| In the processe of cryopreservation food,always contaminated by some low temperature spoilage bacteria,producing heat-resistant proteases and lipases which can cause food spoilage.Lactic acid bacteriocins with the characteristic of natural,non-toxic and efficient antibacterial effect has become the focus of research and development in recent years.However,bacteriocins of Class I such as nisin and the majority of bacteriocins of Class II can only inhibit the activity of Grampositive bacteria instead of Gram-negative bacteria,which is the main source of low temperature spoilage bacteria.Therefore they can not be applied in low temperature food preservation.A plantaricin strain produced by Lactobacillus plantarum was isolated in experiments,which showed excellent antibacterial effect against low temperature spoilage bacteria represented by Pseudomonas fluorescens in the antibacterial tests.In this study,the plantaricin was further isolated and purified and its mechanism of prohibitive effect upon Pseudomonas fluorescens was explored also with the preliminary analysis of the synthesis of QS genes.The specific research result of experiments are as follows:The plantaricin was purified rapidly using the tow-step method.Firstly,the fermentation liquor was purified roughly by SP Sepharose Fast Flow column,and then active fractions were collected and concentrated,with the purification multiple of 133.11 and the yield of 2%.Second,the collected active fractions were further purified by Superdex Peptide 10/300 GL gel column.After two steps of purification,the active fractions were collected and concentrated,reaching the purification mulitiple of 207.8 and the yield of 0.2%.The purity and molecular weight of colleceted active samples were determined preliminarily by Tricine SDS- PAGE,was about 4.6k Da.There was a single absorption peak at 15.341 min,which indicated the high purity of samples.The accurate molecular of 4.88 k Da weight was further determined by LC-ESI/MS.And the amino compositions of purified samples were analysed by amino acid analyzer and the results showed that the plantarcin was a kind of hydrophobic peptide,which provided the foundation for further research of structures and properties.The antibacterial mechanism of plantaricin was investigated mainly in five ways,including the effect on the growth curve of Pseudomonas fluorescens,the changes in cell morphology,the effect on the DNA of bacteria and the damaging degree of cell membrane and bacteria biofilms.The growth curve of plantaricin at different concentrations indicated that the plantaricin at a concentration of25.6μg/mL was able to inhibit the growth absolutely.The antibacterial effect of the plantaricin was realized by binding with intracellular,deducing by streptomycin control.Scanning electron microscopy showed that cell morphology changed significantly after treating with the plantaricin,the rupture of cell membrane and the leakage of extracellular matrix can be seen.It was observed that PI fluorescence absorption intensity enhanced gradually with the increase of plantaricin through the results of Flow cytometry.When the concentration of plantaricin reached25.6μg/mL,the absorption intensity was a near constant.According to the results,it was concluded that plantaricn inhibited the growth of bacterial by damaging cell membrane.The DNA damaging test showed that the DNA bands did not disappear,diffuse or smear, but with the increase of the concentration of plantaricin,strips gradually fades,indicating that the plantaricin had damage effect on DNA,the mechanism needs further exploration.According to the low yield of pantaricin.It was reported that QS system could regulate the synthesis of bacteriocins.The results of screening of primers and the sequence of PCR product concluded that the plantaraum contained the QS gene of regulating the synthesis of palntaricin. |
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