Preparation And Antibacterial Activity Of Antibacterial Peptide From Walnuts(Juglans Regia L.)

Walnut dreg which is generated from the pressing process is fed to animals resulting in alow value-added.Walnut dreg which isriched in abundant protein is of great significance to deepen application of walnut dreg in highly processed products with high biological activity.The research is aimedat obtaining the walnut antibaclerial peptidethoughhydrojysisand purify the peptide to determine aminoacidsequence and protein information.After that,the antibacterial mechanism and bacteriostatic stability of antimicrobial peptide were to preliminarily explore.The contents and results are as follows:(1)Optimize the preparation process of walnut antibacterial peptideThe optimal protease was screened taking the inhibitory activity of enzymolysis solution as an important indicator,In the current research,pepsin hydrolyzed peptide showed a certain inhibitory effect against Staphylococcus aureus,Escherichia coli and Bacillus subtilis.Response surface methodology was designed to optimizetle optimum conditions f'or the optimization of enzymatic hydrolysis of walnut protein by pepsin with the degree of hydrolysis as the evaluation index.The results showed that the optimal conditions were at temperature=54.11?,pH?3.46.enzymes addition 5475.71 U/g,to obtain the best degree of 13.99%.(2)Separation and purification of walnut antibacterial peptideIn the current research,enzyinolysis solution was separated into three size of moleclar weight(3KDa?10KDa and 30KDa)with ultrafiltration in order to determine the bacteriostatic activity,respectively.The 3-10KL)a(H-II)componcnt which showed the best result of bacteriostatic activity was separated and purillcd byreverscd-phase high-performance liquid chroxnatography to obtain seven peptide components(H-II-1-H-II-7)of which the component H-II-6 had the best antibacterial effecton Bacillus subtilis,Escherichia coli and Staphylococcus aureus.(3)Sequence of amtimicrobial peptide H-II-6The molecular and the sequence information of antimicrobial peptide H-II-6 hasbeen identified by matrix-assisted laser desorption tandem time-of-flight mass spectrometry,quadrupole-orbitrap liquid chromatography-mass spectrometry and the website of Sprot which can provide the data of peptide.The results showed that the molecular mass of antimicrobial peptide H-II-6 was 3772.7359Da,the sequence is presumed as Thr-Gly-Ger-Gla-Val-Pro-Ger-Pro-Arg-Gla-Ger-Gla-Thr-Gla-Thr-Met-Glu-Met-Gla?Gla-Gla-M et-Gly-Leu-Met-Pro-Gly-Ger-Pro-Ger-Ger-Val-Ger-Gla-Val-Met-Ger-Pro-PheHydrophobic amino acids includingproline,alanine,leucine,phenylalanine and valine,which account for closely 50%of the total alnino acids and 39 amino acids in length.The seque flce number of the corresponding protein is Q10EL1.(4)Antibacterial stability and mechanism of antibacterial peptide H-II-6The antibacterial stability of H-II-6 antibacterial peptide under ultraviolet irradiation,different pH and temperature was studied.At the same time,the mechanism of H-II-6 antibacterial peptide against three bacteria was explored from the cell morphology.The results showed that pH in the range of 5.0 to 9.0,with the increasingof pH,antibacterial activity of H-II-6 antibacterial peptide decreased,the difference ot inhibition zone diameters were significant(P<0.05);the temperature range of 40 to 121?,there was no significant difference in the antibacterial activity of H-II-6(P>0.05).The inhibitory effect of H-II-6 on UV-irradiation showed no significant change(p>0.05).Therefore,antimicrobial peptide H-II-6 has good bacteriostatic stability against UV and temperature.The results also showed the H-II-6 hasdifferent degrees of surface depression,breakage,and cell disruption,so as to inhibite the bacterial growth.