Characterization and Modification of Scaled-Up Pea and Chickpea Protein Isolates Extracted using Salt Solubilization Coupled with Membrane Filtration in Comparison to Commercial Proteins

Abstract

The demand for plant protein ingredients continues to grow, along with growing interest in plant-based products, such as meat analogues. Soy protein is the most common plant protein source in the market due to availability, low cost, excellent nutritional quality, and good functional properties. In the meat analogue sector, soy protein is commonly combined with wheat gluten to form a meat-like fibrous texture via high moisture extrusion. However, soy protein and gluten are among the “Big Nine” allergens in the U.S., in addition to soy being a genetically modified (GMO) crop, which raises concerns and skepticism among consumers, creating demand for alternative plant protein sources. Among legume crops, pea and chickpea have been known to be good sources of protein. Pea protein is a major protein source that has been used in many food and beverage applications. Chickpea protein is a rather emerging protein source and is less studied compared to pea protein. There remains a limited availability of commercial chickpea protein ingredients in the market. As legumes, pea, chickpea, and soy share similar protein profile and structure, indicating the potential for pea and chickpea protein to substitute soy protein. However, pea and chickpea proteins have inferior functionality compared to soy protein, particularly gel strength. Protein functionality not only depends on the inherent protein structure and amino acid compositions but also on external factors, such as extraction methods. Currently, alkaline extraction coupled with isoelectric precipitation (AE-IEP) is the most common extraction method used in the industry to produce plant protein isolates. Extreme alkaline conditions may cause damage to the protein’s native structure due to excessive denaturation, which can impair protein functionality. Therefore, to compete with soy protein, there is a need to explore and optimize a milder extraction process to produce functional pea and chickpea protein isolates (PPI and ChPI). One method that is gaining traction is salt solubilization coupled with membrane filtration, which has mostly been performed at lab-scale to produce soy and pea protein isolates. To conduct a comprehensive evaluation, the industrial feasibility of such extraction process, following lab-scale optimization for ChPI, and its impact on the protein’s structure and function, must be determined. In addition to the extraction method, structural modification can also be used to further enhance targeted protein functionality. For meat analogue applications, good protein network, demonstrated by high gel strength, is important for the formation of meat-like fibrous texture. Enzyme transglutaminase (TG) has been used in the food industry to catalyze the formation of inter and intra-molecular crosslinks between the amino acids lysine and glutamine. Pea and chickpea are high in lysine, making them suitable substrates for TG. As research on this topic is limited, the selection of treatment parameters, along with protein structural and functional characterization, need to be done to determine the impact of TG on pea and chickpea protein. Therefore, the objectives of this study were: (1) to investigate the feasibility of scaling-up salt extraction coupled with membrane filtration and its impact on the structure and function of chickpea protein in comparison to pea protein and (2) to investigate the impact of transglutaminase treatment on the structure and function of pea and chickpea proteins. For objective 1, the salt solubilization conditions, including salt concentration and temperature were optimized for ChPI on benchtop based on protein purity and yield. Optimized benchtop extraction was then scaled-up at pilot scale to evaluate the scalability based on purity and yield of the chickpea protein. For comparison purposes, scaled-up (SU) PPI was produced following the same extraction method, according to our previous study. Structural characteristics of the SU PPI and SU ChPI were compared to benchtop and commercial isolates by determining the protein profile via SDS-PAGE, protein denaturation via differential scanning colorimetry (DSC), surface charge via zeta potential, and surface hydrophobicity by a spectrophotometric method. Functional characteristics of the SU isolates in comparison to benchtop and commercial counterparts, which included solubility, gel strength, water holding capacity, and emulsification properties, were also assessed. For objective 2, SU isolates were further treated with TG to induce polymerization. Different treatment conditions, including enzyme concentration and treatment time were tested and selected based on degree of polymerization, protein denaturation, and gel strength, as determined by SDS-PAGE, DSC, and thermal-induced gelation. The structural characteristics of the modified isolates (TG PPI and TG ChPI) were determined by evaluating the protein profile via SDS-PAGE, protein molecular weight distribution via SE-HPLC, protein denaturation by DSC, secondary structure via FTIR, surface charge via zeta potential, and surface hydrophobicity by spectrophotometric method in comparison to SU isolates and commercial ingredients. The functional properties of TG PPI and TG ChPI were determined by evaluating the protein’s solubility, gel strength, and emulsion capacity. The optimum salt solubilization conditions for ChPI were 0.5 M NaCl at room temperature, similar to those used for PPI, followed by ultrafiltration and dialysis. The benchtop and SU isolates had comparable protein purity with >90% protein and low ash content. The production of SU ChPI resulted in lower yield (41%) compared to benchtop production (52%), which was also observed for SU PPI, due to unavoidable losses during large scale production. The SU and benchtop isolates shared a similar protein profile. However, SU isolates experienced partial protein denaturation, higher surface hydrophobicity, and some polymerization due to thermal treatments during pasteurization, evaporation, and spray drying. Changes in structural characteristics of the SU isolates significantly impacted their functionality, including superior gel strength compared to their benchtop counterparts. Compared to the commercial pea and chickpea proteins, SU isolates were less denatured and polymerized, confirming that the adopted SE-UF process is milder compared to commercial isolation processes. Further, TG treatment of SU isolates resulted in a high degree of polymerization and a significant increase of intermolecular β-sheet. Heat treatment during enzyme incubation and inactivation step resulted in complete protein denaturation. TG-modified isolates had significantly lower solubility compared to their SU counterparts due to the formation of insoluble high molecular weight (MW) polymers and relatively high surface hydrophobicity. In contrast, TG isolates showed significantly stronger gel strength compared to the SU isolates, which was attributed to TG-induced polymerization. On the other hand, TG ChPI had significantly lower EC compared to SU ChPI, whereas that of TG PPI was not impacted, which was attributed to differences in legumin to vicilin ratio between pea and chickpea proteins. This study, for the first time, successfully demonstrated the scalability of chickpea protein isolate production following a mild SE-UF process. SU extraction resulted in protein isolates with high protein purity and good yield and preserved the protein’s structural integrity in comparison to the commercially produced ingredients, leading to better functionality. In addition, this study uniquely demonstrated the holistic impact of TG on the structure and function of SU PPI and SU ChPI, which could be leveraged to enhance texturization potential for meat analogues applications.