Food Chemistry 122 (2010) 533538

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

Protein isolates from two Mediterranean legumes: Lathyrus clymenum and Lathyrus
annuus. Chemical composition, functional properties and protein characterisation
Elena Pastor-Cavada a, Rocio Juan b, Julio E. Pastor b, Manuel Alaiz a, Javier Vioque a,*
a
b

Instituto de la Grasa (C.S.I.C.), Avda Padre Garca Tejero 4, 41012-Sevilla, Spain

Departamento de Biologa Vegetal y Ecologa, Universidad de Sevilla, 41012-Sevilla, Spain

a r t i c l e

i n f o

Article history:
Received 5 October 2009
Received in revised form 10 December 2009
Accepted 2 March 2010

Keywords:
Lathyrus
Protein isolates
Chemical composition
Nutritional characteristics
Functional properties

a b s t r a c t
Protein isolates were analysed from two Mediterranean legumes, Lathyrus clymenum and L. annuus. Protein isolates were prepared by alkaline extraction, including sodium sulphite and acid precipitation of
Lathyrus proteins at their isoelectric point (pH 4.5). The percentage of proteins recovered from L. annuus
and L. clymenum ours during the preparation of the protein isolates was around 60%. Chemical composition, nutritional parameters, main functional properties and protein composition of Lathyrus protein
isolates were studied. L. annuus and L. clymenum protein isolates contained 81.07% and 82.4% of proteins,
respectively, and they have a balanced content of essential amino acids, except for sulphur amino acids,
with respect to the FAO pattern. The in vitro protein digestibility increased in the protein isolates to 93%
and 95% in L. annuus and L. clymenum, respectively. Functional properties were similar to those observed
in other legumes protein isolates. These results conrm the interest of local crops as sources of high value
protein products obtained after convenient protein extraction procedures and the removal of antinutritional components. These high added value protein isolates are of interest for the food industry and for
the revalorisation of L. annuus and L. clymenum favouring the bioconservation of Lathyrus.
2010 Elsevier Ltd. All rights reserved.

1. Introduction
Plant protein isolates have increasing applications in the food
industry as consumers concerns regarding animal food products
increases. Protein isolates production is also a useful tool for the
exploitation of proteins that otherwise could not be used in human
nutrition. For example, plant protein isolates have been produced
from industrial by-products such as defatted rapeseed (Vioque
et al., 1999), or sunower meal (Snchez-Vioque, Clemente,
Vioque, Bautista, & Milln, 1999; Villanueva et al., 1999). These
proteins cannot be used directly for human nutrition, but after convenient extraction methods protein isolates may be produced and
employed in different food products. Another example are pulses
that cannot be directly consumed because they are damaged, of
small size or hard to cook. These pulses may also be an important
substrate for the generation of protein isolates (Snchez-Vioque
et al., 1999).
Legumes constitute a large family of plants, many of them
cultivated. The nutritional value of legumes is related to the high
protein, mineral and vitamin content of the seeds. Hence, legumes
are consumed in many parts of the world as an essential component of the diet. The Lathyrus genus is a legume belonging to the
tribe Fabeae and represents an ancient crop cultivated broadly in
* Corresponding author. Tel.: +34 954611550; fax: +34 959616790.
E-mail addresses: jvioque@cica.es, jvioque@ig.csic.es (J. Vioque).
0308-8146/$ - see front matter 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2010.03.002

the past for human and animal consumption. Thus, L. sativus is

probably the oldest crop cultivated in Europe (Kislev, 1989). Since
the green revolution a large amount of the worlds phytodiversity has been lost because agriculture has substituted local crops
with others with higher yields and which are genetically uniform.
Legumes in general and the genus Lathyrus in particular are not an
exception to this problem and many of the cultivated species have
seen their cultivated areas reduced dramatically in the last century.
In spite of the potential interest of Lathyrus species as a source of
high quality proteins, studies on their proteins are limited to the
most cultivated species, such as L. sativus or L. cicera. In the present
work, we have studied the production and characterisation of protein isolates from two Lathyrus species widely distributed in the
Mediterranean Region. L. annuus has been locally cultivated for forage and weed, while L. clymenum is a wild specie not cultivated in
the past.
These high added value protein isolates are of interest for the
food industry and for the revalourisation of L. annuus and L. clymenum favouring the bioconservation of Lathyrus.
2. Materials and methods
2.1. Materials
Trypsin, chymotrypsin and peptidase were acquired from Sigma
(Tres Cantos, Madrid, Spain). Diethyl ethoxymethylenemanolate

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E. Pastor-Cavada et al. / Food Chemistry 122 (2010) 533538

was purchased from Fluka. All other chemicals were of analytical

grade. The samples of Lathyrus seeds were taken from wild populations. Voucher specimens of the populations studied are deposited in the Herbarium of the Department of Plant Biology and
Ecology of the University of Seville.
2.2. Preparation of protein isolates
2.2.1. Isolate A
Previously to alkaline protein extraction, Lathyrus ours were
washed with methanol, ethanol and water at pH 4.5 to remove
polyphenols and neurotoxic amino acids such as b-ODAP.
Lathyrus ours were suspended in 0.2% NaOH solution pH 12 in
1/10 (w/v) proportion, and extracted by stirring for 1 h. After centrifugation at 8000g and recovery of the supernatant, two additional extractions were carried out. The supernatants were
pooled and analysed for nitrogen content. The pellet was dried in
an oven at 50 C, weighed and analysed for nitrogen content. The
pH of the soluble proteins was adjusted to the isoelectric point
(pH 4.5) and the precipitate formed was recovered by centrifugation at 8000g. The precipitate was washed with distilled water adjusted to pH 4.5 and freeze-dried.
2.2.2. Isolate B
Lathyrus ours were extracted as earlier with 0.2% NaOH solution but adding also 0.25% Na2SO3 at pH 10.5 to avoid the darkening of the nal product.
2.3. Analytical methods
Moisture and ash contents were determined using AOAC (1999)
945.39 and 942.05 approved methods, respectively. Total nitrogen
was determined by the microKjeldahl method according to AOAC
(1999) 960.52 approved method. Crude protein content was estimated using a conversion factor of 6.25. Total bre was determined
according to the procedure described by Lee, Prosky, and De Vries
(1992). Lipids associated with the our and protein isolates were
extracted and measured following the method of Nash, Eldridge,
and Woolf (1967). Soluble sugars and polyphenols were measured
using standard curves of glucose (Dubois, Gilles, Hamilton, Reber,
& Smith, 1956) and chlorogenic acid (Moores, Demott, & Wood,
1948), respectively.
2.4. Determination of the isoelectric point (pI)
For the determination of the pI, 1.5 g of Lathyrus our were extracted twice with 30 ml of 0.2% NaOH solution and centrifuged for
20 min at 10,000g. Aliquots (4 ml) of the supernatant were titrated
with 0.5 N HCl to various pH values, ranging from 3 to 6. The precipitate formed was separated by centrifugation as above. The percentages of nitrogen in the supernatants in relation to the total
nitrogen extracted were plotted vs. pH to determine the pI.
2.5. Determination of b-ODAP
b-ODAP was determined colourimetrically according to Hussain, Chowdhurry, Haque, Wouters, and Campbell (1994).

Alaiz, Navarro, Giron, and Vioque (1992), using D, L-a-aminobutyric

acid as an internal standard.
2.7. In vitro protein digestibility (IVPD)
In vitro protein digestibility was determined according to the
method of Hsu, Vavak, Satterlee, and Miller (1977).
2.8. Determination of nutritional parameters
The amino acid composition of Lathyrus our and protein isolates was used for the determination of several nutritional
parameters:
(a) Amino acid score (chemical score) was calculated as:
% sample essential amino acids contents/% recommended
essential amino acids (FAO/WHO/UNU, 1985).
(b) Protein efciency ratio values (PER) were calculated from
the amino acid composition of Lathyrus seeds based on the
following three equations (Alsmeyer, Cunningham, & Happich, 1974):

PER1 0:684 0:456  Leu  0:047  Pro

PER2 0:468 0:454  Leu  0:105  Tyr
PER3 1:816 0:435  Met 0:78  Leu
0:211  Hys  0:944  Tyr
(c) Protein digestibility corrected amino acid score (PDCAAS)
(FAO/WHO, 1989) was calculated as:
Lowest individual amino acid score  IVPD.
(d) Predicted biological value (BV) was calculated according to
Morup and Olesen (1976) using the following equation:

BV 102:15  Lys0:41  Phe Tyr0:60  Met Cys0:77

 Thr

2:4

 Trp0:21

where each amino acid symbol represents:

% amino acid/% amino acid FAO pattern (1985), if % amino
acid 6 % amino acid FAO pattern or:
% amino acid FAO pattern (1985)/% amino acid, if % amino
acid P % amino acid FAO pattern.
2.9. Protein determination
For SDSPAGE, method described by Bradford (1976) was used
for protein determination, using bovine serum albumin as standard, in Lathyrus protein extracts.
2.10. Gel ltration chromatography
Gel ltration was carried out in a AKTA purier system
equipped with a Superose 12 HR 10/30 column from GE as previously described (Lqari, Vioque, Pedroche, & Milln, 2002). The
approximate molecular masses were determined using blue dextran 2000 (2000 kDa), b-amylase (200 kDa), bovine serum albumin
(67 kDa) and ribonuclease A (13.7 kDa) as molecular weight standards. With these standards, the resulting calibration curve is:

V=Vo 0:78 logMW 5:66

2.6. Amino acid analysis

2.11. Electrophoresis
Samples (10 mg) were hydrolysed with 4 ml of 6 N HCl. The
solutions were sealed in tubes under nitrogen and incubated in
an oven at 110 C for 24 h. Amino acids were determined after derivatisation with diethyl ethoxymethylenemalonate by high-performance liquid chromatography (HPLC), according to the method of

Lathyrus seed proteins from the our and protein isolate B were
extracted with 0.2% NaOH (1:10 w:v) by shacking the our at
1000 rpm during 30 min at room temperature. Extracted proteins
were recovered by centrifugation at 12,000 rpm during 15 min.

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E. Pastor-Cavada et al. / Food Chemistry 122 (2010) 533538

Protein extracts were diluted to 2 mg of protein per ml and mixed

(1:1 v/v) with a solubilisation buffer Tris HCl 80 mM, 0.57% EDTA,
0.26% DTT, 3.3% SDS, 0.008% blue bromophenol, 20% sucrose pH 6.8
and reduced with 2-mercaptoethanol in boiling water. Proteins
(10 lg) were employed for electrophoresis. Tricine-sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDSPAGE) was
performed following the method of Schgger and von Jagow
(1987) with slight modications. The separating gel consisted of
15% T and 2.6% C. The stacking gel consisted of a 4% T and 3% C
gel. The length of the separating and stacking gels were 6 and
2 cm, respectively, with a gel thickness of 1 mm. Electrophoresis
was performed at a constant voltage of 60 V for stacking and
120 V for separation. Protein bands were xed in a solution containing 20% methanol and 8% acetic acid for 15 min, before they
were stained with 0.25% coomassie brilliant blue G in 45% methanol, 10% acetic acid for 24 h. Destaining of the gel was performed in
10% acetic acid. Molecular masses were determined using the low
molecular weight standards from Pharmacia LKB Biotechnology.

Table 1
Nitrogen balance (%) during the production of protein isolates A and B from L.
clymenum and L. annuus our.
L. clymenum

1st extraction
2nd extraction
3rd extraction
Total N
extracted (a)
Soluble N after
Ip
precipitation
(b)
Total precipited
N (a  b)

2.12.3. Emulsion activity

The emulsion activity was determined according to Naczk, Diosady, and Rubin (1985) with modications. A sample (3.5 g) was
homogenised for 30 s in 50 ml water using a model A Polytron
homogeniser (Brinkmann, Wesbury, NY) at setting six (approximately 10,000 rpm). Canola oil (25 ml) was added, and the mixture
was homogenised again for 30 s. Then, another 25 ml of canola oil
were added, and the mixture homogenised for 90 s. The emulsion
was divided evenly into two 50 ml centrifuge tubes and centrifuged at 1100g for 5 min. The emulsifying activity was calculated
by dividing the volume of the emulsied layer by the volume of
emulsion before centrifugation at pH 7.
3. Results and discussion
3.1. Preparation of Lathyrus protein isolates
Protein isolate A is cheaper to prepare than isolate B because
the former is obtained by extraction of the proteins without sodium sulphite. However, in isolate B production, the extraction
pH is milder (pH 10.5 vs. pH 12) and sodium sulphite inhibits oxidation of residual polyphenols, which may react with proteins,
affecting their nutritional quality and also darkening the product.
Extraction yields were similar for protein isolates A and B in
both Lathyrus species, ranging from 60.71% in protein isolate A
from L. clymenum to 66.28% in protein isolate A from L. annuus (Table 1). Solubility of L. clymenum and L. annuus seed extracted proteins was a minimum between pH 4 and 5 (Fig. 1). Hence, pH 4.5
was used for isoelectric precipitation of extracted Lathyrus proteins. The same pH was also used for the precipitation of proteins
of L. maritimus seeds (Chavan, McKenzie, & Shahidi, 2001). After
acidic precipitation at pH 4.5 of extracted proteins, recovered proteins in the protein isolates ranged between 52.51% in isolate B
from L. clymenum to 62.97% in isolate A from L. annuus (Table 1).
In both species, protein isolate A provided higher protein extraction yields. Similar yields have been reported by other authors

Isolate A
(NaOH, pH
12)

Isolate B
(Na2SO3, pH
10.5)

52.01 0.03
7.50 0.15
1.21 0.01
60.71

59.34 0.30
4.22 0.02
0.93 0.00
64.48

60.35 1.02
4.31 0.85
1.62 0.00
66.28

58.02 3.76
3.08 0.01
1.00 0.01
62.11

1.70 0.03

11.97 0.02

3.31 0.17

3.41 0.09

59.01

52.51

62.97

58.7

L. clymenum

50

L. annuus

% soluble protein

2.12.2. Fat absorption

For the determination of fat absorption the method of Lin, Humbert, and Sosulski (1974) was used.

Isolate B
(Na2SO3, pH
10.5)

60

2.12. Determination of functional properties

2.12.1. Water absorption
Water absorption was determined using the method of Sosulski
(1962).

L. annuus

Isolate A
(NaOH, pH
12)

40
30
20
10
0

pH
Fig. 1. Solubility curve of L. clymenum and L. annuus proteins. Solubility of the
proteins is expressed by measuring the relative percentages of nitrogen solubles at
various pH values.

for legumes such as pigeonpea (AntAnna, Vilela, & Gomes, 1985)

or eld pea (Sumner, Nielsen, & Youngs, 1981). This is expected,
since for isolate A production a higher pH is employed (12 vs.
10.5 in isolate B), increasing the yield of protein extraction. However, this higher alkaline pH may affect the native conformation
of the proteins, affecting their functional and nutritional properties
(Snchez-Vioque et al., 1999).
3.2. Chemical composition of Lathyrus protein isolates
Protein isolates B produced with sodium sulphite were further
characterised for their chemical composition. The rst and most
evident consequence of the enrichment of proteins in isolate B is
the decrease in the contents of other compounds abundant in the
our such as bre or carbohydrates. Both Lathyrus species have
similar our protein contents, around 25% (Table 2). As a result
of protein extraction and precipitation, protein isolates with
82.4% and 81.07% protein contents were produced for L. clymenum
and L. annuus, respectively (Table 2). Minor compounds of high
functional interest whose contents were reduced in the isolates
are polyphenols and the non-protein amino acid b-ODAP (Table 2).
Although polyphenols have recognised antioxidant properties,
their oxidation products may yield quinones that will darken the
protein isolates, limiting their potential application in the food
industry. The presence of b-ODAP has limited for many years the

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E. Pastor-Cavada et al. / Food Chemistry 122 (2010) 533538

Table 2
Chemical composition of L. clymenum and L. annuus our and protein isolates B. Data
expressed as g 100 g1 matter are the mean SD of three analyses.
L. clymenum

Moisture
Ash
Fibre
Proteina
Lipids
Soluble sugars
Polyphenols
b-ODAP
Carbohydrate by
difference
a

Table 4
Nutritional characteristics of L. clymenum and L. annuus our and protein isolates.
L. clymenum

L. annuus

Flour

Protein
isolate B

Flour

Protein
isolate B

11.40 0.14
2.58 0.35
41.10 0.14
21.25 0.2
1.99 0.00
1.38 0.00
0.15 0.00
1.1 0.05
19.05

4.64 0.05
0.26 0.01
9.18 0.03
82.40 0.80
1.16 0.00
2.32 0.01
0.04 0.01
Tr
Tr

10.90 0.14
2.20 0.06
41.00 1.41
19.95 0.07
1.93 0.04
2.82 0.00
0.29 0.00
0.004 0.00
20.9

4.78 0.17
0.20 0.05
7.71 0.55
81.07 0.11
1.09 0.02
5.11 0.00
0.04 0.02
Tr
Tr

IVPDa
E/T (%)b
ASc
BVd
PER1e
PER2
PER3
PDCAASf
a
b
c
d

Total nitrogen  6.25.

e
f

wider use of Lathyrus seeds in human nutrition. This amino acid,

when ingested in high proportions for long periods, is neurotoxic
and may produce neurolathyrism, resulting in paralysis in legs
and arms or even death (Bell, 2003). But the protein isolate, where
this amino acid has been removed, represents a safe product for
human nutrition. It should be noted that the contents of the antinutritional non-protein amino acid b-ODAP, responsible for lathyrism, were reduced to undetectable amounts in both protein
isolates (Table 2).

3.3. Amino acid composition of Lathyrus protein isolates

The amino acid composition of Lathyrus our and protein isolates is shown in Table 3. The protein isolates, as well as the original our are decient in sulphur amino acids, methionine and
cysteine, and in tryptophan. In the protein isolates lower contents
of sulphur amino acids are observed with respect to the our. This
is probably due to the lower contents in albumins in the protein
isolates with respect to the our. Albumins are water soluble and

Table 3
Amino acid composition of L. clymenum and L. annuus our and protein isolates. Data
expressed as g 100 g1 protein are the mean SD of two analyses.
L. clymenum

Aspartic acidb
Glutamic acidc
Serine
Histidine
Glycine
Threonine
Arginine
Alanine
Proline
Tyrosine
Valine
Methionine
Cysteine
Isoleucine
Tryptophan
Leucine
Phenylalanine
Lysine
a
b
c
d
e

FAOa

L. annuus

Flour

Protein
isolate B

Flour

Protein
isolate B

12.8 0.33
19.6 0.13
6.4 0.03
2.2 0.02
4.2 0.02
4.2 0.02
8.8 0.09
4.9 0.03
0.6 0.02
2.7 0.02
4.9 0.03
0.8 0.01
0.7 0.02
4.1 0.02
0.6 0.02
9.4 0.05
5.4 0.05
8.0 0.05

11.6 2.71
18.5 1.69
7.0 0.72
2.3 0.23
4.1 0.43
4.1 0.48
9.6 1.03
4.2 0.03
3.8 1.03
2.3 0.28
5.0 0.18
0.1 0.05
0.5 0.03
4.4 0.20
0.8 0.11
9.4 0.59
5.6 0.57
6.9 0.58

12.6 0.08
21.8 0.08
5.7 0.01
2.4 0.01
4.3 0.01
4.7 0.02
8.7 0.00
4.9 0.01
2.3 0.01
2.3 0.00
4.6 0.01
0.3 0.01
1.1 0.01
3.8 0.01
1.0 0.01
7.9 0.04
4.8 0.03
7.0 0.03

10.8 1.83
19.4 0.87
6.7 0.48
3.0 0.24
4.6 0.29
4.6 0.32
10.4 0.61
4.7 0.03
2.4 0.35
2.5 0.13
5.0 0.20
0.1 0.02
0.5 0.07
4.3 0.17
0.7 0.15
8.9 0.39
5.2 0.28
6.1 0.29

FAO/WHO/UNU Energy and protein requirements (1985).

Aspartic acid + aspargine.
Glutamic acid + glutamine.
Tyrosine + phenylalanine.
Methionine + cysteine.

1.9
3.4

6.3d
3.5
2.5e
2.8
1.1
6.6
5.8

L. annuus

Flour

Protein isolate
B

Flour

Protein isolate
B

80.30 1.53
42.90 0.96
126.40 2.79
38.30 2.98
3.55 0.25
3.50 0.27
3.79 0.06
0.45 0.02

95.00 0.90
41.30 3.17
122.00 9.18
23.70 9.64
3.43 0.31
3.56 0.23
3.85 0.20
0.21 0.02

86.50 2.78
39.80 1.26
117.40 3.74
32.00 2.16
2.81 0.08
2.88 0.04
2.81 0.07
0.52 0.05

93.00 1.66
40.90 1.74
121.00 5.21
17.80 0.66
3.26 0.21
3.31 0.21
3.43 0.54
0.22 0.05

In vitro protein digestibility.

% Essential amino acids/total amino acids.
Amino acids score.
Biological value.
Protein efciency ratio.
Protein digestibility corrected amino acid score.

may be partially removed when washing the our with water previous to the alkaline extraction. It is known that albumins are rich
in sulphur amino acids. Concerning the other amino acids, protein
isolates have percentage contents above FAO recommendations,
especially in the case of lysine an amino acid usually limited in
cereals.
3.4. Nutritional characteristics of Lathyrus protein isolates
In vitro protein digestibility (IVPD) determines the rate at which
a protein may be digested and represents the protein quality of a
protein product. IVPD increases in the protein isolates with respect
to the our (Table 4). Protein digestibility is reduced in the presence of trypsin and chymotrypsin inhibitors. Since these inhibitors
are mostly in the albumin fraction (Richardson, 1991), which is soluble in water, these inhibitors are partially removed during the
washing with water previous to the alkaline protein extraction.
Hence protein isolates have a higher digestibility than the corresponding our, reaching 95% digestibility in L. clymenum. Up to a
70% decrease in the concentration of the trypsin inhibitor has been
reported in the production of soy protein isolates (Waggle, Steinke,
& Shen, 1989). Also, Lathyrus proteins may be partially denatured
during the preparation of the isolates, as they are more accessible
to the proteases, and hence the IVPD increases (Lynch, Rha, & Catsimpoolas, 1977).
Based on the amino acid composition, several nutritional
parameters have been determined for the protein isolates (Table 4).
The proportion of essential amino acids with respect to total amino
acids (E/T) was similar for the our and corresponding protein isolates, as well as the amino acid score. On the contrary, the Biological Value was lower in the protein isolates than in the ours.
Protein efciency ratio (PER) was similar for the our and protein
isolates in L. clymenum, or even higher in L. annuus protein isolate
with respect to the our. In addition, PER values in both protein
isolates were higher than in other food proteins of good quality
such as soybean (2.57) (Wolzak, Elias, & Bressani, 1981) or chickpea (2.8) (Newman, Roth, Newman, & Lockerman, 1987). However,
PDCAAS values were not so favourable in the protein isolates,
mainly due to their lower contents in sulphur amino acids.
3.5. Structural characteristics of Lathyrus protein isolates
Gel ltration chromatography of proteins from Lathyrus protein
isolates and ours is shown in Figs. 2 and 3. In both Lathyrus isolates a disappearance of lower molecular weight proteins with respect to the our is observed. These proteins are probably low

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E. Pastor-Cavada et al. / Food Chemistry 122 (2010) 533538

90

Abs (280 nm) (AU)

Table 5
Functional properties of L. clymenum and L. annuus our and protein isolates.

80
70

L. clymenum

60

Flour

Protein
isolate B

Flour

Protein
isolate B

253.0 15.9

244.0 17.0

279.0 3.4

284.0 35.1

200.0 15.8
80.0 0.0

331.0 11.9
25.0 0.0

206.0 15.3
71.4 0.0

280.0 1.8
38.0 1.8

50

Water
absorptiona
Fat absorptionb
Emulsifying
activityc

40
30
20
10

10

15

20

25

30

Elution volume (ml)

400

Grams of water absorbed per 100 g sample.

Grams of fat absorbed per 100 g sample.
Percentage of fat emulsied (% weight).

L clymenun

350

Abs (280 nm) (AU)

L. annuus

flour

L annuus

protein

flour

isolate

300

protein
isolate

250
200
150
100
50
0

10

15

20

25

94

94

67

67

43

43

30

30

20.1

20.1

30

Elution volume (ml)

Fig. 2. Gel ltration chromatography of proteins extracted from L. clymenum our
(A) and protein isolates (B).

Abs (280 nm) (AU)

35

30
25
20
15
10
5
0

10

15

20

25

30
Fig. 4. SDSPAGE of proteins extracted from L. clymenum and L. annuus our and
protein isolates. Molecular weight standards are indicated on the left.

Elution volume (ml)

Abs (280 nm) (AU)

600

500
400
300
200
100
0

10

15

20

25

30

Elution volume (ml)

Fig. 3. Gel ltration chromatography of proteins extracted from L. annuus our (A)
and protein isolates (B).

molecular weight albumins that are lost during the water washing
of the our previous to the alkaline extraction of proteins. In L.

clymenum and L. annuus protein isolates a main peak of 163 kDa

and 209 kDa is observed, corresponding to the ab trimers of legumin, the main seed storage protein in legume seeds. Also a protein
eluting with the void volume and corresponding to proteins and
aggregates of more than 300 kDa due to the partial denaturation
of proteins with exposition of hydrophobic amino acid groups during the process of protein isolate production. SDSPAGE conrms
the enrichment of protein isolates in legumin like proteins with respect to the our. Hence a predominance of protein bands around
40 and 20 kDa corresponding to a and b subunits of legumin protein was observed (Fig. 4).
3.6. Functional properties of Lathyrus protein isolates
Water absorption capacity was similar for the our and the proteins isolates (Table 5). This has been also observed in chickpea
protein isolates produced in a similar way (Sanchez-Vioque et al.,

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E. Pastor-Cavada et al. / Food Chemistry 122 (2010) 533538

1999). Fat absorption increased about 50% in Lathyrus protein isolates with respect to the our. This has been also observed in Lupinus protein isolates produced in a similar way (Lqari et al., 2002),
and may be due to the partial denaturation of proteins with exposition of hydrophobic amino acids groups during the process of
protein isolate production. Likewise a decrease in the emulsifying
activity was observed in the protein isolates with respect to Lathyrus ours. This has been also observed in chickpea and lupin protein isolates produced in a similar way (Snchez-Vioque et al.,
1999; Lqari et al., 2002).
4. Conclusions
In conclusion, the procedure employed for the production of
Lathyrus protein isolates generates a product mainly consisting of
the globulin fraction. Although the isolates have low contents of
sulphur amino acids, they are well balanced in the other amino
acids with respect to FAO requirements. In addition, the nutritional
quality is high due to the high IVPD, AS and PER values and the removal of antinutritional compounds such as b-ODAP. These protein
isolates may provide an extra amount of proteins in applications
such as balanced nutritional foods and high-protein diet products.
Also, Lathyrus protein isolates could be suitable for the preparation
of cheese, bakery and meat products because of their high water
and fat absorption (Utrilla-Coello, Osorio-Daz, & Bello-Perez,
2007).
Results conrm the interest in studying wild populations of cultivated and non-cultivated Lathyrus species as a source of seeds for
the production of protein isolates with good functional and nutritional characteristics. Produced Lathyrus protein isolates may be
useful for the revalourisation of these species which were more
broadly cultivated in the past or may even help in the domestication of new species or the use of wild populations in breeding programmes, favouring the bioconservation of the Lathyrus genus.
Acknowledgements
This work was nanced by grant AGR-711 from Junta de Andaluca (Spain). Thanks are due to Mara Dolores Garca-Contreras
for technical assistance.
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