| Half-fin anchovy(Setipinna taty),one of the important species of edible mairne fish in China,is rich in nutrients such as protein,carbohydrate,inorganic salts and various mineral elements.However,the research of half-fin anchovy is relatively limited due to its low economic and lack of further processing values.In our previous studies,we reported that the half-fin anchovy hydrolysates(HAHp)prepared by the method of pepsin hydrolysis demonstrated strong antibacterial effects.Moreover,the antibacterial activity of HAHp after Maillard reaction modification was further improved against the growth of Escherichia Coli(E.coli).In the E.coli model,a large amount of hydrogen peroxide(H2O2)was produced after incubation of the Maillard reaction products of HAHp and glucose(named as HAHp(9.0)-G MRPs)at 37? for 3 h as compared with that in the control group.Based on our previous results,the purpose of this paper is to(1)investigate the antibacterial mechanism of HAHp(9.0)-G MRPs by inducing H2O2 production,(2)to separate and identify potential antibacterial peptides from the HAHp(9.0)-G MRPs,(3)to isolate and characterize antibacterial peptides from the HAHp(9.0)-G MRPs by membrane-bind method and(4)to evaluate the interaction of HAHp(9.0)-G MRPs and catalase(CAT).In the E.coli model,the potential H2O2 formation induced by addition of HAHp(9.0)-G MRPs was investigated during the process of E.coli growth,as well as the inhibiton effects against E.coli was evaluated.The results showed that the HAHp(9.0)-G MRPs still showed antibacterial effect on E.coli after addition of CAT.However,the H2O2 formation was detected during the growth of E.coli after addition of HAHp(9.0)-G MRPs,and the total H2O2 production in E.coli cells demonstrated a consistence trend with the inhibiton effects of HAHp(9.0)-G MRPs.The antibacterial effect of HAHp(9.0)-G MRPs on the logarithmic growth phase of E.coli cells was further investigated.Results showed that the addition of HAHp(9.0)-G MRPs in logarithmic E.coli cells induced H2O2 formation.After addition of CAT,the inhibition rate of HAHp(9.0)-G MRPs against E.coli was decreased from 79.26±0.01%to 69.71±0.04%(p<0.01).Also,significantly decreased H2O2 content was found after CAT addition.Based on the results of HAHp(9.0)-G MRPs on the antibacterial activity of logarithmic E.coli cells,potential correlations between the inhibition rate and intracellular oxidase and lipid peroxide(LPO)content of E.coli cells were further determined.The inhibition effect of HAHp(9.0)-G MRPs on logarithmic growth phase of E.coli cells had a linear positive correlation with the LPO content of E.coli(r=0.801,p<0.01);while the LPO content of E.coli displayed a linear positive correlation with the extracellular H2O2 content(r=0.664,p<0.05).These results suggest that the induced H2O2 production should play a crucial role for the bacteriostatic action of HAHp(9.0)-G MRPs.HAHp(9.0)-G MRPs were fractionated by size exclusion chromatography(SEC)into three major fractions(F1-F3).F2,which demonstrated the strongest antibacterial activity against E.coli and showed self-production of H2O),was extracted by SEC.The hydrophobic extract of F2 was further isolated by reverse phase-high performance liquid chromatography(RP-HPLC)into sub-fractions HE-F2-1 and HE-F2-2.Nine peptides were identified from HE-F2-1,and two peptides from HE-F2-2 using liquid chromatography-electrospray ionization/multi-stage mass spectrometry(LC-ESI-MS/MS).Considering the number of amino acid residues,the average confidence of the residues,the confidence of a single residue,the hydrophobicity,and the charge,three peptides,FEDQLR(HGM-Hp1),ALERTF(HGM-Hp2),and RHPEYAVSVLLR(HGM-Hp3),with net charges of-1,0,and+1,respectively,were synthesized.The minimal inhibitory concentration of these synthetic peptides was 2 mg/mL against E.coli.After HGM-Hp1 and HGM-Hp2 were incubated with E.coli in logarithmic growth phase,the results showed that the amount of H2O2 produced by extracellular and intracellular E.coli was significantly increased,and HGM-Hp3 only significantly enhanced E.coli cells.The addition of HGM-Hp1 or HGM-Hp2 increased the potassium ion(K+)content in the E.coli suspension,indicating the destruction of cell integrity via irreversible membrane damage possibly by inducing H2O2 production in E.coli cells.It is report of hydrolysates MRPs-derived peptides that might perform the antibacterial activity inducing intracellular H2O2 production.HAHp(9.0)-G MRPs were added with the logarithmic growth phase of E.coli,incubated at 37? for 1 h,3 h and 5 h,and followed by measuring the absorbance of samples at 630 nm.The results showed that the inhibition rate of sample at 3 h was 95.23±0.32%,significantly higher than those samples of 1 h and 5 h after incubation of HAHp(9.0)-G MRPs.The high performance liquid chromatography(HPLC)profiles of HAHp(9.0)-G MRPs were further evaluated based on molecular weight distribution after incubation of logarithmic E.coli cells at 37? for 1 h,3 h,5 h.Results displayed that the peak areas of F1,F2 and F3 fractions of HAHp(9.0)-G MRPs after incubation of E.coli for 3 h were significantly lower than those corresponding peak fractions of 1 h and 5 h samples.The supernatant of HAHp(9.0)-G MRPs after incubation with E.coli for 3 h at 37? was separated using RP-HPLC,measured at 220 nm and 280 nm.Compared with the original areas of A1,A2,A3 and A4 in HAHp(9.0)-G MRPs,some peaks of A4,the area with the strongest inhibition rate against E.coli,were decreased or disappeared.Therefore,the area of A4 in the HAHp(9.0)-G MRPs was collected to identify potential peptides using LC-ESI-MS/MS technique.More than 200 peptides were identified from the A4.Considering the intensity,hydrophobicity,pI,and net charge,156 peptides were used for cluster analysis based on their molecular weights.Results revealed that the A4 contained 8 positively charged peptides,70 negatively charged peptides and 78 neutral peptides.Furthermore,most of these identified peptides have strong hydrophobicity.In order to investigate the effect of CAT on the antibacterial mechanism of HAHp(9.0)-G MRPs,three sources of CATs including plant source(Ginkgo Biloba),animal origin(mice liver)and bacterial source(E.coli)were studied by in vitro simulated CAT interaction with HAHp(9.0)-G MRPs.Results found that the CAT derived from mice liver(174.70±8.26 U/mg prot)is more sensitive to the HAHp(9.0)-G MRPs than others.The crude CAT of mice liver was further separated into five frations(K1,K2,K3,K4 and K5)using salting-out,dialysis and DEAE Sepharose-CL 6B gel chromatography seperations.The fration of K5 with the highest activity of CAT(CAT-K5)(8737.70±23.45 U/mgprot)was collected and interacted with the HAHp(9.0)-G MRPs.The result of RP-HPLC separation indicated that CAT-K5 could combine with the HAHp(9.0)-G MRPs.Furthermore,the inhibition rate of HAHp(9.0)-G MRPs on logarithmic growth phase of E.coli was decreased from 78.44± 1.31%to 44.87 ± 5.78%after addition of CAT-K5 and incubation of 37? for 3 h.Furthermore,the extracellular H2O2 and intracellular H2O2 contents of E.coli cells were decreased from 48.96± 4.62 mmol/gprot to 25.55 ± 2.41 mmol/gprot,and 85.77 ± 2.76 mmol/gprot to 29.79 ± 3.84 mmol/gprot,respectively(p<0.05).By comparision,the CAT activity of CAT-K5 was increased from 66.17± 3.53 U/mgprot to 105.19± 7.33 U/mgprot after interaction with HAHp(9.0)-G MRPs.The PyMOL molecular simulation software was used to simulate possible binding sites of CAT with HAHp(9.0)-G MRPs.The result of simulation suggests that the active centers of alanine(Ala)75,asparagine(Asn)148 and proline(Pro)358 in CAT might be potential binding sites with peptides indentified from HAHp(9.0)-G MRPs.The binding interactions of peptides derived from HAHp(9.0)-G MRPs in CAT could contribute to enhance the activity of CAT,and increase H2O2 scavenging,and therefore result in decreaed inhibiton effects of HAHp(9.0)-G MRPs.These results futher suggests that the antibacterial mechanism of HAHp(9.0)-G MRPs should have associations with H2O2 formation. |
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