Idenfication,Purification And Characterization Of The Mode Of Action Of Plantaricin PZJ008

At present, China is still extensive use of chemical preservatives, with the raising awareness of chemical preservatives are harmful, it is necessary to look for a natural preservative instead of chemical preservatives. Nisin is one of natural food preservative and it was recognized by global, but Nisin just can inhibit Gram-positive bacteria and cannot inhibit Gram-negative bacteria, therefore, it is very important to purify and characterize the other bacteriocins. A strain of lactic acid bacteria showed antibacterial activity was isolated from fresh raw milk, the main research are as follows:(1) It was identified as Lactobacillus plantarum on the base of physiological and biochemical characteristics and16S rDNA gene sequences, named as Lactobacillus plantarum ZJ008, the bacteriocin named plantaricin PZJ008(2)The bacteriocins are mostly small, hydrophobic and cationic peptides small peptides, we designed three-step purification based on bacterioncins characterization and the conditions of laboratory equipment:①Partial purification of bacteriocin by XAD-2, then collected70%eluation and concentrated. The molecular weight was estimated to be less than4.0kDa by SDS-PAGE;②) Further separated by SP and Superdex PP;③Purification of high purity bacteriocins by reverse-phase HPLC (C18), the molecular weight (2612.4Da) of bacteriocin PZJ008wsa confirmed mass spectrometry;(3) PZJ008showed the strongest antibacterial at pH4-5, PZJ008almost complete loss of antimicrobial activity when pH>8. PZJ008(pH4.5) was heat tolerant and remained active after30min at different temperatures. Bacteriocins ZJ008is sensitive to trypsin, pepsin, a-chymotrypsin, papain, and not sensitive to proteinase K, lysozyme lipase, ribonuclease A, a-amylase.(4) The antibacterial activity of PZJ008was tested, PZJ008displayed a very strong inhibitory activity against gram-negative bacteria and some gram-positive bacteria,such ad M.luteus, S. carnosus, S.citreus, S. aureus, L. monocytogenes, S.Epidermidis, E.coli, P.aeruginosa, PZJ008is a broad spectrum barteriocin. Minimum inhibitory test proved that the bacteriocin PZJ008most sensitive to Staphylococcus aureus, the MIC is33.1IU/mL.(5)Through the restore growth methods by observed indicator strain, we found the mode of action of bacteriocin PZJ008is sterilization. By measuring the indicator bacteria intracellular K+and leakage of nuclear substances,we found the bacteriocin PZJ008can induce massive leakage of K+from the cells in a very short time,it leads 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.