* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Characterisation and functional properties of watermelon (Citrullus
Ribosomally synthesized and post-translationally modified peptides wikipedia , lookup
Signal transduction wikipedia , lookup
Paracrine signalling wikipedia , lookup
Gene expression wikipedia , lookup
Amino acid synthesis wikipedia , lookup
Biosynthesis wikipedia , lookup
G protein–coupled receptor wikipedia , lookup
Expression vector wikipedia , lookup
Point mutation wikipedia , lookup
Genetic code wikipedia , lookup
Ancestral sequence reconstruction wikipedia , lookup
Magnesium transporter wikipedia , lookup
Metalloprotein wikipedia , lookup
Bimolecular fluorescence complementation wikipedia , lookup
Interactome wikipedia , lookup
Biochemistry wikipedia , lookup
Protein purification wikipedia , lookup
Two-hybrid screening wikipedia , lookup
Western blot wikipedia , lookup
Research Article Received: 28 May 2010 Revised: 11 August 2010 Accepted: 11 August 2010 Published online in Wiley Online Library: 7 September 2010 (wileyonlinelibrary.com) DOI 10.1002/jsfa.4160 Characterisation and functional properties of watermelon (Citrullus lanatus) seed proteins Ali Abas Wani,a,b Dalbir Singh Sogi,b∗ Preeti Singh,c Idrees Ahmed Wania,b and Uma S Shivhared Abstract BACKGROUND: People in developing countries depend largely on non-conventional protein sources to augment the availability of proteins in their diets. Watermelon seed meal is reported to contain an adequate amount of nutritional proteins that could be extracted for use as nutritional ingredients in food products. RESULTS: Osborne classification showed that globulin was the major protein (≥500 g kg −1 ) present in watermelon seed meal, followed by albumin and glutelin. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that the polypeptides had low molecular weights ranging from 35 to 47 kDa. Isoelectric focusing revealed that the isoelectric point of most proteins was in the acidic range 4–6. These proteins are rich in aspartic acid, glutamic acid and serine. An increase in pH (5–9) significantly (P < 0.05) decreased the denaturation enthalpy of these proteins. Among functional properties, albumin exhibited a much higher dispersibility index (810.3–869.6 g kg−1 ) than globulin (227.8–245.4 g kg−1 ), glutelin (182.1–187.7 g kg−1 ) and prolamin (162.3–177.7 g kg−1 ). Digestibility was in the ranges 760.6–910.0 and 765.5–888.5 g kg−1 for Mateera and Sugar Baby watermelon protein fractions respectively, while surface hydrophobicity ranged from 126.4 to 173.2 and from 125.8 to 169.3 respectively. The foaming and emulsifying properties of albumin were better than those of the other proteins studied. CONCLUSION: The good nutritional and functional properties of watermelon seed meal proteins suggest their potential use in food formulations. c 2010 Society of Chemical Industry Keywords: watermelon seed protein; thermal properties; protein digestibility; amino acid analysis INTRODUCTION J Sci Food Agric 2011; 91: 113–121 characteristics that affect protein behaviour in food systems during processing, manufacturing, storage and preparation.11 Proteins have unique surface properties owing to their large molecular size and amphiphilic properties. Information on the distribution of seed protein fractions and amino acids of watermelon seed proteins is lacking in the literature. Therefore the objective of this study was to investigate the protein fractions of watermelon seeds for biochemical and functional properties so that they could be effectively used as functional ingredients in different food systems. ∗ Correspondence to: Dalbir Singh Sogi, Department of Food Science & Technology, Guru Nanak Dev University, Amritsar, India. E-mail: [email protected] a Department of Food Technology, Islamic University of Science and Technology, Awantipora, India b Department of Food Science and Technology, Guru Nanak Dev University, Amritsar, India c Chair of Food Packaging Technology, Technical University of Munich, Freising, Germany d Department of Chemical Engineering, Panjab University Chandigarh, India www.soci.org c 2010 Society of Chemical Industry 113 Watermelon is an important but underutilised crop grown in tropical regions of the world. It is used for the production of juices, nectars, fruit cocktails, etc.1 but generates waste in the form of rind and seeds. The seeds are utilised directly for human consumption in various forms, such as snacks in India, Arabian and African countries, as an additive in various dishes, for decorating cakes and as a stuffing in indigenous kheer.2,3 The kernels are used in sweetmeats and toppings as a substitute for almonds and pistachios in India.4 Melon seeds are also used to thicken and emulsify soups and stews that provide proteins in the diet.5 The seeds are also reported to possess medicinal properties and are used to treat chronic or acute eczema.4 Watermelon seeds have been reported to contain high levels of proteins2,3,6 and lipids.7 Arginine, glutamic acid, aspartic acid and leucine are the predominant amino acids in watermelon proteins.8,9 Reports are also available on the biological value, true digestibility, protein efficiency ratio and net protein utilisation of watermelon seeds.3,4 However, the successful use of plant proteins as additives depends greatly on the favourable characteristics they impart to foods. It is therefore essential to investigate the relationship of protein quality with processing parameters that affect the functional performance of protein products. Plant proteins should possess desirable functional properties and provide essential amino acids for their utilisation in different food systems.10 These properties are intrinsic physicochemical www.soci.org MATERIALS AND METHODS Materials Certified watermelon fruits of cultivars Mateera and Sugar Baby were procured from the Central Institute for Arid Horticulture (Bikaner, India) and the Department of Horticulture, Punjab Agricultural University (Ludhiana, India) respectively. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and pI standard protein markers and ampholytes were purchased from Amersham Biosciences (Amersham, UK). All other chemicals were of analytical reagent grade and were obtained from Sisco Research Laboratories (Mumbai, India). Preparation of defatted seed meal Ripe watermelon fruits were cut with a sharp knife and the juice was expelled using a burr mill. Seeds were separated from the pomace using a pilot-scale sedimentation system as described previously.12 The seeds were dried in a cabinet dryer, dehulled and ground using a hammer mill (M/S Narang Scientific Works, New Delhi, India). The meal was extracted four times with n-hexane (60–80 ◦ C) using a meal/solvent ratio of 1 : 10 (w/v). The meal was desolventised at 40 ◦ C in a vacuum oven and ground again to pass through a mesh (212 µm aperture size) to obtain a fine powder, termed defatted seed meal, which was stored at −20 ◦ C until used.6 Protein fractionation A modified Osborne fractionation procedure was used to separate the proteins albumin (water-soluble), globulin (salt-soluble), prolamin (ethanol-soluble) and glutelin (alkali-soluble) from the defatted meal.13 The sample (10 g) was successively extracted with NaCl (0.5 mol L−1 ), aqueous ethanol (700 mL L−1 ) and NaOH (0.1 mol L−1 ). Following each extraction, the slurry was centrifuged (12 600 × g, 15 min, 4 ◦ C) and the supernatant was vacuum filtered to remove insoluble particles. The residues from centrifugation and filtration were mixed and used for the next extraction. The filtrates containing the desired protein fractions were dialysed against deionised distilled (DIDI) water in a dialysing tube (pore size 204 nm; Hi-Media Laboratories Ltd, Mumbai, India) at 4 ◦ C. After dialysis the fraction containing albumin and globulin was centrifuged (12 600×g, 15 min, 4 ◦ C) and the precipitate (globulin) and supernatant (albumin) were separated. All fractions were lyophilised (Heto Dry Winner, Heto-Holten A/S, Allerød, Denmark) directly after dialysis and stored in airtight glass vials at −20 ◦ C until use. AA Wani et al. Isoelectric focusing Isoelectric focusing of non-denatured watermelon seed meal proteins was carried out as described previously.17 Amino acid analysis The amino acid composition of watermelon seed protein fractions was determined after hydrolysis with 6 mol L−1 HCl in the presence of N2 at 110 ◦ C for 24 h. Amino acids were analysed (118BL, Beckman Instruments, Fullerton, Canada) and quantified by reaction with ninhydrin. Amino acid composition was reported as g kg−1 protein. Tryptophan was determined calorimetrically as described previously.18 Analysis was done in triplicate and the amino acid composition of samples was used to determine the nutritional value. The proportion of essential amino acids to total amino acids (E/T) of the test protein was calculated as E/T (%) = [(Ile + Leu + Lys + Met + Cys + Phe + Tyr + Thr + Trp + Val + His)/(Ala + Asp + Arg + Gly + Glu + His + Ile + Leu + Lys + Met + Cys + Phe + Tyr + Pro + Ser + Thr + Trp + Val)] × 100 The amino acid composition of the test protein was compared with that of the FAO/WHO standard protein to calculate the amino acid score (AAS) as AAS (%) = [(mg amino acid g−1 test protein)/(mg amino acid g−1 FAO/WHO standard protein)] × 100 The essential amino acid (g amino acid per 16 g N) pattern of the FAO/WHO standard protein was as follows: Ile, 4.00; Leu, 7.04; Lys, 5.44; Met + Cys, 3.52; Phe + Tyr, 6.08; Thr, 4.00; Trp, 0.96; Val, 4. 96. Differential scanning calorimetry The thermal properties of fractionated watermelon seed proteins were investigated using a Dupont differential scanning calorimeter fitted with a graphic plotter and a Thermal Analyst 2100 system (TA Instruments, New Castle, DE, USA). Thermal denaturation parameters were determined according to previously described methods.19 114 Protein solubility Protein solubility was determined according to the method of Sathe.14 Protein digestibility In vitro protein digestibility was determined by the method of Saunders et al.20 Gel electrophoresis SDS-PAGE SDS-PAGE was carried out with 110 g L−1 acrylamide gels according to the procedure of Laemmli.15 Approximately 15 µg of protein sample was loaded into each well. The electrophoresis (Mini-Protein 3, Bio-Rad Laboratories, Hercules, CA, USA) was run at 100 V until the tracking dye reached the bottom of the gel, which was then removed and stained. Gels were stained with silver nitrate, employing a slight modification of the Amersham Biosciences procedure.16 The stained gels were scanned with a CCD camera (Ultra Lum, Inc., Claremont, Canada), and molecular weights were determined using Gel Pro Analyser 3.1 (Media Cybernetics, Silver Spring, MD, USA). Functional properties Water/oil absorption capacity The sample (2 g) was dispersed in 25 mL of DIDI water or oil in a tube, mixed six times on a vortex shaker for 30 min and centrifuged at 4000 × g for 15 min. The supernatant was carefully decanted and the contents of the tube were allowed to drain at a 45◦ angle for 20 min and then weighed. The water/oil absorption capacity (g g−1 ) was expressed as the average weight gain of four samples. wileyonlinelibrary.com/jsfa Protein dispersibility index The sample (0.5 g) was mixed with 25 mL of DIDI water, stirred for 30 min and centrifuged at 300 × g for 10 min. The supernatant c 2010 Society of Chemical Industry J Sci Food Agric 2011; 91: 113–121 Characterisation and functional properties of watermelon seed proteins was dried at 110 ◦ C for 12 h and then weighed. The dispersibility index was calculated as Dispersibility index (g kg−1 ) = {[weight of dish after drying (g) − weight of empty dish (g)]/[weight of sample (g)]} × 1000 Surface hydrophobicity Surface hydrophobicity (So ) was measured fluorometrically using 8-anilino-1-naphthalene sulfonic acid magnesium salt as a hydrophobic probe according to the procedure described by Paulson and Tung.21 Foaming properties The sample (1 g) was suspended in 50 mL of phosphate buffer (pH 7), stirred in a mixer/blender (HL1606, Phillips, Mumbai, India) for 2 min and then transferred to a 250 mL measuring cylinder. Foaming capacity was recorded as the volume of foam (mL) produced after 30 s. Foam stability was determined by monitoring the decrease in foam volume over time. Both parameters were expressed as the average of four replicates. Emulsifying properties Emulsifying capacity and emulsion stability were determined by the turbidimetric method.22 Statistical analysis Analysis of variance was performed and differences in mean values were determined by Duncan’s multiple range test at P ≤ 0.05 level of significance using SPSS Version 16.0 (SPSS, Inc., Chicago, IL, USA). All experiments were replicated at least three times. Mean values and standard deviations (where necessary) were reported. RESULTS AND DISCUSSION J Sci Food Agric 2011; 91: 113–121 and albumin (83.5 and 68.6 g kg−1 d.b.), in Mateera and Sugar Baby respectively (Table 1). Prolamin was present in minor amounts. Similar results on the distribution of watermelon protein fractions have been reported by other researchers.4,26 The protein content of lyophilised fractions differed significantly (P ≤ 0.05) between cultivars, with values of 953.2 and 924.1 g kg−1 d.b. for albumin, 846.9 and 864.9 g kg−1 d.b. for globulin, 872.4 and 891.2 g kg−1 d.b. for glutelin and 844.3 and 879.1 g kg−1 d.b. for prolamin in Sugar Baby and Mateera respectively (Table 1). Solubility profile The Osborne fractions of watermelon seeds had minimum solubility in the pH range 3–5 (data not shown). Protein solubility increased markedly below pH 3 and above pH 5. The considerable increase in protein solubility in acidic and alkaline environments was attributed to a gain in net negative or positive charge of proteins, with consequent interaction with water molecules.27 The increase in protein solubility was significant (P ≤ 0.05). Maximum protein solubility was observed at pH 11, suggesting that proteins can be best extracted from seed meal under alkaline conditions. The Osborne fractions were least soluble at pH 3–5 for both cultivars, indicating that proteins could be precipitated in this pH range. Ige et al.9 reported minimum protein solubility at pH 4.5–5 for two watermelon (Citrullus vulgaris) cultivars. Sathe14 reported that cashew nut proteins had minimum protein solubility at pH 5, indicating that other oilseed proteins have isoelectric points similar to those of the watermelon seed proteins studied here. The maximum protein solubility in alkaline conditions may be attributed to the higher levels of aspartic acid and glutamic acid. Among the protein fractions, albumin showed the highest solubility for both cultivars, followed by globulin, glutelin and prolamin respectively. The high solubility of albumin may be attributed to its higher content of hydrophilic groups.27 Protein solubility of the Mateera albumin fraction was significantly (P ≤ 0.05) higher than that of the Sugar Baby albumin fraction. However, protein solubility was significantly (P ≤ 0.05) higher for the globulin and prolamin fractions of Sugar Baby seed meal. Protein solubility was non-significant for the glutelin fractions of both cultivars. Similar trends have been observed for melon seed,9 walnut seed24 and cashew nut14 proteins. SDS-PAGE SDS-PAGE of the Mateera and Sugar Baby albumin fractions under non-reducing (without β-mercaptoethanol) and reducing (with β-mercaptoethanol) conditions gave complex banding patterns comprising both low- and high-molecular-weight polypeptides (Figs 1(a) and 1(b), lanes 1 and 2). SDS-PAGE of Mateera albumin under non-reducing conditions showed protein bands in the range 22–194 kDa (Fig. 1(a), lane 1), while for Sugar Baby albumin they were in the range 5–204 kDa. Mateera albumin resolved into at least 16 subunits with major bands at 88.42, 85.93, 65.27, 56.87, 51.39, 41.82 and 22.22 kDa (Fig. 1(a), lane 1), while Sugar Baby albumin resolved into 14 subunits with major bands at 204, 139.05, 90.92, 81.45, 58.33, 55.78 and 13.16 kDa (Fig. 1(a), lane 2). Thus the results revealed that the polypeptide patterns of the Mateera and Sugar Baby albumin fractions differed in both number of subunits and molecular weight distribution. However, densitometric scans revealed that most of the proteins were of low molecular weight in both cultivars. Under reducing conditions, Mateera albumin resolved into nine subunits with major bands at 64.56, 49.47 and 35.46 kDa (Fig. 1(b), lane 1), while Sugar Baby albumin resolved c 2010 Society of Chemical Industry wileyonlinelibrary.com/jsfa 115 Protein fractionation The successive extraction of defatted watermelon seed meal with different solvents yielded albumin, globulin, prolamin and glutelin in varying proportions (data not shown). Globulin was the major protein, accounting for 549.7 and 575.0 g kg−1 of the crude protein (CP) in Mateera and Sugar Baby respectively. Glutelin contributed 176.4 and 248.0 g kg−1 CP respectively, followed by albumin. Prolamin was present at negligible levels of 4.5 and 1.1 g kg−1 CP respectively. Total soluble protein content (g kg−1 CP) was higher in Sugar Baby than in Mateera. Previous studies reported albumin, globulin and glutelin contents of 63.0–126.4, 734.0–779.1 and 15.6–94.0 g kg−1 respectively in watermelon seeds.4,23 The present study revealed a similar pattern, though globulin levels were lower than those reported previously. A similar trend was observed for soluble protein. Other studies on oilseed proteins showed that albumin, globulin, prolamin and glutelin contents were 68, 176, 55 and 701 g kg−1 respectively in walnut24 and 196, 217, 39 and 161 g kg−1 respectively in deoiled tamarind kernel.25 Fractionation of cashew nut proteins revealed that albumin (455.9 g kg−1 ) and globulin (423.7 g kg−1 ) accounted for 879.6 g kg−1 of the total soluble protein, while prolamin (3.6 g kg−1 ) and glutelin (116.8 g kg−1 ) levels were much lower.14 These studies support the present findings, since different plant materials exhibit different composition. Globulin recorded the highest yields of 195.1 and 228.6 g kg−1 dry basis (d.b.), followed by glutelin (133.7 and 192.1 g kg−1 d.b.) www.soci.org www.soci.org AA Wani et al. Table 1. Yield, physicochemical and functional properties of Osborne fractions of Mateera and Sugar Baby watermelon seed proteins (n = 4) Mateera Parameter −1 Yield (g kg d.b.) Protein content (g kg−1 d.b.) Dispersibility index (g kg−1 ) Water absorption capacity (g g−1 ) Oil absorption capacity (g g−1 ) Bulk density (g L−1 ) Total colour difference (E) Digestibility (g kg−1 ) Surface hydrophobicity (So ) Albumin Globulin Prolamin Sugar Baby Glutelin Albumin Globulin Prolamin Glutelin 83.5c ± 0.54 195.1f ± 1.18 2.6a ± 0.02 133.7d ± 1.25 68.6b ± 0.39 228.6g ± 0.60 1.7a ± 0.02 192.1e ± 0.28 924.1g ± 0.18 864.9c ± 0.30 879.1e ± 0.41 891.2f ± 0.46 953.2h ± 0.80 846.9b ± 0.48 844.3a ± 0.82 872.4d ± 0.78 869.6h ± 1.54 227.8e ± 1.65 177.7b ± 1.87 182.1c ± 0.72 810.3g ± 1.04 245.4f ± 0.82 162.3a ± 0.46 187.7d ± 0.51 – 2.4b ± 0.10 3.7a ± 0.24 3.9a ± 0.05 3.8a ± 0.09 2.4b ± 0.05 2.1a ± 0.04 2.5c ± 0.06 – 2.5c ± 0.03 3.9a ± 0.11 3.7a ± 0.06 3.8a ± 0.02 2.6d ± 0.06 2.2a ± 0.03 2.8e ± 0.06 330.0b ± 0.02 550.0f ± 0.02 310.0a ± 0.07 470.0d ± 0.03 360.0c ± 0.02 630.0g ± 0.03 310.0a ± 0.02 540.0e ± 0.02 35.6e ± 1.59 28.3c ± 1.05 24.0b ± 1.39 30.6a ± 0.74 33.1d ± 0.61 28.0c ± 0.62 21.2a ± 0.92 23.9b ± 0.60 910.0g ± 0.10 862.9e ± 0.99 760.6a ± 0.81 783.0b ± 0.10 888.5f ± 0.46 846.0d ± 0.29 795.9c ± 0.93 765.5a ± 0.90 126.4a ± 1.13 167.3e ± 0.95 154.2b ± 1.38 173.2g ± 0.47 125.8a ± 0.87 164.2d ± 1.32 158.3c ± 1.15 169.3f ± 0.73 Values are expressed as mean ± standard deviation. Means in the same row with different letters are significantly different at P < 0.05. 116 into 14 subunits with major bands at 104.34, 73.39, 55.94, 20.43 and 17.38 kDa (Fig. 1(b), lane 2), again indicating that the banding patterns of the two cultivars differed. King and Onuora26 reported only one major band with a molecular weight of 12 kDa for melon seed proteins. Tomato seed and walnut albumins were found to contain peptides of molecular weights 67–360 and 14–20 kDa respectively.24,28 SDS-PAGE of the Mateera globulin fraction under non-reducing conditions gave 18 subunits in the molecular weight range 3–212 kDa with major bands at 43.36, 41.52 and 15.57 kDa (Fig. 1(a), lane 3). The electrophoretic patterns of tomato seed and walnut globulins under non-reducing conditions showed two and four major bands respectively.24,28 SDS-PAGE of globulin under reducing conditions revealed subunits within the ranges 12.31–208.47 and 12.62–58.09 kDa for Mateera and Sugar Baby cultivars respectively (Fig. 1(b), lanes 3 and 4). Mateera globulin resolved into 14 subunits with major bands at 79.85, 54.50, 38.33, 34.38 and 20.43 kDa (Fig. 1(b), lane 3), while Sugar Baby globulin resolved into six subunits with major bands at 58.09, 41.92 and 30.43 kDa (Fig. 1(b), lane 4). These results indicated that globulins of both cultivars were of low molecular weight. King and Onuora26 studied the electrophoretic pattern of melon seed proteins and found by SDS-PAGE that melon globulin resolved into six subunits in the molecular weight range 12.1–58.8 kDa with major bands at 53.0, 33.7, 29.6 and 21.6 kDa. However, SDS-PAGE of tomato seed waste and walnut globulins revealed bands in the molecular weight ranges 19–27 and 44–70 kDa respectively.24,28 These studies showed that the number of bands and molecular weight distribution of the globulin fraction differ according to the protein source. The prolamin fractions of the two cultivars exhibited strong similarity under non-reducing conditions, with only minor differences in molecular weights (Fig. 1(a), lanes 5 and 6). Mateera prolamin resolved into two bands in the molecular weight range 27–212 kDa (Fig. 1(a), lane 5), while Sugar Baby prolamin resolved into two bands in the molecular weight range 25–213 kDa (Fig. 1(a), lane 6). Under reducing conditions, Mateera prolamin resolved into six bands in the molecular weight range 16.11–153 kDa (Fig. 1(b), wileyonlinelibrary.com/jsfa lane 5), while Sugar Baby prolamin resolved into five bands in the molecular weight range 17–108 kDa (Fig. 1(b), lane 6). Densitometric scans revealed that most of the proteins were of low molecular weight in both cultivars. The glutelin fractions of the two cultivars also showed strong similarity under non-reducing conditions, with only minor differences in some bands (Fig. 1(a), lanes 7 and 8). Mateera glutelin resolved into 15 bands in the molecular weight range 30.03–257.00 kDa (Fig. 1(a), lane 7), while Sugar Baby glutelin resolved into eight bands in the molecular weight range 26.46–215.41 kDa (Fig. 1(a), lane 8). Under reducing conditions, Mateera glutelin resolved into five bands in the molecular weight range 15.63–81.27 kDa (Fig. 1(b), lane 7), while Sugar Baby glutelin resolved into four bands in the molecular weight range 20.17–59.53 kDa (Fig. 1(b), lane 8). This study showed that the four fractions of watermelon seed proteins comprised different type of peptides with varying content and molecular weight. It also revealed that the proteins were composed of low-molecular-weight subunits that separated under reducing conditions. Isoelectric focusing Analytical isoelectric focusing allows high resolution of protein mixtures with charge microheterogeneity. Each protein was focused into a number of bands of varying intensity with a range of isoelectric points (pI). Except for albumin, most banding patterns were in the acidic range (pI 4–7). Mateera albumin showed a different banding pattern with intense bands in the pI range 8–9 accounting for 894.1 g kg−1 of the total protein measured by band densitometry (Fig. 1(c), lane 1), while Sugar Baby albumin showed major bands in the pI range 5–7 accounting for 666 g kg−1 of the total protein (Fig. 1(c), lane 2). Thus the albumin fractions of the two cultivars showed different banding patterns. Mateera globulin resolved into 11 subunits in the pI range 5–9 with intense bands in the pI range 5–7 (Fig. 1(c), lane 3), while Sugar Baby globulin resolved into 12 subunits in the pI range 5–9 with intense bands in the pI range 5–7 (Fig. 1(c), lane 4). This indicated that the isoelectric points of globulins in the two cultivars were quite similar, with c 2010 Society of Chemical Industry J Sci Food Agric 2011; 91: 113–121 Characterisation and functional properties of watermelon seed proteins Amino acid analysis The amino acid composition of the watermelon seed protein fractions is presented in Table 2. Hydrophobic and acidic amino acids dominated the amino acid composition of the protein fractions of both cultivars. Arginine was the predominant amino acid in the Mateera protein fractions, while glutamic acid was the predominant amino acid in the Sugar Baby protein fractions. Sugar Baby proteins showed significantly (P ≤ 0.05) higher levels of glutamic acid (191.5–228.3 g kg−1 ) than Mateera proteins (147.1–155.0 g kg−1 ). The other major amino acids in the protein fractions were aspartic acid and leucine. Glycine, valine, phenylalanine and serine were also found in considerable amounts in the protein fractions of both cultivars. Significant differences among protein fractions were found in both cultivars. King and Onuora26 reported the presence of high concentrations of arginine (130–152 g kg−1 ), aspartic acid (70–80 g kg−1 ) and glutamic acid (147–167 g kg−1 ) in different melon seeds. In a study on seed meal proteins of several cucurbits, Jacks et al.8 also observed an abundance of these amino acids. Significant quantitative variation has been reported in the amino acid profiles of Cucumis melo seeds of different origin and variety.29 Lysine and methionine were the most limiting amino acids, followed by tryptophan and threonine, when compared with the recommended amino acid pattern for a weaned child between 2 and 5 years old.30 Lysine and threonine in fluted pumpkin (Telfaria occidentails) are limiting amino acids based on amino acid scores.31 The low level of lysine agrees with a previous report on melon seed protein.26 The ratio of essential to total amino acids (E/T) ranged from 38.19 to 41.60% and from 35.83 to 40.31% in the protein fractions of Mateera and Sugar Baby respectively, while the amino acid score ranged from 74.34 to 95.41 and from 72.40 to 88.98 respectively. Significant (P ≤ 0.05) differences among protein fractions were found for both cultivars. Akobundu et al.32 reported 98.8 g kg−1 arginine, 12.7 g kg−1 tryptophan, 14.2 g kg−1 methionine, 36.2 g kg−1 leucine, 22.0 g kg−1 isoleucine, 24.8 g kg−1 valine, 18.4 g kg−1 threonine, 32.2 g kg−1 phenylalanine and 17.5 g kg−1 tyrosine as the essential amino acids present in defatted hull-free melon (Colocynthis citrullus) flour. Similar results have been reported for fluted pumpkin,31 watermelon seed meal3 and melon seed meal.2 (a) (b) (c) Figure 1. Electrophoregram of Osborne protein fractions under unreduced (A) reduced (B) and isoelectric focusing conditions(C). Lane S represents standard proteins, lane 1 (Mateera albumin), lane 2 (Sugarbaby albumin), lane 3 (Mateera globulin), lane 4 (Sugarbaby globulin), lane 5 (Mateera prolamin), lane 6 (Sugarbaby prolamin), lane 7 (Mateera glutelin) and lane 8 (Sugarbaby glutelin). Thermal denaturation The effect of pH on protein denaturation was studied by differential scanning calorimetry (DSC) of the seed meal protein fractions of the two watermelon cultivars (Table 3). Characteristics associated with the chemical environment, such as pH, ionic strength, etc., can modify both the thermal stability and conformational structure of proteins. The results showed two major endothermic transitions with significant changes in onset (To ), peak (Tp ) and conclusion (Tc ) temperatures and enthalpy (H) of denaturation. Significant changes in thermal transitions of the two peaks were observed with changes in pH. Endothermic peak I of the watermelon protein fractions showed lower denaturation temperatures than endothermic peak II in selected pH ranges. With an increase in pH from 5 to 9, To decreased from 66.4 to 49.2 ◦ C, Tp from 96.8 to 56.2 ◦ C, Tc from 107.5 to 83.1 ◦ C and H from 4.2 to 2.8 J g−1 for endothermic peak I of Mateera albumin. Endothermic peak I of Sugar Baby albumin showed similar results for To , Tc and H, whereas Tp was significantly higher than that of Mateera albumin. With an increase in pH from 5 to 9, To decreased from 109.8 to 102.4 ◦ C, Tp from 123.5 to 105.0 ◦ C, Tc from 129.0 to 123.5 ◦ C and H from 9.2 to 7.1 J g−1 for endothermic peak II of Mateera c 2010 Society of Chemical Industry wileyonlinelibrary.com/jsfa 117 most of the banding pattern in the acidic range. Mateera prolamin resolved into six bands in the pI range 5–7 (Fig. 1(c), lane 5), while Sugar Baby prolamin resolved into eight bands in the pI range 5–9 (Fig. 1(c), lane 6). Mateera glutelin resolved into 12 bands with a complex banding pattern in the pI range 5–9 (Fig. 1(c), lane 7), while Sugar Baby glutelin resolved into 14 bands in the pI range 5–9 (Fig. 1(c), lane 8). Glutelins of both cultivars showed a faint banding pattern in the acidic pI region. J Sci Food Agric 2011; 91: 113–121 www.soci.org www.soci.org AA Wani et al. Table 2. Amino acid composition (g kg−1 ) of Osborne fractions of Mateera and Sugar Baby watermelon seed proteins (n = 4) Mateera Amino acid Essential Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophan Valine Non-essential Alanine Proline Tyrosine Arginine Glycine Cysteine Aspartic acid Histidine Serine Glutamic acid E/T (%) AAS (%) Albumin Globulin Sugar Baby Prolamin Glutelin 29.5c ± 0.04 28.3bc ± 0.04 21.2a ± 0.03 38.2f ± 0.04 72.0c ± 0.02 64.0a ± 0.03 67.7b ± 0.03 86.2f ± 0.03 58.8h ± 0.02 31.7c ± 0.04 51.5f ± 0.02 32.2cd ± 0.03 12.6bc ± 0.02 11.1ab ± 0.03 16.2d ± 0.04 17.7de ± 0.04 52.4d ± 0.04 42.0b ± 0.04 38.4a ± 0.03 53.6de ± 0.04 29.3bc ± 0.08 26.4a ± 0.05 30.5c ± 0.06 29.4bc ± 0.06 20.1abc ± 0.04 19.3ab ± 0.02 19.0a ± 0.05 21.2bcd ± 0.04 50.7bc ± 0.05 51.3c ± 0.04 49.2ab ± 0.06 49.5abc ± 0.05 47.1c ± 0.05 24.1b ± 0.04 35.6b ± 0.05 182.06d ± 0.04 74.6g ± 0.05 18.2d ± 0.04 71.9a ± 0.06 48.1cd ± 0.04 47.8c ± 0.06 152.3b ± 0.08 41.60 90.81 Albumin 31.7d ± 2.83 83.9e ± 0.04 20.9a ± 0.03 10.4a ± 0.01 56.5f ± 0.04 28.2b ± 0.04 22.4d ± 0.03 48.5a ± 0.05 Globulin Prolamin Glutelin 27.5b ± 0.04 28.7bc ± 0.04 36.0e ± 0.13 73.4cd ± 0.03 74.4d ± 0.03 88.4g ± 0.03 25.6b ± 0.04 47.1e ± 0.03 55.2g ± 0.04 10.8ab ± 0.03 14.3c ± 0.04 18.3e ± 0.04 44.7c ± 0.03 40.3b ± 0.03 54.7e ± 0.03 26.1a ± 0.06 30.2c ± 0.05 29.3bc ± 0.03 19.4ab ± 0.01 19.3a ± 0.02 21.6cd ± 0.02 49.2ab ± 0.04 47.9a ± 0.05 48.2ab ± 0.04 39.1a ± 0.05 48.9d ± 0.04 49.6d ± 0.04 50.8d ± 0.07 42.5b ± 0.03 19.0a ± 0.06 32.0e ± 0.04 30.6d ± 0.06 27.8c ± 0.04 20.1a ± 0.04 36.7bc ± 0.04 37.6c ± 0.04 30.6a ± 0.06 36.0bc ± 0.06 36.3bc ± 0.08 221.3j ± 0.06 171.3a ± 0.08 194.7h ± 0.06 185.9e ± 0.04 215.8i ± 0.06 50.0bc ± 0.06 57.9e ± 0.06 48.3a ± 0.04 52.0d ± 0.04 51.7d ± 0.06 18.9de ± 0.04 19.2e ± 0.03 10.5a ± 0.04 13.5b ± 0.04 17.3c ± 0.03 75.8b ± 0.08 87.5d ± 0.17 92.8e ± 0.10 76.1b ± 0.09 76.7b ± 0.06 46.0b ± 0.05 61.2f ± 0.05 46.3bc ± 0.06 43.2a ± 0.05 46.0b ± 0.05 47.9c ± 0.10 47.8c ± 0.07 47.9c ± 0.04 45.2b ± 0.06 44.2ab ± 0.04 155.0c ± 0.11 151.6b ± 0.05 147.1a ± 0.12 206.5e ± 0.08 228.3g ± 0.10 38.19 40.81 40.47 38.03 35.83 74.34 79.96 95.41 79.19 80.26 49.1d ± 0.06 36.2f ± 0.04 38.6d ± 0.04 174.0b ± 0.07 60.5f ± 0.05 17.2c ± 0.04 81.9c ± 0.08 59.0e ± 0.04 44.0ab ± 0.03 222.9f ± 0.05 38.22 72.40 FAO/ WHO pattern 40.0 70.4 54.4 22.0 28.0 40.0 9.6 49.6 49.2d ± 0.08 29.1cd ± 0.05 32.1a ± 0.05 187.3f ± 0.02 49.5ab ± 0.04 11.1a ± 0.03 103.2f ± 0.16 47.1bc ± 0.04 44.8ab ± 0.03 191.5d ± 0.01 40.31 88.98 Values are expressed as mean ± standard deviation. Means in the same row with different letters are significantly different at P < 0.05. Table 3. Effect of pH on thermal denaturation of Osborne fractions of Mateera and Sugar Baby watermelon seed proteins (n = 2) Endothermic peak I Cultivar Protein fraction Mateera Albumin Globulin Prolamin Glutelin Sugar Baby Albumin Globulin Prolamin Glutelin 118 wileyonlinelibrary.com/jsfa Endothermic peak II pH To (◦ C) Tp (◦ C) Tc (◦ C) H (J g−1 ) To (◦ C) Tp (◦ C) Tc (◦ C) H (J g−1 ) 5 7 9 5 7 9 5 7 9 5 7 9 66.4 52.6 49.2 64.4 61.1 55.2 63.1 51.6 47.1 80.4 54.2 69.2 96.8 58.7 56.2 69.5 62.2 59.1 74.4 62.3 57.0 92.6 95.8 81.1 107.5 96.3 83.1 76.0 68.7 64.3 84.5 65.3 66.2 96.2 99.4 85.6 4.2 3.1 2.8 3.4 3.1 3.1 3.2 2.8 2.3 4.1 3.5 3.1 109.8 101.4 102.4 147.2 109.5 96.3 111.3 92.4 83.8 113.1 105.3 101.0 123.5 118.4 105.0 166.4 131.5 129.5 123.9 118.5 101.7 129.2 113.1 108.0 129.0 127.7 123.5 169.9 158.7 151.8 149.5 145.6 123.3 133.1 118.8 113.8 9.2 8.2 7.1 13.4 10.3 8.2 10.3 10.2 8.8 9.1 7.5 7.2 5 7 9 5 7 9 5 7 9 5 7 9 67.8 60.3 48.6 102.4 60.6 52.7 68.2 53.2 47.2 90.9 65.8 56.4 100.2 83.7 73.0 105.1 99.6 58.4 79.7 65.8 59.5 107.0 79.2 71.3 107.2 91.2 79.9 117.9 101.9 65.8 92.6 69.2 66.8 111.4 85.2 81.0 4.4 3.1 3.0 4.8 3.6 3.0 3.4 3.2 2.7 4.1 3.6 3.1 146.0 109.1 102.3 133.2 119.3 106.1 116.2 92.3 102.4 131.6 110.6 100.5 179.6 119.5 114.7 159.9 133.8 113.0 132.3 122.8 105.5 155.6 122.4 112.6 182.9 124.5 119.2 172.7 137.7 116.5 146.8 141.7 127.9 159.4 133.1 120.1 14.1 10.1 6.2 13.7 11.1 10.1 12.1 10.2 9.0 14.1 9.8 8.2 c 2010 Society of Chemical Industry J Sci Food Agric 2011; 91: 113–121 Characterisation and functional properties of watermelon seed proteins J Sci Food Agric 2011; 91: 113–121 Foam volume (mL) 80 60 40 20 0 0 5 0 5 10 Time (h) 15 20 (b) 100 80 60 40 20 0 10 Time (h) 15 20 Figure 2. Foaming capacity and foam stability of Osborne fractions of (a) Mateera and (b) Sugar Baby watermelon seed proteins: , albumin; , globulin; , prolamin; ◦, glutelin. (57.4–90.8 mL) was significantly (P ≤ 0.05) higher than that of the Mateera protein fractions (50.3–88.4 mL). Albumin showed the highest foaming capacity for both cultivars, followed by globulin, glutelin and prolamin. Foam stability, measured as the decrease in foam volume over time, was highest for the albumin fraction of both cultivars, followed by the globulin, prolamin and glutelin fractions. The foam volume of Mateera albumin decreased sharply up to 1 h, after which a gradual decrease was observed. Sugar Baby albumin showed significantly (P ≤ 0.05) higher foam stability than Mateera albumin. The differences in foam stability may be due to differences in the protein structure of the two cultivars. The foam stability of globulin and prolamin showed a similar trend. Glutelin from both cultivars showed the lowest foam stability. Emulsifying properties of the Mateera and Sugar Baby watermelon protein fractions are shown in Figs 3(a) and 3(b) respectively. Emulsifying capacity varied significantly among different protein types of the two cultivars. Globulin showed the highest values of emulsifying capacity, followed by albumin, glutelin and prolamin. Emulsion stability, measured as the decrease in absorbance over time, followed a different trend to emulsifying activity. Globulin showed the lowest stability among all protein types, since the decrease in its emulsifying capacity per unit time was higher. The results further revealed that emulsifying capacity c 2010 Society of Chemical Industry wileyonlinelibrary.com/jsfa 119 Functional properties Functional properties of the watermelon seed protein fractions are presented in Table 1. Albumin showed the highest dispersibility (810.3–869.6 g kg−1 ), followed by globulin (227.8–245.4 g kg−1 ), glutelin (182.1–187.7 g kg−1 ) and prolamin (162.3–177.7 g kg−1 ). The high dispersibility of albumin is attributed to its higher content of hydrophilic groups. Water absorption capacity (WAC) of the protein fractions varied in the range 3.7–3.9 g g−1 . Nonsignificant changes in WAC were observed for different protein fractions in this study. The albumin fraction was completely dissolved and its WAC could not be measured. Oil absorption capacity (OAC) was in the range 2.1–2.8 g g−1 for the protein fractions of both cultivars. The prolamin fraction showed significantly (P ≤ 0.05) lower OAC, while glutelin had a high affinity for oil. This might be due to the varying presence of hydrophobic groups on the surfaces of protein molecules. Bulk density of the protein fractions varied from 310 to 630 g L−1 . Non-significant differences in bulk density were found for the albumin and prolamin fractions of the two cultivars. Total colour difference (E) ranged from 24.0 to 35.6 for the Mateera protein fractions and from 21.2 to 33.1 for the Sugar Baby protein fractions. Albumin showed the highest E and prolamin the lowest E among the protein fractions for both cultivars. Surface hydrophobicity (So ) ranged from 126.4 to 173.2 and from 125.8 to 169.3 for the Mateera and Sugar Baby protein fractions respectively. Albumin showed significantly (P ≤ 0.05) lower So than prolamin, globulin and glutelin for both cultivars. So values in the range 14–426 have been reported for various food proteins.34 The significant changes in So of the samples are due to the varying presence of hydrophobic amino acids on the surfaces of protein molecules. Foaming properties of the Mateera and Sugar Baby watermelon protein fractions are shown in Figs 2(a) and 2(b) respectively. Foaming capacity of the Sugar Baby protein fractions (a) 100 Foam volume (mL) albumin. Endothermic peak II of Sugar Baby albumin showed higher To , Tp , Tc and H at pH 5. However, the DSC thermograms recorded at pH 7 and 9 for Sugar Baby albumin showed similar trends and values to those for Mateera albumin. The DSC thermograms for Mateera globulin also showed two endothermic peaks, with peak I being minor and peak II being major. An increase in pH (from 5 to 9) resulted in decreases in To (from 64.4 to 55.2 ◦ C), Tp (from 69.5 to 59.1 ◦ C), Tc (from 76.0 to 64.3 ◦ C) and H (from 3.4 to 3.1 J g−1 ) for endothermic peak I as well as decreases in To (from 147.2 to 96.3 ◦ C), Tp (from 166.4 to 129.5 ◦ C), Tc (from 169.9 to 151.8 ◦ C) and H (from 13.4 to 8.2 J g−1 ) for endothermic peak II. In the case of Sugar Baby globulin, endothermic peak I had higher To , Tp , Tc and H at pH 5 and 7, whereas at pH 9 these parameters were similar to those of Mateera globulin. The thermal denaturation of watermelon globulin was within the range reported for globulins isolated from melon (C. citrullus Linn.) seed33 and red bean.19 The prolamin fraction showed a similar trend to the albumin and globulin fractions for both cultivars. With an increase in pH from 5 to 7, To , Tp , Tc and H decreased. Sugar Baby glutelin showed higher values of To , Tp , Tc and H for both endothermic peaks I and II at pH 5 than at pH 7 and 9. Endothermic peaks I and II of Mateera glutelin showed lower values at pH 5 but higher values at pH 9 compared with Sugar Baby glutelin. The higher temperatures and enthalpy of denaturation at isoelectric pH 5 were due to repulsive forces inhibiting the unfolding of protein molecules. www.soci.org www.soci.org ACKNOWLEDGEMENTS (a) 0.45 This research work was financially supported by Guru Nanak Dev University, Amritsar. We also acknowledge Professor Subodh Kumar (Department of Chemistry) for allowing us to use the spectrofluorometer for the measurement of protein surface hydrophobicity. Absorbance (500 nm) 0.4 0.35 0.3 0.25 REFERENCES 0.2 0.15 0.1 0.05 0 (b) 10 15 Time (min) 5 20 25 0.4 0.35 0.3 Absorbance (500 nm) AA Wani et al. 0.25 0.2 0.15 0.1 0.05 0 0 3 6 9 12 Time (min) 15 18 21 Figure 3. Emulsifying capacity and emulsion stability of Osborne fractions of (a) Mateera and (b) Sugar Baby watermelon seed proteins: (a) , albumin; , globulin; , prolamin; •, glutelin; (b) , albumin; , globulin; ♦, prolamin; , glutelin. was highest for globulin in both cultivars, followed by albumin. However, Mateera prolamin had higher emulsifying capacity than Mateera glutelin, while in Sugar Baby the opposite was the case. CONCLUSIONS 120 This study showed that different fractions of watermelon seed meal proteins had different properties. Globulin represented the major protein fraction, followed by glutelin and albumin. These proteins were mainly composed of low-molecular-weight polypeptides with minimum solubility in the pH range 4–6, as determined by pH solubility tests and confirmed by isoelectric studies. Amino acid analysis showed that they were rich in aspartic acid, glutamic acid and serine. Globulin had the highest resistance to thermal denaturation; however, an increase in pH led to a decrease in thermal stability irrespective of protein type. Measurements of functional properties of the protein fractions revealed that albumin had better functional properties than globulin, prolamin and glutelin. Overall, the results showed that watermelon seeds are a good source of high-quality proteins that could be extracted from the seed meal and utilised as functional ingredients in various food materials. wileyonlinelibrary.com/jsfa 1 Wani AA, Sogi DS, Grover L and Saxena DC, Effect of temperature, alkali concentration, mixing time and meal/solvent ratio on the extraction of watermelon seed proteins – a response surface approach. Biosyst Eng 94:67–73 (2006). 2 Mello MLS, Narain N and Bora PS, Characterisation of some nutritional constituents of melon (Cucumis melo hybrid AF-522) seeds. Food Chem 68:411–414 (2000). 3 El-Adawy TA and Taha KM, Characteristics and composition of different seed oils and flours. Food Chem 74:47–54 (2001). 4 Teotia MS and Ramakrishna P, Chemistry and technology of melon seeds. J Food Sci Technol 21:332–340 (1984). 5 Wani AA, Sogi DS, Shivhare US, Ahmed I and Kaur D, Moisture adsorption isotherms of watermelon seeds and kernels. Drying Technol 24:99–104 (2006). 6 Wani AA, Kaur D, Ahmed I and Sogi DS, Optimisation of watermelon seed protein using response surface methodology. LWT – Food Sci Technol 41:1514–1520 (2008). 7 Mabaleha MB, Mitei YC and Yeboah SO, A comparative study of the properties of selected melon seed oils as potential candidates for development into commercial edible vegetable oils. J Am Oil Chem Soc 84:31–36 (2007). 8 Jacks TJ, Hensarling TP and Vatsu LY, Cucurbit seeds: characterization and uses of oils and proteins. Econ Bot 26:135–141 (1972). 9 Ige MM, Ogunsua AO and Oke OL, Functional properties of the proteins of some Nigerian oilseeds: conophor seeds and three varieties of melon seeds. J Agric Food Chem 32:822–825 (1984). 10 Wang JC and Kinsella JE, Functional properties of novel proteins: alfalfa leaf protein. J Food Sci 41:286–289 (1976). 11 Kinsella JE, Functional properties of proteins in foods: a survey. CRC Crit Rev Food Sci Nutr 7:219–280 (1976). 12 Kaur D, Sogi DS, Garg SK and Bawa AS, Flotation-cum-sedimentation system for skin and seed separation from tomato pomace. J Food Eng 7:341–344 (2005). 13 Osborne TB, The Proteins of the Wheat Kernel (Publication No. 4). Carnegie Institute, Washington, DC (1907). 14 Sathe SK, Solubilisation and electrophoretic characterisation of cashew nut (Anacardium occidentale) proteins. Food Chem 51:319–324 (1994). 15 Laemmli UK, Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685 (1970). 16 Bollag DM, Rozycki MD and Edelstein SJ, Gel electrophoresis, in Protein Methods, ed. by Bollag DM, Rozycki MD and Edelstein SJ. World Scientific, Singapore, pp. 107–155 (1996). 17 Robertson EF, Dannelly HK, Malloy PJ and Reeves HC, Rapid isoelectric focusing in a vertical polyacrylamide minigel system. Anal Biochem 167:290–294 (1987). 18 Spies JR and Chambers DC, Chemical determination of tryptophan. Anal Chem 10:30–39 (1948). 19 Meng GT and Ma CY, Thermal properties of Phaseolus angularis (red bean) globulin. Food Chem 73:453–460 (2001). 20 Saunders RM, Connor MA, Booth AN, Bickoff EM and Kohler GO, Measurement of digestibility of alfalfa protein concentrates by in vitro and in vitro methods. J Nutr 103:530–535 (1973). 21 Paulson AT and Tung MA, Solubility, hydrophobicity and net charge of succinylated canola protein isolate. J Food Sci 52:1557–1561 (1987). 22 Pearce KN and Kinsella JE, Emulsifying properties of proteins: evaluation of a turbidimetric technique. J Agric Food Chem 26:716–723 (1978). 23 Krishnan PS and Krishnaswamy TK, Proteins and other nitrogenous constituents of watermelon seeds (Citrullus vulgaris). Biochem J 33:1284–1290 (1939). 24 Sze-Tao KWC and Sathe SK, Walnuts (Juglans regia L): proximate composition, protein solubility, protein amino acid composition, c 2010 Society of Chemical Industry J Sci Food Agric 2011; 91: 113–121 Characterisation and functional properties of watermelon seed proteins 25 26 27 28 29 and protein in vitro digestibility. J Sci Food Agric 80:1393–1401 (2000). Bhattacharya S, Bal S and Mukherjee RK, Functional and nutritional properties of tamarind kernel protein. Food Chem 49:1–9 (1994). King RD and Onuora JO, Aspects of melon seed protein characteristics. Food Chem 14:65–77 (1984). Vojdani F, Solubility, in Methods of Testing Protein Functionality, ed. by Hall GM. Blackie Academic and Professional, London, pp. 11–60 (1996). Sogi DS, Arora MS, Garg SK and Bawa AS, Fractionation and electrophoresis of tomato waste seed proteins. Food Chem 76:449–454 (2002). Mello MLS, Bora PS and Narain N, Fatty and amino acids composition of melon (Cucumis melo Var. saccharinus) seeds. J Food Compos Anal 14:69–74 (2001). www.soci.org 30 FAO, Amino Acid Contents of Foods and Biological Data on Proteins. Food and Agriculture Organisation, Rome (1970). 31 Longe OG, Farinu GO and Fetuga BL, Nutritional value of the fluted pumpkin (Telfaria occidentalis). J Agric Food Chem 31:989–992 (1983). 32 Akobundu ENT, Cherry JP and Simmon JC, Chemical, functional, and nutritional properties of egusi (Colocynthis citrullus L.) seed protein products J Food Sci 47:829–835 (1982). 33 Onuora JO and King RD, Thermal transitions of melon seed proteins. Food Chem 13:309–316 (1984). 34 Voutsinas S, Nakai S and Harwalker VR, Relationships between protein hydrophobicity and thermal functional properties of food proteins. Can Inst Food Sci Technol J 16:185–190 (1983). 121 J Sci Food Agric 2011; 91: 113–121 c 2010 Society of Chemical Industry wileyonlinelibrary.com/jsfa