Novel calcium-binding peptides from wheat protein hydrolysates: isolation, identification and mechanistic insights...

Wheat protein hydrolysates (WPHs) can chelate calcium; however, their chelation mechanism remains unclear. This study aimed to isolate and identify novel calcium-chelating peptides using liquid chromatography-tandem mass spectrometry (LC-MS/MS), and then to synthesize them using a solid-phase procedure to investigate their metal-chelating mechanisms.

The calcium-binding properties and bitterness of the WPHs produced via single-enzyme treatment were systematically evaluated. Thermoase PC 10F conferred WPHs with a significantly higher calcium-binding capacity (64.38 mg g^-1) and lower bitterness value (0.82) compared to the other five proteases. Novel calcium-binding peptides from WPHs were systematically purified using ultrafiltration and gel filtration chromatography based on their calcium-binding capacities.

A novel pentapeptide (Lys-Glu-Gly-Val-Gln, KEGVQ) and hexapeptide (Asp-Lys-Val-Ile-Val-Pro, DKVIVP) were identified by LC-MS/MS. The calcium-binding capacity of them reached 110.37 and 114.59 mg g^-1, respectively. The KEGVQ-Ca and DKVIVP-Ca complexes formed new compounds with more compact and denser spherical structures. The carbonyl and carboxyl oxygen atoms of the Glu, Val and Gln residues contributed to the binding of calcium ions to KEGVQ. However, in the chelation of DKVIVP with Ca²⁺, Asp, Lys, Val and Pro residues showed considerable affinity. Moreover, electrostatic interactions were the main driving forces. These findings provide theoretical evidence for the chelation mechanism between KEGVQ, DKVIVP and Ca²⁺. This study not only puts forward a scientific basis for developing new calcium supplements from food resources, but also provides a feasible approach to isolate calcium-binding peptides and to clarify the chelation mechanism of calcium and peptides. © 2025 Society of Chemical Industry.