Screening And Identification Of Microorganisms With Antimicrobial Activity And Study On Its Secondary Metabolites

Microbial drugs have become the main source of natural drugs because of their rich source,novel structure,diverse activity,and short production cycle.In the face of the emergence of drug-resistant strains today,the selection of microbial drugs with high efficiency,low toxicity and new mechanism of action has become a research hotspot.As the main species of bioactive substances,fungi have become one of the major sources of microbial drugs.In this paper,strains with antibacterial activity were screened out from soil samples collected from various regions,and the systemic studies on the identification of antimicrobial activity strains,broad-spectrum,separation and purification of antimicrobial active substances,mutation breeding,and optimization of fermentation processes were conducted to find natural medicines.Its will provide a theoretical basis for development of natural medicines.1.We collected 7 soil samples,isolated and screened 104 fungi from them.Among them,4 strains of fungi had antibacterial activity against S.aureus,E.coli,B.subtilis and B.thuringiensis.Two of the active strains were used as research objects,combined with morphological observation and molecular biological identification to analyze their species status.The strain PA-33 was finally determined to be Penicillium citrinum and named Penicillium citrinum PA-33;the strain PC-5 was finally determined to be Penicillium sp.and named Penicillium sp.PC-5.2.We studied for stability of the fermentation broth,antibacterial spectrum,separation and purification of antibacterial active substances for Penicillium citrinum PA-33.Penicillium citrinum PA-33 had antibacterial activity against 21 kinds of pathogens.The fermentation of Penicillium citrinum PA-33 had strong stability to heat,acid,alkali,natural light and ultraviolet.The MIC of ethyl acetate extract was 7.5 mg/mL for S.aureus,7.5 mg/mL for B.thuringiensis,940 ?g/mL for E.coli,and 1.88 mg/mL for B.subtilis;Finally,active pure product 14 was obtained,and the inhibition zone diameters(1 mg/mL)of against B.thuringiensis,E.coli,S.aureus,and B.subtilis were 16.23 mm,14.43 mm,15.43 mm,and 17.07 mm,respectively.3.Single factor experiments,response surface methodology,ternary quadratic rotation combination design and frequency analysis method were used to optimize the fermentation process of Penicillium citrinum PA-33.The optimal medium composition was 219.91 g/L potato,34.11 g/L mannitol and 6.25 g/L soybean meal.The optimal fermentation conditions were as follows: liquid volume 50 mL,inoculation volume 3.5%,Initial pH value natural,culture temperature 28 ?,rotation speed 150 r/min,fermentation for 12 d.The inhibition zone diameter of fermentation broth on Escherichia.coli reached to 28.99 mm,increased by 10.26 mm compared with the original conditions with the diameter of 18.73 mm.4.With the Penicillium citrinum PA-33 strain used as starting strain and Escherichia coli used as the indicator bacteria,the spore suspension of Penicillium citrinum PA-33 were treated respectively by UV mutagenesis,UV-LiCl mutagenesis,UV-nitrite mutagenesis,microwave mutagenesis and microwave-ultrasound-3% diethyl sulfate(DES)mutagenesis.Five mutant strains(Li-1,N-25,H-14,WB-6 and D-11)producing highly antibiotic were screened.Their antibacterial activity were increased respectively by 39.51%,39.19%,40.26%,32.29% and 28.51% compared with the original strain with the diameter of 18.73 mm.Among them,genetic stability of the mutant strains Li-1,H-14 and N-25 were the better,and the mutant strains WB-6 and D-11 were relatively poor in genetic stability.5.We studied fort the antibacterial activity of Penicillium sp.PC-5 against 22 pathogenic bacteria and the stability of the fermentation broth.And Plackett-Burman design and Box-Behnken response surface method were used to optimize the optimum fermentation medium..The fermentation broth of Penicillium sp.PC-5 had antibacterial activity against 19 kinds of pathogen,and the fermentation broth has low strong stability to temperature,acid and alkali,but has strong stability to natural light and ultraviolet.After optimization,the optimum fermentation medium formulation for Penicillium sp.PC-5 was 342.62 g/L,corn meal 52.42 g/L and MgSO4 0.3 g/L.The inhibition zone diameter of fermentation broth on E.coli reached to 29.44 mm,increased by 10.26 mm compared with the original conditions with the diameter of 18.73 mm,and the antibacterial activity was increased by 61.14%.