.Results showed that the survival rate of 8 LAB strains in simulated intestinal fluid was higher than 60%,and the number of viable bacteria in simulated intestinal juice remained above 10~6CFU/mL.Lactiplantibacillus plantarum C8 had the highest survival rate of 94.83±7.04%when cultured with 0.3%bile salt for 3 h.The auto-aggregation rate of 8 LAB strains ranged from 50%to 81%at 24 h,among which Lactiplantibacillus plantarum C1 had the highest auto-aggregation rate(80.72±1.58%).Lactiplantibacillus plantarum D2 had the best surface hydrophobicity to ethyl acetate(76.56±0.51%).The 8 strains were non-hemolytic and had different drug sensitivity to cephalosporin, β-amide and aminoglycoside antibiotics,as well as possessing sensitive to tetracycline.(3)UPLC-MS/MS was applied to comprehensively profile the lignans,flavonoids and phenolic acids metabolites variations in fermentation cultures by sole LAB strains(Lactiplantibacillus plantarum C1,Lactiplantibacillus plantarum C5,Lactiplantibacillus plantarum SCB0151,Enterococcus durans GW18275)before and after 8-day fermentation.SDG transformation pathway by sole LAB strains was proposed.Results showed that each of the 8 LAB strains was able to deglycosylate SDG and secoisolariciresinol monoglucoside(SMG)to SECO,and the highest SDG conversion was achieved by Lactiplantibacillus plantarum SCB0151.SECO was further converted to metabolites such as demethyl-SECO,demethyl-dehydroxy-SECO and demethyl-dehydroxy-MAT.The biotransformation of SDG by single LAB strains performed fewer metabolic pathways compared with that of colon microbiota.