WHEY PROTEIN ISOLATE NANOSTRUCTURES FOR QUERCETIN ENCAPSULATION: STRUCTURAL CHARACTERIZATION AND THERMAL STABILITY ASSESSMENT

Quercetin (3,3′,4′,5,7-pentahydroxyflavone) is a flavonoid known for its various biological activities, including anticancer, antioxidant, anti-inflammatory, and cardioprotective properties. However, this molecule has low water solubility (0.00215 g∙L^-1) and instability under adverse conditions such as high temperatures, pH variations, and oxygen exposure. These limitations compromise its oral bioavailability and, consequently, its therapeutic efficacy. In this context, the present study evaluated the capacity of nano-structures based on whey protein isolate (WPI) to encapsulate quercetin, aiming to improve its stability and functional properties. The WPI nano-structures were obtained by controlled heating (50 °C for 10 minutes) in the presence of sodium chloride (10 mmol∙L^-1). To incorporate quercetin into the WPI dispersion, seven assays were prepared varying the bioactive concentration (0.015–0.100 mg∙mL^-1), and encapsulation efficiency and binding capacity were evaluated through spectroscopic analyses (373.0 nm). The best encapsulation condition (assay 3) was characterized regarding its structural properties by circular dichroism (CD), fluorescence spectroscopy, atomic force microscopy (AFM), and infrared nano-spectroscopy (AFM-IR), to investigate intermolecular interactions between the protein and the bioactive compound, as well as conformational changes induced by encapsulation. Additionally, the thermal stability of the encapsulated system was evaluated by thermogravimetric analysis (TGA). WPI showed high encapsulation efficiency of quercetin (87.59–96.79%) and binding capacity (11.51–54.59%), both influenced by quercetin concentration. AFM characterization revealed the formation of fibrillar structures after quercetin incorporation. Spectroscopic analyses (CD, AFM-IR, and fluorescence) indicated conformational alterations in the secondary and tertiary structures of WPI. Furthermore, fluorescence data allowed the determination of thermodynamic parameters, which indicated that the main interaction mechanism is predominantly hydrophobic (ΔH > 0 and ΔS > 0), and that the system occurs spontaneously (ΔG < 0). The thermal stability of the nano-structured complex was superior to that of free quercetin, demonstrating that encapsulation increased its resistance to thermal degradation. This behavior suggests a protective mechanism promoted by the protein matrix, related to the organization of the secondary structure, conformational rigidity, and aggregation degree of WPI after encapsulation. The results indicate that nanoencapsulation with WPI is suitable for improving the stability of quercetin, offering potential for the development of nutraceutical foods and pharmaceutical products. Further investigations are underway to evaluate stability in the gastrointestinal system and the applicability of these systems in food formulations.