Study On The Preparation Of Peptide With Calcium-binding Activity From Carp Egg And Its Mechanism Of Binding Calcium

In this thesis, fish egg peptides (FEP) with calcium-binding activity wereprepared from carp eggs through enzymatic technology and the parameters of FEPbinding calcium reaction were optimized to prepare peptide-calcium complex(FEP-Ca). The effects of FEP-Ca on increasing calcium bioavailability were furtherstudied in calcium-deficiency rats. A novel peptide was purified and identified toexplore the mechanism of the peptide binding calcium. The details of the work wouldbe shown as follows:1. Preparation of FEP through enzymatic technology from carp eggsCalcium-binding activity and degree of hydrolysis (DH) were adopted as indexto optimize the enzymatic technology. The trypsin showed the highest ability toprepare peptides with calcium-binding activity from carp eggs. Dephosphorizationtreatment of carp eggs could significantly increase the degree of hydrolysis. However,excessive dephosphorization was disadvantageous to calcium-binding activity. Theoptimization of enzymatic conditions for preparation calcium-binding peptide was asfollows: the degree of dephosphorization of carp eggs30.39%, trypsin dosage3000U/g, substrate concentration2%, pH9.0, temperature49℃and hydrolysis time12h.The DH and calcium-binding activity of the hydrolysate reached31.15%and0.67mmol/g.2. Preparation of FEP-Ca complex and its stabilityBased on binding rate, The conditions of FEP binding calcium were optimized asfollows: FEP concentration5g/L, calcium concentration5mM, temperature45℃, pH8.0and reaction time1h. In these conditions the binding rate of FEP was86%. Thecontents of phosphorus, nitrogen and calcium were3.78,11.15,7.27%respectivelyand the molar ratio of phosphorus to nitrogen was6.53.FEP-Ca complex owned good ability to resist pH with more than90%calciumretention rate at the pH range of5-8. After heated at121.1℃for15min, the calcium retention rate was as high as71.33%. FEP-Ca complex also could resist digestion bypepsin and trypsin with89.69%calcium retention rate after treated with the twoenzymes. However, the calcium retention rate significantly decreased as the additionof phosphate because phosphate can compete with FEP to bind calcium.3. Effect of FEP-Ca on enhancing calcium bioavailability in vivoIn vivo the effect of FEP-Ca on increasing calcium bioavailability were studiedin calcium-deficiency rats. During the experimental period, calcium absorption and itsaccumulation in bone was significantly increased by FEP-Ca supplementation. Thelevels of serum calcium, bone mineral density, bone calcium content andbiomechanical properties of the FEP-Ca group were significantly higher than those ofCaCO3group (p <0.05), but similar to the CPP-Ca group (p>0.05). FEP-Ca isexpected to become a novel calcium nutraceutical additive in food industry due toenhancing Ca bioavailability by its intake.4. Purification and identification of calcium-binding peptideAfter ultrafiltration, the fraction with molecular weight <5kDa (U) was collectedand then purified with hydroxylapatite chromatography (HAC), H3eluted with themaximum concentration of phosphate buffer (400mmol/L) exhibited the highestcalcium binding ability of5.03mmol/g. Amino acid content analysis showed that theSer content of H3is about3times more than that of U but the contents of Thr andTyr are almost identical. H3was further purified using size exclusionchromatography (SEC) and reverse phase high-performance liquid chromatography(RP-HPLC), an oligophosphopeptide with the highest calcium binding ability (7.62mmol/g) was obtained. Its sequence was identified as (pS)S(pS)AF(pS)(pS)ELARthrough ESI-QTOF tandem mass analysis.5. Mechanism of peptide binding calciumThe peptide with the sequence (pS)S(pS)AF(pS)(pS)ELAR (IPP) wassynthesized for further study. MS and FTIR spectrum of IPP and IPP-Ca showed thatphosphate had the priority to binding calcium. Under physiological conditions onemole of IPP could bind four moles of calcium and carboxyl group could not bindcalcium. FTIR and CD spectrum further revealed that regardless of the presence calcium IPP was present in the state of unorded structure in solution underphysiological conditions, without any secondary structures such as α-helix or β-sheet.When pH reached to10with calcium concentration up to3mM, Ca2+boundphosphate and shielded negative charges of it, which made the formation of β-sheet inIPP via hydrophobic interactions and hydrogen bond. Then Ca2+served as asalt-bridge between carboxyl group and induce the formation of nanoparticles.However in the presence of other peptides, Ca2+could induce the peptides solution tofomate nanoparticles at the pH range of6-8.In the supersaturated solution of hydroxylapatite (HAP), IPP could compete withphosphate to bind calcium and adsorb to the surfaces of Ca-P nanoclusters andamorphous calcium phosphate granules (ACP), which inhibited the nuleation ofcalcium phosphate and the aggregation of ACP to crystal. IPP also could adsorb to thesurfaces of calcium phosphate crystals, thus inhibited the growth and precipitation ofcrystals.