Impact of Cold Atmospheric Plasma on the Structure and Functionality of Pea Protein

Abstract

Increased consumer demand for alternative plant protein sources other than soy, which is a GMO crop and “Big Eight” allergen, is driving the growth of the pea protein ingredient market. Yellow field peas (Pisum sativum L.), an easy to grow environment-friendly non-GMO crop, with currently low occurrence of allergenicity, have similar protein profile and nutritional quality compared to soy. Therefore, pea protein has the potential to replace soy protein in the global plant protein ingredient market. The functional properties of pea protein, however, are inferior to that of soy protein counterparts, hindering its expanded use. Current breeding efforts, extraction and processing advances, and traditional modification strategies are limited in improving the functional properties of pea protein while maintaining nutritional quality as well as feasible production cost. Cold atmospheric plasma (CAP), a physical nonthermal processing technology that has been explored in electronics, material science, medicine, and agriculture, is being explored as a novel protein modification approach. Several studies reported unfolding and polymerization of proteins and corresponding improvements in functional properties after CAP treatment. However, the link between different plasma reactive species and observed structural changes, and consequent functional enhancement, has not been demonstrated. Additionally, only plasma sources that produce long-lived species (O3, H2O2, NO2-, and NO3-) have been investigated in protein modification studies. Other plasma sources that can generate various short-lived species (such as OH radicals) are worth investigating to optimize CAP conditions for a directed enhancement in pea protein functionality. Therefore, the objectives of this study were: (1) investigate the impact of plasma reactive species, as well as pH conditions and salt content, on pea protein structure and functionality; (2) investigate the impact of different plasma configurations, gas mixtures, and treatment time on pea protein structure and functionality. For objective 1, the impact of RNS and ROS (O3, NxOy, H2O2 and OH) at two pH conditions (pH 2 and pH 7), on the color, structure, and functionality of pea protein isolate (PPI) was evaluated. Structural characteristics of modified pea protein isolates (mPPIs) and PPI were compared by determining the protein profile using SDS-PAGE and SE-HPLC, protein denaturation by DSC, surface charge by measuring zeta potential, surface hydrophobicity as measured by a spectrophotometric method, and protein secondary structure by FTIR. Protein solubility, gelation, and emulsification properties were evaluated. For the second objectives three different CAP treatments, atmospheric pressure plasma jet (APPJ) coupled with Ar/O2 mixture, two-dimension dielectric barrier discharge (2D-DBD) coupled with Ar/O2 mixture, and nanosecond pulsed discharge (ns-pulsed) coupled with air, on the color, structure, functionality, and amino acid composition of PPI was evaluated. The effect of treatment time (5, 15, 30, and 45 min) was also determined. Structural characteristics and functional properties of PPI samples were determined following the same stated methods. The amino acid profile and non-protein components of the isolates were characterized using UPLC-MS. Pronounced structural and functional changes were observed upon treatment with reactive species at pH 2. All reactive species induced the formation of disulfide-linked soluble aggregates. Protein denaturation was observed after treatment with all reactive species. A significant increase in β-sheet content and surface hydrophobicity was only induced by treatment with O3 and OH, which resulted in the greatest enhancement in gelation and emulsification. While H2O2 enhanced PPI color by increasing whiteness, it had the least impact on protein structure and functionality. Results indicated that the plasma sources that can generate OH and O3 could be used for pea protein functionalization. Accordingly, different plasmas sources that can generate O3 and OH were further investigated in objective 2. All plasma treatments resulted in reduced yellow color of PPI, denaturation of the proteins, formation of disulfide-linked soluble aggregates, and increased surface hydrophobicity. The plasma-induced structural changes resulted in improvement of gel strength and emulsification capacity. The amino acid composition of PPI was not significantly impacted by 2D-DBD treatment, whereas a slight decrease in tyrosine content was observed after APPJ and ns-pulsed treatment. Results indicated that the 30-minute 2D-DBD (Ar + O2) treatment was the most desirable treatment because of moderate changes in protein structure coupled with significant improvement in the gelation and emulsification properties of PPI, with minimal impact on the amino acid composition. Overall, the study successfully demonstrated the link in structural changes induced by plasma reactive species (NxOy/O3, O3, H2O2, and OH) to improvement in functional properties. Results can be used to explain previously reported observations related to the impact of different CAP systems on the functional properties of proteins. Additionally, this work provided a detailed understanding of the potential of different CAP sources and associated reactive species in enhancing pea protein functionality.