Covalent cross-linking of whey protein isolate-chlorogenic acid nanoparticles mediated by laccase: Structural, antioxidant and functional characteristics

To enhance the stability and bioavailability of chlorogenic acid (CHA), laccase (LAC)-catalyzed cross-linking was employed to prepare whey protein isolate (WPI)-CHA nanocomposites. The complexes exhibited sensitivity to variation in pH and temperature. This study investigated the effects of pH (3.0–7.0) and temperature (15–45 °C) on the structural, antioxidant, and functional properties of WPI-CHA (WC) and WPI-LAC-CHA (WLC) complexes. Under the optimal catalytic conditions for LAC (pH 5.0, 35 °C), WLC complexes exhibited the highest structural stability, characterized by the smallest particle size (133 ± 3.93 nm), highest surface hydrophobicity (822.79 ± 1.74), and lowest free amino content (209.99 ± 1.09 mM/mL). The 52 % fluorescence quenching, amide I red shift and >180 kDa aggregates formation confirmed tertiary structure unfolding and covalent cross-linking, indicative of efficient protein-polyphenol conjugation. WLC complexes exhibited optimal radical scavenging activity at pH 5.0 and 35 °C, with DPPH and ABTS scavenging rates of 78.33 ± 5.11 (μmoL TE/g) and 83.99 ± 1.92 %, respectively. Following in vitro gastrointestinal digestion, WLC complexes retained superior antioxidant capacity (48.5 ± 2.21 μmoL TE/g for DPPH and 70.5 ± 1.70 % for ABTS) compared to free CHA and WC complexes. WLC complexes exhibited significantly greater UV protective efficiency of CHA, functional characteristics (solubility, EAI, ESI, FC and FS) compared to WC. These findings highlighted LAC-catalyzed cross-linking as an effective strategy to fabricate high-performance WPI-CHA nanocomposites, which might be applied in nano-carriers and packaging materials in the food industry.