CHAPTER THREE

MATERIALS AND METHODS

3.1Materials

The following materials were used in this research work;

I. Laboratory Oven (Jenlab): This was used for drying the sliced pumpkin fruits at different

drying temperature.

II. Weighing Balance (Sf-400): It was used for measuring the weight of pumpkin fruits before

and after drying and milling.

III. Table Knife: It was used to peeling and slicing of fruits.

IV. Multifunctional vegetable slicer: To cut pumpkin into uniform thin slices for drying.

V. Digital Vernier caliper: For measuring the thickness of sliced pumpkin fruit.

VI. Blender (Gasa blender): To grind dried pumpkin slices into powder (flour).

VII. Sieve: To separate pumpkin flour into different particle sizes.

VIII. Mortar and Pestle: For manual grinding of samples.

IX. Transparent PE polythene: To store pumpkin flour samples.

3.2 Methods

3.2.1Material Sourcing

The raw material used in this research work is pumpkin (cucurbita pepo.). The pumpkin was

purchased from a local market at Ekiti State, Nigeria.

3.2.2 Sample Preparation

Mature, healthy pumpkins (plate 3.1) free from disease or pest damage were selected and washed

thoroughly to remove any dirt, pesticides, or other contaminants. The pumpkin fruit was weighed

using a digital weighing balance after which it was sliced longitudinally (3.2) to remove the seed by
using a table knife. The weight of the seeds, weight of peel, weight of fruit before drying, weight of

fruit after drying and weight of flour was known. A multifunctional slicer was used in cutting the

fruit into different thickness. A vernier caliper was used to measure the thickness of each sliced

samples. Each thickness Of 100 pieces of the sample was measured and the average was found.

1019 g of sliced sample were dried at a temperature of 40 0c – 600c at 50c interval by using laboratory

oven (Jenlab). Plate 3.3, 3.4, 3.5, 3.6, 3.7shows dried uncooked pumpkin fruit at 400c – 600c.

The initial moisture content of the sliced samples before drying and after drying was determined

using equation 3.1.

HHGFC
Mw
[Link] = ×100
M

(3.1)

Where:

[Link] is moisture content (wet basis) (%)

Mw is mass of product after drying (g)

M is total mass of the product (g)

] Each of this experiment was replicated thrice for each of the drying temperature. The dried sample

at different temperature levels were subjected to milling by using Gasa blender. Plate 3.8, 3.9, 3.10,

3.11, 3.12 shows dried uncooked pumpkin flour at 400c – 600c.

3.2.3 Physical Properties Determination

The physical properties such as texture, flowability, odour, solubility, segregation, taste, swelling

capacity was determined. The physical properties was determined using an assessment method in

which a structural questionnaire (Appendix 1) was administered to a 5 man panel. The colour for

each dried sample were determined using colour picker and the swelling capacity was determined

using equation 3.2.

 W s−W d
S . C =
Wd

(3.2)

Where:

S.C is swelling capacity

Ws is weight of the dry sample (g)

Wd is weight of the swollen (hydrated) sample after soaking (g)

3.2.4 Chemical Properties Determination

The proximate composition, mineral composition, nutrient composition and vitamin composition of

the milled samples were determined at Abuad laboratory.

[Link] Proximate Composition of milled Samples

Moisture content

2.0g of the sample(s) were placed in an oven maintained at 100 - 103°C for 16 hours with the weight

of the wet sample and the weight after drying noted. The drying was repeated until a constant weight

was obtained. The moisture content was expressed in terms of loss in weight of the wet sample.

Ash content

2.0g of each of the oven-dried samples in powder form were accurately weighed and placed in

crucible of known weight. These were ignited in a muffle furnace and ashed for 8 hours at 550°C.

The crucible containing the ash was then removed, cooled in a dessicator and weighed and the ash

content expressed in term of the oven-dried weight of the sample.

Protein

The protein nitrogen in 1g of the dried samples were converted to ammonium sulphate by digestion

with concentrated H2SO4 and in the presence of CuSO 4 and Na2SO4. These were heated and the

ammonia evolved was steam distilled into boric acid solution. The nitrogen from ammonia was

deduced from the titration of the trapped ammonia with 0.1M HCl with Tashirus indicator (double
indicator) until a purplish pink color was obtained. Crude protein was calculated by multiplying the

value of the deduced nitrogen by the factor 6.25mg. The protein composition was determined using

equation 3.3

Protein = crude protein x 6.25mg

(3.3)

Crude fibre

2.0g of each sample was weighed into separate beakers, the samples were then extracted with

petroleum ether by stirring, settling and decanting 3 times. The samples were then air dried and

transferred into a dried 100ml conical flask. 200cm 3 of 0.127M sulphuric acid solution was added at

room temperature to the samples. The first 40cm 3 of the acid was used to disperse the sample. This

was heated gently to boiling point and boiled for 30 minutes. The contents were filtered to remove

insoluble materials, which was then washed with distilled water, then with 1% HCI, next with twice

ethanol and finally with diethyl ether. Finally the oven-dried residue was ignited in a furnace at

5500C. The fibre contents were measured by the weight left after ignition and were expressed in term

of the weight of the sample before ignition.

Lipid content

The lipid content was determined by extracting the fat from 10g of the samples using petroleum

ether in a soxhlet apparatus. The weight of the lipid obtained after evaporating off the petroleum

ether from the extract gave the weight of the crude fat in the sample.

Carbohydrate

The carbohydrate content of the samples were determined as the difference obtained after subtracting

the values of protein, lipid, ash and fibre from the total dry matter.

[Link] Mineral Composition Determination

The mineral content of the samples such as calcium, potassium and sodium was determined. The ash

of each sample obtained was digested by adding 5ml of 2 MHCL to the ash in the crucible and

heated to dryness on a heating mantle. 5 ml of 2 MHCL was added again, heated to boil and filtered

through what man No. 1 filter paper into a 100ml 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(PFP7Model) using the filter corresponding to

each mineral element (AOAC, 2025).

Iron content determination (Fe,[Link],Mn,Zn etc)

The iron content of the sample was determined using aas bulk scientific 211 vgp. The digest of the

ash of each sample above as obtained in calcium and potassium determination was washed into

100ml volumetric flask with distilled water and made up to mark. These diluents were aspirated into

the Buck 211 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

[Link] Anti –Nutrients Determination

Tanins, Phytate, and oxalate content of the samples were determined as the anti-nutrient content of

the samples.

Determination of tanins

Tannin was extracted into boiling distilled water for one hour. Colour development was done with

Folin-Dennis reagent and sodium carbonate solution. Absorbance was measured at 750 nm

spectrophotometrically. The tannic acid concentrations were calculated from the tannic acid standard

curve.

Determination of phytate

This was determined using McCance-Widdowcon method as modified by (Wheeler and Ferrel). 2 g

of the samples were extracted with 3% Triochloroacetic acid (TCA) and was precipitated with 4 ml
of ferric chloride solution. The precipitated ferric phytate was converted to ferric hydroxide with 4

ml 1.5M sodium hydroxide each and was then dissolved in hot 40 ml 3.2M HN03 and then diluted

with 20 ml of 1.5M KSCN. The phytate was determined colorimetrically.

Determination of oxalate

This was determined using Dye method. 2 g each of the samples was extracted with dilute HCl, 10ml

concentrated ammonia and then precipitated with calcium chloride as calcium oxalate. The

precipitate was then washed with 25 ml of hot 25% H 2SO4 and dissolved in hot water and titrated

with 0.05M KMnO4 to determine the concentration of oxalate.

[Link] Vitamin Composition Determination

The vitamins such as vitamin A, B1, B2, B3, B5, and C were determined for the samples.

Determination of vitamin A (Beta carotene)

2g of the sample was weighed into a 250 ml volumetric flask, 50 ml of petroleum ether: Acetone

(2:1v/v) mixture was added to the extract the ß-Carotene. The flask containing the mixture was

placed on a shaker to shake at 200 rpm for 20 min to ensure uniform mixing at room temperature.

The mixture was later centrifuged at 4000 rpm for 10 min and the supernatant collected and made up

to 50 ml with the solvent mixture. The supernatant was transferred to a 250 ml separatory funnel to

separate the organic layer (upper layer). The aqueous layer was discarded and the organic layer was

transferred into the 50 ml volumetric flask and made up with solvent mixture for reading of ß-

carotene. Working standard of ß-carotene of range 0-50 ppm or /ml were prepared from stock Beta

carotene solution of 100 ppm concentration. The absorbances of samples as well as working standard

solutions were read on a Cecil 2483 UV Spectrophotometerat a wavelength of 450 nm against blank.

Vitamin A was calculated using equation 3.4

μg Absorbance of sample x Average Gradient x Dilution Factor

B-carotene ( ) =
100 g 1000

(3.4)

Determination of vitamin B1 (Thiamine)

Procedure: 1 g of each sample was weighed into 100 ml volumetric flask; 25 ml of 0.1M H 2SO4 was

added and mixed by careful swirling. Additional 25 ml of 0.1M H 2SO4 was added to rinse off any

adhering sample particle off the flask. The flask was set in a boiling water bath to ensure complete

dissolution of the sample in the acid. The flask was shook frequently in the first 5 minutes and

subsequently every 5 minutes for 3 minutes. 5 ml of taka diastase in 0.5M Sodium acetate solution

was added and flask was set in cold water to cool content below 50 0C. The shaking of the flask was

stopped and kept at 45 - 50ᵒC for 2 hours and thereafter made up to 100 ml mark after mixing

thoroughly. The mixture was then filtered through a No. 42 what man filter paper. The first 10 ml

was discarded and the remaining kept. 10 ml of the remaining mixture (filtrate) was pipette into a 50

ml volumetric flask and 5ml of acid potassium chloride solution was added and shook thoroughly to

mix well. Standard thiamine solution of range 10mg/ml to 50mg/ml was prepared from 100mg/ml

stock and treated the same way prepared from the sample above. The absorbances of the sample as

well as that of standards were read on a fluorescent UV spectrophotometer (Cecil A20 model) at a

wavelength of [Link] B1 in mg/100g was calculated using equation 3.5

mg Absorbance x Average Gradient x Dilution factor

Vitamin B1 ( ) =
100 g Weight of sample

(3.5)

Determination of vitamin B2 (Riboflavin)

1g of each sample was weighed into a 250 ml volumetric flask; 5 mls of 5 NHCL was added,

followed by the addition of 5 mls of dichloroethene. The mixture was shook and deionized with

water. The whole sample was shook thoroughly and heated on a steam bath for 30 minutes to extract

all the Riboflavin. The mixture was cooled and made up to volume with deionized water. It was

filtered, discarding the first 20 ml of the aliquot. 2 ml of the filtrate obtained was pipette into another

250 ml volumetric flask and made up to the mark with deionized water. Standard solution was

prepared by dissolving 0.05 mg Riboflavin into 100 mls of distilled water. Different standard

solution of concentration of between 0 to 5 ppm was prepared from the above to obtain the
equivalence. The absorbance, the standards and samples was read on the Fluorescent

Spectrophotometer at 460nm wavelength. The amount of vitamin B2 in the samples was calculated

using equation 3.6

mg Meter reading x Standard X Dilution of sample x Eqiv . Factor

Vit. B2 ( ) =
100 g Weight of sample

(3.6)

Determination of niacin or nicotinic acid (vitamin B3)

5 g of sample was blended and 100ml of distilled water added to dissolve all Nicotinic acid or Niacin

present.5 ml of this solution was drawn into 100 ml volumetric flask and make up to mark with

distilled water. 10 – 50 ppm of Niacin stock solution was also prepared. The absorbance of the

diluted stock solutions and sample extract were measured at a wavelength of 385 nm on a

spectrophotometer. Different concentrations of standard stock solutions were read on the

spectrophotometer for absorbance at the specified wavelength to obtain the Gradient Factor. The

amount of Niacin in sample was calculated using equation 3.7

Absorbance x dilution X Factor x Stock solution

Mg (100g Niacin) =
10 Gradient

(3.7)

Determination of vitamin B5

2g of sample was added to 25 ml of H 2SO4 (0.1M) solution which was incubated in the oven for 30

mins at 1210C. Then, the contents were cooled and adjusted to pH value of 4.5 with 2.5 M Sodium

acetate and 50 mg takadiastase enzyme was added. The preparation was stored at 350 0C overnight.

The mixture was then filtered through a whatman No 4 filter paper andNthe filtrate was diluted with

50 ml of distilled water and filter again. Quantity of vitamin B5 was accomplished by comparison

with vitamin B5 standards using UV Spectrophotometer T60U PG at 270 nm.

Determination of vitamin B9 (Folate)

1g of each sample was weighed into a 250 ml volumetric flask. 100 ml of distilled water was added

and spinned or shaken for 45min and made up to mark with distilled water. The sample mixture was

filtered into another 250 ml beaker, rejecting the first 20 mls that has been filtered. Another 20 mls

filtrate was collected to the filtrate, 5 mls of 1% Sodium Dithionite solution were added to decolorize

the yellow color. Standard Folic Acid of range 0-10ug/ml was prepared from stock Folic Acid. A

sample blank made up to distilled water was also prepared. The absorbances of sample as well as

standard were read at a wavelength of 445nm on a spectronic 21D spectrophotometer. Equation 3.8

was used to determine the vitamin B9 content.

Vitamin B9 =Absorbance of sample x Gradient Factor x [Link]/ Weight of sample (3.8)

Determination of ascorbic acid (Vitamin C)

10 g of the sample was blended and homogenized with 50 ml of 50% metaphoshonic acid-acetic acid

solution. The mixture was quickly transferred into a 100 ml volumetric flask and shaken gentle until

a homogenous dispersion was obtained then it was diluted up to mark with 5% metaphoric acid-

acetic acid solution. The mixture was filtered and the clear filtrate was stored in another 100 ml

volumetric flask prior to spectrophotometric assay. Few drops of bromine water were added to the

filtrate above until the solution became colored so as to confirm the completion of the oxidation of

the ascorbic acid dehydroascorbic acid. This was followed the addition of few drops of thiourea to

remove excess bromine to give a clear color less solution. A working standard of ascorbic acid of

range 10-15 μg/ml ascorbic acid, standard solution 5 ml of 2, 4-dinitrophenyl hydrazine solutions

was added thoroughly to the sample extracts and working standards to give red color after standing

for 5 mins. The absorbance of working standards as well as sample extract were read on Cecil 2583

spectrophotometer at a wave length of 512 nm. The vitamin C content was determined by using

equation 3.9
 mg Absorbance of sample X gradient factor X dilution factor
Vit. C ( ) =
100 g Weight of sample taken

(3.9)

3. 3. Data Analysis

Inferential analysis with the aid of IBM SPSS 25 version 13 was used to analyse the data collected.

Analysis of variance was carried out to know whether there was any significant difference between

the proximate, mineral, vitamin and the anti-nutrient composition of uncooked pumpkin flour at the

different drying temperature at 95% confidential interval.

3. 4. Bill of Engineering Measurement and Evaluation (BEME)

Table 3.1 Bill of Engineering Materials

SN Materials Quantity Unit cost Total cost

1 Pumpkin fruit 13 1000 13000
2 Moisture content 5 3000 15000
3 Ash content 5 3000 15000
4 Fat content 5 3000 15000
5 Crude fiber 5 3000 15000
6 Protein content 5 3000 15000
7 Carbohydrate 5 3000 15000
8 Sodium 5 300 1500
9 Calcium 5 300 1500
10 Potassium 5 300 1500
11 Iron 5 300 1500
12 Tannins 5 3000 15000
13 Phytate 5 3000 15000
14 Oxalate 5 3000 15000
15 Vitamin A 5 3500 17500
16 Vitamin B1 5 1100 5500
17 Vitamin B2 5 1100 5500
18 Vitamin B3 5 1100 5500
19 Vitamin B5 5 1100 5500
20 Vitamin B9 5 1100 5500
21 Vitamin C 5 1500 7500
Total 206,500