Structure-activity Relationship And Pathway Analysis Of Bile Acids Inhibiting The Growth Of Lactobacilli

Lactobacillus,one of the most common probiotics,is the original bacteria of the human intestine.It has various biological activities such as regulating immunity and maintaining intestinal flora balance,and these activities are dependent on its biomass in the gut.Bile salt is an anionic surfactant that inhibits the growth and reproduction of Lactobacillus,and restricts the exertion of biological activity of strains to some extent.This study first analyzed the composition of the most common three commercial brands of oxgall powders,optimised the bile salt product closest to human bile,and developed a new method to determine the bile sensitivity of Lactobacillus by replacing human bile with this product.The newly developed method could overcome the shortcomings of the traditional methods,and then used this method to study the structure-activity relationship and influencing factors of bile salts inhibiting the growth of Lactobacillus.Finally,the mechanism of bile salt inhibiting the growth of Lactobacillus was analyzed and the main conclusions obtained were as follows.The composition of three commercial brands of oxgall powders was analyzed by high performance liquid chromatography.Nine bile acids were detected in each brand of samples:cholic acid,deoxycholic acid,chenodeoxycholic acid,glycocholic acid,glycodeoxycholic acid,glycochenodeoxycholic acid,taurocholic acid,taurodeoxycholic acid and taurochenodeoxycholic acid.Majority of them were present in the form of conjugate either with glycine or taurine and the content of free bile acid were relatively small.Although there was no significant difference in total bile acid content among the three samples,there was a significant difference in the relative content of individual bile acids.The oxgall powder from Sigma-Aldrich was the most similar to human bile in the parameters of glycine-conjugated bile acids to taurine-conjugated bile acids,dihydroxy bile acids to trihydroxy bile acids,and free bile acids to conjugated bile acids ratios compared to those from Oxoid and BD Difco,therefore,it is more suitable as a substitute for human bile for evaluating the bile sensitivity of Lactobacillus.A new in vitro method was developed to determine the bile sensitivity of Lactobacillus by using of oxgall powder from Sigma-Aldrich.The overnight culture of various Lactobacillus strains was inoculated into sterile,half-strength MRS broth supplemented with and without 0.3%(m/v)oxgall,buffered with 0.1 M sodium phosphate buffer at a final p H of7.3,and incubated at 37 °C for 12 h under anaerobic conditions.The bile sensitivity of strains was expressed as the percentage of the difference propagation generations of the bacterial cells in that both cultures and the propagation generations of the bacterial cells in culture without bile salt.The newly developed method mimics the physiological environment of the human small intestine,and avoids changes in p H of the culture medium and significant dissociation of the conjugated bile salts by bacteria due to the use of buffer culture and an extremely low inoculum concentration,effectively overcoming the drawbacks of the traditional methods.Using the method developed above,the structure-activity relationship and influencing factors of bile salt inhibiting the growth of Lactobacillus were studied.The antibacterial activity of glycine-conjugated bile acids was significantly stronger than that of taurine-conjugated bile acids(P < 0.05);the antibacterial activity of dihydroxy bile acids was significantly stronger than that of trihydroxy bile acids(P < 0.05);the antibacterial activity of free bile acids was significantly stronger than that of conjugated bile salts(P < 0.05).Non-ionic surfactants such as Tween 60,Tween 80,Span 60,Span 80,and lecithin all significantly reduced the bile sensitivity of Lactobacillus(P < 0.05),while the anionic surfactant stearyl sodium lactate significantly increased the bile sensitivity of Lactobacillus(P < 0.05).In addition,the bile sensitivity of Lactobacillus was significantly reduced after heat,acid,and bile salt stress single or in combination(P < 0.05).The pathway analysis of bile salt inhibiting the growth of Lactobacillus was further studied.The effect of bile acids on the intracellular p H of Lactobacillus was determined by intracellular fluorescence probe binding assay.It was found that bile acids reduced the intracellular p H of Lactobacillus with rapid and stepwise kinetic.The role of free bile acids in this area was significantly stronger than that of conjugated bile acids(P < 0.05).Bile acids could also reduce or even disperse the transmembrane potential of Lactobacillus at a certain concentration.Bile acids could destroy the cell membrane integrity of Lactobacillus,and with the increase of bile acids concentration,the degree of destruction increases,and the extent of destruction of the cell membrane integrity of the strains by dihydroxy bile acids was greater than that of to trihydroxy bile acids.Bile salt could also cause the leakage of intracellular nucleic acid and protein substances from the strains,but different bile acids have different effects on the extent of intracellular nucleic acid or protein leakage of Lactobacillus.