Identification And Antimicrobial Spectrum Of A High Inhibiting Penicillium Strain

This experiment strain H317have a good inhibitory effect to the usual bacteria、mould、yeast especially moulds cause deterioration of fruits.So if know the species of the strain clearly, we could utilizate it as a natural food preservative, and fill in the gaps about similar products currently on the market are not enough. Also we could supply the theoretical and practical base for the development of new bacteriostatic agent and effectively further explore the microorganisms resources. To identity the experimental strains, several research was carried out including experimental strains H317morphological observation, physiological and biochemical index determination,16s rDNA sequence analysis and the acid-base stability, thermal stability, the influence of surfactant and antibacterial spectrum. The main results of research were as follows:1. Experimental strains H317in the tablet of beef extract peptone medium,37℃, constant temperature culture3-5d, through observation, colony morphology are:ivory, diameter is about6.75mm, surface not smooth with fold, opaque, convex, rounded, margin wavy or petal shaped. Culture in liquid beef extract peptone medium have a biofilm formation. The bacteria has spores and is gram-positive, short rod(3.75μm×1.45μm), arrangement of features into a single or a chain, and motility. The experimental strain showed positive reaction including nitrate reduction, catalase reaction, determination of V-P, starch hydrolysis, citrate utilization,(2%,5%,7%NaCl) salt resistance, lecithin enzyme, hydrolysis of casein hydrolysis, hippuric acid hydrolysis. The negative reactions including phenylalanine deaminase,10%NaCl salt resistance, indole test, indole pyruvic acid (IPA), gelatin liquefaction, methyl red (M.R). According to the above reaction, therefore, the preliminary judgment was that the experiment strains H317might be Bacillus sp.2. Qiagen method was used to extract experimental strains H317genomic DNA. The extracted DNA of the bacteria under test in bacteria universal primers for amplification primer, to PCR amplification, gene sequencing and homology sequence comparison. The experiment results show that16s rDNA sequence of the strain homology with bacillus amyloliquefaciens, at99%-100%. At the same time drawing the16s rDNA sequence phylogenetic evolution tree. Comprehensive comparison, determined it was Bacillus amyloliquefaciens.3. Based on deterministic experiment of the antimicrobial substance produced by the strain, preliminary determined the antibacterial substances may be hydrogen peroxide with a protein or polypeptide substance. To the strains of antibacterial material for study of physical and chemical properties shows that the antimicrobial substance produced by experimental strains H317can keep the good antibacterial activity at60℃to90℃for15min,30min,100℃heat treatment for15min,30min, bacteriostatic activity still can keep the67%,57%, at121℃for15min after the heat treatment there is still a bacteriostatic activity; pH5-9, bacteriostatic activity stability, pH=2and pH=11, still have good antibacterial activity, as the original77%,81%;Three kinds of surfactants has little influence on its antimicrobial activity; the antimicrobial substance produced by experimental strains H317has broad-spectrum antibacterial activity, for most cause deterioration of moulds has a good inhibitory effect.