Isolation,purification,preliminary Identification And Characterization Of Novel Non-ribosomal Peptides From Bacillus SPP

In recent years,the development of new antibiotics has become increasingly important due to the emergence and widespread spread of drug-resistant bacteria,which pose a great threat to human health and the prevention and treatment of animal diseases.In this study,the secondary metabolites of bacteria were screened,and two new antibacterial substances which could effectively antagonize drug-resistant bacteria were isolated from bacillus,and the structure of the antibacterial substances produced was preliminarily identified and their biological characteristics were analyzed,providing certain reference for the prevention and control of bacterial resistance.This study selected soil of yantai offshore and Marine life,through the plate cultivation method and separation of bacteria,a total of around 355 strains of bacteria,isolation and purification of two strains of bacteria can produce fat peptide antibiotics,bacteriostatic experiment shows that one of the bacteria strains of staphylococcus aureus and methicillinresistant staphylococcus aureus,MRSA T144 have efficient inhibition,The other strain had a high inhibitory effect on staphylococcus aureus,MRSA T144,escherichia coli,and e.coli multimyxin-resistant and carbapenem resistant e.coli e.coli(blandm-5 + McR-1).Two strains of active bacteria were screened and identified as Bacillus amyloliquefaciens 9-14 and Bacillus amyloliquefaciens e14-2(hereinafter referred to as active bacteria 9-14 and active bacteria e14-2)by genome extraction,PCR amplification product sequencing and phylogenetic tree construction.The activity of bacteria in BHI broth liquid medium for bacteria culture,every hour in determination of active bacteria broth in a wavelength of 600 nm ultraviolet absorption value,the growth of bacteria within 24 h curve drawing,determination of the bacterial growth plateau,20 h found active bacterium 9-14 in growth,active bacteria E14-2 in growth after 22 h for optimal growth period of active metabolites.Ethyl acetate was used to extract the secondary metabolites of bacteria.The bacteriostatic test showed that the secondary metabolites had significant inhibitory effects on MRSA T144 and e.coli B2.To obtain relatively pure antibacterial substances,separation and purification of metabolites of bacteria,preparation by gel chromatography and liquid chromatography to carry on the purification,through mass spectrometry for the antimicrobial activity of purified material carries on the preliminary structural analysis,found that it is made up of amino acid chain ring fat peptide,belong to the hay rhzomorph derivatives.This paper also studied the biological characteristics and bacteriostatic spectrum of the metabolites of active bacteria 9-14 and active bacteria e14-2.It was found that the metabolites of active bacteria 9-14 and active bacteria e14-2 had good thermal stability and were resistant to acid and alkali,while the antibacterial substances of active bacteria 9-14 had no significant change in activity after enzyme treatment,and were relatively stable.However,after the active bacteria e14-2 was mixed with trypsin and protease K,the antibacterial activity did not change significantly,and the antibacterial activity was completely lost after the interaction with pepsin and papain.Found in bacteriostatic spectrum measurement,two kinds of antibacterial materials are used to test the gram positive bacteria has certain inhibition,active bacterium 9-14 antibacterial substances,activity of gramnegative bacteria do not have bacteria E14-2 activity against escherichia coli and pseudomonas aeruginosa have antibacterial activity,but do not have to other gram-negative bacteria activity,two bacterial strains of bacillus were not active.