Isolation,Purification And Preliminary Identification Of Effective Antimicrobial Substances From Bacillus Cereus

Since antibiotic drugs were first used to prevent and treat diseases caused by bacteria,the confrontation between bacteria and antibiotic drugs has never stopped.Today we are facing a"post-antibiotic era"where no medicine is available.Therefore,the discovery of new antibiotic drugs has become the obligatory responsibility of relevant scientific researchers.In recent years,the selection of secondary metabolites of specific microorganisms in nature to antagonize pathogenic bacteria has become the focus of many scientific researchers.Aiming at the control of food-borne pathogenic bacteria,two strains of Bacillus cereus that can produce effective antibacterial substances were screened in this paper,and some relevant conditions for the production and extraction of antibacterial substances of the two strains were optimized,and their production the antibacterial substance has been further purified,investigated for stability and preliminary identification.At the same time,with the help of the data analysis software MZmine,the three sets of data of Bacillus cereus,pathogenic bacteria,and the two co-cultured bacteria groups were analyzed for differences in metabolites between groups,in order to find out the differences in Bacillus cereus through the differences between the groups.Effective antibacterial substance.This article is the first attempt to find effective antibacterial substances in Bacillus cereus by analyzing the metabolic differences between groups by data processing software,and correspond to the small molecule antibacterial substances actually extracted to obtain accurate results of the antibacterial substances produced by Bacillus cereus.This article is mainly divided into three modules,and the main research results are as follows:(1)This article uses soil,sea sand,sea water and marine organisms in the Yantai area as the source of bacteria.The soil and sea sand are sampled by the"five-point sampling method",and the seawater and marine organisms are sampled randomly.The bacteria isolated are many After purification until the pure breed,record the source of the strain and number it,and store it in a low-temperature refrigerator at-80°C.Finally,a bacterial sample bank containing 355 bacterial samples was established.The double-layer agar plate method was used to screen 355 bacterial samples that have inhibitory effects on pathogenic bacteria.All bacterial samples were screened twice.The first screening was two typical food-borne pathogenic bacteria E.coli ATCC25922 and golden yellow Staphylococcus ATCC29213was used as the indicator bacteria,and the second screening used the resistant strains of Escherichia coli and Staphylococcus aureus as indicator bacteria,namely E.coli B2(bla NDM-5+mcr-1)and MRSA T144,with the inhibition zone The presence or absence of screening active strains.The results of the screening are as follows:A total of 31 strains with antibacterial activity against ATCC25922 and ATCC29213 standard strains were screened from all bacterial samples.At the same time,5 strains with antibacterial activity against E.coli B2(bla NDM-5+mcr-1)and MRSA T144 were also screened from 31 active strains.Two strains with strong antibacterial activity and relatively stable were selected for the extraction and identification of antibacterial substances.The relationship between the two active bacteria and Bacillus cereus was determined by 16S r DNA,and the two active bacteria belonged to Bacillus cereus.,Respectively named as Bacillus cereus C2-4 and Bacillus cereus 21-1-1-2.(2)Extraction,purification and preliminary identification of the antibacterial substances in the two active bacteria.First,based on the OD₆₀₀ value of the bacterial solution,the growth curves of active bacteria C2-4 and active bacteria 21-1-1-2 were determined.Then carry out antibacterial substance extraction and antibacterial experiments on the fermentation broth and cells of the active bacteria,and determine that the source of the antibacterial substance is in the fermentation broth.At the same time,the secretion and extraction of the antibacterial substances in the active bacteria are used to cultivate the active bacteria Conditions such as time,cultivation method(whether induction factor is added),extraction method and other conditions have been optimized.The optimization results are as follows:In order to ensure the effective extraction of antibacterial substances,the optimal culture time of active bacteria is 20 h,and the production of antibacterial substances is independent of whether the induction factor is added.In terms of extraction methods,the most effective extraction method for antibacterial substances is the chloroform extraction method.Tests have proved that the combination of gel filtration chromatography and preparative liquid chromatography purification can greatly increase the purity of antibacterial substances.Finally,it was preliminarily identified after mass spectrometry analysis that the antibacterial substances of the active bacteria C2-4 and the active bacteria21-1-1-2 are most likely to be the same kind,and they are both Lajollamide A.This substance is a cyclic pentapeptide compound containing three Leu.(3)Based on the high-resolution mass spectrometry data,a method to quickly determine the specific metabolite information of the active bacteria was established.The method test was carried out with the active bacteria in this experiment,and the difference in the metabolites of the active bacteria was analyzed with the help of MZmine analysis software.Information about the specific metabolites of active bacteria.The result is:9 kinds of specific metabolites of the active bacteria C2-4 were found,but only the metabolite with a mass-to-charge ratio of 566.42621 was retrieved in the database,which was Lajollamide A;the active bacteria 21-1-1-2 was found There are 14 specific metabolites,and only the metabolite with a mass-to-charge ratio of 566.42621 was retrieved in the database as Lajollamide A.This result is consistent with the antibacterial substances extracted from the active bacteria in Chapter 3,which proves the feasibility of determining the antibacterial substances of the active bacteria through data processing and difference analysis.