Study On The Preparation, Application And Antibacterial Mechanism Of Tartary Buckwheat Antibacterial Peptides

Tartary buckwheat is a special kind of edible and medicine crop with high research value in China,because it has comprehensive nutritional content and rich protein content.Antimicrobial peptides are peptide antibacterial substance.They have small molecular weight,good solubility,broad antibacterial spectrum,strong antibacterial activity,good selectivity and not easy to produce drug resistance which can be used as a biological preservatives used in the food industry.In this study,Tartary buckwheat was used as raw material,Aspergillus sojae was selected for solid-state fermentation to prepare antimicrobial peptides.The optimization of fermentation conditions,purification of fermentation products and preservation of beef were studied.The main research contents and results are shown as follows:1.Aspergillus sojae was used for the solid state fermentation of tartary buckwheat.The peptide concentration and antibacterial rate were used as indicators.On the basis of single factor,Box-Behnken test was used to optimize the preparation process of antimicrobial peptides.The optimum conditions were as follows:inoculation amount 2.47 mL,fermentation time 39.84 h,glucose addition amount 0.98%,water amount 6.04 mL,peptide concentration prediction value29.12 mg/mL.Under the optimal conditions,the validation test was performed.The peptide concentration was 30.64 mg/mL,which was 9.47%higher than that before optimization?27.99mg/mL?.The antibacterial activity against Staphylococcus aureus was 94.44%,which was higher than that before optimization?87.81%?increased by 7.55%,while the antibacterial rate against E.coli was 19.09%,which was 87.71%higher than that before optimization?10.17%?.2.The beef was stored at 4? coated by different concentrations of tartary buckwheat antibacterial peptides?5,10,and 25 mg/mL?,while Nisin was a positive control.The Sensory evaluation of beef,total volatile base nitrogen?TVB-N?,thiobarbituric acid?TBA?,surface color,texture,and total number of colonies were used as evaluation indicators of freshness.The results showed that the sensory evaluation score of the control group was lower than 4 points when stored for 6 days,and the sensory evaluation score of 10 mg/mL Nisin and 5 mg/mL of tartary buckwheat antimicrobial peptide was lower than 4 points after the 9th day.The sensory evaluation of 10 mg/mL and 25 mg/mL tartary buckwheat antibacterial peptide was less than 4 points after 12days;The TVB-N results showed that the TVB-N values of the 10 mg/mL and 25 mg/mL Tartary Buckwheat antimicrobial peptides did not exceed the standard during the entire storage period,while the the control group exceeded after 12 days.Changes in TBA during storage showed that the effect of the 10 mg/mL Tartary Buckwheat antibacterial peptide treatment group was significantly better than that of 10 mg/mL Nisin?p<0.05?.On the 12th day of the control group,the total number of colonies exceeded 6.0 lg?cfu/g?,belonging to corrupted fleshy.During the entire storage period,the total number of colonies in each treatment group did not exceed 6.0 lg?cfu/g?.In conclusion,the beef preservation period of the control group was 3 days,10 mg/mL Nisin and 5 mg/mL tartary buckwheat antibacterial peptide was 6 days,and 10 mg/mL and 25mg/mL tartary buckwheat antibacterial peptides was 9 days.3.The buckwheat antibacterial peptide solution under the best fermentation conditions was ultrafiltered,peptides less than 5 kDa were collected.After further separation and purification by Sephadex G-25,three highly active antibacterial peptide sequences were sequenced and screened for the highly active elution peaks by shot-gun mass spectrometry:peptide 1(QPEDPR0,peptide2?KETHDHP?,peptide 3?KILISGIIRG?.4.In this experiment,three Tartary Buckwheat antimicrobial peptides were synthesized by chemical synthesis and their antibacterial activity,hemolytic activity and antibacterial mechanism were studied.The minimum inhibitory concentrations?MICs?of peptide 1 against Staphylococcus aureu,E.coli,Shigella sonnei,Listeria monocytogenes,and Salmonella enteritidis were 15.625mg/mL,250mg/mL,31.25mg/mL,125mg/mL,and 125mg/mL,respectively;peptide 2 were15.625mg/mL?62.5mg/mL?62.5mg/m L?125mg/mL and 62.5mg/mL,respectively;peptide 3were 15.625mg/mL?15.625mg/mL?15.625mg/mL?31.25mg/mL and 62.5mg/mL,respectively.The MIC of peptide 1 and peptide 2 against Candida albicans was 3.9?g/mL.The hemolytic rates of peptide 1,peptide 2 and peptide 3 at high solubility?500?g/mL?were 1.49%,1.79%and 1.31%,respectively.The antibacterial mechanism of antimicrobial peptides was investigated by?-galactosidase induction,calcein leakage,cell membrane integrity,and K⁺leakage.?-galactosidase induction test results show that buckwheat antibacterial peptides are more disturbing to Escherichia coli(The OD4₂₀ range was 0.0061 to 0.011 at 190 min)than S.aureus.(The OD4₂₀ range was 0.0024 to 0.0031 at 190 min).Peptide 3 showed the highest release of calcein from Staphylococcus aureus?78.5%?in the calcein leak experiment.Peptide 3 was able to fully bind to E.coli and S.aureus genomic DNA?peptide to DNA mass ratios of 50 and 30,respectively?and DNA was completely blocked in the spotted wells.