| Lactobacillus bacteriocin is a protein or a peptide that produces bacteriostatic activity during the lactic acid bacteria growth and metabolism.It has a broad spectrum of antibacterial activity with no residue in the human body and can be used as a natural preservative.Nisin has been widely used in the food industry.However,with the deepening of research,nisin has been found to have unstable bacteriostatic ability,narrow inhibition spectrum and short bacteriostatic efficacies,which limits its application in food.Application in the field of anti-corrosion.In order to prevent food spoilage and improve food safety and quality,it is urgent to develop a new type of biological preservative with a wide range of antibacterial activity.In this study,the lactic acid bacteria isolated and extracted from Jinhua ham were used as the research object.Firstly,the bacteriocin-producing lactic acid bacteria were screened by excluding organic acid,hydrogen peroxide and protease.The bacteriocin was separated and purified,and then the bacteriocin was purified.The product was identified and the physicochemical properties of the pure bacteriocin were studied,which laid the foundation for the development and application of the new lactic acid bacteria bacteriocin.The detailed research contents and results of this experiment are as follows:1.Screening and identification of broad-spectrum bacteriocin lactic acid bacteria was studied.Among the 162 strains of lactic acid bacteria selected and preserved from Jinhua ham,Escherichia coli and Staphylococcus aureus were used as indicator bacteria,and 159 strains were screened by double-layer agar plate method and turbidity method to inhibit the two indicator bacteria.Four strains of lactic acid bacteria with antibacterial activity were screened out by organic acid and hydrogen peroxide exclusion test.A bacteriocin-producing lactic acid bacteria was obtained by protease digestion test,which inhibited the grouta of Staphylococcus aureus and Escherichia coli.The diameters of the circles were 12.73±0.19 mm and 11.36±0.54 mm respectively.The colony morphology,physiological and biochemical characteristics of the combined strains and 16S rDNA molecules were identified the bacteria as Pediococcus pentosaceus,named Pediococcus pentosaceus Z-1.2.Isolation,purification and identification of bacteriocin produced by Pediococcus pentosaceus Z-1 was investigated.The bacteriocin was crudely extracted by pH adsorption method,and the bacteriocin was separated and purified by cellulose DEAE-52 ion exchange chromatography column and dextran G-50 molecular sieve.The results showed that after ion exchange with cellulose DEAE-52,the activity peak was obtained in PBS buffer(pH=7.4)containing 0.2 M NaCl,and further purified by Sephadex G-50 column to collect active peaks and obtain a single component of bacteriocin.Then Tricine-SDS-PAGE gel electrophoresis showed that the molecular weight of the bacteriocin band was between 4.6-10 kDa,which was classified into class ?a bacteriocin.The molecular weight of the bacteriocin was identified by time-of-flight mass spectrometry as 8227.35 Da.It was named Pediocin Z-1 with a purity of 96.62%and the amino acid sequence was MAITLKTELLDQK MTEVFDCSNDQTPLRDAMCNHVMDDNGHDTMKTIAEAKKWENMNDAE.3.The physical and chemical properties of Pediocin Z-1 was explored.Pediocin Z-1 had good acid-base stability between pH 2-10,and the diameter of the inhibition zone was 24.92±0.14 mm,while Nisin has antibacterial activity only in the acidic environment with pH<7.0.Pediocin Z-1 had an inhibitory effect from 50? to 110?,is sensitive to proteases,and was not sensitive to lipase and amylase(enzyme concentration at 1 mg/mL);it can inhibit Gram-positive bacteria and Gram-negative bacteria in comman foods,the maximum inhibition zone diameter 19.22±0.31 mm,and the mode of action was antibacterial;the results showed that the bacteriocin had a good application prospect in the food industry. |
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