Advances in legume protein extraction technologies: A review
Graphical abstract
Introduction
The challenge of hidden hunger affects over two billion people, especially in developing and underdeveloped countries, mostly because of nutrient deficiency in their diets (Kumar et al., 2021). Food protein has long been linked to the body's growth, development, and maintenance, earning it the status of an essential macronutrient (Campbell, 2019). Protein demand continues to outstrip supply and predicted to quadruple in the long run (Burger & Zhang, 2019; Emkani, Oliete, & Saurel, 2021; Fernando, 2021). By 2050, the world's population is predicted to reach 9.9 billion (Henchion, Hayes, Mullen, Fenelon, & Tiwari, 2017) and maybe difficult to feed in the next years than it can, now. Other key drivers for higher protein demand include improved earnings, rapid urbanization, coupled with recent recognition of the importance of protein in a balanced diet, particularly for growing children and elderly (Detzel et al., 2021; Yagoub, Ma, & Zhou, 2017).
Major sources of protein include animals, plants and microorganisms (Detzel et al., 2021; Ismail, Senaratne-Lenagala, Stube, & Brackenridge, 2020; Jarvio, Maljanen, Kobayashi, Ryynanen, & Tuomisto, 2021; Saw et al., 2020). Animal sourced protein appear to be the most prominent among the three major sources in terms of nutritional quality. However, concerns for such diet sustainability, food security and environmental impact are overwhelming. Several reports argue that animal-based meals negatively impact climate change through the generation and emission of more greenhouse gases (GHG). There is also the challenge of unnecessary pressure on land use, leading to extinction of biodiversity and other important natural ecosystem (Detzel et al., 2021; Karlsson Potter & Röös, 2021). The popularity of plant-based food proteins, especially from legumes and pulses continue to pick momentum. Several benefits outlined by Tripathi et al. (2021) in favor of plant-based proteins mitigate nearly all the challenges presented by the use of animal proteins.
To incorporate legume proteins as food ingredients, firstly, proteins need to be extracted, isolated and purified. Extracted proteins have found wide application and use as foaming agents, emulsifiers, stabilizers and fortificants; as food ingredients, they play enhancement roles in the end product's functional and nutritional qualities (Mudgil, Omar, Kamal, Kilari, & Maqsood, 2019). As people become more aware of the importance of high-quality plant proteins, quest for greener and ecofriendly extraction methods keep rising. Currently, researchers are finding ways to modify and hybridize conventional methods, more still, innovate technologies to meet these demands (Vernes et al., 2019; Zhang, Wen, Zhang, Duan, & Ma, 2020). Also, current extraction methods have not been able to holistically address the presence of residual antinutritional factors (ANF) in pulse proteins. Presence of ANF is one of the fewer problems associated with pulse protein processing, which invariably, play a major role in pulse protein digestibility challenges (Lu, Chen, Wang, Yang, & Qi, 2016; Vernes et al., 2019; Zhang et al., 2020). The intended use of the protein also influences the choice of extraction technique. For example, proteins extracted using enzyme-assisted techniques are mostly applied as emulsifiers in food (Kamal, Le, Salter, & Ali, 2021). Some studies have demonstrated the potentials of green technologies to extract plant proteins with superior functional and nutritional qualities. Fig. 1 highlights the core reasons why the use of green technologies for extraction are currently being advocated for in pulse protein extraction.
For techno-economic sustainability in the use of green technologies for extraction to be achieved, there should be overwhelming acceptance and trust on the safety of protein products resulting from them. There is also need to understand the environmental impact and sustainability challenges, in order to decide the trade-off between these technologies, product quality and economic gains. Therefore, there is need to have current information on studies being done in the area, mechanism of operation of the techniques and areas of gap. These would create further pathways for future research. Previous review discussed advances in plant protein extraction mechanism (Kumar et al., 2021); (Chen et al., 2019b). However, this present study differs because it narrowed the discussion on protein extraction from pulses and legumes, which are the main plant based protein sources from wet and dry fractionation stand points and possible use of green technologies. The review provides more beneficial information for researchers to gauge what has been done using both current and projected novel protein extraction techniques, proffering insight to current findings with regards to mechanism, limitations and future research routes to encourage scale up and popularity of these new technologies in the food industry. When compared with previous reviews on the subject, the present study attempted to identify how yield, recovery and functional properties were affected which is important for decision making with respect to choosing extraction method. Finally, critical discussion on recent advances in pulse protein extraction, their advantages, and major limitations and future outlook to their upscaling were also highlighted.
A typical pulse seed consists of the seed coat (testa), cotyledon, micropyle, and embryo, as seen in Fig. 2. The seed coat, also known as the hull, can be yellow, speckled, black, green, brown, purple, or reddish in appearance. It has a firm, smooth surface and makes up 7 to 15% of the total seed mass. The weakest part of the seed coat's palisade layer is the plumula located at the top of the cotyledons. The hypocotyl serves as a water entrance point during the imbibition period (Byanju, Rahman, Hojilla-Evangelista, & Lamsal, 2020). Cotyledons make up roughly 85% of the seed mass, with the embryo accounting for the remaining 1–4%. The testa is the seed's outermost layer, covering nearly all the seed's surface. The micropyle, located adjacent to the hilum, is a small aperture in the seed coat. Both the micropyle and the hilum have been linked to cellular permeability and water absorption. The embryonic structure remains after the seed coat has been removed from the seed. Two cotyledons and a short axis above and below make up the embryonic structure. Except for the axis and a faint layer of protection given by the seed coat, the two cotyledons are not physically connected. As a result, the seed is particularly prone to breaking. An explicit understanding of legume seed morphology is needed to provide insight into the breakage behavior of their cellular component (Fig. 3), upon impact or deformation (Schutyser, Pelgrom, Van Der Goot, & Boom, 2015). However, the extent of extraction, isolation, and purification is usually hampered by polysaccharides and other polymers present in their cell walls as well as protein location within the cell matrix (Byanju et al., 2020).
To have a more precise extraction, without damaging any of the fractions (protein, starch, and fiber), the structure and composition of the various segments must be investigated and well understood. Also, the exact location and distribution of proteins in the legumes would inform the most appropriate extraction method and optimal extraction parameters. The benefit would be to enhance kinetics of break behavior, increase extraction efficiency and improve key equipment features using such information. Some of the techniques used to explore legume morphology include laser-induced breakdown spectroscopy (LIBS) and atomic force microscopy (Li, Ma, Li, Zhang, & Dai, 2017).
Proteins are mainly classified based on their composition, biological functions, shape or dimensional structure, and solubility (Tripathi et al., 2021). Based on compositional classification, proteins are categorized into simple and conjugated proteins. Simple proteins only yield amino acids and sometimes minute quantities of carbohydrates when hydrolyzed (Tripathi et al., 2021). Examples are albumins, globulins, glutelins, prolamines, and scleroproteins. On the basis of solubility in various solvents, legume and pulse proteins can also be classified as Osborne fractions – globulins, prolamins, albumins and glutelins (Day, 2013). Globulins (also known as storage proteins) make up the majority of legume proteins (about 70%), with albumins and glutelins accounting for 10–20% of total proteins (Roy, Boye, & Simpson, 2010). Table 1 shows some ‘Osborne fractions’ of protein and the type of solvent in which they solubilize.
Finally, based on sedimentation coefficient, globulin proteins are further classified as 7S and 11S, known as conglycinin and glycinin in soybean (Din et al., 2021); while in pea, they are vicilin and legumin, respectively (John, Chandra, Giri, & Sinha, 2021). The amount of these fractions present in the extracted protein will determine the functionality of such proteins in a food system. Also, the knowledge of how different Osborne fractions are affected during extraction will influence the choice techniques.
Legumes and pulses are no doubt popular sources for most dietary nutrients, containing adequate amounts of protein (rich in essential amino acids) and carbohydrate to a wide range of vitamins and minerals, among other nutrients. For example, pea protein is a rich source of branched chain amino acids such as leucine, glutamic acid, lysine, valine and leucine (Hertzler, Lieblein-Boff, Weiler, & Allgeier, 2020; Lu, He, Zhang, & Bing, 2019). For this reason, pea protein extracts can conveniently substitute for whey protein, which is considered to be major ingredient in sports nutrition (Banaszek et al., 2019). However, legumes and pulses also contain anti-nutritional factors (ANF). These are chemical compounds that tend to upset the body's digestive and absorptive processes, leading to certain immune disorders. The prominent ANF in legumes and pulses are chymotrypsin inhibitor, trypsin inhibitors, lectins, anti-fungal peptide and ribosome-inactivating proteins (Kumari & Deka, 2021). Lectin is mostly implicated in pulses such as peas while chymotrypsin inhibitor and trypsin inhibitors are associated with legumes like soybeans. The activities of ANF associated with legumes proceed in two ways: by inactivating the digestive enzymes and hypersecretion of the pancreatic fluid. For lectin, they tend to cleave to epithelial cells of the intestinal mucosa, creating substantive disturbance of the protein digestion process. In contrast to recent findings, however, studies have shown some health benefit potentials of some of these ANF. Kumari and Deka (2021) reported that lectin from peas is able to offer beneficial effect on the immune system by being a source of vitamins and minerals; while chymotrypsin and trypsin due to their possession of anti-inflammatory properties are able to inhibit the growth of tumors and spread of some type of cancers (Roy et al., 2010). The challenge is that these ANF may attach to the fine protein fraction during protein extraction from legumes and pulses especially if dry fractionation is used. Unfortunately, there is paucity of information on the effect of both conventional and newer extraction methods on these ANF in proteins extracted from pulses and legumes. Also, even though recent researchers have found potential health benefits of some of these ANF, there are no sufficient information to ascertain these claims.
Functional properties determine the overall behavior and performance of protein in food systems, particularly during manufacturing and storage. These properties highlight the physicochemical characteristics displayed by proteins as they interact with other components in a multicomponent food system (Awuchi, Igwe, & Echeta, 2019). With regards to the food system, several factors affect protein's functional characteristics. First, noncovalent forces between amino acid side chains interact with covalent disulfide bonds between thiol groups of cysteine residues in an elaborate manner (Awuchi et al., 2019). These series of complex interactions are entirely responsible for any protein's chemical and physical functions. Therefore, changes in physicochemical and functional qualities will occur if these interactions between protein molecules and their conformational structure are altered (Byanju et al., 2020). Secondly, conditions of the immediate environment such as pH, salt concentration, presence of denaturants, surfactants and viscosity of the solvent proteins as contained in Table 2. Lastly, protein structure including protein composition, amino acid sequence and conformation, will influence their behavior in their immediate environment (Taherian et al., 2012).
Generally, it is expected that extraction parameters and conditions be adequately mild to reduce denaturation which induces protein insolubility, while ensuring retention of protein's globular nativity for enhanced functionality. This knowledge is important when making decision for the type of method to use in plant protein extraction. For example, each extraction method has its distinct processing conditions such as pH, solvent use, temperature among other parameters. These conditions will affect the different functional and nutritional property of extracted proteins and consequently their performance in food systems. Protein functional qualities have been extensively explored and reported. Findings from Awuchi et al. (2019), suggested that incorporating proteins in food manufacturing, even in modest amounts, can considerably impact the physical qualities of the Food. Some functional properties of proteins which make them essential ingredients in food processing include emulsification, hydration, water retention ability, gel formation, viscosity, foaming ability, cohesiveness, and color control (Kiosseoglou, Paraskevopoulou, & Poojary, 2021; Yang & Sagis, 2021). Table 2 gives a summary of some functional qualities of protein, typical food application and their mechanism of action.
Section snippets
Overview of pulse and legume protein extraction techniques
The efficiency of any successful protein extraction procedure is influenced by plant protein structure and chemical properties (Byanju et al., 2020); the nature of the protein source (Lee, Show, Ling, & Chang, 2017); extraction methods and condition (Boye, Zare, & Pletch, 2010; Feyzi, Milani, & Golimovahhed, 2018). Peas and many other starch-rich legumes are fractionated by dispersing them in water to form a solution of protein and starch granules. For oil-rich legumes like lupine and soy, the
Extraction of legume proteins using conventional/traditional methods
The conventional methods to be discussed in this section are some currently used wet extraction methods, which are broadly categorized into chemical (acid or alkali), enzymatic, salting-in methods and dry fractionation method. In addition, some of the most common methods and stages for producing pulse / legume protein isolates / concentrates are shown in Fig. 4.
Advances in pulse/legume protein extraction methods
The current interest to reach sustainable protein supplies, coupled with some challenges (which becomes greater on an industrial scale) arising from the use of conventional methods in terms of protein yield, purity, and quality as well as the impact on the environment, has informed researchers to delve into other methods which will improve or replace existing conventional procedures. The methods are mainly physical treatments applied alone or as pretreatments prior to the use of extraction
Future outlook
The world is currently in an environmental chaos, and planetary boundaries are perpetually being infringed due to such unsustainable conventional practices. Therefore, global stakeholders are expected double efforts and advocacy towards developing and using eco-efficient approaches and technologies that could diminish or alleviate consequences related to insufficiency in plant-based protein production. The overall expectation is to meet global consumption in order to restore global health.
Conclusion
The goal of this study is to highlight peer-reviewed articles and reports on plant protein extraction research, with focus on pulses and legumes, and how their protein extraction techniques have developed over time. Employing conventional methods as independent procedures have several disadvantages, ranging from high water, energy, and chemical requirements to consumers' present need for healthier food options free of any trace of chemicals, and eventually, negative environmental consequences.
Funding
The funding for this research was provided by the Sustainable Protein Production Program of the National Research Council Canada (SPP130-A1-019784). This manuscript represents National Research Council Communication # 58298.
Declaration of Competing Interest
None.
Acknowledgment
The authors appreciate the support of Raphael Aidoo in proofreading and editing this paper.
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