Fractional purification process and activity analysis of wheat calcium ion chelating peptides

Calcium-chelating peptide is a new type of calcium supplement with excellent absorption properties and high bioavailability, safety and stability. This study synthesized calcium chelating peptide from gluten by enzymatic hydrolysis, determined peptide sequences with high activity, and analyzed their digestive characteristics and stability. The enzymatic hydrolysis process was optimized using response surface methodology to determine the optimal enzymatic hydrolysis conditions of temperature 55 °C, pH 8.5, and the ratio of alkaline protease to flavor protease (proportion of enzymes) 2.63:1 under a liquid-to-solid ratio of 20:1. The calcium chelation rate of gluten hydrolysate was up to 40.1 % under the optimal conditions. Fractional purification was then carried out and results showed that peptides with a molecular weight below 500 Da exhibited the highest chelation rate (51.1 %). LC-MS/MS analysis was applied to identify 1224 distinct peptide sequences, among which V.YIPPY·C (WCP1) exhibited a higher calcium chelation rate after screening and molecular docking studies. The synthesized WCP1 displayed a calcium chelation rate as high as 53.5 %. Fourier Transform Infrared Spectroscopy (FTIR) confirmed that both carboxyl and phosphate groups play crucial roles in mediating interactions between calcium ions and wheat polypeptides. Circular Dichroism (CD) revealed that the structure of wheat peptide became more compact after chelation. Furthermore, stability experiments indicated that the calcium-chelating peptides displayed notable resistance to digestion as well as excellent pH stability and thermal stability. This study can provide technical support for deep processing and functional product development of gluten flour.