Nutritional and Functional Characteristics of Erophaca Baetica Seeds, a Legume Endemic to the Mediterranean Region
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GRASAS Y ACEITES, 64 (3), ABRIL-JUNIO, 229-236, 2013, ISSN: 0017-3495 DOI: 10.3989/gya.107412 INVESTIGACIÓNINVESTIGACIÓN Nutritional and functional characteristics of Erophaca baetica seeds, a legume endemic to the Mediterranean region By I. Cortés-Giraldo, M. Alaiz, J. Girón-Calle, C. Megías and J. Vioque* Instituto de la Grasa (C.S.I.C.). Avda. Padre García Tejero 4. 41012 Sevilla, Spain * Corresponding author: [email protected] RESUMEN and linoleic acids are the major fatty acids in the seeds. The antioxidant activity of polyphenol extracts was higher than the Características nutricionales y funcionales de semi- activity of the polyphenols extracted from most edible legume llas de Erophaca baetica, una leguminosa endémica de seeds. Hence, E. baetica seeds represent a promising sour- la región Mediterránea ce of functional and nutritional components on the condition that the anti-nutritional alkaloids are previously removed. Erophaca baetica es una leguminosa endémica de la re- gión mediterránea. Aunque los frutos y las semillas son gran- KEY-WORDS: Amino acids – Erophaca baetica – Fatty des, la presencia del alcaloide swainsonina causante de locois- acids – Polyphenols – Protein. mo, ha impedido su uso como alimento para animales o para nutrición humana. El contenido proteico y su perfil cromatográ- fico, la composición de aminoácidos, de ácidos grasos, y con- 1. INTRODUCTION tenido de polifenoles se han determinado con el fin de explorar el potencial de las semillas de E. baetica como una fuente de Legumes are of nutritional interest not only proteínas y de componentes funcionales de la dieta. El conteni- because of their high quality protein, but also do de proteínas es del 36% (w / w), y el análisis de aminoáci- dos reveló deficiencia en aminoácidos azufrados, triptófano y because they are rich in functional components lisina. El contenido de lisina bajo es probablemente debido a la such as polyphenols, fiber, carbohydrates, and abundancia de alcaloides metabólicamente derivados de este unsaturated fatty acids. Polyphenols are minor aminoácido. Los ácidos oleicos y linoleico son los principales components in fruits, vegetables, and legumes ácidos grasos en las semillas. La actividad antioxidante de los and they have become wellknown because of their extractos de polifenoles fue mayor que la actividad de los poli- health promoting properties (Ramos, 2007). fenoles extraídos de la mayoría de las semillas de leguminosas Erophaca baetica (L.) Boiss. the only species of comestibles. Por lo tanto, las semillas de E. baetica represen- tan una fuente interesante de componentes funcionales y nutri- the monotypic genus Erophaca Boiss, is a legume cionales con la condición de que los alcaloides antinutriciona- endemic to the Mediterranean Region. It has les sean quitados antes. been historically included in the Astragalus genus under the denomination A. lusitanicus Lam. until PALABRAS CLAVE: Ácidos grasos – Aminoácidos – it was transferred to Erophaca by Podlech (1993). Erophaca baetica – Polifenoles – Proteínas. It includes two subspecies which are distributed at opposite ends of the Mediterranean region, SUMMARY subsp. baetica in the west and subsp. orientalis in the east. E. baetica is locally well known due Nutritional and functional characteristics of Erophaca to its toxicity to animals because of the presence baetica seeds, a legume endemic to the Mediterranean of swainsonine, an alkaloid responsible for the region so called “locoism” syndrome. This legume and others containing this type of alkaloids are known Erophaca baetica is a legume endemic to the Mediterra- nean region. Although the fruits and seeds are large, the pre- as locoweeds (James et al., 2004). However, sence of the “locoism” which produces the alkaloid, swainso- hepatoprotective, antioxidative, immunostimulant, nine has prevented its use as animal feed or for human and antiviral properties have also been described nutrition. Their protein content and chromatographic profile, for these alkaloids (Ríos and Waterman, 1997) as amino acid composition, fatty acid composition, and polyphe- well as anticancer effects (Sun et al., 2007; Sun nol contents have been determined in order to explore the et al., 2009). The presence of these alkaloids has potential of the E. baetica seeds as a source of dietary pro- prevented the use of E. baetica for animal and tein with functional components. The protein content was found to be 36% (w/w), and an amino acid analysis revealed human nutrition. Nevertheless, removal of the a deficiency in sulphur amino acids, tryptophane, and lysine. anti-nutritional components responsible for toxicity The low lysine content is probably due to the abundance of could allow for the use of E. baetica for feeding alkaloids metabolically derived from this amino acid. Oleic animals or even for human consumption. The 229 I. CORTÉS-GIRALDO, M. ALAIZ, J. GIRÓN-CALLE, C. MEGÍAS AND J. VIOQUE goal of this work was to explore the potential 2.4. Analytical methods nutritional and functional value of E. baetica seeds by determining their chemical composition, amino Moisture, fat, ash, and crude fiber were acid composition, fatty acid composition, and the determined using AOAC (1997) methods 934.01, antioxidant activity of polyphenol extracts. 920.39, 942.05, and 962.09, respectively. Protein was determined by elemental analysis as nitrogen content (%) × 6.25 using a LECO CHNS-932 2. MATERIALS AND METHODS analyzer (St Joseph. MI. USA) or by amino acid analysis according to Hidalgo et al., (2001). Soluble 2.1. Materials sugars were measured according to Dubois et al., (1956), using a standard curve of glucose. Diethyl ethoxymethylenemanolate was Polyphenol content was determined using the purchased from Fluka. b-carotene, 2,2’-Azino-bis Folin-Ciocalteou reagent as described (Singleton et (3-ethylbenzothiazoline-6-sulfonic acid) diammonium al., 1999) using a standard curve of catechin. salt (ABTS), fatty acid standards (ref. 1892, 1894, 1898) were from SIGMA. All other chemicals were of analytical grade. 2.5. Determination of seed oil fatty acid composition 2.2. Preparation of flour and oil. Polyphenol Gas chromatography of fatty acid methyl extraction esters was carried out according to the method of Garcés and Mancha (1993) with modifications. Fully mature E. baetica subsp. baetica seed A methylation reagent (1 mL) consisting of 59% samples were collected from a wild population methanol, 3% sulphuric acid, 8% dimethoxipropane, located in Andalusia (southern Spain). The seeds and 20% toluene (v/v), and heptane (1 mL) was were collected from ten different specimens and added to 50 mg seed flour in a screw-cap tube and stored at –20 °C. Voucher specimens of this incubated at 85 °C for 50 min. The upper phase population are deposited at the Instituto de la Grasa containing the fatty acid methyl esters was taken (C.S.I.C.). Seeds of Lathyrus cicera, Lathyrus to dryness under nitrogen and dissolved in 50 µL sativus, Lens culinaris, Phaseolus vulgaris, Cicer heptane. Two mL of this solution were injected into arietinum, Glycine max, and Vicia faba were a gas chromatograph equipped with an HP-23 purchased from a local market. Whole seeds were FAME capillary column (60 m length and 0.25 mm ground to powder and passed through a 0.2 mm- ID). Hydrogen at 20 psi was used as the carrier mesh sieve. Seed oil was extracted with hexane gas. Temperatures of injector, detector, and oven in a Soxhlet apparatus for 16 h. The extraction of polyphenols was carried out by shaking the defatted were 225 °C, 250 °C, and 180 °C, respectively. Fatty flour (10% w/v) in acetone 75% (v/v) at 4 °C for 1 acid methyl esters were identified by comparing hour three times. The supernatants resulting from retention times with those of standards. centrifugation at 15.000 g for 15 min were taken to dryness under vacuum and dissolved in ethanol. 2.6. Assay of polyphenol antioxidative activity. b-carotene bleaching method 2.3. Extraction and fractionation of proteins The antioxidant activity of polyphenol extracts Defatted, acetone-extracted flour was used was determined according to the b-carotene for the preparation of protein extracts by shaking bleaching method (Marco, 1968) with modifications. a suspension of the flour (10% w/v) in 0.25% The b-carotene reagent was prepared by vigorously mixing 1 mL b-carotene (10 mg/mL in Na2SO3, 0.2 % NaOH pH 10.5 at 4 °C for 1 hour. The resulting extracts were centrifuged at 15.000 g chloroform), 20 mg linoleic acid, and 200 mg tween for 15 min and the pellets were re-extracted twice. 20, followed by the removal of chloroform using a Supernatants containing solubilized proteins were stream of nitrogen, and finally mixing into oxygen- pooled and acidified to the determined isoelectric sparged distilled water (50 mL) to obtain a clear point of globulins, pH 4. For the isoelectric point solution. Polyphenol extracts (4 µg) were added determination, an alkaline protein extract was to 200 µL of b-carotene reagent in 96-well flat- titrated to various pH values using 0.5 N HCl. bottom microtitration plates and incubated at 50 °C Nitrogen was determined in aliquots that were using a microplate reader for the determination withdrawn from the extract and in supernatants of absorbance at 450 nm at fixed time intervals. after centrifugation of these aliquots at 15000 g Four different methods were used to calculate for 15 minutes. Protein solubility was calculated as antioxidative activity as previously described (nitrogen in supernatant / total nitrogen) × 100.