Study Of Purification And Antibacterial Ability Of Peptides Extracted From Xuanwei Ham

Xuanwei ham,Jinhua ham and Rugao ham are the famous Chinese traditional dry-cured hams.The ripening period of dry-cured ham can be as long as more than 6 months.Proteins in dry-cured ham are largely hydrolyzed by endogenous enzymes during the process of fermentation to produce many free amino acids and peptides.These hydrolysis products may have bioactive function.The previous studies from our lab have shown that antioxidant peptides and antihypertensive peptides could be present in Jinhua dry-cured ham and Xuanwei dry-cured ham.However,there is no reports about the antimicrobial ability of crude peptides extracted from Chinese traditional dry-cured ham.In this study,the comparative analysis about the bioactivity of crude peptide extracted from Xuanwei ham,Jinhua ham and Rugao ham were discussed.Then the antibacterial capacity of the peptides extracted from Xuanwei ham was measured under different conditions and the change of bacterial cell permeability was explored.Furthermore,antibacterial peptides were isolated and purified by ion exchange chromatography,dimensional exclusion chromatography,and the peptide sequences were obtained by mass spectrometry.The specific research contents and conclusions are as follows:1.The comparative study on the antioxidant and antibacterial ability of crude peptide extracted from Xuanwei ham,Jinhua ham and Rugao hamIn this study,the antioxidant activity and the antibacterial activity of crude peptide extracted from Xuanwei ham,Jinhua ham and Rugao ham were investigated The results showed that among the three kinds of dry-cured hams,crude peptide extracted from Xuanwei ham had the highest peptide content(67.67±4.65%)and the lowest salt content(7.46±0.68%)(P<0.05).In evaluation of antioxidant capacity,crude peptide from Xuanwei ham had stronger antioxidative abilities of FRAP,ABTS and ORAC than those of the other two dry-cured hams(P<0.05).At the concentration of 2.0 mg/mL,crude peptide from Xuanwei ham had the weaker DPPH free radical scavenging ability compared to that of the other two dry-cured hams(P<0.05).At 5.0 mg/mL,DPPH free radical scavenging ability of crude peptide from Xuanwei ham was 43.40±2.67%,which was significantly higher than that of Rugao ham(P<0.05).In evaluation of bacteriostatic ability,the inhibitory capacity of E.coli 0157 of crude peptides from Xuanwei ham and Rugao ham were significantly higher than Jinhua ham at 0.5-1.5 mg/mL(P<0.05).At 4.0 mg/mL,the inhibitory capacity of L.monocytogenes of crude peptides from Xuanwei ham and Rugao ham were significantly higher than Jinhua ham(P<0.05).Amino acid analysis showed that the content of Leu,Val,Pro,Ala,Trp and other hydrophobic amino acids in crude peptides from Xuanwei ham reached 8.06 g/100 g which was significantly higher than that in other two dry-cured hams(P<0.05).This result could partly explain the higher antioxidant and antibacterial ability of Xuanwei ham.2.Study on the antibacterial activity stability and antibacterial mechanism of crude peptide extracted from Xuanwei dry-cured hamThe stability of inhibitory capacity of E.coli 0157 of crude peptide extracted from Xuanwei ham and the effect of crude peptide on the permeability of E.coli 0157 and L.monocytogenes were studied.The results showed that the bacteriostatic ability of crude peptide extract was affected by temperature.At 25-40?,the bacteriostatic ability of crude peptide extract was increased.At 40?,the bacteriostatic ability was the strongest(43.71±4.12%),which was significantly higher than that of other temperature treatments(P<0.05).At 40-80?,the bacteriostatic ability decreased with the increase of temperature.When the temperature of 40? was maintained,the antibacterial ability of crude peptide extracts decreased gradually with the increase of heating time,and there was no significant difference between 0,5,10 and 15 min treatments(P>0.05).In addition,both acidic and alkaline conditions had a significant effect on the antibacterial ability of crude peptide.The antibacterial ability of crude peptide at pH 7 was the highest(P>0.05).Besides,the inhibition value of pH 7 was significantly higher than that at pH 3 and pH 11(P<0.05).Under the condition of ultraviolet irradiation,the antibacterial activity was enhanced.The antibacterial ability tended to be stable after 60 min irradiation,and the antibacterial effect(63.07±4.68%)was significantly higher than that no ultraviolet treatment(P<0.05).The analysis of the FCM and the loss of nucleic acid and protein showed that crude peptide extract could destroy the cell membrane of E.coli 0157 and L.monocytogenes,while the treatment time of crude peptide solution had no significant effect on the destruction of bacterial cell membrane.The content of alkaline phosphatase showed that crude peptide extract could also affect the integrity of bacterial cell wall and higher concentration of crude peptide extract showed the better bacteriostatic effect.Moreover,the crude peptide had stronger destructive effect on E.coli 0157 than L.monocytogenes.3.Purification and identification of antibacterial peptides extracted from Xuanwei dry-cured hamIn this part,the crude peptide of Xuanwei ham was separated by ion exchange chromatography and desalted by size exclusion chromatography.The efficient antibacterial activities of the separated components were gathered and measured.The ability of component 7 possessed higher inhibitory capacity of E.coli 0157(98.72±1.17%)and L.monocytogenes(56.74±4.91%)than other fractions(P<0.05).Therefore,the three peptides with the highest antibacterial activity were identified by Nano-LC-ESI-MS/MS and the peptide sequences were determined as QYYNGEEHVRFDSDVGEYR,LRNLPNLEVLDLGTNFI and FASFEAQGALANIAVDK.The results showed that all three peptides had strong ability to inhibit E.coli 0157 and L.monocytogenes.The inhibitory capacity of three peptides to inhibit E.coli 0157 was not significantly different(P<0.05).The inhibitory capacity of QYYNGEEHVRFDSDVGEYR to inhibit L.monocytogenes was significantly stronger than LRNLPNLEVLDLGTNFI and FASFEAQGALANIAVDK(P<0.05).