Identification Of An Actinomycete JN18 With Antibacterial Activity And Optimization Of Fermentation Conditions

The issue of food security is a basic issue relate to people’s survival and health.For more than 40 years,The total grain yied of china has been increasing,relying on policies,systems,and science and technology.The contribution rate of agricultural science and technology is as high as 60%,and science and technology have played a huge supporting role on Chinese agriculture.However,Chinese crops are still affected by plant diseases and insect pests.Finding more microbial pesticides with novel structures to replace chemical pesticides is a major direction to solve the food security problem.This article aims to clarify the basic biological information of Actinomycetes JN18,and lay a theorerical foundation for strain JN18 as a microbial resource to be used as a biocontrol agent.1.Strain JN18 is an actinomycete isolated from the rhizosphere soil of camellia in Jiangxi Agricultural University,and deposited in Institute of Applied Microbiology,Jiangxi Agriculturd University.According to traditional identification method and 16s r DNA sequence,the strain JN18 was identified as Streptomyces lilacinus JN18.2.The whole genome of strain JN18 by second and third generation sequencing technologies shows that the total length of the whole gene is 7102713,and the number of genes is 7238,and the proportion of gene length to the length of the whole genome is 86.3%,and the number of sense gene sequences is 3659,and the number of negative genes is 3579.Predictive analysis of the tested whole genome functional gene cluster of strain JN18 through bioinformatics software and website,about whole genome similarity analysis,non-codind RNA analysis,NR annotation,GO analysis,KEGG analysis,GOG annotation statistics,carbohydrate activity enzyme analysis,antibiotic resistance anaysis and predictive analysis of secondary metabolites.Predictive analysis of its antibiotic resistance gene cluster found that it has 152 gene clusters related to antibiotic resistance,including macrolide antibiotic resistance related gene clusters,and the secondary metabolite prediction analysis found that strain JN18.The strain JN18 genome has a guiding pentamycin gene cluster,and it is a macrolide antibiotic;predictive analysis of related functional gene clusters produced by its antibiotic cluster shows that there are a total of 41 functional gene clusters related to the synthesis of secondary metabolites.There are two 100%homologous functional gene clusters including polyketide synthase functional gene cluster to guide the synthesis of pentamycin（pentamycin）and polyketide synthase to guide the production of tetrahydromethylpyrimidine carboxylic acid（ectoine）,and The synthesis of marineosin A and marineosin B guided by polyketide synthase.3.By measuring the fermentation kinetic parameters of strain JN18,and using Origin software to select Logistic model,Luedeking-piret modified model,Luedeking-piret model,the cell growth kinetic model,substrate consumption kinetic model and product synthesis kinetic model were carried out.Fitting to obtain its fermentation kinetic model.The test results show that in this experimental shake flask fermentation culture system,the cells of strain JN18 are continuously growing with the time flying;antibiotics are constantly being synthesized;the p H of the fermentation broth is relatively stable and does not drop to weak acidity.It is always weakly alkaline,which is related to the presence of CaCO₃ in the medium;amino nitrogen and soluble phosphorus are also constantly being consumed;and when the strain JN18 grows in,cell apoptosis causes its content to rise slightly.4.By exploring the inoculation age,inoculation amount,liquid volume,initial fermentation p H,glucose addition,beef extract addition,carbon source,nitrogen source,and inorganic salt of strain JN18 under shaking flask fermentation culture conditions,the inoculation of strain JN18 produce the influence of antibacterial active substances in order to obtain the optimal cultivation plan within the range of experimental conditions.The experimental results show that the optimal inoculation amount of strain JN18 is 8%,and the optimal liquid volume is 25 m L,and the initial p H of the optimal medium fermentation is about 7.0,and the optimal inoculation age is 68 h.The addition of glucose and beef extract is It can improve its antibacterial activity within a certain concentration range,but it will reduce its antibacterial activity if it exceeds a certain concentration.And using the orthogonal test design,the best optimized culture formula within the range of experimental conditions was explored:the optimized formula of the fermentation supernatant of strain JN18:10.0g·L^-1 glucose,20.0 g·L^-1 Corn starch,6.0 m L·L^-1 soybean oil,9.0 g·L^-1 peptone,18.0 g·L^-1soybean meal powder,40.0 g·L^-1 corn flour,15.0 g·L^-1 beef extract,3.0 g·L^-1NaCl,7.0 g·L^-1Na₂CO₃,3.0 g·L-1KNO₃,0.3 g·L^-1KH₂PO₄;the optimal formula of the fermentation equal-volume alcohol extract of strain JN18 is:20.0 g·L^-1 glucose,30.0 g·L^-1 corn starch,5.0 g·L^-1 soybean oil,14.0 g·L^-1 peptone,28.0 g·L^-1 soybean meal powder,10.0 g·L^-1corn flour,9.0g·L^-1 beef Paste,3.0 g·L^-1 NaCl,7.0 g·L^-1 Na₂CO₃,2.5 g·L^-1 KNO₃,0.2 g·L-1 KH₂PO₄.5.Preliminary study on the antibacterial activity of the fermentation extract of strain JN18,and found that it can effectively inhibit the growth of microorganisms such as Escherichia coli,Staphylococcus aureus,yeast,Penicillium italy and filamentous fungi,indicating that the secondary strain produced by strain JN18 the metabolite has broad-spectrum bacteriostasis,and the inhibition zone of the fermentation extract against Penicillium italicum is as high as 21.61 mm.Using the plate confrontation method,it was found that the strain JN18 could effectively inhibit the growth of the rice blast pathogen.The antibacterial active substance of the fermentation extract of strain JN18 was coarsely separated by thin layer chromatography.After the color development with sulfuric acid and iodine,it was found that the developing agent isopropanol:ethanol:ammonia:water=9:2:1:2 can be separated into 5 different components,and they are named JN18-1,JN18-2,JN18-3,JN18-4,JN18-5.Using Penicillium italicum as the indicator bacteria,it was found that the specific shift value of the bacteriostatic active substance was closer to that of the JN18-5 component of thin layer chromatography through bioimaging technology,which was preliminary judged.And using Penicillium italy as the indicator bacteria combined with the Oxford method to further verify the antibacterial activity of the five different separated components of the JN18 fermentation extract after thin-layer chromatography.The results show that the isopropanol extract of JN18-5 it does have antibacterial activity,and it further proves that the experiment of using this unfolding system to isolate the fermentation crude extract of strain JN18 is highly repeatable.