There is an increasing world demand of less expensive proteins with good  nutritional  and  functional  properties, particularly in developing and under-developed countries where the supply of food of animal origin is limited due  to non-availability and high cost (Mune et al., 2013). This situation resulted from the constant increase of the human population and growing interest for protein to industry for application in food and non-food markets (Mune et al., 3013). Legumes are considered as poor man’s meat. They are generally rich in protein (18 to 25%), and good sources of minerals and vitamins (Youseff et al., 1989). Legumes are good sources of cheap and widely available proteins for human consumption. They are staple foods for many people in different parts of the world (Udensi et al., 2007). Legume seeds have an average of twice as much protein as cereals and the nutritive value of the proteins are usually high (Vijayakumari et al., 1998). Studies have shown that the lesser known legumes together with other conventional legumes can be used for combating protein malnutrition prevalent in the third world. This can be achieved by the consumption of the legumes whole and in various processed forms (condiments) (Arisa and Aworh, 2007). Therefore, the use of grain legumes for food is restricted by their beany flavor and the presence of anti-nutritional and toxic factors (Friedman, 1992; Yusuf et al., 2008). There is a wide distribution of biologically-active constituent throughout the plant kingdom, particularly in plants used as animal feeding stuff and in human nutrition (Igile, 1996). The knowledge that these compounds elicit both toxic and advantageous biological responses has given rise to several investigations in recent times as to their possible physiological implications in various biological systems (Igile, 1996). Traditional processing techniques such as soaking, cooking, sprouting (germination) or roasting have limited effects on elimination of anti-nutritional factors, and sometimes could decreased protein quality and affected certain functional properties (Friedman, 1992; Yusuf et al., 2008). However, this work tends to investigate the effects some traditional processing methods on the nutritional composition and anti-nutritional constituent of cowpeas.

 

Description of cowpea (Vigna unguiculata)

Cowpea belongs to the family Leguminosae, other names commonly used include catjang, black-eyed bean or china pea (Udensi et al., 2008), southern pea, clossus, or crowther peas (Uzogara and Ofuya, 1992). In Sudan, it was known as lubia helo or white lubia. Cowpea is one of the most important food legumes crop widely grown in semi-arid tropics as an inexpensive source of protein in both human diet and animal feed (Obasi and Wogu, 2008). Its fresh or dried seeds, pods and leaves are commonly used in human food, since  they  are highly valuable as fodder (Gomez, 2014). Cowpea has great flexibility in use; farmers can choose to harvest them for grain or to harvest forage for the livestock, depending on economical or climatological constraints (Gomez, 2014).

  Dual purpose varieties of cowpea have been developed in order to provide both grain and fodder while suiting the different cropping systems encountered in Africa (Tarawali et al., 1997).

  Cowpea by-products such as cowpea seed waste and cowpea hulls (which result from the dehulling of the seeds for food) have been used to replace conventional feedstuff in some developing countries (Ikechukwu, 2000). Though not as efficient as soybean as a protein sources, cowpea was capable of accumulating useful levels of protein and digestible dry matter under the variable growing conditions of the study (Koratkar and Rao,1997).

 

Determination of proximate composition

The samples were separately analyzed for proximate composition using the official standard methods. Moisture content of the sample was determined by Association of Official Analytical Chemists method (AOAC, 2005). The gross energy values were estimated by multiplying  the  crude  protein,  fat  and  carbohydrate  by  their  at  water  values  of  4,  9  and 4 kcal/g,  respectively (Akubor, 1997). Protein content was estimated from the crude nitrogen content of the sample determined by the micro Kjeldhal method (N × 6.25). Carbohydrate was calculated by difference method. Crude fat, crude fiber and ash content of the samples were determined by the procedure given by Association of Official Analytical Chemists (2005).

 

Crude protein determination

The crude protein in the sample were determined by the routine semi-micro Kjeldahl, procedure/technique. This consists of three techniques of analysis, namely digestion, distillation and titration.

Apparatus: Analytical balance, digestion tubes, digestion block heaters, 50 ml burette, 5 ml pipette, 10 ml pipette, 10 ml measuring cylinder, 100 ml beakers, and fume cupboard.

Reagents: Concentrated H₂SO₄, 0.01 N HCl, 40% (W/V) NaOH, 2% boric acid solution, methyl red–bromocresol green mixed indicator, and Kjeldahl catalyst tablet.

Digestion: 0.5 g of each finely ground dried sample was weighed carefully into the Kjeldahl digestion tubes to ensure that all sample materials got to the bottom of the tubes. To this were added 1 Kjeldahl catalyst tablet and 10 ml of concentrated H₂SO₄. These were set in the appropriate hole of the digestion block heaters in a fume cupboard. The digestion was left on for 4 h, after which a clear colourless solution was left in the tube. The digest was cooled and carefully transferred into 100 ml volumetric flask, thoroughly rinsing the digestion tube with distilled water and the flask was made up to mark with distilled water.

Distillation: The distillation was done with Markham Distillation Apparatus which allows volatile substances such as ammonia to be steam distilled with complete collection of the distillate. The apparatus was steamed out for about 10 min. The steam generator is then removed from the heat source to the entire developing vacuum to remove condensed water. The steam generator is then placed on the heat source (that is, heating mantle) and each component of the apparatus was fixed up appropriately.

Determination: 5 ml portion of the digest above was pipetted into the body of the apparatus via the small funnel aperture. To this was added 5 ml of 40% (w/v) NaOH through the same opening with the 5 ml pipette. The mixture was steam-distilled for 2 min into a 50 ml conical flask containing 10 ml of 2% boric acid plus mixed indicator solution placed at the receiving tip of the condenser. The boric acid plus indicator solution changes colour from red to green showing that all the ammonia liberated have been trapped.

Titration: The green colour solution obtained was then titrated against 0.01 N HCl contained in a 50 ml burette. At the end point or equivalent point, the green colour turns to wine colour which indicates that all the nitrogen trapped as ammonium borate [(NH₄)₂BO₃] have been removed as ammonium chloride (NH₄Cl). The percentage nitrogen in this analysis was calculated using the formula: %N = Titre value × Atomic mass of nitrogen × Normality of HCl used × 4 Or %N = Titre value × Normality/Molarity of HCl used × Atomic  mass  of  N ×  Volume  of  flask  containing  the  digest   ×

100 Weight of sample digested in milligram × Vol. of digest for steam distillation. The crude protein content is determined by multiplying percentage nitrogen by a constant factor of 6.25, that is, % CP = %N × 6.25.

 

Determination of ash

Apparatus: Porcelain crucibles, a desiccator, analytical balances and a furnace.

Determination: 2.0 g of the sample were weighed into a porcelain crucible. This was transferred into the muffle furnace set at 550°C and left for about 4 h. About this time it had turned to white ash. The crucible and its content were cooled to about 100°C in air, then room temperature in a desiccator and weighed. This was done in duplicate. The percentage of ash was calculated from the formula:

Ash content =   Wt. of ash/Original wt. of sample × 100

                                                                 

Fibre determination

Apparatus: Heating mantle, crucibles, furnace, sieve cloth, fibre flask, funnel, analytical weighing balance, and a dessicator.

Reagents: 0.255 N H₂SO₄, 0.313 N NaOH, and acetone.

Determination: 2.0 g of the sample was accurately into the fibre flask and 100 ml of 0.255 N H₂SO₄ was added. The mixture was heated under reflux for 1 h with the heating mantle. The hot mixture was filtered through a fibre sieve cloth. The filtrate obtained was thrown off and the residue was returned to the fibre flask to which 100 ml of 0.313 N NaOH was added and heated under reflux for another 1 h. The mixture was filtered through a fibre sieve cloth and 10 ml of acetone added to dissolve any organic constituent. The residue was washed with about 50 ml hot water on the sieve cloth before it was finally transferred into the crucible. The crucible and the residue were oven-dried at 105°C overnight to drive off moisture. The oven-dried crucible containing the residue was cooled in a dessicator and later weighed to obtain the weight W₁. Crucible with weight W₁ was transferred to the muffle furnace for ashing at 550°C for 4 h.

Crucible containing white or grey ash (free of carbonaceous material) was cooled in the dessicator and weight to obtain W₂. The difference W₁ – W₂ gives the weight of fibre. The percentage fibre was obtained by the formula:

%Fibre = W₁ – W₂ / Wt. of sample × 100

 

Crude fat or ether extracts determination

Apparatus: Soxhlet apparatus and accessories, oven, desiccator and analytical balance.

Reagents: Petroleum spirit or Ether (40 – 60°C boiling point).

 

Determination: 1 g of each dried sample was weighed into fat free extraction thimble and pug lightly with cotton wool. The thimble was placed in the extractor and fitted up with reflux condenser and a 250 ml Soxhlet flask which has been previously dried in the oven, cooled in the desiccator and weighed. The Soxhlet flask is then filled to ³/₄ of its volume with petroleum ether (b.pt. 40 – 60°C), and the Soxhlet flask. Extractor plus condenser set was placed on the heater. The heater was put on for 6 h with constant running water from the tap for condensation of ether vapour. The set is  constantly watched for ether leaks and the heat source is adjusted appropriately for the ether to boil gently. The ether is left to siphon over several times say over at least 10 to 12 times until it is short of siphoning. It is after this is noticed that any ether content of the extractor is carefully drained into the ether stock bottle. The thimble containing sample is then removed and dried on a clock glass on the bench top. The extractor, flask and condenser is replaced and the distillation continues until the flask is practically dry. The flask which now contains the fat or oil is detached, its exterior cleaned and dried to a constant weight in the oven. If the initial weight of dry Soxhlet flask is Wo and the final weight of oven dried flask + oil/fat is W₁, percentage fat/oil is obtained by the formula:

W₁ – Wo / Wt. of Sample taken ×   100

 

Dry matter and moisture determination

Apparatus: Oven, crucibles, dessicator and balance.

Reagents: Silical gel, grease.

Determination: 2 g of the sample was weighed into a previously weighed crucible. The crucible plus sample taken was then transferred into the oven set at 100°C to dry to a constant weight for 24 h overnight. At the end of the 24 h, the crucible plus sample was removed from the oven and transferred to dessicator, cooled for 10 min and weighed.

If the weight of empty crucible is Wo, weight of crucible plus sample is W₁, weight of crucible plus oven-dried sample W₃.

(%DM) %Dry Matter   =   W₃ – Wo / W₁ – Wo × 100

%Moisture  =   W₁ – W₃ / W₁ – Wo  × 100     

Or

%Moisture   = 100 – % DM.

 

Determination of methionine (A Spectrophotometric method)

Lunder (1973) recommended an improved method for the colorimetric or spectrophometric determination of methionine in acid hydrolysates of biological products. Methionine was determined using the method described by Lunder (1973).

Sample (0.2 g) was weighed into a 30 ml test tube. 10 ml of Tris buffer reagent of pH 7.2 was added with 0.1 mg papain and incubated at 55°C for 18 h overnight. It was removed from incubator and 4 drops of orthophosphoric acid (88%) added to stop enzymatic hydrolysis. The mixture was transferred into a 25 ml volumetric flask and made up to volume with distilled water. The solution obtained was shaken thoroughly and filter through a Whatman No. 41 Filter paper into a test tube or conical flask. 5 ml aliquot was pipetted out of this into another test tube to which 1 ml of 5 N NaOH, 1 ml of 1% aqueous glycine and 1 ml of 1% sodium nitroprusside were added. The tubes were kept in the water bath at 40°C for 10 min after thorough shaking.

The contents were allowed to cool and 6 ml of 88% orthophosphoric acid added with thorough shaking to obtain a red colour. Methionine standards of range 0.5 to 2.5 mg/ml were prepared and treated as mention earlier. Absorbances of samples and standards were read using Spectronic 21D Spectrophotometer at a wavelength of 520 nm. % Methionine or Methionine in g/100 g protein can be calculated using the formula: %Methionine   =   Factor × Absorbance of sample/Sample weight ×% Protein in sample × 100%Protein in sample was determined by the Kjeldahl method.

 

Determination of lysine

Lysine was evaluated using the method prescribed by Jambunathan et al. (1983) in which the rapid methods was applied for estimating lysine and protein in sorghum.

Sample (1 g) was weighed into a 5 ml centrifuge tube. 40 ml of acid orange dye solution was added and shaked for 1 h. The mixture was filtered through a Whatman No. 41 filter paper into a 50 ml volumetric flask and made up to mark with distilled water and incubated at 60°C for 18 h overnight. Standards Lysine of range 0.35 to 0.85 mg/ml were prepared and treated with acid orange dye to develop colour. The absorbances of the standards as well as samples were read on a Spectronic 21D Spectrophotometer at a wavelength of 415 nm.

%Lysine or Lysine in g/100 g protein can be calculated using the formula:

Factor × Absorbance / Sample weight × % Protein in sample × 100

 

Chemical analysis of mineral elements concentration (Na, K, Mg, Ca, P {Fe, Zn, Cu})

Samples were analyzed chemically according to the official methods of analysis described by the Association of Official Analytical Chemist (AOAC, 2005). All analyses were carried out in triplicate.

Calcium, potassium and sodium determination

Apparatus: Heating mantle, crucible, glass rod, flame photometer, 100 ml volumetric flask, Whatman No. 1 Filter paper, wash bottle, 10 ml pipette, and funnel.

Reagent: 2 M HCl.

Determination: The ash of each sample obtained was digested by adding 5 ml of 2 M HCl to the ash in the crucible and heat to dryness on a heating mantle. 5 ml of 2 M HCl was added again, heat to boil, and filtered through a Whatman No. 1 filter paper into a 100 ml volumetric flask. The filtrate  was made up to mark with distilled water stoppered and made ready for reading of concentration of calcium, potassium and sodium on the Jenway Digital Flame Photometer (DFP7 Model) using the filter corresponding to each mineral element.

The concentration of each of the element was calculated using the formula:

%Ca or %K or %Na = Meter Reading (MR) × Slope × Dilution factor / 1000

where MR × slope × dilution factor = Concentration in part per million (ppm or mg/kg). You get concentration in % when you divide by 10000.

 

Phosphorus determination (Spectrophotometric method)

Phosphorus was determined routinely by the vanado-molybdate colorimetric or spectrophotometric method.

Apparatus: Spectrophotometer or colorimeter, 50 ml volumetric flask, 10 ml pipette, filter paper, funnel, wash bottle, glass rod, heating mantle, and crucibles.

Reagents: Vanadate – Molybdate yellow solution, 2 M HCl.

Determination: The ash of each sample obtained was treated with 2 M HCl solution as described for calcium determination earlier. 10 ml of the filtrate solution was pipetted into 50 ml standard flask and 10 ml of vanadate yellow solution was added and the flask was made up to mark with distilled water, stoppered and left for 10 min for full yellow development. The concentration of phosphorus was obtained by taking the optical density (OD) or absorbance of the solution on a Spectronic 20 spectrophotometer or colorimeter at a wavelength of 470 nm.

The percentage phosphorus was calculated from using the formula:

%Phosphorus = Absorbance × Slope × Dilution factor / 10000

 

Determination of Mg, Cu, Mn, Fe and Zn using BUCK200 AAS

The digest of the ash of each sample earlier as obtained in calcium and potassium determination  was washed into 100 ml volumetric flask with deionised or distilled water and made up to mark. This diluent was aspirated into the Buck 200 Atomic Absorption Spectrophotometer (AAS) through the suction tube. Each of the trace mineral elements was read at their respective wavelengths with their respective hollow cathode lamps using appropriate fuel and oxidant combination (AOAC, 2005).

 

Determination of trypsin inhibitor activity (TIA)

The casein digestion method

Defatted ground sample (0.2 g) was weighed into a centrifuge tube. 10 ml of 0.1 M phosphate buffer was added and shaked on a shaker at room temperature for 1 h. The suspension was centrifuged at 5000 rpm in a centrifuge for 5 min. The content was later filtered through a Whatman No 42 filter paper into a 250 ml conical flask.

0.2, 0.4, 0.6, 0.8, and 1.0 ml of the filtrate were pipette into a set of triplicate set of test-tubes (one set for each level of extract). The final volume is adjusted to 2 ml by the addition of 0.1 M phosphate buffer. These test-tubes were arranged into a water bath maintained at 37°C. A blank was prepared by adding 6 ml of 5% TCA solution to one set of triplicate tubes. 2 ml of 2% casein solution was added to all the tubes which is previously kept at 37°C to incubate for 20 min. The reaction of casein will be stopped by the addition of 6 ml of 5% TCA solution and this was allowed to proceed for 1 h at room temperature. The mixture was later filtered at room temperature through a Whatman No. 42 filter paper into 100 ml conical flask. 0.2, 0.4,  0.6, 0.8, and 1.0 ml of stock trypsin solution were also pipetted into a triplicate set of test-tubes (one set for each level of trypsin) as mentioned earlier and treated similarly as sample to the point of filtration.

The absorbance of the filtrates of both samples and standard trypsin solution were read on a spectrophotometer at a wavelength of 280 nm. The actual absorbance of sample is the difference between absorbance of stock trypsin filtrate and that of sample filtrate. The absorbance of blank was also read. One trypsin inhibitor unit (TIU) is arbitrarily defined as an increase of 0.01 absorbance units at 280 nm in 20 min per 10 ml of the reaction mixture under the conditions mentioned herein.

Trypsin Inhibitor Unit for each sample was calculated using the formula: Change in absorbance of sample extract/ 0.01 × mg protein in sample

 

Determination of phytate

Phytate was determined according to the method described by Maga (1983). 2 g of each sample was weighed into  250 ml  conical flask. 100 ml of 2% hydrochloric acid was added to soak each sample in the conical flask for 3 h. This was filtered through a double layer of hardened filter paper. 50 ml of each filtrate was placed in 0.50 ml conical flask and 107 ml distilled water was added in each case to give proper acidity. 10 ml of 0.3% ammonium thiocyanate (NH₄SCN) solution was added into each solution as indicated. This was titrated with standard iron (III) chloride solution which contained 0.00195 g iron per ml. The end point was slightly brownish-yellow which persisted for 5 min. The % phytic acid was calculated using the formula:

%Phytic acid = Titre value  ×  0.00195  ×  1.19  ×  100  ×  3.55 / Wt. of sample

 

Determination of tannin

Sample (0.20 g) was measured into a 50 ml beaker 20 ml of 50% methanol was added and covered with parafilm and placed in a water bath at 77 to 80°C for 1 h. It was thoroughly shakied to ensure a uniform mixing. The extract was quantitatively filtered using a double layered Whatman No 41 filter paper into a 100 ml volumetric flask, 20 ml water added, 2.5 ml folin-Denis reagent and 10 ml of 17% Na₂CO₃ were added and mixed properly. The mixture was made up to mark with water mixed well and allow to stand for 20 min. Bluish-green color was develop at the end of range 0 to 10 ppm where similar treatment was given as 1 ml of the sample earlier.

The absorbance of the tannic acid standard solutions as well as samples was read after color development on a spectronic 21D spectrophotometer at a wavelength of 760 nm. Percentage tannin was calculated using the formula.

%Tannin = Absorbance of sample × average gradient factor × dilution factor/ Wt. of sample × 10,000

 

Data analysis

Data obtained were analyzed by the one-way analysis of variance (ANOVA) and means were compared using the Least Significant Difference (t-Test). Significant difference between the treatments was accepted at 5% probability

Effect of treatment on the micro elements content of cowpea

The result in Table 5 showed that heat treatments (boiling and pressure cooking) recorded decreased level of micro elements (Fe, Zn, Cu, and  Mn), while germination had increased the micro elements by 4.66, 3.78, 13.85 and 6.38% for Fe, Zn, Cu and Mn, respectively. This result agreed with the report of Hefnawy (2011) that cooking in boiling water caused great losses of copper and iron. It was also reported by Hefnawy (2011) that pressure cooked mature cowpeas had 30% loss of copper. Mean values with the same superscript in a column are not significantly different (p<0.05).

 

 

This research showed that heat processing amounted to loss of nutrients; this may be due to leaching during heat application. There was drastic reduction of anti-nutrients content of boiled and pressure cooked cowpeas and this probably is because the anti-nutrients are heat labile. On the other hand, germination had increased the nutrients and anti-nutrients composition of cowpeas. Germination as a biochemical process had induced enzymatic reactions that resulted in the bioavailability of some nutrients. This result indicated that in addition to protein, cowpea is rich in macro and micro minerals and if properly processed, it can alleviate malnutrition.

  Further research should be carried out to determine the effect of heat treatment in combination with germination on cowpea flour as to reduce the anti-nutrient factors. 

The authors have not declared any conflict of interests.