Study On The Identification And Antibacterial Components From Pseudomonas Tolaasii DL001

Objective A strain of Pseudomonas DL001 was identified and its secondary metabolites were screened for antimicrobial activity and chemical screening.The stability of the compounds and the optimal fermentation conditions of the strain DL001 were preliminarily explored.Methods A strain of Pseudomonas DL001 was identified by multi-phase taxonomy(based on 16 S r DNA sequence,phenotype and physiological and biochemical characteristics);its secondary metabolites were screened by high performance liquid chromatography and ultra-high performance liquid chromatography tandem mass spectrometry for antimicrobial activity and chemical screening;the stability of active products was tested from four aspects: p H,temperature,ultraviolet irradiation and protease.The fermentation conditions of strain DL001 were optimized by single factor test.Results 1.Identification of strain DL001 Polyphasictaxonomy was used to identify the strain DL001.Phylogenetic analyses,based on 16 S r DNA gene sequence,revealed that the bacterial strain belonged to the Pseudomonas tolaasii,and showed the highest similarity to the type strain P.tolaasii KX186992.1 with an identity of 100%.The strain was Gram-positive and could grow utilizing glucose and sucrose as carbon sources.Positive for V-Preaction,methyl red and indole test,and negative for gelatin liquefaction,starch hydrolysis,the lactose and cellulose hydrolysis.These characteristics were basically consistent with the description of P.tolaasii in Berger’s Bacterial Identification Manual.Taking the above phylogenetic,phenotypic and chemotaxonomic results into account,it is suggested that the bacterial strain is affiliated to P.tolaasii,for which the name Pseudomonas tolaasii DL001 is proposed.2.Research on antibacterial components from strain DL001 The antibacterial activity of secondary metabolites from strain DL001 was studied.The antibacterial activity,estimation of chemical structure,chemical stability of bioactive fraction and fermentation conditions optimiz of strain DL001 were conducted.It was found that strain DL001 could produce a new type of antimicrobial tolaasins-derived polypeptides,which showed inhibition for the growth of Staphylococcus aureus,Aspergillus Niger,Penicillium rubens,Rhizobium subtilis,Bacillus subtilis and Micrococcus luteus with MIC values ranging from 6.25 to 25 μg/m L.Meanwhile,the ethyl acetate extract of the culture broth of strain DL001 exhibited the strongest antibacterial activity for Staphylococcus aureus with MIC value of 6.25 μg/m L.The chemical durability of secondary metabolites of strain DL001 was tested under the guidance of antibacterial bioassay.The results revealed that the bioactive fraction was mostly stable when the p H is 6-10,and the temperature falls below 60 °C,and sensitive to ultraviolet radiation and protease.The optimization fermentation of strain DL001 were determined by single factor experiment,which showed that strain DL001 could highly yield antibacterial compounds under the temperature of 28 °C and the p H of 4-5,the fermented volume of 90 m L(250 m L conical bottle)and the fermentation time of 48 hours,and utilize glucose and yeast extract as carbon source and nitrogen source,respectively.Conclusions 1.Polyphasictaxonomy was used for the identification of P.tolaasii DL001,which provided reliable species information for further research on secondary metabolites of this strain.2.Strain DL001 could produce tolaasins-derived polypeptides with potent antibacterial activity.This discovery provided bacterial resources and antibacterial candidates for the research and development of new antibiotics.3.the chemical durability of these tolaasins analogues is greatly affected by external factors.In addition,the optimal fermentation conditions of strain DL001 have been preliminarily determined.4.This study will provide theoretical support and technical guidance for the subsequent research of new antibiotic compounds from strain DL001.