Study On The Purification And Bioavailability Of Calcium-binding Peptides From Tilapia Scale Protein Hydrolysate

With the rapid development of fish processing in China, byproducts, like scales,haven’t been highly utilized today. In this paper, calcium-binding peptides wereprepared from Tilapia scale by controlled enzymatic technology, and a novel peptidewas purified and identified, which could be used to explore the relationship betweencalcium binding capacity and the amino acid sequence of peptide. Otherwise, theeffects of calcium-peptide complex on calcium absorption and bone quality of ratswere investigated, which contributed to clarify the bioavailability and safety of fishscale peptide-calcium compound in vivo. The details of the article are as follows:1. Optimization of enzymatic hydrolysis conditions for preparation of calcium--binding peptide.Based on calcium binding capacity and degree of hydrolysis (DH), pepsin, trypsinand flavourzyme were used alone or in combination to hydrolyze fish scales. Thepeptide that obtained from hydrolysates produced by pepsin, trypsin and flavourzymesequentially showed the highest calcium binding capacity (1.04mmol/g) with the DHof22.24%. The optimization of enzymatic hydrolysis conditions for preparationcalcium-binding peptide were as follows: the order of the addition of enzyme waspepsin, trypsin and flavourzyme, and the suitable enzyme dosage and hydrolysis timeof the three proteases were2.0%,2.5%,1.0%and4.5h,5.0h,4.5h, respectively. TheDH and calcium binding capacity of the hydrolysate reached28.27%and1.33mmol/g, increasing27.11%and27.88%respectively, compared to those of originalcondition. The main hydrolysis components were confirmed to be small peptides withmolecular weight under3kDa by HPLC.2. Purification and identification of calcium-binding peptide.The hydrolysate of scale was collected and further purified by hydroxyapatiteaffinity chromatography (HA), and F3fraction with the strongest calcium bindingactivity (2.6mmol/g) was gathered, which increased by95.18%compared with that of the origin condition. Gel chromatography was taken to separate the F3fraction andtwo peaks were collected, of which F31showed higher activity (2.84mmol/g). Then itwas injected into RP-HPLC and three major fractions were pooled, F312(2.91mmol/g)was collected and further identified by MALDI-TOF--MS/MS, whose amino acidsequence was Asp-Gly-Asp-Asp-Gly-Glu--Ala-Gly-Lys--Ile-Gly with1033Damolecular weight. The FTIR spectrum of calcium-binding peptides (FSP) andpeptides-calcium complex (FSP-Ca) showed the shift, intensity enhancement anddisappear of characteristic peaks before and after calcium binding, which indicatingthe–NH2and–COOH of the fish scale peptides were the major binding site for Ca2+.3. Optimization of binding condition of fish scale peptide and calcium.The factors affecting the binding reaction between fish scale peptide and calciumwere optimized. The conditions were as follows: temperature50℃, pH6.0, the massratio of peptides to calcium salt6:1, and reaction time30min. Through determinationof BC50(the concentration of peptides at half of calcium binding ratio) of threedifferent fractions after ultrafiltration,1-5kDa fraction possessed the lowest BC50value (0.83mg/mL) followed by <1kDa and>5kDa fraction. The BC50value of thetwo fractions (<1kDa&1-5kDa fractions) were significant lower than that of>5kDa fraction (P<0.05), which indicating that peptides with lower molecular weightshowed the stronger the ability to bind calcium ions.4. Calcium absorption and bioavailability of peptide-calcium complex.The calcium absorption and bioavailability of different calcium supplements in ratswere investigated. The results indicated that the body weight growth ratio ofcalcium-deficient rat fed with FSP-Ca and CPP-Ca supplement was25.53%and25.04%, significantly higher than that of low calcium group (P<0.05).The experiment of metabolism showed the calcium absorption ratio and serumcalcium of rats in FSP-Ca group were significnatly higher than that of inorganiccalcium group (P<0.05), and alkaline phosphatase activity was significantly lowerthan that of low calcium group (P<0.05), but similar to that of CPP-Ca group, whichindicating that the absorption ratio of FSP-Ca similar to that of CPP-Ca. The weight,calcium content, and intensity of rat femur were also improved by FSP-Ca and CPP-Ca treatment, and both of them have the same effect on improving the quality ofbone.