Optimising Enzymatic Cross-Linking: Impact on Physicochemical and Functional Properties of Lupin Flour and Soy Protein Isolate

The growing demand for plant-based protein alternatives has driven interest in protein modifications to enhance their functional properties in food applications. Enzymatic cross-linking using laccases derived from Rhus vernicifera (LR) and transglutaminase (TG) offers a promising strategy to enhance protein solubility, emulsifying properties, and foaming properties of food proteins. This study varied the enzymatic reaction conditions, including enzyme concentration, pH, temperature, incubation time, and ferulic acid addition, for the most effective cross-linking between proteins in lupin flour (LF) and soy protein isolate (SPI), resulting in changes in physicochemical and functional properties of the cross-linked proteins. LR-induced cross-linking in lupin and soy proteins was most favourable at 142.5 U/100 mg protein, pH 6, and 20 °C, where ferulic acid enhanced cross-linking efficiency with prolonged incubation (20 h). TG-induced cross-linking in lupin and soy proteins was most favourable at 1.25 U/100 mg protein, pH 6 and 30 °C, where high-molecular-weight aggregates were observed. Cross-linking modified protein surface characteristics, increasing ζ-potential and particle size due to protein aggregation, while ferulic acid further enhanced polymerisation. Morphological analysis revealed a porous powder structure across all samples with increased porosity in cross-linked samples as evidenced by the predominance of small fragments within the particles. Prolonged incubation led to partial disaggregation in LR-treated samples unless they were stabilised by ferulic acid. Under mild conditions (1 h, pH 6, 20 °C), LR and ferulic acid-added samples showed minor and significant improvements in protein solubility and foaming stability, respectively. Additionally, a significant increase in foaming ability was observed in ferulic acid-added LR samples after prolonged incubation (20 h), compared to the corresponding control. In contrast, prolonged incubation (20 h) or TG treatment had a lower foaming stability compared to the mild LR treatment. Emulsifying ability and emulsion stability showed limited variation across treatments. These findings suggest that cross-linking conditions influence specific functional properties, highlighting the need for further optimisation to achieve desired protein functionality in food applications.