Plant Foods for Human Nutrition 58: 1–10, 2003. © 2004 Kluwer Academic Publishers. Printed in the Netherlands.

Fatty acid, amino acid and trace mineral composition of Eleusine coracana (Pwana) seeds from northern Nigeria ∗ DIANE R. FERNANDEZ1, DOROTHY J. VANDERJAGT1, MARK MILLSON2, YUNG-SHENG HUANG3, LU-TE CHUANG3, ANDRZEJ PASTUSZYN1 and ROBERT H. GLEW1∗ 1 Department of Biochemistry and Molecular Biology, School of Medicine, University of New Mexico, Albuquerque, NM; 2 National Institute of Occupational Health and Safety, Cincinnati, Ohio; 3 Lipid Research Laboratory, Ross Products Division, Abbott Laboratories, Columbus, OH; (∗ author for correspondence; e-mail: [email protected])

Abstract. In northern Nigeria the seeds of the cereal Eleusine coracana (finger millet), called ‘pwana’ by the Birom and ‘tamba’ by the Hausa, are used as a supplemental food taken in the form of tea or a porridge-like meal. Seeds were analyzed for fatty acid, amino acid and mineral contents. They contained 12 mg/g total fatty acid, 42% of which was oleic acid (C18:1n-9), 21% palmitic acid (C16:0), 25% linoleic acid (C18:2n-6) and 4% α-linolenic acid (C18:3n3). The linoleic acid/α-linolenic acid ratio (6.5:1) was within the World Health Organization (WHO) recommendation (5:1 to 10:1). Although the total protein content of E. coracana was relatively low (6.9% dry weight), its protein composition compared favorably to that of the WHO standard for all of the essential amino acids except lysine. E. coracana seeds contained only 49% of the WHO ideal for lysine, but 436% of the WHO ‘ideal’ for tryptophan. In terms of mineral content, E. coracana is a useful source of calcium, phosphorous, and copper, and an excellent source of chromium, iron, magnesium, manganese, molybedenum and selenium. These data indicate that while E. coracana does not provide a complete food source in terms of its qualitative and quantitative fat, protein or mineral contents, it does contain appreciable quantities of a number of essential nutrients that make it a useful food supplement for the people of northern Nigeria. Key words: Eleusine coracana, Essential amino acids, Essential fatty acid (EFA), Finger millet, Minerals, Nigeria, Pwana

Introduction Eleusine coracana is an edible grass commonly found in northern Nigeria. It is an annual tetraploid comprised of two subspecies, one wild (africana) ∗ This study was supported by a Minority International Research Training (MIRT) grant from the Fogarty International Center of the National Institutes of Health.

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2 and the other domesticated (coracana) [1]. E. coracana, also known as finger millet, is an important cereal crop widely distributed throughout the semi-arid regions of Africa and India [2]. Previous studies indicate E. coracana is an allopolyploid resulting from the combination of the diploids E. indica (from India) and an as-of-yet-undetermined second genomic donor [2]. E. coracana is called ‘pwana’ by the Birom people and ‘tamba’ by the Hausa of northern Nigeria. Pwana is considered a famine food, which is defined as food eaten yearround or seasonally when available as a supplemental source of nutrients [3]. In northern Nigeria E. coracana is milled into a powder and mixed with boiling water. It can be prepared as a tea after a meal, used as an appetizer or eaten as a whole meal. Potatoes, corn, spices or sugar may be added, depending on which meal is being prepared. E. coracana is used extensively in northern Nigeria and eaten by people of all ages, particularly children. Most published reports regarding the nutritional properties of E. coracana have involved specimens grown in India [4, 5]. Sridhar and Lakshminarayana [6] performed a detailed analysis of lipid content, lipid subclasses and fatty acid composition in small millets from India. Barbeau and Hilu [7] analyzed the calcium, iron and amino acid contents of selected wild and domesticated cultivars of finger millet from India and East Africa. Also, the Food and Agriculture Organization (FAO) [8], as well as the National Research Council (NRC) [9], have each compiled reports from various sources regarding the nutrient composition of finger millet. Since the literature showed wide variations in the nutrient content of various E. coracana cultivars, as well as differences related to geography, it was deemed necessary to directly determine the fatty acid, amino acid and mineral contents of finger millet grown in northern Nigeria [7,8]. This nutritional information can then be used in comparison to data determined in previous studies as well as a means of comparison to the nutrient compositions of other Nigerian cereals. Also, although previous works report selected information regarding the fatty acid, amino acid and mineral contents of E. coracana, the information presented in this paper has not previously been collected into one publication. Furthermore, the present study of E. coracana included an analysis of 17 trace minerals. A literature search did not uncover any publication containing this information. There are ways in which the nutrient composition of E. coracana could be useful not only to the scientific community, but also to the peoples of northern Nigeria. Knowledge of the nutritional content of E. coracana from Nigeria would allow the people of this region to select appropriate food sources to supplement their diet. This is especially important in the western Sahel where malnutrition is common. Furthermore, the wider scientific community could utilize the data from an analysis of E. coracana to increase the body of

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3 knowledge regarding nutrient composition of edible, indigenous plants of Africa and to allow for comparison with the same species grown in India and other countries. Reseachers therefore sought to ascertain the fatty acid, amino acid and mineral composition of this popular plant food and to compare it to data in prior publications. This report contains the results of the study of E. coracana seeds harvested in northern Nigeria.

Methods and materials Source of plant material E. coracana seeds were obtained in the market at Bassa Local Government, Nigeria. They were grown in the Lower Plateau Region, harvested, sun dried and then pounded. Prior to analysis, E. coracana was ground to a powder using a Krups type 203 stainless steel mill. Fatty acid analysis The dried, powdered specimens were extracted with chloroform:methanol (2:1, v/v) and the solid, non-lipid material was removed by filtration [10]. The total lipid fraction was recovered after solvent removal in a stream of nitrogen. The samples were then redissolved in anhydrous chloroform and clarified by centrifugation. Transmethylation was performed using 14% (w/v) BF3 in methanol [11]. Fifty nanograms of heptadecanoic acid (internal standard) and a 1 ml aliquot of each sample was transferred to a 15 ml teflon-lined screw-cap tube. After removal of solvent by nitrogen gassing, the sample was mixed with 2 ml of BF3 reagent, placed in a warm bath at 100 ◦ C for 30 min and cooled. After the addition of 2 ml of saline solution, the transmethylated fatty acids were extracted into hexane. Aliquots of the hexane phase were analyzed by gas chromatography. A Hewlett-Packard gas chromatograph (5890 Series II) with a Flame-Ionization Detector was used to separate and quantify fatty acids. One to two microliter aliquots of the hexane phase were injected in split-mode onto a fused-silica capillary column (Omegawax; 30 m × 0.32 mm I.D., Supelco, Bellefonte, PA). The injector temperature was set at 200 ◦ C, detector at 230 ◦ C, oven at 120 ◦ C initially, then 120–205 ◦ C at 4 ◦ C per min, 205 ◦ C for 18 min. The carrier gas was helium and the flow rate was approximately 50 cm/sec. Electronic pressure control in the constant flow mode was used. The internal standard (heptadecanoic acid, 17:0) and calibration standards were used for quantification of fatty acids in the various lipid extracts. Fatty acid calibration standards and heptadecanoic acid were procured from Nu-chek, Elysian, MN.

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4 Solvents were purchased from EM Science, Gibbstown, NJ. The data reported represent the average of three determinations. Amino acid analysis Two to three mg of the seeds, in triplicate, were transferred into a tared glass ampoule then weighed and analyzed. Norleucine was the internal standard used in all determinations. After 1.0 ml of 6 N HCl was added, the samples were flushed with nitrogen, evacuated, sealed and placed in an oven at 110 ◦ C for 24 hrs. Following hydrolysis, a 10 µl aliquot was withdrawn and subjected to derivatization. Samples to be used for the determination of cysteine were first oxidized with performic acid (formic acid:30% hydrogen peroxide, 9:1, v:v) for 18 hrs at room temperature [12]. Performic acid was removed in an evaporative centrifuge and the samples were hydrolyzed with HCl as described above. The tryptophan (Trp) content was determined separately. With regard to the Trp analysis, 450 µL of 4.67 M KOH containing 1% (w/v) thiodiglycol were added to each sample. Hydrolysis was performed in plastic tubes within an evacuated ampoule at 110 ◦ C for 24 hrs. After allowing the hydrolysate to cool, 0.5 ml of 4.2 m perchloric acid (PCA) and 50 µl of acetic acid were added to neutralize the solution. The samples were mixed thoroughly using a Thermolyne Maxi mixer, chilled on ice, and centrifuged. Fifteen microliters of the supernatant were transferred to 6 × 50 mm glass tubes and dried in a speedvac in preparation for derivatization. Two lysozyme controls and two calibration mixtures were prepared as well. The samples were dried using 20 µl of ethanol:triethylamine:water (2:1:2, v/v) and derivatized with 20 µl of ethanol:triethylamine:water: phenylisothiocyanate (7:1:1:1 v/v). The speedvac was used to remove excess reagent. Derivatized, dried samples were dissolved in 100 µl of equilibration buffer. Analysis of the amino acids was performed with a Waters C18 column (3.9 × 150 mm). The gradient solution was the same as that described by Bidlingmeyer et al. [13]. Amino acids were detected at 254 nm. The solvents utilized were the sodium acetate buffer and acetonitrile (300 ml ACN, 200 ml water, 0.2 ml CaEDTA). Twenty microliter aliquots were injected onto the column. Tryptophan analysis was performed according to Hariharan et al. [14]. Elution of the amino acids was achieved by increasing the acetronitrile concentration in the eluent. Minerals The seeds were dried overnight at 110 ◦ C. They were then stirred and allowed to cool to room temperature. Three replicate portions containing approxim-

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5 ately 0.100 g of each sample were weighed into 125 ml Phillips beakers and then digested using 20 ml concentrated nitric acid and 1 ml concentrated perchloric acid. The samples were covered with watch glasses and set on a hot-plate at 120 ◦ C for one hour. The hot-plate temperature was then increased to 150 ◦ C and the samples were refluxed overnight. The watch glasses were removed and the samples taken to near dryness (approximately 1 ml) at the same temperature. At that point, the samples were taken off the hot-plate, treated with 2.5 ml of 4:1 nitric-perchloric acid, and a minimal amount of deionized water to rinse down the side walls of the beakers. After cooling, the solutions were quantitatively transferred to graduated centrifuge tubes and diluted to a 50 ml final volume with deionized water. The samples were analyzed by ICP-AES for trace mineral contents. This digestion technique makes no attempt to solubilize any silicate-based materials that may have been in the samples. Statistics Tables contain information from selected sources which allowed descriptive comparison to data ascertained in this study.

Results The fatty acid content of E. coracana from Nigeria is summarized in Table 1. Total fatty acid content of E. coracana was relatively low at 12 mg/g (dry weight). Almost half of the fatty acids were oleic acid (C18:1n-9). Saturated fatty acids, mostly palmitic acid (C16:0), accounted for 24% (2.9 mg/g dry wt.) of the total fatty acids. With regard to the essential fatty acids; linoleic acid and α-linolenic acid accounted for 26% (3.0 mg/g dry wt.) and 4% (0.5 mg/g dry wt.) of the fatty acid total, respectively. The linoleic acid/linolenic acid ratio was 6:1, which is within the range of values (5:1 to 10:1) recommended by the World Health Organization (WHO) [15]. The amino acid content of E. coracana is summarized in Table 2. The seeds contained 6.9% protein on a dry weight basis. In order to assess the quality of the protein contained in the Nigerian finger millet, the amino acid composition of the seeds was compared to that of an ‘ideal’ protein defined by the World Health Organization (WHO), which is based on the amino acid needs of the preschool child (Table 3) [16]. E. coracana contained the same proportion or more of the essential amino acid standards as set by WHO, except for lysine which was only 49% of the WHO ideal. Most remarkable was the tryptophan content of E. coracana; the proportion of tryptophan was more than four-fold that of the WHO standard.

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6 Table 1. Fatty acid composition of E. corocana (pwana) seeds Fatty acid

Palmitic acid Stearic acid Arachidic acid Oleic acid Linoleic acid α-Linolenic acid a b c d e

16:0 18:0 20:0 18:1n-9 18:1n-7 18:2n-6 18:3n-3

Pwanaa (mg/g dry wt)

Pwanaa

Sridharb Mahadevappac (weight percent)

2.56 (0.17)d 0.25 (0.02) 0.05 (0.01) 5.09 (0.39) 0.37 (0.03) 3.04 (0.21) 0.47 (0.03)

21.6 2.11 0.42 43.0 3.13 25.7 3.97

23.3 1.80 0.50 47.6 NRe 22.4 4.40

24.7 Tr NR 49.8 NR 24.2 1.29

Average of three samples. Data from reference 6. Data from reference 4. The number in parentheses indicates SD. NR, not reported.

E. coracana seeds contained significant amounts of calcium, chromium, copper, iron, magnesium, manganese, molybdenum and phosphorous (Table 4). Noteworthy was the presence of sizable amounts of selenium, a component of the antioxidant enzyme glutathione peroxidase. Discussion The main purpose of this study was to assess the qualitative and quantitative aspects of the nutrient content of E. coracana seeds. Most noteworthy was the finding that they contained large amounts of linoleic acid (C18:2n-6) and tryptophan, as well as beneficial quantities of several minerals, including calcium and iron. However, the total protein content of E. coracana was low (6.9%) and the seeds lacked sufficient lysine. Consequently, whereas E. coracana would be an inadequate plant to use as a main protein source, it would be useful as a supplemental food. In order to place the present study of E. coracana in perspective, researchers compared it to similar research conducted by other investigators. All studies regarding the nutrient composition of E. coracana have shown this plant to be poor source of the essential amino acid lysine, as are most cereal grains. Amino acid determinations of E. coracana in the present study support these findings. Relative to the WHO ideal, analysis in this study, as well as those of the NRC [9] and Malleshi et al. [17] indicated that E. coracana protein contained a low amount of lysine (49%–58%). Two previous studies of the amino acid composition reported relatively high values of methionine in

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7 Table 2. Amino acid composition (mg/g dry weight) of E. corocana seeds

a b c d

Amino acid

Pwanaa (mg/g dry wt) (Range)

Malleshib (mg/g dry wt)

NRCc (mg/g dry wt)

Asp Glu Ser Gly His Arg Thr Ala Pro Tyr Val Ile Leu Phe Lys Cys Met Trp Total protein

4.2 14.1 3.6 2.2 1.4 2.9 3.0 3.5 4.6 2.8 4.3 3.1 6.5 3.8 1.8 1.6 2.4 3.0 68.9

5.3 14.7 3.8 3.2 2.2 3.1 3.0 4.9 4.5 2.7 3.4 1.9 6.1 3.8 2.2 1.4 2.4 ND 68.6

NRd NR NR NR NR NR 3.1 NR NR 4.1 6.4 4.0 7.8 4.1 2.5 1.7 5.0 1.3 88.0

4.02–4.43 14.1 –14.2 3.56–3.63 2.14–2.17 1.42–1.46 2.77–3.03 2.98–2.99 3.54–3.56 4.43–4.71 2.79–2.83 4.30–4.39 3.08–3.08 6.43–6.59 3.80–3.85 1.80–1.88 1.58–1.63 2.28–2.45 2.88–3.12

Average of determinations. Data from reference 17. Data from reference 9. NR, not reported.

finger millet [9, 18]. Present analysis showed that methionine levels exceeded the WHO ideal [16]. Alternatively, while the NRC highlighted E. coracana because it had a tryptophan value almost double that of maize, present analysis revealed a tryptophan value in Nigeria finger millet that was twice again higher than the value reported by the NRC [9]. The E. coracana analyzed also contained useful amounts of copper and relatively high amounts of chromium, magnesium, molybdenum and selenium when compared to the Recommended Daily Allowances (RDA) [20]. As mentioned in the FAO report [8], environmental conditions often affect the mineral content of cereals more than genetic factors; therefore, soil conditions of northern Nigeria could be responsible for this variability. Mineral analysis provided more information than had been reported previously, and when compared to published values, certain minerals were found

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8 Table 3. Comparison of amino acid composition of E. corocana to WHO ideal Amino acid

WHO Ideala (% of total)

Pwana/Ideal (× 100)

Malleshi/Idealb (× 100)

NRC/Idealc (× 100)

Ile Leu Lys Met+Cys Phe+Tyr Thr Trp Val

4.0 7.0 5.5 3.5 6.0 4.0 1.0 5.0

112 135 49 164 160 108 436 126

70 128 58 158 159 109 ND 99

114 127 52 218 155 88 148 146

a Data from reference 16. b Data from reference 17. c Data from reference 9.

Table 4. Mineral composition (mg/100 g dry wt) of E. corocana (pwana) Mineral

Pwanaa

Antonyb

FAOc

NRCd

Al Ba Ca Cd Co Cr Cu Fe K Mg Mn Mo P Se Sr Ti Zn

24.1 4.40 407 0.05 0.05 0.45 0.57 31.8 416 182 36.9 0.16 274 0.50 1.90 0.66 2.27

NRe NR 313 NR NR NR 1.01 6.53 NR NR 9.86 NR 467 NR NR NR 2.02

NR NR 398 NR NR 0.03 0.47 3.90 NR 137 5.49 0.1 320 NR NR NR 2.30

NR NR 431 NR NR NR 0.60 11.9 378 169 2.29 0.00 301 NR NR NR 1.81

a Average of three samples. b Data from reference 19. c Data from reference 8. d Data from reference 9. e NR, not reported.

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9 in high concentration. Several studies of the nutrient composition of E. coracana seeds have shown them to be an excellent source of calcium, especially when compared to other cereal grains [7–9, 18]. The FAO [8] reported a high manganese content for E. coracana while Vadivoo [18] and Antony [19] reported a high phosphorous content in this cereal. Current mineral analysis also revealed high calcium, manganese and phosphorous contents. For example the calcium content of E. coracana from Nigeria (407 mg/100 g dry wt) was higher than that reported by Antony (313 mg/100 g dry wt) [19], but lower than the average reported by Barbeau (430.7 mg/100 g dry weight) [7]. Vadivoo [18] found the mean calcium content of 36 genotypes of E. coracana to be 320.8 mg/100 g dry wt. With regard to manganese, the present analysis showed a manganese content that was six-fold greater than that reported by the FAO [8] and more than three-fold that found by Antony [19]. Although the phosphorous content of E. coracana (274 mg/100 g dry wt) was less than that of Antony, the FAO and the NRC (467, 320, and 301 mg/100 g dry wt, respectively) [8, 9, 18], it still appeared to be a good source of this mineral. The FAO [8] also reported that high tannin levels in darkly pigmented finger millet could decrease the bioavailability of iron. The iron content of the E. coracana seeds from northern Nigeria exceeded the iron content of every cereal grain analyzed by the NRC [9] and the FAO [8], as well as the finger millet analyzed by Barbeau [7] and Antony [19]. The importance of identifying an iron-rich cereal grain in Nigeria, where iron deficiency anemia is common, cannot be over emphasized. Several published reports of the fatty acid composition of E. coracana seeds have identified linoleic, oleic and palmitic acids as the major fatty acids present in this seed. Current results are in general agreement with these investigations [4, 6]. There are many published reports regarding the bioavailability of the minerals in E. coracana and the digestibility of all nutrients therein. Malleshi [17] and Antony [19] both compared nutritional components in unfermented finger millet with finger millet that had been fermented. Antony [19] reported a decrease of phytates, phenols, tannins and trypsin inhibitors in E. corocana that had been fermented for 24 hours. Concommitantly, an increase in calcium, phosphorous, iron and zinc availability was noted, as well as an increase in the protein content. Malleshi [17] cited an increase in lysine, riboflavin, niacin and ascorbic acid with fermentation; however, as fermentation time increased, an overall decrease in total protein content occurred. Therefore, fermentation of E. coracana offers a means of increasing the overall availability of nutrients and improving the diet of people who consume this cereal grain.

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