Experiment was conducted at Department of Soil Science and Agriculture chemistry, N. M. College of Agriculture, Navsari Agricultural University, Navsari during
Kharif- 2022. Four cultivars of cowpea NCK-15-01, NCK-15-05, NCK-15-08 and NCK-15-09 had been chosen for analysis in experiment. The raw cowpeas have been amassed from Pulse Research Station, Navsari Agricultural University, Navsari. After collection, samples were analysed under three exceptional processed states like raw, pressure cooking and sprouting. For the study the analysis of variance of the observed data was done using factorial complete randomized design (Factorial concept). The observations for every parameter had been taken with three repetitions. The critical difference at 5% level of the significance was worked out to compare the treatment means. Among the biochemical parameters some nutrient compositions like crude protein, albumin, globulin, prolamine, glutelin, total soluble sugar, crude fat, antioxidant activity, moisture content and crude fiber were analysed.
Sample processing techniques
After collection, sample was taken to lab and washed properly before processing. Processing techniques such as pressure cooking, sprouting as well as raw cowpea used in the experiment. Pressure cooking treatment was performed pressure cooker. For that 100 gm seeds were soaked for 12 hr in water and then pressure cooked at 105°C for 15 min. LPG gas cylinder was used for the treatment having gas pressure 5.5 kg/m
2. Diameter of burner head was 85 mm and burner pore size was 1.7mm. For sprouting treatment, 100 gm seeds were soaked overnight in fresh water for 12 hr. On the following morning, the seeds were rinsed and water drained off. Then it was allowed to germinate in petri-plate with moistened germination paper until the sprouts were seen (16-18 hr). Raw cowpea seeds was analysed as it was.
Crude protein
Protein analysis was done by micro-Kjeldahl method as described in
Sadasivam and Manickam (1992) with minor modification. Powdered sample of raw, cooked and sprouted seeds (0.5 g) was taken in a conical flask and 10 ml of H
2SO
4 was added and incubated overnight. Digestion of sample was done on hot plate. Sample was cooled at room temperature and 2.5 ml of 50% chromic acid was added. Sample was again heated until fumes produced and transferred the solution in 1000 ml volumetric flask. One molar NaOH (100 ml) was added with some amount of water. Few drops of mix indicator were added and 1000 ml volumetric flask was attached with distillation chamber on burner. On the other side distillation chambers outlet was dipped in 25 ml 4% boric acid. Distillate was collected after turning into blue colour. Distillate was titrated against 0.1NH
2SO
4.
Protein fractions: albumin, globulin, prolamine and glutelin
Protein fractions determination was done by standard method of
Makeri et al., (2017). 100 g sample was suspended in 600 mL distilled water and extracted at room temperature (~25°C) for 2 hr and then centrifugedat 1250 rpm for 10 min to obtain albumin fractions. The supernatant was decanted and the residue extracted with 600 ml of 25 g/kg NaCl for 2 hr to yield globulin extract. Resulting residue was further extracted with 600 ml of 0.1 MNaOH (adjusted to pH 9.0), then followed with 70% ethanol extraction for 2 hr each to obtain glutelin and prolamin fractions, respectively. Prolamin was precipitated by adding threefold acetone. Precipitated proteins were washed twice with distilled water, neutralized, then stored below 4°C until analysis. True protein was quantified by Folin Lowry’s method. In this, 0.5 g fresh sample was extracted in 10 ml borate buffer of pH 7.0. The extracted sample was centrifuged @ 1000 rpm for 30 minute and the supernatant was used for estimation of protein. Sample aliquot of 0.1 ml was pipetted out into two test tubes and diluted to 1 ml with distilled water. A separate tube containing 1 ml distilled water was used as blank. To this 5 ml of alkaline copper solution was added, mixed thoroughly and was allowed to stand for 10 minutes.(prepared by mixing 50 ml of 2% Na
2CO
3 in 0.01N NaOH and 1 ml 0.5% CuSO
4.5H
2O in 10% potassium sodium tartrate). Then 0.5 ml of Folin-Ciocalteau reagent was added and mixed thoroughly. The tubes were kept in dark for 30 minutes for the development of colour. The absorbance was read subsequently at 660 nm and the amount of protein was estimated from standard curve prepare using standard solution of bovine serum albumin (200 µg/ml).
Total soluble sugar content
Total soluble sugar was analysed by spectrophotometer with the reaction of anthrone as described by
Hedge and Hofreiter (1962). Powdered sample (100 mg) was mixed with 5 ml of 2.5N HCl in test tube and boiled over water bath at 85-90°C for 3 hours followed by cooling of the sample material. After that the sample was crushed in a mortar and centrifuged the mixture @ 5,000 rpm for 10 min. Supernatant were collected and the volume was made up to 100 ml with water. Aliquot (0.1 ml) taken in test tube and volume was made up to 1 ml with distilled water. Distilled water (1 ml) in another test tube was also taken as blank. Anthrone (4 ml) reagent (200 mg anthrone was dissolved in 100 ml of ice cold 95% H
2SO
4) was added to the tube and heated for 8 min in boiling water bath. Then the tube was cooled and the intensity of the green colour was read at 630 nm in spectrophotometer. The amount of soluble sugar was determined from the standard curve of glucose and expressed as mg/g dry weight.
Crude fiber
Crude fiber estimation was carried out according to the method described by
(Maynard, 1970). 2g of sample was extracted with ether or petroleum ether to remove fat and boil 2 g of dried sample with 200 ml of H
2SO
4 for 30 min and then filtered through muslin cloth and washed with boiling water until washing was free of acid. The residue was boiled with 200 ml of NaOH for 30 min and filtered through muslin cloth again and washed with 25 ml of boiling H
2SO
4, three 50 ml portion of water and 25 ml alcohol. The residue was removed and transfered into pre-weighed ash dish. Then the residue was dried for 2 h at 130±20°C, cooling a desiccator and weighed. Ignited for 30 min at 600±150°C then cooled in a desiccator and reweighed.
Crude fiber content was calculated by the following formula:
Where,
W1 = Weight (g) of residue with pre-weighed ash dish.
W2 = Weight (g) of dried residue with ash dish.
W3 = Weight (g) of ignited with ash dish.
Crude fat
Fat content was determined by using the soxhlet apparatus as described by the
(Sadasivam and Manickam, 1992). Cowpea powder samples of 2 gm were weighed in a thimble and then placed it in the soxhlet apparatus. Connected a dry pre-weighed solvent flask beneath the apparatus and added the required volume of solvent (petroleum ether or ethyl ether or hexane) and connect the condenser. The heating rate was adjusted to give a condensation rate of 2-3 drops and extracted for 16 hr. The thimble was removed and the solvent along with dissolved fat was preserved. The ether from solvent flask was evaporated on the hot water bath and then the flask was dried at 105°C for 30 min. Flask was then cooled in desiccator and weigh.
Crude fat content was calculated by the following formula.
Where,
a = Weight (g) of dry pre-weighed solvent flask.drying.
Antioxidant activity (DPPH)
The total antioxidant activity was determined using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging assay by
Sombie et al. (2018). DPPH solution (0.004% w/v) was prepared in 95% methanol. The crude extracts of seeds were mixed with 95% methanol to prepare the stock solution (10 mg 100 ml
-1). The concentration of extract solution was 10 mg 100 ml
-1. From stock solution 2 ml, 4 ml, 6 ml, 8 ml and 10 ml of this solution were taken in five test tubes and by serial dilution with methanol and was made the final volume of each test tube up to 10 ml whose concentration was then 20 μg ml
-1, 40 μg ml
-1, 60 μg m
-1, 80 μg ml
-1 and 100 μg ml
-1 respectively. Freshly prepared DPPH solution (0.004% w/v) was added in each of these test tubes containing extract of cowpea seed sample (20 μg ml
-1, 40 μg ml
-1, 60 μg ml
-1, 80 μg ml
-1 and 100 μg ml
-1 ) and after 10 min, the absorbance was taken at 517 nm using a spectrophotometer. Ascorbic acid was used as a reference standard and dissolved in distilled water to make the stock solution with the same concentration (100 μg ml
-1) of extract of tissues. Control sample was prepared containing the same volume without any extract and reference ascorbic acid. 95% methanol was used as blank. The means of three values were obtained, expressed as mg of ascorbic acid equivalent per 100 g of dry seeds weight (mg AAE/100 g seeds dw).
Moisture content
Moisture was determined by the method of
AOAC (1965). Clean and dried empty dishes with open lid were kept at oven at 105°C for 3 hours and after that transfer to the desiccator for cooling. Weigh the empty dish and lid. Weigh about 3 g of sample to the dish and spread the sample uniformly and place the dishes in oven for drying at 105°C for 3 hours. After drying transfer the dishes with partially covered lid to the desiccator to cool. Reweigh the dishes and its dried sample.
Moisture content was analysed by the following formula.
Where,
W1 = Weight (g) of sample before drying.
W2 = Weight (g) of sample after drying.
