Protein and amino acid composition of select wild legume species of tribe Fabeae

Food Chemistry 163 (2014) 97–102

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Protein and amino acid composition of select wild legume species of tribe Fabeae Elena Pastor-Cavada a,⇑, Rocío Juan b, Julio E. Pastor b, Manuel Alaiz a, Javier Vioque a a b

Instituto de la Grasa (C.S.I.C.), Avda Padre García Tejero 4, 41012 Sevilla, Spain Dpto. de Biología Vegetal y Ecología. Universidad de Sevilla, 41012 Sevilla, Spain

a r t i c l e

i n f o

Article history: Received 30 January 2014 Received in revised form 19 April 2014 Accepted 21 April 2014 Available online 2 May 2014 Keywords: Nutritional quality Proteins Seed Wild legume Fabeae

a b s t r a c t The nutritional characteristics of seed proteins of 50 Spanish wild taxa of Lathyrus, Lens, Pisum and Vicia have been compared. The highest average protein richness and the in vitro protein digestibility have been observed in the genus Vicia and Lens, respectively, whereas the genus Pisum showed the lowest proteindigestibility corrected amino acid score. Using the K-means algorithm three clearly distinguished groups of taxa have been established in relation to their essential amino acid contents, protein richness, digestibility and nutritional parameters. The most adequate protein profile was observed in the taxa of group 1. This group includes four taxa of genus Lathyrus and nine taxa of genus Vicia. It should be noted that seven of the thirteen taxa included in this group have never been used as crops. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction The legumes have a high content of good quality proteins (Morrow, 1991), but legume intake has decreased in last decades in many western countries (Hellendoorn, 1976), despite the fact that they include carbohydrates, dietary fibre and have a low content of saturated fats. Legume proteins are rich in lysine and complement the proteins in cereals, which are deficient in this amino acid (Anjum, Ahmad, Butt, Sheikh, & Pasha, 2005; Sai-Ut, Ketnawa, Chaiwut, & Rawdkuen, 2009). The consumption of legumes has health benefits related to components such as proteins, fibre or some minor compounds, such as certain lipids, polyphenols or bioactive peptides (Ng & Ng, 2013; Rochfort & Panozzo, 2007). These positive effects have been related to the prevention of colon cancer, diseases like diabetes mellitus or coronary heart diseases (Duranti, 2006; Kui Du, Jiang, Yu, and Jane, 2014). The tribe Fabeae presents in Spain (Talavera et al., 1999) the genus: Lathyrus, Lens, Pisum and Vicia. These plants, as other legumes, may grow under drought stress conditions and on poor soils due their capacity to fix atmospheric nitrogen (Campbell et al., 1994; Nemecek et al., 2008). Some of the species studied are broadly cultivated for human consumption, such as Pisum sativum and Lens culinaris, whereas some Lathyrus species are locally cultivated as forage crops for livestock feeding (Granati, Bisignano, ⇑ Corresponding author. Tel.: +34 954611550; fax: +34 954616790. E-mail addresses: [email protected], [email protected] (E. Pastor-Cavada). http://dx.doi.org/10.1016/j.foodchem.2014.04.078 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.

Chiaretti, Crinó, & Polignano, 2003). There is evidence suggesting that Lathyrus sativus is probably the oldest crop in Europe (Kislev, 1989). Lathyrus species are also well known for the presence of neurotoxic and osteotoxic non protein amino acids that have limited their use in human nutrition (Akalu, Johansson, & Nair, 1998; Yang et al., 2006). In Vicia, the best known specie is the faba bean (V. faba), which is an important pulse, fodder crop and vegetable (Madar & Stark, 2002). Other Vicia species sometimes cultivated are V. ervilia, V. narbonensis, V. sativa, V. benghalenis, V. articulata, V. pannonica or V. Villosa. Among these, V. sativa, the common vetch, is the most cultivated in the Mediterranean region. The protein fraction of V. faba has been extensively characterised, including the amino acid composition (Lattanzio, Bianco, Crivelli, & Miccolis, 1983), functional properties (Cepeda, Villaran, & Aranguiz, 1998), the production of proteins isolates (Macarulla et al., 2001) and protein hydrolysates (Chakraborty, Bramsnaes, & Bose, 1979). Recently, a study of the amino acid composition and nutritional characteristics of seed proteins in 28 Vicia species from southern Spain was carried out (Pastor-Cavada, Juan, Pastor, Alaiz, & Vioque, 2011a). In Lens there are some studies on seed proteins of L. culinaris (Carbonaro, Cappelloni, Nicoli, Lucarini, & Carnovale, 1997; De Almeida Costa, Da Silva Queiroz-Monici, & Reis, 2006; Iqbal, Khalil, Ateeq, & Khan, 2006; Martín-Cabrejas et al., 2009; Monsoor & Yusuf, 2002; Solanki, Kapoor, & Singh, 1999), and in Pisum of P. sativum (Abreu & Bruno-Soares, 1998; Palander, Laurinen, Perttila, Valaja, & Partanen, 2006; Yáñez-Ruiz,

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Martín-García, Weisbjerg, Hvelplund, & Molina-Alcaide, 2009). The studies on their proteins, with respect to Lathyrus, are limited to the most cultivated species, but there are also some studies on wild species (Pastor-Cavada, Juan, Pastor, Alaiz, & Vioque, 2011b). In recent decades a large amount of the world’s phytodiversity has been lost because local varieties and species have been substituted by commercial ones with a high yield and genetic uniformity. To recover and maintain this biodiversity, a diversification of plant species is necessary, and this can be achieved by increasing our knowledge of local species. In this work 50 legume wild taxa of tribe Fabeae, from southern Spain, and belonging to the genera Lathyrus, Lens, Pisum and Vicia, have been studied. The aim of the present research was both to compare the nutritional characteristics of seed proteins among the four genera examined and to establish the group of taxa which show the most favourable composition from a nutritional and functional point of view, regardless of their genus.

from A/B (86:14) to A/B (69/31); 30.0–35.0 min, elution with A/B (69:31). The column was maintained at 18 °C. Tryptophan was analysed by HPLC after basic hydrolysis according to Yust et al. (2004). Protein contents of studied samples were determined after amino acid analyses. 2.5. In vitro protein digestibility (IVPD) In vitro protein digestibility was determined according to the method of Hsu, Vavak, Satterlee, and Miller (1977). Samples containing 62.5 mg of protein were suspended in 10 ml of water and the pH was adjusted to 8.0. An enzymatic solution containing 1.6 mg trypsin (17.7 BAEE U mg 1), 3.1 mg a-chymotrypsin (43 U mg 1) and 1.3 mg peptidase (50 U g 1) per ml was added to the protein suspension in a 1:10 v/v ratio. The pH of the mixture was measured after 10 min and the in vitro digestibility was calculated as a percentage of digestible protein using the equation: % digestible protein = 210.464 18.103  pH.

2. Materials and methods 2.6. Determination of nutritional parameters 2.1. Materials Trypsin, chymotrypsin and peptidase were from Sigma (Tres Cantos, Madrid, Spain). Diethyl ethoxymethylenemanolate was purchased from Fluka (Buchs, Suiza). All other chemicals were of analytical grade. Fully matured seed samples were collected from wild populations located in Andalusia (southern Spain). The seeds were collected from ten specimens in a given population and stored at 20 °C. The seeds were stored for less than 3 months, which is the harvesting period for wild seeds in the country. After this period, lab analysis began. Voucher specimens were deposited in the Herbarium of the University of Seville. All other reagents were of analytical grade. 2.2. Flour preparation Select grains were milled using a Cyclone sample mill (Udy Corporation, Fort Collins, Colorado, USA) to obtain a uniform flour with 10  = 92 lm size mesh. 2.3. Protein estimation method Total nitrogen was determined by the micro Kjeldahl method according to AOAC (1999) 960.52 approved method. Crude protein content was estimated using a conversion factor of 6.25 for legumes.

The amino acid composition of studied species was used for the determination of several nutritional parameters of seed protein: (a) Amino acid score (chemical score) was calculated % Sample essential amino acids contents/% recommended essential amino acids (FAO/WHO/UNU, 1985). (b) Protein efficiency ratio values (PER) were calculated from the amino acid composition of 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 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 + Tyr)0.60  (Met + Cys)0.77  Thr2.4  Trp0.21 where each amino acid symbol represents: % Amino acid/% amino acid FAO pattern (1985), if % amino acid 6 % amino acid FAO pattern % Amino acid FAO pattern (1985)/% amino acid, if % amino acid 6 % amino acid FAO pattern. 2.7. Statistical analysis

2.4. Amino acid analysis and protein content 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 derivatization with diethyl ethoxymethylenemalonate by highperformance liquid chromatography (HPLC), according to the method of Alaiz, Navarro, Giron, and Vioque (1992), using D, L-aaminobutyric acid as internal standard. The HPLC system consisted of a Model 600E multi-system with a 484 UV–Vis detector (Waters) equipped with a 300  3.9 mm i.d. reversed-phase column (Novapack C18, 4 lm, Waters). A binary gradient was used for elution with a flow of 0.9 ml/min. The solvents used were (A) sodium acetate (25 mM) containing sodium azide (0.02% w/v) pH 6.0 and (B) acetonitrile. Elution was as follows: time 0.0– 3.0 min, linear gradient from A/B (91:9) to A/B (86/14); 3.0– 13.0 min, elution with A/B (86/14); 13.0–30.0 min, linear gradient

Results are expressed as the mean values ± standard deviation of several samples except for species with only one population. The data were statistically analysed by one-way analysis of variance (ANOVA). Means were compared by Scheffe’s test. The Kmeans algorithm (Hartigan & Wong, 1979) was used to group the taxa studied. Lastly, a discriminating analysis was performed in order to verify whether the parameters used to separate the taxa are actually effective. 3. Results and discussion The genera Lens and Pisum have shown high protein amount (Table 1) similar to the content observed in the other two genera which were included in the tribe Fabeae (Pastor-Cavada et al., 2011a, 2011b). Considering the joint results obtained from these four genera in the Southern Spain, the highest protein richness is

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Table 1 Seed protein contents (g/100 g flour) and essential amino acids composition (g/100 g protein) in the genera studied. Data are average ± standard deviation. Superscript letters indicate significant differences between values in the same row (Scheffe’s test).

Protein** Cysteine* Phenylalanine Isoleucine** Leucine*** Lyisine*** Methionine Histidine Tyrosine Threonine* Tryptophan Valine** T essential aa

Lathyrus

Lens

Pisum

Vicia

22.38 ± 2.56a 1.09 ± 0.31b 5.04 ± 0.31 3.62 ± 0.50a 8.26 ± 0.77b 7.55 ± 0.47b 0.58 ± 0.26 2.65 ± 0.30 2.51 ± 0.24 4.49 ± 0.40a 0.69 ± 0.11 4.48 ± 0.30a 40.96 ± 2.58

22.93 ± 1.27ab 0.62 ± 0.17a 5.40 ± 0.22 4.04 ± 0.33ab 8.28 ± 0.31ab 7.28 ± 0.14ab 0.69 ± 0.18 2.63 ± 0.22 2.29 ± 0.41 4.92 ± 0.38ab 0.62 ± 0.14 5.20 ± 0.54b 41.97 ± 2.67

23.30 ± 1.76ab 0.42 ± 0.03a 4.95 ± 0.31 4.00 ± 0.17ab 7.55 ± 0.17ab 7.41 ± 0.12ab 0.53 ± 0.05 2.65 ± 0.08 2.57 ± 0.05 4.18 ± 0.20ab 0.65 ± 0.08 5.00 ± 0.10ab 39.91 ± 2.54

24.17 ± 2.95b 1.08 ± 0.34b 5.09 ± 0.40 3.87 ± 0.31b 7.86 ± 0.45a 7.14 ± 0.44a 0.56 ± 0.23 2.81 ± 0.41 2.49 ± 0.21 4.75 ± 0.63b 0.68 ± 0.10 4.75 ± 0.41b 41.08 ± 2.48

FAO

2.8 6.6 5.8 2.5A 1.9 6.3B 1.1 3.5

A

Met + Cys. Phe + Tyr. * P < 0.05. ** P < 0.01. *** P < 0.001. B

observed in the genus Vicia, but it is only significantly different from the genus Lathyrus (Table 1). Although the average total essential amino acid content is similar in the four genera, a further detailed analysis reveals some differences in most of these amino acids (Table 1). The most relevant differences have been observed between Lathyrus and Vicia, since these are the genera with a higher number of taxa. Thus, Lathyrus features significantly higher quantities of leucine and lysine (P < 0.001) than Vicia, whereas contents of isoleucine and threonine are fairly lower. With respect to valine, the lowest content was observed in the Lathyrus genus, in this case distinguishing itself (P < 0.01) from the contents in Lens and Vicia, which were the highest. Additionally, cysteine was the only essential amino acid with a significantly higher content (P < 0.05) in both Lathyrus and Vicia, as compared to Lens and Pisum. As in other legumes, the only limitation detected in the seeds of the four genera examined is in both sulphur-containing amino acids (cysteine and methionine) and tryptophan, whose levels do not reach the recommendations by FAO/WHO/UNU (1985). The poor levels in sulphur-containing amino acids in Lens, Pisum, and Vicia seeds can be compensated by mixing them with cereals which have rich contents of methionine and cysteine, as do other leguminous plants (Plahar, Annan, & Nti, 1997). Thus, legumes provide the lysine which the cereals lack. On the other hand, the reduced contents of tryptophan may be improved by mixing the seeds with Amaranthus seeds, since the species from this genus exceed the minimum amount recommended by FAO/ WHO/UNU (1985) (Juan, Pastor, Alaiz, & Vioque, 2007). Therefore, by complementing the general amino acid contents in leguminous plants with cereals and amaranthus, a fairly balanced source of vegetal amino acids can be obtained. In relation to the in vitro protein digestibility (IVPD), it should be noted that the highest average content is found in the genus Lens, even though the difference is notable only with Lathyrus (Table 2). The results obtained in the present study are consistent with those provided by other authors for different legumes (Carbonaro et al., 1997; Lqari, Vioque, Pedroche, & Millán, 2002; Martín-Cabrejas et al., 2009). The nutritional value of the seeds studied was assessed using both their amino acid composition and their IVPD (Table 2). In general terms, even though the essential amino acid proportion of the total amount (%EEA/TAA), such as the essential amino acid percentage as recommended by FAO/WHO/UNU (1985) (AAS) is lower in Pisum, there are not significant differences (Table 2). In relation

Table 2 In vitro protein digestibility (IVPD) and nutritional parameters of studied seed proteins. Results are the average ± standard deviation of different populations. Superscript letters indicate significant differences between values in the same row (Scheffe’s test). Lathyrus IVPD* % EAA/TAA AAS BV PER1** PER2** PER3* PDCAAS*

Lens

Pisum

Vicia

80.71 ± 3.00a 84.10 ± 0.89b 83.83 ± 0.28ab 83.12 ± 2.31b 41.01 ± 1.84 41.75 ± 1.62 39.90 ± 0.55 41.18 ± 1.65 120.94 ± 5.48 123.03 ± 4.61 117.67 ± 1.75 121.43 ± 4.89 38.41 ± 9.85 26.40 ± 5.33 29.57 ± 5.36 35.87 ± 13.84 3.01 ± 0.36b 2.89 ± 0.07ab 2.66 ± 0.07ab 2.80 ± 0.20a b ab ab 3.03 ± 0.34 2.98 ± 0.11 2.69 ± 0.08 2.85 ± 0.20a 3.07 ± 0.63b 3.06 ± 0.13ab 2.44 ± 0.20ab 2.83 ± 0.41a 0.47 ± 0.07b 0.45 ± 0.11ab 0.32 ± 0.01a 0.46 ± 0.07b

% EAA/TAA = % Essential amino acid/total amino acids. AAS = Amino acids store. BV = Biological value. PER = Protein efficiency ratio. PDCAAS = Protein digestibility corrected amino acid score. * P < 0.05. ** P < 0.01.

to the biological value (BV), no differences are observed either among the four genera examined (Table 2). Nevertheless, the theoretical protein efficiency ratio (PER) and the protein-digestibility corrected amino acid score (PDCAAS) for these genera do show some differences. Moreover, the average theoretical PER values obtained for Lathyrus are significantly higher than for Vicia, and although the average PDCAAS is similar in both genera, it is significant higher (P < 0.05) than in the genus Pisum which showed a lower PDCAAS result (Table 2). Among the genera studied, certain trends have been determined. For example, the contents of leucine and lysine in the genus Lathyrus are higher and the digestibility in this genus is the lowest. However, considering the heterogeneity observed among some species in the same genus, it is necessary to analyse the 50 taxa in the Fabeae tribe jointly, in order to determine which species show a more adequate protein profile, regardless of their genus. To do so, using the K-means algorithm three clearly distinguished groups of taxa have been established in relation to their essential amino acid contents, protein richness, digestibility and nutritional parameters (Table 3). In order to verify whether the parameters used to divide the taxa are actually effective, a discriminating analysis has been performed. An overview of this analysis in which only the first

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Table 3 Groups of taxa studied established in relation to their essential amino acids composition, seed protein contents, digestibility and nutritional parameters. Taxa Group 1

Lathyrus amphicarpos L. annuus L. cicera L. latifolius Vicia altísima

V. V. V. V.

Group 2

Lathyrus angulatus L. aphaca L. clymenum L. filiformis L. ochrus L. pratensis L. sativus L. setifolius L. sphaericus

Group 3

Lathyrus hirsutus Lens lamottei Lens nigricans Vicia disperma

Table 4 Summary of discriminant analysis based on the essential amino acids composition, seed protein contents, digestibility and nutritional parameters. Functions

Wilks Landa

Chi-square

Df

P-value

Cumulative percentage

1 2

0.0668098 0.564101

101.4714 21.4696

40 19

0.0000 0.3114

90.59 100

discriminating function of the two available is statistically relevant (P < 0.001) is shown in Table 4. The distribution of the taxa into groups is illustrated in Fig. 1. Even though the protein richness in the three groups is similar, the amino acid content shows some significant differences (Table 5). Therefore, group 1 is clearly the group with the highest values in cysteine (P < 0.001). It also has the highest content of methionine, so this group of taxa is the richest in sulphur-containing amino acids, although as in other legumes, they do not reach the minimum FAO requirements (1985). As observed in Table 5, the three groups exceed the FAO minimum required levels (1985) of lysine, although the highest content is found in group 2 (P < 0.05). In relation to phenylalanine and threonine, the taxa in

Group 1

Group 2

Group 3

V. V. V. V.

hirsuta lathyroides monantha ssp. calcarata narbonensis

L. tingitanus Lens culinaris Pisum sativum ssp. elatius Vicia dasycarpa V. eriocarpa V. ervilia V. glauca ssp. giennensis V. incana

V. V. V. V. V. V. V. V.

lutea ssp lutea var. hirta lutea ssp. lutea var. lutea lutea ssp. vestita parviflora peregrina pseudocracca pyrenaica sativa ssp. sativa

V. V. V. V.

V. V. V. V.

faba hybryda lutea ssp. cavanillesii monardii

faba hybryda lutea ssp. cavanillesii monardii

Table 5 Seed protein contents (%) and essential amino acids composition (g/100 g protein) for the three groups of taxa belonging to tribe Fabeae from Southern Spain. Data are average ± standard deviation. Superscript letters indicate significant differences between values in the same row (Scheffe’s test).

Protein Cysteine*** Phenylalanine** Isoleucine Leucine Lysina* Methionine* Histidine Tyrosine Threonine*** Tryptophan Valine

Group 1

Group 2

Group 3

FAO

23.00 ± 3.00 1.36 ± 0.30b 4.92 ± 0.34a 3.67 ± 0.24 7.86 ± 0.43 7.12 ± 0.31a 0.65 ± 0.20b 2.77 ± 0.30 2.53 ± 0.24 4.38 ± 0.24a 0.71 ± 0.10 4.55 ± 0.38a

23.48 ± 2.95 0.98 ± 0.28a 5.10 ± 0.3ab 3.82 ± 0.45 8.12 ± 0.67 7.43 ± 0.50b 0.54 ± 0.24a 2.75 ± 0.42 2.48 ± 0.63 4.52 ± 0.41a 0.67 ± 0.10 4.68 ± 0.33ab

24.22 ± 2.34 0.89 ± 0.26a 5.24 ± 0.48b 3.85 ± 0.38 7.90 ± 0.61 7.16 ± 0.50a 0.58 ± 0.24ab 2.70 ± 0.31 2.48 ± 0.30 5.47 ± 0.55b 0.66 ± 0.10 4.82 ± 0.55b

2.8 6.6 5.8 2.5A 1.9 6.3B 1.1 3.5

A

Met + Cys. Phe + Tyr. * P < 0.05. ** P < 0.01. *** P < 0.001. B

Centroids

Table 6 In vitro protein digestibility (IVPD) and nutritional parameters for the three groups of taxa belonging to tribe Fabeae from Southern Spain. Results are the average ± standard deviation of different populations. Superscript letters indicate significant differences between values in the same row (Scheffe’s test).

4.1

2.1

Function 2

articulata angustifolia benghalensis cordata

DPIV* E/T (%)* % Aa FAO VBT*** CEPT1* CEPT2 CEPT3 PDCAAS***

0.1

-1.9

-3.9 -6

-3

0

3

6

9

Funci Function 1 Fig. 1. Distribution in groups of taxa studied according to discriminant analysis.

Group 1

Group 2

Group 3

83.39 ± 2.25b 40.53 ± 1.27a 119.49 ± 3.75a 49.32 ± 9.61c 2.80 ± 0.20a 2.85 ± 0.20 2.81 ± 0.38 0.51 ± 0.05b

81.98 ± 3.06a 41.21 ± 1.76ab 121.55 ± 5.22ab 34.76 ± 9.43b 2.93 ± 0.30b 2.96 ± 0.30 2.97 ± 0.55 0.45 ± 0.07a

82.16 ± 2.28ab 41.60 ± 1.95b 122.73 ± 5.78b 22.81 ± 5.52a 2.81 ± 0.28ab 2.88 ± 0.28 2.89 ± 0.50 0.43 ± 0.08a

% EAA/TAA = % Essential amino acid/total amino acids. AAS = Amino acids store. BV = Biological value. PER = Protein efficiency ratio. PDCAAS = Protein digestibility corrected amino acid score. * P < 0.05. *** P < 0.01.

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group 3 have the highest values (P < 0.01 and P < 0.001, respectively), but the tyrosine content compensates the proportion of aromatic amino acids (phenylalanine + tyrosine) in the three groups (Table 5). The highest IVPD was observed in the taxa of group 1, although only a significant difference (P < 0.05) with the taxa in group 2 was detected (Table 6). On the other hand, the results obtained for multiple nutritional parameters in the three groups are shown in Table 6. As described in paragraphs above, the most reliable nutritional parameter is the protein-digestibility corrected amino acid score (Sarwar, 1997; Schaafsma, 2005). Group 1 has the highest values for this index (PDCAAS), distinguishing itself clearly (P < 0.001) from the other two groups. Moreover, this group also has the highest biological value BV (P < 0.001). With respect to the theoretical PER, differences (P < 0.05) have only been found in PER1, and group 2 has the highest score. 4. Conclusion The results obtained indicate that the taxa in group 1 show the most adequate protein profile jointly. It should be noted that seven of the thirteen taxa included in this group have never been used in crops. Additionally, even though the taxa in group 2 and 3 feature a slightly less adequate profile, it should be noted that they include species which are very commonly used in crops, such as L. culinaris, P. sativum or V. faba, so any of the species examined in the present study might be used in future crops. Acknowledgement This work was financed by Grant AGR-711 from Junta de Andalucía (Spain). Thanks are due to María Dolores García-Contreras for technical assistance. References Abreu, J. M. F., & Bruno-Soares, A. M. (1998). Chemical composition, organic matter digestibility and gas production of nine legume grains. Animal Feed Science and Technology, 70, 49–57. Akalu, G., Johansson, G., & Nair, B. M. (1998). Effect of processing on the content of b-N-oxaly-a, b-diaminopropionic acid (b-ODAP) in grass pea (Lathyrus sativus) seeds and flours as determined by flow injection analysis. Food Chemistry, 62, 233–237. Alaiz, M., Navarro, J. L., Giron, J., & Vioque, E. (1992). Amino acid analysis by highperformance liquid chromatography after derivatization with diethyl ethoxymethylenemalonate. Journal of Chromatography, 591, 181–186. Alsmeyer, R. H., Cunningham, A. E., & Happich, M. L. (1974). Equations predict PER from amino acid analysis. Food Technology, 28, 34–38. Anjum, F. M., Ahmad, I., Butt, M. S., Sheikh, M. A., & Pasha, I. (2005). Amino acid composition of spring wheats and losses of lysine during chapatti baking. Journal of Food Composition and Analysis, 18, 523–532. AOAC (1999). Official methods of analysis (16th). Arlington, Washington DC: Association of Official Analytical Chemists. Campbell, C. G., Mehra, R. B., Agrawal, S. K., Chen, Y. Z., Abd El Moneim, A. M., Khawaha, H. I. T., et al. (1994). Current status and future strategy in breeding grasspea (Lathyrus sativus). Euphytica, 73, 167–175. Carbonaro, M., Cappelloni, M., Nicoli, S., Lucarini, M., & Carnovale, E. (1997). Solubility–digestibility relationship of legume proteins. Journal of Agricultural and Food Chemistry, 45, 3387–3394. Cepeda, E., Villaran, M. C., & Aranguiz, N. (1998). Functional properties of faba bean (Vicia faba) protein flour dried by spray drying and freeze drying. Journal of Food Engineering, 36, 303. Chakraborty, P., Bramsnaes, F., & Bose, A. N. (1979). Characteristics of enzymatic hydrolysate of faba bean (Vicia faba minor) protein. Journal of Food Science and Technology – Mysore, 16, 137–142. De Almeida Costa, G. E., Da Silva Queiroz-Monici, K., Pissini Machado Reis, S. M., & Costa de Oliveira, A. (2006). Chemical composition, dietary fibre and resitan starch contents of raw and cooked pea, common bean, chickpea and lentil legumes. Food Chemistry, 94, 327–330. Duranti, M. (2006). Grain legume proteins and nutraceutical properties. Fitoterapia, 77, 67–82. FAO/WHO (1989). Protein quality evaluation. Report of the joint FAO/WHO expert consultation. Food and nutrition paper No. 51. Rome: Food and Agriculture Organizations and the World Health Organization.

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