Food Hydrocolloids 46 (2015) 233e243

Contents lists available at ScienceDirect

Food Hydrocolloids
journal homepage: www.elsevier.com/locate/foodhyd

Effect of globular pea proteins fractionation on their heat-induced

aggregation and acid cold-set gelation
Jean-Luc Mession*, Mohammed Lazhar Chihi, Nicolas Sok, Re
mi Saurel
Agrosup Dijon, UMR PAM 02.102, Equipe PAPC (Proc
ed
es Alimentaires et, PhysicoChimie), 1 Esplanade Erasme, 21000 Dijon, France

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

Article history: This report focuses on cold-set gelation of pea (Pisum sativum L.) proteins and related globulin fractions,
Received 18 April 2014 namely Vicilin 7S and Legumin 11S. Protein thermal denaturation and aggregation were investigated
Received in revised form using differential scanning calorimetry (DSC) sodium dodecyl sulfate polyacrylamide gel electrophoresis
17 November 2014
(SDS-PAGE) analysis, and sulfhydryl (S
)/disulfide (SeS) bond assessment. While the denaturation
Accepted 24 November 2014
temperature (Td) of pea proteins increased from about 69 to 77  C with increasing legumin content, the
Available online 15 December 2014
disulfide-linked acidic (a) and basic (b) legumin subunits (56e58 kDa) denatured and aggregated in a
temperature range of 75e85  C. Dissociation of legumin oligomers and their rearrangements via hy-
Keywords:
Legumin
drophobic interactions and sulfhydryl/disulfide bonds exchange reactions would occur concomitantly
Vicilin during the heat-treatment, giving rise mainly to high-molecular weight aggregates of random structure.
Thermal denaturation and aggregation However thermal denaturation and aggregation of vicilin molecules would involve solely non-covalent
Sulfhydryl/disulfide bond exchange interactions. Then glucono-d-lactone (GDL) acid-induced gelation of protein thermal aggregates was
reactions evaluated by means of G0 and G00 moduli. The preheated mixed pea globulins and vicilin-enriched
GDL-induced cold-set gelation samples gave rise to increased final moduli values of the acid gels, while legumin-enriched samples
displayed low gelling properties. Improving functional properties of pea proteins would help to promote
their application in plant-based gelled products such as tofu, alternatively to their soy counterparts.
© 2014 Elsevier Ltd. All rights reserved.

1. Introduction Soybean represent two-thirds of the world plant protein con-

sumption (USDA, 2014). Despite its widespread utilization in many
Demographic change in combination with rapid urbanization “vegetarian” meat-like products, the dependence on soy importa-
and economical growth tend to increase dramatically worldwide tions for many countries is in discordance with improving effi-
pressure on land, water and natural resources. (Lundqvist et al., ciencies of food production (O'Kane, Happe, Vereijken, Gruppen, &
2007). In spite of the wide inequalities in access to water and van Boekel, 2004a, 2004b, 2004c). Mainly used as protein source in
food, increases in income in most of developing countries are animal feed, pea (Pisum sativum L.) proteins could represent an
associated with modifications in food consumption, with a marked alternative to soybean as functional ingredient in human food
preference for animal-based food to the detriment of vegetarian (Marcone, Kakuda, & Yada, 1998a).
products. However, given the heavy ecological debt of meat pro- Dry pea seeds contain about 20e25% crude protein, among
duction systems, the need to develop alternative protein sources of them 70% are storage globulins (Boye et al., 2010). Salt-soluble
reduced bioenergy and water demands for human diet is of primary globulins are composed of two fractions, namely legumin 11S and
interest. Thus one crucial challenge for food researchers would be vicilin/convicilin 7S (Tzitzikas, Vincken, de Groot, Gruppen, &
to increase the awareness on the utilization of sustainable plant Visser, 2006). These are constituted of heterogeneous subunits
proteins, displaying both satisfying nutritional value and func- assembled into high molecular weight (Mw) oligomers. Pea legu-
tionality (Boye, Zare, & Pletch, 2010). The development of novel and min 11S is hexameric (330e410 kDa), and its subunits (mainly
attractive plant protein-based foodstuffs would ultimately 60e65 kDa) dissociate into acidic a (40 kDa) and basic b (20 kDa)
encourage the partial replacement of ingredients provided by ani- polypeptides by disulfide bond reduction (O'Kane et al., 2004c).
mal production (meat, eggs, milk). Vicilin and convicilin 7S are trimeric (150 kDa and 180e210 kDa,
respectively) (Tzitzikas et al., 2006). Vicilin is a combination of
heterogeneous polypeptides, since the major subunits (48e52 kDa)
* Corresponding author. Tel.: þ33 380 774 051; fax: þ33 380 774 047.
E-mail address: [email protected] (J.-L. Mession). can be cleaved in vivo into low-Mw fragments (12e16, 20, 25e30

http://dx.doi.org/10.1016/j.foodhyd.2014.11.025
0268-005X/© 2014 Elsevier Ltd. All rights reserved.
234 J.-L. Mession et al. / Food Hydrocolloids 46 (2015) 233e243

and 30e36 kDa) (O'Kane et al., 2004a, 2004b). The convicilin sub- b.). The GDL powder was obtained from Prolabo (99.5% Fontenay-
units (70 kDa) display about 80% amino-acids sequence homology sous-Bois, France).
with the uncleaved vicilin subunits, though are distinguishable by All other reagents and chemicals purchased from Sigma-Aldrich
their highly charged N-terminal extension region close to the C- (St-Quentin Fallavier, France) were of analytical grade.
terminus, and also the absence of in vivo cleavage (Tzitzikas et al.,
2006). 2.2. Methods
Focusing on one essential functional property, gelation of
globular proteins is based on their ability to form three- 2.2.1. PP extraction and purification
dimensional network (Bryant & McClements, 1998). To induce Mixed globular pea proteins (PP) isolate was prepared from the
proteineprotein interaction, a denaturing thermal process is usu- defatted pea flour using a salt-extraction at pH 8, followed by
ally applied. This leads to protein unfolding, exposure of initially tangential ultrafiltration/discontinuous diafiltration with deionized
buried reactive residues and subsequent aggregation caused by water (UF/DF), using a polyethylenesulfone membrane of 100 kDa,
different molecular interactions, possibly involving non-covalent as previously described (Mession, Assifaoui, Lafarge, Saurel, &
and/or covalent bonds. Many recent reports pointed out the Cayot, 2012). During the UF step, a volume concentration ratio of
lower textural features of pea protein heat-set gels than those ob- 3 (VCR, mL/mL) was applied. The further discontinuous DF step
tained with their soy counterparts, though processed under the consisted of a re-VCR of 3 (Taherian et al., 2011). Separation of the
same conditions (O'Kane et al., 2004c; O'Kane, Vereijken, Gruppen, globulin fractions Vic and Leg was achieved by a chromatography
& van Boekel, 2005; Shand, Ya, Pietrasik, & Wanasundara, 2007; column, pre-packed with DEAE Sepharose® Fast Flow anion-
Sun & Arntfield, 2010, 2011, 2012). The type and composition of exchanger (150 mL, 15 cm bed height, GE Healthcare Amersham
the pea proteins used and the different procedures of each study Biosciences Corp., Uppsala, Sweden). The pea protein concentrate
affected gelation properties. As reported by many authors, the obtained by UF as described above underwent the discontinuous DF
commercial pea protein isolates utilized exhibited poor gelling step with 0.1 M Na2HPO4 e citric acid buffer, pH 7. Elution was
properties, since proteins were extensively denatured during their performed at 20  C using a stepwise gradient of NaCl (0, 0.06, 0.1,
large-scale production (Shand et al., 2007). Besides, pea proteins 0.25 and 0.5 M) in the phosphate-citrate buffer. Each eluate
heat-induced gelation would require concentrated pea protein (150 mL) was monitored at 280 nm and analyzed by SDS-PAGE for
suspensions in the presence of high salt concentrations (Sun & purity. Polypeptide bands at 70, 50, 36e30 and lower than 14.2 kDa
Arntfield, 2010, 2011, 2012). Moreover, slow heating/cooling rates were attributed to vicilin/convicilin subunits, while the large band
applied would be required for pea protein molecules to form gel at 56e58 kDa belonged to legumin ab subunits (Mession et al.,
network of enhanced elasticity (Sun & Arntfield, 2011). Neverthe- 2012; O'Kane et al., 2004a, 2004b, 2004c). Eluates of identical
less, Shand et al. (2007) indicated that the number of cross-links electrophoretic pattern were pooled together, and underwent for
within the gelled network increased with the availability of solu- each an UF (VCR of 4) and discontinuous DF (re-VCR of 3). At the
ble pea protein aggregates provided by heat-treatment. end, dried protein samples were obtained by freeze-drying, sealed
From these observations, pea proteins cold-set gelation could and stored at 4  C until further use. These dry samples contained
represent an alternative route (Bryant & McClements, 1998). This mixed globular pea proteins, enriched-fractions vicilin/convicilin
procedure would enable better control of soluble protein aggre- and legumin and were named PP, Vic and Leg, respectively.
gation; indeed heat-denaturation and gelation are divided into two
steps. First, a low-concentrated protein solution (<10 wt%) at a pH 2.2.2. Chemical analyses
far from its isoelectric point and in the absence of salt is heated to Total moisture and ash contents were evaluated according to
produce soluble aggregates. After cooling, gelation of the thermal AOAC (1990) procedures. Total nitrogen (TN) and non-protein ni-
aggregates is carried out by lowering electrostatic repulsions, trogen (NPN) were determined according to EN ISO 20483:2006
allowing them to assemble into structured network. The second and Chavan, McKenzie, and Shahidi (2001) methods, respectively.
step could be achieved by adding an acidifying agent, glucono-d- This allowed calculation of protein content, using a (protein)
lactone (GDL). Acid-induced cold gelation is well documented for nitrogen-to-protein conversion factor of 6.25 (Marcone et al.,
whey proteins (Alting, Hamer, de Kruif, & Visscher, 2003). In 1998a; Shand et al., 2007).
contrast, no data was available concerning pea proteins.
In a previous work, heat-treatment of a pea globulins solution 2.2.3. Preparation of heat-induced aggregates
resulted in high-Mw and soluble thermal aggregates (Mession, Sok, The freeze-dried PP, Vic and Leg samples were suspended in
Assifaoui, & Saurel, 2013). In this regard, pea proteins extraction by deionized water, stirred at room temperature for at least 2 h, and
an ultrafiltration/diafiltration procedure in addition with careful then centrifuged (12,000 g, 25 min, 4  C) to obtain protein super-
control of heat-treatment would promote the usefulness of aggre- natants. To ensure that protein samples were at low ionic strength
gates as “building blocks” for cold-set gels. Hence, the present study prior to heat-treatment, the supernatants were extensively dia-
aimed at investigating the thermal denaturation and aggregation of lyzed against a 10 mM Na2HPO4 buffer at pH 7.2, (ratio protein
the different pea globulin fractions (mixed globular pea proteins: solution-to-buffer of 1:10, 4  C, 24 h), and again centrifuged
PP, legumin: Leg fraction and vicilin: Vic fraction), in terms of (12,000 g, 25 min, 4  C). The PP, Vic and Leg stock solutions at 4.2,
protein thermal stability and molecular interactions involved. 6.2 and 3.7 wt% protein concentration, respectively, were diluted
Thereafter cold-gelation properties upon acidification of the ther- with the phosphate buffer used for dialysis to obtain various pro-
mal aggregates were compared according to the globulin fraction. tein concentrations: 1, 2, 4 wt% for PP and Vic samples and 1, 2,
3.5 wt% for Leg samples. Protein samples (5 mL) were poured in PX/
2. Material and methods 1636/04 MP tubes, hermetically sealed (SciLabware Ltd, Stafford-
shire, U.K.). They were placed in a water bath that was preheated at
2.1. Materials 40  C, then heated at 1  C/min from 40 to 85  C, and incubated at
85  C for 60 min. Heated tubes were subsequently cooled in ice for
Pea proteins were extracted from smooth yellow peas (P. sat- 30 min and stored at 4  C overnight until further use. Some protein
ivum L.), supplied by Roquette SA (Lestrem, France). Dehulled and samples were also heated at various temperatures (70e85  C) and
ground peas contained 4.1 wt% total nitrogen (TN) on a dry basis (d. times (0e60 min) for SDS-PAGE analysis (Section 2.2.5).
 J.-L. Mession et al. / Food Hydrocolloids 46 (2015) 233e243 235

Additionally, 20 mM N-ethylmaleimide (NEM, powder) was dis- calibration. The INR/IR ratio served as correction factor (1),
solved by stirring at room temperature in Leg samples especially, enabling the apparent amount (concentration) of polypeptides
and then heated as described above. migrating in the running gel under NR condition. Calibration curves
were then established, plotting the band area of a polypeptide of
2.2.4. Differential scanning calorimetry (DSC) interest as a function of its apparent concentration.
Thermal parameters of the PP, Vic and Leg protein extracts in
solution were assessed with a MicroDSC III calorimeter (Setaram,
Caluire, France). An accurate weight (500 mg) of protein stock so- 2.2.6. Free sulfhydryl content determination
lution diluted to 3 wt% protein was hermetically sealed in a 1 cm3 The free sulfhydryl (S
free) contents of pea protein samples were
stainless steel vessel. The sample was heated from 25 to 100  C with evaluated using 5,50 -dithio-bis-2-nitrobenzoic acid (DTNB), accord-
a heating rate of 0.5  C/min, using the 10 mM phosphate buffer in ing to Ellman (1959). The reagents used were DTNB (1 mM) and
the reference vessel. Onset temperature (Tonset), temperature of guanidium hydrochloride (GuHCl, 6 M) solutions, previously dis-
denaturation (Td) and enthalpy of denaturation (DHd) were solved in a 0.1 M phosphate buffer, pH 7.5. Unheated and heated
computed from the thermograms by the thermal analysis SETSOFT (85  C/60 min) protein samples were extensively dialyzed at 4  C
2000 software (Setaram). In addition, protein samples were heated against the same 0.1 M phosphate buffer, pH 7.5 (ratio protein
in the presence of 20 mM NEM. solution-to-buffer 1:20). The reaction mixture was prepared by
mixing 650 mL of the protein sample containing either 9 or
2.2.5. SDS-PAGE 18 mgprotein/mL (depending on initial protein concentration) with
Non reducing (NR) or reducing (R)-SDS-PAGE was performed on a 750 mL GuHCl and 100 mL DTNB solutions, stirred vigorously and kept
discontinuous buffered system, using a triseHCl polyacrylamide in dark for 10 min at 25  C. Absorbance was read at 412 nm, using a
(C ¼ 2.7 wt%) stacking gel (T ¼ 4%, wt/v, pH 6.8) and running gel molar extinction coefficient 3 for DTNB of 12,800 M
1/cm, as calcu-
(T ¼ 10%, wt/v, pH 8.9) in the presence of SDS (0.1%, wt/v) (Laemli, lated from calibration curves with N-acetyl-cysteine in the range of
1970). The dimensions of the gel were 14  16 cm. Unheated and 0e60 mM. The free S
content was expressed as mM S
free/gprotein.
preheated samples prepared in Section 2.2.3 were centrifuged
(12,000 g, 25 min, 4  C). Supernatants (soluble fraction) were diluted 2.2.7. Gelation experiments
with deionized water on the basis of 1 wt% protein concentration for Acid-induced cold gelation of thermally-aggregated protein
the unheated sample. When noticed, insoluble protein sediment in samples (85  C/60 min) was performed at 20  C using GDL. The GDL
heated samples was collected and mixed at room temperature with a powder was added to preheated protein samples, then stirred for
denaturant solution (ratio 1:20, w/w) containing 2% SDS (wt/v), 6 M 1 min (Table 1). Depending on protein initial concentration and/or
urea and 100 mM triseHCl, pH 8.5, till complete solubilization. Pro- composition, the GDL concentration was adjusted in the range of
tein samples were then mixed (ratio 1:1) with the sample buffer 0.2e0.75 wt% to reach a pH of 4.5 after 4 h incubation. The pH was
62.5 mM triseHCl, pH 6.8, 10% glycerol (wt/v), 0.005% bromophenol measured at regular intervals with a pH meter model C561 (Con-
blue (wt/v) and 0.2% (wt/v) SDS, without (NR) or with (R) 1% (wt/v) sort, Turnhout, Belgium), till pH reached its steady-state value.
dithiothreitol (DTT). For R-SDS-PAGE, samples were heated for Gel formation under isothermal conditions (20  C) was moni-
10 min in boiling water. Twenty microliter of sample (5 mgprotein/mL) tored by measurements of the elastic (G0 ) and loss (G00 ) modulus of
was loaded in each well. Wide-range Mw standards (S8445, Sigma) the protein samples under acidification at 20  C, using a controlled-
were deposited in a separated lane to identify polypeptides according stress rheometer (SR-5 Rheometric Scientific e Piscataway, NJ,
to their apparent Mw. Fixed proteins were stained with Coomasie USA), managed by the RSI Orchestrator software (V.6.5.8). Samples
Blue R-250 (0.125%, wt/v) in 20% (v/v) ethanol, and destaining was (1 mL) were placed into the parallel plateeplate geometry (25 mm
performed using 5% (v/v) acetic acid and 20% (v/v) ethanol. diameter, gap set at 1 mm) immediately after dispersion of GDL,
The destained gels (colorless background) were scanned with a and edges of the sample were covered with mineral oil to prevent
ChemiDoc XRS þ System (Bio-Rad Lab., Hercules, CA, USA), and the water evaporation. Both moduli were measured as a function of
density of individual polypeptide bands in a lane was expressed as time at an oscillation frequency of 0.5 rad/s and oscillatory stress of
series of staining peaks as a function of the running distance from 0.5 Pa within the linear viscoelastic region, as previously deter-
the border (distance 0) separating the stacking and the running gel, mined by a stress-sweep test (0.3e10 Pa) on gels. The gel point Gp
using ImageLab (v. 3) software. The areas (intensities) under the was considered as the time/corresponding pH value when the G0
peaks were integrated and summed. This allowed calculation of and G00 cross-over occurred (tan d ¼ G00 /G0 ¼ 1). Gelation was
total lane intensity and thus the relative proportion of each poly- considered as achieved when each modulus plateaued around a
peptide constituting the protein sample. stable value (after about 4 h of acidification, pH in the range of
Upon heat-treatment of a protein sample, sulfhydryl-containing 4.5e4.3). Then a frequency-sweep test (from 0.06 to 6 rad/s) was
polypeptides such as 11S globulins could denature and form co-
valent aggregates via disulfide linkages (Choi, Mine, & Ma, 2006).
Under NR conditions, the amount of these polypeptides of interest Table 1
could be quantified by densitometry, since their related band area Composition of pre-heated protein samples prepared at the concentrations indi-
cated, for cold gelation experiments in the presence of GDL at 20  C.
would decrease upon heat-treatment. On the same gel, a range of
protein concentrations (0.5e5 mgprotein/mL, i.e. 10e100 mg protein) Sample Protein (wt%) GDL/protein weight ratio (w/w)
of the unheated sample (reference) and related samples collected PP 1 0.22
during heat-treatment (Section 2.2.3) were applied in separated PP 2 0.21
lanes. Within this protein amount loading range for sample appli- PP 4 0.19

cation, it was considered that all the polypeptides (<200 kDa) un- Vic 1 0.18
der R conditions could migrate in the running gel, and total lane Vic 2 0.17
Vic 4 0.17
intensity was correlated to the initial protein amount loaded in the
well. Because staining may vary from gel to gel, the ratio of total Leg 1 0.16
lane intensity I under NR and R conditions was calculated for each Leg 2 0.15
Leg 3.5 0.13
protein concentration of the unheated sample applied for
236 J.-L. Mession et al. / Food Hydrocolloids 46 (2015) 233e243

conducted at fixed stress within the linear viscoelastic domain, to broad endothermic peak that was attributed to thermal denatur-
determine frequency-dependence of both moduli. ation of 7S and/or 11S globulin fractions. The Tonset value is the
temperature from which unfolding of the globular protein structure
2.2.8. Statistical analysis is initiated. This value was of about 64  C for both PP and Vic
The presented results were obtained from two repetitions of samples, 5  C lower than that measure for Leg. The Td value is the
complete sample preparation and measurements for each prepa- peak thermal denaturation temperature; a high value indicates
ration were at least duplicated. Analysis of variance (one-way usually a high thermal stability for globular proteins (Choi & Ma,
ANOVA) and Tukey's procedure were carried out at the p < 0.05 level 2005). Both PP and Leg samples displayed a Td value in a narrow
to evidence significant differences between mean values, using range of 75e77  C, at least 6  C higher than that measured for Vic.
Statistica software (version 7.1, StatSoft Inc, Maisons-Alfort, France). The DHd value is the content of ordered structure of a protein, or
can be related to the proportion of non-denatured proteins before
3. Results and discussion heat-treatment (Sun & Arntfield, 2010). There was no significant
difference between the protein samples investigated, despite mo-
3.1. Protein extracts characterization lecular structure differences of the extracted pea globulins.
Both Td and DHd values could be indicative of the preservation of
The fat content in the pea flour was 1.3 wt% (d. b.). Regarding TN the “native” globulin structure during the extraction procedure. To
content in the defatted pea flour, about 83% was recovered in the compare with previous data, the PP sample had a Td value of about
protein crude extract (pH 8), while 22% consisted of NPN (Mession 10  C lower than that reported for pea globulins prepared by Shand
et al., 2012). Thus the salt-extractable protein content in the et al. (2007) and Sun and Arntfield (2010). The PP sample had a DHd
defatted pea flour was 18.1 wt% (d. b.), in agreement with data value close to that measured by Shand et al. (2007). The Td value
reported by Boye et al. (2010). After the UF/DF procedure, protein measured for the Vic sample was in agreement with that found in
yield from the crude extract was around 75%, while protein content similar conditions by O'Kane et al. (2004a) for pea vicilin fractions,
in the diafiltrated pea proteins concentrate represented 85e90% of however the Leg sample had lower thermostability of about 7  C
total solids content. Hence, non-protein material was efficiently than that reported by Marcone, Kakuda, and Yada (1998b). Mean-
removed by UF/DF. while in the present study, higher pea proteins thermostability
By ion-exchange chromatography applied on the diafiltrated could be indicative of higher legumin molecules content in the
pea proteins concentrate, vicilin and legumin proteins eluted at order Vic < PP < Leg samples (Table 3), since legumin hexamers
0.1 M and 0.25 M NaCl in the phosphate-citrate buffer, respectively were of higher conformational compactness e thus harder to
 & Gue
(Larre guen, 1986). As reported by these authors, it was thermally-unfold e than that for vicilin trimers (Marcone et al.,
observed that non-globulin proteins (mainly albumins) and non- 1998b). Additionally, the presence of disulfide bonds within legu-
protein components eluted as broad peaks of strong UV- min subunits Lab would contribute to the thermal stability of the
absorption at 0 and 0.5 M NaCl, respectively (elution profile not protein, as reported for its buckwheat legumin counterpart (Choi
shown). Protein yields for both Vic and Leg-enriched fractions were et al., 2006). However discrepancies regarding thermal parame-
in the range of 8e10% of the starting PP content loaded in the ters of pea globulins were particularly related to the pea cultivar
column. Protein contents in the freeze-dried PP, Vic and Leg- and the legumin-to-vicilin ratio; while vicilin proteins were prac-
enriched fraction samples were 85.8 wt%, 54.2 wt% and 56.5 wt% tically devoid of sulfur-amino acids, the number of cysteine residue
(d. b.), respectively. Salt contents were 4 wt% for PP and around per legumin subunit could vary between 2 and 7 (O'Kane et al.,
30e35 wt% for both enriched-fractions (d. b.). Despite the UF/DF 2004c, 2005; Sun & Arntfield, 2012). Moreover, extrinsic factors
procedure performed on either Vic or Leg-enriched fraction, the enhancing protein compact packing and/or proteineprotein in-
salts brought during the purification step remained in large teractions could be mentionned in particular the stabilizing effect
amounts in their related freeze-dried samples (concentrates). of NaCl (at pH higher than 6) towards globulin quaternary structure
Thereby the dialysis step following the dissolution of freeze-dried and/or an increase of the level of energy required for protein
samples in the 10 mM phosphate buffer (pH 7.2) was a cautious unfolding with an higher heating rate applied (Mession et al., 2013;
approach (Section 2.2.3). Ultimately total protein accounted for Sun & Arntfield, 2011). The different experimental conditions used
more than 90% of total solid in protein stock solutions. rendered difficult comparisons between the authors regarding
heat-denaturation of pea and other plant proteins.
With respect to the possible involvement of sulfhydryl/disulfide
3.2. Differential scanning calorimetry (DSC)
bonds (S
/SeS) exchange reactions during pea globulins denatur-
ation and aggregation, NEM (20 mM) was added to protein samples
Thermal properties of PP, Leg and Vic samples at pH 7.2 were
prior to DSC analysis (Table 2). NEM is a sulfhydryl-blocking agent
evaluated using DSC, firstly in the absence of NEM additive
(O'Kane et al., 2004c). All thermograms showed one endothermic
(Table 2). All the thermograms (data not shown) displayed one

Table 2
Thermal parameters of pea protein extracts (3 wt%, heated at 0.5  C/min) in 10 mM Na2HPO4 buffer, pH 7.2, in the absence (
NEM) and in the presence (þNEM) of 20 mM N-
ethylmaleimide.

Sample Tonset ( C)a Td ( C)a DHd* (J/gprotein)a

NEM þNEM
NEM þNEM
NEM þNEM

PP 63.3 ± 2.6bA 62.2 ± 1.4bA 74.5 ± 1.6aA 73.7 ± 1.4aA 7.9 ± 1.5aA 6.2 ± 0.0aA
Leg 69.3 ± 1.3aA 70.6 ± 0.5aA 76.6 ± 0.4aA 72.1 ± 0.3aB 10.3 ± 1.4aA 7.5 ± 0.6bB
Vic 64.9 ± 2.5bA 63.0 ± 2.7bA 68.5 ± 1.0bA 71.1 ± 2.8aA 7.6 ± 1.1aA 8.3 ± 0.8bA

Column values followed by the same lowercase letter (a and b) are not significantly different (p < 0.05).
Effect of NEM addition on each thermal parameter of a same pea protein extract: row values followed by the same capital letter (A and B) are not significantly different
(p < 0.05).
a
Mean ± SD.
 J.-L. Mession et al. / Food Hydrocolloids 46 (2015) 233e243 237

peak (data not show). For PP, the overlapping thermal transitions of under R conditions (Lane V*). Since any polypeptide band assign-
pea proteins may hide small differences in their respective Td values able to either La or Lb polypeptides was noticed in V*, there would
(Shand et al., 2007). In the presence of NEM, the Tonset value of Leg be no cross-contamination of the Vic fraction by Lab. Nonetheless,
was higher by 5  C as those for PP and Vic, while the Td values close remaining legumin in Vic could not be totally ruled out; the very
to 72  C were not found to differ. Concerning DHd, the PP þ NEM small increase under R conditions of staining intensity just below
sample displayed the lowest value, however in the same range than the Vi1 bands would suggest the presence of a minor acidic legumin
that measured for the PP e NEM sample. By comparing each protein polypeptide of slightly higher Mw (faint band around 44 kDa) than
sample in the presence or in the absence of NEM, it appeared that Td that determined for La (Table 3). The NV band could be otherwise
and DHd values were solely affected for the Leg þ NEM sample, assigned to disulfide-bonded albumins co-extracted with vicilin
decreasing of about 4.5 and 3 J/gprotein, respectively. These results molecules during the chromatography procedure, of Mw lower
were indicative of the limited effect of NEM on PP thermal dena- than 14.2 kDa for the monomers under R conditions (Larre  &
turation, whereas there was no apparent effect for Vic. Firstly, NEM Gueguen, 1986; Shewry, Napier, & Tatham, 1995).
reactivity would be lowered since sulfhydryl groups were deeply From the electrophoretic patterns, a densitometric analysis was
buried within legumin structure (O'Kane et al., 2004c). Secondly, performed to evaluate the relative amounts of the polypeptides
non-covalent interactions were solely involved in the stabilization encountered in unheated samples (Table 3). For PP in NR condi-
of the pea globulins oligomeric structure (Marcone et al., 1998a); tions, ratios of vicilins/legumin (Vi1e3/Lab) and vicilins/convicilin
heat-induced dissociation of pea globulin subunits would be a (Vi1e3/Cv) were of about 2.4 and 5.7, respectively. Besides, a legumin
prerequisite to allow sulfhydryl exposure and thus protein struc- content close to 25% was found in most of pea cultivars (O'Kane
tural changes by covalent bonding. Therefore NEM could induce et al., 2005; Tzitzikas et al., 2006). The enrichment of the Leg and
slight conformational change of legumin molecules. As NEM Vic samples by Lab and Vi1e3, respectively, were of about 2.5 and 1.5-
inhibited disulfide bonds formation, it could be hypothesized that fold higher than their relative amounts in PP. About 25% of the total
non-covalent bonds, of lower energy than the former ones, were amount of La and Lb polypeptides (R conditions) was not disulfide-
promoted during heat-denaturation of legumin molecules thus bonded in the Leg sample under NR conditions. The increase in the
facilitating their unfolding and subsequent aggregation via non- relative amount of Vi3 in Vic under R conditions compared to that
specific interactions. This assumption is supported by the very found under NR conditions was possibly attributable to traces of
narrow temperature range of 2  C between Tonset and Td values of dissociated basic legumin polypeptides (18e22 kDa), and/or albu-
Leg þ NEM, indicative of a highly cooperative endothermic tran- min monomers.
sition, i.e. occurring simultaneously at several levels of structure Prior to electrophoresis, the heated Leg samples developed
(Marcone et al., 1998b). Besides, this was consistent with the lower strong turbidity at 80  C, while PP and Vic remained slightly
DHd value for Leg þ NEM than that for Leg e NEM sample, possibly yellowish. By centrifugation, precipitation was evidenced for
reflecting the higher contribution of exothermic events than heated Leg; it was assessed that around 85e90% of the initial
endothermic ones during thermal denaturation of Leg þ NEM (Choi protein content in unheated Leg samples remained soluble in su-
et al., 2006). Meanwhile, the decreasing thermal stability of pernatant of heated ones, while this was more than 95% for both PP
Leg þ NEM was not evidenced by O'Kane et al. (2004c) for purified and Vic.
pea legumin. This may account for the less native state of the The NR-SDS-PAGE profiles of soluble proteins in heated PP and
fraction Leg in this study, probably increasing legumin molecules Leg samples (Fig. 1aeb, from the lanes SP-75  C and SL-75  C,
sensitivity towards NEM. respectively) exhibited components of low electrophoretic mobility
By reheating one replicate of each protein sample, no thermal (Mw > 200 kDa), since they entered hardly in the running gel.
transition was detected; pea protein denaturation was complete Additionally, a new polypeptide band at 85 kDa (noted LA) was
and irreversible. detected in the case of heated Leg. During heat-treatment, the Lab
band intensity decreased for both PP and Leg samples. This was
3.3. SDS-PAGE indicative of pea proteins heat-induced denaturation initiated at
70e75  C.
Polypeptide composition of unheated pea protein samples was Under R conditions, the disappearance of the high Mw-
investigated by NR- or R-SDS-PAGE (Fig. 1a: PP, lanes P and P*; 1b: aggregate and LA bands was related to their dissociation, and
Leg, L and L*; 1c: Vic, V and V*). Electrophoretic patterns displayed electrophoretic patterns of unheated and heated samples were
numerous polypeptide bands ranging from 90 kDa to less than comparable; the heated PP and Leg samples exhibited the charac-
14.2 kDa. Band identification was performed on the basis of Shand teristics bands attributed to La and Lb polypeptides (Fig. 1aeb, lanes
et al. (2007), and O'Kane et al. (2004a, 2004c) reports. SP*-, SL*-85  C/60 min). Therefore thermal denaturation of legumin
The polypeptides of Mw 85e90 kDa were attributed to lip- subunits Lab would lead to the formation of non-covalent and co-
oxygenases Lox1e2. The legumin subunit Lab (Fig. 1a and b, lanes P valent aggregates, with the possible involvement of intermolecular
and L, Mw 56e58 kDa) was characterized by its dissociation under disulfide bonding. Besides, soluble and insoluble legumin fractions
R conditions (þDTT) into acidic La (38e42 kDa) and basic Lb1e2 had similar patterns, except the low band resolution for the latter
polypeptides (24 and 18 kDa, noted Lb in the following for simpli- one, which could be related to enhanced protein denaturation
fication, lanes P* and L*). The band caught at the top of the stacking (lanes IL- and IL*-85  C/60 min).
gel was reported to be disulfide-bonded legumin polymers; these The occurrence of disulfide linkages in heated Leg samples was
are usually encountered in “native” (unheated) pea protein samples further investigated by heating the fraction Leg with added NEM
and were dissociated under R conditions (Marcone et al., 1998a). (Fig. 1b0 ). Protein precipitation was markedly noticeable from 75  C,
Additionally, some La and Lb2 polypeptides appeared not to be due to the insolubilization of the Lab subunits (lane ILN-
disulfide-bonded in both unheated PP and Leg samples. The vicilin 85  C/0 min). Remaining soluble polypeptides were residual vicilin
polypeptides were composed of convicilin (Cv), uncleaved Vi1 Vi2 polypeptides (from the lane SLN-75  C) (36e20 kDa). Surpris-
subunits (52e48 kDa), and cleaved ones Vi2 (37e30 kDa) and also ingly, further heat-treatment induced the breakup of intermolec-
Vi3 (22e14.2 kDa) (1c, lane V). A polypeptide of Mw 60 kDa was ular disulfide linkages between La and Lb polypeptides of insoluble
detected (namely NV: non-vicilin) and though vicilin subunits were legumin (lanes ILN- and ILN*-85  C/60 min). The present results
not held together by disulfide bonds, the former one dissociated suggested restricted intramolecular S
/SeS exchange reactions
238 J.-L. Mession et al. / Food Hydrocolloids 46 (2015) 233e243

Fig. 1. Electrophoretic patterns (SDS-PAGE, 12%) of unheated (40  C, prior to heat-treatment) PP (a, lanes P and P*), Leg (b, lanes L and L*), Leg þ 20 mM NEM (b0 , lane LN) and Vic
(c, lanes V and V*) samples in non-reducing (NR) or reducing (R) conditions (in the presence of DTT, as indicated by the asterisk“*”). A protein amount lower than or equal to 100 mg
was deposited in each lane. Under the lanes, were soluble (S) proteins after centrifugation of heated PP, Leg, Leg þ NEM and Vic samples at various temperature (70e85  C) and
incubation time at 85  C (0e60 min), while IL and ILN were insoluble protein material encountered in the Leg and Leg þ NEM samples, respectively. On the lanes, Mw M: molecular
weight markers, polypeptide band Lox: lipoxygenases 1e2, Cv: convicilin, NV: non-vicilin protein, Lab: main legumin subunit, Vi1e3: bands attributed to vicilin 7S, La and Lb1e2: acidic
and basic polypeptides constitutive of the main legumin subunit, respectively, LA and VA: disulfide-bonded protein aggregates (<200 kDa) formed in heated Leg and Vic samples,
respectively.

deep within La and Lb polypeptides that were not inhibited by NEM, NEM modified thermal denaturation of Lab, the newly-formed
owing to low accessibility of sulfhydryl groups and/or probably intermolecular disulfide bonds between denatured legumin mol-
their closeness on polypeptide chains. The lower thermostability as ecules would be required to maintain a stable conformation of the
measured by DSC (Table 2) for the Leg þ NEM sample could arise heat-induced aggregates.
from easier unfolding and higher polypeptide chain flexibility in The heated Vic samples displayed from 75  C several novel
the presence of NEM that enhanced aggregation via non-specific polypeptide bands noted VA (Fig. 1c, from lane SV-75  C, Mw
interactions, whereas the reduction in DHd would be related to 100e200 kDa), while intensity of the polypeptide band NV (60 kDa)
the formation of insoluble aggregates (Choi & Ma, 2005). Since started to decrease between 80 and 85  C/0 min; thus the NV
 J.-L. Mession et al. / Food Hydrocolloids 46 (2015) 233e243 239

Table 3
Densitometric analysis of the electrophoretic patterns (Fig. 1aec) of unheated PP (1a, lanes P and P*), Leg (1b, lanes L and L*) and Vic (1c, lanes V and V*) samples in either non-
reducing (NR) or reducing (R) conditions. Polypeptide band assignation is displayed in Fig. 1. The relative amounts of the different polypeptides were calculated from the
intensity of the band of interest relatively to the sum of total migrating polypeptide bands in the same lane of the running gel electrophoresis.

Polypeptide Mw (kDa) % PPa % Vica % Lega

NR R NR R NR R

Lox 87 7 ± 0.35 5.7 ± 0.5 2.5 ± 0.7 2.9 ± 1.5 5.4 ± 0.2 5.8 ± 0.3
Cv 66 8.7 ± 2.3 8.6 ±2 13.3 ± 0.3 12.4 ± 1.5 3.4 ± 1.1 2.2 ± 0.0
NV 60 e e 9.5 ± 0.4c 3.7 ± 0.8 e e
Lab 56e58 21.2 ± 3.2b e e e 65.1 ± 0.8b 3.1 ± 0.8
Vi1 52e48 13.8 ± 2.9 12.2 ± 1.8 24.5 ± 2.7 25.6 ± 2.4 e e
La 42 5.2 ± 1.6 16.2 ± 1.3 e e 12.2 ± 0.8 42.4 ± 1.1
Vi2 36e30 16.2 ± 0.9 17.9 ± 0.7 21.8 ± 0.4 21.6 ± 3.3 6.7 ± 1.3 8.3 ± 1.6
Lb1 24 3.2 ± 0.6 8.3 ± 1.3 e e 3.1 ± 0.8 13.0 ± 0.2
Lb2 18 5.0 ± 1.0 8.8 ± 1.6 e e 4.8 ± 0.4 25.1 ± 0.8
Vi3 22e20; 14.2 20.2 ± 1.4 21.9 ± 1.1 27.3 ± 3.0 34.3 ± 2.6 e e
a
Means ± SD.
b
The NV polypeptide band could not be quantified separately from the Lab band, owing to close running distance and thus Mw.
c
The NV band could be evaluated accurately for the Vic sample.

concentration decreased from 0.9 to 0.2 mgNV/gsolution at temper- during the heating ramp (Fig. 2). For all the protein samples, it was
atures below (or equal to) 80  C and 85  C/60 min, respectively worth noting that half of the initial Lab subunits content was
(densitometric analysis not shown). The vicilin bands Vi1e3 were already denatured at 80  C (Fig. 3a). From 85  C/0 min, the decrease
not affected by the heat-treatment. Patterns of unheated and in the Lab concentration slowed down, and plateaued at 85  C/
heated Vic samples were found similar under R conditions (lanes V* 30 min toward values in the range of 10e20% of the initial con-
and SV*-85  C/60 min). Thus thermal denaturation of vicilin mole- centration in the unheated sample, independently of the total
cules and their aggregation may involve solely non-covalent in- protein amount which underwent heat-treatment. Moreover,
teractions, whereas the formation in low amounts of disulfide- calculating the ratio 1
INR/IR of total lane intensity I could estimate
bonded covalent aggregates would be attributable to NV and/or the proportion of protein material involved into soluble disulfide-
sulfur-containing albumins. bonded aggregates (Fig. 3b). The breakup under R conditions of
A densitometric analysis was conducted on SDS-PAGE patterns intermolecular disulfide linkages would allow the migration of
of soluble proteins to study change in protein composition during polypeptides involved into high-Mw aggregates (>200 kDa) that
heat-treatment of PP (1a, lanes SP-) and Leg (1b, lanes SL-) samples, could not be quantified under NR conditions in the running gel of
from 1 to 4 wt% and 1 wt% protein concentration, respectively. The the electrophoresis. It was checked that INR remained lower than IR
thermal denaturation and aggregation of Lab subunits was of for a same protein amount deposited (100 mg) in the lane and pre-
peculiar interest (Fig. 2). For gel calibration, a linear relation was treated under each condition, in a same electrophoresis gel; this
established between the Lab band intensity and its relative con- was significant of the increased electrophoretic mobility and thus
centration in the lane according to different dilution factors used higher content in migrating polypeptides of the reduced sample.
for the unheated samples. Thus the decrease in the Lab band in- Therefore the noticeable decrease in INR with increasing tempera-
tensity was related to its decreasing concentration by thermal ture, in parallel to the Lab denaturation, was interpreted as the in-
denaturation, leading to either covalent aggregation or precipita- crease in the relative amount of covalent aggregates in heated
tion. The Lab concentration was shown to decrease drastically

Fig. 3. Change in protein composition (left axis) during heat-treatment (dotted line,
Fig. 2. Evolution of the legumin concentration (Lab subunit, z56e58 kDa, mgprotein/ right axis), as evaluated by densitometric analysis of the electrophoretic patterns of
gsoluble fraction, left axis) during the heat-treatment applied (dotted line, right axis: 1  C/ unheated and heated PP (1, 2 and 4 wt%) and fraction Leg (1 wt%) samples. (a): con-
min from 40 to 85  C, then 85  C from 0 to 60 min), as measured by densitometric centration ratio of the Lab subunit (z56e58 kDa), relative to that in the unheated
analysis of the electrophoretic patterns under NR conditions of unheated and heated sample (time 0, 40  C, Fig. 2). (b): calculated ratio 1
INR/IR of total lane intensity for a
PP (1, 2 and 4 wt% initial protein concentration; Fig. 1a for 1 wt%; 2 and 4 wt% are not same protein sample collected at given temperature and then pre-treated under NR
shown), and fraction Leg (Fig. 1b, 1 wt% protein). and R conditions, respectively.
240 J.-L. Mession et al. / Food Hydrocolloids 46 (2015) 233e243

samples; this was particularly prominent during the heating ramp buckwheat counterpart (Choi et al., 2006). Based on a mean average
(75e85  C/0 min). For heated Vic, PP and Leg samples at 1 wt%, the Mw of 58 kDa for the Lab subunit and thus 350 kDa for the hexamer,
calculated relative amounts of high-Mw covalent aggregates were legumin in the Leg sample would contain z1.2 mol S
free/mol. By
around 13%, 25% and 52%. Consequently these were more promi- comparison, O'Kane et al. (2005) reported for pea protein isolates
nent with increasing the initial legumin content in heated pea from various cultivars a S
free content in the range of 3e70 mmol
protein samples. Regarding the occurrence of the LA aggregates S
free/gprotein, strongly suggesting that the pea proteins used here
(85 kDa) in the heated Leg sample, their concentration at 85  C/60 contained a few sulfur-amino acids.
was practically ten-fold lower (0.4 mgLA/gsolution) than that for non- The S
free content of heated and aggregated PP, Vic and Leg
migrating ones (>200 kDa). samples at various concentrations was evaluated, to determine the
From the densitometric profiles of SL and SP lanes, it should be formation of new disulfide bridges (Table 4). For all the initial
noted that band intensities under NR conditions of unbound La protein concentrations investigated, heated Vic samples had the
and Lb polypeptides were not affected upon heating; there was no lowest S
free content, while contents were not found to differ be-
significant dissociation of legumin subunits by extensive dena- tween heat-treated PP and Leg. By considering separately each
turation, thus no preferential involvement of La or Lb polypeptides protein fraction, the S
free content was unaffected for PP and Vic
into covalent aggregation. This constituted a crucial difference samples, while it increased two-fold for Leg. Such a tendency was
with heat-treated 11S from buckwheat or soy glycinin (Choi et al., independent of the initial protein concentration that was pre-
2006). However, the hypothesized mechanism of pea legumin heated. The relatively constant S
free content for unheated and
thermal aggregation would be compared with that reported for heated PP samples was related to the balance between breakup of
oat globulin 11S, despite the use of less severe heat-treatment and existing disulfide bridges in non-denatured proteins and the new
lower ionic strength in the present study (Ma & Harwalkar, 1987); ones established by thermal denaturation and covalent aggregation
with respect to Td values of PP and Leg samples (Table 2), loss of (Choi et al., 2006). For the fraction Vic, the low S
free content in the
the legumin oligomeric structure by weakening of hydrophobic unheated sample would minimize potentially disulfide bonding by
interactions and subsequent S
/SeS exchange reactions between heat-treatment. Concerning heated Leg samples, the increase in
the Lab subunits were suggested to be initiated from 75  C. S
free content would originate from the disruption of preexisting
Intermolecular disulfide bonding between the Lab subunits in the disulfide bridges. Therefore upon heat-treatment, the denatured
early stages of heat-treatment would decrease the extent of their legumin subunits would rearrange into large aggregates via hy-
denaturation, and consequently further molecular rearrangements drophobic interactions and/or disulfide linkages, suggested to occur
via non-specific interactions were lowered. The unachieved legu- concomitantly (O'Kane et al., 2005). However a few released S
free
min unfolding was assumed to result in stiff protein aggregates, groups would be involved in the formation of intermolecular di-
which remained in majority soluble contrary to that was observed sulfide bonds, since the simultaneous exposure of hydrophobic
in the presence of NEM (Fig. 1b0 ). By incubating at 85  C, the de- groups would result in non-specific interactions between unfolded
natured legumin molecules would further aggregate covalently by polypeptide chains (Choi & Ma, 2005). Consequently the propensity
incorporating more Lab subunits, which involvement reached ul- for random aggregation of denatured legumin molecules would
timately (85  C/60 min) about 80e90 % of their initial amount decrease the accessibility of S
free groups.
(Fig. 3). In the following, the cold-set gelation procedure was performed
For heated PP at either 1 or 2 wt% protein concentration, Fig. 3 on the total (soluble þ insoluble) protein content of preheated
showed that disappearance and subsequent covalent aggregation samples (no centrifugation of the preheated Leg samples), to avoid
of the Lab subunits occurred similarly from 80  C, while these were protein loss and thus to allow direct comparisons between samples
slightly alleviated at 4 wt%. From this, it was suggested that both prepared at the same initial protein concentration.
steric hindrance and electrostatic repulsions caused by vicilin and
convicilin molecules in the PP samples could decrease aggregation 3.5. GDL-induced acid gelation of pea globulins
of denatured legumin via non-covalent bonds, possibly preventing
macro-aggregates formation. In fact the presence of convicilin was 3.5.1. Kinetics of acidification
previously shown to reduce gelation ability of pea protein samples Cold-gelation of preheated pea protein samples (85  C/60 min)
and thence aggregation via non-specific interactions, especially for was performed with added GDL. As extensively reported for whey
protein isolates of low cysteine content (O'Kane et al., 2005). proteins, the protons released with time by GDL hydrolysis led to
the progressive reduction of electrostatic repulsion between pro-
3.4. Sulfhydryl content tein aggregates. These were incorporated into growing clusters by
physical (non-covalent) interactions, which in turn could interact
Free sulfhydryl groups content (S
free, located on surface and
inside globulin structure) of unheated PP, Vic and Leg samples was
Table 4
evaluated in the presence of the denaturant GuHCl (Table 3). The PP
Free sulfhydryl contents (mmol S
free/gprotein) in unheated and heated pea protein
sample displayed the highest content, three-fold higher than that samples, prepared at various initial protein concentrations as indicated.
measured in both Leg and Vic samples. However legumin mole-
Sample Protein wt%
cules were reported to be the main sulfur-amino acids source
among pea globulins (O'Kane et al., 2005). Thus regarding the S
free Unheateda 1a 2a 3.5a 4a
content of PP, these could originate from pea albumins 2S of high PP 8.9 ± 0.8aA 6.5 ± 0.4aA 7.4 ± 1.0aA ND b
7.0 ± 0.3aA
cysteine content (Shewry et al., 1995). In turn, the S
free found in Leg 2.8 ± 0.4bB 5.4 ± 0.3aA 5.4 ± 0.1aA 5.3 ± 0.8A NDb
the Vic sample could arise from remaining albumins, the presence Vic 2.9 ± 0.4bA 2.3 ± 0.1bB NDb NDb 2.4 ± 0.2bB
of the disulfide-bonded NV polypeptide, and possibly convicilin Column values followed by the same lowercase letter (a and b) are not significantly
which could contain one or two cysteine residues (O'Kane et al., different (p < 0.05).
2005). The low S
free content in Leg may be on account of the Effect of the initial protein concentration (of the same protein fraction) that un-
derwent thermal denaturation and aggregation: row values followed by the same
pea cultivar from which the pea seeds originated from, and also the capital letter (A and B) are not significantly different (p < 0.05).
involvement to a large extent of sulfhydryl groups of legumin a
Means ± SD.
b
subunits in disulfide bridges, as has been mentioned for its 11S Not determined.
 J.-L. Mession et al. / Food Hydrocolloids 46 (2015) 233e243 241

physically with each other to form a space-filling network (Alting Table 5

et al., 2003). Gelation properties at 20  C of pre-heated protein samples (85  C/60 min) in the
presence of GDL, prepared at various initial protein concentrations.
The amount of GDL was adjusted owing to the buffering capacity
of proteins and their initial concentrations (Table 1). Hence, the Sample Viscoelastic Protein wt%
preheated PP, Vic and Leg samples displayed comparable kinetics of properties
1a 2a 3.5a 4a
acidification profiles (Fig. 4, the preheated PP samples were given b e
PP Gp (min) 10.1 ± 2.9bA 9.2 ± 2.3bA ND 5.6 ± 1.2bB
as an example); a pH decrease of 2 units occurred in the first 2 h, Gp (pH)b 6.7 ± 0.2aA 6.6 ± 0.2aA 6.7 ± 0.1aA
followed by a slower decrease of 0.7 units within 2 h. A pH value of G0 (Pa)c 52 ± 7aC 178 ± 13aB 567 ± 74aA
about 4.5e4.3 was measured after 4 h incubation with GDL, within tan dc 0.2 ± 0.0bA 0.2 ± 0.0bA 0.21 ± 0.01bA
the pH range (4e5) of minimum solubility as measured for pea Leg Gp (min)b No geld No geld 7.7 ± 1.5 NDe
proteins (Shand et al., 2007). The steady-state pH values of around Gp (pH)b 6.6 ± 0.0
4.0e4.3 were reached after 6 h incubation with GDL. It was ensured G0 (Pa)c 200 ± 55.9
tan dc 0.2 ± 0.0
that higher amounts of GDL were inadequate, since proteins re-
solubilized at pH values at equilibrium below 3.8; this was Vic Gp (min)b 20.7 ± 2.1aA 19.5 ± 2.1aA NDe 20.0 ± 2.0aA
Gp (pH)b 6.1 ± 0.2aA 5.9 ± 0.2bA 6.0 ± 0.1bA
ascribed to the increasing solubility of pea proteins at acidic pH
G0 (Pa)c 44 ± 12aB 84 ± 12bB 619 ± 40aA
(Mession et al., 2012). Regarding concentration ratios GDL/protein tan dc 0.23 ± 0.01aA 0.24 ± 0.01aA 0.23 ± 0.01aA
(wt/wt), it appeared that the heated fraction Leg had the lowest
To compare each viscoelastic property, column values followed by the same
buffering capacity. lowercase letter (a and b) are not significantly different (p < 0.05).
To overcome the effect of GDL hydrolysis, the time scale during Effect of initial protein concentration (of the same protein fraction) that underwent
gelation experiments was converted into pH values. thermal denaturation and aggregation: row values followed by the same capital
letter (A
C) are not significantly different (p < 0.05).
a
Means ± SD.
3.5.2. Rheological measurements b
Time (and corresponding pH) to observe the gelation point Gp, which was
Acid-induced gelation at 20  C of preheated/aggregated pea defined as the last cross-over of G0 and G00 moduli (tan d ¼ 1).
protein samples (listed in Table 1) was characterized using oscil- c
Elastic modulus (G0 ) and loss factor (tan d) plateau values.
d
latory rheological measurements. At the beginning of the acidifi- The rheological test evidenced wide discrepancies in measurements between
replicates; sample instability would arise from protein coagulation (no true gel).
cation process, all the protein exhibited a predominant viscous e
Not determined.
behavior (G00 > G0 , tan d > 1), till the elastic component increased
dramatically (G0 > G00 , tan d < 1) from the gel point noted Gp
(tan d ¼ 1), indicative of the solegel transition (Sun & Arntfield,
2010). Time to observe Gp and corresponding pH (as obtained concentration of 1 wt%, with final tan d values in the same range as
from the acidification profiles, Fig. 4) were measured (Table 5). that measured at higher protein concentration (Table 5). Structural
Preheated Leg samples at either 1 or 2 wt% initial protein concen- rearrangement within the acidified sample were suggested to occur
tration were unstable during the rheological measurements; at the onset of gelation, giving rise to clusters of protein aggregates
among replicated samples, some apparently evidenced gelation, via physical interactions, and consequently an increase in G0 value.
however G0 plateaued at values below 10 Pa, while tan d remained This was noticed previously for acid gels made of preheated whey
around 0.35; this could reflect coagulation rather than gelation, proteins (Alting et al., 2003). For both PP and Vic samples at 1 wt%
since the presence of macro-aggregates of low solubility were re- concentration, the protein amount was too low to form a self-
ported to be detrimental for gel formation (Sun & Arntfield, 2010). supporting gel (G0 at plateau lower than 100 Pa, Table 5). Mean-
It appeared that gelation was triggered at higher pH values for all while at 2 wt%, the increase in the G0 plateau value was of about
the PP samples than those found for Vic samples, around 6.6 and 6, four-fold and two-fold for PP and Vic, respectively; a longer gelation
respectively, with no significant difference regarding the various time (larger pH range) for PP than that for Vic would allow more
concentrations of each preheated protein samples. cross-links between the thermal aggregates, which arranged into
Surprisingly preheated PP and fraction Vic samples could form more structured network. In contrast, the Vic sample required a
weak gels (low G0 value at apparent plateau) from a protein higher level of acidification prior to Gp, possibly attributable to the
higher amount of repulsive charges between aggregates to screen.
The increase in G0 was more marked from 2 to 4 wt% preheated
protein concentration, than that observed from 1 to 2 wt%; a higher
amount of denatured protein were incorporated in the gel network
since the number of possible cross-links would increase (Fig. 5).
From Gp, a one hundred-fold G0 increase was observed in a pH
range of 6.7e6, 6.6e5.7 and 6e5.8 for preheated PP, Leg and Vic
samples at 4, 4 and 3.5 wt% initial protein concentration, respec-
tively. Therefore network formation was particularly straightfor-
ward from pH 6 concerning the Vic fraction. Protein network
strengthened while pH decreased, as G0 increased more rapidly
than G00 . The tan d values stabilized at pH 5.7, close to 0.2 for both PP
and Leg samples, whereas it was slightly higher for Vic. At pH 4.8,
both G0 and G00 moduli stabilized. The preheated PP and Vic samples
had close G0 plateau values (Table 5). Meanwhile, the G0 plateau
value for the Leg sample was shown to vary markedly between
replicated samples; as shown by O'Kane et al. (2004c) for heat-set
gels, legumin had the propensity to assemble into heterogeneous
network of coarse structure. As the fraction Leg displayed a higher
Fig. 4. Change in pH at 20  C in the presence of GDL, of pre-heated/aggregated PP minimum protein aggregation concentration to allow cold-gelation
samples prepared at various initial concentrations (protein wt%, Table 1). than those obtained for both PP and Vic, this was indicative of the
242 J.-L. Mession et al. / Food Hydrocolloids 46 (2015) 233e243

decrease protein aggregation via non-covalent interactions upon

thermal denaturation, and probably during acidification of the
cold-set gelation procedure (Table 3).

4. Conclusion

A GDL-induced cold-set gelation procedure was applied to pea

globulins extracted by ultrafiltration. Protein denaturation
occurred in a range of 75e85  C, while the S
/SeS interchange
reactions occurred between partially-denatured legumin subunits.
The legumin fraction in this study could produce insoluble macro-
aggregates that were detrimental for acid gel formation, whereas
the presence of vicilin and convicilin in the PP sample would alle-
viate the propensity of denatured legumin to form coagulum.
Acid gelation of preheated PP was triggered at pH higher than 6,
even at low protein concentrations; given that predominance of
non-covalent interactions within and between pea proteins ther-
Fig. 5. Variation of (a) storage modulus G0 and (b) tan d at 20  C as a function of mal aggregates, those would be easily destabilized upon acidifica-
decreasing pH in the presence of GDL, for preheated/aggregated PP, fractions Vic and tion, and their local rearrangements into large clusters would led to
Leg samples prepared initially at 4, 4 and 3.5 wt% protein concentration, respectively
a rapid increase in gel strength. Since it occurred in the early stages
(Table 1).
of acidification, rapid pea proteins gelation at low temperatures
would provide evidence of the versatility of preheated pea proteins
lower gelling abilities of preheated Leg. From Figs. 2e3 and Table 3, as an alternative food ingredient.
it was suggested that the concomitancy of thermal unfolding and
aggregation of legumin molecules led to random arrangements Acknowledgments
between partially-denatured proteins. Moreover, protein precipi-
tation in heated Leg samples would be indicative of macro- The Plateau de Rheologie des Materiaux Biologiques is thanked
aggregates formation, which was reported to be detrimental for for its technical support. The stakeholders in the food field that are
the formation of a structured gel network; more likely coagulum involved in a proactive approach to change food consumption
was obtained (Sun & Arntfield, 2010). models are as well gratefully acknowledged.
When viscoelastic moduli of acid gels were found to be stable,
frequency sweep tests were carried out (Fig. 6). Spectra displayed a References
G0 modulus higher than G00 in the whole frequency range. Due to the
relaxation of protein molecules that were not incorporated into the Alting, A. C., Hamer, R. J., de Kruif, C. G., & Visscher, R. W. (2003). Cold-set globular
gel network (viscous contribution), both moduli decreased at low protein gels: interaction, structure and rheology as a function of protein con-
centration. Journal of Agricultural and Food Chemistry, 51, 3150e3156.
frequencies. This was typical of food protein gels, which loose- Boye, J., Zare, F., & Pletch, A. (2010). Pulse proteins: processing, characterization,
gelled network exhibited viscoelastic properties (Alting et al., functional properties and application in food and feed. Food Research Interna-
2003); for acid gels, the protein network involved predominantly tional, 43, 414e431.
Bryant, C. M., & McClements, D. J. (1998). Molecular basis of protein functionality
physical bonds between pea protein thermal aggregates, which in
with special consideration of cold-set gels derived from heat-denatured whey.
turn could rearrange extensively as their structure would be mainly Trends in Food Science and Technology, 9, 143e151.
held by non-covalent forces. Additionally, the tan d plateau values Chavan, U. D., McKenzie, D. B., & Shahidi, F. (2001). Functional properties of protein
isolates from beach pea (Lathyrus maritumus L.). Food Chemistry, 74, 177e187.
of about 0.2 were significant of weak acid gels, in particular
Choi, S.-M., & Ma, C.-Y. (2005). Conformational study of globulin from common
significantly weaker for Vic than that for PP at the same initial buckwheat (Fagopyrum esculentum Moench) by Fourier transform infrared
protein concentration (Table 5). As reported by O'Kane et al. (2005), spectroscopy and differential scanning calorimetry. Journal of Agricultural and
the higher convicilin content in Vic than that in PP was supposed to Food Chemistry, 53, 8046e8053.
Choi, S.-M., Mine, Y., & Ma, C.-Y. (2006). Characterization of heat-induced aggregates
of globulin from common buckwheat (Fagopyrum esculentum Moench). Inter-
national Journal of Biological Macromolecules, 39, 201e209.
Ellman, G. L. (1959). Tissue sulfhydryl groups. Archives Biochemistry Biophysics, 82,
70e77.
Laemli, U. K. (1970). Cleavage of structural proteins during the assembly of the head
of bacteriophage T4. Nature, 227, 680e685.
, C., & Gue
Larre guen, J. (1986). Large-scale purification of pea globulins: comparison
between six anion exchangers in medium pressure liquid chromatography.
Journal of Chromatography, 361, 169e178.
Lundqvist, J., Barron, J., Berndes, G., Berntell, A., Falkenmark, M., Karlberg, L., et al.
(2007). Water pressures and increases in food & bioenergy demand implica-
tions of economic growth and options of decoupling. In Scenarios of economic
growth and resource demand, Background to the Swedish Environmental Advisory
Council memorandum (Vol. 1).
Ma, C.-M., & Harwalkar, V. R. (1987). Thermal coagulation of oat globulin. Cereal
Chemistry, 64, 212e218.
Marcone, M. F., Kakuda, Y., & Yada, R. (1998a). Salt-soluble seed globulins of
dicotyledonous and monocotyledonous plants e I: isolation, purification and
characterization. Food Chemistry, 62, 27e47.
Marcone, M. F., Kakuda, Y., & Yada, R. (1998b). Salt-soluble seed globulins of
dicotyledonous and monocotyledonous plants e II: structural characterization.
Fig. 6. Frequency-dependence at 20  C of storage modulus (G0 ) and loss modulus (G00 ) Food Chemistry, 63, 265e274.
for pre-heated/aggregated PP, fractions Vic and Leg samples at 4, 4 and 3.5 wt% initial Mession, J.-L., Assifaoui, A., Lafarge, C., Saurel, R., & Cayot, P. (2012). Effect of pea
protein concentration, respectively, at the end of the acid-induced gelation (about 4 h proteins extraction and vicilin/legumin fractionation on the phase behavior in
incubation with GDL, pH z4.5e4.3). admixture with alginate. Food Hydrocolloids, 29, 335e346.
 J.-L. Mession et al. / Food Hydrocolloids 46 (2015) 233e243 243

Mession, J.-L., Sok, N., Assifaoui, A., & Saurel, R. (2013). Thermal denaturation of Pea Shewry, P. R., Napier, J. A., & Tatham, A. S. (1995). Seed storage proteins: structure
globulins (Pisum sativum L.) e molecular interactions leading to heat-induced and biosynthesis. The Plant Cell, 7, 945e956.
protein aggregates. Journal of Agricultural and Food Chemistry, 61, 1196e1204. Sun, X. D., & Arntfield, S. D. (2010). Gelation properties of salt-extracted pea protein
O'Kane, F., Happe, R. P., Vereijken, J. M., Gruppen, H., & van Boekel, M. A. J. S. induced by heat treatment. Food Research International, 43, 509e515.
(2004a). Characterization of pea vicilin. 1. Denoting convicilin as the a-subunit Sun, X. D., & Arntfield, S. D. (2011). Gelation properties of salt-extracted pea protein
of the Pisum Vicilin Family. Journal of Agricultural and Food Chemistry, 52, isolate induced by heat treatment: effect of heating and cooling rate. Food
3141e3148. Chemistry, 124, 1011e1016.
O'Kane, F., Happe, R. P., Vereijken, J. M., Gruppen, H., & van Boekel, M. A. J. S. Sun, X. D., & Arntfield, S. D. (2012). Molecular forces involved in heat-induced pea
(2004b). Characterization of Pea Vicilin. 2. Consequences of compositional protein gelation: effects of various reagents on the rheological properties of
heterogeneity on heat-induced gelation behavior. Journal of Agricultural and salt-extracted pea protein gels. Food Hydrocolloids, 28, 325e332.
Food Chemistry, 52, 3149e3154. Taherian, A. R., Mondor, M., Labranche, J., Drolet, H., Ippersiel, D., & Lamarche, F.
O'Kane, F., Happe, R. P., Vereijken, J. M., Gruppen, H., & van Boekel, M. A. J. S. (2011). Comparative study of functional properties of commercial and mem-
(2004c). Heat-induced gelation of pea legumin: comparison with soybean brane processed yellow pea isolates. Food Research International, 44,
glycinin. Journal of Agricultural and Food Chemistry, 52, 5071e5078. 2505e2514.
O'Kane, F. E., Vereijken, J. M., Gruppen, H., & van Boekel, M. A. J. S. (2005). Gelation Tzitzikas, E. N., Vincken, J.-P., de Groot, J., Gruppen, H., & Visser, R. G. F. (2006).
behavior of protein isolates extracted from 5 cultivars of Pisum sativum L. Genetic variation in pea seed globulin composition. Journal of Agricultural and
Journal of Food Science, 70, 132e137. Food Chemistry, 54, 425e433.
Shand, P. J., Ya, H., Pietrasik, Z., & Wanasundara, P. K. J. P. D. (2007). Physicochemical USDA. (2014). A reference guide to important soybean facts & figures. Soystats.com.
and textural properties of heat-induced pea protein isolate gels. Food Chemistry, Website.
102, 1119e1130.