The various materials which were utilized in this investigation and the experimental procedure adopted are discussed under the following heads and sub heads: 3.1 Procurement of raw materialCowpea and refined wheat flour for the research work were procured from local market, Pantnagar, Uttarakhand. Apart from these other raw materials used in the preparation of biscuits viz. castor sugar, vegetable oil (dalda), eggs, baking powder, vanilla essence and sodium bicarbonate were purchased from the local market of Pantnagar and sodium bicarbonate was procured from the local bakery of Pantnagar.3.2 Cleaning and milling of cowpea seedsFor cleaning, cowpea were sun dried for one day and manually cleaned to remove stones, grit, chaff and other impurities. After sun drying, the seeds were roasted in oven at 120ºC for 2 hours in order to remove its anti-nutrients before milling (Udensi et al., 2007). Roasted seeds were then milled in flour mill to give finer flour. Cowpea flour thus prepared was used for making biscuits in the Phoolbagh bakery, Pantnagar.3.3 Selection of biscuitsEqui-calorie biscuits providing about 500 Kcal of energy/day/child were chosen for supplementation purposes in case of pre-school children. As a result, 100g each of refined wheat flour biscuits and cowpea biscuits with 70 per cent cowpea flour incorporation were used for supplementation each day each child. Both the biscuits were produced assuming the yield of biscuits to be double the weight of major raw ingredient i.e. flour. Biscuits were prepared with cowpea and refined wheat flour as per recipe of Whitley (1970) given in Appendix- I, with some modifications.3.4 Formulation of biscuitsBiscuits were prepared at Phoolbagh bakery every 15 days for 3 months in 6 lots throughout supplementation period. The creaming method was used to make biscuits, in which ghee and sugar were creamed first and then flour, liquid etc. were added to make dough of rolling consistency (Raina et al., 2002).The basic recipe which was used in present investigation for biscuits preparation is given below:Table 3.1: The recipe used for 100 g biscuit preparation
Raw ingredients Amount   Blend (cowpea + refined wheat flour)                                             64 g   Sugar 32g   Hydrogenated fat (Dalda)                               32g   Egg 1/2 number   Sodium bicarbonate 0.5g   Baking powder 0.3g   Vanilla 0.2ml   Water As per consistency  MethodA known weight of sugar, hydrogenated fat (Dalda) and vanilla flavour were mixed at medium speed until fluffy, then composite flour (cowpea + refined wheat flour), baking powder, sodium bicarbonate and eggs were slowly added into the mixture and mixed until a uniform smooth paste was obtained. The dough was uniformly spread over the board to a thickness of about 2 cm using a rolling pin. Circular biscuits of about 4 cm in diameter were cut using a circular cutter. The biscuits were placed on a baking mesh and baked at 200°C for 10-15 minutes in furnace (tandoor).The baked biscuits were cooled to room temperature and stored in sealed polyethylene sachets of appropriate thickness and permeability.3.5 Sensory evaluation of biscuitsBoth types of biscuits were evaluated for sensory quality characteristics on a nine point hedonic scale and score card (Amerine et al., 1965).  Hedonic rating relates to pleasurable or unpleasurable experiences. The hedonic test is used to measure the consumer acceptability of food products. All samples were evaluated by 15 semi trained panelist from the Department of Foods and Nutrition, Pantnagar.
Plate 1: Biscuits with 70 per cent cowpea flour incorporation
Plate 2: Refined wheat flour biscuits
Both the biscuits were taken up for chemical analysis and supplementation.3.6 Chemical analysis of biscuitsThe chemical analysis of 70 per cent cowpea incorporated biscuit and 100 per cent refined wheat flour biscuits were done in triplicates. 3.6.1 Proximate compositionThis include the determination of the percentages of moisture, total ash, crude protein, crude fat, crude fiber, carbohydrate by difference and energy (Kcal) in biscuits. MoistureMoisture content was determined as per AOAC (1995) procedure. Two gram of powdered biscuit sample was taken in a clean, dried (at 130±3⁰C for 20 min) and pre weighed aluminum dish. The content was dried in oven at 130±3⁰C for 1 hour till a constant weight was obtained and cooled in desiccators. After cooling, the loss in weight was taken as moisture content and expressed in terms of percentage.                                     Where,W1      =    Weight of empty aluminum dish                                   W2      =   Weight of aluminum dish + sample before drying                            W3    =   Weight of aluminum dish + sample after drying Total ash The ash content was determined using AOAC (1995) method. Five gram powdered biscuit sample was weighed in a pre-dried silica dish and ignited on hot plate till the fuming ceased and then was cooled in desiccators, and weighed soon after reaching room temperature. The dish was then transferred to a muffle furnace at 550⁰C until light grey ash results, and subsequently cooled in desiccator. The weight of the residue was then noted and the per cent ash was calculated as follows:           W1 = Weight of silica dish (before incineration)           W2 = Weight of silica dish + Weight of sample (after incineration)          W = Weight of sample3.6.1.3 Crude proteinCrude protein content was determined by the Kjeldahl method of AOAC (1995). Two gram powdered biscuit sample was digested with 10 g of digestion mixture (potassium sulphate and copper sulphate in the ratio 96:4), and 25 ml of concentrated sulphuric acid. The contents were then digested till a carbon free liquid was obtained and clean light green colour was obtained. The volume of the digested material was made up to 100 ml with distilled water. A 20 ml aliquot of digested sample was distilled with 40 per cent sodium hydroxide solution for 15-20 minutes. The ammonia liberated was collected in a conical flask containing 25 ml of 4 per cent boric acid added with few drops of mixed indicator (bromocresol green and methyl red in the ratio 2:1), and the distillate was then titrated against 0.1N H2SO4 until the end point (light pink colour) was reached. Blank determination was done by taking sucrose in place of sample.Nitrogen content in the sample was calculated using the following formula:  Protein per cent = Nitrogen per cent x Conversion Factor (6.25) Crude fatCrude fat was estimated by A.O.A.C. (1995) procedure. Two gram of powdered biscuit sample was transferred to a thimble and extraction was carried out for 16 hrs in Soxhlet extraction assembly using petroleum ether with a boiling range of 40-60º C. The extracted fat and petroleum ether was collected in a pre weighed glass beaker. The petroleum ether was removed by evaporation. Per cent crude fat was calculated as follows: Crude fibreCrude fibre determination was done as per the method described in AOAC (1995). The residue (2g) from crude fat determination was taken and transferred in a spoutless beaker containing 200 ml of 1.25 percent H2SO4 and boiled for 30 min. After 30 minutes, the beaker was removed and the solution was filtered through Whatman No. 54 filter paper and the residue washed with hot distilled water. The residue was then boiled in 1.25 per cent sodium hydroxide solution for exactly 30 minutes. After 30 min of boiling, the contents were filtered through Whatman No. 54 filter paper and washed with hot distilled water using Buchners funnel under gentle suction. The filter paper with the residue was dried in oven at 105⁰C for 3 to 4 hours or till constant weight. It was cooled in a dessiccator and weighed. The loss in weight represented the crude fiber content. It was calculated using the following expression:                   Where,                 W1   = Weight of filter paper (g)                              W2   = Weight of residue + filter paper (g) Carbohydrate by differenceThe carbohydrate content was determined by difference i.e., by subtracting the sum of the values (per 100 g) for moisture, total ash, crude fat, crude fibre and crude protein from hundred.  Physiological energyThe physiological energy (Kcal/100g) of sample was calculated by summing up the product of multiplication of percent crude protein, crude fat and carbohydrate present in the sample by 4, 9, and 4 respectively.3.6.2 Estimation of minerals   Iron and ZincIron and Zinc were estimated using atomic absorption spectrophotometer.Ash solution was prepared using wet ashing procedure as described by Raghuramulu (2003). Three gram of dry ground sample was placed in a 100 ml Kjeldahl flask and 25 ml of the mixture of nitric acid, perchloric acid and sulphuric acid (3:2:1) were added and mixed well to ensure that no dry sample lump remains. It was left aside for 3-4 hrs or preferably overnight in a fume cupboard. Then it was heated for about 30 min cautiously until the initial vigorous reaction has subsided (dense yellow fumes will evolve).Heating was continued for four hrs until most of the nitrous fumes were ceased off and the white fumes of perchloric acid evolved. The contents were cooled and then transferred quantitatively with 3-4 washings of deionised water to the 15 ml graduated test tube and the contents were then transferred to 100 ml volumetric flask to made the volume up to 100 ml with deionised water and mixed thoroughly. Ash solution was stored in a cool place prior to mineral analysis. Blank determination was done with each set of samples.Stock solution 1mg/ml: One gram mineral equivalent of mineral salt was dissolved in a minimum amount of HCl (about 10 ml). It was then diluted to 1liter with deionised water.Working standard solution: Aliquots of the stock solution were diluted with deionised water to make at least 4 working standard solution of each mineral within the range of determinations procedure.Representative sample in a suitable liquid form was sprayed into the flame of an atomic absorption spectrophotometer and the absorbance or emission of the mineral to be analyzed was measured at specific wavelength. Depending upon the mineral to be determined, standards and sample solutions, (wet digestion in this case) were aspirated to the flame after suitable dilutions to attain working range of instrument. The optimum operating conditions recommended by the instrument manual were used. At least 3-4 ranges of standard solution were read before and after sample reading. Burner was flushed with deionised water between samples and checked for zero setting. Calibration curve were prepared from the readings of the standards. Concentrations of samples were determined from standard curve.Calculation: 3.7 Cost calculation of formulated biscuitsCost of the formulated biscuits was calculated on the basis of the cost of raw ingredients, at which they were purchased from the local market, as well as the cost of production of biscuit i.e. cost of electricity, water, labour and packaging, which was Rs. 22.50/per kg of biscuit in our case. The market cost of raw ingredients used in the preparation of biscuits is given in Table 3.2.Table 3.2: Market cost of main raw ingredients (per 100 g) used in the preparation of dough
Raw ingredients Cost (Rs. per 100 g)   Cowpea grain 6.4   Refined wheat flour 1.8   Sugar 3.8   Dalda 6.0   Eggs 6/egg   Baking powder 23   Vanilla essence 10  3.8 Randomized control trial to study the effect of cowpea biscuit supplementation on nutritional status of preschool children
3.8.1 Locale and period of StudyThe research was carried out in the School of Govind Ballabh Pant University of Agriculture and Technology, Pantnagar, Udham Singh Nagar, Uttarakhand. Out of 3 schools of Pantnagar where pre-schoolers were available Saraswati Shishu Mandir had been selected. The study was carried out for three months from February 12, 2011 to May 12, 2011.3.8.2 Selection of subjectsFirst of all written consent (Appendix- II) was obtained from principal of Saraswati Shishu Mandir to assess anthropometry of children. A total of 86 children were examined for the presence of malnutrition by weight for age criteria using Gomez classification (Appendix- IV). 60 children were selected for study who met the criteria of preschool age and presence of malnutrition according to weight for age. After selection of subjects, written consent was drawn from parents of the subjects before intervention and the procedure adopted in present investigation was explained to the parent or guardian (Appendix- III).A total of 60 malnourished pre-schoolers selected from the school to form the sample subjects. Out of total sampled children, three groups of each 20 children were randomly allocated to three groups namely Control I, Control II and Experimental group, to whom no biscuits, refined wheat flour biscuits and 70 per cent cowpea incorporated biscuits were fed respectively. Before starting intervention deworming process of three groups was done by using Albandazole (prescribed by Medical Officer incharge of University). 3.8.3 Background information of children and parentsInformation on child’s date of birth, its father name and their home address was collected from school record. Information regarding the general profile of families, such as socio- economic status, type of family, family size, food habits, income profile, type of house, education and occupation of parents were collected from the mother by visiting the homes of subjects.3.8.4 Anthropometric measurementsAnthropometric measurements viz. height, weight and MUAC of each subject were recorded before supplementation and at every month supplementation for three months of each of three groups. HeightA vertical anthropometric rod with the least count of 0.1 cm was used for measuring height of subjects. Subject was asked to stand erect and look straight in front with head, shoulder blades, buttock and heels in vertical plane touching the measuring rod. Footwear and headwear were taken off before taking measurement (Gibson, 1990).WeightWeight was recorded in kilograms using digital weighing balance with minium divison of 0.5 Kg. Machine was placed on leveled surface and when it showed zero, subjects were asked to stand straight, erect, relaxed with minimum clothing and without footwear (Gibson, 1990).Mid Upper Arm Circumference (MUAC)Fiber glass tape was used for arm circumference measurement with the minimum count of 0.1 cm. the left arm was measured while hanging relaxed. The measurement was taken at the mid way between the tip of the acromion process of the scapula and the olecranon process of the ulna. While taking measurement, tape was placed gently, but firmly around the limb to avoid compression of the soft tissues (Gibson, 1990).3.8.5 Dietary assessmentIn the present study, dietary intake of children was determined by 24- hour recall method. The mother was asked to recall the child’s exact food intake during the previous 24- hour period. She was also asked to tell the cooked amount of the food in terms of standard measuring cups or number. Number of the persons taking the meal was recorded. Major nutrients such as protein, fat, carbohydrate, energy, crude fiber, iron, calcium, vitamin C and vitamin A value were calculated using the food composition table (Gopalan et al., 1989). Average dietary intake of preschool children was thereafter compared with ICMR (1990) recommendations.3.8.6 Food Frequency Questionnaire (FFQ)FFQ contained the frequency with which certain food items or food groups were consumed by subjects during specified time period i.e. daily, twice/weekly, weekly frequently and monthly.  3.9 Supplementation using cowpea and refined wheat flour biscuits
Daily serving of 100g cowpea biscuits and refined wheat flour biscuits were given to experimental and control- II group. Each supplemented group received ten biscuits comprising the weight of 100 grams, five in the morning at 9 o’ clock and five in the mid morning at 11 o’ clock. No supplementation in control group- I.3.10 Effect of supplementation on nutritional statusSupplementation effect on nutritional status in term of height, weight and MUAC of the children was assessed after each supplementation i.e. after every month supplementation of three groups for three months. Plate 3: A glimpse of location of study


Plate 4. Weight measurement   Plate 5. Height measurement

Plate 6: Control- I Group (No supplementation)


Plate 7: Control- II Group (Refined wheat flour biscuits)

Plate 8: Experimental Group (cowpea biscuits supplementation)



Plate 9: Subjects having biscuits
3.11 Statistical analysisThe data was analyzed for percentage, mean and standard deviation. Paired ‘t’ test was used to determine the differences in the anthropometric parameters (weight, height and MUAC) at each month for period of three months in three groups. ANOVA was also used to find significant difference in anthropometric parameters among the groups (Snedecor and Cochran, 1967). Chi square test was used to determine the differences in background information of children and parents of different groups.


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