Abstract
The Dipteryx lacunifera plant is a native of the states of Piauí and Maranhão in Brazil. The fruit kernel, in spite of having a good potential for food use, is still unexplored. The present work investigates the kernel composition, electrophoretic behavior, amino acids composition, and antinutritional components in defatted kernel flour. The kernel constituted about 6.5% of the fruit. The average weight, length, width, and thickness of the kernel were 1.03 g, and 2.45, 1.44 and 0.58 cm, respectively. The lipid and the protein contents of whole kernel flour were about 41.8 and 13.3 g per 100 g, respectively, while in defatted kernel flour, the protein content was 26.4 g per 100 g on dry weight basis. The polyacrylamide gel in the presence of sodium dodecyl sulfate and 2β-mercapto-ethanol (PAGE-SDS-2βMe) revealed the presence of 12 proteins with molecular weights ranging from 7.8 to 97.3 kDa. The proteins with molecular weights 18.0, 39.6, 57.6, 69.2, and 97.3 kDa were the prominent ones, while those corresponding to 7.8, 12.0, 14.7, 25.3, 31.3, 33.7, and 83.9 kDa were present in small concentrations. Defatted kernel flour did not present trypsin inhibitory and hemagglutinating activity. The tannin content of the flour was 0.63 mg/g. In comparison to FAO reference protein, the defatted flour showed all essential amino acids (except methionine) in good concentrations.
La planta Dipteryx lacunifera es nativa de los estados de Piauí y Maranhão en Brasil. La almendra de esta fruta, a pesar de tener un buen potencial como alimento, se encuentra aun inexplorada. El presente trabajo investiga la composición de la almendra de la planta D. lacunifera, su comportamiento electroforético, composición de aminoácidos y componentes nutricionales en harina hecha con esta almendra a la que se le ha eliminado la grasa. La almendra constituyó alrededor del 6,5% de la fruta. El peso promedio, medida, anchura y espesor de la almendra fue de 1,03 g; 2,45; 1,44 y 0,58 cm respectivamente. El contenido de lípidos y proteínas en la harina entera de esta almendra fueron de alrededor de 41,8 y 13,3 g por 100 g, respectivamente; mientras que en la harina a la que se le eliminó la grasa, el contenido de proteína fue de 26,4 g por 100g en peso seco. El PAGE-SDS-2βMe reveló la presencia de 12 proteínas con pesos moleculares en el rango de 7,8 a 97,3 kDa. Las proteínas con pesos moleculares 18,0; 39,6; 57,6; 69.2 y 97,3 kDa fueron las más prominentes, mientras que aquéllas correspondientes a 7,8; 12,0; 14,7; 25,3; 31,3; 33,7 y 83,9 kDa estuvieron presentes sólo en pequeñas concentraciones. La harina de la almendra a la que se le eliminó la grasa, no presentó actividad inhibitoria a trypsin, ni actividad hemaglutinante. El contenido de tannin en la harina fue de 0,63 mg por g. En comparación a la referencia de proteína de la FAO, la harina a la que se le eliminó la grasa mostró todos los aminoácidos esenciales (excepto metionina) en buenas concentraciones.
Introduction
Foods from plant origin are used for human nutrition throughout the world. On a global basis, 80% of the calories and 65% of the proteins are supplied by foods of plant origin (Sathe, Citation2002). Kinsella (Citation1981) predicted that the high cost of the animal proteins could lead the food industries to utilize proteins from plant origin. According to the same author, functional properties, biological value, and the cost will be the principal criteria that will determine the acceptability of plant proteins. The search for alternative non-conventional protein sources is indispensable because of the high cost of animal proteins and the necessity for the introduction of inexpensive new protein food products for the population of the low-income group.
Brazil possesses a large number of native plants, among which the fruits of some species are rich sources of nutrients. The genus Dipteryx belongs to the family of Leguminosae faboideae, which includes 14 species. The plants of this genre are distributed in the Amazon region and the central and northeastern regions of Brazil, Venezuela, Costa Rica, and Panama (Mendes & Silveira, Citation1994). The Dipteryx lacunifera is a native of the mountainous region of the states of Piauí and Maranhão in Brazil. The fruits of D. lacunifera contain lengthy and flat oleaginous kernels (locally known as fava de morcego or fava de boi) with a brown pellicle that gets loosened during roasting. The kernels are rich in oil. In the region of its production, the kernels are commercialized in weekly open markets for use as snacks in roasted form in place of cashew nuts. To conserve this important plant species from unscrupulous exploitation, the EMBRAPA – Meio Norte (Empresa Brasileira de Pesquisa Agropecuária), has started a systematic plantation of this species.
However, scientific information on its nutritional components is scarce. Therefore, the present work was undertaken to explore the possibility of using D. lacunifera seed kernel protein as an alternative plant protein source. Investigation was carried out with respect to the electrophoretic behavior, amino acids composition of its protein, and the presence of antinutritional factors in the seed kernel. The weight and dimensions and proximate analysis of the seed kernel were also carried out to gain an idea of the economic feasibility of the exploration of D. lacunifera seed kernel.
Material and methods
Material
The D. lacunifera kernels from mature fruits were collected from the county of Bom Jesus de Gurgüeia, in the State of Piauí, Brazil. The kernels were separated manually and subjected to the process of cleaning, washing under running water and drying in an air circulation oven at 45–50 °C for 24 h. The kernels were divided in three lots, packed in polyethylene bags and stored in a refrigerator at about 5 °C.
Methods
Weight and dimensions of D. lacunifera fruit and seed kernels
The analysis was carried out by measuring weight and dimensions: maximum and minimum length, and thickness utilizing semi-analytical balance, and Vernier calipers. The maximum length was considered as distance between the apex while, the minimum length was measured at the center of the polar axis.
Preparation and proximate analysis of defatted kernel flour
The kernel flour was obtained by grinding the seeds without pellicle in a domestic mixer and passing them through the sieve of 25-mesh sizes. The flour was defatted by extraction with n-hexane in a Soxhlet apparatus for about 24 h till free of lipids. After this period, the defatted flour was spread in a tray for the removal of residual solvent at room temperature for 24 h, triturated again and passed through the screen of 40 mesh sizes.
The proximate composition of defatted D. lacunifera flour was determined according to the methods described in AOAC (Citation1993). Total carbohydrates (including the fiber) were calculated by difference.
Electrophoretic analysis
Electrophoresis of the proteins was carried out utilizing polyacrylamide gel in the presence of sodium dodecyl sulfate and 2β-mercapto-ethanol (PAGE-SDS-2βMe), as described by Laemmli (Citation1970). Mini-protean® 3 Cell electrophoresis apparatus with 13.8 cm × 7.9 cm × 0.75 cm plates from Biorad Co. was used.
The application gel containing 3.5% polyacrilamide was mounted in Tris-HCl 0.5 M, pH 6.8 buffer while the separating gel with a gradient of 3.5 a 17.5% polyacrilamide on Tris (hydroxymethylaminomethane) – HCl 3 M, pH 8.8. Both the buffers contained 1% SDS. The sample (mg/ml) was suspended in a sample buffer (0.35 ml water milli-q, 0.125 ml Tris-HCl 0.5 M (pH 6.8), 0.2 ml SDS 1% (p/v), 0.25 ml glycerol, 0.02 ml de bromophenol blue 0.5% (p/v), and 0.05 ml de (2βMe). The suspension was heated to 100 °C for 10 min and centrifuged at 5000 rpm. The supernatant was separated and 10 μl aliquot was applied. The voltage was adjusted to 200 V. After the completion of the run, the gel was stained with Coomassie blue R-250, 0.05% in a mixture of methanol, acetic acid, and water (1:3.5:8, V/V). Molecular weight markers (Sigma, Co., St. Louis, MI, USA) were used to determine the molecular weight of the protein bands.
The stained gel was scanned in a scanner, and the image was processed through a software Scion Image (Scion Corporation, MA). The area of the peak corresponding to the density of protein band was used to determine its relative concentration.
Amino acids composition
The analysis of the amino acids composition of the protein of defatted kernel flour was carried out in a High Performance Liquid Chromatograph (Waters Corporation, Milford, MA), equipped with a reverse phase column (C18) System Pico-Tag (Waters Division, Millipore Co. Milford, MA), according to the method proposed by Bindlingmeyer, Cohen, and Tarvin (Citation1984).
About 1 mg sample was hydrolyzed with 6N HCl containing 1% formaldehyde, in an atmosphere of nitrogen at 110 °C for 24 h. After concentration, the hydrolyzed sample was neutralized with methanol–water–triethanolamine (2:2:1, v/v/v) and dried in a vacuum. In the dried residue, 20 μl of the amino acids derivatizing solution (methanol–water–triethanolamine–phenilisothiocynate (7:1:1:1, v/v/v/v) was added. The mixture was vigorously agitated and kept at rest for 20 min at room temperature. The phenylthiocarbamyl-amino acids (PTC-AA) derivatives were vacuum dried. The PTC-AA derivatives were re-suspended in 100 μl acetate/acetic acid (pH 6.4) buffer with agitation and centrifuged at 10,000 rpm for 10 min. The supernatant containing amino acids derivatives were separated, and (10 μl) aliquots were injected in reverse phase column (C-18) Pico Tag (Waters Corporation, Milford, MA). The temperature of the column was maintained at 38 °C by using a controlled temperature module (Waters Corporation, Milford, MA). The column was equilibrated with acetate buffer (0.14 M, pH 6.4). The PTC-AA was eluted using acetonitrile gradient (0 to 60%), for 12 min maintaining a flux of 1.0 ml/min in the Chromatograph (Alliance, Waters Corporation, Milford, MA) and monitored at 254 nm in a detector 486 (Waters Corporation).
The determination (in triplicate) of the composition of amino acids in the sample was made by a comparison of the retention time and the peak area obtained for each amino acid with that of standard amino acids (Sigma, Co).
Antinutritional factors
Antinutritional factors such as tannin, trypsin inhibitor and lectin (hemagglutinating activity) were determined as follows. The determination of tannins was based on the reduction of phosphotungstomolybdic acid (Folin–Denis reagent), a spectrophotometric method, as described by Ranganna (Citation1970). The trypsin inhibitor activity of the kernel seed protein was evaluated through its ability to prevent the casein hydrolysis by standard trypsin solution (Kakade, Citation1969). The hemagglutinating activity was measured by adding rabbit blood to diverse dilutions of kernel seed protein solution in Tris buffer, 0.1 M, pH 7.6, and observing the agglutination (Moreira & Perrone, Citation1977).
Results and discussion
Physical and proximate composition of fruit and seed kernels
The physical parameters such as weight, diameter and thickness of the fruit and seed kernel are shown in and the results of the chemical analysis of the whole seed kernel flour of D. lacunifera are shown in the .
Table 1. Weight and measurements of the fruit and seed kernel of D. lacunifera.
Tabla 1. Peso y medidas de la almendra del fruto de D. lacunifera.
Table 2. Proximate analysis of D. lacunifera seed kernel.
Tabla 2. Análisis aproximado de la almendra del fruto de D. lacunifera.
The knowledge of the principal constituents of the food is important for the formulation of nutritionally balanced diets. The table of food composition is utilized at macro level for the planning of the demand and supply of the foods, while at micro level for the development of prescribed diets as well as for the nutritional evaluation of diet.
Lipid and protein constituted the principal components, being present at 41.8 and 13.3%, respectively. There is no information in scientific literature on the chemical composition of the seed kernel of this fruit. The results obtained in this study are similar in lipids but inferior in proteins when compared with the results of baru (Dipteryx alata) seed kernel (lipid 40.3 and protein 29.6%), a member of the same family, reported by Togashi and Sgarbieri (Citation1994). Vallilo et al. (Citation1990) and Takemoto et al. (Citation2001) also reported lipid contents of 41.6 and 38.2%, respectively, and protein contents of 23.4 and 23.9%, respectively. Factors such as the species, maturity, and the ones related to the ecological and geographical areas promote differences in the proximate composition.
Compared with other seed kernels normally used in human nutrition, the D. lacunifera seed kernel presented a higher lipid content than soy (Deshpande & Damodaran, Citation1990), chichá (Sterculia striata) (Oliveira et al., Citation2000), similar to groundnut (Sathe, Citation2002), but inferior to the cashew nut (Neto, Silva, Narenda, & Bora, Citation2001), the Brazil nut (Ramos & Bora, Citation2003), and macadamia kernels (Bora & Ribeiro, Citation2004).
The protein content of D. lacunifera seed kernel was similar to that of macadamia (Bora & Ribeiro, Citation2004), but inferior to soy (Deshpande & Damodaran Citation1990), sunflower and groundnut (Sathe, Citation2002), the cashew nut (Neto et al., Citation2001), and the Brazil nut (Ramos & Bora, Citation2003).
Electrophoretic analysis
represents the electrophoretic pattern of the proteins obtained with PAGE-SDS-2βMe. The polypeptide bands were subjected to densitometric analysis, and the quantity of individual proteins in total defatted flour proteins was calculated on a percentage basis.
Table 3. Molecular weight and percentage of constituent protein in D. lacunifera seed kernel flour.
Tabla 3. Peso molecular y porcentaje de la proteína constituyente en la harina hecha a base de la almendra del fruto de D. lacunifera.
The defatted kernel flour presented a total of 12 protein bands, with a molecular weight varying from 7.8 to 97.3 kDa. According to the results, the prominent proteins with molecular weights 18.0, 39.6, 57.6, 69.2, and 97.3 kDa constituted individually 15.6, 26.3, 13.5, 21.9, and 11.1% of the total proteins, respectively. The proteins of molecular weights of 7.8, 12.0, 14.7, 25.3, 31.3, 33.7, and 83.9 kDa were present in smaller concentrations, being present in trace quantities, 0.4, 3.8, 0.4, 3.5, 1.6, and 1.9%, respectively.
The protein of the defatted flour presented molecular weights similar to the proteins of various plant species. In lupin seed protein, Sgarbieri and Galeazzi (Citation1978) reported the existence of proteins with 12.0, 15.0, 18.0, 24.5, and 38.5 kDa molecular weights. Protein of 18.0 kDa (Santos, Ferreira, & Teixeira, Citation1997) and 85.0, and 97.3 kDa were reported in Lupinus mutabilis (Sathe, Deshpande, & Salunkhe, Citation1982). In cashew nut protein, the presence of proteins of molecular weights of 12.0 and 14.13 kDa (Sathe, Citation1994), 18.86 (Sathe, Sze Tao, Wolf, & Hamaker, Citation1997), 24.83, and 31.99 kDa (Sathe, Citation1994) have been reported. In black mucuna pruriens proteins of 21.0, 30.0 (Machuka, Citation2000), and 97.0 kDa (Adebowale & Lawal, Citation2003) have been reported. Mnembuka and Eggum (Citation1993) identified in the groundnut the presence of proteins with molecular weights 12.0, 13.2, 18.6, 33.1, and 39.8 kDa. Basha and Cherry (Citation1976) reported proteins of 84 kDa. In winged bean protein, of weights 12.3, 14.7 (Sathe & Salunkhe, Citation1981), 67.0, and 82.0 kDa (Meng & Ma, Citation2001) have also been reported. The Brazil nut also presented proteins of 14.0 and 17.0 kDa (Sun, Leung, & Tomic, Citation1987).
Amino acids composition
The amino acids composition of defatted D. lacunifera seed kernel flour is shown in . In comparison to the amino acids composition of the FAO (1992) reference protein, the seed kernel flour has shown better essential amino acids concentration with the exception of sulfur-containing (methionine and cysteine) amino acids. These amino acids were the limiting amino acids containing about 61% of what is recommended by the FAO (1992). A similar deficiency of sulfur-containing amino acids has been observed in other plant proteins such as baru (Togashi & Sgarbieri, Citation1994), fava bean, and pea (Fernández-Quintela, Macarulla, Barrio, & Martinez, Citation1997), groundnut (Andersen, Hill, Gorbet, & Brodbeck, Citation1998), etc. Other essential amino acids ranged from 100 to 152% of the corresponding essential acids of the FAO reference protein.
Table 4. Amino acids composition of D. lacunifera kernel flour proteins.
Tabla 4. Composición de aminoácidos de las proteínas en la harina hecha a base de la almendra del fruto de D. lacunifera.
The non-essential amino acids found in good quantities in defatted seed kernel flour were glutamic (187.1 g/kg protein) and aspartic acid (88 g/kg protein), as was also reported earlier by Cavalcante (Citation1998) for plant proteins, Oliveira et al. (Citation2000) for chichá (S. striata), Vasconcelos et al. (Citation2001) for soy, etc., among various other authors who also observed high concentrations of these amino acids in various plant proteins.
The proportion of essential/total amino acids for defatted kernel flour was 41.3%, which could be considered satisfactory for being superior to the limit 33.9% recommended by the FAO (1992). The higher the ratio of essential/total amino acids, the higher the biological value or nutritional quality of a protein will be. The essential/total amino acids ratio of defatted kernel flour is better than that of the groundnut (Andersen et al., Citation1998) and fava bean (Fernández-Quintela et al., Citation1997) but inferior to that of soy (Siddhuraju, Becker, & Makkar, Citation2000), chichá (Oliveira et al., Citation2000), etc. A similar ratio was observed in baru (41.11%) (Vallilo et al., Citation1990) and Mucuna monosperma (Mohan & Janardhanan, Citation1993).
Proteins, deficient in one or more amino acids, as in a majority of the proteins of plant origin, necessitate supplementation to overcome the deficiency. In the case of D. lacunifera, defatted kernel flour supplementation with sulfur-containing aminoacids or with soybean protein concentrate (Brumano et al., Citation2006) in a proportion of 70:30, can produce a flour mixture having a better amino acids composition than the FAO reference protein. The supplemented flour could be used as an ingredient in the formulation of diverse food preparations.
Antinutritional factors
Tannins
The tannin content of the defatted kernel flour with and without (brown) pellicle is shown in . The defatted kernel flour with pellicle contained about 18.3 mg/g tannins against that of 0.63 mg/g of defatted kernel flour without pellicles. The removal of pellicles from the seed kernels eliminated a large concentration of tannins. Esteves (Citation2000) also reported that in light-colored beans the concentration of polyphenols was smaller than that in dark-colored beans. Contrary to our results, Togashi and Sgarbieri (Citation1994), in a study of defatted kernel flour of baru seed kernel, a plant of the same species, did not detect the presence of tannins. However, lower concentrations of tannins in defatted flours of soy (0.8 mg/g) (Fernández-Quintela et al., Citation1997), groundnut (0.0088 to 0.0098 mg/g) (Mnembuka & Eggum, Citation1993), common bean (2.6 mg/g) (Carbonaro, Grant, Cappelloni, & Pusztai, Citation2000) have been reported. Similarly, Mnembuka and Eggum (Citation1993) reported a tannin content varying from 11.0 to 14.7 mg/g in fava beans. In the defatted flour of the same species, Carbonaro et al. (Citation2000) reported 7.5 mg/g tannin, while Siddhhuraju and Becker (Citation2001) reported a tannin content of 1.3 and 2.1 mg/g in sword (Canavalia gladiata) and virotic (Canavalia virosa) beans.
Table 5. Tannin content of whole and defatted kernel flours of D. lacunifera.
Tabla 5. Contenido de tannin en harina entera y harina a la que se le eliminó la grasa, hechas a base de la almendra del fruto de D. lacunifera.
Trypsin inhibitor
No trypsin inhibitor activity was observed in the defatted flour of D. lacunifera seed kernel (with or without pellicles). In baru seed kernel flour, high trypsin inhibitor activity (38.60 UTI/mg) was reported by Togashi and Sgarbieri (Citation1994). Trypsin inhibitor activity in soy (Fernández-Quintela et al., Citation1997; Trugo, Donangelo, Trugo, & Bach Knudsen, Citation2000), groundnut (Mnembuka & Eggum, Citation1993), mesquite beans (Ortega-Nieblas, Moreno, & Burgueno, Citation1996), etc., to cite a few, has been reported. However, in white and blue lupin seed (Hudson, Citation1979; Ruiz-Lopes, García-Lopes, Castaneda Vazquez, Zamora, & Garzon De La Mora, Citation2000; Ruiz-Lopes & Sotelo, Citation2001), and in defatted chicha (S. striata) seed kernel flour (Oliveira et al., Citation2000) no trypsin inhibition activity was observed.
Lectin (hemagglutinating activity)
No hemagglutinating activity was observed in either of the two defatted seed kernel flours. Togashi and Sgarbieri (Citation1994) also did not report hemagglutinating activity in defatted baru seed kernel flour. Oliveira et al. (Citation2000) also did not observe lectin in chichá seed flour.
Conclusions
The proximate composition characterizes the kernel as a rich source of lipids and protein. The PAGE-SDS-2βMe revealed the presence of 12 protein sub-units, which are also reported in various other plant proteins. The protein presented excellent amino acid composition, except the sulfur-containing amino acids. The kernel flour did not show the hemagglutinating and trypsin inhibitor activity. The tannins were present in very low concentration. Considering all these factors it could be concluded that the D. lacunifera seed kernel can probably be used in human nutrition.
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