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


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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.
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Composition and structure of carob (Ceratonia siliqua L.) germ proteins
C. Bengoechea
, A. Romero
, A. Villanueva
, G. Moreno
, M. Alaiz
F. Milla
, A. Guerrero
, M.C. Puppo
Departamento de Ingenierıa Quımica, Facultad de Quımica, Universidad de Sevilla, c/P. Garcıa Gonza
´lez, 1, 41012 Sevilla, Spain
Instituto de la Grasa (CSIC), Av. Padre Garcıa Tejero 4, 41012 Sevilla, Spain
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
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
(Alimcarat S.L., Spain). Carovit
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.
Corresponding author. Fax: +54 221 4254853.
E-mail address: (M.C. Puppo).
Available online at
Food Chemistry 107 (2008) 675–683
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
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
2.4.2. Analysis of carob protein isolate under different
extraction conditions 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. 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). 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
´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
´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
. 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
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,
N6.25) (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
´n, 1999) and in a pseudo cereal with a protein
content similar to that of legumes, as amaranth (Martı
´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
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
Results are expressed as the mean ± standard deviation of two
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 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
´n, 1996; O’Kane,
2004, chapter 5; Puppo & An
´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
´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). Analysis of the carob proteins under different
extraction conditions 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
´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
´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 4 8 10 12 14
Soluble protein (%)
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
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
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. 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
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
= 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
´n, 1996; Puppo
´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)
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
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
3±0 78
Tyrosine 2 ± 0 1.95 ± 0.07
Methionine 0 ± 0 32
0.05 ± 0.07 24
Cysteine 0.8 ± 0.0 0.55 ± 0.07
Phenylalanine + tyrosine.
Methionine + cysteine.
Results are expressed as the mean ± standard deviation of two determinations.
Temperature (ºC)
40 60 80 100 120
Heat flow (mJ/seg)
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.
This work was supported by Secretarı
´a de Estado de
Universidades e Investigacio
´n of Ministerio de Educacio
y Ciencia of Spain (SAB2003-0314).
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... The carob pulp is characterized by high sugar content (40-60%), predominantly sucrose, appreciable amounts of protein (3-6%) and low levels of fat (0.2-1%) [2,3]. The carob seeds contain more protein (50%), more fat (3-6%) and less total sugars (1-5%) compared to the carob pulp [3,4]. ...
... It is a valuable natural food additive for its strong gel characteristics [5]. Other products, such as carob germ flour is used as dietetic human food or as a potential ingredient in cereal-derived foods for celiac people [4]. In Western countries, carob powder is produced from the roasted and milled pulp. ...
... The protein fraction of carob pods was composed mainly of seeds proteins that present an interesting biological value. In fact, Dakia, Wathelet, et Paquot [30] and Bengoechea et al. [4] detected an interesting amount of essential amino acids in carob germ seeds. The mineral fraction of carob pods (3.35 g/100 g dry matter) was predominated by potassium (332.47 mg/100 g dry matter), followed by calcium (147.91 mg/100 g dry matter), magnesium (101.34 mg/100 g dry matter) and sodium (52.65 mg/100 g dry matter). ...
This study presented the main nutritional composition of Tunisian carob pods, some properties of carob juice obtained by hot water extraction (50°C for 190 min) and the effects of thermal pasteurization (70°C for 15 min). The carob pods showed high amounts of sugar (~65 g/100 g dry matter), an appreciable content of protein (~10 g /100 g dry matter), significant content of ash (3.35 g/100 g dry matter) and low levels of lipids (0.28 g/100 g dry matter). The corresponding juice was characterized in terms of physical characteristics, nutritional composition, microbiological characteristics and sensory properties. Results showed high viscosity, high content of soluble sugars and absence of pathogenic. The sensory test presented high overall acceptability of carob juice by elders (80%) compared with a reference juice (fruit cocktail juice). Raw carob juice was thermally pasteurized at 70°C for 15 min. The effects of pasteurization on color and clarity, microflora and vitamin C content were investigated. An important reduction in microflora number was observed, especially the total flora from 1900 to 270 CFU/ml. Significant increases were also observed in color values from 2.87 to 3.01 and clarity values from 0.87 to 1.04. A significant decrease was detected in vitamin C content in pasteurized juice compared to raw juice.
... Owing to their excellent flavouring properties, the non-fleshy carob pods are utilized in many countries of the Mediterranean area to produce popular beverages, homemade cakes, pastries and confectionery Yousif & Alghzawi, 2000). Ground roasted carob pods constitute natural sweeteners, commonly employed as caffeine-and theobromine-free cacao and coffee surrogates (Bengoechea et al., 2008). Carob pod flour has been evaluated as a functional ingredient for wheat bread (Salinas et al., 2015) and successfully used as a suitable ingredient to produce gluten-free baked products as well (Tsatsaragkou et al., 2014). ...
... Seeds account for 10-15 % in weight of the carob pods, germ representing one third-to-half of the seed dry weight (Sciammaro et al., 2016a). Carob seed germ flour (SGF) is a protein-rich edible matrix, containing 48-55 % protein with a balanced content of essential amino acids (Fidan et al., 2020) and a nutritional profile similar to other Leguminosae such as soy (Bengoechea et al., 2008). The structural and functional properties of SGF proteins have been investigated (Dakia et al., 2007;Smith et al., 2010). ...
... SGF also contains other functionally and nutritionally interesting components, including unsaturated lipids, phospholipids and phenolic compounds (Custódio et al., 2011;Sciammaro et al., 2016aSciammaro et al., , 2016bSiano et al., 2018). By virtue of the exclusive composition and nutritional properties, SGF has potential to be used as an ingredient for special food formulations (Bengoechea et al., 2008;Dakia et al., 2007;Sciammaro et al., 2016b). The idea of using SGF as an ingredient for pasta or baked products is ancient, as it was proposed for increasing the level of glutenlike proteins in a US patent dating back to 1935 (Bienenstock et al., 1935). ...
Carob (Ceratonia siliqua L.) seed germ flour (SGF) is a by-product resulting from the extractionextraction of locust bean gum (E410), which is a texturing and thickening ingredient used for food, pharmaceutical and cosmetic preparations. SGF is a protein-rich edible matrix and contains relatively high amounts of apigenin 6,8-C-di- and poly-glycosylated derivatives. In this work, we prepared durum wheat pasta containing 5 and 10 % (w/w) of SGF and carried out inhibition assays against type-2 diabetes relevant carbohydrate hydrolysing enzymes, namely porcine pancreatic α-amylase and α-glycosidases from jejunal brush border membranes. Nearly 70-80% of the SGF flavonoids were retained in the pasta after cooking in boiling water. Extracts from cooked pasta fortified with 5 or 10% SGF inhibited either α-amylase by 53% and 74% or α-glycosidases by 62 and 69%, respectively. The release of reducing sugars from starch was delayed in SGF-containing pasta compared to the full-wheat counterpart, as assessed by simulated oral-gastric-duodenal digestion. By effect of starch degradation, the SGF flavonoids were discharged in the water phase of the chyme, supporting a possible inhibitory activity against both duodenal α-amylase and small intestinal α-glycosidases in vivo. SGF is a promising functional ingredient obtained from an industrial by-product for producing cereal-based foods with reduced glycaemic index.
... Furthermore, pulp (90% of the pods) contains high amounts of carbohydrates and dietary fiber, in addition to a variety of macro and microelements and minerals 6 . Carob fruits are free from gluten, therefore, they are used in producing gluten-free foods 7 , or as a cocoa substitute [8][9][10] . ...
... The scientific name (Ceratonia siliqua L.) derived from Greek keras (horn), and Latin siliqua, which refer to the form and the hardness of the pods 4,45 . In different Arabian countries, carob fruits are used for preparing traditional beverages and confectionery 8 . Carob syrup, traditionally prepared from the deseeded mature pods, is considered as an important energetic food in many carob producer countries, including Morocco 46 , Cyprus 4,47 and Turkey 48 . ...
... Carob powder from the pods is a natural sweetener that tastes and looks like chocolate, which is why it is often used as a cocoa substitute. The advantage of using carob is that, unlike chocolate, it does not contain stimulants because it is devoid of caffeine and theobromine [76]. Currently, carob is being investigated as a source of new natural antioxidants, specifically those found in the seed coat and the pulp of the fruit. ...
Full-text available
The carob tree (Ceratonia siliqua L.) is currently considered one of the most valuable fruit and forest trees in various fields and sectors of activity. It is a versatile plant, belonging to the Fabaceae family. It is widely used in traditional medicine to treat many diseases such as diabetes, hypertension, and gastrointestinal disorders, given that all its parts (leaves, flowers, pods, seeds, wood, bark, and roots) are useful and hold value in many areas. Its importance has increased significantly in recent years. Originating from the Middle East, it is recognized for its ecological and industrial significance. Previous studies conducted on Ceratonia siliqua L. have revealed the presence of several compounds, including polyphenols, flavonoids, carbohydrates, minerals, and proteins. The carob tree demonstrates antihypertensive, antidepressant, anti-obesity, and antihyperglycemic activities. This plant is known for its medicinal and therapeutic virtues. Moreover, it is particularly interesting to consider the pharmacological activities of the major phytochemical compounds present in the different extracts of this plant, such as phenolic acids, for example, coumaric and gallic acids, as well as flavonoids such as kaempferol and quercetin. Therefore, this review aims to analyze some aspects of this plant, especially the taxonomy, cytogeography, traditional uses, phytochemical constituents, and pharmacological activities of Ceratonia siliqua L., in addition to its biological properties.
... Due to its sweetness and flavor similar to chocolate, as well as its low price, the carob milled into flour is widely used in the Mediterranean region as a cocoa substitute for sweets, biscuits and processed drinks production [20][21][22][23]. Additionally, the advantage of using carob powder as a cocoa substitute is that it does not contain caffeine and theobromine [24]. ...
Full-text available
The aim of this research was to improve the physical-chemical properties and processability of wheat durum pasta while adding supplementary nutritional benefits. This was accomplished by incorporating carob powder into the conventional wheat pasta recipe. The study investigated the properties of pasta made with different proportions of carob powder (2%, 4%, 6% w/w) and evaluated its nutritional profile, texture, dough rheological properties and the content of bioactive compounds such as phenolic compounds. The physical and chemical properties (total treatable acidity, moisture content, and protein content), compression resistance, rheological properties of the dough and sensory analysis were also analyzed. Results showed that incorporating up to 4% carob powder improved the sensory and functional properties of the pasta. Additionally, the study found that the pasta contained phenolic compounds such as Gallic, rosmarinic, rutin and protocatechuic acids, ferulic, coumaric, caffeic acid, resveratrol and quercetin, and increasing the percentage of carob powder improved the polyphenolic content. The study concluded that it is possible to create innovative value-added pasta formulas using carob powder. Thus, the information revealed by this study has the potential to expand the portfolio of functional pasta formulations on the food market.
... Τέτοιες επικαλύψεις έχουν ένα λαμπρό τεχνολογικό μέλλον στις εξειδικευμένες αγορές των αεροναυτικών, διαστημικών, ναυτικών, κτηριακών, αυτοκινητοβιομηχανικών και βιοϊατρικών τομέων. Με πειράματα στο εργαστήριο αποκαλύφθηκαν οι υδρόφοβες και οι υπερυδρόφοβες ιδιότητες καθώς και η χημική σύσταση των επιφανειακών κηρών στις άνω και κάτω επιφάνειες του σύνθετου φύλλου (αποτελείται από φυλλάρια) της χαρουπιάς (Ceratonia siliqua L.), ενός μεσογειακού δέντρου με καρπούς που έχουν σημαντική εμπορική αξία 58 το οποίο αναπτύσσεται ως αυτοφυές σε χώρες της Μεσογείου και το όνομά του φτάνει ετυμολογικά μέχρι τα καράτια 59 (Bengoechea et al. 2008, Oziyci et al. 2014, El Batal et al. 2016, Rasheed et al. 2019. Οι γωνίες επαφής που έχουν μετρηθεί στα φύλλα της χαρουπιάς (Εικόνα 3.3) αποκαλύπτουν συνάφεια σταγόνων του νερού στη νανοδομημένη υφή. ...
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Η περί τη βιομιμητική (Biomimetic) έρευνα αντλεί έμπνευση από ιδιότητες ζωντανών οργανισμών που υπάρχουν στη φύση και ζουν σε διαφορετικούς βιοτόπους και διακριτά ενδιαιτήματα. Η βιομιμητική βασίζεται στη μελέτη, την ανάδειξη, την αντιγραφή και την προσομοίωση ιδιοτήτων και δομικών χαρακτηριστικών έμβιων οργανισμών που έχουν αποδεδειγμένα αντέξει περιβαλλοντικές καταπονήσεις. Έτσι, δομές και ιδιότητες ζωντανών οργανισμών (υδρόφοβες, οπτικές και άλλες ιδιότητες) που αποκαλύπτονται με τη βασική έρευνα χρησιμοποιούνται από την τεχνολογία δημιουργώντας ανταλλακτικές αξίες, θέσεις εργασίας, χρηστικά και πρωτοποριακά προϊόντα. Η βιομίμηση (Biomimicry) έχει σχέση με την απατηλή μίμηση χαρακτηριστικών ενός οργανισμού από έναν άλλο οργανισμό για ξεγέλασμα, για προσέλκυση ή άμυνα. Για παράδειγμα, τα άνθη ορισμένων φυτών όπως οι ορχιδέες μιμούνται, καθώς ανοίγουν και επιδεικνύουν τον φαινότυπό τους (χρώματα, σχήματα και συμμετρίες) όσον αφορά τη μορφολογία, ορισμένα έντομα για να τα προσελκύσουν. Επίσης, πεταλούδες και έντομα μιμούνται το φύλλωμα δέντρων και θάμνων για να μην γίνουν αντιληπτές/αντιληπτά από θηρευτές. Τόσο η βιομιμητική όσο και η βιομίμηση σχετίζονται με την αποκάλυψη χαρακτηριστικών ιδιοτήτων της ύλης της φύσης από την ερευνητική δραστηριότητα των ανθρώπων. Ωστόσο, η ανάπτυξη των ηλεκτρονικών μικροσκοπίων ήταν μια κομβική υποδομή αναφοράς για τη βασική έρευνα που πυροδότησε πολλές από τις ιδέες της βιοέμπνευσης και των μετέπειτα εφαρμογών. Κατά συνέπεια, στη βιομιμητική τεχνική καθρεφτίζονται επιλεκτικά ιδιότητες της ύλης της φύσης που προβάλλονται με προσομοίωση σε τεχνητές, ανθρώπινες κατασκευές. Η πρωταρχική προσέγγιση θεμάτων βιομιμητικής (Biomimetics) συνδέεται με την άποψη ότι οργανισμοί που ζουν σε βιοτόπους έχουν ήδη αποκριθεί με επιτυχία σε περιβαλλοντικές καταπονήσεις, με την πάροδο του χρόνου και πρόκειται για εκατομμύρια χρόνια εξέλιξης και προσαρμογής. Δηλαδή, η επιβίωση και η ανάπτυξή τους έχει αποδεδειγμένα δοκιμαστεί και βασίζεται στις προσαρμογές τους στο φυσικό τους περιβάλλον. Η βιομιμητική μελετά και αναζητά τις επιτυχημένες αποκρίσεις στο περιβάλλον από τους ζωντανούς οργανισμούς, με απώτερο στόχο να προτείνει λύσεις, έτσι ώστε επιλεγμένα αντικείμενα, υλικά και δομές που θα βοηθήσουν την ανθρώπινη και ανθρωποκεντρική δραστηριότητα να βρίσκονται σε δυναμική ισορροπία με το περιβάλλον του πλανήτη μας. Στις μέρες μας είναι γεγονός ότι από επίμονες, επίπονες και μακρόχρονες μελέτες φυσικής ιστορίας, οραματισμό και έμπνευση προκύπτουν σύγχρονες τεχνολογικές εφαρμογές και νέα προϊόντα με ποικίλες εφαρμογές. Η ανάδειξη της σημασίας της βιομιμητικής, της βιομίμησης, της βιομιμητικότητας ή του βιομιμητισμού σχετίζεται με τη συμβολή την οποία μπορεί να προσφέρει η ενσωμάτωση “πληροφορίας” που προϋπήρχε και προϋπάρχει στη φύση σε καινοτομίες και ανθρωπογενείς επεμβάσεις στο περιβάλλον, με στόχο τη βιώσιμη ανάπτυξη. Στο πλαίσιο αυτό ενθαρρύνεται η διεπιστημονικότητα, η αλληλεπίδραση ιδεών και καινοτόμων απόψεων. Πρόκειται για ταχύτατα αναπτυσσόμενες διεργασίες διεθνώς, σε πανεπιστήμια και ερευνητικές δομές. Με εφόδιο το έργο που διεξάγεται και δημοσιοποιείται, θετικές επιστήμες, κοσμετολογία, αθλητισμός, φαρμακευτική, γεωπονία, αρχιτεκτονική, αρχαιολογία, καλές τέχνες, επιστήμες υλικών, κατασκευών, υφασμάτων, υγείας, περιβάλλοντος και διαστήματος, επιστήμες αγωγής και φυσικής αγωγής αποβλέπουν σε ανάλογα αποτελέσματα. Η έμβια ύλη της φύσης, η οποία αποτελεί “καλούπι” για την τεχνολογία. Επίσης, προσεγγίζονται με παιδεία νέες πτυχές έρευνας και τεχνολογικών δυνατοτήτων. προκλήσεων και προοπτικών για τις επερχόμενες γενιές. Εννοείται ότι τα κεφάλαια του παρόντος συγγράμματος δεν καλύπτουν όλη την έκταση της βιομιμητικής και της βιομίμησης, αλλά είναι ένα πρώτο, σημαντικό βήμα για την ανάδειξη του θέματος και της έμπνευσης καθώς και την προώθηση των ερεθισμάτων ή των παρατηρήσεων από την έμβια ύλη της φύσης.
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Carob (Ceratonia siliqua L.) is a tree species native to the Mediterranean region and belongs to the Fabaceae family. Te tree is well-known for its sweet and nutritious fruits, which have been used for long time as a nutritious food. In addition to the edible fruits, the carob tree also produces seeds that are highly prized for their ability to produce carob gum (locust bean gum). Te carob seed consists of three main components: the shell, the endosperm, and the embryo. Te shell is the outermost layer of the seed, followed by the endosperm, which is the largest part of the seed and contains high levels of carbohydrates and proteins. Te embryo is the smallest part of the seed and is rich on bioactive compounds. Carob seed constituents have attracted considerable attention due to their exceptional nutritional and therapeutic properties in various industries, including food, medicine, pharmaceuticals, cosmetics, and textiles. Te high content of bioactive compounds in carob seeds, such as polyphenols, tannins, and favonoids, is believed to be responsible for their antioxidant and anti-infammatory properties. Te use of carob seed constituents in the food industry is mainly due to their ability to act as thickeners and stabilizers in various foods. Tey are used as a substitute for other thickening agents such as guar gum and carrageenan, due to their superior properties. In the pharmaceutical industry, carob seeds have been found to have antidiabetic, antihyperlipidemic, and anticancer properties, among others. Te cosmetics industry is also interested in the ingredients of carob seed, as they can improve hydration and elasticity of the skin. Tey are also used as a natural alternative to synthetic thickeners in cosmetic formulations. Te textile industry has also recognized the potential of carob seed constituents, as they can be used as a natural dye and as a sizing agent to improve the strength and durability of textiles. In summary, carob seed constituents ofer a wide range of applications in various industries, owing to their high content of bioactive compounds, excellent nutritional and therapeutic profle, and ability to act as thickeners, stabilizers, and antioxidants. Tis review has highlighted the latest fndings on the chemical composition, applications, and health benefts of carob seed constituents.
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Carob flour and its main constituents have been shown to possess nutritional benefits and might be considered as low-cost competitor to other food ingredients in enriched food products. In this study, we performed a DSC characterization of the thermal properties of carob flour and derived fractions (carob protein fraction and locust bean gum) at various moisture percentages, aiming at understanding their behaviour in more complex matrices. Furthermore, wheat/carob ingredient blends were investigated at different moisture content and components ratios to asses and dissect the interplay between carob and wheat flour macromolecules following their thermal transitions. The results indicated that only the carob protein fraction is adequate as ingredient for enriched wheat-based baking products since it does not significantly influence the water partition between the starchy and protein phases. Such a prediction was confirmed by technological trials, i.e., by preparing and comparing reference wheat loafs and ones enriched with 5% w/w of carob protein.
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Biologie, écologie, techniques culturales, biens et services, importance économique, analyse de la chaine de valeur et éléments de réflexion pour l’élaboration d'un plan stratégique de développement du caroubier.
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Protein isolates of beach pea were prepared using sodium hydroxide (NaOH) and sodium hexametaphosphate (SHMP). Functional properties of the isolates so prepared were investigated and compared with those of other pea samples. Protein isolate of beach pea, prepared via NaOH extraction, had a protein content of 86.6%, while SHMP-extracted isolates contained 85.1% protein. Corresponding values for NaOH- and SHMP-extracted green pea and grass pea were 90.6, 89.9, 90.6 and 88.3%, respectively. Sulphur-containing amino acids were more prevalent in SHMP-extracted beach pea and green pea, while they were higher in NaOH-extracted grass pea. Tryptophan content was higher in NaOH-extracted than SHMP-extracted isolates. The predicted biological value and protein efficiency ratio of beach pea protein isolates indicated the high quality of products so prepared. Beach pea protein isolates exhibited a minimum solubility at pH 4.5. The pH and NaCl concentration effectively changed the functional properties of protein isolates. Beach pea protein isolates (NaOH- and SHMP-extracted) had in-vitro digestibility of 80.6 to 82.6% for pepsin-trypsin and 78.6 to 79.2% for pepsin-pancreatin.
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The present investigation was undertaken in order to study the influence of the dehusking procedure on the germ meal composition of carob (Ceratonia siliqua L.) seeds and also to investigate its detailed composition and nutritive value. Meals of carob seed germ were obtained by acid treatment or boiling water treatment of the whole seeds. These procedures allowed to separation of the tight-fitting brown coat of the seed and the removal of the endosperm. Results indicated that the carob germ meal composition could be affected by the isolation procedures. Small reductions were observed in protein and lipid contents in germ meals from acid extraction. The analysis of the carob germ meal (containing fine fragments of husk and endosperm), which could be really obtained industrially, showed the following composition: moisture 8.3%, ash 6.5%, lipids (neutral and polar) 6.6% containing ∼21% of polar lipids, crude proteins 54.7% and energy value 17.5 kJ/g.Oleic acid (34.4%) and linoleic acid (44.5%) were the major fatty acids, while palmitic acid (16.2%) and stearic acid (3.4%) were the main saturated ones. Essential amino acids were present in very interesting amounts, according to the FAO standard except for a low content of tryptophan. The abundant protein fraction bands detected by SDS polyacrylamide gel electrophoresis showed that the carob germ protein fraction was extremely heterogeneous.
A joint AOAC/AACC (American Association of Cereal Chemists) collaborative study of methods for the determination of soluble, insoluble, and total dietary fiber (SDF, IDF, and TDF) was conducted with 11 participating laboratories. The assay Is based on a modification of the AOAC TDF method 985.29 and the SDF/IDF method collaboratively studied recently by AOAC. The principles of the method are the same as those for the AOAC dietary fiber methods 985.29 and 991.42, Including the use of the same 3 enzymes (heat-stable α-amylase, protease, and amyloglucosldase) and similar enzyme Incubation conditions. In the modification, minor changes have been made to reduce analysis time and to Improve assay precision: (1) MES-TRIS buffer replaces phosphate buffer; (2) one pH adjustment step Is eliminated; and (3) total volumes of reaction mixture and filtration are reduced. Eleven collaborators were sent 20 analytical samples (4 cereal and grain products, 3 fruits, and 3 vegetables) for duplicate blind analysis. The SDF, IDF, and TDF content of the foods tested ranged from 0.53 to 7.17, 0.59 to 60.53, and 1.12 to 67.56 g/100 g, respectively. The respective average RSDR values for SDF, IDF, and TDF determinations by direct measurements were 13.1, 5.2, and 4.5%. The TDF values calculated by summing SDF and IDF were in excellent agreement with the TDF values measured independently. The modification did not alter the method performance with regard to mean dietary fiber values, yet It generated lower assay variability compared with the unmodified methods. The method for SDF, IDF, and TDF (by summing SDF and IDF) has been adopted first action by AOAC International.
In the present work, guava seed storage proteins have been fractionated and characterized. Glutelins (86–90 g/100 g) and globulins (≈10 g/100 g) are the main components of the protein extract. Albumins and prolamins are minor components (≈2 g/100 g). Guava seed glutelin extracts, like rice and amaranth glutelins, are legumine-like proteins that, due to their solubility properties, have to be extracted using extreme pH (borate buffer, pH 10, Gt-Bo; NaOH pH 12 Gt-Na), denaturing (borate buffer plus sodium dodecyl sulfate, Gt-BoSDS) or reducing conditions (borate buffer plus 2-mercaptoethanol, Gt-BoME; borate buffer plus sodium dodecyl sulfate and 2-mercaptoethanol, Gt-BoSDSME). The highest yield was obtained with SDS extraction, suggesting that proteins in the seeds form aggregates stabilized mainly by non-covalent interactions. Glutelins are mainly composed of 65 and 67 kD subunits, with a lower proportion of 55 kD subunits. These subunits are formed by disulfide bond-linked polypeptides with molecular masses 40–45 kD, 22–27 kD and 23–25 kD, respectively. The guava seeds protein isolate (GSI) exhibited a polypeptide profile very similar to that of the glutelin fraction.The guava seed could be an alternative source of protein for human and animal consume, additional to this to solve at least in part the pollution problem that fruit processing industry has for discarding this material.
The Dumas combustion procedure using Leco's CNS 2000 analyzer has been shown to give nitrogen values comparable to those obtained with the Kjeldahl procedure for soil and plant products (slightly higher for most samples). These procedures were compared for a range of samples more typical for an animal nutrition laboratory (feed, excreta, carcass, egg yolk, milk and urine). For the 36 samples compared, the Kjeldahl procedure gave N values slightly lower than the Dumas procedure: NKjeldahl=0.0103+0.9855×NCNS. The coefficient of variation for standard AAFCO broiler and cattle feeds (measured repeatedly over an 18-month period) were 2.23 (n=90) and 2.12g/100g (n=177), respectively. It was concluded that the Dumas combustion procedure is capable of replacing the Kjeldahl procedure for routine animal nutrition laboratory N analyses. The Dumas procedure has the advantages of using fewer strong reactants, requiring less labor, and uses a more efficient temperature to release the nitrogen from the samples than the Kjeldahl procedure.
Amaranth protein isolates were prepared by (a) extraction at different alkaline pHs and precipitation at pH 5 and (b) extraction at pH 9 and precipitation at different pHs. The isolates were compared with protein fractions. DSC analysis showed that albumin-1 was composed of several species of Td below 80 °C and ΔH below 4.0 J/g. Glutelin had two species of different Td. Both, globulin and albumin-2, had a main component of Td of 94 °C and ΔH of 19.7 ± 3.2 J/g. The thermal behavior and composition of isolates prepared by method a depended on the extraction pH. The isolate extracted at pH 8 was mainly composed of albumin-1 and globulin, whereas at pHs 9, 10, and 11, albumin-2 and glutelin were also present. The increase of the extraction pH led to a decrease in the thermal stability of proteins from pH 8 on and to a decrease in ΔH at pH 11. With method b, different isolates were obtained. At pH 6 and 7, most of the albumin-2 and some of the globulins precipitated, whereas at pHs 4 and 5, all protein fractions precipitated. Acidification at pH 5 and lower lead to denaturation of the proteins, which was only partially reversed by returning to pH 7. Keywords: Protein isolate; amaranth proteins; calorimetry; protein structure
Determination of the tannins, pectins, hemicellulose, cellulose, nitrogen, mineral elements, total and reducing sugars and fat contents was carried out on carob pods from Mallorca, Spain. The results are compared with data from the literature. A survey of papers on the composition and practical applications of carob pods is included.