Study On Antimicrobial Activity And Mechanism Of Protein From Ginkgo Biloba Seeds

Taking Ginkgo biloba seeds as material,the antimicrobial activity andmechanism of the purified proteins were studied, and its property, composition andstructure were analyzed.The results would provide a reference for the developmentof natural food preservative and the utilization of ginkgo seeds. The main resultswere as follows:(1)There were three kinds of antimicrobial substances existed in Ginkgoseeds,they were phenolic acids, polysaccharides and protein. Ginkgolic acidsconsists of C13:0、C15:1、C17:2、C15:0and C17:1which had a stronger antimicrobialactivity against E.coli, B.subtilis which were bacterias, and penicillium which wasfungi and the MIC were7.5mg/mL,15mg/mL,25mg/mL, respectively. Thepolysaccharide that is50～80%fraction of ethanol precipitation in Ginkgo seedswas a kind of glycoprotein compounds that contained87.4%polysaccharide and1.03%protein.Its molecular weight was about40kDa,and it has antimicrobialactivity against B.subtilis and the MIC was50mg/mL and it has almost no activityto fungis.The protein that is40～80%fraction of ammonium sulfate precipitationin Ginkgo seeds consists of at least17kinds of amino acids and it hasbroad-spectrum antimicrobial activity,and could inhibit the activity of somegram-positive and gram-negative bacterias, in which the activity is trongest toK.pneumoniae and the MIC was100mg/mL and the MIC also were100mg/mLagainst Penicillium, T.delbrueckii and A.niger which were fungis.(2) Phosphate buffer was used to extract Ginkgo biloba seeds protein(GBSP).Theextraction yield of protein was used as an index, RSM was performed to study theeffect of ratio of material to liquid,extraction concentration and extraction time onprotein yield rate. The results showed that solid-liquid ratio, extract concentrationand extraction time were highly significant influence (P<0.01) for protein extractionrate, and the interaction among them were significantly affected (P<0.05) on theextraction rate. And the optimum extraction conditons were as follows: ratio ofliquid to material20mL/g, extraction concentration0.07mol/L, and extraction time14.0h and under the condition the one-time extraction rate is up to86.58%, and theanalysis results was within the scope of credibility.(3) Taking K.pneumoniae and S.aureus which were bacterias and T.delbrueckii,A.niger which were fungis as tested strains, GBSP was purified by ammoniumsulfate precipitation, dialysis, DEAE-cellulose DE52ion-exchange and SephadexG-75gel filtration successively.The suitable saturation of ammonium sulfate was 40～80%, and purified protein was obtained named GBSP-A which has betterantimicrobial activity.The MIC of GBSP-A against above four strains were20mg/mL,20mg/mL,20mg/mL and12mg/mL, respectively. SDS-PAGE analysissuggested that the protein was homogeneous and its apparent molecular mass was41.8kDa. Schiff staining showed that the protein was a glycoprotein and the ratioof protein to polysaccharide was70:1. The results of chromatographicidentification by Superdex G-75and HPGPC showed a single and symmetrical peakand its molecular weight was39.316kDa.(4) The structure of GBSP-A was characterized. β-elimination reactiondemonstrated the existence of O-glycosidic linkage in it. IR analysis showedGBSP-A was a pyran glycoprotein compound contained α-and β-glycosidic bond.Congo red test showed that there was no three-helix in the polysaccharide chains ofGBSP-A. LC-MS-MS analysis indicated that GBSP-A had a high match rate with a11S-binding protein (ginkbilobin-2precursor)from ginkgo, and10matching peptidesequences were obtained. Amino acid sequence alignment of the peptide fragmentsby CAMP antimicrobial peptide database showed that GBSP-A was a newantimicrobial protein.(5) The studies about physical and chemical properties of GBSP-A showed thatthere was no starches, free polysaccharides and amino acids, having typicalreaction of protein and polysaccharide and UV absorption peak at280nm and220nm. GBSP-A had better stability of pH, temperature and NaCl. DSC analysisshowed that its thermal denaturation temperature was63.7℃.Amino acidcomposition analysis of GBSP-A suggested that it was consisted of16amino acidsat least, in which the content of Asp(7.269%）was the highest，the content ofhydrophobic amino acids was36.2%which play an important role in maintainingprotein stability.(6) Taking K.peneumoniae(G-) and S.aureus(G+) as tested bacterias,effect ofGBSP-A on the growth of bacteria was studied, the result showed that the growthof K.peneumoniae and S.aureuswas were inhibited by GBSP-A and the growthcurve was changed and different from the normal bacteria. SEM and TEM showedthat the wall and membrane of the tested bacterias treated by GBSP-A thawed,disappeard, and the cytoplasmic leakaged, and eventually the bacterials collapsedand leading to the cells death. Effect of GBSP-A on membrane permeability wasresearched and the results showed that GBSP-A could make the conductivity of thebacteria suspension rise, odium ions and other macromolecules leak, andextracellular β-galactosidase activity increase. GBSP-A could also make bacterialcell surface electronegativity and hydrophobicity increase and eventurally lead tothe cells flocculation and death.(7) Taking K.peneumoniae and S.aureus as tested bacterias, the respiratoryinhibition pathway of GBSP-A on bacteria was studied, and the effect of GBSP-A on the content of proteins, DNA, ATP and some related enzymes activity wasanalyzed. The results showed that the respiratory metabolism could be inhibited byGBSP-A, but the pathway was not the one of TCA, EMP and HMP.The synthesisof protein, DNA and ATP was suppressed, but the expression of any protein was notinhibited and DNA was also not ruptured. The content of ATP enzyme,β-galactosidase and AKP was reduced and the normal physiological metabolism ofthe bacterias was disturbed. The concentration of GBSP-A showed no significantdose-effect relationship with the inhibitory effect.