Review Lentil protein: a review of functional properties and food application. An overview of lentil protein functionality

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1 892 International Journal of Food Science and Technology 2018, 53, Review Lentil protein: a review of functional properties and food application. An overview of lentil protein functionality Marcela Jarpa-Parra* Universidad Adventista de Chile, Casilla 7-D, Chillan, Chile (Received 11 July 2017; Accepted in revised form 1 November 2017) Summary Keywords There is an increased interest, driven by environmental sustainability and food security points of view, in seeking new protein sources as alternatives to replace animal proteins (Day, Trends in Food Science and Technology, 32, 2016, 25). Pulse proteins, including lentil proteins (LP), are promising good substitutes as the wide variety of functional properties shown by them (Alsohaimy et al., World Journal of Agricultural Sciences, 3, 2007, 123; Boye et al., Food Research International, 43, 2010a, 537). Interest in LP has grown due to its high nutritional value, good Leu/Ile and Leu/Lys ratios ( and , respectively) (Urbano et al., Lentil: An Ancient Crop for Modern Times, 2007, 47, Berlin: Springer), high digestibility (~83%) (Barbana & Boye, Food & Function, 4, 2013, 310), and its potential use in food product applications (Farooq & Boye, Novel Food and Industrial Applications of Pulse Flours and Fractions. Pulse Foods, 1st edn, 2011, Amsterdam: Elsevier Ltd; Aider et al., Journal of Food Research, 1, 2012, 160; De la Hera et al., LWT Food Science and Technology, 49, 2012, 48; Turfani et al., LWT Food Science and Technology, 78, 2017, 361). On the other hand, great progress has been made lately to reveal the good functionality of LP such as solubility and emulsifying, foaming and gelation capacities (Avramenko et al., Food Research International, 81, 2016, 17; Jarpa-Parra et al., Food Hydrocolloids, 61, 2016, 903; Primozic et al., Food Chemistry, 237, 2017, 65). However, the relatively unknown relationship between its molecular structure and functionalities and the lack of knowledge of the impact of the extraction and environmental conditions on those properties has hindered the exploitation of their full potential. This review describes the current knowledge of the LP structure, physical chemistry and functional properties, and its potential role as an ingredient for the development of food grade products. The gap between the current knowledge and what remains to be known is emphasised. Functionality, lentil, protein, structure. Introduction Mature pulse seeds are normally high in protein because, throughout their development, pulse seeds accumulate these compounds (Roy et al., 2010). They are principally storage proteins that are classified as albumins, globulins and glutelins according to their solubility behaviour, and the salt-soluble globulins are the main fraction present in pulses (Kiosseoglou & Paraskevopoulou, 2011). Based on their sedimentation coefficient, two types of globulin usually prevail in pulses, vicilin or legumin, which sediments at 7S or 11S, respectively. Albumins that are soluble in water account for 10 20% of the total protein, while *Correspondent: Fax: ; marcelajarpa@unach.cl glutelins, soluble in dilute acid and base solutions, account for 10 20% of the total protein found in the pulses (Shewry, 1995). Besides the water-soluble storage proteins, there are other proteins that constitute part of the defensive mechanism of the seed, mainly enzymes, enzyme inhibitors, such as angiotensin I-converting enzyme (ACE) inhibitor, and lectins, which are considered as antinutritional factors for the human diet (Roy et al., 2010). As for other proteins, the molecular structure of the pulse proteins influences the physicochemical properties of the various protein constituents. For example, the albumin fraction is characterised by a low to medium molecular weight and a hydrophilic surface that renders the proteins water soluble, whereas the globulins that are multisubunit molecules of high molecular weight have a relatively hydrophobic surface that limits their solubility doi: /ijfs Institute of Food Science and Technology

2 An overview of lentil protein functionality M. Jarpa-Parra 893 in aqueous media (Kiosseoglou & Paraskevopoulou, 2011). Lentil protein chemistry and structure Lentil (Lens culinaris) is a leguminous plant high in fibre and low in fat. As demonstrated by Brummer et al. (2015) lentil is richer in total soluble fibre than peas and chickpeas. Also, its content of dietary fibre is higher than beans and chickpeas. Like most legumes, lentil is a rich source of protein, having between 20.6% and 31.4% proteins (Urbano et al., 2007). Most of these are storage proteins located in the cotyledon, containing a low percentage of sulphur-containing amino acids. Lentil proteins are comprised of around 16% albumins, 70% globulins, 11% glutelins and 3% prolamins (Boye et al., 2010b). Albumins, glutelins and prolamins have a molecular weight of about 20, 17 46, and kda, respectively, and they are comprised of approximately 13, 4 and 10 polypeptides each. Globulins contain both legumin- and vicilin-like proteins. The first group consists of six polypeptide pairs that interact noncovalently and have a molecular weight (Mw) of kda. Each of these polypeptide pairs is comprised of an acidic subunit of about 40 kda and a basic subunit of about 20 kda, linked by a single disulphide bond (Shewry, 1995). Traditionally, this group is identified as 11S proteins. However, Jarpa-Parra et al. (2015) classified it as 13S, as a result of a rate-zonal centrifugation study. They also identified three bands of legumin-like protein with relative molecular weights of 32, 42 and 47 kda, corresponding to the acidic polypeptide chains and two bands at 18 and 20 kda corresponding to the basic polypeptide chains, which coincides with Aydemir & Yemenicioglu (2013) study for a crude lentil protein extract. The second group of proteins is referred to as 7S proteins and are generally isolated from seed extracts as trimers of glycosylated subunits with a Mw of kda (Argos et al., 1985). The isoelectric points of LP vary from ph (Bhatty, 1988; Aydemir & Yemenicioglu, 2013). All protein fractions are glycosylated, particularly the vicilins, containing about 2.8% carbohydrate (Urbano et al., 2007; Bamdad et al., 2009; Barbana & Boye, 2011). Lentil protein quality and health benefits The nutritional characteristics of lentil have been associated with cholesterol- and lipid-lowering effects in humans, along with reducing the incidence of colon cancer and type-2 diabetes (Roy et al., 2010). In addition, lentils have high phenolic, flavonoid and condensed tannin contents (6.56 mg gallic acid equivalents g 1, 1.30 and 5.97 mg catechin equivalents g 1, respectively). Pulses with the highest total phenolic content such as lentil may exert the highest antioxidant capacity as demonstrated in in vitro systems such as DPPH, ABTS, FRAP, ORAC methods (Campos-Vega et al., 2010; Yeo & Shahidi, 2016; Jamdar et al., 2017). In a study of the antioxidant activities of the hydrophilic extracts from nine selected legumes based on copper-induced human LDL oxidation model in vitro, Xu et al. (2007), found that the extracts of lentils had significant (P < 0.05) longer LDL oxidation lag times (124.2 min) than the LDL control group (94.9 min). Also, showed higher antioxidant capacities than other pulses such as peas, chickpea and soybeans in both LDL-conjugated dienes assay and LDL- TBARS assay. Additionally, Garcıa-Mora et al. (2017) research showed that the presence of certain type of peptides produced by gastrointestinal digestion of LP results in dual antioxidant and angiotensin I-converting enzyme (ACE) inhibitory activities. The results were as follows: lmol Trolox eq./lmol peptide and IC 50 = lm, respectively. Jamdar et al. (2017) studied hydrolysates obtained from red and green LP by sequential digestion with Alcalase/ Flavourzyme, simulated human gastrointestinal digestion and hydrolysis with papain and bromelain. The relatively low IC 50 values obtained indicate a good ACE inhibitory activity. A downside of lentils, as other pulses, might be the presence of antinutritional compounds (ANCs) and allergens that may reduce their potential health benefits and bioavailability (Derbyshire, 2011; Nosworthy et al., 2017). Some ANCs found in pulses include lectins (carbohydrate-binding proteins), protease inhibitors and enzyme inhibitors. However, in many instances, the effects of ANCs may be diminished after different food processes (Derbyshire, 2011). For example, Nosworthy et al. (2017) studied the effect of extrusion, baking and cooking on the protein quality of red and green lentils. They found that protein digestibility-corrected amino acid score (PDCAAS) and the average digestible indispensable amino acid score (DIAAS) were higher in red lentils (PDAAS:55.0; DIAAS:0.54) than in green lentils (PDAAS:50.8; DIAAS:0.49). Also, that extruded lentil flour had a higher PDCAAS (63.01 red, green) than either cooked (57.40 red, green) or baked (53.84 red, green) flours. Additionally, the protein efficiency ratio of the extruded lentil flours (1.30 red, 1.34 green) was higher than that of the baked flour (0.98 red, 1.09 green). The presence of allergens in pulses is also a growing concern because some of them seem to be an important cause of IgE-mediated hypersensitivity in the Mediterranean region and India (Sanchez-Monge et al., 2000). Protease inhibitors found in lentils have been characterised as members of the Bowman-Birk 2017 Institute of Food Science and Technology International Journal of Food Science and Technology 2018

3 894 An overview of lentil protein functionality M. Jarpa-Parra family. Lectin is found in high amount in lentils, but it can be completely removed after a treatment of 72-h fermentation at 42 C. Yet, lectins from lentil are nontoxic (Campos-Vega et al., 2010). The content of a- amylase inhibitors differs greatly among legumes. In lentils, a-amylase inhibitor activity was undetected (Campos-Vega et al., 2010). However, the presence of these ANCs in lentil may have a positive effect too. For example, they may have potential use as antihypertensive agents (Barbana & Boye, 2011) because of their relatively good ACE inhibitory activity or they may be used as protein markers of cancer, as in the case of lectins (Campos-Vega et al., 2010). Lentil protein extraction Lentils and other pulses are traditionally consumed whole and mainly prepared as salads and soups (Yadav et al., 2007). Thus, novel applications need to be identified to increase its use in the food industry. Fibre, starch and protein concentrate or isolate can be extracted and separated from lentil and other pulses (Boye et al., 2010a), and then, they can be utilised as ingredients in the preparation of diverse food products. Extraction of pulse proteins may be relatively easy using wet processes, as they are highly soluble under alkaline and acidic conditions. They are commonly extracted by alkaline solubilisation, first dispersing the pulse flour in water at ph , followed by stirring of the dispersion. After that, the insoluble material is removed by centrifugation and proteins are recovered by adjusting the supernatant ph to a value around 4.5, where proteins are precipitated isoelectrically. The final concentrate or isolated protein is then dried using a spray-, drum- or freeze-drying method (Lee et al., 2007a). Most of the studies have focused on LP extracted with diluted sodium hydroxide (Bhatty, 1988) under a single ph condition that varies from one study to another (Table 1). For example, Joshi et al. (2012) produced LP isolate by alkaline extraction at ph 8.0, room temperature and 1:10 solid-to-solvent ratio, and compared the physicochemical characteristics of the isolated proteins obtained by the three methods of drying (freeze drying, spray drying and vacuum drying). Similarly, Boye et al. (2010a) extracted LP using isoelectric precipitation or ultrafiltration after extraction at ph 9.0 and 25 C, using 1:10 solid-to-solvent ratio from red and green lentils and compared their yields and protein contents. Their concentrates had aprotein content between 78.2% and 88.6% with a yield of % based on the protein content of the starting dehulled flour. On the contrary, Alsohaimy et al. (2007) extracted LP using seven different ph values ( ) and three different methods of protein recovery (isoelectric precipitation, ammonium sulphate precipitation and alcohol precipitation) at room temperature and 5:100 solid-to-solvent ratio. The optimum ph of extraction was 12.0 with ammonium sulphate and alcohol precipitation giving the highest protein recovery (93% and 100%, respectively). Lee et al. (2007b) performed a comprehensive investigation of extraction conditions. They determined the best parameters of protein extraction of green and red lentils, considering five ph values (distilled water and ph 8.0, 8.5, 9.0 and 9.5) and four temperatures (22, 30, 35, and 40 C), while keeping the solid-to-solvent ratio constant (1:10) and analysed the results according to protein content and percentage of starch damage. Upon evaluation of all extraction conditions, ph 9.0 at 30 C was chosen as the optimum extraction condition for green lentil (56.6% of protein), while ph 8.5 at 35 C was chosen for red lentil (59.3% of protein). Regarding the structure of the protein, most of the studies just determined the type of protein fraction present based on Mw determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS- PAGE). Lentil protein functionality Depending on the method of extraction and the conditions used to isolate or concentrate the proteins, their functionality may vary due to their compositional and physicochemical characteristics and the magnitude to which these characteristics are affected (Farooq & Boye, 2011). Generally, the studies related to protein functionality evaluate their gelling, emulsifying, foaming, solubility and water- and oil-absorption capacity (OAC) (see Table 2). However, most of the studies assess the functional properties only at one ph, which is generally neutral ph. In addition, only studies of Boye et al. (2010) and Aydemir & Yemenicioglu (2013) compare the functional properties performance of LP with other proteins under similar process conditions. Although, Aydemir and Yemenicioglu s research was more extensive as they studied six different varieties of red and green lentil; three of each one. Protein solubility is one of the important functional properties as most of the other functional properties depend on it (Joshi et al., 2012). Thus, the solubility profile at different ph levels may serve as a useful indicator of the performance of the protein in food systems, and of the extent of protein denaturation caused by the extraction method. According to Boye et al. (2010) and Jarpa-Parra et al. (2014) regardless of the extraction process, LP shows a minimum solubility at the ph range of , and two regions of maximum solubility at ph levels lower or higher than this range. Similar results were obtained by Bora (2002). Ma et al. (2011) study the solubility of red and green lentil flours with or without hull at ph 3.0, 5.0 International Journal of Food Science and Technology Institute of Food Science and Technology

4 An overview of lentil protein functionality M. Jarpa-Parra 895 Table 1 Lentil protein extraction studies Authors Study Conditions of extraction Alsohaimy et al. (2007) Lee et al. (2007b) Boye et al. (2010) Joshi et al. (2012) Jarpa-Parra et al. (2014) Comparison of protein recovery of lentil protein concentrate obtained by alkaline extraction and three methods of protein recovery: isoelectric precipitation ammonium sulphate precipitation or alcohol precipitation Comparison of protein content and percentage of starch damage in lentil protein concentrate obtained by alkaline extraction and isoelectric precipitation Comparison of yield and protein content of lentil protein concentrate obtained by alkaline extraction and two methods of protein recovery: isoelectric precipitation or ultrafiltration Comparison of physicochemical characteristics of isolated lentil proteins obtained by alkaline extraction and three methods of drying: freeze drying, spray drying or vacuum drying Study of the impact of extraction ph and environmental ph on the molecular structures and functionalities of lentil protein ph: 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, and 12.0 Temperature: Room temperature Solid-to-solvent ratio: 5:100 ph: 8.0, 8.5, 9.0, 9.5 Temperature: 22, 30, 35, 40 C Solid-to-solvent ratio: 1:10 ph: 9.0 Temperature: 25 C Solid-to-solvent ratio: 1:10 ph: 8.0 Temperature: Room temperature Solid-to-solvent ratio: 1:10 ph: 8.0, 9.0, 10.0 Temperature: Room temperature Solid-tosolvent ratio: 1:10 and 7.0. The solubility values ranged from 17% to 49% at ph 3.0 and from 55% to 57% at ph 7.0. Both, red and green lentil with or without hull had less than 10% of solubility at ph 5.0. Other authors like Joshi et al. (2011) and Can Karaca et al. (2011) only determined the solubility at neutral ph obtaining values that ranged from 78% to 91%. When compared with other proteins, the solubility of LP was approximately 1.3 fold higher than that of chickpea proteins, it was significantly higher than soy proteins, and it had a comparable value with WPI (Aydemir & Yemenicioglu, 2013). Water-absorption capacity (WAC) and OAC are the terms often used to refer to the amount of water and oil that can be absorbed per gram of sample, respectively. WAC values constitute useful indices of the ability of the protein to prevent fluid leakage from a product during food storage or processing (Farooq & Boye, 2011; Kiosseoglou & Paraskevopoulou, 2011). WAC and OAC values for LP isolates or concentrates vary in a wide range as different authors use different units to express the results. For example, Bora (2002) and Boye et al. (2010) obtained WAC values between 1.1 and 4.2 ml g 1 of protein, Joshi et al. (2011) found values from 0.43 to 0.48 g g 1 of protein, while Lee et al. (2007b) got values ranging from 2.1% to 3.8% (w/w). Additionally, where Bora (2002) obtained OAC values ranging from 2.0 to 2.6 ml g 1, Boye et al. (2010) found values from 120% to 225% depending on the lentil variety and the extraction conditions. All these studies were carried out at a single value of ph that varied from one study to another. In addition, Aydemir & Yemenicioglu (2013) found that LP exhibited better OAC than soy protein extract, and egg white proteins ( , 8.23, 6.37 g g 1, respectively), being only surpassed by chickpea protein ( g g 1 ). However, the WAC value was inferior to bovine gelatin (8.84 g g 1 ), chickpea ( g g 1 ) and soy protein isolate (7.94 g g 1 ), although better than egg white protein (0.14 g g 1 ). There is not much current work done regarding the mechanisms and molecules involved in water and oil-adsorption capacities. According to Joshi et al. (2011), there are several factors that might explain the differences in water-adsorption capacity: extraction protein method, protein shape and size, surface topography and polarity. Also, it seems that the presence of carbohydrates and other components may hinder water adsorption, although not a mechanism has been suggested for this phenomenon (Suliman et al., 2006). By the other hand, the change in the protein structure may increase or reduce the oil-adsorption capacity depending on the effect that is produced. For example, (Bora (2002) related the increase in hydrophilic groups at the oil water interface with a low oil-adsorption capacity. Meanwhile, Suliman et al. (2006) explained an improved OAC with the exposition of nonpolar groups as a result of a change in the conformation of the protein. The emulsifying ability of a protein is its ability to help in the preparation of an emulsion with oil droplets of a relatively small size by forming a film around them while they are dispersed in an aqueous medium, thereby preventing droplet aggregation that might lead to phase separation (Karaca et al., 2013). Emulsifying properties of proteins are normally described by two parameters: emulsifying activity index (EAI) and emulsifying stability index (ESI). EAI is the maximum surface area created per unit of protein, and it is usually determined by applying turbidimetry to highly diluted emulsion samples. ESI measures the ability of the emulsion to hold its structure over a defined time period, and it is determined by monitoring the decrease in turbidity with time 2017 Institute of Food Science and Technology International Journal of Food Science and Technology 2018

5 896 An overview of lentil protein functionality M. Jarpa-Parra Table 2 Lentil protein functional properties studies Functional property Description Authors Solubility Native lentil protein Maximum values: 15% to 35% at Bora (2002) ph 2.0; 98% at ph 8; Isoelectric point: 3% at ph 4.5 Succinylated lentil protein Maximum values: 95% at ph 2.5; 98% at ph 8; Isoelectric point: 5% at ph 3.5 Prepared by isoelectric precipitation Max. values: 70% (ph 1); 82% (ph 10); Min.: 4% (ph 5) Prepared by ultrafiltration Max. values: 85% (ph 2); 89% (ph 10); Min.: 5% (ph 5) Prepared by isoelectric precipitation Max. values: 69% (ph 2); 73% (ph 10); Min.: 2% (ph 5) Prepared by ultrafiltration Max. values: 83% (ph 2); 83% (ph 10); Min.: 5% (ph 5) Dehulled red lentil protein 30% at ph 3; 57% at ph 7 Ma et al. (2011) Red lentil protein (with hull) 31% at ph 3; 55% at ph 7 Dehulled green lentil protein 49% at ph 3; 56% at ph 7 Green lentil protein (with hull) 17% at ph 3; 56% at ph 7 All samples have less than 10% at ph 5 Freeze-dried lentil protein isolate 78% at ph 7 Joshi et al. (2011) Spray-dried lentil protein isolate 81% at ph 7 Vacuum-dried lentil protein isolate 50% at ph 7 Lentil protein isolate Can Karaca et al. (2011) Prepared by isoelectric precipitation 91% at ph 7 Prepared by salt extraction 90% at ph 7 Lentil protein extract Aydemir & Yemenicioglu (2013) Red and Green Lentil 0.65 g g 1 (average; ph 9.5) Lentil protein concentrate Jarpa-Parra et al. (2014) Extracted at ph 8 Max. values: 78% (ph 2); 96% (ph 10); Min.: 5% (ph 5) Extracted at ph 9 Max. values: 82% (ph 1); 97% (ph 10); Min.: 5% (ph 5) Extracted at ph 10 Max. values: 96% (ph 1); 97% (ph 10); Min.: 5% (ph 5) Water-absorption capacity (WAC) Oil-absorption capacity (OAC) Native lentil protein 1.1 ml g 1 protein (N.M) Bora (2002) Succinylated lentil protein ml g 1 protein (N.M) Prepared by isoelectric precipitation 3.8 ml g 1 protein (N.M) Prepared by ultrafiltration 4.2 ml g 1 protein (N.M) Prepared by isoelectric precipitation 3.9 ml g 1 protein (N.M) Prepared by ultrafiltration 3.4 ml g 1 protein (N.M) Red lentil protein (15 mg ml 1 ) 2.7% to 3.1% at ph 7 Lee et al. (2007b) Green lentil protein (15 mg ml 1 ) 2.1% to 3.8% at ph 7 Freeze-dried lentil protein isolate 0.48 g g 1 of protein at ph 7 Joshi et al. (2011) Spray-dried lentil protein isolate 0.43 g g 1 of protein at ph 7 Vacuum-dried lentil protein isolate 0.47 g g 1 of protein at ph 7 Lentil protein extract Aydemir & Yemenicioglu (2013) Red and Green Lentil 1.04 g g 1 of protein (N.M) Native lentil protein 2.6 ml g 1 protein (N.M) Bora (2002) Succinylated lentil protein ml g 1 protein (N.M) Prepared by isoelectric precipitation 120% (N.M) Prepared by ultrafiltration 225% (N.M) International Journal of Food Science and Technology Institute of Food Science and Technology

6 An overview of lentil protein functionality M. Jarpa-Parra 897 Table 2 (Continued) Functional property Description Authors Emulsifying activity index Emulsifying stability index Prepared by isoelectric precipitation 125% (N.M) Prepared by ultrafiltration 175% (N.M) Lentil protein extract Aydemir & Yemenicioglu (2013) Red and Green Lentil 8.62 g g 1 of protein (N.M) Native lentil protein (15 mg ml 1 ) 54% (N.M) Bora (2002) Succinylated lentil protein (15 mg ml 1 ) 60% to 63% (N.M) Red lentil protein (15 mg ml 1 ) 41% to 47% at ph 7 Lee et al. (2007b) Green lentil protein (15 mg ml 1 ) 43% to 46% at ph 7 Prepared by isoelectric precipitation 5.0 m 2 g 1 protein at ph 7 Prepared by ultrafiltration 5.9 m 2 g 1 protein at ph 7 Prepared by isoelectric precipitation 4.9 m 2 g 1 protein at ph 7 Prepared by ultrafiltration 5.1 m 2 g 1 protein at ph 7 Lentil protein isolate (0.25% w/w) Can Karaca et al. (2011) Prepared by isoelectric precipitation 44 m 2 g 1 protein at ph 7 Prepared by salt extraction 37 m 2 g 1 protein at ph 7 Lentil protein isolate (10 mg ml 1 ) Joshi et al. (2012) 90 m 2 g 1 protein at ph 7 Lentil protein extract Aydemir & Yemenicioglu (2013) Red and Green Lentil Max. value: 330 NTU Native lentil protein (15 mg ml 1 ) 52% (N.M) Bora (2002) Succinylated lentil protein (15 mg ml 1 ) 54% to 59% (N.M) Red lentil protein (15 mg ml 1 ) 82% to 90% at ph 7 Lee et al. (2007b) Green lentil protein (15 mg ml 1 ) 82% to 89% at ph 7 Prepared by isoelectric precipitation 18 min at ph 7 Prepared by ultrafiltration 19 min at ph 7 Prepared by isoelectric precipitation 17 min at ph 7 Prepared by ultrafiltration 19 min at ph 7 Lentil protein isolate (0.25% w/w) Can Karaca et al. (2011) Prepared by isoelectric precipitation 87 min at ph 7 Prepared by salt extraction 11 min at ph 7 Lentil protein isolate (10 mg ml 1 ) Joshi et al. (2012) 245 h at ph 3.0; 27 h at ph 5.0; 92 h at ph 6.0; 101 h at ph 7.0 Lentil protein extract Aydemir & Yemenicioglu (2013) Red and Green Lentil Max. value: 325 NTU Foaming capacity Native lentil protein (5 mg ml 1 ) ml from ph 2.5 to 7.0 Bora (2002) Succinylated lentil protein (5 mg ml 1 ) ml from ph 2.5 to 7.0 Red lentil protein (15 mg ml 1 ) 24% to 43% at ph 7 Lee et al. (2007b) Green lentil protein (15 mg ml 1 ) 42% to 68% at ph 7 Prepared by isoelectric precipitation 63% at ph 7 Prepared by ultrafiltration 63% at ph 7 Prepared by isoelectric precipitation 79% at ph 7 Prepared by ultrafiltration 69% at ph 7 Lentil protein concentrate Jarpa-Parra et al. (2014) 680% at ph 3 (average); 570% at ph 5 (average); 600% at ph 7 (average) Lentil legumin-like protein Jarpa-Parra et al. (2015) 425% at ph 3; 403% at ph 5; 410% at ph Institute of Food Science and Technology International Journal of Food Science and Technology 2018

7 898 An overview of lentil protein functionality M. Jarpa-Parra Table 2 (Continued) Functional property Description Authors Lentil legumin-like protein and polysaccharides Jarpa-Parra et al. (2016) With Guar Gum 475% at ph 3; 445% at ph 5; 450% at ph 7 With Xanthan Gum 425% at ph 3; 455% at ph 5; 390% at ph 7 With Pectin 420% at ph 3; 380% at ph 5; 440% at ph 7 Foaming stability Native lentil protein (5 mg ml 1 ) 34 s to 77 s ph 2.5 to 7.0 Bora (2002) Succinylated lentil protein (5 mg ml 1 ) 9 s to 56 s from ph 2.5 to 7.0 Red lentil protein (15 mg ml 1 ) 21% to 32% at ph 7 Lee et al. (2007b) Green lentil protein (15 mg ml 1 ) 13% to 62% at ph 7 Prepared by isoelectric precipitation 37% at ph 7 Prepared by ultrafiltration 38% at ph 7 Prepared by isoelectric precipitation 42% at ph 7 Prepared by ultrafiltration 36% at ph 7 Lentil protein concentrate Jarpa-Parra et al. (2014) 79% at ph 3 (average); 82% at ph 5 (average); 84% at ph 7 (average) Lentil legumin-like protein Jarpa-Parra et al. (2015) At ph 3: 25.5 min; at ph5: 50.7 min; at ph 7: 84.1 min Lentil legumin-like protein and Jarpa-Parra et al. (2016) polysaccharides With Guar Gum 26 min at ph 3; 125 min at ph 5; 49 min at ph 7 With Xanthan Gum 88 min at ph 3; 112 min at ph 5; 50 min at ph 7 With Pectin 38 min at ph 3; 275 min at ph 5; 65 min at ph 7 Minimum gelling concentration Prepared by isoelectric precipitation 12% (w/v) (N.M) Prepared by ultrafiltration 10% (w/v) (N.M) Prepared by isoelectric precipitation 12% (w/v) (N.M) Prepared by ultrafiltration 8% (w/v) (N.M) Freeze-dried lentil protein isolate 11% (w/v) at ph 7 Joshi et al. (2011) Spray-dried lentil protein isolate 11% (w/v) at ph 7 Vacuum-dried lentil protein isolate 14% (w/v) at ph 7 Lentil protein extract Red and Green Lentil 13% (w/w) (N.M) Aydemir & Yemenicioglu (2013) Lentil protein concentrate Jarpa-Parra et al. (2014) Extracted at ph 8, 9, and % (w/v) (no s.d at ph 3, 5 or 7) N.M: ph value not mentioned or not adjusted; s.d: significant difference. of a diluted emulsion during short-term storage (Kiosseoglou & Paraskevopoulou, 2011). Studies related to emulsifying properties of LP show great variability depending on the conditions of testing and protein extraction. Thus, making very hard to compare the results. In general, LP emulsifying properties are similar to those of other pulses, with values ranging from 5 to 90 m 2 g 1 (Boye et al., 2010; Can Karaca et al., 2011; Joshi et al., 2012) for EAI and 19 min to several days for ESI depending on test conditions. Most of these studies were done at ph 7.0, with the exception of Joshi et al. (2012) who tested both, EAI and ESI in the ph range of 3.0 to 7.0. They found that LP isolate has a higher EAI compared to whey protein isolate, but lower ESI than sodium caseinate and whey protein isolates, which is not in agreement with the findings of Aydemir International Journal of Food Science and Technology Institute of Food Science and Technology

8 An overview of lentil protein functionality M. Jarpa-Parra 899 & Yemenicioglu (2013), who found opposing results at ph 7.0. They also found that LP showed similar behaviour than soy protein, lower EAI and ESI values than chickpea and fish gelatin, but higher performance than bovine gelatin. None of the teams considered different conditions of protein extraction. On the contrary, Lee et al. (2007b) tested emulsifying properties of proteins extracted at different ph levels, while Can Karaca et al. (2011) compared proteins recovered by isoelectric precipitation and salt extraction, although both were tested only at neutral ph. Additionally, Avramenko et al. (2013) studied the emulsifying properties of LP as a function of the degree of hydrolysis (DH: 4%, 9% and 20%) following exposure to trypsin/heat. They reported that enzymatic treatment reduced LP emulsifying properties and that no significant difference was found between DH 4% and 20%. On the other hand, Primozic et al. (2017) demonstrated that LP might be utilised as an emulsifier in nanoemulsions, as well as in emulsion gels at high concentrations (more than 2 wt%), which may find applications in the pourable foods and beverages sectors, gelled-food products and controlled delivery of nutraceuticals or pharmaceutical compounds. Foams are formed when proteins diffuse and adsorb to air water interface reducing the surface tension, while partially or totally unfolding and forming an interfacial film around the air bubbles, which helps to prevent the foam from collapsing immediately after formation and, sometimes, during storage (Boye et al., 2010a). The foaming properties of proteins are usually expressed in terms of the foaming capacity index (FC), which represents the relative increase in the volume of a protein solution resulting from air incorporation. Also, the foam stability index (FS), which measures the ability of the system to retain the air in the form of bubbles during ageing (Bamdad et al., 2009; Boye et al., 2010b; Kiosseoglou & Paraskevopoulou, 2011). This functionality is not extensively studied and overall, the results are difficult to compare as they differ in the method of calculation. Boye et al. (2010) compared foaming properties of LP at ph 7.0 with those of chickpea and pea recovered by isoelectric precipitation and ultrafiltration. The method of extraction had no impact on the FC of the LP, but it made a difference on the FS, with LP performing fairly well compared to the other pulses. Aydemir & Yemenicioglu (2013) study showed that FC of bovine gelatin and fish gelatin were slightly higher than that of LP, which was similar to chickpea protein. Similarly, both lentil and chickpea protein formed the most stable foams. Bora (2002) determined the FC and FS of LP at several ph values, but his results are not comparable to those of Boye et al. (2010) or Aydemir & Yemenicioglu (2013). There were also significant differences between the values at different ph levels, but a correlation neither was found nor was any explanation offered. Lee et al. (2007b) also measured FC and FS at ph 7.0 for LP extracted at different ph values, but once again, the results are not comparable to those of other authors due to the different measurement conditions. Jarpa- Parra et al. (2014) studied the ph dependence of the FC of LP extracted at ph 8.0, 9.0 and No significant difference in FC values was observed between the samples extracted at those values. However, significant differences occurred when the ph of solution was changed from 3.0 to 7.0 with values ranging from 550% to 680%. Additionally, all samples showed FS values ranging from 77% to 84% regardless of the protein extraction ph or the environmental ph, which indicates a very high stability. Additionally, the same research team studied the structural properties of lentil legumin-like protein in relation to its air water interfacial behaviours. They found that the FS was closely related to the surface conformation of the protein, which was strongly influenced by the environmental ph (Jarpa-Parra et al., 2015). Lately, they also studied the foaming properties of lentil legumin-like protein in the presence of different polysaccharides (guar gum, pectin and xanthan gum) and at different ph values. Their findings included that the protein FC was not significantly impacted by adding polysaccharides, whereas FS was greatly enhanced at ph 3.0 and 5.0 due to stability mechanisms dependent of the structures formed at the air water interface (Jarpa-Parra et al., 2016). Protein gelation normally occurs when proteins form a three-dimensional network after heating to a temperature higher than the protein denaturation temperature followed by subsequent cooling. Gelling capacity is usually expressed by the minimum gelling concentration (MGC), which may be defined as the minimum protein concentration required for a self-supporting gel to form. The lower the MGC, the better is the ability to form gels. Pulse protein gel formation is frequently favoured by hydrophobic interactions and, in some cases, covalent disulphide bridges may also contribute to the network development. Similar to the foaming properties, there are only a few studies related to the gelation of LP. Joshi et al. (2011) studied the effect of different drying methods after protein extraction on gelation properties at ph 7.0. MGC values ranged from 11% to 14% w/v of protein. Jarpa-Parra et al. (2014) determined the MGC values for LP extracted at different ph and found values of 8 10 g/100 g of protein. The extraction ph did not significantly affect MGC values neither did the gelation ph. Compared to other proteins, in the study of Aydemir & Yemenicioglu (2013), LP exhibited the poorest gelling performance (12 14 g/100 g), below chickpea, soy, fish gelatin, bovine skin gelatin and egg white protein; all of them with MGC lower than 10 g/100 g under similar gelling conditions. However, another comparative 2017 Institute of Food Science and Technology International Journal of Food Science and Technology 2018

9 900 An overview of lentil protein functionality M. Jarpa-Parra study showed that LP might have the best performance (8% w/v) when compared to proteins from yellow pea and two kinds of chickpeas, depending on its source and extraction method (Boye et al., 2010a). Lentil protein and food applications Modern food production greatly relies on animal protein to provide energy, nutrients and functionality to ensure sustainable living. Yet, the market is changing and during the last decade, the per capita meat consumption has been declining, while sales of plant protein-based products have increased due to consumers economic, environmental and sustainability apprehensions (Aiking, 2011; Earth Policy Institute, 2012). Undoubtedly, this is leading the food scientists to incorporate pulse-derived ingredients into food formulations (Day, 2016). Because of their diverse range of functionalities, pulse-derived proteins can be incorporated into novel food products, such as milk substitute, curd-like products, meat products, extruded products and baked goods (Boye et al., 2010b). However, this is a big challenge for the food industry for both technological and consumer acceptance reasons (Aiking, 2011; Day, 2016). As already explained above, lentil is an excellent source of several nutritional factors and its consumption is associated with several positive effects on human health (Urbano et al., 2007; Campos-Vega et al., 2010; Roy et al., 2010). In addition, LP has high nutritional value, good Leu/Ile and Leu/Lys ratios ( and , respectively) and high digestibility (~83%) (Boye et al., 2010b). As LP has a high lysine content, they can also satisfy nutritional requirements when incorporated into cereal-based products or combined with cereal proteins that contain adequate amounts of sulphur-containing amino acids (Gomez et al., 2008). So far, lentil flour and LP alone or in combination with other pulse proteins have been incorporated with more or less success in tofu-like product, imitation milk, meatballs and bread, among others. According to Swanson (1990), LP isolate produced a milk of intermediate quality equivalent to milk prepared from soy protein isolates. Cai et al. (2001) prepared a curd (tofulike product) from LP that was of inferior quality compared to that of soy and pea proteins. Serdaroglu et al. (2005) formulated meatballs with lentil flour as an extender, resulting in greater cooking yield, fat retention and moisture retention values than those with chickpea flour. A salad dressing was also supplemented with raw and thermally treated lentil flour, that is from ground roasted seeds, roasted flours, precooked, ground and freeze-dried seeds; precooked, ground and spray-dried seeds (Ma et al., 2011). They measured the rheological, physical stability, microstructural and sensory properties and found that the thermally processed pulse flours may be suitable as value-added ingredients in salad dressing applications. Han et al. (2010) reported the development of a gluten-free, 100% pulsebased cracker snack using several pulse fractions that included green and red lentil, among other flours and proteins. The acceptance of the products scored highly by consumers. In addition, good crispness of crackers containing red lentil flour and other legumes was reported. However, the developed products showed a strong beany flavour and their gluten-free nature produce an unfamiliar texture for many consumers. Similarly, Ryland et al. (2010) formulated a snack bar partially replacing oats with micronized flaked lentils (MFL), and they identified the sensory attributes that contribute to consumer acceptability. Three of six MFL formulations obtained high mean acceptability mean values, while external preference mapping determined that sweetness, grainy and lentil flavours, hardness, cohesiveness, cohesiveness of mass and moistness had the greatest influence on consumer acceptability. In addition, Morales et al. (2015) studied the effects of extrusion processing on fibre (soluble and insoluble) and phenolic components, as well as on the antioxidant capacity of different fibre-enriched lentil flours. Extrusion partially reduced the dietary fibre, which was related with a significant increase in the soluble fibre fraction. Additionally, it had an effect on hydrolysis of polyphenols bound to fibre and proteins that increased most of polyphenols fractions, also increasing antioxidant activity. As a result, they developed snack-type products, gluten-free with a balanced nutritional and antioxidants composition. In the bakery field, Aider et al. (2012) substituted wheat flour in bread with LP. They found that the substitution produced a bread of intermediate quality with lower volume than the control and a greener colour. Depending on the level of supplementation, it had similar or higher hardness than the control bread. Similarly, De la Hera et al. (2012) substituted wheat flour with lentil flour to produce a layer cake and a sponge cake and studied its effect on the characteristics of the batters and of the final products. The addition of lentil flour changed the density of both cakes and reduced layer cake volume, symmetry index, cohesiveness and springiness. In sponge cakes, no clear trends were observed in volume or symmetry index, but the total substitution of wheat flour with lentil flour resulted in harder and less cohesive cakes, as it also increased hardness in layer cake (Aider et al., 2012). Lately, Turfani et al. (2017) enriched wheat bread with green lentils flour (5 6%), which reduced tenacity, stability and strength of wheat dough, although it still gave acceptable loaves. It seems that LP, which reduces dough stability and increase dough development, dilutes gluten. It is also probably that LP competes with gluten protein for water, thus delaying hydration process. International Journal of Food Science and Technology Institute of Food Science and Technology

10 An overview of lentil protein functionality M. Jarpa-Parra 901 Also, Jarpa-Parra et al. (2017) replaced egg and milk as the principal sources of proteins in angel food cakes and muffins. They found that replacement by LP affected the specific gravity and consistency index of batters, which reduced the mean area of air cells but increased the number of air cells per unit area, and thus, the volume of the final product was not affected. Also, that LP can totally or partially substitute egg white protein in cakes producing final products with appropriate appearance, flavour and moisture. Yet, further improvement of the formulations may be achieved by increasing the amount of water added, especially in muffins. Mechanical properties were affected in a different way, according to the type of bakery product. In angel food cakes, cohesiveness and springiness were not affected by the presence of LP in the formulation, which indicated that LP did not impact egg or gluten functionalities and a sufficiently strong protein network was developed. Whereas in muffins, cohesiveness was impacted suggesting some kind of interaction between LP and other ingredients that need to be further investigated. Although, under the process conditions, LP did not perform with the same effectiveness as egg white foaming agent in angel food cake; in muffins, it seems to perform as efficiently as egg/milk proteins. Except for the study of Ma et al. (2011), Turfani et al. (2017) and Jarpa-Parra et al. (2017), there are no other comprehensive studies that include a lentil-based food product. In terms of studying structure, texture, appearance, colour and sensory analysis, there are no other studies focusing on a product where LP has been utilised as the principal source of protein for food application. Additionally, in another field of application, Bamdad et al. (2006) prepared an edible film from LP and they determined their mechanical, optical and barrier properties. Characteristics of the lentil protein-based edible films were similar to other edible protein films; it had good mechanical properties and water vapour permeability, besides good solubility. Avramenko et al. (2016) utilised lentil protein-based maltodextrin microcapsules to entrap flaxseed oil, using native (n-lpi) and pretreated [heated, un-hydrolysed (u-lpi) and heated, hydrolysed (h-lpi)] lentil proteins. The results showed that capsules prepared using n-lpi with 10% oil loading had the lowest surface oil content (~3.7%) and highest efficiency entrapment (~62.8%) for all formulations. An oxidative storage stability test over a 30 days period proved encapsulation process effective at lowering the production of primary and secondary oxidative products compared to free oil. Conclusions and future recommendations Protein functional properties are largely dictated by protein s physicochemical and structural properties, which are influenced by the extraction method and processing conditions. Protein extraction conditions vary from one study to another without providing a full justification for it. In addition, there is a lack of systematic studies showing how extraction parameters influence LP yield and purity, and how the extraction conditions influence the LP conformational structure. Even though some studies have addressed the effect of different conditions of extraction or testing on LP functionality, those studies are still lacking in making a deeper connection between these conditions and their influence on the LP structure and the subsequent functional properties of these extracts. The lack of studies that discuss functional properties of LP related to a structure-function approach is evident. Also, the impact of other environmental factors that are also important for extraction and potential protein applications, such as the ionic strength and temperature, is not addressed. It would be beneficial to understand the molecular structure of the LP and its functional properties as impacted by such factors. As globulin proteins are soluble in salt solution, investigating ionic strength as a factor would help to determine the appropriate concentration of salt to add to the alkaline solution to maximise protein recovery and purity. However, there is at least one study where functional properties of LP are compared with other proteins. Aydemir and Yemenicioglu s study demonstrated that LP solubility is higher than that of chickpea proteins; its gelling property, EAI, ESI, OAC and WAC values are inferior, while FS and FC have similar performance. Soy proteins show lower values for solubility, FC, FS, and OAC, better results for WAC and similar performance in EAI and ESI. Compared with whey protein isolate, LP had a comparable solubility value, a higher FS value, and lower values of FC, EAI and ESI. LP also exhibited better OAC and WAC than egg white proteins. In addition, further investigation on the impact of the other components present in the LP concentrate is essential. As environmental conditions, protein treatment, physicochemical and structural properties influence protein functional properties, more fundamental research that addresses this gap is critical to facilitate wider applications of LP. In addition, some of these functional properties have received less attention than others such as the foaming properties. Thus, more research is also needed to increase the knowledge in this field, considering that a large segment of the food market, such as bakery, confectionery and dairy desserts, is comprised of products for which foaming is essential. References Aider, M., Sirois-Gosselin, M. & Boye, J.I. (2012). 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