Formation mechanism and functional properties of walnut protein isolate and soy protein isolate nanoparticles using the pH-cycle technology

Abstract

Walnut protein isolate (WPI) is a nutritious protein with poor solubility, which severely limits its application. In this study, composite nanoparticles were prepared from WPI and soy protein isolate (SPI) using the pH-cycle technology. The WPI solubility increased from 12.64 to 88.53% with a WPI: SPI ratio increased from 1: 0.01 to 1: 1. Morphological and structural analyses illustrated that interaction forces with hydrogen bonding as the main effect jointly drive the binding of WPI to SPI and that protein co-folding occurs during the neutralization process, resulting in a hydrophilic rigid structure. In addition, the interfacial characterization showed that the composite nanoparticle with a large surface charge enhanced the affinity with water molecules, prevented protein aggregation, and protected the new hydrophilic structure from damage. All these parameters helped to maintain the stability of the composite nanoparticles in a neutral environment. Amino acid analysis, emulsification capacity, foaming, and stability analysis showed that the prepared WPI-based nanoparticles exhibited good nutritional and functional properties. Overall, this study could provide a technical reference for the value-added use of WPI and an alternative strategy for delivering natural food ingredients.

Keywords: functional properties; interaction; nanoparticles; pH-cycle; soy protein isolate; walnut protein isolate.

FIGURE 1

(A) Schematic illustration of the pH-cycle technique for fabricating protein-based composite nanoparticles with hydrophobic WPI and hydrophilic SPI. (B) Photographs of protein solutions and composite nanoparticles after the pH-cycle treatment. (C) Photographs of sample supernatants by centrifuging at 10,000 × g and 4°C for 20 min (1:0.1, 1:0.5, 1:1 represents walnut protein and soy protein isolate at the ratio of WPI:SPI = 1:0.1, 1:0.5, 1:1, respectively).

FIGURE 2

SDS-PAGE profiles of supernatant (A) and precipitates (B) of prepared composite nanoparticles, respectively (lane 1 is marker, lanes 2–10 are WPI, SPI, composite nanoparticles prepared by WPI: SPI = 1:0.01, 1:0.05, 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, and 1:1 (w/w), respectively). (C) Solubility of WPI in composite nanoparticles with WPI: SPI (w/w) from 1:0.01 to 1:1 by the pH-cycle technique (WPI represents walnut protein isolation solution without the pH-cycle treatment, control represents walnut protein isolation solution with the pH-cycle treatment).

FIGURE 3

(A) Transmission electron microscopy (TEM) image of WPI solution. (B) TEM image of SPI solution. (C) TEM image of composite nanoparticles prepared by WPI: SPI (w/w) mass ratio of 1: 0.1. (D) TEM image of composite nanoparticles prepared by WPI: SPI (w/w) mass ratio of 1: 0.5. (E) TEM image of composite nanoparticles prepared by WPI: SPI (w/w) mass ratio of 1: 1.

FIGURE 4

(A) Atomic force microscopy (AFM) image of composite nanoparticles prepared by WPI: SPI (w/w) mass ratio of 1: 0.1. (B) AFM image of composite nanoparticles prepared by WPI: SPI (w/w) mass ratio of 1: 0.5. (C) AFM image of composite nanoparticles prepared by WPI: SPI (w/w) mass ratio of 1: 1. (D) Particle size of composite nanoparticles with WPI: SPI (w/w) from 1:0.01 to 1:1.

FIGURE 5

(A) Emission spectra of composite nanoparticles with WPI: SPI (w/w) from 1:0 to 1:1. (B) Emission spectra of composite nanoparticles (WPI: SPI = 1:1, w/w) at pH 12, 11, 10, 9, 8, and 7. (C) Emission spectra for ANS binding to composite nanoparticles with WPI: SPI (w/w) from 1:0 to 1:1. (D) Emission spectra for ANS binding to composite nanoparticles with WPI: SPI (WPI: SPI = 1:1, w/w) at pH 12, 11, 10, 9, 8, and 7.

FIGURE 6

(A) Fluorescence spectra of composite nanoparticles WPI: SPI = 1:1 (w/w) with bond-disrupting agents added. (B) Nitrogen solubility index (NSI) of WPI with the addition of bond-disrupting agents (10 mM). (C) Far-UV CD spectra of composite nanoparticles with WPI/SPI (w/w) from 1:0.01 to 1:1. (D) Far-UV CD spectra of a representative composite nanoparticles (WPI: SPI = 1:1, w/w) prepared at pH 12, 11, 10, 9, 8, and 7. (E) Near-UV CD spectra of a representative composite nanoparticles (WPI: SPI = 1:1, w/w) prepared at pH 12, 11, 10, 9, 8, and 7. (F) Near-UV CD spectra of composite nanoparticles with WPI: SPI (w/w) from 1:0.01 to 1:1.

FIGURE 7

(A) The hydrophobicity of WPI, SPI and the composite nanoparticles at pH 7; (B) the zeta-potential of WPI, SPI and the composite nanoparticles at pH 7; (C) the hydrophobicity of the composite nanoparticles prepared at WPI: SPI ration of 1:1 at pH 12, 11, 10, 9, 8, and 7; (D) the zeta-potential of the composite nanoparticles prepared at WPI: SPI ration of 1:1 at pH 12, 11, 10, 9, 8, and 7.