Effects of protease-assisted aqueous extraction on almond protein profile, digestibility, and antigenicity

Abstract

Almonds (Prunus dulcis) are one of the most consumed tree nuts worldwide and have been recognized as a healthy and nutritious food. Nevertheless, almonds are also a source of allergenic proteins that can trigger several mild to life-threatening allergic reactions. The effects of selected extraction conditions (aqueous vs. protease-assisted aqueous extraction) on the protein profile determined by proteomics analysis of excised SDS-PAGE gel bands, in vitro protein digestibility, and immunoreactivity of almond protein extracts, were evaluated. Proteolysis altered almond protein sequential and conformational characteristics thus affecting digestibility and antigenicity. Proteomics analysis revealed that enzymatic extraction resulted in the reduction of allergen proteins and epitopes. While complete hydrolysis of Prunin 1 and 2 α-chain was observed, Prunin 1 and 2 β-chains were more resistant to hydrolysis. Protein in vitro digestibility increased from 79.1 to 88.5% after proteolysis, as determined by a static digestion model. The degree of hydrolysis (DH) and peptide content of enzymatically extracted proteins during gastric and duodenal digestion were significantly higher than the ones from unhydrolyzed proteins. Proteolysis resulted in a 75% reduction in almond protein immunoreactivity as determined by a sandwich enzyme-linked immunosorbent assay and a reduction in IgE and IgG reactivities using human sera. The present study shows that moderated hydrolysis (7% DH) using protease can be used as a strategy to improve almond protein digestibility and reduce antigenicity. This study's findings could further enhance the potential use of almond protein hydrolysates in the formulation of hypoallergenic food products with improved nutritional quality and safety.

Keywords: Almond protein in vitro digestibility; Antigenicity; Enzymatic extraction; Proteolysis; Proteomics.

Graphical abstract

Fig. 1

SDS-PAGE protein profile of AEP and EAEP almond samples indicating the gel slices used for proteomics analysis (black squares) (A). Relative abundance of protein identifications (numbers in the middle of pie charts represent the number of proteins identified) (B) identifications of α- and β-chains in prunin 1 and prunin 2 (C) analyzed by LC-MS/MS-based proteomics analysis.

Fig. 2

Total in vitro protein digestibility of casein, unhydrolyzed (AEP) and hydrolyzed (EAEP) almond protein extracts and soybean isolated protein. Different letters indicate a significant difference between samples at p < 0.05 (A). SDS-PAGE of digestion kinetics for the AEP (B) and EAEP (C) almond proteins samples, arrows indicate the pepsin in the gastric phase and the pancreatin in the intestinal phase. Peptide concentration (D) and degree of hydrolysis (DH) (E) of AEP and EAEP proteins samples.

Fig. 3

Total almond protein immunoreactivity by sandwich ELISA (A). Different letters indicate a significant difference between samples at p < 0.05. Western blot of AEP (unhydrolyzed samples - A1 to A3) and EAEP (B1 to B3): Human serum sample P4C, Primary: human sera 1:20, Secondary: mouse anti-Human IgE Fe 1:5000 (B); Human serum sample P196b, Primary: human sera 1:20, Secondary: mouse anti-Human IgE Fc 1:5000 (C); Human serum sample P4C, Primary: human sera 1:200, Secondary: goat anti-Human IgG Fc 1:10,000 (D); Human serum sample P196b, Primary: human sera 1:200, Secondary: goat anti-Human IgG Fe 1:10,000 (E).

Fig. 4

Western blot of P196b IgE highlighting the more reactive bands (A); Average of the lane area for the AEP and EAEP samples (B), Integration of the highlighted bands by Image J (C).