Separation And Identification Of Antimicrobial Fractions From Pickled And Dried Mustard And Its Preservation Effect On Minced Pork

With the improvement of people’s living standards,more and more attention has been paid to food safety issues.The development of green and safe food additives from natural products has become a research hotspot for scientists.In this paper,the pickled and dried mustards were used as experimental materials,and the common spoilage bacteria and pathogenic bacteria in food were used as test microorganisms.The diameter of inhibition zone,the minimum inhibitory concentration(MIC)and the minimum bactericidal concentration were used as the activity monitoring parameter.The antibacterial substances from pickled and dried mustard were extracted,isolated and identified in this thesis.The antibacterial activity,antibacterial mechanism of the antibacterial fraction and its preservative effect on minced pork were investigated.This research provides a theoretical basis for the better utilization of the pickled and dried mustard and the development of natural antibacterial agents.The main results were shown as follows:1.Antibacterial activity of the ethyl acetate extract of pickled and dried mustard was inveatigated.The antibacterial activity of ethyl acetate extracts against spoilage bacteria and pathogenic bacteria commonly found in pork was determined by tube-disc method and double dilution method.The results showed that the antibacterial activity of the ethyl acetate extracts against the tested strains has the following order:Staphylococcus aureus>Pseudomonas fluorescens>Listeria monocytogenes.Since the ethyl acetate extracts has weak antibacterial activity against Listeria monocytogenes,Staphylococcus aureus and Pseudomonas fluorescens were selected as tested strains for the following experiments.2.Isolation,identification and antibacterial activity of the ethyl acetate extract of the pickled and dried mustard were performed.Silica gel column chromatography was used to separate the ethyl acetate fraction.The antimicrobial activity of obtained samples against tested strains were determined by the tube-dish method and double dilution method.The structure of the highly antibacterial fraction was identified by UV,FT-IR,HPLC,1H NMR,13C NMR and GC-MS.The ethyl acetate fraction was separated into 7 sub-fractions,with the third fraction(Fr.3)possessing the strongest antimicrobial activity against Staphylococcus aureus and Pseudomonas fluorescens,and the MICs were 0.3125 mg/mL and 0.625 mg/mL,the MBCs were 0.3125 mg/mL and 0.625 mg/mL,respectively.The Fr.3 was identified as succinic acid.3.The antibacterial mechanism of succinic acid against Staphylococcus aureus and Pseudomonas fluorescens was nivestigated.The antibacterial mechanism was explored mainly by measuring the time-kill curve,intracellular component leakage,cell size and transmission electron microscopy of Staphylococcus aureus and Pseudomonas fluorescens treated with succinic acid.The results showed that after treatment with succinic acid,the cells of the tested strains were shrunk,the permeability of the cell membrane increased,or fine pores were formed,which caused the cell contents released,thereby inhibiting or killing microorganisms.4.The preservative effect of succinic acid on raw minced pork was investigated.The raw minced pork was treated with 0.25%,0.5%,1%succinic acid,0.1%potassium sorbate,and 0.5%succinic acid+0.1%potassium sorbate,respectively.The preservative effect on raw minced pork was evaluated by measuring the total bacterial count,instrumental color(L,a*,b*values),pH and TVB-N value.Results showed that groups treated with 0.5%and 1%succinic acid can prolong the shelf life of raw minced pork at 4℃ for 4 and7 days,significantly better than 0.1%potassium sorbate treated group;combined treatment with 0.5%succinic acid and 0.1%potassium sorbate had a synergistic effect,and the total number of colonies would still be within the standard of second grade fresh meat when stored at 4℃ for 15 days.However,the addition of succinic acid has an adverse effect on the color of raw minced pork,and the higher the concentration of succinic acid,the greater the effect on the color of raw minced pork.