Download Characterisation and functional properties of watermelon (Citrullus

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
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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
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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
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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.
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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
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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
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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.)
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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),
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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
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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
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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
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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
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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
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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.
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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
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0.1
0.05
0
0
3
6
9
12
Time (min)
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Figure 3. Emulsifying capacity and emulsion stability of Osborne fractions
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, globulin; , prolamin; •, glutelin; (b) , albumin; , globulin; ♦,
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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
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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.
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