Ultrasonic engineering of bovine serum albumin nanoparticles for high internal phase Pickering emulsions: Interfacial behavior, microstructural evolution and stabilization enhancement

Abstract

This study investigated the influence of ultrasonic treatment on the physicochemical properties of bovine serum albumin (BSA) and its applicability in stabilizing high internal phase Pickering emulsions (HIPPEs). Under optimized sonication conditions (250 W, 12 min), stable ultrasonically modified BSA (UBSA) particles were generated, exhibiting a small particle size (41.39 nm), a polydispersity index < 0.17, a higher absolute zeta potential (> 20 mV), and favorable wettability (three-phase contact angle: 78.71°), accompanied by reduced surface hydrophobicity. Intrinsic fluorescence spectra and circular dichroism (CD) analysis confirmed that ultrasonication altered the secondary structure of BSA, leading to the exposure of hydrophobic groups. Dynamic interfacial tension and adsorption kinetics analyses revealed that UBSA particles exhibited lower interfacial tension and significantly higher diffusion coefficient (K_diff) and penetration coefficient (K_p) than native BSA, indicating enhanced diffusion and adsorption capabilities of UBSA at the oil-water interface. Rheological analyses demonstrated that UBSA-stabilized HIPPEs possessed higher viscosity and larger storage (G') and loss (G″) moduli. Optical and confocal laser scanning microscopy confirmed the successful formation of HIPPEs at UBSA concentrations ≥ 1.0 % (w/v). UBSA-stabilized HIPPEs displayed a reduced droplet size (10.59 µm) and a more densely packed droplet structure, which conferred enhanced resistance against droplet coalescence compared to emulsions stabilized by native BSA. Moreover, stability assessments indicated that the centrifugal, freeze-thaw and storage stability of the prepared HIPPEs were significantly improved. Importantly, UBSA-based HIPPEs serving as a delivery vehicle also effectively enhanced the thermal processing stability of β-carotene. The findings demonstrate the potential of ultrasound-modified BSA nanoparticles as effective stabilizers for HIPPEs, providing valuable insights for the development of healthy and safe food-grade emulsion systems.

Keywords: Bovine serum albumin; Interfacial adsorption; Microstructure; Pickering emulsions; Stability; Ultrasonic treatment.

Fig. 1

Physicochemical properties of BSA after ultrasonic power treatments: (A) Three-phase contact angle, (B) particle size and PDI, (C) particle size distribution, (D) turbidity, (E) zeta potential, (F) intrinsic fluorescence spectroscopy, (G) surface hydrophobicity, (H) circular dichroism (CD), and (I) protein secondary structure changes. Different letters indicate statistically significant differences (P < 0.05).

Fig. 2

(A) Three-phase contact angle, (B) particle size and PDI, (C) particle size distribution, (D) turbidity, (E) zeta potential, (F) intrinsic fluorescence, (G) surface hydrophobicity, (H) circular dichroism (CD), and (I) protein secondary structure changes of BSA after different ultrasonic time treatments. Note: Different letters on the bars indicate significant differences (P < 0.05).

Fig. 3

Characterization of stabilized high internal phase Pickering emulsions (HIPPEs) at different protein particle concentrations. Visual appearance of HIPPEs stabilized by (A) BSA and (B) UBSA particles; optical microscopy images of HIPPEs stabilized by (C) BSA and (D) UBSA particles; droplet size distribution of HIPPEs stabilized by (E) BSA and (F) UBSA particles; and dynamic interfacial tension at the oil–water interface for (G) BSA and (H) UBSA versus time. Notes: (a) Upright position and (b) inverted position in panels (A) and (B); scale bar in panels (C) and (D) represents 50 μm.

Fig. 4

Fitting of the surface pressure (π) during the first diffusion process at the oil–water interface according to Equation (2) (A-1 to A-5) and fitting of π during the late adsorption stage at the oil–water interface according to Equation (4) (B-1 to B-5).

Fig. 5

Confocal laser scanning microscopy (CLSM) images of high internal phase Pickering emulsions (HIPPEs) stabilized with (A) BSA and (B) UBSA particles at concentrations of 1.0 %–5.0 % (w/v). Notes: Images were acquired at 40 × magnification; scale bar represents 50 μm. Green fluorescence corresponds to the oil phase, while red fluorescence indicates protein particles adsorbed at the interface. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

Stability of HIPPEs stabilized with different concentrations (1.0 %–5.0 %) of protein particles. (A) Appearance after centrifugation (10,000 rpm, 20 min): (A-1) BSA-stabilized HIPPEs (a: before; b: after), (A-2) UBSA-stabilized HIPPEs. (B) Appearance before and after freeze–thaw cycles: (B-1) BSA-stabilized HIPPEs, (B-2) UBSA-stabilized HIPPEs. (C) Appearance and optical microscopy (OPM) images after 30 d storage: (C-1) BSA-stabilized HIPPEs, (C-2) UBSA-stabilized HIPPEs. Scale bar: 50 μm (micrographs).

Fig. 7

Rheological properties of emulsions: viscosity (A), storage modulus (B), loss modulus (C).

Fig. 8

Improvement of the thermal processing stability of β-carotene using UBSA-stabilized HIPPEs. (A) Appearance of β-carotene-loaded HIPPEs before and after thermal treatment. (B) β-Carotene retention under pasteurization conditions (65 °C for 30 min). (C) β-Carotene retention under cooking conditions (100 °C for 10 min). Note: Different letters on the bars indicate significant differences (P < 0.05).