Purification,Identification And Characterization Of A Novel Antibacterial Substance Produced By Lactobacillus Rhamnosus

There is a lot of lactic acid bacteria contained in traditional fermented food in China,which can produce more than one antibacterial substance,including the protein,polypeptide or other small molecule natural substances.These antibacterial substances have become the object of research as new bio-preservatives in food because of security.In this study,a novel natural product with high antimicrobial activity is purified and characterized from the metabolites of a strain named LS-8 screened from traditional fermented foods.This substance with broad-spectrum antibacterial activity,simultaneously,its physicochemical properties and structure are analyzed and identified,and its antibacterial mechanism is preliminary deduced.?1?An antibacterial substance designated as LS-8-25 was obtained from the metabolites of the Lactobacillus rhamnosus LS-8 strain screened from the traditional fermented food.The pure sample was purified by performing ethyl acetate,ethanol alcohol precipitation,a polypeptide column,and HPLC separation.?2?According to MS spectrum result,it confirmed that LS-8-25 was a small molecule substance with the molecular mass of 157.092 Da,and its molecular formula structure obtained by the high resolution mass spectrometry result was C₆H₁1N₃O₂.The result of LC-MS showed that LS-8-25 existed isomers,in addition,it had a symmetrical structure,and comprised amide linkage and existed enol interconvert.Combined with ¹H NMR and ¹³C NMR analysis,it presumed that the structural formula of LS-8-25 may have the following two situations:4,6-dimethyl-1,2,5-triazepane-3,7-dione: 1H-NMR?500MHz,D2O?,??ppm?:1.33-1.35?d,6H,J=7Hz,-CH3?,4.27-4.31?q,2H,J=7Hz,-CH-?,4.7-5.0?m,3H,-NH-and D2O solvent residual signals?;13C-NMR?125 MHz,D2O???ppm?:19.32,66.56,178.86;HRMS?ESI+?m/z calculated for C₆H₁₂N₃O₂ [M+H]+ 158.0851,found 158.0923.4,6-dimethyl-1,2,5-triazepane-3,7-dione: 1H-NMR?500MHz,D2O?,??ppm?:1.33-1.35?d,6H,J=7Hz,-CH3?,4.27-4.31?q,2H,J=7Hz,-CH-?,4.7-5.0?m,3H,-NH-and D2O solvent residual signals?;13C-NMR?125 MHz,D2O???ppm?:19.32,66.56,178.86;HRMS?ESI+?m/z calculated for C₆H₁₂N₃O₂ [M+H]+ 158.0851,found 158.0923.?3?LS-8-25 was identified as a heat-stable substance,its antibacterial activity had not been significantly influenced even heated 20 min at 121 ?.LS-8-25 had antibacterial activity only under acidic conditions,and was performed the best inhibitory activity at pH4.0,however,the activity of LS-8-25 gradually decreased as the pH increased,and completely lost activity at neutral and alkaline conditions.It was not sensitive to pepsin,papain,trypsin,proteinase K and ?-amylase.LS-8-25 had a broad antimicrobial spectrum,it not only inhibited common Gram positive and negative food-borne pathogens,but also could inhibit drug-resistant strains of foodborne pathogens.?4?Scanning electron microscopy?SEM?and transmission electron microscopy?TEM?were used to observe the indicator bacteria which treated by LS-8-25,the destruction of cell membrane inferred the site of action of LS-8-25 may be located on the bacterial cell membrane,it would be affected the potential of the membrane.