Pickering emulsions stabilized by plant derived particles align with clean label demands yet faba bean protein isolate (FBPI) often shows limited interfacial performance due to low solubility and compact globular structure. Non covalent interactions with polyphenols can modulate protein conformation but the cooperative effects of dual polyphenols remain insufficiently understood. This study aimed to enhance Pickering emulsion stability by forming FBPI complex nanoparticles with gallic acid (GA) as a small phenolic acid and rutin (RU) as a bulky flavonoid glycoside. The objective was to clarify how the complementary molecular characteristics of GA and RU generate cooperative structural and functional modulation of FBPI, thereby reshaping its molecular conformation, particle level properties, and interfacial assembly within a moderately unstable emulsion system with an oil volume fraction of 0.6.

Pretreated FBPI was mixed with GA at a fixed concentration followed by RU addition at increasing RU to GA ratios (0.0:1.0 ~ 1.0:1.0). Non covalent FBPI-RU/GA complex nanoparticles were characterized for particle size, ζ-potential, polyphenol binding content, solubility, exposed sulfhydryl (SH) and amino residues, and secondary structure. Pickering emulsions were prepared using canola oil and analyzed for droplet size (D_4,3), ζ-potential, interfacial protein adsorption, Turbiscan stability index (TSI), and microscopic features. Statistical differences were determined by analysis of variance with Duncan test at p < 0.05.

GA reduced FBPI particle size (Fig. 1A), increased solubility (Fig. 1C), and transformed disordered secondary structures into more ordered α-helix and β-sheet forms (Fig. 1F), improving dispersion and interfacial affinity. Incorporation of RU progressively increased polyphenol binding (Fig. 1B) and exposure of functional residues (Fig. 1D-E) while altering the balance between compaction and unfolding. At an intermediate RU to GA ratio of 0.4 to 1.0, nanoparticles exhibited high solubility, more negative surface charge, and a compact yet flexible secondary structure. These features enabled efficient migration to the oil water interface and formation of dense particulate films. The resulting emulsions showed smaller D_4,3, higher magnitude of ζ-potential (Fig. 1G), continuous fluorescent interfacial rings in microscopy (Fig. 1K), and the lowest TSI (Fig. 1I), reflecting strong resistance to coalescence and creaming. When RU proportion exceeded the optimal level, over unfolding and steric congestion occurred, reducing effective interfacial packing and weakening the particulate film. These effects were evident in larger D_4,3, decreased interfacial protein concentration, and increased TSI values. Collectively these findings indicate that Pickering stabilization depends on achieving an optimal balance in FBPI structure where GA driven compaction and RU driven unfolding act cooperatively rather than independently.

Dual polyphenol complexation offers a tunable route to engineer FBPI for clean label Pickering emulsions. A GA RU ratio of 0.4 to 1.0 produced nanoparticles forming cohesive and elastic interfacial films that markedly enhanced stability, whereas excessive RU impaired assembly. This work clarifies how dual polyphenols modulate FBPI and provides a design framework for next generation plant-based stabilizers.