Preparation, Purification And Characterization Of Calcium-binding Peptide From Whey Protein

The loss of calcium has been the health issue in the whole world. Inorganic calcium is prone to become insoluble salt such as phytic acid calcium, oxalic acid calcium and phosphoric acid calcium resulting in the low bioavailability. However, organic calcium is easier to be absorbed and utilized because of its sepcific chelate and transportation mechanism. Whey protein is the residual protein of the whey in the casein production process, its hydrolysate has certain calcium binding activity. In the study, calcium-binding peptides were isolated and purified from the whey protein hydrolysate. Through the structural property identification, the chelate interaction mechanisms were further invested which layed the foundation of the third generation calcium supplement in the future.The main aim of this study was that the preparation, purification and characterization of calcium-binding peptide from whey protein. The whey protein was digested by compound protease and the compound flavor protease, the calcium binding capacity of the hydrolysate was 33.92 μg/mg. The hydrolysate was named as WPH (Whey protein hydrolysate). DEAE anion exchange chromatography, G-25 gel filtration chromatography and C18 reversed high performance liquid chromatography were investigated to isolate peptides from WPH and four specific calcium-binding peptides were obtained, respectively named WPH1A1, WPH I A2, WPH1B and WPH2. Corresponding calcium-binding activities of WPH1A, WPH1B and WPH2 were 73.34 ug/mg,79.51μg/mg and 67.81 μg/mg. Compared to WPH, their calcium-binding activities enhanced 116.21%,134.38% and 99.88%. The molecular weight of them respectively were 237.99 Da,279.97 Da,396.98 Da and 204.10 Da, the amino acid sequences were, Gly-Tyr (GY), Phe-Asp (FD), Tyr-Asp-Thr (YDT) and Glu-Gly (EG) through the liquid mass analysis.Characterization methods including ultraviolet spectrum scan, fluorescence spectra, infrared spectroscopy, nuclear magnetic resonance (NMR) one-dimensional hydrogen spectrum, X-ray scattering techniques, TG-DSC. Zeta potential and calcium releasing percentage were applied to analyze calcium chelate property to investigate the calcium-binding sites of the GY. FD, YDT and EG. Moreover, physicochemical property containing the pH stability and thermal stability were also discussed. The results showed that the combination pattern between the calcium-binding peptide and calcium ion was one or several moles of peptides surround one mole calcium to form penta cyclic compound or hexa cyclic compound. The animo acids participating in chelation included glutamic acid, aspartic acid and glycine. In addition, the carbanyl group and imino group of amido bond also participated in incorporation with calicum ion. The primary binding sites were oxygen atom of carbanyl group and nitrogen atom of amino group. The peptide calcium complex exhibited high pH stability and thermal stability. The experiments of clacium-releasing percentage indicated that the peptide calcium complexes were stable in basic intestinal tract of human body, which was benefical to be absorbed and transported to avoid becoming precipitation.Conclusion:The calcium-binding dipeptides GY, FD, EG and tripeptide YDT purified from the whey protein hydrolysates possessed high calcium binding activities. The pH stability, thermal stability and calcium releasing percentage of peptide calcium complexes exhibited well. If the calcium-binding peptides in this study were used in commercial process, they will be great development potential as the third generation calcium supplements.