Food Chemistry 128 (2011) 902–908

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Functional properties of protein fractions obtained from commercial yellow

field pea (Pisum sativum L.) seed protein isolate
Abayomi P. Adebiyi, Rotimi E. Aluko ⇑
Department of Human Nutritional Sciences and The Richardson Centre for Functional Foods and Nutraceuticals, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2

a r t i c l e i n f o a b s t r a c t

Article history: Commercial pea protein isolate was separated into water-soluble (WS), salt-soluble (SS), alkaline-soluble
Received 6 November 2010 (AS) and ethanol-soluble (ES) fractions. AS fraction was the most abundant, constituting about 87% of the
Received in revised form 26 February 2011 proteins in PPI followed by WS, SS and ES fractions in decreasing order. ES fraction consistently formed
Accepted 30 March 2011
emulsions with a narrow range of smaller oil droplet sizes (0.6–19 lm) at pH 4.0, 7.0 or 9.0 compared to a
Available online 3 April 2011
wider range of sizes for emulsions stabilised by WS, SS and AS fractions. Emulsions formed with ES frac-
tion were also the most stable (p < 0.05) over the 3 h test period at all the pH values used in this work. The
Keywords:
WS fraction had significantly highest (p < 0.05) protein solubility and foaming capacity at all the pH val-
Pea protein isolate
SDS–PAGE
ues when compared to solubility of PPI, SS, and ES. Except for AS and ES fractions, foaming capacities of
Functional properties the protein fractions were higher at pH 9.0 than at pH 4.0 or 7.0.
Water-soluble proteins Ó 2011 Elsevier Ltd. All rights reserved.
Salt-soluble proteins
Alkaline-soluble proteins
Alcohol-soluble proteins

1. Introduction McKenzie, & Shahidi, 2001). Although, pea is a widely consumed

legume seed, protein-based ingredients of pea are not yet well uti-
Legumes are the edible fruits or seeds of pod-bearing plants lised in food applications. This is probably due to limited informa-
belonging to the family Leguminosae and are widely cultivated tion on the structural and functional properties of pea proteins. For
throughout the world (Kaur, Sandhu, & Singh, 2007). Yellow field plant proteins to be useful and successful in food application they
pea is an important grain legume, both as human food and animal should ideally possess several desirable characteristics, referred to
feed (Koyoro & Powers, 1987; Sun & Arntfield, 2010). The increased as functional properties (Adebiyi et al., 2007; Ragab, Babiker, &
interest in plant proteins for feed and food led to the evaluation of Eltinay, 2004).
yellow field pea seeds (Pisum sativum L.) as one of the valued crops Proteins are often used as food ingredients for their functional
in the world market (Tian, Kyle, & Small, 1999) and Canada has be- properties and/or to impart certain specific characteristics to the fi-
come a world leader in field pea export over the past 10 years nal product. These properties are intrinsic physicochemical charac-
(Agboola, Mofolasayo, Watts, & Aluko, 2010; Wang, Daun, & Mal- teristics, which affect the behaviour of proteins in food system
colmson, 2003). Protein fractions are valuable constituents of the during processing, manufacturing, storage and preparation (Kinsel-
pea seed and they can be extracted together to give a heterogenous la, 1979). Therefore, the technological uses of pea proteins depend
product called pea protein isolate (PPI) or each protein can be iso- largely on the physicochemical properties which are necessary for
lated based on solubility characteristics in aqueous solutions. their successful incorporation into food systems.
Like other legume seeds, yellow field pea seed is characteristi- Previous reports have investigated the functional properties of
cally rich in proteins (18–30%) (Kaur et al., 2007; Shand, Ya, Pietr- plant protein fractions. These include soybean proteins (Kinsella,
asik, & Wanasundara, 2007; Wang et al., 2003) and contains high 1979); oat protein fractions (Ma & Harwalkar, 1984), salt-soluble
levels of lysine which can be used to balance the deficiencies of this (SS) fraction from pea seeds (Koyoro & Powers, 1987), water-solu-
essential amino acid in cereal-based diets (Chel-Guerrero, Scilingo, ble (WS) and SS fractions from tepary bean flour (Idouraine, Yen-
Tintore, Davila, & Anon, 2007; Tian et al., 1999). In addition to pro- sen, & Weber, 1991), WS fraction from pea seeds (Lu, Quillien, &
viding essential amino acids, the ultimate success of utilising any Popineau, 2000), SS fraction from red bean (Meng & Ma, 2002),
seed protein as a food ingredient depends largely on its functional WS and SS fractions from African locust bean (Lawal, Adebowale,
properties (Adebiyi, Adebiyi, Ogawa, & Muramoto, 2007; Chavan, Ogunsanwo, Sosanwo, & Bankole, 2005), alcohol-soluble (AS) frac-
tion from Australian rice (Agboola, Ng, & Mills, 2005), rice bran
⇑ Corresponding author. Tel.: +1 204 474 9555; fax: +1 204 474 7593. protein fractions (Adebiyi et al., 2007), rice endosperm protein
E-mail address: alukor@cc.umanitoba.ca (R.E. Aluko). fractions (Pinciroli, Vidal, Anon, & Martinez, 2009), as well as WS,

0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2011.03.116
 A.P. Adebiyi, R.E. Aluko / Food Chemistry 128 (2011) 902–908 903

SS and AS fractions from wheat germ (Tomoskozi, Lasztity, Sule, 2.3. Sodium dodecyl sulphate polyacrylamide gel electrophoresis
Gaugecz, & Varga, 1998). These efforts were aimed at effective util- (SDS–PAGE)
isation of inexpensive protein materials for nutritional and func-
tional purposes. Reducing (with mercaptoethanol) and non-reducing (without
In the formulation and development of traditional and novel mercaptoethanol) SDS–PAGE were performed on 8–25% gradient
foods, emulsification and foaming are two most important func- gels using the PhastSystem Separation and Control and Develop-
tionalities of proteins and other amphoteric molecules (Aluko, ment Units according to the manufacturer’s instructions (GE
Mofolasayo, & Watts, 2009; Rangel, Domont, Pedrosa, & Ferreira, Healthcare, Montreal, PQ) as previously described (Aluko, Yada,
2003). Many formulated foods come as foams or emulsions, thus Lencki, & Marangoni, 1997). Gels images were acquired with Lab-
proteins having good surface properties and solubility are desir- scan on ImageScanner (GE Healthcare) and the approximate
able as food ingredients. Changes in pH often occur during the pro- molecular weights and proportions of the proteins in each lane
cessing of foods; therefore it is important to study the effects of were analysed using the ImageQuant TL software program.
this factor on the functional properties of food proteins. Thus,
knowledge of the functional properties of a particular pea protein 2.4. Emulsion formation
fraction is an important step towards their evaluation and utilisa-
tion. In earlier work (Aluko et al., 2009), the emulsifying and foam- Oil-in-water emulsions were prepared as previously described
ing properties of commercial yellow pea seed flours was by Aluko, McIntosh, and Reaney (2001) with modifications. Aque-
investigated. While previous works have reported the functional ous dispersions of the PPI and PPFs were prepared in 0.1 M sodium
properties of PPI (O’Kane, Vereijken, Gruppen, & van Boekel, phosphate solutions, pH 4.0, 7.0 and 9.0 such that the final protein
2005; Shand et al., 2007), to date there is little information on concentration for each was 1% (w/v). Emulsions were prepared by
the relative functional attributes of pea protein isolate fractions adding 1 ml of pure commercial canola oil to 5 ml of the protein
(PPFs). solution followed by homogenisation for 1 min using a Polytron
Therefore, the objective of this work was to separate commer- PT 10-35 homogenizer (Kinematica AG, Lucerne, Switzerland)
cial PPI into different protein fractions based on their solubility equipped with a 12-mm non-foaming generator. The mean oil
in water, NaCl solution, aqueous ethanol, and dilute NaOH solution. droplet size (d3,2) of the emulsions was determined in a Mastersiz-
The PPFs were then compared to PPI in terms of functional proper- er 2000 (Malvern instruments Ltd., Malvern, UK) with Milli-Q
ties at different pH values. water as dispersant. Emulsion sample was added (under constant
shearing) to about 100 ml of water contained in the small volume
wet sample dispersion unit (Hydro 2000) attached to the
2. Materials and methods
instrument until the required level of obscuration was attained.
The instrument was set to measure each emulsion in triplicate
2.1. Materials
and to calculate the mean value. Emulsion stability was deter-
mined by measuring the d4,3 values (volume weighted mean) of
Commercial PPI (a gift from Nutri-Pea Ltd., Portage la Prairie,
the emulsion at hourly intervals for a period of 3 h after emulsion
Manitoba, Canada) was produced using a proprietary method
formation.
(Nickel, 1981). Briefly, the peas were dehulled and ground into
flour, which was passed through a screen to separate the coarse
2.5. Foam formation
fibre particles. The flow-through flour was then extracted with
NaOH solution, centrifuged and the supernatant adjusted to pH
Slurries were prepared in 10 ml of 0.01 M phosphate buffer pH
4.5 (with HCl solution) to precipitate proteins, which were recov-
4.0, 7.0, or 9.0 followed by homogenisation at 20,000 rpm for 1 min
ered by centrifugation, washed with water and then spray-dried.
using 20 mm foaming generator on the Polytron PT 3100 homoge-
The dried powder contains approximately 80% protein content
nizer (Kinematica AG, Lucerne, Switzerland). The foam was formed
(as is basis) and is referred to as PPI.
in a 50 ml graduated centrifuge tube, which enabled determination
of foam volume (ml). The volume of foam remaining after 30 min
2.2. Preparation of PPFs at room temperature was expressed as a percent value of original
foam volume to obtain foam stability.
Functional properties of food proteins are dependent on their
environment and therefore, the use of various aqueous extraction 2.6. Solubility
solutions would yield fractions that differ in protein functionality.
The procedure was similar to that described by Adebiyi, Adebiyi, Protein solubility was determined by the method of Aluko and
Hasegawa, Ogawa, and Muramoto (2009) for rice bran protein frac- Yada (1997). A 1% (w/v) solution of the PPI, albumin, globulin
tions. Modifications were made in the sequential extraction steps and glutelin in 0.01 M phosphate buffer. Then, the pH of the solu-
due to high protein content of PPI (80%) compared to that of rice tions was carefully adjusted from pH 3 to 8 with either 1 M HCl or
bran (15%). In addition, ES fraction was obtained by dialysis of eth- 1 M NaOH. After 30 min of stirring (with a magnetic stirrer at room
anol soluble extract instead of precipitating the extract with ace- temperature), the pH of the solutions was again measured and cen-
tone. In brief, sequential extraction of WS and SS fractions from trifuged at 10,000g for 20 min. For total soluble protein content
PPI were carried out by using 2% NaCl, followed by extraction with (control), the samples were dispersed in 0.1 M NaOH, to solubilise
70% ethanol to obtain ES fraction. The AS fraction was extracted the protein, and also centrifuged. The protein content in the super-
from the residue by using 0.05 M NaOH. All the extracts were natants was then determined Markwell, Haas, Biebar, and Tolbert,
extensively dialysed against water at 4 °C. The WS and SS fractions 1987) using bovine serum albumin (BSA) as the standard. The ES
were separated from the 2% NaCl extract by centrifugation after fraction was not analyzed due to complete insolubility in aqueous
dialysis, with the supernatant and sediment collected, respectively solutions.
(Fig. 2). All protein extracts were centrifuged at 5600g for 30 min, Protein solubility (PS %) was calculated as:
freeze-dried separately and stored at 20 °C until required for
analysis. Protein content was determined using the modified % protein content of sample
 100%
Lowry method of Markwell, Haas, Biebar, and Tolbert, 1987. % protein content of control
904 A.P. Adebiyi, R.E. Aluko / Food Chemistry 128 (2011) 902–908

2.7. Least gelation concentration (LGC) 2.8. Statistical analysis

This was determined by preparing 10 ml dispersions between Each determination was performed in triplicate and the data
1% and 20% (w/v) solids concentration in test tubes. The dispersion subjected to analysis variance and Duncan’s multiple range tests
was thoroughly mixed on a vortex mixer for 5 min and then heated to determine significant differences (p < 0.05) between mean val-
in a boiling water bath for 1 h. The mixture was cooled in a cold ues within each group using the Statistical Analysis Systems
room at 4 °C for 2 h after which the tube was inverted. The lowest (SAS) desktop software, version 9.1.
concentration at which the sample did not fall down or slip from
an inverted tube was taken as the LGC (Lawal et al., 2005). The 3. Results and discussion
ES fraction was not analyzed due to complete insolubility in aque-
ous solutions. 3.1. SDS–PAGE

PPI and PPFs molecular weight profiles were analysed by SDS–

PAGE with and without reduction of the protein disulfide bonds
(Fig. 1). As observed in the non reducing patterns, PPI had nine
polypeptides (68, 47, 35, 29, 25, 22, 19, 15 and 12 kDa) with the
35 kDa band in the highest proportion. The WS and SS fractions
have slightly different polypeptide composition profiles. The WS
fraction appear as smears of three bands around the 21, 15 and
11 kDa regions while the SS fraction had six bands at 43, 25, 21,
18, 12 and 10 kDa with the 25 kDa in the highest proportion. The
AS fraction had seven bands at 66, 45, 35, 28, 21, 18, and 12 kDa
which were similar to the polypeptide composition of the PPI.
The ES fraction was not properly separated into distinguishable
bands, probably as a result of limited solubility in the electropho-
resis reagent. Under the reducing condition, PPI, AS and WS pro-
teins had similar peptide compositions as the non-reducing gel,
which suggests minimal concentrations on disulfide bonds in the
proteins. The polypeptide composition of the PPI under reducing
conditions is similar to the results of Shand et al. (2007).

3.2. Yield of protein fractions

Fig. 1. SDS–PAGE profiles of pea protein isolate (PPI) and pea protein fractions (PPF)
in the absence and presence of 2-mercaptoethanol. Lane M indicates standard
Table 1 shows the relative proportions of PPFs fractionated
protein marker, lane 1: PPI, lane 2: water soluble (WS) fraction, lane 3: salt soluble according to their solubility in different solvent systems, which
(SS) fraction, lane 4: alkaline soluble (AS) fraction, lane 5: ethanol soluble (ES) has been illustrated in Fig. 2. Yield is reported as gross weight
fraction. and was obtained as the percentage ratio of the freeze-dried

Fig. 2. Scheme of sequential extraction of pea protein fractions (PPF) from pea protein isolate (PPI). RT = room temperature.
 A.P. Adebiyi, R.E. Aluko / Food Chemistry 128 (2011) 902–908 905

Table 1 The data showed that the WS fraction was almost completely sol-
Gross yield and protein content of pea protein fractions isolated from pea protein uble over a wide pH range. This is similar to the solubility observed
isolate (PPI).a
in WS fraction from oat (Ma & Harwalkar, 1984) and sunflower
Protein fraction Yield per 100 g PPI Protein content (%) (Gonzalez-Perez, Vereijken, Van Koningsveld, Gruppen, & Voragen,
WS 7.01 ± 0.74b 86.26 ± 3.94a 2005). The AS fraction showed the typical bell-shaped curve with
SS 2.47 ± 0.25c 80.44 ± 3.00a minimum solubility at pH 5.0. PPI exhibited low solubility (below
AS 87.47 ± 3.40a 79.03 ± 4.54a 30%) with the highest at pH 8.0. This is similar to the reported
ES 1.52 ± 0.12c 57.72 ± 2.43b
nitrogen solubility index obtained by Shand et al. (2007) who also
a
Mean values with different letters within the same column are significantly worked on commercial PPI. The reason for the low solubility even
different (p < 0.05): WS, water soluble; SS, salt soluble; AS, alkaline soluble; ES, at alkaline pH is not very clear but may be due to formation of
ethanol soluble.
insoluble protein complexes during isoelectric protein precipita-
tion. It should also be noted that the PPI contained ES fraction,
protein fraction to the original weight of PPI (Sathe & Venkatacha- which are completely insoluble in aqueous solutions and would
lam, 2007). The AS fraction was the predominant protein fraction have reduced the overall solubility of the protein isolate. Thus,
(87.5%) followed by WS fraction (7.0%), SS fraction (2.47%) and ES the PPFs had better solubility properties than PPI at all the pH
fraction (1.52%). Unlike most legume seed proteins, the major pro- examined. Except for the WS fraction, PPI, SS and AS fractions
teins of PPI are AS fraction rather than the SS fraction (Chel-Guer- exhibited reduced solubility in the pH range from 4.0 to 5.0, which
rero et al., 2007; Meng & Ma, 2002; Sathe & Venkatachalam, 2007) is similar to results reported for other legume proteins (Chau, Che-
or WS fraction (Ragab et al., 2004; Tjahjadi, Lin, & Breene, 1988). ung, & Wong, 1997; Chavan et al., 2001; Rangel et al., 2003). The
This observation may be due to the possibility of the yield being af- solubility of SS fraction was significantly higher at the alkaline
fected by the original PPI preparation method. The low contents of pH than acidic pH, which may be due to increase net protein
WS and SS fractions may be due to the fact that the protein raw charge as pH was increased. It is important to note that compared
material (PPI) was produced from isoelectric precipitation at pH to PPI, all the PPFs examined showed higher solubilities at pH P 7,
4.5 (intense protein–protein interactions), which could have re- indicating that they could be easily incorporated into products that
duced the number of water and/or salt soluble proteins. However, have neutral or basic pH values. Possible uses of the WS fraction in-
the fact that WS proteins could still be separated from PPI indicates clude formulation of acidic drinks, desserts (Rangel et al., 2003),
that some of the precipitated proteins possessed hydrophilic prop- non-acidic beverages and other liquid beverages (Meng & Ma,
erties that enhanced their interactions with water. Table 1 also 2002).
shows the protein content of the protein fractions and the WS frac-
tion had the highest value (86.3%) while ES fraction had the signif- 3.4. Least gelation concentration
icantly lowest (p < 0.05) value of 57.7%. The relatively low protein
content of ES fraction may be due to a combination of low solubil- Gel formation by globular proteins is a complex process that of-
ity in the NaOH solution that was used in the Lowry assay and also ten involves several reactions such as denaturation, aggregation
the presence of non-protein components such as polyphenols that and network formation (Gonzalez-Perez & Vereijken, 2007). A
may have been extracted by the alcoholic solution used during low value of LGC is an indication of better gelling ability of the pro-
fractionation. tein ingredient because small amount is required (Kaur et al.,
2007). Gel-forming ability and viscoelastic properties of proteins
3.3. Protein solubility (PS) largely depend on modes of interaction and bonding such as
hydrogen and covalent bonds as well as electrostatic and hydro-
Solubility is an important prerequisite for a protein in order to phobic interactions. The ES fraction was not evaluated due to com-
be useful as a functional ingredient in foods (Kinsela, 1981). It plete insolubility in water. The WS and SS fractions were unable to
may confer an advantage of ease of addition and ease of uniform form a firm gel but AS fraction formed a firm gel at the LGC of 10%
distribution of proteins within a food product. Solubility is a phys- compared to that of 20% for PPI. Agboola et al. (2010) similarly re-
icochemical property of a protein that critically affects texture, col- ported that PPI produced a paste rather than a cohesive gel, an
our and sensory properties of products (Meng & Ma, 2002), indication that the intensity of inter-molecular interactions was
including emulsifying, foaming and gel forming properties (Shand not strong enough to overcome repulsive forces. The results con-
et al., 2007). In many protein-based formulations such as emul- firm that gelation is not only a function of protein quantity but also
sions, foams and gels, good protein solubility is usually required. related to the type of protein(s), the non-protein components and
The PS profiles of PPI and PPFs (except ES fraction, which is insol- solubility (Ragab et al., 2004; Sathe & Salunkhe, 1981). In their
uble in aqueous solutions) as a function of pH are shown in Fig. 3. study, Sathe and Salunkhe (1981) similarly observed that SS frac-
tion from great northern bean did not form a firm gel up to 20%
(w/v) concentration range. Sun and Arntfield (2010) reported that
100
the LGC of commercial PPI was 14.5%. However, in our experiment
80 we observed gelation of PPI only at 20%. The discrepancy may be
PPI accounted for by the differences in the preparation method. Sun
Solubility (%)

WS
60 and Arntfield (2010) dissolved the PPI in 0.3 M NaCl while we used
SS
distilled water to prepare our protein solution. Probably, the pres-
AS
40 ence of NaCl favoured gel formation by increasing the intermolec-
ular hydrophobic interactions, reducing electrostatic repulsion and
20 alteration of water structure around the protein thus enhancing
hydration of the protein molecules in addition to network forma-
0 tion (Shand et al., 2007). The fact that only AS fraction formed a
2 3 4 5 6 7 8 9
gel among the PPF examined indicate that AS fraction may be
pH
responsible for the gel forming property of PPI. This is not surpris-
Fig. 3. Protein solubility profiles of pea protein isolate (PPI) and pea protein ing since AS fraction constitutes the majority of the proteins in PPI
fractions (PPF) as a function of pH. as shown in Table 1. Hence, AS fraction of PPI may not find
906 A.P. Adebiyi, R.E. Aluko / Food Chemistry 128 (2011) 902–908

application in the food industry as a gelling agent due to its high interact with the lipid phase and reduce surface tension. The pres-
LGC, especially in the formation of salt-free gelled products. ence of hydrophobic non-protein components that enhance oil
droplet formation may also have contributed to the observed
emulsion quality of the ES fraction. Also important is that changes
3.5. Effect of pH on oil droplet size distributions of PPI and PPFs
in pH did not have any substantial effect on the oil droplet size dis-
tribution of emulsions stabilised by ES fraction, which indicates
Oil droplet size distributions in the emulsions at different pH
that the protein probably has similar structural conformation at
values are shown in Fig. 4. With the exception of the ES fraction,
pH 4.0, 7.0, and 9.0. In contrast, the emulsions stabilised by PPI,
all the emulsions showed bimodal particle size distribution that re-
AS, WS and SS factions had different oil droplet distribution pat-
flects the inefficiency of the proteins in producing uniform oil
terns at pH 4.0 in comparison to pH 7.0 and 9.0.
droplets during homogenisation. In particular, at all the pH values
examined, emulsions stabilised by ES fraction had the narrowest
range of oil droplet size distributions. Therefore, ES fraction is a 3.6. Effect of pH on emulsion mean oil droplet size
better emulsifier probably as a result of its hydrophobic character
that enhanced interactions with the lipid phase. Aluko et al. (2009) The effects of pH on oil droplet size of emulsions stabilised by
reported that increased protein content of pea flours contributed to PPI and PPFs are shown in Fig. 5. At all pH values, the ES fraction
lowering of surface tension thereby enhancing formation of emul- had the lowest emulsion oil droplet size (d3,2 values), with no sig-
sions containing small oil droplet coalescence. This is contrary to nificant difference at pH 4.0 and 9.0. However, in terms of its use as
the present observation because ES fraction had the lowest protein functional food ingredient, the low yield of ES fraction is a disad-
content but formed emulsions with a narrower range of oil drop- vantage. PPI and AS fractions had better emulsification (reduced
lets size distribution. The results indicate that it is not just the pro- oil droplet size) at neutral and alkaline condition when compared
tein content that is important to the formation of high quality to pH 4.0, which reflects the solubility pattern at pH 7.0. It is pos-
emulsions but the quality is also important in terms of ability to sible that the proteins in PPI, SS, and AS fractions have a more
folded structure at pH 4.0 which limits its ability to encapsulate
oil droplets efficiently when compared to a probably more open
12 structure at pH 7.0. This is supported by the fact that the WS frac-
pH 4.0 tion that is highly soluble at pH 4.0 formed a better emulsion than
10
PPI, SS and AS fractions. The emulsifying property of WS fraction
Volume (%)

8 PPI also did not change substantially with variation in pH values,

AS which is also consistent with the fact that the proteins had similar
6
SS solubility values at acidic, neutral and alkaline pH range.
4 WS
ES
2 3.7. Effect of pH on emulsion stability

0 Generally, the term ‘‘emulsion stability’’ indicates the ability of

0.01 0.1 1 10 100 1000
an emulsion to resist changes in its physicochemical properties
Oil droplet size (µm) over time (McClements, 2007). Emulsifying stability was measured
in kinetic modes using d4,3 (volume weighted mean) as an indica-
12
tor of long term stability of emulsion. Fig. 6 shows the effect of pH
10 pH 7.0 on emulsion stability over a period of 3 h. Our data showed that
emulsions stabilised by ES, WS and SS fractions were very stable
Volume (%)

8 PPI at pH 4.0, in comparison to PPI- and AS fraction-stabilised emul-

6 AS sions. However, at pH 7.0 and 9.0 the SS fraction-stabilised emul-
SS sions showed time-dependent loss of stability as indicated by
4 WS increases in oil droplet size from 1 to 3 h. The loss of emulsion sta-
ES bility could indicate that as pH was increased, there was an in-
2
crease in net charges on the SS fraction proteins, which disrupted
0 the cohesiveness of the interfacial protein films and resulted in a
0.01 0.1 1 10 100 1000 higher rate of oil coalescence. In contrast, the PPI- and AS frac-
Oil droplet size ( µ m)

12 a
Oil droplet size-d 3,2 (µm)

50 b
10 pH 9.0 PPI
40
WS
Volume (%)

8 PPI
30 SS
c
6 AS d AS
ef de de f
SS 20 h h g ES
i
4 WS j
k j
10
2 ES
0
0 4.0 7.0 9.0
0.01 0.1 1 10 100 1000
pH
Oil droplet size (µ m)
Fig. 5. Effect of pH on the emulsifying capacity (oil droplet size) of pea protein
Fig. 4. Oil droplet size distribution of emulsions stabilised by pea protein isolate isolate (PPI) and pea protein fractions (PPFs) at pH 4.0, 7.0 and 9.0. Bars with
(PPI) and pea protein fractions (PPF) at pH 4.0, 7.0 and 9.0. different letters have mean values that are significantly different (p < 0.05).
 A.P. Adebiyi, R.E. Aluko / Food Chemistry 128 (2011) 902–908 907
D[4,3] Volume weighted mean

17.5 a
A PPI
250 15.0
b b b WS
a pH 4.0 0h

Foam volume (ml)

12.5 b
200 1h cc c SS
c cc cd cd
2h 10.0 de AS
150 b e ES
c
3h
de cd 7.5
ef
100 f
5.0
50 hg hg g 2.5
hi hg hg hg hg hg
i i i i
0 0.0
PPI WS SS AS ES 4.0 7.0 9.0
pH
D[4,3] Volume weighted mean

100
a
b b B b b PPI
60 b b b

Foam stability (%)

80 c WS
a 0h
50 d SS
1h d
pH 7.0 b b 60 e AS
40 c c 2h e
f ES
3h
30 ddd d d 40 g
e e
f f ff
20 20
g g g g
10 0
4.0 7.0 9.0
0
PPI WS SS AS ES
pH

Fig. 7. Effect of pH on the foaming ability (A) and foam stability (B) of pea protein
D[4,3] Volume weighted mean

isolate (PPI) and pea protein fractions (PPF). Bars with different letters have mean
50
a a values that are significantly different (p < 0.05).
pH 9.0 a 0h
40 b 1h
b
c 2h
d ed d to increased charge density that prevented rapid coalescence of
30 ed 3h
ef gf the air bubbles. The increase in charge density could have stabi-
gh gh
20 h h lized the foams by increasing electrostatic repulsions which re-
i duced the rate of coalescence of foam particles.
i i i
10
4. Conclusions
0
PPI WS SS AS ES
The results from this work have shown that protein type may be
Fig. 6. Effect of pH on the emulsion stability (changes in d4,3) of pea protein isolate as important as protein content in contributing to high emulsion
(PPI) and pea protein fractions (PPF) over a period of 3 h. Bars with different letters quality. The superior emulsion forming ability of the hydrophobic
have mean values that are significantly different (p < 0.05).
ES fraction supports previous reports that indicate increased inter-
actions with the lipid phase contributes to enhanced oil droplet
formation. Increased solubility in aqueous solutions as shown by
tion-stabilised emulsions showed increased stability at pH 7.0 and
the WS fraction contributed to better foaming ability. The presence
9.0, probably due to increased cohesiveness of the interfacial
of non-AS fractions may be responsible for the poor gel forming
proteins.
ability of PPI, since AS fractions alone had excellent gelation ability.
However, it should be noted that the observed functional proper-
3.8. Effect of pH on foam formation and stability ties may be dictated by synergistic interactions between constitu-
ent proteins because the fractions are not homogenous.
Foaming capacity and stability was greatly influenced by pH as
shown in Fig. 7. One of the requirements in the manufacture of Acknowledgements
foods such as ice cream, cakes and meringues is foam formation.
Compared to PPI, ES, AS and SS fractions, foaming capacity of WS Operating and equipment research grants for this project were
fraction was significantly higher (p < 0.05) at pH 4.0 and 7.0 but provided to Dr. R.E. Aluko by the Natural Sciences and Engineering
not at pH 9.0. Higher foaming capacity of WS fraction may be Research Council of Canada (NSERC) and the Advanced Foods and
due to its higher protein solubility index. This is because solubility Materials Network of Centre of Excellence (AFMNet) Canada. We
enhances protein unfolding and formation of interfacial protein thank our industrial partner, Nutri-Pea Ltd (Portage la Prairie, Man-
membranes at the air–water interface which enhances encapsula- itoba) for supply of the yellow pea seed protein isolate
tion of air bubbles. A previous report has also shown that increase
in foaming capacity of certain protein isolates might be due to in-
creased solubility, rapid unfolding at the air–water interface, lim- References
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