Effect of processing conditions and in vitro protein digestion on bioactive potentials of commonly consumed legumes

Author’s Accepted Manuscript Effect of processing conditions and in vitro protein digestion on bioactive potentials of commonly consumed legumes Sahayog N. Jamdar, Deshpande Rajalakshmi, Sushama A. Marathe www.elsevier.com/locate/sdj

PII: DOI: Reference:

S2212-4292(17)30402-9 http://dx.doi.org/10.1016/j.fbio.2017.07.007 FBIO213

To appear in: Food Bioscience Received date: 9 May 2016 Revised date: 31 May 2017 Accepted date: 17 July 2017 Cite this article as: Sahayog N. Jamdar, Deshpande Rajalakshmi and Sushama A. Marathe, Effect of processing conditions and in vitro protein digestion on bioactive potentials of commonly consumed legumes, Food Bioscience, http://dx.doi.org/10.1016/j.fbio.2017.07.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Effect of processing conditions and in vitro protein digestion on bioactive potentials of commonly consumed legumes Sahayog N. Jamdar1, Deshpande Rajalakshmi, Sushama A. Marathe

Food Technology Division, Bhabha Atomic Research Centre, Trombay Mumbai 400085

[email protected]

Abstract The antioxidant properties and ACE inhibitory activity of green gram, horse gram, lentil, chickpea, cowpea, black pea and white pea were evaluated before and after different processing conditions and in vitro protein digestion (IVPD). Chemical assays indicated that unprocessed horse gram had the highest antioxidant activity. The process of soaking decreased radical scavenging activity (RSA), total antioxidant capacity (TAC), ferric ion reducing power (FRAP) and metal ion chelation activity (MICA) in all the legumes. Cooking of the soaked seeds led to further decreases in these activities. Germination improved MICA in chickpea and lentil as well as TAC in all the legumes except for white pea. IVPD increased RSA and TAC of all the legumes by 2-4 folds, while FRAP and MICA in selective legumes showed a significant contribution from the

peptides released due to hydrolysis of proteins. The scavenging and reducing activities in this legume as well as other legumes were correlated with free phenolics content. However, in contrast to the chemical assay, biological assays showed no such correlation. Significant ACE inhibitory activity was observed only after IVPD of the legumes.

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Food Technology Division, FIPLY, Bhabha Atomic Research Centre, Trombay Mumbai 400085, India, Tel: +91-022 25595375, Fax: +91-022 25505151 1

Keywords: Legumes; Horse gram; Lentil; Cow pea; Chickpea; Black pea; White pea; Green gram; Radical scavenging activity; ACE inhibitory activity

1. Introduction Legumes are an excellent source of protein, carbohydrates and fiber as well as many essential vitamins and minerals (Roy et al., 2010). They are consumed worldwide and are a staple in South Asian countries including India. In these regions, legumes are primarily eaten as a source of proteins. Their consumption has been correlated with many beneficial health promoting properties like management of high cholesterol and type-2 diabetes and also in prevention of various types of cancers as well as heart diseases (Menotti et al., 1999; Duranti 2006, Boye et al., 2010). Many of these aforementioned beneficial effects have been attributed to the antioxidant potential of the seed legumes (Wang et al., 2011). Besides this, angiotensin converting enzyme (ACE, EC 3.4.15.1) inhibitory activity responsible for an antihypertensive effect has also been reported for many leguminous protein hydrolysates (Lopez-Barrios et al., 2014). Oxidative stress is basically overproduction of reactive oxygen species (ROS) causing damage to cellular functionality by harming lipids, proteins and DNA, which ultimately results in development of different human diseases like cancer, diabetes, atherosclerosis and neurodegenerative diseases (Pisoschi & Pop, 2015). The protection of cells against oxidative damage is brought about by antioxidants. In mammalian system, the oxidative stress is addressed by different endogenous antioxidant mechanisms, which are strengthened by antioxidants in the diets (Pisoschi & Pop, 2015). This is the reason that the demand for intake of antioxidant food or dietary antioxidant has increased in the 21st century. Generally, plant

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derived products like fruits, vegetables, cereals and legumes are considered as excellent source of dietary antioxidants. Hypertension is a chronic medical condition considered as the main risk factor for stroke, myocardial infarction, heart failure and peripheral arterial diseases (Mentz et al., 2013). It is associated with abnormal activity of the rennin-angiotensin-aldosterone system. ACE converts angiotensin I to angiotensin II causing an increase in vasoconstrictor activity, which in turn is responsible for increased blood pressure (Mentz et al., 2013). ACE inhibitory compounds such as captopril and enalapril are thus used as drugs for lowering the blood pressure and to help protect heart muscle. However, these compounds cause serious side effects (Spiller, 2014). Hence, natural ACE inhibitors, which could serve as cheaper and secure alternatives for the synthetic drugs are being investigated. In this regard, dietary proteins containing different peptide sequences could exert antioxidant and antihypertensive effects. They are released during enzymatic digestion of these proteins (Carbonaro et al., 2015). Numerous efforts are being made to purify these bioactive peptides derived from food proteins (Li-Chan, 2015; Barbana & Boye, 2011; Boschin et al., 2014; Duranti, 2006). The antioxidant activity of the seed legumes has been primarily associated with its phenolics content (Marathe et al., 2011; Wang et. al., 2011), which are present in abundance in raw or unprocessed seeds. However, consumption of raw seeds may cause deleterious effects due to the presence of antinutrional compounds (Boye et al., 2010). Hence, different processing conditions like soaking, germination and cooking are used to inactivate these factors in the seeds (Tharanathan & Mahadevamma, 2003). These processes, however, alter the overall composition of the seed legumes as well as the bioactive potential of these commodities. In fact a few studies report the influence of processing conditions on the antioxidant activity of legumes and the change was attributed to the processing losses and to the degradation or formation of free phenolics (López-Amorós et al., 2006; Kim et al., 2015;

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Kalpanadevi & Mohan, 2013; Xu et al., 2008). In the past few years, legume protein isolates as well as protein hydrolysates have been studied for their bioactive potential (Boye et al., 2010; Carbonaro et al., 2015). However, information regarding the changes in protein composition with respect to processing conditions as well as gastrointestinal digestion and their effect on bioactive potential of the seed legumes is scarce. It is worth mentioning that the legume proteins are acted upon by proteolytic enzymes either during germination or during gastrointestinal digestion after consumption. Thus, there exists a strong possibility of release of peptides showing biological activities. Hence, the present study aims to evaluate the effect of processing conditions [soaking, germination (24, 48, and 72 h) and soaking and cooking] and in vitro protein digestion (IVPD) on antioxidant potential and ACE inhibitory activity of seed legumes. Further, an attempt was made to establish the correlation between the compositional changes (phenolics and protein breakdown) and the bioactive potentials of legume seeds. Moreover, all the activities were measured in the aqueous extract after individual treatment, which is more relevant in view of the bioavailability of the compounds imparting the effects.

2. Materials and methods 2.1 Samples Selected legumes viz. cowpea

(CWP) (Vigna unguiculata), black pea (BLKP) (Pisum

sativum), white pea (WHTP) (Pisum sativum), horse gram (HG) (Macrotyloma uniflorum), green gram (GG) (Vigna radiata), lentil (LN) (Lens culinaris), chickpea (CHKP) (Cicer arietinum) grown in India (dried mature seeds, crop of 2014) were purchased from three outlets of local grocery stores in Mumbai, India, which served as triplicate samples. 2.2 Processing of samples

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Unprocessed samples (DR) were pulverised in a laboratory mill (Perten Equipment, Hägersten, Sweden) to get fine powders. Ten g of each legume sample was processed by soaking (SK) (16 h in excess of water); germination [24 (G1 D), 48 (G2 D) and 72 h (G3 D)] and pressure cooking (15 lb psi at 121°C) of soaked seeds (SKCK) for 10 min in a pressure cooker. After processing the samples were freeze dried (ScanVac, LaboGen, Industrivej 6-8, Lynge, Denmark) and pulverised in a laboratory mill (Perten, Hagerstein, Sweden) to get fine powders. A 2% aqueous extract of all the powdered samples was prepared in nanopure water (Water purification system, Millipore, Burlington, MA, USA) with shaking for 2 h at ambient temperature (25°C). The soluble fraction was then separated by centrifuging (Lynx 4000, Thermo Scientific, Waltham, MA, USA) the suspension at 12000 g for 20 min. The supernatant was used for investigating different activities, while the sediment was used for estimation of nitrogen content using the Kjeldahl method using a conversion factor of 5.3 (Kel Plus, Classic Dx, Pelican Equipment, Chennai, India) (AOAC, 2002). 2.3 In vitro protein digestion (IVPD) IVPD was carried out according to method suggested by Hamaker et al. (1987) with slight modifications. A 200 mg sample was suspended in 10 ml deionised water and the pH was adjusted to 2.0 with 0.1N HCl. Pepsin (2250 AU/mg) (Sigma, St Louis, MO, USA) at a concentration of 1.0 mg/ml was added to each sample. The samples were then incubated at 37°C for 2 h with intermittent mixing. After incubation, the pH of the individual sample was increased to 6.8 using 0.1N NaOH. Trypsin (94 U/mg) (Sigma) and chymotrypsin (52 U/mg) (Sigma) each at a concentration of 1.0 mg/ml were then added. The samples were further incubated at 37 °C for 2 h with intermittent mixing. After incubation the samples were centrifuged at 10,000 g for 10 min. at 4 °C. The supernatant, as such was used for

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investigating different activities, while the sediment was used for estimation of nitrogen content using the Kjeldahl method. 2.4 Estimation of protein, free amino nitrogen (FAN) and free phenolics (FP) content All protein measurements in the samples were carried out using the Kjeldahl method. The unprocessed legume powders as well as the residues after extraction with water were subjected to nitrogen estimation for determining total protein (TP) and residual protein (RP), respectively. The percent soluble protein (SP) content was calculated using the following formula: SP (%) = (TP-RP)/TP X 100 The concentration of FAN in the legume extract was estimated using the modified ninhydrin colorimetric method (Doi et al., 1981) using glycine as a standard. The assay system consists of an aqueous extract (80 µl) and 320 µl of ninhydrin reagent (0.08 g of ninhydrin [Himedia, Mumbai, India] in 8 ml of distilled ethanol and 10 ml of acetic acid, to which 100 mg of CdCl2 dissolved in 0.1 ml of water, was added). The reaction mixture was heated for 5 min in boiling water bath Absorbance was measured at 505 nm (V-630 Jasco, Tokyo, Japan) after cooling the solution. The concentration of FAN was estimated using the following equation: A=0.018C; r2=0.991 [Where A is the absorbance at 505 nm and C is concentration as glycine (Sigma) equivalent (nmol/ml)]. The concentration of FP in legume extracts was measured using the Folin-Ciocalteu assay (Singleton & Rossi, 1965). The concentration of the FP was calculated using the following equation: A=0.016C; r2=0.997 [Where A is the absorbance at 750 nm and C is the concentration as gallic acid (Sigma) equivalent (µg/ml)]. FP was expressed as mg of gallic acid equivalent (GAE) per g legume powder prior to extraction. 2.5 Determination of antioxidant activity

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2.5.1 ABTS+ Radical scavenging The assay was done as described by Ma et al., (2010) with some modifications. A mixture of ABTS solution (7 mM) and potassium persulphate (2.45 mM, final concentration) was left to stand overnight (16 h) at ambient temperature, in the dark to form radical ABTS·+. The mixture was suitably diluted with distilled water to make a working solution of ABTS radicals with absorbance of 1.00 ± 0.02 at 734 nm An aliquot of 40 µl of sample extracts diluted 1:10 (subjected to IVPD) or 1:2 (for all other samples) with nanopure water was mixed with 160 µl of ABTS working solution. The decrease in absorbance value was measured at 734 nm after 6 min The percent scavenging of ABTS radical was calculated as follows: ABTS radical-scavenging activity (%) = [(Acontrol-Asample)/Acontrol] × 100 The scavenging activity was expressed as µM Trolox (Sigma) equivalent per mg of sample using following equation: A=1.882C, r2=0.996 [Where A is the absorbance at 734 nm and C is the concentration as µM Trolox]. 2.5.2 FRAP (Ferric Reducing Antioxidant Power)

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The Fe+2 chelating activity was determined by measuring formation of Fe+2 ferrozine complex (Carter, 1971) with some modifications. Freshly prepared FRAP reagent (160 µl) was mixed with 40 µl of 1:1 nanopure water diluted sample extracts. The FRAP reagent was prepared by mixing 0.3 M sodium acetate buffer (pH 3.6), aqueous 10 mM 2,4,6-Tris (2pyridyl)-s-triazine (TPTZ) (Sigma) in 40 mM HCl and 20 mM FeCl3.6H2O in the ratio of 10:1:1. After 10 min of incubation at room temperature (25°C), the absorbance was measured at 593 nm. A freshly prepared standard solution of FeCl2.6H2O was used for preparation of calibration curve and FRAP activity was expressed as mM per mg of sample. A=0.003C, r2=0.998 [Where A is the absorbance at 593 nm and C is concentration as Fe+2 (mM)].

2.5.3 Total antioxidant capacity This assay is based on the reduction of Mo (VI) to Mo (V) by the sample and the subsequent formation of a green phosphate Mo (V) complex at acidic pH (Prieto et al. 1999). An aliquot of 0.1 ml of sample extract was combined in a microfuge tube with 1 ml of reagent solution (0.6 M sulphuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate). The tubes were capped and incubated at 90°C for 90 min., after that, each sample was allowed to cool to room temperature and the absorbance was measured at 695 nm against a blank. The activity of the legumes was expressed as α-tocopherol (Sigma) equivalent using the following linear equation: A= 0.011C + 0.0049, r2=0.987 [Where A is the absorbance at 695 nm and C is concentration as α-tocopherol (µmol/ml)].

2.5.4 Estimation of metal ion (Fe+2) chelation The Fe2+ chelating activity of the extract was measured as reported by Jamdar et al., (2010) with modifications. The reaction mixture consisted of extract (10 µl), nanopure water (185 8

µl), 2 mM FeCl2.4H2O (10 µl) and 5 mM ferrozine (Hi-media, Mumbai, India)

(10 µl).

Absorbance was measured at 562 nm after 10 min. Fe+2 chelating activity (%) was calculated as follows and was expressed as µM of EDTA equivalent/mg legume using an EDTA (50 mM EDTA stock was prepared using deionised water maintained at pH 8.0) calibration curve. Chelating activity (%) = [(Acontrol-Asample)/Acontrol] × 100 The equation obtained from the calibration curve was: A=1.57C, r2=0.987 [Where A is the absorbance at 562 nm and C is concentration as EDTA (mM)]. 2.5.5 Reactive Oxygen Species (ROS) Scavenging The assay was done according to method suggested by Patwardhan et al. (2013). MCF7 human breast cancer cells (Sigma) were treated with 50 µmolar H2O2 and incubated at 37°C for 15 min and then 20 µM oxidation-sensitive dichlorofluoresceindiacetate (DCF-DA) (Sigma) was added. After incubation for 20 min, 200 µl of these treated cells were incubated with 30 µl of legume extract and 770 µl of phosphate buffered saline which was used as the dilution medium. The mixture was further incubated for 1 h. Increased fluorescence from the oxidation of H2DCF to DCF (485 nm excitation/535 nm emission) was measured using a spectrofluorimeter (FP8200 Jasco). The fluorescence intensity of the cells in the absence of sample was considered as 100%, while that in the presence of legume extract was calculated as percent reduction in the fluorescence. 2.6 Angiotensin Converting Enzyme (ACE) inhibitory activity A crude preparation of ACE was prepared according to the procedure described by Jamdar et al., 2010. The starting material, i.e., pig lungs, were purchased from the slaughter house immediately after slaughtering the animal and brought to the laboratory on ice. The tissue was washed using physiological saline to remove the superficial dirt. The lung tissue (only

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the lobe portion) was then diced into small pieces using a kitchen knife and subsequently homogenized (Polytron, Kinematica, Luzern, Switzerland) in 10 volumes of ice cold 10 mM potassium phosphate buffer (pH 8.3) containing 0.1 mM pepstatin (Sigma) and 0.1 mM phenyl methyl sulfonyl fluoride (Sigma). The homogenate was centrifuged at 5000 g for 10 min and the resulting supernatant was dialyzed against 20 volumes of the same buffer at 4 °C. The dialyzed supernatant was used as a source of ACE. The activity of the preparation was checked using a specific substrate (hippuryl (Hip)-His-Leu) (Sigma) as described in the following section. ACE inhibitory activity was measured using the spectrophotometric assay of Cushman & Cheung (1971) with some modifications. Each sample (50 µl) was incubated with 50 µl of ACE at ambient temperature for 10 min. To this mixture 250 µl of 8.3 mM Hip-His-Leu was added and further incubated at 37 °C for 30 min. The reaction was stopped using 250 µl of 1N HCl. The hippuric acid released was extracted with ethyl acetate (1000 µl). The ethyl acetate was evaporated by incubating the tubes in boiling water bath for 20 min and hippuric acid was redissolved in distilled water (1000 µl) and its amount was measured spectrophotometrically at 228 nm. The assay mixture without sample was used as the control. The percent ACE inhibitory activity was determined as follows: % ACE inhibition = (Acontrol- Asample//Acontrol) x 100 The ACE inhibitory activity was determined by regression analysis of ACE inhibition (%) versus concentration of weight of legume (mg) and the IC50 value, defined as the legume dry weight (mg) /ml required to produce 50% ACE inhibition with the described conditions. 2.7 Statistical methods All data were expressed as mean ± standard deviation and analysed using Analysis of Variance (one way) (ANOVA). Further, the means were compared using Tukey’s multiple comparison test using Origin Pro 8.0 software (Origin Lab Corp. Northampton, MA, USA). P

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<0.05 was considered to be statistically significant. Correlation and regression analyses were done using Pearson correlation test. The data for processing conditions and antioxidant activities of different grains was subjected to Principal Component Analysis (PCA) using XLSTAT 2014 software (Addinsoft Inc., New York, NY, USA). Loading and score plots were then drawn using principal components 1 and 2. 3. Results and discussion 3.1 Soluble protein (SP), free phenolics (FP) and free amino nitrogen (FAN) content The effect of processing conditions and in vitro protein digestion on SP (expressed as a percentage of total protein), FAN and FP of legume seeds is shown in Tables 1 and 2. The values in the parenthesis show total protein content of each legume. Native proteins of green gram, cowpea, white pea and lentil showed higher (P <0.05) solubility (≥60%) as compared to that of others like horse gram (51 ± 2%), chickpea (55 ± 2%) and black pea (53 ± 3%). This suggests that the bioavailability of proteins from green gram, cowpea, white pea and lentil is more than that of other legumes (Garcés-Rimón, 2016). Soaking and germination increased (P<0.05) the solubility of proteins in all the legume seeds, except for chickpea. Thus, the soaking and germination processes are definitely useful as far as protein availability is concern. This could be due to action of endogenous proteases in the seeds that were activated during the germination process, acting on storage proteins and thus increasing their solubility. The steady increase in FAN during soaking and germination also indicated this. However, the increase in FAN content was variable with different legume seeds with the highest level observed in lentil. Nevertheless, the relative increase with respect to initial content was highest in black pea and white pea (3 fold) at the end of the 3rd day of germination, while the corresponding values for green gram, lentil and horse gram were higher by 2.5 fold (Table 1). Chickpea and cowpea showed the least increase (1.6 fold) in

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FAN at the end of the 3rd day of germination. The changes in FAN content during germination has been reported in different seed legumes by Kuo et al. (2004). The authors report that the increased amino acid content as well as the profile of the amino acids during germination is characteristic of individual legumes. Moreover, the conditions used for germination also affected the content of different amino acids (Kuo et al., 2004). Interestingly, soaking followed by cooking decreased (P <0.05) the content of soluble protein to ≤41% in all the legume seeds with horse gram and chickpea showing minimum values of 20±4% and 29±3%, respectively. Similar trends were also observed for FAN content. Cooking has been reported to cause denaturation of proteins thereby decreasing their solubility (Carbonaro et al. 1997; Le Gall et al. 2005). IVPD increased (P<0.05) the FAN as well as the percent soluble protein of all the samples irrespective of the processing conditions used prior to digestion (Table 2). However, the solubility of cooked samples, although improved with IVPD, was still less than other digested samples. The lower digestibility of the cooked samples, could be due to lower solubility of the proteins after cooking. Carbonaro et al. (2000) showed a reduction in digestibility of proteins of faba beans and common beans upon cooking. IVPD increased the FAN of dry seeds by 2.3-4.2 fold, while that of germinated seeds by 1.5-2.5 fold. Since germination itself led to higher initial levels of FAN, the relative increase in these samples after IVPD was less. However, the final levels of FAN were observed to be highest in germinated samples after IVPD. Among all the legumes studied, lentil, green gram and horse gram showed relatively higher FAN content after IVPD. The FP content of unprocessed seeds was found to be higher as compared to soaked and germinated seeds (Table 1). Legumes lost 20-60% of their phenolic content with soaking. However, germination did not cause further decreases (P≥0.05) in FP content. In fact a small increase in FP was observed during germination, which could be due to synthesis of new

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compounds during germination (Lope-Amoros et al. 2006). Because FP content was determined using Folin’s method, other compounds, particularly, amino acids, also could interfere in the estimation of total phenolics (Everette, et al. 2010) The content of phenolics in soaked and subsequently cooked seeds was lower (P<0.05) than that of raw seeds; however, it was higher (P<0.05) than that of just soaked seeds. This is in contrast to Xu et al. (2008), who reported decreased FP after cooking due to thermal degradation of the compounds. Moreno-Jiménez et al. (2015) reported both increased and decreased phenolics due to thermal processing depending upon the variety of common beans as well as the type of thermal treatment. Nevertheless, positive changes phenols (Dueñas et al. 2016) as well as increased FP due to release of bound phenolics after heat treatment has also been documented (Kim & Cheung 2015, Sreeramulu et al. 2013). 3.2 ACE inhibitory activity The data shown in Table 3 shows ACE inhibitory activity of different legume samples expressed in terms of IC50 values (mg of powder/ml). The IC50 values of ≥5 mg/ml obtained for different unprocessed as well as processed (soaking, germination and cooking) samples were mentioned as ND (not detected) indicating insignificant hypotensive potential of these commodities (Table 1). Nevertheless, legumes like horse gram, black pea and white pea showed comparatively better ACE inhibitory activity with IC50 values in the range of 1-5 mg/ml. Enzymatic hydrolysis of protein is known to produce ACE inhibitory peptides (LiChan, 2015). Surprisingly, the process of germination, which is known to activate the endogenous proteases leading to increase in concentration of peptides, did not alter ACE inhibitory potential of the seeds. On the contrary, enzymatic processing by GI enzymes significantly increased the ACE inhibitory activity of these seed legumes (P<0.05). The IC50 values after the aforementioned enzymatic digestion decreased to 0.2-0.7 mg/ml. Moreover, the increased ACE inhibitory activity of individual legume seeds was independent of the

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processing condition. However, it depended on the type of seed legume. For instance, IC50 values obtained for lentil, horse gram and white pea were in the range of 0.1-0.3 mg/ml, which are significantly (P<0.05) lower than those obtained for green gram, cowpea, chickpea and black pea (IC50: 0.5-0.7 mg/ml). Considering the average protein content of the legume seeds to be 20-25%, the respective IC50 values for lentil, horse gram and white pea could be in the range of ≈ 0.03-0.06 mg protein/ml, while for green gram, cowpea, chickpea and black pea the range would be ≈ 0.1- 0.15 mg protein/ml. Although it is difficult to compare the IC50 values obtained in the present study to those reported earlier by other authors for a variety of reasons, which includes variation in enzymes and conditions used for protein hydrolysis as well as method used in determining ACE inhibitory activity. Nevertheless, the values (IC50) reported here in terms of mg protein for lentil, chickpea, cowpea and white pea are in the range of those reported earlier with use of GI enzymes (Barbana & Boye 2010; LopezBarrios et al. 2014, Jakubczyk et al. 2013). Overall, the results thus suggest that the hypotensive effect of legumes is facilitated by digestive enzymes by releasing the specific peptides showing ACE inhibitory activity. 3.3 Antioxidant Activity 3.3.1 ABTS+ Radical Scavenging Activity (RSA) The ABTS+ radical scavenging activity can be used with both lipophilic and hydrophilic compounds and has been used widely for evaluation of radical scavenging activity of protein hydrolysates (Jamdar et al. 2011, Jamdar et al. 2010, Valdez-Ortiz et al. 2012). As shown in the Fig. 1A, B and C, dry legume seeds of horse gram showed the highest activity (214 µM Trolox equivalent/mg), which was followed by black pea (84 µM/mg) and white pea (64 µM/mg), while others including chickpea, cowpea, green gram and lentil showed ≤50 µM/mg activity. Moreover, this activity was reduced further upon processing like soaking, germination or cooking of the seeds. This trend of decreasing RSA correlates with the FP

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content at least in horse gram, green gram and lentils (r2= 0.88-0.75, P<0.05) and showed no correlation with FAN content during processing. It thus suggests that FP content could be affecting the ABTS+ RSA in these commodities. In other commodities like cowpea, chickpea, black pea and white pea no significant correlation was observed either with FP or FAN content. Interestingly, IVPD of the samples irrespective of the processing conditions increased the RSA values by 2-4 folds depending upon the type of legume seeds (Fig. 1A-C). The increase was thus correlated with FAN content (r2=0.8-0.9, P<0.001) except for horse gram (r2=0.55, P=0.09) (Fig. 1A). Thus, digestion of the seed storage proteins must be forming scavenging peptides after degradation by gastrointestinal proteases. However, even after digestion, the maximum activity was still observed for horse gram (also containing the highest amount of FP), which could be attributed to both peptides formed due to action of digestive enzymes as well as FP content of the seeds. In some of the commodities like black pea, white pea or lentil the RSA was observed to be much higher after subjecting to GI digestion as compared to their unhydrolysed counterpart. Increases in oxygen radical absorbance activity was reported by López-Barrios et al. (2016) after protein hydrolysis of raw as well as germinated black bean by GI digestive enzymes. It thus indicates contributions by peptides after IVPD is significant as far as RSA of seed legumes are considered. Both phenolics as well as proteins of different legumes have been shown to have the ABTS radical scavenging activity (Marathe et al. 2011; Siddhuraju & Becker 2007; Arcan & Yemenicioglu 2010). The values reported by them are comparable to those reported in this study. 3.3.2 Total antioxidant capacity (TAC) The phosphomolybdenum assay is a quantitative method to evaluate the antioxidant activity of both FP and protein as well as protein hydrolyastes (Arabshahi-Delouee & Urooj 2007). Figure 2A, B and C shows TAC of unprocessed and processed legume seeds as well as each

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of those after subjecting them to gastrointestinal digestion. Overall, the trend was similar to that observed for RSA. However, in contrast to the RSA, the relative decrease in TAC was not very large after soaking and germination of the legumes. On the contrary, some of the legumes seeds like green gram, lentil, cowpea and black pea, showed increased TAC during germination (Fig. 2A-C). The profile of TAC in these seeds was correlated with concurrent changes in FAN (r2=0.78-0.92, P<0.05). However, in legume seeds like horse gram and white pea, correlation was observed with FP as well as FAN but with poor significance values. For chickpea, neither FP nor FAN content showed any correlation with TAC. The results thus suggest that at least in green gram, lentil, cowpea and black pea seed, storage proteins and their proteolysis during germination make a significant contribution to the TAC of these legumes. In horse gram as well as white pea, both FP and FAN contributed to the activity, while in chickpea the contribution by both of them was less. Cooking after soaking significantly decreased the TAC in horse gram, lentil, cow pea, black pea and white pea. Sowndhararajan et al. (2011) also reported similar observation for Vigna vexillata, an underutilized legume. As observed for RSA, legumes seeds when subjected to gastrointestinal digestive enzymes showed nearly 2-3 fold increase in the TAC values (Fig. 2A-C). Even cooked samples showed similar relative increases in the activity. The activity after IVPD was well correlated with FAN for all the legumes (r2=0.78-0.95, P<0.05. However, for each legume, the highest activity was observed for those samples in which unprocessed seeds (where FP content was the highest) were subjected to digestion with GI enzymes, suggesting a significant contribution even by FP. Overall, the TAC values denoted the increased electron donating capacity of the legume seed proteins after digestion with GI enzymes, which could thus act as better radical chain terminators or free radical stabilizers. 3.3.3 Ferric reducing antioxidant power (FRAP) assay

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The assay gives an indication of the reducing ability of the compounds. The data shown in Figure 3A, B and C shows Fe+3 ion reducing capacity of different legumes and also shows how different processing conditions applied to these seeds affected the FRAP values. The activity shown by dry seeds of all the legume types was comparatively higher than those subjected to different processing conditions. The FRAP values for different legumes were in the range of 25-215 µM of Fe+2/mg, which is lower than that reported (96-681 µM of Fe+2/mg) by Sreeramulu et al., (2013). The difference in the range could be attributed to different media used for extraction of the active component. Sreeramulu et al., (2013) used 80% methanol as the extraction media, while in this study aqueous extracts were used for estimation of FRAP. Among all the legumes, horse gram and green gram showed relatively higher FRAP values of 215 ± 9 and 159 ± 6 µM of Fe+2/mg respectively (Fig. 3A). The process of soaking reduced the activity (P<0.05), which was further reduced upon germination of these seeds. The activity correlated well with the FP content in all the seeds (r2=0.85-0.98, P<0.05) except for black pea as well as white pea samples (Fig. 3C). Thus, the FRAP activity could be attributed to the FP content of these legume seeds. In contrast to the observed effect of cooking of the soaked seeds on FP content, FRAP activity was found to decrease after this treatment. This probably was due to degradation of those phenolics contributing to the FRAP activity. Digestion with GI enzymes did not improve FRAP of any of the legumes except for green gram and black pea (Fig. 3A, C). Thus, peptides generated during germination or digestion with GI enzymes were found to be ineffective for Fe3+ reduction. Hence, it can be concluded that reduction of Fe3+ ions of legumes mainly dependent on FP content of the legume and protein breakdown products do not contribute much to this activity. 3.3.4 Metal ion chelating activity (MICA)

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Transition metal ions can generate hydroxyl and superoxide radicals that lead to lipid peroxidation, protein modification and DNA damage. Chelating agents make these ions unavailable for such reactions and thus potentially inhibit the metal ion dependent processes (Finefrock et al. 2003). Data shown in Figure 4A, B and C shows that all the seed legumes showed MICA. Among unprocessed legume seeds, chickpea showed relatively higher activity (210 µM EDTA equivalent/mg) (Fig. 4B), while the respective values for horse gram, green gram, cowpea, black pea and white pea were in the range of 120-162 µM EDTA equivalent/mg. Lentil showed least activity of 62 µM EDTA equivalent/mg (Fig. 4A). The values reported for MICA in this study are much higher than those reported (0.4-2.0 µM EDTA equivalent/mg) for 80% methanolic extract, which mainly contained phenolic compounds, of different legumes by Marathe et al. (2011) and Nithiyanantham, et al. (2012), while the values are comparable to those reported for chick pea protein isolates (156 µM EDTA equivalent/mg) as well as chickpea protein hydrolysates (122 µM EDTA equivalent/mg) by Arcan & Yemenicioglu (2010). It thus suggests that aqueous extract of legumes as reported in the present study showed higher MICA as compared to 80% methanolic extracts. Hence, use of aqueous extracts of legumes for testing MICA is more relevant than 80% methanolic extract. The correlation analysis showed poor correlations between MICA and FP or FAN of the legume seeds during processing. The process of soaking decreased (P<0.05) the activity in white pea, horse gram and chickpea by 16-17%, while in chickpea the decrease was up to 33% (Fig.4 A-C). Germination decreased the activity in all the legumes except chickpea and lentil, while on the other hand the activity in these two legumes increased. Cooking surprisingly increased (P<0.01) the chelating activity in all the legumes except in black pea. However, the extent to which the increase was observed varied from 10 to 50%.

18

The process of protein digestion by GI enzymes improved (P<0.05) MICA in legumes like black pea, white pea, cowpea and lentil (Fig.4A-C). The activity in blackpea after protein digestion was improved by 2.5-3 folds, while that in the white pea by 2 fold. The correlation analysis showed fair correlation between MICA and FAN in black pea, white pea and lentil (r2=0.75-0.83, P<0.05). In fact the highest amount of MICA (416 µM EDTA equivalent/mg) was observed in black pea after subjecting it to protein digestion. In green gram (r2= - 0.75, P<0.05), chickpea (r2= - 0.55, P <0.05) and horse gram (r2= 0.38, P <0.05) on the other hand the activity decreased after protein digestion. Overall, it suggests that MICA might be associated with more than one type of water soluble compounds, which could be phenolics, proteins, peptides or some other legume specific compounds and their availability could thus be the deciding factor for the MICA of the individual legume. 3.3.5 Reactive Oxygen Species (ROS) scavenging activity In vitro chemical assays although showing the antioxidant capacities of different legumes, do not predict there values in biologically relevant systems. Hence, it is important to carry out cellular antioxidant activity studies along with chemical assays. Several researchers have reported the application of the dichlorofluoresceindiacetate (DCF-DA) method for evaluation of natural antioxidants and their capacities to scavenge intracellular ROS (Xu & Chang 2012, Eberhardt et al. 2005, Wolf & Riu 2007). Moreover, the cell based antioxidant assay may be regarded as a more biologically relevant method than the popular chemical assay methods, as it accounts for uptake, metabolism, and location of antioxidant compounds within cells (Wolfe & Riu 2007). In the cellular assay involving DCF-DA, the compound is de-esterified intracellularly and turns into highly fluorescence DCF upon oxidation. The ROS generated due to H2O2 oxidizes non-fluorescent dichlorofluorescein (H2DCF) into fluorescent dichlorofluorescein (DCF). The degree of inhibition of oxidation of H2DCF by antioxidants which scavenge ROS is thus used as a measure of antioxidant capacity of the legumes.

19

Among all the food legumes, cowpea in the unprocessed form, after soaking or after soaking and cooking showed the highest activity (P<0.05) (Fig. 5). Germination decreased activity of this legume. In vitro protein digestion affected the activity negatively in soaked as well as in two day germinated seeds. In horse gram, the highest activity was seen with soaked seeds after in vitro digestion (Fig. 5A). Interestingly, processing methods like soaking and germination did not affect ROS scavenging activity of horse gram, in fact the process of germination increased this activity slightly. Chick pea and lentil showed moderate ROS scavenging activity, while green gram showed none. While in chickpea the activity was negatively affected due to both soaking and germinaition, in lentil it decreased after soaking and one day germination and increased further after the 2nd and 3rd day of germination. Both black and white pea showed <20% ROS scavenging activity per mg, which was increased by >2 fold in black pea due to processing. Xu & Chang (2012) have reported ROS scavenging activity expressed in terms of IC50 values in the range of 0.6-1.4 mg/ml for different legumes like soybean, black bean, pinto bean, lentil and green pea, while no activity was found for legumes like mung bean, chickpea, small red bean, red kidney bean, etc. The IC50 values obtained in this study for cowpea and horsegram were 0.8 and 1.3 mg/ml, respectively, were in the same range as reported by Xu & Chang (2012). Interestingly, the process of cooking did not affect the ROS scavenging activity in most of the legumes, while the process of in vitro protein digestion did not increase the activity. On the other hand, the process of GI digestion or the conditions during the same decreased ROS scavenging activity in legumes like black pea, white pea or chickpea (P<0.05). It thus suggested that peptides generated due to GI digestion of legume proteins do not have a significant role in ROS scavenging activity. Thus, unlike the chemical antioxidant assays, the cellular antioxidant assay shows poor antioxidant capacity for peptides. Jahandideh et al. (2016) have also identified nearly 16 antioxidant peptides from egg protein ovotransferrin

20

using the ORAC method, which were ineffective in anti-inflammatory and antioxidant activities in cells Moreover, this study also showed that ROS scavenging activity did not correlate with FP (P≥0.05). Thus it indicates that chemical assays may not be a good measure of antioxidant activity in biological models (Wolf & Rui 2007). 3.3.6 Principal Component Analysis (PCA) Analysis of data on processing conditions and antioxidant activity by PCA showed the changes in antioxidant activity due to processing conditions (Fig.6). Two main PCs for the analysed grains processing conditions accounted for 79.4% variability. PC1 for 58.6% and PC2 for 20.7%, PC1 was primarily responsible for differences in processing conditions and antioxidant activities like ABTS radical scavenging activity and TAC, while PC2 was responsible for different grains and their processing conditions verses FRAP and MIC. The data suggested that the process of IVPD is positively correlated with RSA and total reducing ability of the grains irrespective of other processes like soaking, germination or cooking. The IVPD correlation with MIC is grain specific and is observed only for black pea and white pea samples. 4. Conclusions

Dry legumes have the highest bioactive potential (antioxidant activity and ACE inhibitory activity) compared to their soaked, germinated and cooked forms. In vitro protein digestion with GI enzymes (IVPD) of legumes (dry or processed) showed higher bioactivity compared to non-IVPD counterparts. The bioactive potential can be broadly summarized as horse gram > lentils > white pea > black pea > green gram > cowpea > chickpea. The soaking process significantly decreased antioxidant activity in all legumes, which remained more or less similar during germination or cooking. Thus, based on chemical antioxidant activity assays unprocessed legumes are a better source of nutraceutical compounds. IVPD improved the protein solubility and increased FAN without affecting the FP content of the legumes, 21

which in turn increased RSA as well as total antioxidant capacity in all the legumes. However, FP and peptides released during GI digestion showed poor ROS in cellular systems suggesting that the poor contribution of legume proteins in scavenging the ROS in cellular system. This in turn suggests that the real antioxidant capacity of a bioactive compound goes beyond its reducing or scavenging activity and in this system the adaptive responses involving activation of the antioxidant responsive signaling pathways could be playing an important role in combating ROS. As far as ACE inhibitory activity is concerned, FP as well as proteolysis during germination did not contribute significantly towards ACE inhibitory activity in any of the legume, while IVPD led to peptides with potent ACE inhibitory activity especially in lentil, horse gram and white pea.

Conflict of interest statement There are no conflicts of interest to report.

Acknowledgement We thank Mr. R. Patawardhan, Radiation Biology and Health Science Division of Bhabha Atomic Research Centre, Mumbai, India for his assistance and guidance in measuring the ROS scavenging activity of our samples.

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Figure 1. Radical (ABTS) scavenging activity (RSA) of different legumes after using different processing conditions and in vitro protein digestion. RSA was expressed as Trolox equivalent (µM) per mg of legume powder. GG: green gram; HG: horse gram; LN: lentil; CHK: chickpea; CWP: cowpea, BLKP: black pea; WHTP: white pea; IVPD: in vitro protein digestion; DR: dry, SK: soaked, G1D: germinated 1 day, G2D: germinated 2 days, G3D: germinated 3 days, SKCK: soaking followed by cooking. The data represents the Mean ± S.D. of three independent experiments. Different letters above the individual bars show significant differences (P<0.05) in the values in a given data set. Figure 2. Total antioxidant capacity (TAC) of different legumes after using different processing conditions and in vitro protein digestion. TAC was expressed as µmole of αTocopherol per mg of legume powder. GG: green gram; HG: horse gram; LN: lentil; CHK: chickpea; CWP: cowpea, BLKP: black pea; WHTP: white pea; IVPD: in vitro protein digestion; DR: dry, SK: soaked, G1D: germinated 1 day, G2D: germinated 2 days, G3D: germinated 3 days, SKCK: soaking followed by cooking. The values are expressed as Mean ± S.D. of three independent experiments. Different letters above the individual bars show significant differences (P<0.05) in the values in a given data set. Figure 3. Ferric ion reducing antioxidant power (FRAP) of different legumes after using processing conditions and in vitro protein digestion. FRAP activity was expressed as µM of Fe2+ formed per mg of legume powder. GG: green gram; HG: horse gram; LN: lentil;

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CHK: chickpea; CWP: cowpea, BLKP: black pea; WHTP: white pea; IVPD: in vitro protein digestion; DR: dry, SK: soaked, G1D: germinated 1 day, G2D: germinated 2 days, G3D: germinated 3 days, SKCK: soaking followed by cooking. The values are expressed as Mean ± S.D. of three independent experiments. Different letters above the individual bars show significant differences (P<0.05) in the values in given data set. Figure 4. Metal (Fe2+) ion chelation activity (MICA) of different legumes after employing processing conditions and subjecting to in vitro protein digestion. MICA was expressed as µM of EDTA per mg of legume powder. The data represents Mean ± S.D. of three independent experiments. GG: green gram; HG: horse gram; LN: lentil; CHK: chickpea; CWP: cowpea, BLKP: black pea; WHTP: white pea; IVPD: in vitro protein digestion; DR: dry, SK: soaked, G1D: germinated 1 day, G2D: germinated 2 days, G3D: germinated 3 days, SKCK: soaking followed by cooking. Different letters above the individual bars show significant differences (P<0.05) in the values in given data set. Figure 5. ROS scavenging activity of different legumes after employing processing conditions and subjecting to in vitro protein digestion. ROS scavenging activity was expressed as percent scavenging per mg of legume powder. GG: green gram; HG: horse gram; LN: lentil; CHK: chickpea; CWP: cowpea, BLKP: black pea; WHTP: white pea; IVPD: in vitro protein digestion; DR: dry, SK: soaked, G1D: germinated 1 day, G2D: germinated 2 days, G3D: germinated 3 days, SKCK: soaking followed by cooking. The data represents Mean ± S.D. of three independent experiments. Different letters above the individual bars show significant differences (P<0.05) in the values in given data set. Figure 6 PCA map showing relationships among different grains, type of processing and antioxidant activities. GG: green gram; HG: horse gram; LN: lentil; CHK: chickpea; CWP:

cowpea, BLKP: black pea; WHTP: white pea; I: In vitro protein digestion; 1: dry, 2: soaked,

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3: germinated 1 day, 4: germinated 2 days, 5: germinated 3 days, 6: soaking followed by cooking.

Table 1: Effect of Processing Conditions on Soluble Protein (SP), Free Amino Nitrogen (FAN) and Total Free Phenolics (FP) Content of Legumes Legumes GREEN GRAM

HORSE GRAM

LENTIL

CHICK PEA

COW PEA

BLACK PEA

WHITE PEA

DR

SK

G1D

G2D

G3D

SKCK

SP (%) (26±1%)

61±3a

65±3b

67±2b

68±3bc

72±2d

40±5e

FAN (nmol/mg)

84±15a

120±20b

150±30c

130±20d

160±30e

70±8f

FP (mg/g)

0.7±0.1 a

0.3±0.0 b

0.4±0.0 c

0.4±0.0 d

0.4±0.0d

0.6±0.1e

SP (%) (21±2%) FAN (nmol/mg)

51±2a 110±20a

63±3b 160±20b

65±4b 190±30c

65±3b 270±30d

65±4b 250±20d

20±4c 110±20a

FP (mg/g)

2.4±0.0a

0.8±0.1b

0.6±0.0c

0.6±0.1c

0.7±0.1d

1.4±0.1e

SP (%) (27±1%) FAN (nmol/mg)

60±4a 110±20a

67±3b 150±20b

66±3c 180±20c

67±4c 240±30d

67±4d 260±30e

42 ±4e 120±10f

FP (mg/g)

0.6±0.0a

0.4±0.0b

0.2±0.0c

0.2±0.0d

0.3±0.0c

0.4±0.1e

SP (%) (19±1%) FAN(nmol/mg)

55±2a 54±9a

56±4b 68±11b

60±3c 86±14c

63±3c 81±12c

67±3d 89±14c

29±3e 46±6d

FP (mg/g)

0.6±0.1a

0.4±0.0b

0.3±0.0b

0.3±0.1b

0.3±0.0b

0.5±0.0c

SP (%) (22±2%) FAN (nmol/mg)

62±3a 110±10a

67±3b 140±10b

67±3b 180±20c

68±3b 180±20c

78±5c 150±20b

35±2d 75±10d

FP (mg/g)

1.3±0.4a

0.7±0.0b

0.3±0.0c

0.3±0.0c

0.3±0.0 c

0.4±0.0d

SP (%) (22±2%)

53±3a

58±3b

58±2b

60±2bc

61±2c

41±2d

FAN(nmol/mg)

67±9a

91±15b

120±20c

160±20d

200±20e

80±10f

FP (mg/g)

0.7±0.1a

0.4±0.0bd

0.3±0.0c

0.5±0.1d

0.3±0.0c

0.5±0.0d

SP (%) (19±1%)

61±2a

68±2b

67±3b

68±3b

68±2b

40±3c

a

FAN (nmol/mg)

60±8

FP (mg/g)

0.6±0.1a

97±12

b

0.4±0.0b

110±14

b

0.43±0.04c

150±10

c

0.5±0.0d

210±20

d

74±12e

0.4±0.0b

0.5±0.0e

The values in parenthesis indicate total protein content of the individual legume.

31

SP: Soluble protein; FAN: Free amino nitrogen; FP: Free phenolics; N-IVPD: Before subjecting to in vitro protein digestion; D: Dry, SK: Soaked, G1D: Germinated 1 day, G2D: Germinated 2 days, G3D: Germinated 3 days, SKCK: Soaking followed by cooking. The values are expressed as the Mean ± S.D. of three independent experiments. Values in each row with different superscript letters are significantly different (P<0.05).

Table 2: Effect of Processing Conditions and in vitro Protein Digestion on Soluble Protein (SP), Free Amino Nitrogen (FAN) and Total Free Phenolics (FP) Content of Legumes Legumes GREEN GRAM

HORSE GRAM

LENTIL

CHICK PEA

COW PEA

BLACK PEA

DR

SK

G1D

G2D

G3D

SKCK

FP (mg/g)

72±3e 320±40a 0.7±0.1a

77±4b 370±40ab 0.4±0.0b

77±4b 460±40c 0.4±0.0b

80±5c 420±30cd 0.5±0.0d

83±5d 430±40c 0.5±0.0d

65±4e 360±40ab 0.5±0.1d

SP (%) (21±2%) FAN (nmol/mg) FP (mg/g)

72±4a 280±30a

71±5ab 340±40b

69±5b 350±40b

71±5ab 400±40c

72±4a 380±50d

60±5c 310±20a

SP (%) (27±1%)

2.6±0.2a

1.0±0.1b

0.7±0.0c

0.6±0.1c

0.7±0.0c

1.3±0.1d

FAN (nmol/mg) FP (mg/g)

75±4a 410±40a

80±4b 470±40b

79±4a 460±40b

67±4c 481±30b

67±4c 510±40c

42±4d 370±30d

SP (%) (19±1%)

0.7±0.1a

0.3±0.0b

0.4±0.1bc

0.4±0.1c

0.4±0.0c

0.6±0.0d

67±4a

72±4b

72±3b

76±5db

80±7d

59±4e

FP (mg/g)

210±20a

220±20a

220±20a

190±20ab

180±10b

140±10c

SP (%) (22±2%)

0.5±0.0a

0.4±0.0b

0.3±0.0b

0.4±0.1bc

0.4±0.0c

0.4±0.0c

FAN (nmol/mg) FP (mg/g)

68±3a 240±30a

70±3a 260±20b

70±4a 310±20c

72±3ab 300±30c

73±4b 300±20c

62±3c 240±20a

SP (%) (22±2%)

1.3±0.4a

0.7±0.1b

0.4±0.0c

0.4±0.1c

0.3±0.0c

0.7±0.0b

FAN(nmol/mg)

77±2a 220±20a 0.8±0.1a

82±2b 280±20b 0.4±0.0b

85±3c 310±30c 0.5±0.0c

83±3c 360±40d 0.4±0.0d

70±3d 210±20a 0.6±0.0e

76±3a 250±20a 0.6±0.0a

80±4b 310±30b 0.6±0.0b

82±5b 320±30b 0.6±0.0a

86±5c 400±30c 0.5±0.0b

61±3d 240±20a 0.4±0.1c

SP (%) (26±1%) FAN (nmol/mg)

FAN(nmol/mg)

FP (mg/g) SP (%) (19±2%)

WHITE PEA

FAN (nmol/mg) FP (mg/g) SP (%) (19±1%)

85±3c 300±30bc 0.5±0.0c 86±4c 310±20b 0.6±0.1b

The values in parenthesis indicate total protein content of the individual legume.

32

SP: Soluble protein; FAN: Free amino nitrogen; FP: Free phenolics; N-IVPD: Before subjecting to in vitro protein digestion; D: Dry, SK: Soaked, G1D: Germinated 1 day, G2D: Germinated 2 days, G3D: Germinated 3 days, SKCK: Soaking followed by cooking. The values are expressed as the Mean ± S.D. of three independent experiments. Values in each row with different superscript letters are significantly different (P<0.05).

33

3 4 5 6 7 8

1 2

ND 0.7±0.1b 4.3±1.0b 0.5±0.1 b 2.4±0.6b 0.2±0.0b

ND 0.1±0.0a ND 0.7±0.1a ND 0.8±0.1a 2.7±0.8a 0.7±0.1a 2.8±0.5a 0.3±0.0a

Non-IVPD IVPD

Non-IVPD IVPD

Non-IVPD IVPD

Non-IVPD IVPD

Non-IVPD

IVPD

CHICK PEA

COW PEA

BLACK PEA

WHITE PEA

0.2±0.0b

3.1±0.5ac

2.9±0.8a 0.6±0.1a

ND 0.7±0.0b

ND 0.6±0.1b

ND 0.1±0.0a

2.9±0.5c 0.2±0.0b

ND 0.6±0.1a

G 1D

0.2±0.0b

2.0.±0.6db

1.6±0.5c 0.7±0.1a

0.3±0.0a

1.2±0.1e

3.9±0.6b 0.7±0.1a

ND 0.6±0.1c

2.8±0.9a 0.6±0.1b

3.0 ±0.9a 0.6±0.1b ND 0.7±0.1ab

ND 0.1±0.0a

1.2±0.2ab 0.2±0.0ab

ND 0.6±0.1a

G 3D

ND 0.2±0.0ab

3.5±0.3d 0.2±0.0ab

ND 0.6±0.1a

G 2D

0.2±0.0b

ND

3.5±0.8db 0.7±0.1a

ND 0.6±0.1c

ND 0.6±0.0c

1.7±0.5 0.2 ±0.0b

ND 0.7±0.1b

1.1±0.9b 0.6±0.1a

SKCK

34

ND: No inhibitory activity was detected (i.e., IC50 > 5mg/ml); IVPD: In vitro protein digestion; N-IVPD: Before in vitro protein digestion; DR: Dry, SK: Soaked, G1D: Germinated 1 day, G2D: Germinated 2 days, G3D: Germinated 3 days, SKCK: Soaking followed by cooking. 1 IC50: Inhibitory concentration expressed as mg of sample per ml assay volume. 2 The values are expressed as Mean ± S.D. of three independent experiments. Values in each row with different superscript letters are significantly different (P<0.05).

ND 0.6±0.0a

ND 0.2±0.0b

0.9±0.1b 0.2±0.0b

1.4±0.3a 0.2±0.1a

Non-IVPD IVPD

HORSE GRAM

LENTIL

ND 0.5±0.1a

2.4±0.5a 0.6±0.1a

Non-IVPD IVPD

SK

GREEN GRAM

DR

ACE Inhibitory Activity (IC50)1,2

Table 3: Effect of processing conditions following in vitro protein digestion on ACE inhibitory activity of legumes

Figure 1

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Figure 2

36

Figure 3

37

Figure 4

38

Figure 5

39

Figure 6

40