Physico‐chemical and functional properties of legume protein, starch, and dietary fiber—A review

Legumes have gained increased dietary importance in recent years due to their recognized health benefits. Recent plant protein revolution has elevated legumes to the forefront from consumers' and food industry's perspective. Unlike cereal proteins and starches, there is a scarcity of information on the structural properties of legume starches. Consumption of legume‐derived dietary fibers have a positive impact on the human health, in particular, gut health, which is a current research focus for nutrition and health professionals. Knowledge of legume ingredients properties (e.g., protein denaturation, starch gelatinization, pasting, and thermal properties) could aid in understanding functionality and potential uses of these materials. The physicochemical, thermal, and the functional properties of legume proteins, starches, and dietary fibers are elucidated. Both the food ingredient manufacturers and research and development professionals in the food industry can benefit from the information provided in this review article.


| INTRODUCTION
Legumes (Fabacea family) are dicotyledonous seeds, rich in proteins, carbohydrates, and dietary fibers (DFs). In recent years, legume-based ingredients have steadily increased in use in various food applications.
Legumes have significantly higher protein content than cereal grains, making legumes among the richest food sources of proteins and amino acids for human nutrition. In addition to offering a source of essential amino acids and bioactive peptides, legume proteins influence many functional properties, which could help expand their potential use in the development of a wide variety of food products (Boye et al., 2010;Dhull, Punia, Sandhu, et al., 2020).
The carbohydrate fraction of legumes is primarily composed of starch (65%-72%) and DF (10%-20%) (Haytowitz et al., 2011). Legume starches are characterized by a higher percentage of slowly digestible resistant starch (RS), resulting in low glycemic index, and act as functional foods. The hypoglycemic effects of legumes have been further supported by high contents of DF (Trinidad et al., 2010).
In comparison to wheat, the predominant flour/ingredient used for many food products, legumes offer improved nutritional quality.
Legumes have higher proteins and higher total DF content, and lower carbohydrates (Dhull, Punia, Kidwai, et al., 2020;Siddiq & Uebersax, 2012). In addition, legume-based ingredients can be used to develop gluten-free products, which has been a growing segment of food industry in recent years. Furthermore, legume-extracted proteins are emerging as a major source of continued global demand for plant proteins as meat alternative. However, despite many nutritional benefits, the per capita consumption of legumes is very low in the developed countries. The superior functionality of legume-based ingredients can play an important role in expanding legumes consumption beyond traditional products and uses, as shown in Figure 1. There is considerable variation in legume ingredients by pulse type; therefore, an understanding of species-specific functional properties is important. Our objective is to provide a review of research on the physico-chemical and functional properties of legume ingredients (starch, protein, and DFs) and their functional role in food product development.
F I G U R E 1 Diverse application of legume protein, starch and fiber ingredients T A B L E 1 Amino acid profiles of selected legume seeds  (Baptista et al., 2017). The seed proteins can be classified as structural, storage, and biologically active. The main biologically active proteins are enzymes, lectins, and enzyme inhibitors. Globulins (35%-80%) and albumins (2%-37%) are the major protein fractions in legume seeds (Hall et al., 2017). Legumin (11S) and vicilin (7S) are the major globulins, whereas enzymes, enzyme inhibitors, and lectins belong to albumins (Boye et al., 2010;Venkidasamy et al., 2019). Albumins (rich in lysine and sulfurcontaining amino acids) and globulins (containing higher content of aspartic acid and glutamic acid) have different overall amino acid profiles (Venkidasamy et al., 2019).

| Amino acid profiles of legume seeds
Amino acid profiles are indicators of proteins' nutritional qualities and functionalities. Essential/nonessential amino acid content is one of the parameters that provide important nutritional details about the protein quality of legumes (Table 1). Moreover, functional groups of amino acids also affect the physicochemical and functional properties and the protein behavior in foods. Glutamic acid and aspartic acid, nonessential amino acids, are the most abundant amino acids in the selected legumes except chickpea, whereas limiting amino acids vary according to legume species (Table 1).

| Physicochemical and functional properties of legume protein isolates (PIs)
Two of the important properties of PIs are their thermal stability and thermal behavior during the processing. Thermal behavior of protein denaturation (T d ) has been discussed in detail in a separate review (Ahmed et al., 2021), with a brief discussion here. The T d of chickpeas at 70% purity and faba beans at 88% purity were 205 C and 183 C, respectively; bean isolates at 55% and 75% purity had T d of 211.5 C and 193.8 C, respectively; and lentil isolates at 45% and 75% purity had T d of 199.5 C and 183.4 C, respectively (Ricci et al., 2018). Thermal stability of legume PIs increases with the purity. The T d and denaturation enthalpies of various bean PIs ranged from 90 C to 152 C and 32.9 to 134 J/g, respectively (Gundogan & Karaca, 2020).
The water holding capacity (WHC) and oil holding capacity (OHC) of legume PIs range between 1.8-6.8 g/g and 3.5-6.8 g/g, respectively. Legume PIs exhibit good foaming capacity, emulsion capacity, solubility, and emulsifying activity index (Gundogan & Karaca, 2020;Lafarga et al., 2020), and those properties are retained even at extreme pH (2.0 and 10.0) (Lafarga et al., 2020). Bara c, Peši c, Stanojevi c, Kosti c, and Bivolarevic (2015) observed that the native isolates of soybean had the highest and adzuki isolates had the lowest solubility at selected pH (3.0, 5.0, 7.0, and 8.0) while the most stable foams were observed for native soy PIs.
In addition to food applications, legume proteins have found extensive use in the encapsulation. Legume PIs (e.g., from pea and chickpea) are used to encapsulate vitamin B 9 (folate), α-tocopherol, ascorbic acid, and phytase separately; with significantly high encapsu-  Structural and functional characteristics of these glucan polymers influence the functionality and the end use of starch.
In comparison with cereal grains, legumes predominantly possess slowly digestible starch (SDS), which is the most desirable form of dietary starch because it elicits slow glycemic response and attenuates plasma insulin levels (Chung et al., 2009). This functional property of legume starch makes it a perfect ingredient for use in healthy food products.

| Starch structure: Amylose and amylopectin
The proportion of amylose (AM) to amylopectin (AMP) in legume starches depends upon the starch source, that is, variety, growing condition, and origin; however, amylopectin remains the significant component . The accepted structure of amylopectin comprises short amylopectin chains forming double helices and combining into clusters (Aberle et al., 1994). These clusters yield a structure that consists of alternating crystalline and amorphous lamellae. The amylose content of legume starches varies from 18.7% to 49.7% (Table 2), which differs widely due to genotypic variation, growth conditions, enzymatic activity during biosynthesis of starch, procedures of starch isolation, and so forth (Kossmann & Lloyd, 2000;Ovando-Martínez et al., 2011;Zhou et al., 2004). A range of molecular weights (M w ) have been reported for amylose (1.0 Â 10 5 to 5.45 Â 10 6 Da) and amylopectin (4.34 Â 10 6 to 8.31 Â 10 8 Da) ( Table 2). The average chain length of amylopectin (13-24 DP, degree of polymerization) is responsible for the crystallinity for legume starches (Hoover et al., 2010;Ma et al., 2017). The chain lengths affect the enzymatic susceptibility and functional properties of starches (Du et al., 2014). The percent amylopectin chain length distribution in legume starches (presented in Table 3) followed the order of DP 13-24 > DP 6-12 > DP 25-36 ≥ DP ≥ 37. Chickpea starches, however, are found to be exceptional as it contained very high amount of shorter amylopectin chains DP (6-12) compared with other legumes.

| Gelatinization and rheological properties of legume starches
Starch rheology is a vast area of research as it has significant impact on food product development. Starch granules gelatinize in the presence of water at the appropriate temperature followed by gel formation (Ahmed, 2012). The gel rigidity depends on the concentration of the starch and many other factors. The gelatinization and the glass transition temperature of starch have been described in another review article (Ahmed et al., 2021).  Ma et al. (2017) subjected to small/large amplitude oscillatory shear, steady flow, or creep measurements during rheological measurements (Acevedo et al., 2020;Ahmed et al., 2016;Doublier, 1987;Phrukwiwattanakul et al., 2014). Ahmed (2012)    Abbreviations: GI, glycemic index; ΔH gel , gelatinization enthalpy; ΔH r , retrogradation enthalpy after 7 days' storage at 4 C; RS, resistant starch; T p , peak gelatinization temperature of starches.
Steady flow measurements of starch dispersions/gels are characterized by non-Newtonian behavior and described by a power law or the Herschel-Bulkley models. Most of the starches demonstrate shear-thinning behavior (flow index, n < 1), with yield stresses and thixotropy (Ahuja et al., 2020). The n values for a pool of legume starches range from 0.37 to 0.76 (Byars & Singh, 2016). Thixotropic breakdown of navy bean starch has been reported at 10% and 12% concentrations during shearing at 85 C and 95 C (Lee et al., 1995).

| Retrogradation of legume starches
The gelatinized starches retrograde during storage and shorten the shelf life and acceptability of food products. Retrogradation in pulse starches have been studied extensively using various techniques (Betancur-Ancona et al., 2002;Hoover et al., 2010). However, syneresis is the most extensively studied method for pulse starches with a significant variation of data due to variations in measuring conditions. Syneresis is directly related to amylose content of starches, which reassociates rapidly to form a hard gel and expels out water present between the adjacent chains (Betancur-Ancona et al., 2002).
Syneresis is a concentration-dependent phenomenon and the syneresis index decreases with increasing the starch concentration for many cultivars of black bean, chickpea, lentils, and navy bean starches (Byars & Singh, 2016). Thermal analysis reflect reduction in retrogradation enthalpy (ΔH r ) of stored starches after gelatinization, which ranged from 3.86 to 8.07 J/g ( Table 4). The ΔH r corresponds to melting of crystals formed through recrystallization of outer branch chains of amylopectin, which fail to regain the same degree of order as present in native starch resulted in ΔH r < ΔH gel (Gunaratne & Corke, 2007). Retrograded starches show reduced mobility compared to gelatinized starches due to formation of crystallites caused by AM-AM, AM-AMP, and AMP-AMP interactions. Ahmed (2012) recommended that a precise rheometric measurement has more advantages than BVA/RVA to investigate starch characteristics by not rupturing the gel structure during the measurement.

| Pasting properties of legume starches
A representative rheometric pasting properties of mung bean starch is illustrated in Figure 3, where the complex viscosity is plotted against  Table 5.
Rotational viscometric measurement of gelatinization of navy bean starch demonstrated that the torque response of swelling of starch granules approached equilibrium after an increase to a peak followed by a decline during cooking and the viscosity of the paste increased as the starch concentration and/or cooking temperature increased (Lee et al., 1995). The RVA pasting parameters of stored carioca bean starches at 5 C-15 C for 360 days showed no difference in the PT and the PV (Rupollo, 2011). The sample stored at 5 C showed higher SBV and lower BDV because of the lower crystallinity and the presence of higher amylose contents that reassociate easily.
Influence of germination, soaking-cooking, and microwave treatments on pasting properties of pigeon pea, dolichos bean, and jack bean flours reduced the process of retrogradation (Acevedo et al., 2017).
Hydrocolloids, xanthan and konjac gums, incorporation, conversely improved the PV, BDV, and FV of mung bean RS (Lin et al., 2021). The PV of two varieties of 9% common bean starches did not appear while adjusted to pH 5, 7 and 9; however, the viscosity improved after holding the paste at 90 C for 15 min (Paredes-L opez et al., 1988). At acidic and alkaline pH, the paste exhibited a marginal higher degree of setback, or retrogradation pattern, compared with the control paste.
The HP treatment of lentil starch showed pressure-dependent changes in the pasting properties (Ahmed et al., 2016). At 400 MPa, the PV increased from 958 BU to 981 but decreased to 520 BU at 600 MPa. The PT also decreased from 64.1 C to 56.2 C with pressurization. The FV reduced more than 50% after 600 MPa. Such a decrease in the pasting parameters at 600 MPa was attributed to the complete gelatinization of starch granules. Both the BDV and the SBV decreased systematically as a function of pressure. The decrease in BDV break-down indicates that pressure-modified gel network is more heat-stable compared with control sample.

| RS content and glycemic index (GI)
Legumes are frequently incorporated in food products to reduce postprandial plasma glucose response after ingestion, which is the key element for dietary management of people suffering from diabetes mellitus and cardiovascular diseases. Both in vitro and in vivo studies indicate that the legume starches are capable of lowering the GI because of the presence of higher RS and SDS content (Hoover & Zhou, 2003;Zhang et al., 2016). Legume flours have lower GI compared with extracted/isolated starches (Chung & Liu, 2012;Chung, Liu, Donner, et al., 2008;Chung, Liu, Pauls, et al., 2008) as the former contain both RS-1 (physically inaccessible starch due to presence of proteins) and RS-2 (ungelatinized/semicrystalline form of starch) whereas the latter only contains RS-2 form. For native legume starches, RS ranges between 3.2% and 80.78% (Table 4).
The differences in RS content among legumes occur due to variation in amylose contents, amylopectin chain length distribution, surface morphology, ratio of A/B polymorphic content, degree of molecular order on granular surface, and packing of double helices in crystalline region (Ambigaipalan et al., 2014;Hoover et al., 2010). The size of the starch granule also influences the enzyme accessibility.
Legumes with large granular diameters compared with cereals exhibit lower GI due to reduced surface area (Acevedo et al., 2020). The GI values for legumes are listed in Table 4. Extrusion has been found to decrease the RS content of all legume starches due to gelatinization, which unveils the whole structure of starch making it susceptible to enzymes. However, the RS content in legume starches was still found to be higher compared with corn starch after extrusion (Zhang et al., 2016). A higher RS-3 content is achieved after 24 h in cold storage for chickpea and lentil starches (Tovar et al., 2002).

| Dietary fiber (DF)
DF, a bioactive component of legumes, has proven health benefits.
Legume fibers have ability to change textural, rheological, and sensorial characteristics of foods related to their physicochemical properties (Tosh & Yada, 2010). DFs are classified into soluble dietary fiber (SDF) and insoluble dietary fiber (IDF). The concentration of DF fractions differs in the hull (seed coat) and the cotyledons, which affects the physicochemical properties of legumes (Table 6). Legume hulls mainly consist of IDF, cellulose and hemicelluloses, and smaller amounts of lignin (Tiwari & Cummins, 2011

| Physicochemical and functional properties of DFs
Major physicochemical properties of legume fibers/hull are summarized in Table 7. The solubility indexes of legume hulls vary widely.
The highest and the lowest swelling capacity were observed in black gram and green gram, respectively. Black gram and dolichos hulls have the highest WHC, whereas soybean showed the highest OHC (Mannuramath & Jamuna, 2012). The amount of SDF and IDF fractions influences the physicochemical properties of legume ingredients. SDF has a significantly higher WHC than the IDF because of its hydrophilic character (Capuano, 2017). Pectic substances (soluble fractions) are known to be the major fraction responsible for legumes fiber's water binding. Conversely, the insoluble fractions (pectic polysaccharides, lignin, cellulose, and hemicellulose) have the ability to enhance the oil-binding capacity of legume fibers (Huang et al., 2009;Vaz Patto et al., 2015).
DFs have the ability to change physical, rheological/textural, and sensorial properties of food systems according to their physicochemical properties (Martens et al., 2017). Pea fiber addition to meat products increased cooking yield without affecting sensorial properties of the products (Anderson & Berry, 2001;Besbes et al., 2008 pH, and temperature of the environment, and the bile acid type (G orecka et al., 2003).

| CONCLUSIONS
Development and commercialization of legume ingredients, especially novel starches, PIs/fractions, and dietary fibers can offer economic benefits to the food industry and boost legume growers' revenues as well. The bioactive properties of legume-derived proteins and peptides have gained interest in recent years. Further, being a low glycemic index product, legume starch contributes to a slow release of glucose. Legume dietary fibers are effective in normalizing bowel function and gastrointestinal health. Given the current trends, the demand for various legume ingredients will continue to grow in the future. The superior functionality of legume-based products will contribute to these trends since legume ingredients not only provide the daily nutritional requirements but are also capable of producing specialty food products. In summary, legumes will continue to play a key role in human nutrition, health, and increasingly recognized potential for enhanced crop environmental sustainability.