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Analytical nutritional characteristics of seed proteins in six wild Lupinus species from Southern Spain
Food Chemistry 117 (2009) 466–469
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Short communication
Analytical nutritional characteristics of seed proteins in six wild Lupinus species from Southern Spain 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 García Tejero 4, 41012 Sevilla, Spain Departamento 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 3 December 2008 Received in revised form 14 April 2009 Accepted 15 April 2009
Keywords: Lupinus Seed proteins Amino acids Nutritional quality
a b s t r a c t The nutritional characteristics of seed proteins of Spanish wild populations of Lupinus angustifolius, L. cosentinii, L. gredensis, L. hispanicus, L. luteus and L. micranthus have been studied. Protein contents in this genus ranged from 23.8% in L. gredensis to 33.6% in L. luteus. On the one hand, L. cosentinii showed the most balanced amino acid composition, being only deficient in lysine. On the other hand, L. gredensis showed the worst amino acid composition. The in vitro protein digestibility (IVPD) was high in all species examined, ranging from 82.3% in L. gredensis to 89.0% in L. cosentinii. In addition to the amino acid composition and IVPD, other nutritional parameters, such as amino acid score, calculated biological value, predicted protein efficiency ratio or protein digestibility corrected amino acid score, were studied. These data yielded L. luteus, L. hispanicus and L. cosentinii as the species with seed proteins with the best nutritional properties, similar to those observed in other legumes with recognised high quality proteins, such as soybean. Results confirm the importance of studying wild populations of cultivated and non-cultivated Lupinus species as sources of seeds with good nutritional characteristics. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Legumes represent, together with cereals, the main plant source of proteins in human diet. Beans are generally rich in high quality proteins, dietary fibre and carbohydrates and have a low content of saturated fats. For these reasons, beneficial health effects derived from legume consumption have been recognised and related to legume components, such as fibre or proteins and other minor compounds, such as certain lipids, polyphenols or bioactive peptides (Rochfort & Panozzo, 2007). These health promoting effects have been related to the prevention of diseases like diabetes mellitus, coronary heart diseases, or colon cancer (Duranti, 2006). However, legume consumption has decreased in recent decades in many Western countries, although the chemical and nutritional composition of beans show that they could play a much more important role in human nutrition. The genus Lupinus is not an exception and many of the cultivated species have seen their areas of cultivation reduced in the last century. However, the conservation of biodiversity may be, especially in developing countries, an important factor for their development. In order to recover and maintain this biodiversity the diversification of crops is necessary and this can be achieved by increasing our knowledge of local plants. Legume
* Corresponding author. Tel.: +34 954611550; fax: +34 959616790. E-mail addresses: [email protected], [email protected] (J. Vioque). 0308-8146/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2009.04.039
seeds are one of the most promising alternative sources of protein for human and animal nutrition. Among the main cultivated legumes, soybean is by far the most important. Other legumes, such as lupins, are being studied as potential alternative sources of protein in order to reduce the need to import soybean. Lupinus is an ancient crop belonging to the tribe Genisteae that was widely cultivated in the Mediterranean Region and South American Andes. The major cultivated species of lupins are L. albus (white lupin), L. angustifolius (blue lupin), L. luteus (yellow lupin) and L. mutabilis (pearl lupin). On average, lupin seeds have total protein content similar to soybean and acceptable amounts of essential amino acids. The chemical, functional and nutritional properties of lupin proteins mean that lupin seeds can be considered as a source of high quality protein (Lampart-Szczapa, Obuchowski, Czaczyk, Pastuszewska, & Buraczewska, 1997), with the protein content of cultivated lupin seeds ranging from 33.8% in L. angustifolius (Lqari, Vioque, Pedroche, & Millán, 2002) to 44% in L. albus (Duranti, Restani, Poniatowska, & Cerletti, 1981). Another interesting aspect of lupins as a new source of protein is that they can be grown in soils and in climates where soybean cannot grow. It has been suggested that lupin proteins supplemented with methionine could replace soy concentrate in countries where soybean must be imported (Ruiz & Hove, 1976). Moreover, lupin seeds have a lower content of the main anti-nutritional components often contained in legumes, such as phytates, oligosaccharides, trypsin inhibitors and lectins (El-Adawy, Rahma, El-Bedawey, & Gafar, 2001). Furthermore, Lupinus seed flours have been used for the production of
E. Pastor-Cavada et al. / Food Chemistry 117 (2009) 466–469
protein isolates with good functional and nutritional properties (Lqari et al., 2002). The aims of this study were to evaluate the nutritional characteristics of seed proteins of six lupins species which grow in Southern Spain. 2. Materials and methods
467
2.5. Cluster analysis Cluster analysis of different taxa was performed using PRIMERpc program, employing the Bray–Curtis index of dissimilarity (Bray & Curtis, 1957). The dissimilarity index was transformed to the index of similarity (1 dissimilarity index 100).
2.1. Materials
3. Results and discussion
Trypsin, chymotrypsin and peptidase were acquired from Sigma (Tres Cantos, Madrid, Spain). Diethyl ethoxymethylenemanolate was purchased from Fluka. All other chemicals were of analytical grade. The samples of Lupinus seeds were collected from wild populations in Spain.
Seed protein contents in studied lupins ranged from 23.8% in L. gredensis to 33.6% in L. luteus (Table 1). The percentages observed in wild populations are lower than those observed in seeds belonging to cultivated samples from the same species. Hence, L. angustifolius samples studied contained as an average 26.6% protein in contrast to 33.8% in commercial samples (Lqari et al., 2002). However, protein contents of the studied lupins were higher than those observed in other commercial legumes such as chickpea (24.7%) (Sánchez-Vioque, Clemente, Vioque, Bautista, & Millán, 1999). The amino acid composition of studied wild lupin populations is shown in Table 1. All species show an amino acid pattern characteristic of legumes. Thus, except L. cosentinii, all taxa are deficient in sulphur amino acids and tryptophan according to FAO recommendations (FAO/WHO/UNU, 1985). Also, some species were deficient in aromatic amino acids and valine. Besides, all taxa, except L. hispanicus, were deficient in lysine, while in legumes the lysine content is usually above that suggested by FAO (1985). On the one hand, from the amino acid composition point of view, the most equilibrated species are L. cosentinii which is only deficient in lysine, and L. hispanicus which is also deficient in sulphur amino acids and tryptophan. On the other hand, L. angustifolius and L. luteus appear as the species which have the worst amino acid composition. This deficiency of lysine, tryptophan and valine has also been reported for commercial samples of L. luteus, L. angustifolius and L. albus (Sujak, Kotlarz, & Strobel, 2006). The importance of protein composition in the diet has been well known for many years. Less clear is how to determine the nutritional quality of protein intake, which depends mainly on the amino acid contents. The first step is to determine the amino acid composition of proteins. To make use of these amino acids proteins must be digested in order to release them. Many different factors may impair digestibility, which would result in lower protein quality. Hence, in addition to amino acid composition, protein digestibility has been proposed as another protein characteristic which determines the protein quality. In vitro protein digestibility (IVPD) in studied Lupinus ranged from 82.3% in L. gredensis to 89.0% in L. cosentinii. This is a high IVPD, similar to those observed in other lupins, such as L. albus (86.9–88.8%) (El-Adawy et al., 2001), or to soybean (85.8%), rice (84.8%), or wheat (83.7%) (Wolzak, Elias, & Bressani, 1981). Moreover, it is higher than those observed in other legumes, such as chickpea (76.2%) (Sánchez-Vioque et al., 1999). The highest predicted biological value was in L. cosentinii and L. luteus with values of 85.9 and 90.2, respectively (Table 2). The lowest predicted biological values were observed in L. hispanicus with 42.3. The best predicted biological values observed in studied Lupinus were lower than those described for lactoalbumin, with 97%, but higher than those reported for other crops, such as triticale (65.3) or wheat (61.6) (Friedman, 1996). All Lupinus species studied here showed PER theoretical values over 2 and many even close to 3, corresponding to proteins of high nutritional value. Better calculated PER values were observed in L. luteus with indexes around 3. The lowest values were calculated for L. gredensis and L. angustifolius. Calculated PER values in wild populations of L. Luteus studied (Table 2) were higher than those previously observed in cultivated L. luteus (between 2.17 and 2.67) (Sujak et al., 2006), and those in many meat-containing foods
2.2. Amino acid analysis Duplicate 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 Alaiz, Navarro, Giron, and Vioque (1992), using D,L-a-aminobutyric acid as an internal standard. Tryptophan was analysed by HPLC after basic hydrolysis according to Yust et al. (2004). 2.3. In vitro protein digestibility (IVPD) In vitro protein digestibility was determined according to the method of Hsu, Vavak, Satterlee, and Miller (1977). 2.4. Determination of nutritional parameters The amino acid composition of studied lupins was used for the determination of several nutritional parameters of lupin seed proteins: – Amino acid score (chemical score) was calculated as: % sample essential amino acids contents/% recommended essential amino acids (FAO/WHO/UNU, 1985). – Protein efficiency ratio values (PER) were calculated from the amino acid composition of lupin seeds based on the following three equations (Alsmeyer, Cunningham, & Happich, 1974):
PER1 ¼ 0:684 þ 0:456 Leu 0:047 Pro PER2 ¼ 0:468 þ 0:454 Leu 0:105 Tyr PER3 ¼ 1:816 þ 0:435 Met þ 0:78 Leu þ 0:211 His 0:944 Tyr – Protein digestibility corrected amino acid score (PDCAAS) (FAO/ WHO, 1989) was calculated as: The lowest individual amino acid score IVPD. – Predicted biological value (BV) was calculated according to Morup and Olesen (1976) using the following equation:
BV ¼ 102:15 Lys0:41 ðPhe þ TyrÞ0:60 ðMet þ CysÞ0:77 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.
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Table 1 Seed protein amino acids composition of studied lupins. Data expressed as g/100 g protein are the average ± standard deviation of indicated number of populations studied.
b
n Aspc Glud Ser His Gly Thr Arg Ala Pro Tyr Val Met Cys Ile Trp Leu Phe Lys Protein a b c d e f
L. angustifolius
L. cosentinii
L. gredensis
L. hispanicus
L. luteus
L. micranthus
4 11.8 ± 0.6 22.9 ± 0.2 6.4 ± 0.3 2.9 ± 0.2 4.6 ± 0.1 4.1 ± 0.1 11.4 ± 0.3 3.9 ± 0.0 3.1 ± 0.3 2.9 ± 0.0 3.4 ± 0.0 0.5 ± 0.1 1.0 ± 0.1 3.3 ± 0.0 1.0 ± 0.1 7.4 ± 0.2 4.0 ± 0.0 5.2 ± 0.1 26.6 ± 1.6
1 11.7 24.2 5.2 2.6 4.4 4.1 10.3 3.9 3.4 2.5 3.8 0.7 1.8 3.6 1.1 7.8 3.9 5.2 24.7
1 16.0 22.4 7.8 2.7 6.0 3.0 11.2 4.5 2.1 2.4 1.9 0.7 1.3 2.2 0.7 6.9 3.4 4.9 23.8
3 12.0 ± 0.1 22.0 ± 0.7 6.4 ± 0.0 2.8 ± 0.1 4.6 ± 0.1 4.3 ± 0.2 11.2 ± 0.4 3.8 ± 0.1 2.3 ± 0.2 2.7 ± 0.1 4.0 ± 0.1 0.1 ± 0.2 1.2 ± 0.1 3.7 ± 0.0 0.8 ± 0.2 7.9 ± 0.1 4.3 ± 0.1 5.9 ± 0.2 33.2 ± 1.2
3 11.6 ± 0.3 23.7 ± 0.7 6.5 ± 0.0 2.8 ± 0.1 4.5 ± 0.1 3.8 ± 0.2 11.5 ± 0.7 3.6 ± 0.2 2.3 ± 0.2 2.3 ± 0.1 3.3 ± 0.1 0.2 ± 0.1 2.0 ± 0.3 3.1 ± 0.0 0.8 ± 0.2 8.2 ± 0.0 4.0 ± 0.1 5.4 ± 0.1 33.6 ± 1.8
3 12.1 ± 0.1 22.4 ± 0.6 5.7 ± 0.1 2.6 ± 0.0 4.4 ± 0.1 4.2 ± 0.1 13.0 ± 0.9 4.0 ± 0.1 2.2 ± 0.2 2.8 ± 0.1 3.7 ± 0.1 0.6 ± 0.0 1.2 ± 0.3 4.0 ± 0.0 0.5 ± 0.1 7.8 ± 0.1 3.9 ± 0.1 4.9 ± 0.1 31.1 ± 1.4
FAOa
1.9 3.4
6.3e 3.5 2.5f 2.8 1.1 6.6 5.8
Suggested pattern of amino acid requirements (FAO/WHO/UNU, 1985). Number of populations studied. Asp + Asn. Glu + Gln. Tyr + Phe. Met + Cys.
Table 2 Nutritional characteristics of studied lupin seed proteins. Results are the average of populations indicated in Table 2 for each taxa.
a
IVPD % EAA/TAAb AASc BVd PER1e PER2 PER3 PDCAASf a b c d e f
L. angustifolius
L. cosentinii
L. gredensis
L. hispanicus
L. luteus
L. micranthus
87.6 34.7 102.4 47.3 2.54 2.60 2.05 0.53
89.0 35.3 104.1 85.92 2.72 2.82 2.81 0.80
82.3 29.3 86.4 67.9 2.36 2.39 2.15 0.45
88.5 36.5 107.7 42.3 2.81 2.85 2.51 0.46
88.5 33.9 100 90.2 2.95 3.03 3.09 0.64
87.5 35.0 103.2 50.2 2.77 2.80 2.43 0.40
In vitro protein digestibility. % Essential amino acids/total amino acids. Amino acids score. Biological value. Protein efficiency ratio. Protein digestibility corrected amino acid score.
(Alsmeyer et al., 1974). The Lupinus angustifolius calculated PER reported here were similar to those calculated for cultivated samples (between 2.10 and 2.77) (Sujak et al., 2006). Similar values to those reported here for Lupinus have been described for other legumes, such as soybean (2.57) (Wolzak et al., 1981) or chickpea (2.8) (Newman, Roth, Newman, & Lockerman, 1987). Lower PER values were observed in other legumes such as peanut (between 1.45 and 1.76) (Ghuman, Mann, & Hira, 1990), or Vigna radiata (between 1.6 and 2.1) (Savage & Deo, 1989). The use of amino acid scores has been proposed as a more accurate alternative to PER parameters (Sarwar et al., 1984). The amino acids score was the highest in L. hispanicus and L. cosentinii with average values of 107.7 and 104.1, respectively (Table 2). The lowest value was observed in L. gredensis with average values of 86.4. Similarly, the% essential amino acids / total amino acids ratio was the highest in L. hispanicus and L. cosentinii with average values of 36.5 and 35.3, respectively. The PDCAAS is nowadays the most recommended theoretical parameter for evaluating the nutritional quality of food proteins. It is based on FAO recommendations of amino acid requirements (FAO/WHO/UNU, 1985) and in vitro protein digestibility. The highest PDCAAS value for a given protein is 1.0. The lowest PDCAAS was observed in L. micranthus with 0.4, while the highest PDCAAS was
observed in L. cosentinii with 0.80. This index was higher than that observed in other legumes, such as peas (0.69), beans (0.68) or lentils (0.52). The analysis of similarity based on a profile of seed protein amino acid composition shows two major groups (A and B, Fig. 1) with 91.7% affinity. The first includes only L. gredensis, while the remaining species studied form the other group (group B). Within group B, two groups (C and D) are distinguished with 95.8% similarity. Group C includes only L. cosentinii, while the remaining species form group D. In the latter group two other groups are recognised (E and F) with 96% affinity. Not considering L. gredensis, the separation of L. cosentinii (group C) from the other species (group D) supports the delimitation of the Lupinus species on the basis of the seed coat texture into two distinct groups: the rough-seeded (L. cosentinii) and the smooth-seeded species (the remaining species) (Naganowska, Wolko, Sliwinska, & Kaczmarek, 2003; Przybylska & Zimmniak-Przybylska, 1995). With respect to group D the subdivision (with 96% affinity) in group E (L. micranthus) and group F is in accordance with molecular data (Naganowska et al., 2003). According to the amino acid composition L. gredensis is the most divergent specie. In conclusion, the analysis of similarity of studied Lupinus based on the amino acid composition shows some discrepancy with the established interspecific taxonomical
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References
Fig. 1. Clustering based on the amino acid composition of studied Lupinus species, according to the Bray–Curtis similarity index (1 dissimilarity index 100).
relationships. However, amino acid composition was useful to group in single clusters populations belonging to the same specie. From the studied species L. cosentinii appears as the one with the most equilibrated amino acid composition. It was only deficient in lysine. It is used for livestock feeding and soil fertilisation and has been used in breeding programmes in Australia since 1954 (Weder, Salmanowicz, & Köhler, 1997) due to its successful adaptation to alkaline soils. With respect to the nutritional parameters, L. luteus, L. hispanicus and L. cosentinii showed the best results. On the contrary, L. gredensis showed the worst nutritional characteristics. Results confirm the interest in studying wild populations of cultivated and non-cultivated Lupinus species as a source of seeds with good nutritional characteristics. This may help in the domestication of new species or the use of wild populations in breeding programmes, favouring the bio-conservation of Lupinus. 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.
Alaiz, M., Navarro, J. L., Giron, J., & Vioque, E. (1992). Amino acid analysis by highperformance liquid chromatography after derivatization with diethylethoxymethylenemalonate. 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. Bray, R. J., & Curtis, J. I. (1957). An ordination of the upland forest communities of southern Wisconsin. Ecological Monographs, 27, 325–349. Duranti, M. (2006). Grain legume proteins and nutraceutical properties. Fitoterapia, 77, 67–82. Duranti, M., Restani, M., Poniatowska, M., & Cerletti, P. (1981). The seed globulins of Lupinus-albus. Phytochemistry, 20, 2071–2075. El-Adawy, T. A., Rahma, E. H., El-Bedawey, A. A., & Gafar, A. F. (2001). Nutritional potential and functional properties of sweet and bitter lupin seed protein isolates. Food Chemistry, 74, 455–462. 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. FAO/WHO/UNU (1985). Energy and protein requirements, report of the joint FAO/ WHO/UNU expert consultation. Technical report series no. 724. Geneva: FAO, WHO and the United Nations University. Friedman, M. (1996). Nutritional value of proteins from different food sources. A review. Journal of Agricultural and Food Chemistry, 44, 6–29. Ghuman, P. K., Mann, S. K., & Hira, C. K. (1990). Evaluation of protein quality of peanut (Arachis hypogaea) cultivars using Tetrahymena pyriformis. Journal of Agricultural and Food Chemistry, 52, 137–139. Hsu, H. W., Vavak, D. L., Satterlee, L. D., & Miller, G. A. (1977). A multienzyme technique for estimating protein digestibility. Journal of Food Science, 42, 1269–1273. Lampart-Szczapa, E., Obuchowski, W., Czaczyk, K., Pastuszewska, B., & Buraczewska, L. (1997). Effect of lupine flour on the quality and oligosaccharides of pasta and crisps. Nahrung-Food, 41, 219–223. Lqari, H., Vioque, J., Pedroche, J., & Millán, F. (2002). Lupinus angustifolius protein isolates: Chemical composition, functional properties and protein characterization. Food Chemistry, 76, 349–356. Morup, I. K., & Olesen, E. S. (1976). New method for prediction of protein value from essential amino acid pattern. Nutritional Report International, 13, 355–365. Naganowska, B., Wolko, B., Sliwinska, E., & Kaczmarek, Z. (2003). Nuclear DNA content variation and species relationships in the genus Lupinus (Fabaceae). Annals of Botany, 92, 349–355. Newman, C. W., Roth, N. R., Newman, R. K., & Lockerman, R. H. (1987). Protein quality of chickpea (Cicer arietinum L.). Nutrition Reports International, 36, 1–5. Przybylska, J., & Zimmniak-Przybylska, Z. (1995). Electrophoretic patterns of seed globulins in the Old-World Lupinus species. Genetic Resources and Crop Evolution, 42, 69–75. Rochfort, S., & Panozzo, J. (2007). Phytochemicals for health, the role of pulses. Journal of Agricultural and Food Chemistry, 55, 7981–7994. Ruiz, L. P., Jr, & Hove, E. L. (1976). Conditions affecting production of protein isolate from lupin seed kernel. Journal of the Science and Food Agriculture, 27, 667–674. Sánchez-Vioque, R., Clemente, A., Vioque, J., Bautista, J., & Millán, F. (1999). Protein isolate from chickpea (Cicer arietinum L.): Chemical composition, functional properties and protein characterization. Food Chemistry, 64(23), 7–243. Sarwar, G., Blair, R., Friedman, M., Gumbmann, M. R., Hackler, L. R., Pellet, P. L., et al. (1984). Inter- and intra-laboratory variability in rat growth assays for estimating protein quality in foods. Journal of Association of Official Analytical Chemists, 67, 976–981. Savage, G. P., & Deo, D. (1989). The nutritional value of mung bean and urd (Vigna radiata var. Aureus and var. mungo). Nutrition Abstracts and Review, 59, 639–662. Sujak, A., Kotlarz, A., & Strobel, W. (2006). Compositional and nutritional evaluation of several lupin seeds. Food Chemistry, 98, 711–719. Weder, J. K. P., Salmanowicz, B. P., & Köhler, P. (1997). Primary structure of 2S albumin from seeds of Lupinus cosentinii. Zeitschrift fur Lebensmittel Untersuchung und Forschung A, 205, 452–456. Wolzak, A., Elias, L. G., & Bressani, R. (1981). Protein quality of vegetable proteins as determined by traditional biological methods and rapid chemical assays. Journal of Agriculture and Food Chemistry, 29, 1063–1068. Yust, M. M., Pedroche, J., Girón-Calle, J., Vioque, J., Millán, F., & Alaiz, M. (2004). Determination of tryptophan by high-performance liquid chromatography of alkaline hydrolysates with spectrophotometric detection. Food Chemistry, 85, 317–320.
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Short communication
Analytical nutritional characteristics of seed proteins in six wild Lupinus species from Southern Spain 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 García Tejero 4, 41012 Sevilla, Spain Departamento 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 3 December 2008 Received in revised form 14 April 2009 Accepted 15 April 2009
Keywords: Lupinus Seed proteins Amino acids Nutritional quality
a b s t r a c t The nutritional characteristics of seed proteins of Spanish wild populations of Lupinus angustifolius, L. cosentinii, L. gredensis, L. hispanicus, L. luteus and L. micranthus have been studied. Protein contents in this genus ranged from 23.8% in L. gredensis to 33.6% in L. luteus. On the one hand, L. cosentinii showed the most balanced amino acid composition, being only deficient in lysine. On the other hand, L. gredensis showed the worst amino acid composition. The in vitro protein digestibility (IVPD) was high in all species examined, ranging from 82.3% in L. gredensis to 89.0% in L. cosentinii. In addition to the amino acid composition and IVPD, other nutritional parameters, such as amino acid score, calculated biological value, predicted protein efficiency ratio or protein digestibility corrected amino acid score, were studied. These data yielded L. luteus, L. hispanicus and L. cosentinii as the species with seed proteins with the best nutritional properties, similar to those observed in other legumes with recognised high quality proteins, such as soybean. Results confirm the importance of studying wild populations of cultivated and non-cultivated Lupinus species as sources of seeds with good nutritional characteristics. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Legumes represent, together with cereals, the main plant source of proteins in human diet. Beans are generally rich in high quality proteins, dietary fibre and carbohydrates and have a low content of saturated fats. For these reasons, beneficial health effects derived from legume consumption have been recognised and related to legume components, such as fibre or proteins and other minor compounds, such as certain lipids, polyphenols or bioactive peptides (Rochfort & Panozzo, 2007). These health promoting effects have been related to the prevention of diseases like diabetes mellitus, coronary heart diseases, or colon cancer (Duranti, 2006). However, legume consumption has decreased in recent decades in many Western countries, although the chemical and nutritional composition of beans show that they could play a much more important role in human nutrition. The genus Lupinus is not an exception and many of the cultivated species have seen their areas of cultivation reduced in the last century. However, the conservation of biodiversity may be, especially in developing countries, an important factor for their development. In order to recover and maintain this biodiversity the diversification of crops is necessary and this can be achieved by increasing our knowledge of local plants. Legume
* Corresponding author. Tel.: +34 954611550; fax: +34 959616790. E-mail addresses: [email protected], [email protected] (J. Vioque). 0308-8146/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2009.04.039
seeds are one of the most promising alternative sources of protein for human and animal nutrition. Among the main cultivated legumes, soybean is by far the most important. Other legumes, such as lupins, are being studied as potential alternative sources of protein in order to reduce the need to import soybean. Lupinus is an ancient crop belonging to the tribe Genisteae that was widely cultivated in the Mediterranean Region and South American Andes. The major cultivated species of lupins are L. albus (white lupin), L. angustifolius (blue lupin), L. luteus (yellow lupin) and L. mutabilis (pearl lupin). On average, lupin seeds have total protein content similar to soybean and acceptable amounts of essential amino acids. The chemical, functional and nutritional properties of lupin proteins mean that lupin seeds can be considered as a source of high quality protein (Lampart-Szczapa, Obuchowski, Czaczyk, Pastuszewska, & Buraczewska, 1997), with the protein content of cultivated lupin seeds ranging from 33.8% in L. angustifolius (Lqari, Vioque, Pedroche, & Millán, 2002) to 44% in L. albus (Duranti, Restani, Poniatowska, & Cerletti, 1981). Another interesting aspect of lupins as a new source of protein is that they can be grown in soils and in climates where soybean cannot grow. It has been suggested that lupin proteins supplemented with methionine could replace soy concentrate in countries where soybean must be imported (Ruiz & Hove, 1976). Moreover, lupin seeds have a lower content of the main anti-nutritional components often contained in legumes, such as phytates, oligosaccharides, trypsin inhibitors and lectins (El-Adawy, Rahma, El-Bedawey, & Gafar, 2001). Furthermore, Lupinus seed flours have been used for the production of
E. Pastor-Cavada et al. / Food Chemistry 117 (2009) 466–469
protein isolates with good functional and nutritional properties (Lqari et al., 2002). The aims of this study were to evaluate the nutritional characteristics of seed proteins of six lupins species which grow in Southern Spain. 2. Materials and methods
467
2.5. Cluster analysis Cluster analysis of different taxa was performed using PRIMERpc program, employing the Bray–Curtis index of dissimilarity (Bray & Curtis, 1957). The dissimilarity index was transformed to the index of similarity (1 dissimilarity index 100).
2.1. Materials
3. Results and discussion
Trypsin, chymotrypsin and peptidase were acquired from Sigma (Tres Cantos, Madrid, Spain). Diethyl ethoxymethylenemanolate was purchased from Fluka. All other chemicals were of analytical grade. The samples of Lupinus seeds were collected from wild populations in Spain.
Seed protein contents in studied lupins ranged from 23.8% in L. gredensis to 33.6% in L. luteus (Table 1). The percentages observed in wild populations are lower than those observed in seeds belonging to cultivated samples from the same species. Hence, L. angustifolius samples studied contained as an average 26.6% protein in contrast to 33.8% in commercial samples (Lqari et al., 2002). However, protein contents of the studied lupins were higher than those observed in other commercial legumes such as chickpea (24.7%) (Sánchez-Vioque, Clemente, Vioque, Bautista, & Millán, 1999). The amino acid composition of studied wild lupin populations is shown in Table 1. All species show an amino acid pattern characteristic of legumes. Thus, except L. cosentinii, all taxa are deficient in sulphur amino acids and tryptophan according to FAO recommendations (FAO/WHO/UNU, 1985). Also, some species were deficient in aromatic amino acids and valine. Besides, all taxa, except L. hispanicus, were deficient in lysine, while in legumes the lysine content is usually above that suggested by FAO (1985). On the one hand, from the amino acid composition point of view, the most equilibrated species are L. cosentinii which is only deficient in lysine, and L. hispanicus which is also deficient in sulphur amino acids and tryptophan. On the other hand, L. angustifolius and L. luteus appear as the species which have the worst amino acid composition. This deficiency of lysine, tryptophan and valine has also been reported for commercial samples of L. luteus, L. angustifolius and L. albus (Sujak, Kotlarz, & Strobel, 2006). The importance of protein composition in the diet has been well known for many years. Less clear is how to determine the nutritional quality of protein intake, which depends mainly on the amino acid contents. The first step is to determine the amino acid composition of proteins. To make use of these amino acids proteins must be digested in order to release them. Many different factors may impair digestibility, which would result in lower protein quality. Hence, in addition to amino acid composition, protein digestibility has been proposed as another protein characteristic which determines the protein quality. In vitro protein digestibility (IVPD) in studied Lupinus ranged from 82.3% in L. gredensis to 89.0% in L. cosentinii. This is a high IVPD, similar to those observed in other lupins, such as L. albus (86.9–88.8%) (El-Adawy et al., 2001), or to soybean (85.8%), rice (84.8%), or wheat (83.7%) (Wolzak, Elias, & Bressani, 1981). Moreover, it is higher than those observed in other legumes, such as chickpea (76.2%) (Sánchez-Vioque et al., 1999). The highest predicted biological value was in L. cosentinii and L. luteus with values of 85.9 and 90.2, respectively (Table 2). The lowest predicted biological values were observed in L. hispanicus with 42.3. The best predicted biological values observed in studied Lupinus were lower than those described for lactoalbumin, with 97%, but higher than those reported for other crops, such as triticale (65.3) or wheat (61.6) (Friedman, 1996). All Lupinus species studied here showed PER theoretical values over 2 and many even close to 3, corresponding to proteins of high nutritional value. Better calculated PER values were observed in L. luteus with indexes around 3. The lowest values were calculated for L. gredensis and L. angustifolius. Calculated PER values in wild populations of L. Luteus studied (Table 2) were higher than those previously observed in cultivated L. luteus (between 2.17 and 2.67) (Sujak et al., 2006), and those in many meat-containing foods
2.2. Amino acid analysis Duplicate 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 Alaiz, Navarro, Giron, and Vioque (1992), using D,L-a-aminobutyric acid as an internal standard. Tryptophan was analysed by HPLC after basic hydrolysis according to Yust et al. (2004). 2.3. In vitro protein digestibility (IVPD) In vitro protein digestibility was determined according to the method of Hsu, Vavak, Satterlee, and Miller (1977). 2.4. Determination of nutritional parameters The amino acid composition of studied lupins was used for the determination of several nutritional parameters of lupin seed proteins: – Amino acid score (chemical score) was calculated as: % sample essential amino acids contents/% recommended essential amino acids (FAO/WHO/UNU, 1985). – Protein efficiency ratio values (PER) were calculated from the amino acid composition of lupin seeds based on the following three equations (Alsmeyer, Cunningham, & Happich, 1974):
PER1 ¼ 0:684 þ 0:456 Leu 0:047 Pro PER2 ¼ 0:468 þ 0:454 Leu 0:105 Tyr PER3 ¼ 1:816 þ 0:435 Met þ 0:78 Leu þ 0:211 His 0:944 Tyr – Protein digestibility corrected amino acid score (PDCAAS) (FAO/ WHO, 1989) was calculated as: The lowest individual amino acid score IVPD. – Predicted biological value (BV) was calculated according to Morup and Olesen (1976) using the following equation:
BV ¼ 102:15 Lys0:41 ðPhe þ TyrÞ0:60 ðMet þ CysÞ0:77 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.
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Table 1 Seed protein amino acids composition of studied lupins. Data expressed as g/100 g protein are the average ± standard deviation of indicated number of populations studied.
b
n Aspc Glud Ser His Gly Thr Arg Ala Pro Tyr Val Met Cys Ile Trp Leu Phe Lys Protein a b c d e f
L. angustifolius
L. cosentinii
L. gredensis
L. hispanicus
L. luteus
L. micranthus
4 11.8 ± 0.6 22.9 ± 0.2 6.4 ± 0.3 2.9 ± 0.2 4.6 ± 0.1 4.1 ± 0.1 11.4 ± 0.3 3.9 ± 0.0 3.1 ± 0.3 2.9 ± 0.0 3.4 ± 0.0 0.5 ± 0.1 1.0 ± 0.1 3.3 ± 0.0 1.0 ± 0.1 7.4 ± 0.2 4.0 ± 0.0 5.2 ± 0.1 26.6 ± 1.6
1 11.7 24.2 5.2 2.6 4.4 4.1 10.3 3.9 3.4 2.5 3.8 0.7 1.8 3.6 1.1 7.8 3.9 5.2 24.7
1 16.0 22.4 7.8 2.7 6.0 3.0 11.2 4.5 2.1 2.4 1.9 0.7 1.3 2.2 0.7 6.9 3.4 4.9 23.8
3 12.0 ± 0.1 22.0 ± 0.7 6.4 ± 0.0 2.8 ± 0.1 4.6 ± 0.1 4.3 ± 0.2 11.2 ± 0.4 3.8 ± 0.1 2.3 ± 0.2 2.7 ± 0.1 4.0 ± 0.1 0.1 ± 0.2 1.2 ± 0.1 3.7 ± 0.0 0.8 ± 0.2 7.9 ± 0.1 4.3 ± 0.1 5.9 ± 0.2 33.2 ± 1.2
3 11.6 ± 0.3 23.7 ± 0.7 6.5 ± 0.0 2.8 ± 0.1 4.5 ± 0.1 3.8 ± 0.2 11.5 ± 0.7 3.6 ± 0.2 2.3 ± 0.2 2.3 ± 0.1 3.3 ± 0.1 0.2 ± 0.1 2.0 ± 0.3 3.1 ± 0.0 0.8 ± 0.2 8.2 ± 0.0 4.0 ± 0.1 5.4 ± 0.1 33.6 ± 1.8
3 12.1 ± 0.1 22.4 ± 0.6 5.7 ± 0.1 2.6 ± 0.0 4.4 ± 0.1 4.2 ± 0.1 13.0 ± 0.9 4.0 ± 0.1 2.2 ± 0.2 2.8 ± 0.1 3.7 ± 0.1 0.6 ± 0.0 1.2 ± 0.3 4.0 ± 0.0 0.5 ± 0.1 7.8 ± 0.1 3.9 ± 0.1 4.9 ± 0.1 31.1 ± 1.4
FAOa
1.9 3.4
6.3e 3.5 2.5f 2.8 1.1 6.6 5.8
Suggested pattern of amino acid requirements (FAO/WHO/UNU, 1985). Number of populations studied. Asp + Asn. Glu + Gln. Tyr + Phe. Met + Cys.
Table 2 Nutritional characteristics of studied lupin seed proteins. Results are the average of populations indicated in Table 2 for each taxa.
a
IVPD % EAA/TAAb AASc BVd PER1e PER2 PER3 PDCAASf a b c d e f
L. angustifolius
L. cosentinii
L. gredensis
L. hispanicus
L. luteus
L. micranthus
87.6 34.7 102.4 47.3 2.54 2.60 2.05 0.53
89.0 35.3 104.1 85.92 2.72 2.82 2.81 0.80
82.3 29.3 86.4 67.9 2.36 2.39 2.15 0.45
88.5 36.5 107.7 42.3 2.81 2.85 2.51 0.46
88.5 33.9 100 90.2 2.95 3.03 3.09 0.64
87.5 35.0 103.2 50.2 2.77 2.80 2.43 0.40
In vitro protein digestibility. % Essential amino acids/total amino acids. Amino acids score. Biological value. Protein efficiency ratio. Protein digestibility corrected amino acid score.
(Alsmeyer et al., 1974). The Lupinus angustifolius calculated PER reported here were similar to those calculated for cultivated samples (between 2.10 and 2.77) (Sujak et al., 2006). Similar values to those reported here for Lupinus have been described for other legumes, such as soybean (2.57) (Wolzak et al., 1981) or chickpea (2.8) (Newman, Roth, Newman, & Lockerman, 1987). Lower PER values were observed in other legumes such as peanut (between 1.45 and 1.76) (Ghuman, Mann, & Hira, 1990), or Vigna radiata (between 1.6 and 2.1) (Savage & Deo, 1989). The use of amino acid scores has been proposed as a more accurate alternative to PER parameters (Sarwar et al., 1984). The amino acids score was the highest in L. hispanicus and L. cosentinii with average values of 107.7 and 104.1, respectively (Table 2). The lowest value was observed in L. gredensis with average values of 86.4. Similarly, the% essential amino acids / total amino acids ratio was the highest in L. hispanicus and L. cosentinii with average values of 36.5 and 35.3, respectively. The PDCAAS is nowadays the most recommended theoretical parameter for evaluating the nutritional quality of food proteins. It is based on FAO recommendations of amino acid requirements (FAO/WHO/UNU, 1985) and in vitro protein digestibility. The highest PDCAAS value for a given protein is 1.0. The lowest PDCAAS was observed in L. micranthus with 0.4, while the highest PDCAAS was
observed in L. cosentinii with 0.80. This index was higher than that observed in other legumes, such as peas (0.69), beans (0.68) or lentils (0.52). The analysis of similarity based on a profile of seed protein amino acid composition shows two major groups (A and B, Fig. 1) with 91.7% affinity. The first includes only L. gredensis, while the remaining species studied form the other group (group B). Within group B, two groups (C and D) are distinguished with 95.8% similarity. Group C includes only L. cosentinii, while the remaining species form group D. In the latter group two other groups are recognised (E and F) with 96% affinity. Not considering L. gredensis, the separation of L. cosentinii (group C) from the other species (group D) supports the delimitation of the Lupinus species on the basis of the seed coat texture into two distinct groups: the rough-seeded (L. cosentinii) and the smooth-seeded species (the remaining species) (Naganowska, Wolko, Sliwinska, & Kaczmarek, 2003; Przybylska & Zimmniak-Przybylska, 1995). With respect to group D the subdivision (with 96% affinity) in group E (L. micranthus) and group F is in accordance with molecular data (Naganowska et al., 2003). According to the amino acid composition L. gredensis is the most divergent specie. In conclusion, the analysis of similarity of studied Lupinus based on the amino acid composition shows some discrepancy with the established interspecific taxonomical
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References
Fig. 1. Clustering based on the amino acid composition of studied Lupinus species, according to the Bray–Curtis similarity index (1 dissimilarity index 100).
relationships. However, amino acid composition was useful to group in single clusters populations belonging to the same specie. From the studied species L. cosentinii appears as the one with the most equilibrated amino acid composition. It was only deficient in lysine. It is used for livestock feeding and soil fertilisation and has been used in breeding programmes in Australia since 1954 (Weder, Salmanowicz, & Köhler, 1997) due to its successful adaptation to alkaline soils. With respect to the nutritional parameters, L. luteus, L. hispanicus and L. cosentinii showed the best results. On the contrary, L. gredensis showed the worst nutritional characteristics. Results confirm the interest in studying wild populations of cultivated and non-cultivated Lupinus species as a source of seeds with good nutritional characteristics. This may help in the domestication of new species or the use of wild populations in breeding programmes, favouring the bio-conservation of Lupinus. 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.
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