Caseinate nanoparticles co-loaded with quercetin and avenanthramide 2c using a novel two-step pH-driven method: Formation, characterization, and bioavailability

The pH-driven method is a green and efficient encapsulation technology to incorporate hydrophobic polyphenols. However, this method has never been used to prepare nanoparticles loaded with multiple polyphenols of different water solubility and chemical stability. In the present study, the pH-dependent water solubility and chemical stability of quercetin and avenanthramide 2c (AV 2c) were characterized to develop a novel two-step pH-driven method to co-load quercetin and AV 2c in sodium caseinate (NaCas) nanoparticles (Q-2c-N). Quercetin-loaded NaCas nanoparticles (QNN) were fabricated by increasing the mixture to pH 12.0, followed by acidification to pH 7.0. Subsequently, AV 2c was dissolved in the QNN dispersion that was further acidified to pH 6.0 to prepare Q-2c-N. The encapsulation efficiency of quercetin and AV 2c in the Q-2c-N was up to 94.3% and 80.6%, respectively, and remained stable in 21 days at 21 °C. Based on solubility, particle structure, zeta potential, and fluorescence spectroscopy assays, molecular binding between NaCas and deprotonated quercetin was critical to quercetin encapsulation during acidification to pH 7.0, and the gradual loss of AV 2c solubility during further pH adjustment to 6.0 enabled the embedment of AV 2c in QNN to form Q-2c-N with reduced particle size. Moreover, quercetin and AV 2c were almost molecularly distributed in Q-2c-N based on XRD patterns and FT-IR spectra. Co-loading quercetin and AV 2c in Q-2c-N significantly improved the bioaccessibility and Caco-2 cell monolayer uptake. The present study may be significant to the utilization of polyphenols with distinctly different physical properties. • Quercetin and avenanthramide 2c (AV 2c) had different pH-dependent water-solubility and stability. • The two-step pH-driven method firstly enabled the co-encapsulation of quercetin and AV 2c in sodium caseinate (NaCas). • Co-encapsulation reduced the particle size and enabled dispersion stability within 21 days at room temperature. • Co-loading quercetin and AV 2c in NaCas significantly improved the bioaccessibility and Caco-2 cell monolayer uptake.