Purification Of Antimicrobial Components Produced By Paenibacillus Sp.Strain BD3526 And Study On Its Antibacterial Mechanism

As more attention has been paid to food safety issues,it is expected earnestly tofind a new kind of natural preservative to replace those chemical ones.Since Nisin wasapproved as high safety food preservative,bacteriocins research has become an activefield in the study of microbiological research.Bacteriocins are a class of protein orpeptide substances produced by microorganisms through ribosomal mechanism in theprocess of metabolism.Based on their high efficiency,non-toxic,no residue,and noresistance,bacteriocins have been the focus in the study of biological food antisepticand are considered to be the most effective alternative to antibiotics.At present,researches on bacteriocins mainly involve in screening bacteriocin-producing strain,optimization of culture conditions,isolation and purification ofantibacterial substances,structure identification and antibacterial mechanism.Thepresent studies on species of bacteriocin-producing strain are mainly concentrated inlactic acid bacteria rather than other species.In this experiment,Paenibacillus sp.Strain BD3526 is a new source used to screen out antibacterial substances.Therefore,the range of sources of bacteriocins has been expanded and it is hoped thatPaenibacillus sp.could become important sources in the isolating of efficientbroad-spectrum bacteriocins in the future,which might be applied in food and otherindustries.BD3526 strain is a new kind of Paenibacillus,which wasseparated from the yak milk in Tibet.BD3526 was cultured in TYC medium and the fermentation supernatant had significant antimicrobial activity on Staphylococcus aureus,Micrococcus luteus,and Listeria monocytogenes,it was found that fermentation supernatant had unstable antibacterial activity,which might be influenced by temperature,time,oxygen content and other factors,while the Paenibacillus sp.BD3526 itself had strong antimicrobial activity,which had good repeatability Thus,we choose BD3526 cells as the source toprepare crude extract,which would be used to separate antibacterial substances in the following study.Crude antimicrobial substances were extracted using organic solvent.Set antagonistic bacteria flat at 4 ?for 6 d and extracted with acetone.The minimum inhibitory concentrations(MIC)of the crude extracts against Micrococcus luteus,Staphylococcus aureus and Listeria monocytogenes were 12.5,25 and 25 mg/m L,respectively.Then the crude antibacterial substances were preliminarily purified By Sephadex LH-20.The minimum inhibitory concentrations of Sephadex LH-20 purified samples against Micrococcus luteus,Staphylococcus aureus and Listeria monocytogenes were 1,2 and 2 mg/m L,respectively.After Sephadex LH-20 separation,MIC values of the sample reduced 12.5 times which indicated the sample had been purified effectively.For further purification,RP-HPLC was used with methanol-water system to get more pure active ingredient.In thermal stability experiment,samples were treated respectively in 50 ?,60 ?,70 ?,80 ?,and 90 ? for 2 h,100 ? for 15 min,and 121 ? for 5 min.Compared with the control group,the activity of the treatment group had no significant difference,indicating samples had good thermal stability.In order to the optimum p H of the active ingredient,samples were dissolved in buffer solutions with different p H and,that in p H 7 was choosed as control samples.The results showed that the activities of samples were inhibited in acidic mediums while the highest activity was obtained in the neutral or slightly alkaline conditions.In enzyme sensitivity experiment,samples were treated by trypsin,pepsin,lipase,and proteinase K and the activity did not changed.After digestion by pronase,the activity disappeared.Within 0?18 h,the maximum inhibition rate of sample without enzyme processing was 90.43%while that was 17.45% after digestion by pronase.In antibacterial mechanism experiment,at the first,research on the effect of different sample concentrations on the Micrococcus luteus growth curve showed that samples could increase the lag phase of Micrococcus luteus.At low concentrations,the mode of action was inhibition while the mode of action was sterilization at high concentrations.Then,analysis of relative viability of Micrococcus luteus by LIVE/DEAD Baclight Bacterial Viability Kits,the results showed viability level of Micrococcus luteus is 54.38% after disposed by samples,while the control group is 98.52%.Finally,the detection of extracellular nucleic acid and protein,showed that the samples may destroy the bacterial cells by inducing the cell membrane forming holes and then causing leakage of cell contents.