Food Chemistry 172 (2015) 528–536

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Quality traits analysis and protein profiling of field pea (Pisum sativum) germplasm from Himalayan region Shagun Sharma a, Narpinder Singh a,⇑, Amardeep Singh Virdi a, Jai Chand Rana b a b

Department of Food Science and Technology, Guru Nanak Dev University, Amritsar 143005, Punjab, India National Bureau of Plant Genetic Resources (NBPGR), Regional Station, Phagli, Shimla 171004, HP, India

a r t i c l e

i n f o

Article history: Received 2 June 2014 Received in revised form 21 August 2014 Accepted 18 September 2014 Available online 28 September 2014 Keywords: SEM Antioxidant activity Minerals Resistant starch SDS–PAGE Trypsin inhibitor

a b s t r a c t The grain and flour characteristics of different field pea (FP) accessions were evaluated. Accessions with higher grain weight had less compact structure with a greater proportion of large-sized starch granules. Accessions with higher protein content had lower starch content, blue value and kmax whereas accessions with higher amylose showed higher resistant starch (RS) and final viscosity and lower rapidly digestible starch (RDS). Ca, Zn, K and Fe content vary significantly amongst different accessions and creamish green and white seeds accessions showed higher Fe and Zn content. Yellow coloured accessions (1.36–3.71%) showed lower antioxidant activity as compared to brownish and green coloured accessions (4.06– 9.30%). Out of 21 major polypeptides observed (9–100 kDa), 11 showed differential trypsin inhibitory activity (TIA) under non-reducing conditions. Polypeptides of 68, 46, 33 and 22 kDa showed prominent TIA. Ó 2014 Published by Elsevier Ltd.

1. Introduction Peas (Pisum sativum L.), the earliest domesticated crop in the world, are widely cultivated in temperate zones (Ambrose, 1995; Zohary & Hopf, 2000). Peas are considered to be an inexpensive source of proteins (especially rich in the essential amino acids tryptophan and lysine; 21–25%), complex carbohydrates, high fibre (soluble and insoluble), B vitamins, folate and mineral content, such as calcium, iron and potassium, 86–87% total digestible nutrients and very low sodium and fat content (Tiwari & Singh, 2012). Peas are also considered to be a rich source of biologically active components that have potentially beneficial health and therapeutic effects, such as lowering high LDL-cholesterol, risk of heart disease, risk of type-2 diabetes and in the prevention of various forms of cancer (Roy, Boye, & Simpson, 2010). These beneficial properties

Abbreviations: FP, field pea; AOA, antioxidant activity; TPC, total phenolic content; TIA, trypsin inhibitory activity; SEM, scanning electron microscopy; SW, seed weight; CT, cooking time; SS, soaked seeds; CS, cooked seeds; HC, hydration capacity; SC, swelling capacity; H, hardness; Coh, cohesiveness; Gum, gumminess; BV, blue value; PT, pasting temperature; PV, peak viscosity; BDV, breakdown viscosity; FV, final viscosity; SBV, setback viscosity; RDS, rapidly digestible starch; SDS, slowly digestible starch; RS, resistant starch; TS, total starch; TG, total glucose; PP, polypeptides; TI, trypsin inhibitor; UPGMA, unweighted pair group method with arithmetic mean; ANOVA, analysis of variance. ⇑ Corresponding author. Tel.: +91 183 2258802 3216; fax: +91 183 2258820. E-mail address: [email protected] (N. Singh). http://dx.doi.org/10.1016/j.foodchem.2014.09.108 0308-8146/Ó 2014 Published by Elsevier Ltd.

make the peas an important agricultural commodity. They are widely used in soups, breakfast cereals, processed meats, health foods, pastas and purees or processed into pea flour, pea starch, or pea protein concentrates (Agriculture & Agri-Food Canada, 2008; Slinkard, Bhatty, Drew, & Morrall, 1990). However, raw pulses seed or flour contains antinutritional proteins, such as lectins, protease inhibitors and the non-antinutritional compound, angiotensin I-converting enzyme (ACE) inhibitor and ingestion of these antinutritional substances may result in various mild to severe deleterious effects, such as hemagglutination, bloating, vomiting and pancreatic enlargement, in humans and livestock (Roy et al., 2010). Several studies demonstrate that cooking under high pressure and extrusion cooking significantly reduces the deleterious effect of antinutritional substances (Morrison, Savage, Morton, & Russell, 2007). Garcia-Cerreno (1998) demonstrated that protease inhibitors negatively affect the gel-formation, waterholding capacity, foaming and whipping ability of legume products. Additionally, the physical properties of legumes, such as seed weight, volume, seed coat and cotyledon characteristics, hydration capacity and storage temperature, have also been reported to affect the cooking quality (Bishnoi & Khetarpaul, 1993; Sefa-Dedeh & Stanley, 1979). Evaluation of functional characteristics of legume flour is necessary before considering it as a good food ingredient. The demand of pea starches and flours in frozen foods, extruded snacks, cookies, crackers, sauces and soups is increasing. However,

S. Sharma et al. / Food Chemistry 172 (2015) 528–536

information on the physicochemical, textural properties, pasting, AOA, TIA, digestibility and proteins characteristics of FPs grown in India is scanty. Additionally, starch and proteins are major constituents of flour (22–45%) and widely used as an important ingredient for the modification of textural and sensory attributes in food products. Therefore, the evaluation of these properties will improve our knowledge, allowing development of texturally improved food products with better consumer acceptance. Therefore, this study was undertaken to evaluate the diversity in quality traits of the seed and flour of FP germplasm in terms of microstructure of cotyledon and hull, AOA, TPC, mineral composition, RS, SDS–PAGE and TIA along with physical, pasting and textural properties. 2. Materials and methods 2.1. Materials Seeds of fourteen different FP accessions (IC279082, IC279125, IC276171, IC291541, IC291544, IC299013, IC342025, IC342026, IC342028, IC342033, IC381453, IC469135, IC469137 and IC469145) were procured from NBPGR, Shimla, India in 2011– 2012. These were selected on the basis of agronomic superiority and genetic variability for various characteristics, particularly for number of pod clusters/plant, pods/plant, pod length, seed size, colour and yield plant. 2.2. Seed characteristics 2.2.1. Scanning electron microscopy (SEM) Scanning electron micrographs of FP grain cotyledon and hull sections (1 mm  1 mm  3 mm) were measured by SEM (Carl Zeiss AG, Oberkochen, Germany) using an accelerating potential of 15 kV. 2.2.2. Physical, hydration and cooking properties L⁄, a⁄ and b⁄ values were determined using a Hunter colorimeter (Hunter Associates, Laboratory Inc, Reston, VA, U.S.A) and CT as described in the literature (Kaur et al., 2013). Hydration and swelling properties were determined as described by William, Nakoul, and Singh (1983). 2.2.3. Textural properties of soaked seeds (SS) and cooked seeds (CS) Hydration and texture profile analysis of SS and CS was done as described in the literature (Singh, Kaur, Rana, & Sharma, 2010). Textural parameters, such as H (maximum height of the force peak on the first compression cycle), coh (ratio of the positive force areas under the first and second compressions) and gum (H * Coh) were determined. 2.3. Flour characteristics 2.3.1. Composition Grains were ground to pass through sieve no. 60 (BIS). Protein, ash and fat content were determined by using AOAC (1990) methods. 2.3.2. Hunter colour L⁄, a⁄ and b⁄ values were determined as described in Section 2.2.2. 2.3.3. AOA AOA was measured using a modified version of the method described by Brand-Williams, Cuvelier, and Berset (1995). Briefly, flour (100 mg) was extracted with methanol (1 ml) for 2 h at room temperature and centrifuged (3000 g, 10 min). The supernatant (100 ll) was mixed with 6  105 mol/l DPPH (3.9 ml) solution.

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Absorbance at 515 nm was recorded at 0 and 30 min using methanol as blank. AOA was calculated as% discolouration.

% AOA ¼ ð1  ðA of samplet¼30 =A of controlt¼0 ÞÞ  100 2.3.4. TPC Total phenolics in extracts were determined with the Folin–Ciocalteu (FC) reagent as described by Xu and Chang (2007). Briefly, sample (500 mg) was weighed and phenolic compounds were extracted in methanol (80%) for 2 h, followed by centrifugation (3000 rpm, 10 min). Supernatant (100 ll) was mixed with distilled water (900 ll) and FC reagent (0.5 ml). The mixture was allowed to equilibrate (5 min) followed by addition of 20% sodium carbonate (1.5 ml) and incubation in the dark at room temperature for 2 h. The volume was made up to 10 ml and absorbance was taken at 765 nm. Gallic acid was used as a standard and the total phenolics were expressed as mg gallic acid equivalents (GAE)/g. 2.3.5. BV and kmax BV and kmax were measured as described by Takeda, Takeda, and Hizukuri (1983). Briefly, flour (100 mg, db) was suspended in ethanol (1.0 ml) to which 1.0 M NaOH (9 ml) was added, followed by heating in a boiling water bath for 10 min with shaking to completely dissolve the sample. The suspension was adjusted to pH 6.5 with 1.0 M HCl and diluted to 100 ml with distilled water. An aliquot (5 ml) of the solution was added to 0.2% iodine solution (1 ml) and made up to 100 ml with distilled water. The mixture was kept at room temperature for 15 min and kmax values were determined by measuring the wavelength of maximum absorbance from 450 to 800 nm with a spectrophotometer (Lambda 25 UV/Visible spectrophotometer, PerkinElmer). BV was calculated as:

BV ¼ 4x

Abs680 C

where Abs680: absorbance at 680 nm, and C: concentration of starch or flour in the solution in units of 1 mg/100 ml. 2.3.6. Mineral composition Samples (1 g) were placed in a porcelain crucible and heated to 600 °C in a furnace. The resultant ash was dissolved in 1 N nitric acid (2.5 ml), filtered and analysed for Na, K, Fe, Zn, Mg, Mn, Cu and Ca content, determined using an Atomic Absorption Spectrometer (Agilent, Malaysia). The instrument was calibrated with standard stock solutions of all the minerals estimated. 2.3.7. In vitro digestibility RDS, SDS and RS were determined using the method of Englyst, Kingman, and Cummings (1992). Flour (0.5 g) was taken and guar gum (50 mg), 5 glass beads and sodium acetate buffer (20 ml, pH 5.2) were mixed by vortexing. This was followed by incubation with (P7545, 8 USP Specifications, Sigma–Aldrich Co., St. Louis, MO) and amyloglucosidase from Aspergillus niger (A7095, 300 U/ ml, Sigma–Aldrich Co., St. Louis, MO) and Invertase from Baker’s yeast (I4504, Sigma–Aldrich Co., St. Louis, MO) mixture (5 ml) at 37 °C. After 20 min (G20) and 120 min (G120) of incubation 0.5 ml aliquots of hydrolyzate were removed and added to 60% ethanol (20 ml) to stop the enzymatic reaction. The glucose released in each measurement was determined by using a glucose oxidase peroxidase diagnostic kit (Autopak, Pd. Code-763, Siemens Ltd.). The remaining content of each tube was heated in a boiling water bath, treated with 7 M KOH, and hydrolysed further with amyloglucosidase (50 U/ml) to determine total glucose. RDS, SDS, and RS percentage of the total starch were calculated from the values of G20, G120 and TG (total glucose) as follows:

RDS ¼ G20  0:9

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S. Sharma et al. / Food Chemistry 172 (2015) 528–536

SDS ¼ ðG120  G20 Þ  0:9 TS ¼ TG  0:9 RS ¼ TS  ðRDS þ SDSÞ 2.3.8. Pasting properties Pasting properties (PT, PV, BDV, SBV and FV) of flours were evaluated by Rapid Visco Analyzer (Newport Scientific Pvt. Ltd., Australia) as described in the literature (Singh et al., 2010). 2.4. Starch isolation and amylose content

of Laemmli (1970). A total of 20 lg of each protein was resolved on SDS–PAGE under reducing and non-reducing conditions. For non-reducing conditions, the b-mercaptoethanol was omitted from sample buffer [100 mM Tris buffer (pH 6.8); 4% SDS; 20% glycerol; 0.04% bromophenol blue]). The electrophoresis was carried out at 35 mA constant current. Immediately after electrophoresis, the gels were soaked in 100 ml of trypsin solution [50 mM Tris–HCl pH 8.3, 0.2 mg/ml trypsin (w/v)] for 60 min at 4 °C. After trypsin treatment, the gels were washed thrice with deionized water and soaked in casein solution [100 mM phosphate buffer pH 6.0, 2% casein (w/v)] at 37 °C for 90 min and gels were washed thrice with deionized water, fixed, and stained with Coomassie Brilliant Blue R-250 staining solution and scanned with HP4210 scanner at 600 dpi to visualise the inhibitory PP. Phylogenetic analysis was also carried out for the establishment of relationship among different FP accessions. AlphaEase Software™ v 6.0.0, from Alpha Innotech Corporation was used for the development of dendrogram under default parameters. Gels were saved in 8-bit grayscale mode and selection of line was carried out by auto lane selection command under analysis tool, whereas, bands were selected manually and UPGMA cluster method and Pearson coefficient matrix were selected before the construction of final dendrogram.

The starch from FP accessions was isolated as described in the literature (Schoch & Maywald, 1968). Briefly, FP (100 g) was steeped overnight in toluene solution (2.2 ml/500 ml distilled water) at 40 °C and then washed with tap water, peeled and ground with distilled water (1:10). The slurry was then passed through a nylon cloth to remove fibre and the residue obtained was again ground with distilled water. The slurry was then allowed to stand for 2–3 h, after which the supernatant was discarded. Washing was continued until the supernatant became clear followed by centrifugation and then drying at 40 °C. Amylose content of starch was determined according to the method of William, Kuzina, and Hlynk (1970). To the starch sample (20 mg), 0.5 N KOH (10 ml) was added and the dispersed sample was made up to 100 ml with distilled water. An aliquot of the starch solution (10 ml) was taken followed by the addition of 0.1 N HCl (5 ml) and iodine reagent (0.5 ml). The volume was diluted to 50 ml with distilled water and the absorbance was measured at 625 nm.

The data reported are averages of triplicate observations, except for the textural parameters which were averaged over five replicates. Data were subjected to ANOVA and Pearson correlation using Minitab Statistical Software (MINITABÒ v 14.12.0, State College, PA).

2.5. SDS–PAGE analysis and trypsin inhibitory staining

3. Results and discussion

Inhibitory activity staining was carried out as described by Wati, Theppakorn, and Rawdkuen (2009) and Wati, Theppakorn, Benjakul, and Rawdkuen (2010) with minor modifications. In total, 12.5% resolving gel and 5% stacking gels were casted for better resolution of complex globular proteins. SDS–PAGE analysis of seed storage proteins was carried out according to a modified method

3.1. Seed characteristics

2.6. Statistical analysis

3.1.1. Physical properties Scanning electron microscope analysis of different accessions of FP cotyledons showed difference in structure, shape and alignment of starch granules and thickness of protein matrix. FP accessions

a (IC469135)

b (IC279125)

c (IC342028)

d (IC279082)

Fig. 1. Scanning electron micrographs of representatives of FP cotyledon: (a-IC469135, b-IC279125, c-IC342028 and d-IC279082).

531

Gum-CS (N)

C C LiB CG OG DW CB ChB CG BrC CG CB YB HW 15.53b 14.69b 23.65l 20.67j 18.28h 16.94e 17.55f 21.88k 13.39a 19.55i 18.12g 35.00m 15.04c 17.61f IC267171 IC279082 IC279125 IC291541 IC291544 IC299013 IC342025 IC342026 IC342028 IC342033 IC381453 IC469135 IC469137 IC469145

Means with similar superscript in a column do not differ significantly (p 6 0.05). SW – seed weight, HC – hydration capacity, HI – hydration index, SC – swelling capacity, SI – swelling index, CT – cooking time; cream (C), light brown (LiB), creamish green (CG), organic green (OG), Dutch white (DW), creamish brown (CB), chocolate brown (ChB), brownish cream (BrC), yellowish brown (YB), hansa yellow (HY); H – hardness, Coh – cohesiveness, Gum – gumminess, Chew – chewiness, SS – soaked seeds, CS – cooked seeds.

0.11c 0.09b 0.20f 0.08b 0.01a 0.01a 0.02a 0.14d 0.10c 0.17e 0.09b 0.08b 0.16e 0.20f

Coh-CS H-CS (N)

25.47f 25.69g 17.16b 32.90j 47.53l 43.15k 22.04d 23.60e 27.26h 16.62a 27.78i 53.18m 19.73c 17.25b 12.87a 11.03a 27.05e 15.28b 16.92b 15.65b 31.81f 24.12d 18.60b 21.44c 25.15d 31.45f 21.67c 22.44c

Gum-SS (N) Coh-SS

0.20c 0.18a 0.30i 0.21d 0.20c 0.20c 0.36j 0.19b 0.24g 0.20c 0.23f 0.22e 0.25h 0.25h 64.35b 62.67a 90.17i 74.18c 82.94e 77.49d 88.85h 125.63l 77.52d 107.17j 110.31k 144.24m 88.09f 88.32g 75d 74c 111h 111h 81e 59a 106g 114i 64b 74c 81e 120j 80e 101f 1.48e 1.72g 1.08b 1.13b 1.39d 1.54e 1.61f 1.22c 1.83h 1.48e 1.32c 0.94a 1.67f 1.52e 0.64b 0.64b 0.73h 0.60a 0.67d 0.66c 0.70f 0.68e 0.66c 0.71g 0.68e 0.88i 0.68e 0.66c 0.77b 0.91g 0.89f 0.77b 0.80c 0.86d 0.99j 0.55a 0.92h 0.92h 0.92h 0.87e 1.06k 0.98i 0.12a 0.13b 0.21g 0.16d 0.15c 0.15c 0.17e 0.12a 0.12a 0.18f 0.17e 0.30h 0.16d 0.17e 14.90h 16.02m 14.38f 13.92d 11.64b 14.69g 14.91i 11.34a 15.11k 15.25l 15.08j 14.88h 14.32e 13.81c 2.92b 4.00f 5.17j 3.34c 1.84a 4.86h 5.35l 3.81e 4.05g 5.00i 3.70d 5.71m 3.73d 5.31k

Textural properties of grains

H-SS (N)

CT(mins) SI SC (ml/seed) HI HC (g/seed) b⁄ a⁄

Hunter colour parameters

L⁄

Seed colour 100 SW (g) Accessions

Table 1 Physical and textural properties of grains of different FP lines.

3.1.2. Hydration and cooking properties Among the accessions, the highest HC and SC was observed for IC469135 with 0.30 g/seed weight and 0.88 ml/seed volume. Whereas, the lowest HC and SC was depicted for IC342028, IC342026, IC267171 and IC291541 which was 0.12 g/seed and 0.60 ml/seed, respectively (Table 1). Additionally, IC279125 and IC469135 had significantly higher HC and SC (0.21–0.30 g/seed and 0.73–0.88 ml/seed, respectively) than other accessions. HC was positively correlated with SW (r = 0.870, p 6 0.005) whereas SC showed positive correlation with both SW and HC (r = 0.835 and 0.883, respectively, p 6 0.005). Hydration index ranged between 0.55 (IC342026) and 1.06 (IC469137). IC469135 showed the lowest swelling index (0.94) and IC342028 showed the highest (1.83). IC469135 had less compact structure with a high proportion of starch granules compared to IC342028, which may have led to more absorption of water causing higher HC and SC. Additionally, hull of IC469135 and IC279125 had a loose network of cells with spaces in between (Fig. S1a and b) in comparison to IC342028 and IC279082 (Fig. S1c and d). Typical microstructures of cotyledons of two representative accessions are shown in Fig. 1a and c. Water-absorbing capacity of seeds depends on cell wall structure, composition of seed and compactness of the cells in the seeds along with the architecture and composition of hull (Muller, 1967).

61.00g 62.43i 59.35e 61.01g 57.16c 63.20j 57.57d 50.12a 61.55h 59.51e 64.51k 57.14c 60.17f 55.38b

with low 100 SW (13.4–15.0 g) were inversely proportional to the number of small-sized granules per unit area embedded in thick protein matrix and vice versa (Fig. 1a–d). IC469135 and IC279125 showed loose network of cells with spaces in between (Fig. S1a and b), whereas, IC342028 and IC279082 showed compact networking of cells bound together along with absence of fissures (Fig. S1c and d) which may have to lead to a difference in hydration properties. The surfaces of hull had tile-shaped fibro vascular cells with a network of interwoven hair-like projections. Among the various accessions, IC469135 had the highest 100 SW (35 g) whereas, IC342028 showed the least (13.4 g) (Table 1). Singh et al. (2010) reported 100 SW of FP accessions in the range of 4.3–29.3 g. L⁄ (lightness), a⁄ (red–green) and b⁄ (yellow–blue) values of different accessions ranged from 50.1 (IC342026) to 64.5 (IC381453), 1.8 (IC291544) to 5.7 (IC469135) and 11.3 (IC342026) to 16.0 (IC279082), respectively (Table 1). IC381453, IC299013 and IC279082 showed significantly higher L⁄ value than the other accessions indicating lighter colour grain accessions were suitable for preparing composite flours. IC291544 and IC267171 showed lower a⁄ values between 1.8 and 2.9 in comparison to 3.3 and 5.7 observed for other accessions. The majority of accessions were yellowish with b⁄ value greater than 14. All of the accessions showed the presence of a black tip at the hilum. The a⁄ value showed higher variation followed by b⁄ and L⁄ (Table S1). IC291541, IC342028 and IC381453 grains were creamish green with L⁄, a⁄ and b⁄ values varying from 61 to 64.5, 3.3 to 4.1 and 13.9 to 15.1, respectively against 50.1 to 60.2, 3.7 to 5.7 and 11.3 to 14.9, respectively for brown coloured accessions (IC279125, IC342025, IC342026, IC469135 and IC469137). Cream and Dutch white accessions showed L⁄, a⁄ and b⁄ values from 61 to 63.2, 2.9 to 4.9, and 14.6 to 16.0, respectively against values between 55.4 to 59.5, 5 to 5.3 and 13.8 to 15.3, respectively for brownish cream (IC342033) and hansa yellow (IC469145) coloured. IC291544 grains were organic green with L⁄, a⁄ and b⁄ values of 57.2, 1.8 and 11.6, respectively. Colour to the FP grains is contributed by chlorophyll and carotenoid pigments. Around 17 pigments including xanthophylls, chlorophyll a and b related compounds, and (allE)-b-carotene were identified in pea accessions (Edelenbos, Christensen, & Grevsen, 2001). Lutein and b-carotene have also been identified in FP with green, yellow and orange cotyledons (Holasová, Dostálová, Fiedlerová, & Horácˇek, 2009).

2.75b 2.36b 3.50e 2.70b 0.67a 0.60a 0.35a 3.26d 2.78c 2.89d 2.39b 4.15f 3.08d 3.45e

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Water absorption by seeds during soaking determines the % denaturation of protein and the degree of starch gelatinization during heating, which eventually affect the texture of cooked grains (Tiwari & Singh, 2012). CT for different accessions ranged from 59 (IC299013) to 120 min (IC469135) (Table 1) that may be related to the rate at which cell separation occurs, due to loosening of the intercellular matrix of the middle lamella upon cooking (Rockland & Jones, 1974). The longer CT of IC279125, IC291541, IC342025, IC342026, IC469135 and IC469145 could be attributed due to their larger SW, as seed size governs the distance to which water must penetrate in order to reach the innermost portion of seeds (SefaDedeh & Stanley, 1979). CT was positively correlated with SW, HC and hardness of soaked seeds (H-SS) (r = 0.718, p 6 0.005 and 0.544 and 0.540, p 6 0.05, respectively). CT has been reported to vary with permeability of the seed coat and internal structure of the cotyledon, which was determined by the rate of soaking of water (Seena & Sridhar, 2005). Among various hydration and cooking parameters, CT showed the greatest variation followed by HC, SI, SC and HI as indicated by ANOVA (Table S1).

3.1.3. Textural parameters H-SS was ranged from 62.7 N (IC279082) to 144.2 N (IC469135). IC469135, IC342026, IC381453 and IC342033 showed significantly higher (107.2–144.2 N) H-SS compared to other accessions (Table 1). H-SS was positively correlated with SW, SC and HC (r = 0.777 and 0.786, p 6 0.005 and r = 0.652, p 6 0.05, respectively). H-CS varied from 16.6 (IC342033) to 53.2 N (IC469135). IC469135, IC291544 and IC299013 had significantly higher H-CS (43.2–53.2 N) than other accessions (Table 1). Werker (1997) proposed that the seed hardness may be attributed to the presence of some impermeable part of the palisade layer that restricts the water uptake. Coh-SS ranged between 0.18 (IC279082) and 0.36 (IC342025). Coh-CS ranged between 0.01 (IC291544, IC299013) and 0.20 (IC469145 and IC279125). Cohesiveness is an index indicating how well the food material withstands a second deformation, relative to its behaviour under first deformation. Gum-SS and Gum-CS varied from 11.03 (IC279082) to 31.81 N (IC342025) and 0.35 (IC342025) to 4.15 N (IC469135), respectively (Table 1). Gum-SS was positively correlated with SW, HC and SC (r = 0.597, 0.669, p 6 0.05 and 0.731, p 6 0.005, respectively). The lower values of textural parameters of CS as compared to SS indicate softening of cotyledon during heating. The rate and degree of softening differed significantly among different accessions. Softening during cooking was accompanied by structural changes in the seed such as separation of cells, gelatinization of cell starch

and consequent deformation of the spherical granules (Rockland & Jones, 1974). Ca and Cu content were related to hardness and Coh-CS, however, were significantly correlated with Coh-CS (r = 0.702, p 6 0.005 and r = 0.581, p 6 0.05, respectively) indicating that accessions with higher Ca and Cu content could withstand second deformation well and required more energy for mastication. This could be attributed to formation of calcium pectate. Though various textural parameters differed significantly amongst accessions, ANOVA showed greater variation in H-SS (Table S1). 3.2. Flour characteristics 3.2.1. Proximate composition Average protein, fat and ash contents were 25.2%, 1.7% and 2.8%, respectively. Protein showed the highest variation amongst accessions followed by ash and fat content as indicated by ANOVA (Table S2). Difference in protein content among accessions may be attributed to their genetic makeup. Interestingly, IC469145, which has the highest protein and the lowest fat and amylose content, also showed superiority for agronomic traits such as no. of pods clusters/plant (19.0), pods/plant (61), pod length (6.5 cm) and seed yield/plant (92.4 g). Average protein, fat and ash content of 25.2%, 1.9% and 2.8%, respectively in FP flours has been reported earlier (Kaur, Sandhu, & Singh, 2007). 3.2.2. Hunter colour parameters L⁄, a⁄ and b⁄ values ranged from 88.1 (IC291544) to 93.1 (IC469137), 5.02 (IC291544) to 1.8 (IC299013) and 13.4 (IC342026) to 17.9 (IC291544), respectively (Table 2). Higher L⁄ values indicate that the accessions were lighter in colour. IC291544, IC279082, IC342026, IC342025, IC469137 and IC342028 showed lower a⁄ values between 5.02 and 0.98 in comparison to other accessions (1 and 1.83). Average L⁄, a⁄ and b⁄ values of 81.5, 4.3 and 17.3, respectively in FP flours has been reported (Kaur et al., 2007). Amongst colour parameters, a⁄ showed greater variation followed by b⁄ and L⁄ as indicated by ANOVA (Table S2). 3.2.3. AOA and TPC Average AOA was 5.1% (Table S2). AOA of creamish green accessions varied between 4.1% and 7.7% and from 4.4% to 9.3% for brown accessions AOA of cream and Dutch white accessions varied between 1.9% and 3.7% in comparison to 9.2% for organic green accession. Hansa yellow and brownish cream accessions had lower AOA (1.4% and 2.4%). AOA showed significant negative correlation

Table 2 Proximate composition and physico-chemical properties of flours of different FP lines. Accessions

Protein content (%)

Ash content (%)

Fat content (%)

Hunter colour parameters L

IC267171 IC279082 IC279125 IC291541 IC291544 IC299013 IC342025 IC342026 IC342028 IC342033 IC381453 IC469135 IC469137 IC469145

24.21d 23.49c 25.81j 25.64h 27.18l 24.78e 22.58a 26.48k 24.19d 25.09f 25.54g 23.20b 25.74i 28.77m

3.55l 2.82g 2.54d 2.30b 2.42c 2.89i 3.12k 2.87h 2.86h 2.64e 2.73f 2.23a 2.96j 2.55d

2.2i 2.0h 1.5c 1.9g 2.3j 1.9g 1.8f 1.5c 1.7e 1.9g 1.9g 1.6d 1.2b 0.2a

Means with similar superscript in a column do not differ significantly (p 6 0.05). BV – blue value, AOA – antioxidant activity, TPC – total phenolic content.

⁄

92.20b 92.72f 92.49c 92.60e 88.10a 92.92i 92.84g 93.08j 92.89h 92.88h 92.56d 93.06j 93.11k 92.18b

⁄

BV

kmax (nm)

AOA (%)

TPC (mg GAE/g)

0.14f 0.15g 0.13e 0.14f 0.13e 0.12d 0.15g 0.12d 0.11c 0.10b 0.10b 0.11c 0.11c 0.08a

575e 578g 571c 570b 570b 570b 570b 568a 573d 574e 571c 576f 571c 570b

1.87b 3.37d 6.41j 7.69k 9.24m 3.71e 4.37g 7.95l 4.06f 2.42c 4.96h 9.30h 5.20i 1.36a

81.62h 78.42g 99.46m 83.59i 68.15d 96.00k 99.20l 90.62j 68.35e 66.39c 73.89f 62.45a 73.86f 63.02b

⁄

a

b

1.14j 0.56b 1.00g 1.30k 5.02a 1.83m 0.91d 0.72c 0.98f 1.32l 1.03h 1.09i 0.96e 1.32l

17.95l 16.69g 15.52b 17.10j 17.97l 16.78h 15.89f 13.41a 15.42c 15.86a 17.06i 15.23b 15.22b 17.41k

533

63.0 61.0i 42.3e 55.2h 37.6c 43.0e 39.5d 35.7b 33.0a 47.7g 35.9b 31.8a 40.1d 45.3f 7.0 4.7b 7.1f 6.4d 6.7e 4.1a 4.1a 8.1i 7.3g 6.5d 8.0h 5.7c 7.1f 7.0f 845 871m 701i 745j 751k 555g 1666n 647h 405e 377c 370b 417f 390d 295a 37 38b 104e 53c 91d 121g 7a 118f 124h 160j 166k 124h 138i 198l 1995 2022m 1942i 1971k 1945j 1887g 2723n 1911h 1780e 1730c 1725b 1789f 1762d 1623a 1188 1188b 1345e 1278c 1284d 1452g 1066a 1383f 1500i 1513k 1522l 1495h 1510j 1526m 73 76h 76h 75f 74e 75g 73c 79l 74e 74d 77j 76i 73a 78k 25.2 25.5g 24.4f 25.1g 24.7f 25.2f 26.1h 23.2e 19.8c 18.7b 18.2b 20.1d 19.3c 16.5a 20.8 21.1m 18.1i 19.0k 18.9j 16.8g 22.4n 17.2h 14.0e 12.7c 12.0b 15.5f 13.5d 9.9a 11.3 11.9e 12.1f 11.6c 11.7d 13.0h 10.3a 12.7g 15.0j 15.4l 16.0m 14.3i 15.1k 18.0n

36.91 37.0m 35.65i 36.66k 35.92j 32.59g 38.34n 33.67h 28.90e 27.48c 26.70b 29.78f 28.14d 23.13a IC267171 IC279082 IC279125 IC291541 IC291544 IC299013 IC342025 IC342026 IC342028 IC342033 IC381453 IC469135 IC469137 IC469145

Means with similar superscript in a column do not differ significantly (p 6 0.05). RDS – rapidly digestible starch, SDS – slowly digestible starch, RS – resistant starch, PT – pasting temperature, PV – peak viscosity, FV – final viscosity, BDV – breakdown viscosity, SBV – setback viscosity. * Amylose content of starch.

14.4 10.8c 14.1j 20.6m 13.2h 12.0f 17.7l 10.4b 11.0d 13.5i 13.0g 8.60a 11.5e 13.5i 56.9 76.6m 63.5j 68.8k 69.4l 78.0n 49.0a 61.1i 52.9d 52.8c 52.4b 57.9f 59.8g 60.4h 46.0 37.5g 40.3i 43.3i 33.2d 45.4l 26.3b 29.6c 36.0e 36.9f 45.0k 25.8a 43.4j 40.1h 61.5 61.7g 55.3d 70.4j 60.1e 63.9h 47.7a 48.9b 65.4i 75.5l 74.8k 55.3d 60.4f 53.4c 18.1 9.8a 13.2f 15.4h 13.4f 12.1d 11.4c 12.4e 14.2g 11.0b 17.3j 13.3f 16.2i 13.4f

g

Fe

k

Mn

f l b l b b g l b l

BDV (cP) FV (cP) PT (°C)

PV (cP)

Pasting properties

SDS (%)

RS (%) Starch digestibility

RDS (%)

Amylose* (%) Accessions

3.2.6. RDS, SDS and RS content RDS, SDS and RS content ranged from 10.3% to 18%, 9.9% to 22.4% and 16.5% to 26.1%, respectively. RDS, SDS and RS differed significantly and a larger difference was observed for SDS than RDS and RS (Table S3). The accessions with higher amylose content (IC342025, IC279082, IC267171, IC291544, IC291541 and IC279125) had higher SDS and RS content and showed positive cor-

Table 3 Digestibility, pasting properties and mineral composition of flours of different FP lines.

3.2.5. Mineral composition Different FP accessions showed average macronutrients content i.e., K, Mg, Na and Ca was 61.4 ppm, 13.2 ppm, 43.7 ppm and 78.6 ppm, respectively, whereas, Cu, Mn, Fe and Zn were 6.4 ppm, 13.7 ppm, 61 ppm and 37.8 ppm, respectively (Table S3). Highest Cu (8.1 ppm), K (78 ppm), Fe (75.5 ppm), Mg (20.6 ppm) and Ca (94.1 ppm) were observed in IC342026, IC299013, IC342033, IC291541 and IC469137, respectively, whereas IC267171 had the highest Mn (18.1 ppm), Zn (46 ppm) and Na content (63 ppm) (Table 3). IC267171 was found rich in Mn and Na along with a high number of pods clusters/plant (18.3), pods/plant (48.9), pod length (5.9 cm) and seed yield/plant (44.5 g) while IC299013, which was rich in Zn and K had medium agronomic performance with 16.2 g seed yield/plant. Average Cu, Fe, Zn and Mn content of 11.9 ppm, 59.7 ppm, 45.7 ppm and 13.2 ppm, respectively was observed in FP accessions (Warkentin, Sloan, & Ali-Khan, 1997). Accessions with higher Zn also showed high Mn and Fe content as both were positively correlated (r = 0.547 and 0.577, p 6 0.05, respectively). Cu and Mn were positively correlated (r = 0.578, p 6 0.05). Fe and Zn were positively correlated with L⁄ value (r = 0.711, p 6 0.005 and r = 0.644, p 6 0.05, respectively) indicating that accessions with lighter colour had more Fe and Zn. Accessions showed significant variation in Ca, Zn, K and Mg content, however, they were highest in Ca followed by Zn, K, Mg, Fe, Mn, Cu and Na as indicated by ANOVA (Table S3).

SBV (cP)

3.2.4. BV and kmax BV and kmax varied from 0.08 (IC469145) to 0.15 (IC342025, IC279082) and 568 nm (IC342026) to 578 nm (IC279082), respectively (Table 2). The kmax ranged between 570 nm and 575 nm for the majority of accessions. The higher BV and kmax might be due to the large branching chains that exist in molecules of amylose and amylopectin. BV was positively correlated with amylose (r = 0.974, p 6 0.005) and negatively with protein (r = 0.540, p 6 0.05), indicating that the accessions with higher protein had lower starch content and vice-versa. Accessions showed significant variation in BV and kmax, however, in kmax it was greater as indicated by the higher F value (Table S2).

Cu

Mineral composition (ppm)

Zn

m

K

e

Mg

k

Na

j

Ca

with b⁄ value (r = 0.566, p 6 0.05). Average TPC was 78.9 mg GAE/ g (Table S2). IC279125, IC299013, IC342025 and IC342026 showed between 90.6 and 99.2 mg GAE/g compared to 62.5–83.6 mg GAE/g for other accessions. Sreeramulu, Reddy, and Ragunath (2009) and Turkmen, Sari, and Velioglu (2005) also reported TPC in different FP as 126.6 mg/100 g and 183.3 mg/ 100 g, respectively, whereas elsewhere it was 16.2–32.5 mg/100 g (Wang et al., 1998). These results suggest that TPC might not contribute significantly to AOA of FP accessions. Our results are in agreement with results reported for pulses (Sreeramulu et al., 2009). The lack of correlation could be due to variable responses of different phenolic compounds in different assay systems. AOA did not necessarily correlate with TPC, since the molecular antioxidant responses of phenolic compounds vary remarkably depending on their chemical structure (Kahkonen et al., 1999). AOA and TPC differed significantly among accessions, however, larger F value of TPC than AOA indicated large effect of accessions on TPC as compared to AOA (Table S2).

68.9d 82.1g 91.0i 92.1l 66.1c 63.0b 58.0a 69.1e 91.5k 91.0j 69.1e 77.1f 94.1m 87.1h

S. Sharma et al. / Food Chemistry 172 (2015) 528–536

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S. Sharma et al. / Food Chemistry 172 (2015) 528–536

relation with amylose content (r = 0.984 and 0.971 respectively, p 6 0.005). Contrarily, RDS was negatively correlated with amylose content (r = 0.992, p 6 0.005). Lower swelling of FP accession after heating resulted in higher FV during cooling. A significant positive correlation of RS with FV (r = 0.712, p 6 0.005) and negative with PV (r = 0.849, p 6 0.005) was observed. RDS showed positive correlation with PV (r = 0.885, p 6 0.005). Additionally, RS and SDS were positively correlated with fat content and RDS showed negative correlation (r = 0.612, 0.615, and 0.663, respectively, p 6 0.05). The formation of amylose–lipid complexes and their effects on decreasing starch susceptibility to enzymatic degradation cannot be ruled out. The lower bio-availability of starch and presence of higher RS in legumes can be due to the presence of intact cell structures enclosing starch granules, a high level of amylose, viscous soluble dietary fibres, and the presence of amylase inhibitors, B-type crystallites and strong interactions between amylose chains (Deshpande & Cheryan, 1984). 3.2.7. Pasting properties Average PV, BDV, FV, SBV and PT were 1375cP, 106cP, 1915cP, 645cP and 75 °C, respectively (Table S3). Among various accessions, IC342025 showed the highest SBV (1666cP) and FV (2723cP) and the lowest PV (1066cP), and BDV (7cP). On the contrary, IC469145 showed the lowest SBV (295cP), indicating its lower tendency to retrogradation and displayed the highest BDV (198cP) that indicates its greater susceptibility to disintegrate. IC267171, IC279082, IC291541, IC291544 and IC342025 with lower BDV (7-91cP) had higher thermal stability. The lower BDV observed may be attributed to lower disintegration of the swollen starch granules in the presence of higher amylose content (Singh, Nakaura, Inouchi, & Nishinari, 2008). PV and FV were negatively and positively correlated with amylose content (r = 0.904 and 0.750, p 6 0.005, respectively). Proteins also affect the viscosity by increasing water binding, so restricting its availability to starch during gelatinization. FV and BDV were negatively and positively correlated with protein content (r = 0.545 and 0.599, p 6 0.05, respectively). These results demonstrated that the starch granules swell and disrupt to a lesser extent in the presence of higher amylose during heating, thus resulting in reduced PV and BDV. The statistical analysis showed higher variation in FV, SBV, PV and PT than BDV (Table S3). The behaviour of the viscosity during heating (from

50 to 90 °C) reflects the capacity of the starch to absorb water and swell. On cooling, viscosity of the paste increased due to retrogradation. SBV and BDV are measures of retrogradation tendency on cooling of cooked starch pastes and the tendency of swollen granules to disintegrate upon shearing. Kaur et al. (2007) reported PV, BDV, FV, SBV, and PT between 1314 and 1472cP, 192 and 196cP, 1801 and 1832cP, 615 and 710cp and 73.9 and 75.4 °C, respectively for FP accessions. The difference in pasting properties may be due to variation in extent up to which the granule structure was broken down and amylose was dispersed (Loh, 1992). 3.3. Amylose content Average amylose content of starch was 32.2% among all the accessions. It differed significantly among different accessions as indicated by F value (Table S3). IC299013, IC291544, IC291541, IC267171, IC279082, IC279125, IC342026 and IC342025 had significantly higher amylose content (32.638.3%) as compared to other accessions. Amylose content for different FP accessions represented a wide range between 21.4% and 58.3% (Singh et al., 2010). 3.4. SDS–PAGE analysis of FP accessions A total of 14 accessions of FP were evaluated by SDS–PAGE to find out possible diversity among different FPs at PP level under reducing and non-reducing conditions (Fig. 2). A total of 21 major PP ranging from 9 to 100 kDa were visualised (Fig. 2). Most of the accessions showed identical banding pattern except for 11 kDa PP that was least accumulated in IC279082 and IC342025 (Fig. 2). Banding pattern of PP under reducing (in presence of b-me) and non-reducing conditions (absence of b-me) was not significantly different and only one PP of 59 kDa was observed in non-reduced conditions (Fig. 2). Phylogenetic analysis resulted in scattering of all FP accessions into two major clusters. Cluster one consisted of eight accessions, whereas, second major cluster consisted of six accessions (Fig. S3). Additionally, the majority of the accessions from cluster one showed higher accumulation RDS, except for IC342026, whereas, accessions grouped into the second cluster showed higher accumulation of SDS and RS except for accession IC299013 (Table 3 and Fig. S3). Studies carried out by Ghafoor and Muhammad (2008) demonstrated that three Indian FP varie-

Fig. 2. Coomassie brilliant blue R-250 stained SDS–PAGE of different filed pea proteins. Lanes: TI UR – trypsin inhibitor unreduced; TI R – trypsin inhibitor reduced.

535

TI- R

IC 469145

IC 469137

IC 469135

IC 381453

IC 342033

IC 342028

IC 342026

IC 342025

IC 299013

IC 291544

IC 291541

IC 279125

IC 279082

IC 267171

TI-UR

Marker

S. Sharma et al. / Food Chemistry 172 (2015) 528–536

97.4 79

66.0

68 63 62 56 46

43.0

41 33

29.0 24

20.0

22 17 15

Fig. 3. Coomassie brilliant blue R-250 stained SDS–PAGE of different filed pea proteins after tryptic digestion. Lanes: TI UR – trypsin inhibitor unreduced; TI R – trypsin inhibitor reduced. Trypsin inhibitor was used as negative control and markers were used as positive controls to monitor the tryptic digestion of proteins by trypsin.

ties were entirely different from the germplasm of rest of the world, which was likely to be due to their unique genetic makeup. Our study further strengthens the findings of Ghafoor and Muhammad (2008) to validate the diversity among Indian FP germplasm.

3.5. TIA SDS–PAGE analysis of total proteins under reduced and nonreducing conditions revealed the presence of high molecular weight proteins in the latter conditions. Earlier studies carried out by Godbole, Krishna, and Bhatia (1994), Macedo, Garcia, Freire, and Richardson (2007) also demonstrated that TIA of several proteins was observed due to the presence of s–s-bonds under non-reducing conditions, therefore, TIA was accomplished only under non-reducing conditions. However, the banding pattern of all major PP was identical under both reduced and non-reduced conditions. Wati et al. (2009, 2010) demonstrated the presence of three PP of 132, 118 and 13 kDa that had TIA. We used 12.5% resolving gel instead of 15%, used in earlier studies that resulted in better resolution of high molecular and low molecular weight proteins. TIA of total proteins showed the presence of 11 PP of 79, 68, 63, 62, 56, 46, 41, 33, 24, 22, 17 and 15 kDa that were differential among all of the FP accessions (Fig. 3). PP of 68, 46, 33 and 22 kDa showed highest TIA. PP of 68 kDa and 46 kDa showed the highest TIA in IC267171, IC469145, IC279125, IC342033 and IC267171, IC342025, IC469137, respectively (Figs. 3 and S2). On the contrary, 22 kDa PP showed very high TIA in IC267171 and IC469145 (Figs. 3 and S2). PP of 33 kDa was highly sensitive to trypsin digestion in IC279082 and showed the least TIA. IC267171, IC342026 and IC469137 had high resistance towards tryptic digestion, whereas IC279082 accumulated highly trypsin sensitive proteins (Fig. 3). TI enzyme (20 lg in 50 mM Tris HCl, pH 8.3) was also loaded in two wells to check the activity of trypsin both under reduced and non-reduced conditions (Fig. 3). Our results demonstrate that TI was not digested with trypsin under both conditions. On the contrary, the molecular weight markers (94.3, 66, 43, 29, 20 and 14.3 kDa) undergo significant tryptic

digestion under same conditions. Therefore, these results further validated the TIA activity of FP proteins. 4. Conclusion Accessions with higher SW had a greater proportion of large size starch granules per unit area in cotyledon and loose intercellular network in hull, leading to higher hydration and swelling characteristics. Accessions with higher amylose content and FV had higher RS and lower RDS content so can be beneficial for health, whereas, accessions with higher protein and lower starch content had lower BV. AOA and TPC varied significantly among the accessions. IC469145, IC299013, IC267171 and IC342026 were not only rich in micronutrients but also had superior agronomic traits. Brown and green coloured accessions with higher antioxidant potential can be useful to reduce the risk for chronic diseases. TIA showed differential accumulation of trypsin inhibitor PP in different accessions. Accessions with low TIA can be used as donors in the quality improvement of FP. Acknowledgements S.S. wishes to thank Department of Science and Technology, New Delhi, for providing financial assistance in the form of INSPIRE Fellowship. N.S. acknowledges the financial support by Department of Biotechnology (102/IFD/SAN/2398/2011-12), Government of India. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.foodchem.2014. 09.108. References Agriculture & Agri-Food Canada. (2008). Dry peas: Situation and outlook. Bi-weekly Bulletin, 21(2). Retrieved 20.08.08. Ambrose, M. J. (1995). From near east centre of origin the prized pea migrates throughout world. Diversity, 11, 118–119.

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