Legume Protein: Properties and Extraction for Food Applications

Abstract

Grain legumes are important sources of protein for nutritional and techno-functional applications. Their protein content is 18-50% protein on dry matter basis. Most of the protein is of the storage type, of which 70% are globulins. The globulin proteins are mainly legumins and vicilins, which are also known as 7S and 11S globulins, respectively. Several methods comprising wet and dry processes are used to extract protein from legumes. Choice of extraction method mainly depends on legume type and desired purity and functionality of extracted protein. Dry processing is suitable for starch-rich legumes, and involves fine milling and air classification. Wet processing uses solubility differences to extract and separate protein from non-protein components. The major extracted protein products are protein concentrate and isolate. Functional properties of protein depend on its amino acid profile, protein structure, hydrophobic, and hydrophilic effects. The major functional properties for food applications are solubility, water absorption capacity, oil absorption capacity, gelling, texturization, emulsification and foaming. They indicate ability of a protein to impart desired physico-chemical characteristics to food during processing, storage and consumption. The food products where isolated legume protein can be used include bakery products, plant based dairy alternative products, beverages and meat analogues.

1. Introduction

Grain legumes are important sources of protein for food at household level and also for applications during food processing. Legume protein has nutritional and techno-functional benefits and uses. In terms of nutrition, the protein is a rich and low-cost source of some of the essential amino acids, which has relevance for food, nutrition security and health. As the world population continues to increase, they are sustainable and affordable alternatives to animal protein. They are also increasingly being used in processed food as functional ingredients to impart desirable characteristics such as texture. This emanates from techno-functional properties of protein such as solubility, emulsification, water holding capacity, foaming, gelation, and oil holding capacity. These properties are important in formulation of food products such as plant based milk products, bakery products, beverages, meat analogues, and other categories of food products which require incorporation of protein as functional ingredient to achieve a optimal product. The functional properties of legume protein varies, and is influenced by type of legume, cultivar, molecular weight, amino acid composition, and charge distribution, and processing [1, 2].

Grains legumes are contained in pods at harvest, and are used as food either as dry or immature seeds. They are in the class Leguminosae. Their major common characteristic is ability to fix nitrogen from the atmosphere as a result of nitrogen-fixing microorganisms that is present in their nodules. They have been important sources of food for humans for over a thousand years in most parts of the world. Grain legumes are generally classified as either pulses or oilseed. Pulses include dry beans (Phaseolus spp.), dry peas (Pisum sativum), dry broad beans (Vicia faba), lentil (Lens culinaris), chickpea (Cicer arietinum), lupins (Lupinus spp), dry cow pea (Vigna unguiculata), pigeon pea (Cajanus cajan), bambara groundnuts (Vigna subterranea), vetch (Vicia sativa), and other minor pulses. Oilseeds are soybean (Glycine max), peanut (Arachis hypogaea), rapeseed (Brassica napus), sunflower (Helianthus annuus), sesame (Sesamum indicum) and other minor oilseed crops. They form key part of human food, as they are generally good sources of protein, energy, and micronutrients [3, 4]. Grain legumes generally have a common characteristic of being rich in protein. Their protein content range from about 18 to 50% on dry matter basis [5]. About 40 species of grain legumes exist in the world [6]. Besides their use as food, protein from legumes is also added to food during processing to achieve desired functional properties in the end product. They are therefore good sources of protein for food applications. Soybean and pea are among the major legumes that have been most explored and used in food applications. The other legumes have generally found lesser applications in industrial use. Their extraction and characterization continue to generate more knowledge on their properties and food applications. This chapter discusses protein from grain legumes, their characteristics, functional properties, and extraction for food applications.

2. Grain legume composition and protein characteristics

Grain legumes content of protein, carbohydrates, lipid, and micronutrients vary with type of legume, environment and agronomic factors. The macro-nutrient content of major grain legumes is approximately 18–50%, 0.8–21%. 60–69% and 0.9–7.2%, representing protein, oil, carbohydrates and fiber, respectively [6, 7]. Composition of grain legumes is presented in Table 1.

Legume	Composition (g/100 g)					Ref.
	Protein	Carbohydrate	Crude fiber	Fat	Ash
Dry bean (Phaseolus spp)	16–25	39–56	6.3–18.9	1.5	2.9–4.3	[8]
Black gram (Vigna mungo)	24	51–59	5.5	1.44	1.4	[9]
Green gram (Vigna radiata)	26–27	62–70	4.6	1.6–1.8	1–3.7	[10, 11]
Dry peas (Pisum sativum)	20–30	41–46	3.3–6.5	1–4	2–3	[12, 13, 14]
Dry broad beans (Vicia faba)	26–38	55–60	6.4–8.4	0.7–1.1	2.7–3.5	[15, 16]
Lentils (Lens culinaris)	23–29	60	13–14	0.8	2.4–2.8	[17, 18, 19, 20]
Chickpea (Cicer arietnum)	17–22	56–63	4.8–8	4.5–5.7	1.9–3.2	[21, 22]
Cowpea (Vigna unguiculata)	27	69.8–76	1.4–14	5	3.2–4.1	[23, 24, 25]
Pigeon peas (Cajanus cajan)	19–21.7	54	9.8–13	1.2–1.3	3.9–4.3	[26, 27]
Lupin (Lupinus spp)	40	47.7	10.8	8.9	3.15	[28]
Soybean (Glycine max)	40–44	30–34	4.5	18–19	3–4.8	[29, 30, 31]
Peanut (Arachis hypogea)	24–28	38	4.1–4.8	44–49.5	2.3–2.5	[32, 33]

Table 1.

Composition of selected legume sources of protein.

Legume protein are generally classified into three groups, namely storage, biologically active, and structural. Storage protein are the most abundant in legumes. They are a store of nitrogen for germination. Biologically active protein include lectins, enzymes and enzyme inhibitors, while structural proteins are ribosomal, chromosomal and membrane proteins. The storage proteins can further be classified into albumins, globulins, prolamins and glutelins. At the basic level, they can be differentiated by their solubility in various solvents. Albumins are soluble in water, globulins in dilute salt solution, prolamins in 75% ethanol, and glutelins show difficulty to solubilize but are soluble in dilute alkali. They can also be differentiated by their occurrence in monocotyledonous, cereals; and dicotyledonous plants, legumes. Albumins and globulins are the major storage protein in legumes, whereas prolamins and glutelins are the major protein in cereals. Globulin and albumin fractions account for about 70% and 10–20% of total protein in legumes, respectively [34]. The molecular weight of albumins is about 5–80 kDa, and is therefore generally low. Trypsin inhibitors, lectins, and amylase inhibitors may in some cases be part of albumins. As nitrogen source for germination, the storage proteins are rich in nitrogen containing the amino acids asparagine, arginine and glutamine [35].

The major classes of globulin proteins in legumes are legumins and vicilins. They are also referred to as 7S and 11S globulins respectively, based on their Svedberg sedimentation values (S) [36]. This is similar for peas, soybean and other grain legumes. But they occur in different ratios in legumes, but both are oligomers. The 11S type is a hexamer made up of six subunits of molecular weight of about 340–360 kDa; whereas the 7S fraction has molecular weight of about 145–180 kDa made up of trimers of molecular weight of about 60–70 kDa [34, 37, 38]. Vicilins generally vary among the legumes in terms of molecular weight. Legume protein is low in cysteine, cysteine, methionine and tryptophan, which are sulfur containing amino acids. Only soybean contains all the essential amino acids required for human nutrition. Its protein also has good digestibility. Thus it has high quality protein almost comparable to protein from animal sources in terms of nutritional quality. But legumes in general have high nutritional value due to their high protein content, and content of other macro- and micro-nutrients.

Protein occur in structures described as primary, secondary, tertiary and quaternary. The primary structure denotes the linear sequence of the amino acid of the protein but providing little information that explains protein functionality. The higher structures are determined by conformational fold and helps in understanding protein molecular characteristics such as net charge, size, shape and other properties.

3. Technologies for extracting legume protein

Several technologies exist for extraction of protein from legumes. Some have found success in commercial processing, while others have not mainly due to economics of the technologies. The methods include several wet extraction and dry processes. Extraction of protein from legume to an appropriate level of purity is necessary for its use in food formulations. Choice of extraction process depends on amount of protein required in the protein extract, legume type, functionality desired in the protein and nature of previous processing [35]. The processing methods can generally be classified into two categories, namely aqueous and dry processing. The various legumes differ in composition as some are lipid rich, while other are starch rich. Each is suitable for protein extraction using a particular method. Dry processing which involves fine milling and air classification is suitable for starch-rich legumes as pea and faba bean, but generally not used to process oil-rich legumes such as soybean, peanut and similar seeds [5, 39].

3.1 Aqueous extraction

Aqueous processing involves using water or alkaline solution to solubilize protein from legume flour or flakes in the initial processing step. It uses solubility differences to separate protein from carbohydrates, fiber and other non-protein components. This is then followed by purification and drying. This method has been used for several decades in soy protein extraction. Extraction efficiency depends on pH, temperature, ionic environment and solvent to flour ratio. It has variations depending on raw material which is being used for protein extraction. It is desired to achieve highest protein recovery without the process being detrimental to protein functionality. The major extracted protein products in terms of application in food are protein concentrate and isolate.

3.1.1 Protein concentrate

Processing of grain legumes into protein concentrate follows the following general steps, namely cleaning, de-hulling, flaking, defatting, protein extraction, neutralization and drying. Production of protein concentrate from legumes is illustrated in Figure 1. De-fatted soy flakes or flour is the starting raw material. Protein extraction can be carried out using three methods. The methods are washing with (1) aqueous alcohol (60–90%), (2) acid leaching at pH 4.5 and (3) moist heat water leaching. If extraction was done using alcohol, solvent is removed in a process step referred to as desolventization. The protein may then be neutralized to pH 7 then dried. Drying options include drum drying and spray drying, with the latter being preferred and most commonly used. Protein concentrate products contain about 70% protein.

Figure 1.

Process flow diagram for production of protein concentrate.

3.1.2 Protein isolate

Protein isolate is more refined than concentrate. Its processing generally involves alkaline extraction of protein followed by protein recovery at their isoelectric pH of about 4.0–5.0, depending on the legume. The general principles of this extraction approach can be applied, with appropriate modifications, to extract protein from different legumes. Process options exist for unit operations such as preparation, defatting, pH of carrying out protein recovery and drying. The general process and its possible modifications are described here. Figure 2 illustrates processing steps involved in production of protein isolate from legumes.

Figure 2.

Process flow diagram for production of protein isolate.

Processing starts with cleaning of legume seeds to remove impurities such as stones, mold infected seeds, discolored seeds, sand, soil and other foreign matter. Grains such as soybean, chickpea, and pigeon pea are du-hulled to remove the fiber-rich seed coat. The seed coat is also referred to as hull, husk, testa, or husk depending on grain legume. The grain is then prepared into flakes or flour. Then defatting is carried out. The most commonly used method to remove fat is by hexane extraction. It can be carried at either low or high temperature of around 60–80°C. The high temperature also inactivates lipoxygenase, which contributes to causing off-flavors in the resultant extracted protein. Defatting using hexane leaves residual oil of about 0.5–1% (w/w) of the extracted protein. The residual lipid is composed of phospholipids and polar lipids. They cannot be extracted with hexane. They are sources of off-flavors as they participate in auto-oxidation and lipoxygenase mediated reactions. Following defatting, the solvent is the recovered from the meal and oil. Alternative defatting processes include supercritical carbon dioxide extraction, enzymatically aided extraction, organic solvent extraction, and aqueous extraction [40]. They have various degrees of efficiency and cost implications. They have however not been used for commercial processing due to cost and efficiency.

The intermediate product obtained at the above step can be toasted and cooled to make defatted soy flour. Toasting is applied to inactivate trypsin inhibitors. The product obtained from removal of solvent is also called flakes, which can also be milled into flour, as starting materials for aqueous extraction into protein concentrate and isolate.

To extract proteins, the flakes is milled into flour and mixed with water at a ratio of 1:6. The pH is adjusted to 9 using lye so that protein goes into solution. Legume proteins are generally soluble in aqueous media. In solutions where pH is less or greater than isoelectric pH, a net positive or negative charge occurs resulting in repulsion and protein staying in solution [41]. Clarification is then carried out to remove non-protein insoluble materials such as carbohydrates and insoluble fiber. Clarification can be done using centrifugation, filtration or membrane process. Protein is then recovered from the solution. Recovery can be done using ultra-filtration or precipitation. Precipitation can be achieved using isoelectric precipitation, salting out, salting in, heating or organic solvents. Isoelectric precipitation is the most commonly used method during commercial scale processing. It is generally not detrimental to protein functionality. But it may cause protein to aggregate and also result in changes in solubility as a result of non-covalent interactions. Isoelectric precipitation is carried out by adjusting the acidity of the protein solution using dilute acid to pH 4.0–5.0. Separation is then done by centrifugation or decanting. The resultant protein is washed, then may be neutralized to pH 7. Final processing step involves drying by spray or drum drying. In the former method, a thin layer is applied to a heated drum to evaporate water. The preferred method is however spray drying because it gives protein that has less heat damage. Some aggregation may however occur. Protein isolate contain at least 90% protein.

3.2 Dry extraction

Dry fractionation can produce protein-enriched products which possess native functional properties. This is mainly because the process is mild [5]. Dry processing involves physical separation of starch and protein. It relies on the principle that milling can separate protein bodies from other seed components to give flour streams that is fractionated into different components. This has applications in starch rich legumes such as pea and faba bean. The method is however not applicable to grains that are rich in oil. In general, size of starch granules partly determines suitability of a particular legume for dry fractionation [5]. Milling is considered effective when it removes the protein bodies from starch granules, the latter being bigger in size. For pea and faba bean, starch and protein can be separated by milling followed by air classification. During air classification, protein separate as fine particles while starch are the coarse particles. Protein are separated as the light fraction, and starch as the heavy fraction based on their different shape, density, and size. This results in protein and starch enriched fractions.

Dry process involves milling and air classification. Starch granules are separated from protein bodies during fine milling usually by pin milling. This enables their separation during fractionation by air classification. Two processes are therefore involved in dry processing, namely, reducing particle size by milling and separation of the particles using their shape, density, size, and electrostatic properties [42]. The starch rich fraction is re-milled and fractionated. Particle size and shape are the main properties which are manipulated during dry extraction. The particle size can be varied by size reduction in the range, coarse, >500 μm; fine, 50–500 μm and ultrafine, <50 μm. Particles sizes selection enhance protein and starch enrichment and also reduce content of undesirable components such as anti-nutrients. Impact mill can produce the above range of particle size, while hammer milling can be used to produce only coarse and fine particle size. Ultrafine milling is used for protein and starch enrichment with pulses such as peas and faba beans. Air classification is applied after fine milling to obtain protein enriched fraction. The resulting end product has its protein content doubled compared to the raw material. Dry milling of faba bean and Lima bean have been reported to give protein yields of 63–75% and 43–50%, respectively [5, 42]. Air classification has not been found to be successful with oilseeds due to generation of free lipids [5].

4. Functional properties and food applications of legume proteins

Legume proteins have diverse application in foods due to their functional properties. They contribute to food having desirable textural characteristics during processing, storage and consumption. The major functional properties that are relevant for food applications are solubility, water absorption capacity, oil absorption capacity, gelling property, texturization, flavor binding, emulsification and foaming. These properties depend on amino acid profile, protein structure, hydrophobic, and hydrophilic effects. The functional properties that are key to their application in food are reviewed below.

4.1 Functional properties

4.1.1 Solubility

Solubility of legume protein is important in formulation of products such as plant based dairy alternative products and beverages among many other similar products. Protein possess minimum solubility at pH corresponding to their p1. This is because of a zero net charge at their pI which results in aggregation of proteins. In general, legume proteins have minimum solubility in the range pH 4–4.5, and solubility maxima at above pH 8 and below pH 2.5. There solubility is high at low and conditions, and high alkaline conditions. Solubility is lowest at the isoelectric points of proteins. This is taken advantage of during protein extraction when purifying proteins. Solubility of proteins is affected during processing depending on level of heat treatment. Protein of high solubility is achieved by mild processing conditions mainly in terms of heat treatment. Solubility of legume proteins generally vary with heat treatment and pH [43].

4.1.2 Water absorption capacity

Water absorption capacity (WHC) refers to the amount of water that protein can absorb. It is also be referred to water binding capacity. Commercially prepared soy protein isolate can absorb water at 4–5.5 times its weight, and 2.4–3.4 for concentrates [4]. In fava bean, lentils, cow peas, chickpeas, soybean, beans and peas WHC was reported to be in the range 2.39–6.78 g of water per gram of protein concentrate [44]. This generally indicates wide variation in WHC, and suggests that some of the protein isolates are better suited than others for use where WHC is intended to be achieved. Soybean and pea protein isolates have been reported to improve viscosity and prevent syneresis in cultured dairy products [45]. These are partly functions of their WHC. In another study [46], WHC was found to be 4.09–6.13 g/g for pinto bean, lima bean, red bean, kidney bean, black bean, navy bean, red bean, mung bean, lentil and chickpea. WHC of flour produced from 21 legume samples which included green gram, chickpea, lentil, soybean and several bean varieties was found in another study to be 1.32–3.14 g/g [47]. Protein products from legumes are in the form of flour, protein concentrates and isolates. They differ in level of refining, with flour being less refined and isolate being highly refined source of protein. The various forms of protein have different uses. Refining whole flour such as by defatting and removal of other non-protein components as much as possible improve functional properties such as WHC and oil holding capacity [48]. Compared to other legumes, soybean and pea have generally been extensively characterized for functional properties and also used in food applications. WHC of 7 protein isolates from 7 pea cultivars were reported to range be in the range 1.88–2.37 g/g [49].

4.1.3 Oil absorption capacity

Oil absorption capacity (OAC) refers to the weight of oil absorbed per unit weight of protein. Legume proteins have been reported to have OAC in the range 1–4 g/g protein [50, 51]. OAC has been reported to be in the range 3.46–6.37 grams oil per gram of protein concentrate in fava bean, lentils, cow peas, chickpeas, soybean, beans and peas [44]. Like WHC, OAC also varies among legumes, thus they vary in their effectiveness in achieving OAC. Lentil and chickpea and several types of common beans was reported to have OAC in the range 0.93–1.38 g/g [46]. In another study, flour prepared from 21 legume samples comprising green gram, chickpea, lentil, soybean and several bean varieties had OHC of 0.62–2.57 g/g [47]. OHC as well as other functional properties will vary with type of legume as well as form of protein. In a study, OHC of protein isolates from 7 pea cultivars ranged from 1.07 to 1.40 g/g [49].

4.1.4 Emulsification

Plant protein exhibit emulsifying properties. This relates to their ability to stabilize oil in water and water in oil emulsions thereby reducing interfacial tension and phase separation [15, 52, 53]. Thus they play a role as emulsifiers due to hydrophobic and hydrophilic balance at the interface of oil and water. Proteins align at interface so that the hydrophobic part face the oil phase, while its hydrophilic part face the water phase. Emulsification properties can be measured using methods such as turbidimetric method and droplet size measurement. The former comprise emulsifying activity index (EAI) and emulsifying stability index (ESI). Emulsification properties of legume protein affected by pH, temperature, protein concentration, and ionic environment [46]. Bean and pea protein isolates were reported in a study to have similar emulsifying capacity of about 27%, but emulsion stability was higher for pea protein isolate [54]. A study reported emulsifying activity index and emulsifying capacity index of protein isolates from 7 cultivars of pea to be 31.09–39.05 m²/g and 10.97–11.26 min, respectively [49]. Pea and soybean protein products are one of the most characterized and used in food applications.

4.1.5 Foam formation and stability

Legume protein are also important in foam formation and stability. When protein unfold to form a interfacial skin that hold air, this is referred to as foam. Foaming property of a given legume protein can be measured by homogenizing known concentration of protein dispersion to form foam. Foam capacity (FC), also known as form expansion (FE), is calculated as percent increase in volume from whipping, and foam stability (FS) measures change in volume over time [39]. Foaming is utilized in food applications such as whipped toppings, cakes, meringues, leavened breads and beverages. In a study, protein from fava beans had high foaming capacity than protein from lentils, cow peas, chickpeas, soybean, beans and peas [44].

4.1.6 Gelation

Legume proteins, as other proteins, can also form gels upon heating. Heat induced gelation involves unfolding of proteins, and exposure of reactive groups leading to aggregation into a gel. Gelation imparts characteristics such as water holding capacity to the food. It also holds ingredients. This has applications in foods such as meat and desserts. Gelation affected by pH and ionic environment. Gelling effectiveness is evaluated using the parameter, least gelling concentration (LGC) which is the lowest concentration of protein required to form a stable gel [19]. Legume protein can provide suitable gels for food applications [55, 56, 57].

4.2 Food applications

Legume proteins applications in food formulation are in the areas of nutritional enhancement, technological and functional properties. The advantages of using plant proteins include their being abundant, low cost, and healthful compared to alternatives such as chemical based ingredients. Legume proteins can be used in the form of flours, concentrates and isolates depending on the particular food application. Soybean is currently the most widely used legume protein. The areas where the proteins can be used include bakery products, plant protein based dairy type products, meat analogues, and emulsifiers [5, 58, 59, 60]. Bambara groundnuts can have applications in acidified high acid beverages owing to properties of vicilin fraction of its protein [61].

5. Conclusions

Grain legumes are important sources of protein and other macro- and micro-nutrients. Besides having a significant role in nutrition and food security where they are grown, they are also source of proteins whose functional properties have diverse food applications. As the world population continues to increase, legume protein have potential to play a significant role to fill the gap for demand for more protein. Methods for extracting legume protein for food applications can generally be classified into dry and aqueous methods. The various legume proteins still need to be characterized for their ability to impart desired physico-chemical properties in food systems during processing, storage and consumption.

Conflict of interest

The authors declare no conflict of interest.