The formation of non-heat-treated whey protein cold-set hydrogels via non-toxic chemical cross-linking

A new cold-set gelation method based on the cross-linking of non-heat-treated whey protein upon alkali-catalysis in the presence of citric acid and at low temperature was established in the current study. For this purpose, different concentrations of citric acid were applied to the WPI solution at various pH values (7.0–11.0), to induce protein cross-linking reaction with time. Cross-linking increased both the WPI solution turbidity and protein aggregates size over time. It was postulated according to spectroscopy results that proteins citric acid-mediated cross-linking decreased inter-protein interactions and that histidine was the most reactive amino acid during the cross-linking reaction. In the absence of citric acid, the WPI solution with 100 mg mL⁻¹ concentration did not gel regardless of the pH value applied, while a concentration of citric acid applied equal or more than 55 mM led to self-supporting gel at pH values 8.0–11.0. Increasing the WPI concentration resulted in gel formation at pH 7.0 and in the presence of 55 mM citric acid. Scanning electron microscopy images indicated that the gel formed at alkaline condition had a more porous microstructure than that prepared at pH 7.0.