Heat Induced Gelation of Pea (Pisum sativum) Mixed
Globulins, Vicilin and Legumin
PUSHKAR S. BORA, CLARK J. BREKKE, and JOSEPH R. POWERS
ABSTRACT
Mixed globulins (MG) were extracted from ground dry peas (Pisum
sntivum, B-160) with 0.5M NaCl, 50 mM potassium phosphate, pH
7.2, and isolated by precipitation at pH 4.5. Crude vicilin and legumin
were fractionated from the MG by dialysis against 0.2M NaCl, pH 4.8,
and centrifugation, then further purified using DEAE-cellulose chro-
matography. Conditions for maximum gel hardness of heat induced
MG gel, as determined with an Instron Universal Testing Machine,
were heating for 20 min at pH 7.1 at 87°C. Purified vicilin, but not
legumin, formed heat induced gels. The relationship was linear be-
tween protein (globulin) concentration and log gel hardness. At all
protein concentrationsstudied, as proportion of legumin decreased,gel
hardnessincreased.
Key Words: pea protein, globulins, gelation, vicilin, legumin
INTRODUCTION
PROTEIN GELATION is an aggregation in which polymer to pol-
ymer and polymer to solvent interactions (both attractive and
repulsive) are balanced, and an ordered network or matrix ca-
pable of holding water is formed (Schmidt, 1981). The capac-
ity of gels to act as a matrix for holding water, lipids, sugars,
flavors and other ingredients is useful in food applications and
for development of new products (Kinsella, 1979).
Thermal gelation of soybean protein has been studied ex-
tensively (Mori et al., 1982a; Nakamura et al., 1984a, 1985a).
Mori et al. (1982b) and Nakamura et al. (1984b, 1985b) have
reported the contributions of 7s and 11s soy globulins in the
process of gelation. Utsumi and Kinsella (1985) indicated that
electrostatic interactions and disulfide bonds are involved in
the formation of 11s globulin gel, mostly hydrogen bonding
in 7s globulin gel, and hydrogen bonding and hydrophobic
interactions in soy isolate gel. Similar studies on the involve-
ment of molecular forces in the process of gelation of soy
protein were reported by Nakamura et al. (1986a,b) and Mori
et al. (1986). The 11s and 7s globulins may interact with each
other in gels made from soy isolate proteins (Babajimopoulos
et al., 1983; Utsumi and Kinsella, 1985).
Pea seeds contain 20 to 30% protein, most of which are
storage proteins. The two major proteins, legumin and vicilin,
are classical globulins and represent 65 to 80% of the total
buffer-extractable protein (Schroeder, 1982). Legumin and vi-
cilin proteins are similar to the 11s and 7s proteins of soybean,
respectively (Derbyshire et al., 1976), and, thus, potentially
may be important as functional protein ingredients in proc-
essed foods. However, information on the gelation behavior of
such proteins is limited (Bacon et al., 1990; Gueguen and Le-
febvre, 1983). Our objective was to examine thermal gelation
of mixed pea globulins at various conditions and compare the
results to those for crude and column purified legumin and
vicilin at standardizedconditions.
Authors Brekke and Powers are with the Dept. of Food Science
& Human Nutrition, Washington State Univ., Pullman, W A
99164-6378. Author Bore’s present address: Universidade Fed-
eral da Paraiba, Department0 de Tecnologia Q&mica e de
Alimentos, Centro de Tecnologia, Campus Universithrio I,
58.059 - Joao Pessoa/PB, Brasil. Address inquiries to Dr. C. J.
Brekke.
594-JOURNAL OF FOOD SCIENCE-Volume 59, No. 3, 1994
MATERIALS & METHODS
Extraction of pea globulins
The extraction method was that of Koyoro and Powers (1987).
Whole dry green peas (Pisum sat&urn, B-160) from a single bag, pro-
vided by Dumas Seed Co., Pullman, WA, were ground into flour using
a cyclone sample mill equipped with a 0.5 mm screen. The flour was
extracted at room temperature (-23°C) with 0.5 M NaCl, 50 mM
potassium phosphate buffer, pH 7.2, in a ratio of 6 mL buffer/g of
flour for 1 hr with constant agitation using a propeller stirrer. The
extract was centrifuged (19,000 X g, 15 min, 4”C), and the supernatant
was filtered through pads of glass wool. This filtrate was diluted with
5 vol of cold distilled water (4°C) and pH adjusted to 4.5 with 2N HCl
to precipitate salt soluble proteins. The protein floe was allowed to
settle overnight at 4°C. After decanting much of the supernatant,the
remaining suspension was centrifuged, the pellet resuspended and
washed with water, and re-centrifuged. The resulting pellet was re-
extracted twice with extraction buffer in a ratio of 5 mL buffer/g of
pellet for 1 hr at 4”C, followed by re-centrifugation. The supematants
resulting from the re-extraction were combined and divided into three
parts. The pH of one-third of the supernatantwas adjusted to 4.5 to
precipitate mixed globulins, which were recovered by centrifugation
(19,000 X g, 15 min, 4’C), washed with water, centrifuged and freeze-
dried. The remaining two-thirds of the supematantwas dialyzed against
Mcllvaines buffer, 493 mL 0.2M Na,HPO, * 2H,O + 507 mL O.lM
citric acid, containing 0.2M NaCl, pH 4.8 (Scholz et al., 1974), at 4°C
for 42 hr with two changesof buffer in a ratio of 10 mL of buffer per
mL of original supematant.The precipitate (legumin) was collected by
centrifugation, suspendedin distilled water at a ratio of l:lO, agitated,
re-centrifuged (19,000 X g, 15 min) and the precipitate freeze-dried.
The supematant from dialysis was adjusted to pH 4.5 and the precip-
itate (vicilin) recovered, washed with distilled water, centrifuged and
freeze-dried. The freeze-dried protein samples, representing several
separateprotein extractions, were stored in screw cap glass scintillation
vials at -20°C until used.
Chromatography of legumin and vicilin fractions
Legumin and vicilin preparations were further purified by DEAE
cellulose column chromatographyusing 35 mM potassium phosphate,
0.075M NaCl, 0.02% sodium azide buffer, pH 8.0. Freeze-driedprotein
was re-solubilized in column buffer at a concentration of 12 to 15 mgl
mL by sonication for 1 min and placed on the column. Vicilin and
legumin were eluted stepwise with buffer containing 0.15M and 0.4M
NaCl, respectively (Grant and Lawrence, 1964). Eluted fractions were
isoelectrically precipitated, centrifuged, washed with distilled water,
centrifuged and frozen (-20°C).
Elution patterns of mixed globulin, crude legumin and crude vicilin
samples from the DEAE column were also used as a measure of the
composition of those preparations.Thus, total protein eluting at 0.15M
NaCl was recorded as a measure of vicilin and that at 0.4M NaCl was
as legumin content.
Denaturation temperature of mixed globulins by differential
scanning calorimetry (DSC)
DSC was performed in a Perkin-Elmer DSC 4 fitted with a 3600
Thermal Analysis Data Station. Protein samples in 35 mM phosphate
buffer, 0.4M NaCl, pH 7.2, were hermetically sealedin aluminum pans
(Perkin-Elmer kit No. 219-0062) and ‘scanned at lO”C/min over the
range 25-100°C at a sensitivity of 1.0 mW using phosphatebuffer in
the referencepan. The instrument was calibrated for temperatureusing
indium (m.p. 156.6O”C). Tm was determined with data analysis pro-
grams supplied by Perkin-Elmer.
 i.oT 17.4% globulin8
lieatlng The (mln)
Fig. l-Heating time and protein concentration effects on peak
force of gels prepared from pea mixed globulin at 87’C and pH
7.2.
0.0
5.5 6.0 6.5 7.0 7.5 8.0
px of Gel
Fig. 2-Effect of pH on peak force of gels prepared from pea
mixed globulins. Gels were made by heating 15% mixed glob-
ulin solutions 20 min at 87°C in 30 mM Tris-HCI buffers.
Preparation of gels
Gelationof proteinsampleswas carriedout accordingto the method
describedby Utsumi and Kinsella (1985). Freeze-driedprotein was
suspendedin 30 mM Tris-HCI buffer, pH 7.2, in a test tube, and the
suspensionwas sonicatedfor 1 min. The suspensionwas centrifuged
for 30 min, and the supematant (protein solution) was used for prep
arationof the gels. Aliquots of protein solution (25 to 3OpL) in 30
mM Tris-HCl buffer weretransferredto Carawayblood tubes(75 mm
X 2.5 mm i.d) (FisherScientific),the tube endswere sealedwith po-
lyvinylidene chloride film and O-rings,andcentrifugedat 500 X g for
10 min to remove air bubbles. Heating time of the tubes ranged from
0 to 45 min in a water bath at 87°C followed by immersion in cold
water. The tubes were then held at 4°C for 24 hr, prior to gel peak
force determinations.
In a secondexperiment,pH wasvariedfrom 5.95
to 8.60, with heating for 20 min at 87°C in the water bath. All other
procedures and conditions were as before.
Gel peak force and protein
The gels formed in blood tubes were carefully removed by blowing
on the narrow end, taking care to collect the expelled gel on a glass
slide without damage,cut into 1 cm pieces, a piece laid lengthwise on
the stage of an Instron Universal Testing Machine, Model 1350 (In-
stron Corp., Canton, MA), and the gel peak force was determined. The
speed of the plate (76 mm diam) was adjusted to 4 cm/min and the
compression to 70%. Peak force refers to that obtained during the first
compression cycle (Boume, 1978). Two to five gels were used for
replicatedeterminations.Proteinwas determinedby the biuret method
(Gomall et al., 1949),usingbovine serumalbuminas standard.
OS-
0.01 I
0 0.1 0.2 0.3 0.4 0.5
Sodium Chloride Concentration (Y)
Fig. 3-Sodium chloride concentration and peak force of gels
made with pea mixed globulin. Gels were prepared by heating
15% mixed globulin solutions 20 min at 87°C in 30 mM Tris-HCI,
pH 7.1, with variable concentrations of NaCI.
Table l-Composition of crude globulin fractions a8 calculated from
DEAE cellulose chromatography profiles according to Grant and
Lawrence (1964)
Legumin Vicilin
Fraction (%) (%)
Mixed globulins 35.7 84.3
Crude legumin 88.5 31.5
Crude vicilin 9.1 90.9
RESULTS & DISCUSSION
DSC OF MIXED GLOBULINS indicated one transition between
74 and 9S’C with a maximum at 86.2”C (thermogram not
shown). This was essentially the same temperature (86’C) pre-
4
viously reported for field peas by Arntfield and Murray (1981),
although the solvent system they used was not specified. Time
of heating at 87°C had a marked effect on mixed globulin gel
peak force (Fig. 1). At pH 7.2, gel strength increased with
heating time up to 20 min, but heating beyond that period
caused a decreasein gel peak force. Nakamura et al. (1985a)
noted similar behavior for soybean glycinin (11s) when heated
at 100°C for different times. They also reported maximum gel
strength at 20 min heating. We adopted 20 min heating time
at 87°C as our standard gelation conditions, based on observed
maximum transition of 86.2’C and data presented in Fig. 1.
To study the effect of pH on gel formation and gel peak
force, and further define standard conditions, pH of the mixed
globulin solutions was varied from 5.95 to 8.60 (Fig. 2). We
observed that at pH 5.95 no gel formed. Gel formation oc-
curred at 2 pH 6.4, with the greatest gel peak force at pH 7.1.
Increasing pH to > 8.0 resulted in a translucent and sticky gel
which was difficult to remove from the tubes.
Sodium chloride had an adverse effect on mixed globulin
gel peak force. Within the concentration range 0 to 0.5M NaCl,
gel strength was maximum at OM NaCl and decreased with
increasing NaCl concentration (Fig. 3). Utsumi and Kinsella
(1985), using soybean isolate and 11s protein, also reported a
decrease in gel strength with increase in NaCl concentration.
Wang and Damodaran (1991) noted that in the presence of
0.5M NaCl, soy 11s did not form a gel when heated at 9O”C,
and the peak force of soy isolate gels was reduced. Conversely,
they did not observe a reduction in peak force of soy 7s gels
when OSM NaCl was added.
The make-up of the mixed globulin, crude legumin and
crude vicilin preparations, based on elution patterns from the
DEAE column was determined (Table 1). The data are based
on the definition of Grant and Lawrence (1964) that vicilin
elutes at 0.15M NaCl and legumin at 0.4M NaCl from DEAE
cellulose under the conditions used. At the conditions used for
gelation, i.e. pH 7.1, 20 min at 87”C, and at the protein con-
centrations studied, legumin fractions, either crude or further
Volume 59, No. 3, 1994JOURNAL OF FOOD SCIENCE-595 I
PEA GLOBULIN GEUTION. ..
0.1
8 10 12 14 16 18 20
Protein Concsntratlon (al/v)
Fig. bPeak force of gels from solutions of several pea protein
fractions of variable protein concentration. Gels were made by
heating protein solutions for 20 min at 87°C at pH 7.01-7.04. W
= mixed globulins, 0 = crude vicilin, Ir, = DEAE-purified vicilin.
purified by DEAE cellulose column-chromatography, did not
gel. At the highest concentration, neither of these fractions
showed any evidence of a gel hard enough to measure. How-
ever, further-purified vicilin from the DEAE column formed a
gel of greater peak force than for gels from crude vicilin or
mixed globulins. While both 7s and 11s soy globulin fractions
have the capacity to form gels, the pea vicilin (7s) fraction
had that capacity while pea legumin (11s) did not. Saio and
Watanabe (1978) reported that the 11s soy protein produced
a much harder gel than the 7s soy protein.
Data for vicilin fractions and mixed globulins demonstrated
a linear relationship (semi-log scale) between gel peak force
and protein concentration (Fig. 4). At equal protein concentra-
tions, peak force of the gel from column purified vicilin was
greatest, followed by the gels made from crude vicilin and
mixed globulins. That is, the greater the proportion of vicilin,
the greater was the gel peak force. That all three lines (Fig. 4)
were nearly parallel indicated that the effect of legumin on gel
peak force was not protein concentration dependent. The de-
crease in gel peak force with increase in legumin may not be
simply a dilution or vicilin sparing effect, but may be due to
protein to protein interaction. For example, with soybean 7s
and 11s globulins, the 7s protein interacts electrostatically
with the basic subunits of 11s globulin to form soluble com-
plexes during gelation (Damodaran and Kinsella, 1982; Ut-
sumi et al., 1984). However, unlike vicilin and legumin in our
study, the interaction of soybean 11s and 7s proteins was
affected by the proportion of globulins.
CONCLUSIONS
VICILIN, the 7s component of pea globulin, undergoes heat
gelation in a model system while legumin, the 11s component,
does not gel under the same conditions. The optimal conditions
for gelation of a pea mixed globulin system, as assessedby
gel hardness, were pH 7.1 and heating time 20 min at 87°C.
Addition of NaCl at concentrations of 0.05M or greater re-
sulted in mixed globulin sets of reduced hardness. Legumin
concentration in a range of pea globulin preparations, as esti-
mated by DEAE ion exchange chromatography elution profile,
was inversely related to gel hardness in the model system.
596-JOURNAL OF FOOD SCIENCE-Volume 59, No. 3, 1994
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MS received 6128193;revised 12/18/93; accepted l/19/94.
Author Rors gr&foUy achowledges the fellowship awarded by Cmselho Nackmal de
Desenvolvimento Cientillco e Tecnologico, Brazil and the Univmkhde Federal da Fnr&a
for study leave. The technical advice of Dr. Youling I. Xiong regarding the @ation and
DSC studies is much appreciated Washington State University, A@iculhual Research Cen-
tar, Fullman, WA project No. 0626. /