Study On Purification And Characterization Of A Novel Bacteriocin PZJ5in Lactobacillus Plantarum

The preservatives is one of the most important additives in the food industry. The preservatives is an effective way to prevent food spoilage. At present, China is extensive use of chemical preservatives, which may cause food safety risks. Nisinis used widely as one of a natural preservative, but it can inhibit G+bacteria only and cannot inhibit G-bacteria, yeast and fungus. Plantaricin, produced by Lactobacillus plantarum, has a high resistance of low temperature and acid. Plantaricin, which can be biodegradable in body, was considered safe and has a strong antibacterial activity. Our group screened a Lactobacillus plantarum, which has an antibacterial effect, and separated a novel bacteriocin named plantaricin PZJ5. Therefore, we study on purification, cloning and biological information, physic-chemical characteristic and production condition optimization of bacteriocin were developed. The results are as follows:(1) Under acidic conditions (pH≤5), bacteriocins PZJ5have significant inhibitory effect on Gram-positive bacteria and gram-negative bacteria. PZJ5performance good thermal stability, and very sensitive to protease, which the antibacterial activity can be decreased by pepsin and trypsin.(2) After the gradient ammonium sulfate precipitation, ion exchange chromatography, hydrophobic interaction chromatography, gel permeation chromatography and reverse-phase HPLC purified, the PZJ5was separation and collected by a single vitality peak. This indicates that the PZJ5has reached a single component. The mass spectrometry of PZJ5showed a molecular weight of2573.8Da. Edman method for sequencing of the N-terminal amino acid sequence of PZJ5, the sequence is KTKQQFLIKAQTQLFKVFGYTLKK.(3) Through the restore growth methods by observed indicator strain, we found the mode of action of bacteriocins PZJ5is sterilization. By transmission electron microscopy and scanning electron microscopy, we found the bacteriocins PZJ5could leading to cell contents leak of the indicator bacteria by causing cell walls break down, and the cell metabolism can not be continue, that eventually leading to the death of the indicator bacteria. By measuring the indicator bacteria intracellular K+and leakage of nuclear substances, the lactobacillus PZJ5can bacterial indicator bacteria in a very short time.(4) Cloning and sequencing analysis found that the mature peptide of PZJ5has22amino acid residues:KTKQQFLIKAQTQLFKVFGYTL, and there is a signal peptide in the N-terminal:MSLVKENKTLVDELIAQITFGG. Blast alignment found PZJ5is a new bacteriocins. By the bioinformatics analysis, the mature peptide of PZJ5with four positively charged amino acids had an amphiphilic alpha-helix structure at the position of3to18residues. These comply with the principle of membrane-bound.(5) To optimize the fermentation conditions by the different culture conditions, medium composition, the use of PB test and response surface methodology, the best conditions is glucose2.00%, yeast extract1.02%and inoculum2.06%. After the optimization, the titer of plantaricin ZJ5increased from474IU/ml to872IU/ml.