British Biotechnology Journal 6(2): 43-50, 2015, Article no.BBJ.2015.026 ISSN: 2231–2927

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Compositional and Amino Acid Profile of Nicker Bean ( gigas)

H. N. Ogungbenle1* and O. T. Oyadipe1

1Department of Chemistry, Ekiti State University, P.M.B. 5363, Ado - Ekiti, Nigeria.

Authors’ contributions

This work was carried out in collaboration between both authors. Author HNO designed the study, performed the statistical analysis, wrote the protocol, managed the literature searches and wrote the first draft of the manuscript. Author OTO managed the analyses of the study. Both authors read and approved the final manuscript.

Article Information

DOI: 10.9734/BBJ/2015/14681 Editor(s): (1) Alok Adholeya, Biotechnology and Management of Bio-resources Division, The Energy and Resources Institute, India. (2) Kuo-Kau Lee, Department of Aquaculture, National Taiwan Ocean University, Taiwan. Reviewers: (1) Fagbemi, Tayo Nathaniel, Food Science and Technology, Federal University of Technology, Nigeria. (2) Anonymous, USA. (3) Anonymous, Nigeria. (4) Anonymous, Mexico. (5) Anonymous, Brazil. Complete Peer review History: http://www.sciencedomain.org/review-history.php?iid=806&id=11&aid=7900

Received 14th October 2014 th Original Research Article Accepted 6 December 2014 Published 27th January 2015

ABSTRACT

The proximate, anti-nutritional factors, functional properties, minerals and amino acid composition of nicker bean (Entada gigas) were determined. The sample contained crude protein and carbohydrate of 24.8±0.02% and 47.2±0.10% respectively. The sample exhibited good emulsion capacity and emulsion stability of 62.3±0.03% and 36.2±0.01% respectively. The foaming capacity was 35.0±0.02% while the least gelation concentration was 4.00±0.01%. It contained nutritionally valuable minerals. The values of the anti-nutritional factors were: phytate (6.59±0.50 mg/g), oxalate (3.50±0.10 mg/g) and tannin (0.26±0.02%). Glutamic acid was the most abundant amino acid with the value of 51.1 g/100 g crude protein while methionine (4.10 g/100 g crude protein) was the least abundant amino acid in the sample. Entada gigas is a potential source of nutrients that should be cultivated and its consumption encouraged.

Keywords: Proximate; anti-nutritional; functional; minerals; amino acid.

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*Corresponding author: E-mail: [email protected];

Ogungbenle and Oyadipe; BBJ, 6(2): 43-50, 2015; Article no.BBJ.2015.026

1. INTRODUCTION 200 ml of 1.25% H2SO4 in 500 ml conical flask and boiled for 30 minutes. The mixture was later Global alliance for improved nutrition is filtered through muslin cloth, rinsed with hot paramount to food scientists. Food scientists are distilled water and scrapped back into the flask always in search for newly improved and and added 200 ml of 1.25% NaOH and allowed underutilized legumes in order to proffer to boil again for another 30 minutes. The mixture solutions to the global food shortages and was filtered, rinsed with 10% HCl twice with consequent malnutrition which has lead to series industrial methylated spirit and allowed to drain of prevalent diseases. Entada gigas has not and dry. The residue was scrapped into a been consumed as food in Africa but as crucible, later dried in an oven at 105ºC, cooled medicinal for local treatment of some in a desicator, re-weighed and finally incinerated ailments. It is known as fever nut; spreading in muffle furnace at 300ºC for 30 minutes, again vine-like in nature which grows as tall as 6-7 m in cooled at room temperature and re-weighed [4]. length and uses other vegetation for support. The The carbohydrate value was calculated by stem is covered by sharp spines which may grow difference. to 0.4 or more metres in diameter [1] and the grows in a flat pod with length of 0.04 m. 2.2 Determination of Functional Properties Two or three hard smooth seeds of 0.01 m in length are contained in each pod. The seeds The water and oil absorption capacities of the inside pod are olive green in colour and later sample were determined using the methods of become dark-red brown at maturity. Entada Beuchat [5]. Ten milliliters was added to 1.0 g gigas is very common in coastal habitats sample in a centrifuge tube. The suspension was including beaches and dry areas in West Africa mixed vigorously using Marlex mixer and then [1]. The data obtained from the analyses would centrifuged at 15,000 rpm for 15 minutes and the help us to know whether the sample has good volume of the supernatant was recorded. The nutritional values as other existing edible bound water was calculated from the difference or may be processed for future consumption in the initial volume of the solvent used and the because the seed is known in Africa to contain final volume after centrifuging. The same certain toxins which hinder its edibility. Functional procedure was used for oil absorption capacity properties of any food must be known before it by replacing King’s vegetable oil of density 0.880 can be incorporated into food system such as g/ml with water in the method described above. emulsification, foamability and gelation [2]. Therefore, the aim of this work is to determine The emulsion capacity and stability were the proximate, functional properties, anti- determined according to methods of described nutrients and amino acid composition of the by Lin et al. [6]. Two grams sample flour was sample. added to 100 ml distilled water and blended for 30 seconds using Marlex food mixer at a high 2. MATERIALS AND METHODS speed. After complete dispersion, 5 ml portion of King’s vegetable oil of 0.880 g/ml density was The sample used for the present work was added from a burette with continuous blending bought in Ado-Ekiti central market, Ekiti State until the emulsion break point (i.e. a separation Nigeria in Africa continent. The 5 kg of the into two layers) at room temperature. The value sample was separated from the shells, later obtained was expressed as gram of oil emulsified dried and ground into flour. The flour was per 1 gram of the sample. packaged and stored in freezer (-4°C) prior to the analyses. The emulsion stability was determined as the volume of the water separated after 24 hours at 2.1 Proximate Analysis room temperature.

The slightly modified method of Sathe et al. [7] The moisture and total ash contents were was used to determine the least gelation determined using the air oven and muffle furnace concentration. Sample slurries of 2, 4, 6, 8, 10, as described by Pearson [3]. The sample was 12, 14, 16, 18 and 20% were prepared in 5 ml analyzed for crude fat and crude protein using portions of distilled water. The test tubes methods of AOAC [4] and the percentage containing these slurries were heated for one nitrogen was converted to crude protein by hour in boiling water followed by rapid cooling for multiplying by 6.25. The crude fiber was 2 hours at -4ºC. The least gelation concentration determined by adding 2 g of the sample into

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was determined as concentration which did not 2.5 Determination of Amino Acid slip when the test tubes were inverted. The amino acid profile was determined using the The method of Coffman and Garcia [8] was method described [13]. The sample was dried to employed to determine foaming capacity and constant weight and defatted using Soxhlet stability. One gram of the sample was whipped extractor. After the defatting process, the with 50 ml distilled water for 5 minutes in a defatted sample (2 g) was weighed into a glass Marlex food mixer and later poured into a 100 ml ampoule; 7 ml of 6MHCl was added and oxygen graduated flask to study the foaming capacity was expelled by passing nitrogen into the (per cent increase in volume). The foaming ampoule so as to avoid possible oxidation of stability was determined as the difference some amino acids during hydrolysis. The glass between the foam and the water level after 2 hrs. ampoule was then sealed with bunsen burner flame and placed in an oven at 105±5ºC for 22 2.3 Mineral Analysis hours. The ampoule was allowed to cool before broken at the tip and the content was filtered to remove the organic matters. The filtrate was then The minerals were analyzed by dry ashing the evaporated to dryness at 40ºC under vacuum in sample at 550ºC in a Carbolite, Sheffield muffle a rotavapor (BÜCHI Rotavapor, R110). The furnace to constant weight and dissolving the ash residue was dissolved in 5 ml of acetate buffer in 100 ml standard flask using distilled deionized (pH 2.0) and stored in specimen bottles which water with 3 ml of 3 M HCl. Sodium and were kept in the freezer. The hydrolysate (7.5 potassium were determined using a flame µL) was dispensed into the cartridge of the photometer (model 405, corning, U.K). All other Technicon Sequential Multi-Analyser (TSM) minerals were determined using Atomic using a syringe. The TSM analyser is designed Absorption Spectrophotometer (Perkin & Elmer to separate and analyse neutral, acidic and basic model 403, USA) [9]. amino acids of the hydrolysate. The amount of amino acid was obtained from the chromatogram 2.4 Determination of Anti Nutrients peaks. The whole analysis lasted for 76 minutes and the gas flow rate was 0.50 ml/min at 60ºC with reproducibility consistent within ±3%. Tannin: A 10 ml of 70% aqueous acetone was added to 200 mg of the sample, tightly covered 3. RESULTS AND DISCUSSION and put into an ice bath then shaken for 2 hours at 30°C, later centrifuged at 3,600 rpm. A 0.2 ml 3.1 Proximate Composition of the mixture was pipetted into a test tube and 0.8 ml of distilled water was added. Standard Proximate composition of the Entada gigas is tannic acid solutions were prepared from 0.5 presented in Table 1. The crude fat content of mg/ml stock solution and made up to 1 ml with the Entada gigas was lower than those of distilled water. Folin Wu reagent (0.5 ml) and 2.5 legume seeds [14] and Colocynthis citrullus ml of 20% Na CO were added to the sample 2 3 (51.9%) [11]. The value of carbohydrate was mixture and the standard solutions; both 47.1±0.10% and that of total ash was solutions were incubated for 40 minutes at room 2.55±0.12%. The crude protein was higher than temperature and the absorbance was measured that of cereal, millet (11.4%) [15]. But this at 725 nm [10]. present value was higher than those of lima bean flour (22. 7%) [16], pigeon (22.4%) [16], Oxalate: One gram of the sample was weighed African mango full fat (10.6%) and defatted into 100 ml conical flask, 75 ml of 1.5 M H2SO4 (14.8%) [17]. The sample contained crude was added and stirred for 1 hour then filtered. protein of 24.8±0.02%. The high crude protein The sample filtrate (25 ml) was titrated against content may be valuable for food formulation. 0.1 MKMnO4 solution until faint color persisted The moisture content (8.94±0.02%) was higher for 30 seconds [10]. than those of Parinari curatillefolia (4.40%) [18], full fat (5.76%) and defatted (5.44%) of cashew Phytate was determined on Spectronic 20 nut kernel [19]. The crude fiber content colorimeter (Gallenkamp, UK). The amount of (4.87±0.01%) was lower than that of Parinari phytate in the sample was determined as curatillefolia (5.45%) [18], while the crude fat hexaphosphate equivalent [11,12]. content of the sample (15.7±0.03%) was higher

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than those of L. leucocephalia (6.80%) [20] and in the use of the Entada gigas for formulation scarlet runner bean (5.3-6.9%) [21]. and as an additive to other gel- foaming material in food products [27]. This value was lower than Table 1. Proximate composition of those of pigeon pea (12%) [2], benniseed [15], Entada gigas lupin seed flour (14%) [28], Afzelia africana (6%) [29] but higher than that of African nutmeg [23]. Parameter % This indicates that the sample may be a useful Moisture 8.94±0.02 additive to other materials for gel formation in Total ash 2.55±0.12 food products. Crude fat 15.7±0.03 Crude fiber 4.87±0.01 Crude protein 24.8±0.02 The oil absorption capacity (250±0.30%) was Carbohydrate by difference 47.2±0.10 higher than those of benniseed (45.5%) [15], millet 55% [30] and conophor nut (108.13%) [14], 3.2 Functional Properties and lower than that of African nutmeg (256%) [23].

The results of functional properties are presented in Table 2. The emulsion capacity for Entada 3.3 Mineral Composition gigas is 62.3±0.03% while the emulsion stability was 36.2±0.01%. The value of the emulsion The results of the mineral analysis of Entada capacity was higher than those of benniseed gigas are presented in Table 3. The highest (30%), cowpea (28.0%) [22], dehulled African concentrated mineral element was sodium with nutmeg (45.6%) [23], dehulled African yam bean the value of 362±0.30 mg/100 g followed by (10.0-20.0%) [24] and soy flour (18.0%) [6]. It calcium (361±0.20 mg/100 g) while selenium implies that Entada gigas may be good for food (1.00±0.03 mg/100 g) was the least abundant. additives and emulsified foods like ice cream and This indicates that the value of calcium in the salad dressings. The foaming capacity sample is adequate for infant development of (35.0±0.02%) was lower than those of Luffa bones and teeth. Therefore, suggests that cylindrica (60%) [25] and African nutmeg (50%) Entada gigas flour might be a good supplement [23]. The foaming stability was lower than that for human food formulations in future if accepted reported for African nut meg (8.5%) [23]. The as food. foaming capacity and stability make the sample good for food formulations and stabilizing The value of calcium (361±0.20 mg/100 g) in colloidal food system. Entada gigas was higher than those of dehulled

Afzelia Table 2. Functional properties of African nutmeg (203.7 mg/100 g) [31] and Entada gigas africana (208 mg/100 g) [32]. The value of Fe (10.5±0.04 mg/100 g) was also higher than that Parameter % of dehulled African nutmeg (3.0 mg/100 g) [31]. Water absorption capacity 145±0.20 Fe is an important component in the transfer of Oil absorption capacity 250±0. 30 oxygen and it is most closely related to anaemia. Iron is highly useful for the blood formation [22]. Foaming capacity 35.0±0.02 + + Foaming stability 5.00±0.15 The Na /K ratio is an indicator for mineral Emulsion capacity 36.2±0.01 recommendation. The Na/K ratio of 1.0 is Emulsion stability 62.3±0.03 recommended as reported by Niemann [33]. Na Least gelation concentration 4.00±0.01 and K control water equilibrium level in body ( W/V) tissue and are also important in the transportation of some non-electrolytes. Mg also activates enzymatic process in the body. Cu and The least gelation concentration was Zn are known to be associated with enzyme 4.00±0.01%. The ability of the bean to form gels, systems particularly the biochemical oxidation- provides a structural matrix for holding water, reduction processes. The results showed that the flavor, sugar and food ingredients is useful in sample is rich in most of the essential minerals food application and in new product and could therefore serves as additional source development, thereby providing an added for such minerals. On comparative basis, Entada dimension to food functionally [26]. The least gigas is a better source of Ca, Mg, Zn. Mg and gelation concentration observed may be an asset Fe respectively.

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Table 3. Mineral composition of Entada gigas g/100 g) and bulma cotton flour (189.9 g/100 g) [41]. The total essential amino acids (TEAA) of Mineral mg/100 g the sample amounted to 206.3 mg/g (with Fe 10.5±0.04 histidine), 199.3 mg/g (without histidine) Zn 75.8±0.01 (Table 5). The TEAA (with histidine) value of the Cu 1.33±0.02 Entada gigas was lower than that of soy bean; Mn 25.7±0.10 444 g/100 g [42]. Entada gigas is also a good Ca 361±0.20 source of leucine (Leu) and arginine (Arg). Na 362±0.30 Table 5 also shows that Entada gigas has high K 62.2±0.08 quality protein and a good source of essential As N.D amino acid for children [42,43]. One can also Cd N.D conclude that the sample will also meet the Ni 1.20±0.10 amino acid requirement for adult since the amino Se 1.00±0.03 acid composition that meets the requirement for Pb 1.50±0.01 a child will also meet that for an adult [43,44]. Co N.D Maize food products are highly deficient in lysine N.D – Not Detected and they are the most common weaning foods for children in most African countries [45], 3.4 Anti-nutritional Factors Entada gigas may not be therefore suitable for the enrichment of maize food products since it The results of oxalate, tannin and phytate contains low amount of lysine (12 g/100 g). The contents are presented in Table 4. The phytate quality of dietary protein can be measured in level (6.59±0.50 mg/g) was higher than those of various ways [46] but basically as the ratio of the sorghum (5.34 mg/g) and millet (4.41 mg/g), available amino acids in the food or diet Afzelia africana (13.59%) [29] and date palm fruit compared with the body needs, expressed as a (4.87 mg/g) reported by Ogungbenle [34]. ratio [47-49]. Methionine and cysteine were the Oxalate (3.50±0.10 mg/g) was lower than those limiting amino acids in the present sample. This of sorghum (5.22 mg/g) and millet (4.06 mg/g) was also observed by Salem [50] for faba bean [35] but higher than those of Afzelia africana materials. Apata and Ologhobo [51] also (2.84 mg/g) [29] and date palm fruit (3.0 mg/g) reported low amounts of methionine and cysteine [34]. The value of tannin (0.26±0.02%) was lower in legumes. The values of these amino acids than those of date palm fruit (3.0%) [34], millet were higher than those of faba bean materials (4.06%) [12], sorghum (0.44%) [35] and cooked [50] and velvet tamarind pulp methionine (4.30 walnut (2.33%) [36]. Tannin has been shown to g/100 g), cysteine (5.83 g/100 g) [52]. contribute some degrees of resistance to pre- harvest germination [37]. The high value of Table 4. Anti-nutritional factors of phytate, indicates that the Entada gigas may be Entada gigas processed chemically as a source of phytic acid, which binds polyvalent cations and makes them Parameter Value unavailable in the body. Tannin which was very Tannin (%) 0.26±0.02 low makes the sample important medicinally as a Phytate (mg/g) 6.59±0.50 detersive and astringent in intestinal tubules [38]. Oxalate (mg/g) 3.50±0.10 It can also prevent alcohol intoxication in the body system. The percent TNEAA was 35.6% and percent TEAA was 64.4% (with histidine), 62.2% (without 3.5 Amino Acid Profile histidine) showing that TNEAA was less concentrated in the sample than TEAA The result of the amino acid analysis of the (%TNEAA< %TEAA), thus making it a good Entada gigas is presented in Table 5. Glutamic source of essential amino acids (Table 6). The % acid (Glu) was the highest amino acid with the TEAA for the sample (64.4%, with histidine) and value of 51.1 g/100 g while methionine was (62.2%, without histidine) was higher than those found to be least concentrated amino acid with of germinated millet (50.2%, with histidine) and the value of 4.10 g/100 g. This is in agreement (46.3%, without histidine) [40] and Afzelia with the observation of Olaofe et al. [39]. The africana (48.5%, with histidine) and (41.6%, glutamic acid value of the sample was higher without histidine) [29]. Histidine is an important than those of cooked walnut (151.6 g/100 g) [36], amino acid required by children for growth and raw millet (67.4 g/100 g) [40], white melon (180.9 development.

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Table 5. Amino acid composition of COMPETING INTERESTS Entada gigas (g/ 100 g crude protein) Authors have declared that no competing Amino acid g/100 g crude protein interests exist. Lysine* 12.0 Histidine* 7.00 REFERENCES Arginine* 46.1 Aspartic acid* 23.0 1. Irvine R. West African Crops, 3rd edition. Threonine* 6.60 London Oxford University, Press; 1980. Serine 12.0 2. Oshodi AA, Ekperigin MM. Functional Glutamic* 51.1 properties of pigeon pea (Cajanus Cajan) Proline 6.10 flour. Food Chem.1989;34:187-191. Glycine 15.6 3. Pearson D. Chemical analysis of foods. 7th Alanine 10.0 ed. Churchill Livingstone. London; 1979. Cystine 5.50 4. AOAC. Official methods of analysis 15th Valine 23.0 edition. Association of Official Analytical Methionine* 4.10 Chemists Washington D.C.; 2005. Isoleucine* 11.2 5. Beuchat LR. Functional and Leucine* 50.2 electrophoretic characteristics of Tyrosine 13.6 succylated peanut flour. J Agric Food Phenylalanine* 23.1 Chem. 1977;25:258 -261.  Essential amino acid 6. Lin MYY, Humbert ES, Sosulki FO. Certain functional properties of sunflower meal Table 6. Essential, non essential, neutral, products. J Food Sci. 1974;39:368-370. acidic and basic amino acids 7. Sathe SK, Desphande SS, Salunkhe DK.

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