Sequential Ultrasound-Assisted Enzymatic Hydrolysis and Microwave Treatment of Gluten–Starch Complexes: Functionality, Volatile Profiles, In Vitro Immunoreactivity and Interactions
Abstract
Sequential non-thermal and thermal processing offers a route to jointly adjust the functionality, color/volatile profile and immunoreactivity of gluten-containing systems. This study aimed to evaluate the effects of sequential ultrasound-assisted enzymatic hydrolysis, wheat/rice starch incorporation and microwave heating on soluble gluten hydrolysate–starch composites, and to probe the underlying starch–gluten interactions. Gluten hydrolysates were blended with wheat or rice starch and microwave-heated to induce the Maillard reaction. Sequential processing enriched the <1 kDa peptide fraction (2.34%→13.05–14.99%) and raised ABTS·+ scavenging (0.25→0.40 mM Trolox/g). After starch compounding and microwaving, the composites exhibited increased protein-normalized TEAC (0.07–0.08 to 0.10–0.13 mM per mg/mL protein) and released more ninhydrin-reactive amino groups (>55 μmol/mg protein) after in vitro digestion. All microwave composites remained pale (L* > 94), with the coupled treated rice-starch composite showing the lowest color difference (ΔE = 0.25). GC-FID/electronic-nose profiling with PCA–DFA showed that the coupled treated composites retained a native cereal-like volatile profile, marked by the green/malty aldehyde 3-methylbutanal and hexanal, with only mild roasted-type Maillard volatiles and the clearest separation for rice starch. The lowest residual R5-reactive gluten reached 8.31–9.82 ppm for the coupled treated groups. Docking provided putative models of multi-site starch binding near immunodominant gliadin sequences (e.g., QQPYP, LQPFP) via Glu/Gln–glucose hydrogen bonds and hydrophobic contacts, with more diverse binding modes for rice starch. Overall, the sequential treatment improved functional and volatile properties and lowered R5-detectable immunoreactivity for composites, offering a process reference for the development of balanced gluten-derived functional ingredients and special dietary food formulations.