Revealing flavor improvement in whey-based cold brew coffee: characteristic flavor compounds and formation mechanisms.

This study aimed to enhance the flavor of cold brew coffee (CB) by replacing water with whey (pH 5.2 ± 0.2) in the cold brew system, and elucidated the formation mechanism of characteristic flavor compounds. Whey cold brew coffee (WCB) refers to a CB that is significantly enhanced in flavor by using whey extract as the extraction medium and continuously extracting for 12 h at 4 °C. Headspace solid phase microextraction gas chromatography-mass spectrometry (HS-SPME-GC-MS) analysis revealed significantly elevated concentrations of pyrazines, furans, and ketones in WCB, imparting distinct nutty, caramel, and creamy notes. Odor activity value (OAV) analysis identified 2,5-dimethylpyrazine (OAV = 208.68) and 2,3-pentanedione (OAV = 70.63) as key flavor contributors, synergistically enhanced by 2-thiophenecarboxaldehyde and 2-acetylpyrazine for cocoa-nutty flavors. Sensory evaluation indicated that WCB significantly outperformed CB in terms of enzymatic flavor (floral/fruity) and Maillard reaction products (nutty/chocolate flavors), with a significantly higher overall score compared to CB (p < 0.05). Ultraviolet-visible spectroscopy (UV-Vis) and Circular dichroism (CD) analyses and molecular docking confirmed that β-lactoglobulin (β-LG) binds flavor compounds via hydrogen bonds and hydrophobic interactions (binding energy: -4.12 to -5.22 kcal/mol). This induced a structural transition toward higher order (α-helix + β-sheet content), enhancing complex stability (ΔG = -5.22 kcal/mol) and suppressing off-flavors. This study advances the high-value utilization of whey resources and innovation in cold brew processes through flavor optimization technology driven by "whey protein-flavor synergy enhancement".