Composition and structure of carob (Ceratonia siliqua L.) germ proteins

Abstract

This study was focused on the analysis of the chemical composition of defatted carob germ flour and the protein isolate. The amino acid composition and the nature of the subunits that compose carob germ proteins were also studied. Isolate was obtained by alkaline extraction followed by isoelectric precipitation of proteins. Results showed that an isolate of 96.5% of protein content was obtained. A high amount of amino acids like glutamic acid, aspartic acid and arginine was detected. Carob proteins were found to be composed by aggregates formed by a 131 and 70kDa subunits linked by non-covalent bonds, and other peptides strongly bounded by disulfide interactions. Both, aggregates and subunits were formed mainly by 100 and 48kDa monomers linked by disulfide bonds. A considerable content of high molecular mass peptides (HMWP) strongly bounded were also found. Proteins became partially denatured and thermally stabilized at acid pH (pH 2). These results could be useful in the study of different functional properties of carob germ proteins, and the application of these proteins as nutritional ingredients in formulated food.

Full text

Composition and structure of carob (Ceratonia siliqua L.) germ proteins
C. Bengoechea
a
, A. Romero
a
, A. Villanueva
b
, G. Moreno
b
, M. Alaiz
b
,
F. Milla
´n
b
, A. Guerrero
a
, M.C. Puppo
c,*
a
Departamento de Ingenierıa Quımica, Facultad de Quımica, Universidad de Sevilla, c/P. Garcıa Gonza
´lez, 1, 41012 Sevilla, Spain
b
Instituto de la Grasa (CSIC), Av. Padre Garcıa Tejero 4, 41012 Sevilla, Spain
c
CIDCA (CONICET), Facultad de Ciencias Agrarias y Forestales (UNLP), 47 y 116 s/n, 1900 La Plata, Argentina
Received 17 April 2007; received in revised form 23 August 2007; accepted 24 August 2007
Abstract
This study was focused on the analysis of the chemical composition of defatted carob germ flour and the protein isolate. The amino
acid composition and the nature of the subunits that compose carob germ proteins were also studied. Isolate was obtained by alkaline
extraction followed by isoelectric precipitation of proteins. Results showed that an isolate of 96.5% of protein content was obtained. A
high amount of amino acids like glutamic acid, aspartic acid and arginine was detected. Carob proteins were found to be composed by
aggregates formed by a 131 and 70 kDa subunits linked by non-covalent bonds, and other peptides strongly bounded by disulfide inter-
actions. Both, aggregates and subunits were formed mainly by 100 and 48 kDa monomers linked by disulfide bonds. A considerable con-
tent of high molecular mass peptides (HMWP) strongly bounded were also found. Proteins became partially denatured and thermally
stabilized at acid pH (pH 2). These results could be useful in the study of different functional properties of carob germ proteins, and
the application of these proteins as nutritional ingredients in formulated food.
Ó2007 Elsevier Ltd. All rights reserved.
Keywords: Carob proteins; Chemical analysis; Amino acid composition; Protein solubility; Protein subunits; Thermal properties
1. Introduction
The carob tree (Ceratonia siliqua L.) is a typical Medi-
terranean plant (Avallone, Plessi, Baraldi, & Monzani,
1997; Dakia, Wathelet, & Paquot, 2007; Yousif & Algh-
zawi, 2000). In many Arabian countries, the fruit is used
for preparing popular beverages and confectionery. In
Western countries, carob powder is produced by deseeding
carob pods. The kibbled carob thus obtained is finally
roasted and milled (Yousif & Alghzawi, 2000). Carob pods
are characterized by high sugar content (more than 50%)
mainly composed of sucrose. Carob powder is a natural
sweetener with flavor and appearance similar to chocolate;
therefore it is often used as cocoa substitute. The advantage
of using carob as a chocolate substitute resides in that
carob is an ingredient free from caffeine and theobromine.
In Europe several carob commercial products can be found
as Carovit
TM
(Alimcarat S.L., Spain). Carovit
TM
is roasted
carob flour used as a cocoa substitute in baking, cereal
bars, chocolate confectionery, ice creams and light prod-
ucts. Other products, such as carob germ flour contains
high protein content, almost 50%, with a high content of
lysine and arginine. Carob germ flour is used as dietetic
human food (Dakia et al., 2007) or as a potential ingredient
in cereal-derived foods for celiac people (Feillet & Roul-
land, 1998).
The protein content of carob germ flour obtained from
seeds is higher than those observed for other beans such
as faba bean (Viciafaba L.), pea (Pisum stivum L.) and soy-
bean (Glycine max. Merr.). Maza et al. (1989) found values
of 48.4% for protein content for carob germ defatted flour,
while Marcone, Kakuda, and Yada (1998) determined pro-
tein values of 18.83% and 34.35% for pea and soybean
seeds, respectively. Caroubin, the water-insoluble protein
0308-8146/$ - see front matter Ó2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2007.08.069
*
Corresponding author. Fax: +54 221 4254853.
E-mail address: mcpuppo@quimica.unlp.edu.ar (M.C. Puppo).
www.elsevier.com/locate/foodchem
Available online at www.sciencedirect.com
Food Chemistry 107 (2008) 675–683
Food
Chemistry

isolated from carob bean embryo, is a mixture composed of
a large number of polymerized proteins of different size
(Wang et al., 2001). This protein system has been reported
to possess similar rheological properties to gluten, though
caroubin has a more ordered structure, with minor changes
in secondary structure when hydrated.
The chemical composition of carob pods (Avallone
et al., 1997; Calixto & Canellas, 1982; Yousif & Alghzawi,
2000) and carob germ meals (Dakia et al., 2007) has been
studied by several authors. Nevertheless, the chemical com-
position in relation to structural and thermal properties of
carob germ proteins has not yet been deeply studied. It is
well known that carob germ proteins have a well-balanced
amino acid composition. These proteins could be used as
healthy ingredients in nutraceutical foods and can consti-
tute a new food source for different population sectors.
Therefore, the aim of this work is to study the chemical
composition and structure properties of a carob germ pow-
der and a carob protein isolate.
2. Materials and methods
2.1. Preparation of carob protein isolate
Carob protein isolate (CI) was prepared from commer-
cial carob germ flour (Caratina) produced by Alimcarat
(Alimcart S.L., Mallorca, Spain) with a composition of
46% protein, 5% fiber, 7% soluble sugars and 25% other
carbohydrates, 7% lipids and 6% ash. Carob germ flour
was defatted by hexane. Aliquots of 350 g of flour were
inserted in cylindrical cartridges (3 cm diameter 20 cm
height) made with filter paper. All cylinders were intro-
duced in a bag that was perfectly sealed. Samples were per-
colated three times at 50 °C with hexane (150 L) and then
macerated during four days. Flour was put into trays and
excess of hexane was eliminated by evaporation into the
air at room temperature and then in air oven 48 h at
30 °C. Flour was stored at room temperature until used.
Sifted (mesh size 0.9 mm) flour was dispersed in water
(8% w/v). The original pH of this flour dispersion was
6.8. The dispersion was adjusted to pH 10.5 with 25%
NaOH, stirred at room temperature for 2 h and centrifuged
at 9000gfor 15 min at 4 °C in a RC5C Sorvall centrifuge
(Sorvall Instruments, Wilmington, DE, USA). The super-
natant was then adjusted to pH 4.0 with 6 N HCl and cen-
trifuged at 9000gfor 10 min at 4 °C. The pellet was washed
once and then resuspended with distilled water. Protein dis-
persion was introduced in a lab S1 spray dryer (ANHY-
DRO, Copenhagen, Denmark) at a 7 L/min flow rate
with an intake temperature of 190 °C. The isolate was only
1 min inside the spray dryer and the temperature at the out-
flow was 90 °C.
2.2. Determination of protein isoelectric point (IEP)
For determination of the IEP of germ flour proteins,
aqueous flour dispersions (0.96 g protein/40 mL) were
prepared and pH of different aliquots were adjusted with
6 N NaOH to pH 6, 8, 10 and 12; and with 6 N HCl to
pH 2. Dispersions were centrifuged and supernatants ana-
lyzed (%N 6.25) using a LECO CHNS-932 nitrogen
micro analyzer (Leco Corporation, St. Joseph, MI, USA)
(Etheridge, Pesti, & Foster, 1998). Percentages of soluble
protein in the supernatants in relation to the total protein
extracted were plotted vs. pH. We assume the IEP as the
minimum protein solubility.
2.3. Chemical composition of carob flour and isolate
Protein, lipid, moisture and ash contents were deter-
mined using AOAC (1990) approved methods. Protein
content of carob germ flour, carob protein isolate and
aqueous flour dispersions was determined as %N 6.25
using a LECO CHNS-932 nitrogen micro analyzer (Leco
Corporation, St. Joseph, MI, USA) (Etheridge et al.,
1998). Soluble sugars and polyphenols were measured
using a method described previously by Maza et al.
(1989). Standard curves of glucose and chlorogenic acid
were used. Total fiber was determined according to the pro-
cedure described by Lee, Prosky, and De Vries (1992).
2.4. Protein composition of carob germ flour and carob
protein isolate
2.4.1. Protein comparison of carob germ flour and isolate
(SDS–PAGE)
Protein composition was analyzed by sodium dodecyl
sulfate–polyacrylamide gel electrophoresis (SDS–PAGE).
Carob germ flour (CF) and carob protein isolate (CI) pro-
teins were extracted for 2 h at 20 °C with a buffer containing
0.025 M Tris–HCl and 0.1% SDS, 5% b-mercaptoethanol at
pH 8.4. Electrophoresis was performed according to the
method of Laemmli (1970) using a 20% gel and a 5% stack-
ing gel prepared with 30% Acrylamide/Bisacrylamide
solution.
2.4.2. Analysis of carob protein isolate under different
extraction conditions
2.4.2.1. Extraction of proteins. Proteins present in carob iso-
late were extracted and analyzed by electrophoresis under
different denaturing (method A), denaturing and reducing
(method B) and native (method C) conditions. Isolate was
extracted for 2 h at 20 °C with a 0.086 M Tris-base,
0.045 M mM glycine, 2 mM EDTA, 0.5% SDS pH 10 buffer
in method A; while in method B, proteins were dissolved in
the same buffer containing 1% DTT. In method C, proteins
were extracted at pH 2 with 2 N HCl and at pH 10 with
sodium borate buffer (0.1 M). With the aim of knowing the
species that form proteins extracted at these extreme pH val-
ues, these extracts were also treated with b-mercaptoethanol.
Dispersions were centrifuged at 10,000gfor 10 min at 15 °C.
2.4.2.2. SDS–PAGE. These assays were performed using
a 10% continuous running gel with a 4% stacking gel. A
676 C. Bengoechea et al. / Food Chemistry 107 (2008) 675–683

dissociating buffer system was used, containing 1.5 M Tris-
base, 0.5% SDS, pH 8.8 for the separating gel and 0.125 M
Tris-base, 0.96 M glycine, and 0.5% SDS, pH 8.3, for the
running buffer. Electrophoresis was performed in a Mini
Protean III at a constant voltage of 60 V (stacking gel)
and 120 V (continuous gel) with a Power-Pack 300 (Bio-
Rad, Richmond, CA, USA). Low MW markers (Pharma-
cia calibration kit) used included phosphorylase b
(94 kDa), albumin (67 kDa), ovalbumin (43 kDa), carbonic
anhydrase (30 kDa), trypsin inhibitor (20.1 kDa) and a-
lactalbumin (14.4 kDa).
2.4.2.3. Native-PAGE. Electrophoretic mobility of proteins
extracted by method C was analyzed by native electropho-
resis. Native electrophoresis was performed in a 7% poly-
acrylamide running gel with a 3.5% polyacrylamide
stacking gel. A non-dissociating buffer system containing
1.5 M Tris-base, pH 8.8 for the separating gel and
0.125 M Tris-base, 0.96 M glycine pH 8.3, for the running
buffer was used. Soybean proteins isolate (SPI) and soy-
bean 11S globulins (11S), prepared according to Puppo
and An
˜o
´n (1999), were used as standards.
2.5. Amino acid analysis
Samples containing 2 mg of protein were hydrolyzed
using 6 N HCl at 110 °C for 20 h under inert nitrogen
atmosphere. Derivatization of amino acids was performed
at 50 °C during 50 min with an excess of diethyl ethoxym-
ethylenemalonate. Amino acids were analyzed by reversed-
phase high-performance liquid chromatography (HPLC)
using D,L-a-aminobutyric acid as internal standard as
described (Alaiz, Navarro, Giron, & Vioque, 1992). Tryp-
tophan was analyzed by HPLC after basic hydrolysis
according to Yust et al. (2004).
2.6. Thermal analysis of carob proteins
The extent of thermal denaturation of carob germ pro-
teins was evaluated by differential scanning calorimetric
(DSC) measurements (Puppo & An
˜o
´n, 1999). Dispersions
(8% w/v) of isoelectric isolate (CI4) were adjusted to pH
2 (CI2) and pH 10 (CI10) with 2 N HCl and 2 N NaOH,
respectively; and then freeze-dried. Aqueous dispersions
of carob flour (CF) and isolates (CI2, CI4, CI10) at 20%
w/v were analyzed. Tests were performed in duplicate in
hermetically sealed aluminum capsules, where sample and
reference were gradually heated between 20 and 130 °C,
at 10 °C min
1
. Capsules were punctured after each run
and kept overnight in an oven to determine dry matter con-
tent. Assays were performed in a Rheometric Scientific
DSC calorimeter (Polymer Laboratories, UK). The equip-
ment was calibrated using indium, lauric acid and stearic
acid as standards (Rheometric Scientific, Polymer Labora-
tories, UK). Denaturation temperature (Td) and transition
enthalpy (DH) were obtained by analyzing the thermo-
grams with a Software Plus V5.41 (UK).
3. Results and discussion
3.1. Chemical composition of carob germ flour and carob
isolate
A protein isolate may be an attractive ingredient to use
in the production of human food, as it has reduced levels of
undesirable components, like fiber, lipids or darkening
agents (Maza et al., 1989). Compositional data for both
carob flour and isolate are shown in Table 1. The isolate
and the flour have a protein content of 96.5% and 48.6%,
respectively. The results for the protein isolate are in con-
cordance with the criteria of the minimum protein content
used in obtaining a protein isolate (higher than 85%). In
the case of other legumes, like soybean, a protein concen-
trate must have at least a protein content of 70%, while a
protein isolate should have at least 85% (dry basis,
N6.25) (Pearson, 1983, chapter 20). Different factors,
such as the high percentage of protein obtained or the drop
in the content of lipid, ash, humidity, soluble sugars and
total fiber, show that the procedure based on alkaline sol-
ubilization and isoelectric precipitation was an effective
way to get a carob isolate with a high protein content. This
method was used previously for other legumes, such as
soybean, lupine and beach pea (Chavan, McKenzie, &
Shahidi, 2001; Pozani, Doxastakis, & Kiosseoglou, 2002;
Puppo & An
˜o
´n, 1999) and in a pseudo cereal with a protein
content similar to that of legumes, as amaranth (Martı
´nez
&An
˜o
´n, 1996).
3.2. Solubility profile of carob proteins
The solubility profile of carob germ flour was obtained
in order to know the pH corresponding to the isoelectric
point of carob proteins. The knowledge of the minimum
protein solubility is essential to carry out the isolation pro-
cess. Flour dispersion (8% w/v) has a pH of 6.8. Fig. 1
shows that the isoelectric point (IEP) of carob germ pro-
teins is located near pH 4.0. This means that the proteins
are more soluble at acidic (pH < 2.5) and especially at alka-
line pH values (pH > 7.0). Solubility is an important prop-
erty when functional properties as gelation, emulsification
or foaming are considered.
Table 1
Chemical characterization of defatted flour and the isolate obtained from
carob germ meal
a
Flour Isolate
Protein content (%) 48.2 ± 0.24 96.5 ± 0.71
Lipids (%) 2.26 ± 0.13 0.58 ± 0.01
Moisture (%) 5.76 ± 0.32 2.81 ± 0.03
Ash (%) 6.34 ± 0.15 1.61 ± 0.23
Polyphenols (%) 0.45 ± 0.01 0.56 ± 0.01
Soluble carbohydrates (%) 2.92 ± 0.03 0.27 ± 0.03
Total fiber (%) 24.3 ± 0.09 0.95 ± 0.02
a
Results are expressed as the mean ± standard deviation of two
determinations.
C. Bengoechea et al. / Food Chemistry 107 (2008) 675–683 677

As a result, the carob protein isolate was obtained by
precipitation at pH 4.0 before spray drying.
3.3. Protein composition of carob germ flour and carob
protein isolate
3.3.1. Protein profile
3.3.1.1. Comparison between flour and isolate. SDS–PAGE.
Treatment with denaturing and dissociating agents
(SDS + b-mercaptoethanol) made it possible to extract
from both the flour and the isolate four protein fractions
(131, 102, 48 and 24 kDa), with the 131 and the 48 kDa
being the more abundant fractions (Fig. 2). There were
no qualitative or quantitative differences between the pro-
tein fractions of the flour or the isolate. The 48 kDa band
of carob proteins possesses a molecular weight similar to
those presented by the subunit of 11S globulins present in
vegetable proteins, as soybean conglycinin, pea legumin,
fababean, lupine globulin and globulin-P of amaranth
(Abdellatif et al., 2005; Martı
´nez & An
˜o
´n, 1996; O’Kane,
2004, chapter 5; Puppo & An
˜o
´n, 1999). Nevertheless, more
data are needed to confirm that this subunit belongs to an
11S-type globulin. The 11S globulins are formed by sub-
units of approximately 50 kDa linked by non-covalent
bonds (AB subunits). These subunits are known to be con-
stituted by acidic (A polypeptide) of 30 kDa and basic (B
polypeptide) of 20 kDa polypeptides linked by disulfide
bonds (Puppo & An
˜o
´n, 1999). Therefore, in dissociation
and reducing conditions, the AB subunit should be absent,
and a great proportion of A and B polypeptides should be
found. Due to the absence of the 30 and 20 kDa polypep-
tide, the 24 kDa band could be a new kind of low molecu-
lar mass polypeptide, that is not present in other legumes.
Similar electrophoretic profiles were obtained by other
authors for carob seed germ proteins (Dakia et al., 2007)
and caroubin (Feillet & Roulland, 1998).
3.3.1.2. Analysis of the carob proteins under different
extraction conditions
3.3.1.2.1. SDS-PAGE. Extraction with SDS. Electropho-
retic profile of isoelectric carob proteins (pI= 4) extracted
at pH 10 with SDS and in the absence and presence of a
reducing agent (DTT) are shown in Fig. 3a. Soluble aggre-
gates of high molecular mass (HMMA) (>138 kDa) and
the 131 kDa polypeptide previously described was
observed. A polypeptide of 70 kDa and small amount of
the 48 kDa protein were also detected (Fig. 3a-1). The
incorporation of DTT (Fig. 3a-2) produced the rupture
of disulfide bonds and therefore the proportion of HMMA
diminished considerably. Proteins of HMMA were there-
fore strongly aggregated by covalent disulfide linkages.
These aggregates were mainly composed of monomer sub-
units of 100 and 48 kDa mainly linked by disulfide bonds.
Moreover, a high amount of the aggregates of high molec-
ular mass were retained in the stacking gel. The disappear-
ance of the 70 kDa band and the appearance of the 48 kDa
subunit indicate that the 70 kDa protein would be a dim-
mer protein stabilized by the 48 and 24 kDa subunits (the
last one did not appear in the gel) linked by disulfide bonds.
A polypeptide of 70 kDa was also obtained in an amaranth
protein isolate extracted at pH 10, that belongs to the 11S
globulin fraction (Martı
´nez & An
˜o
´n, 1996). Amaranth is a
cereal-like crop with high seed protein content composed
mainly by globulin-P, globulin 11S and glutelins. In a
guava seeds protein isolate, analyzed under dissociating
conditions, a 70 kDa polypeptide was also observed (Ber-
nardino-Nicanor, Scilingo, An
˜o
´n, & Da
´vila-Ortiz, 2006).
In reducing conditions, this peptide was dissociated in sev-
eral peptides including 48 kDa and 24 kDa monomers.
On the other hand, carob proteins presented a
high amount of HMMA formed mainly by the 100 kD
0
10
20
30
40
50
60
70
80
0 4 8 10 12 14
pH
Soluble protein (%)
26
Fig. 1. Solubility profile (Solubility, % vs. pH) of carob germ flour.
Fig. 2. SDS–PAGE of carob germ flour and carob isolate proteins
analyzed under reducing conditions: CF, carob flour; CI, carob isolate;
LMW: low molecular weight markers.
678 C. Bengoechea et al. / Food Chemistry 107 (2008) 675–683

monomer. This monomer is not present in soybean, neither
in amaranth and guava seeds. Therefore, carob proteins
presented different structure than those observed in other
seeds.
Extraction without SDS. Electrophoretic profile (SDS–
PAGE) of carob proteins extracted without denaturing
agents, like SDS, is shown in Fig. 3b-1. Proteins that
are linked by non-covalent bonds such as electrostatic,
Fig. 3. SDS–PAGE of proteins extracted from carob germ isolate. (a) Proteins extracted under dissociating (SDS – lane 1) and reducing (SDS + DTT –
lane 2) conditions. (b) Proteins extracted at pH 10 (CI10) and pH 2 (CI2): (gel 1) electrophoretic sample buffer with 0.5% SDS; (gel 2) electrophoretic
sample buffer with 0.5% SDS + 5% b-mercaptoethanol. LMW: low molecular weight markers.
C. Bengoechea et al. / Food Chemistry 107 (2008) 675–683 679

hydrogen, van der Waals and hydrophobic interactions,
are able to be extracted at extreme pH. At pH values above
and below the pI, where protein has negative and positive
charge, protein unfolds interacting with water and increas-
ing its solubility (Vodjani, 1996, chapter 2). The extraction
performed at pH 10 and pH 2 (CI10 and CI2) showed a
great proportion of HMMA (>120 kDa) and high amounts
of the 70 kDa polypeptide. Lower amount of 120, 45 and
32 kDa subunits were observed. Extracts at extreme pH
values presented the same protein profile of that observed
for the SDS extract of the isoelectric isolate (Fig. 3b-1).
Results suggest that a high amount of these proteins are
greatly stabilized by non-covalent bonds such as hydrogen
bonds, van der Waals bonds, ionic and hydrophobic inter-
actions. Proteins of 48 and 100 kDa (Fig. 3a) present in the
aggregates were only observed in the presence of the reduc-
ing agent, indicating that they would be linked by disulfide
bonds.
No differences in the electrophoretic profile between
proteins extracted with an SDS buffer containing b-
mercaptoethanol (Fig. 3a-2) and proteins extracted only
with SDS and then reduced by b-mercaptoethanol
(Fig. 3b-2), were observed. Both extraction procedures
showed that HMMA and peptides of 70 kDa were
mainly made up of the 48 kDa subunit through disulfide
bonds.
3.3.1.2.2. Native-PAGE. Carob protein isolate, at acid
(CI2) and mainly at alkaline pH (CI10), presented only
one protein fraction with lower electrophoretic mobility
in comparison to fractions observed for the 11S soybean
glycinin of pH 8. Soybean isolate contains low and a
high electrophoretic mobility proteins corresponding to
the 11S and 7S subunits, respectively (Fig. 4 – SPI,
11S). No differences were observed between CI2 and
CI10 profiles. In addition, the typical 7S fraction was
not present. These results indicate that proteins present
in carob germ would belong to a different kind of protein
group, distinct from the 11S globulins observed in other
legumes.
3.3.2. Amino acid profile
Table 2 shows the amino acid profile of both the carob
germ flour and carob protein isolate. They both show a
high content of the non-essential amino acids, specially glu-
tamic or aspartic acid and arginine. Values of glutamic and
aspartic acid, for both samples (CF and CI), were higher
than those obtained by Maza et al. (1989). On the other
hand, in carob seed germ flours, our values were lower than
those obtained by Dakia et al. (2007). Low values of sul-
phur amino acids, methionine and cysteine were obtained;
with a Met + Cys content approximately 30% and 24% of
the values prescribed by the FAO–WHO (1991) for the
flour and the isolate, respectively. In addition, our values
of these amino acids were lower than those observed by
Dakia et al. (2007). The very low content of Met + Cys
may be due to the absence of oxidation before hydrolysis
in order to avoid the partial degradation of these amino
acids. The high content of Met + Cys (3.4%) reported by
the carob germ flour manufacturer (Caratina, Alimcart
S.L., Mallorca, Sapin), agree with the fact that this flour
is not limited in sulphur amino acids content. The content
in aromatic amino acids (Phe and Tyr) was also deficient,
showing values about 25% lower than the recommended
ones. The amount of the amino acid lysine was slightly defi-
cient in the isolate.
The quality of these proteins was estimated calculating
the chemical score (CS) as the ratio between the percent-
age of the essential amino acid of the sample (CF or CI)
to this amino acid in the standard (FAO–WHO (1991)).
The lowest number of these ratios is the limiting amino
acid. In our case, in addition to sulphur amino acids,
the other limiting amino acids were found to be phenyl-
alanine and tyrosine. Our results contrast with values
obtained by Dakia et al. (2007) in which tryptophan
was the limiting amino acid. On the other hand, compar-
ing the chemical score of carob germ proteins with those
obtained for other legumes, limiting amino acids of soy
proteins (Pearson, 1983, chapter 20; Seligson & Mackey,
1984) were valine and mainly sulphur amino acids, while
for lupin proteins (Sujak, Kotlarz, & Strobel, 2006) the
first and second limiting amino acids were Met + Cys
and tryptophan.
However, the high content in glutamic acid and arginine
would allow the use of carob proteins as a suitable ingredi-
ent for functional foods for sportspeople. These amino
acids play an important role in the nutrition of sportspeo-
ple, as they increase the muscular matter, the collagen syn-
thesis, and the glycogen production (Flynn, Meininger,
Haynes, & Wu, 2002; Varnier, Leese, Thompson, & Ren-
nie, 1995; Wernerman, 2002).
Fig. 4. Native-PAGE of isolate carob proteins extracted at pH 2 (CI2)
and pH 10 (CI10). Soybean proteins isolate (SPI) and 11S globulins (11S)
were used as standards.
680 C. Bengoechea et al. / Food Chemistry 107 (2008) 675–683

3.4. Thermal analysis of carob proteins
Fig. 5 shows the thermograms for the carob flour (CF)
and the isolates prepared at different pH values (CI2, CI4,
CI10). Both samples, flour and isolates, presented an
endotherm at different temperatures. Carob flour (pH 6.8)
presented an endotherm at 105.7 ± 0.3 °C with a denatur-
ation enthalpy of 16.6 ± 4.1 mJ/mg flour. Protein in the
isolate of pH 10 (CI10) was still in native form (DH=
17.7 ± 0.1 mJ/mg isolate) almost at the same denaturation
temperature (T
d
= 103.1 ± 1.2 °C) as that observed for
CF. At acid pH, the carob isolate (CI2) was denatured
and the endotherm was shifted to high temperatures at
extreme acid pH, as it can be deduced from the enthalpy
(5.0 ± 0.7 mJ/mg isolate) and the Td (115.2 ± 1.7 °C) val-
ues. This great thermal stabilization could be due to the
formation of a new protein structure due to the acid pH
effect. The isoelectric isolate (CI4) presented the lowest val-
ues of enthalpy and temperature of denaturation, being
3.8 ± 0.1 mJ/mg isolate and 90.5 ± 1.5 °C, respectively.
The low values of DHof proteins at the isoelectric point
have been reported previously for other legumin proteins,
like soy or fababean (Arntfield, Ismond, & Murray, 1990,
chapter 4). At the pI, there is an intense aggregation as a
consequence of hydrophobic interactions. This aggrega-
tion produces an increase in the exothermic processes
that would contribute to a decrease in the enthalpy. Other
leguminous proteins have been reported to present, at neu-
tral pH, endotherms in the same temperature range, like
soybean (92.7 °C), fababean (91.0 °C), field pea (94.5 °C)
and amaranth, a globulin-rich pseudocereal (94.0 °C) (Arnt-
field et al., 1990, chapter 4; Mart
ınez & An
˜o
´n, 1996; Puppo
&An
˜o
´n, 1999). This endotherm could be attributed to the
denaturation of the 11S globulin. Nevertheless, to confirm
the presence of an 11S type globulin in this isolate calorimet-
ric assay on an 11S fraction, purified from carob germ flour,
should be performed.
4. Conclusions
An isolate with protein content higher than 95% from
carob flour, applying the procedure of alkaline solubiliza-
tion followed by isoelectric precipitation, can be obtained.
Table 2
Amino acid composition (AAC, g amino acid/100 g protein)
c
and chemical score (CS % of FAO) of defatted flour and the isolate obtained from of carob
germ meal
Amino acids Carob flour (CF) Carob isolate (CI) FAO–WHO (1991) standards
AAC CS AAC CS
Aspartic acid 8.75 ± 0.07 – 8.55 ± 0.07 –
Glutamic acid 28.1 ± 0.07 – 30.2 ± 0.57 –
Arginine 11.5 ± 0.21 – 13.7 ± 0.28 –
Serine 5.05 ± 0.07 – 5.0 ± 0.3 –
Glycine 5 ± 0 – 4.9 ± 0.1 –
Alanine 4.4 ± 0.0 – 4.1 ± 0.0 –
Proline 8.2 ± 0.3 – 5.1 ± 0.3 –
Histidine 2.3 ± 0.0 121 2.4 ± 0.2 126 1.9
Threonine 3.5 ± 0.0 103 3.3 ± 0.2 97 3.4
Valine 3.05 ± 0.07 87 2.5 ± 0.3 71 3.5
Isoleucine 2.3 ± 0.0 82 2.15 ± 0.07 77 2.8
Leucine 5.9 ± 0.0 89 6.35 ± 0.071 96 6.6
Lysine 5.5 ± 0.0 95 4.9 ± 0.0 84 5.8
Tryptophan 0.9 ± 0.0 82 1.05 ± 0.07 95 1.1
Phenylalanine 2.9 ± 0.0 78
a
3±0 78
a
6.3
a
Tyrosine 2 ± 0 1.95 ± 0.07
Methionine 0 ± 0 32
b
0.05 ± 0.07 24
b
2.5
b
Cysteine 0.8 ± 0.0 0.55 ± 0.07
a
Phenylalanine + tyrosine.
b
Methionine + cysteine.
c
Results are expressed as the mean ± standard deviation of two determinations.
Temperature (ºC)
40 60 80 100 120
Heat flow (mJ/seg)
-5
-4
-3
-2
-1
CF
CI2
CI4
CI10
Fig. 5. DSC thermograms of aqueous dispersions (20% w/v) of carob
germ flour (CF) and carob germ isolate. CI2: protein precipitated at pH 4
and dissolved at pH 2, CI4: protein precipitated at pH 4, CI10: protein
precipitated at pH 4 and dissolved at pH 10.
C. Bengoechea et al. / Food Chemistry 107 (2008) 675–683 681

This isolate thus obtained presented, as in flour, high con-
tent of aspartic and glutamic acids, and arginine amino
acids. According to our results, the limiting amino acids
were methionine + cysteine and phenylalanine + tyrosine.
The isolate presented proteins with low electrophoretic
mobility and were mainly stabilized by HMMA. These
aggregates were formed by the 131 and 70 kDa subunits
linked by non-covalent bonds, and other peptides strongly
bounded by disulfide interactions. Both, aggregates and
subunits were formed mainly by 100 and 48 kDa mono-
mers linked by disulfide bonds. A considerable content of
high molecular mass peptides (HMWP) strongly bounded
were also found. These HMWP were not dissociated by
the combined effect of the SDS and DTT. No differences
in the nature of proteins present in the acid or alkaline iso-
lates were observed. Nevertheless, proteins presented
higher denaturation temperature and became more dena-
tured at acid pH (pH 2) than at pH 10.
Carob is a legume whose composition analysis, protein
characterization and thermal properties vary from results
obtained by other authors for other crops as soybean,
pea, lupine, amaranth and guava. The knowledge of the
nature of proteins that form carob materials like germ
flours and isolates, and its thermal properties would be
important from the point of view of the application of this
crop as ingredient in formulated foods.
Acknowledgement
This work was supported by Secretarı
´a de Estado de
Universidades e Investigacio
´n of Ministerio de Educacio
´n
y Ciencia of Spain (SAB2003-0314).
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