CHAPTER 3 MATERIALS AND METHODS

CHAPTER-3

MATERIALS AND METHODS

3.1 Materials
The seeds of four hybrids of chillies namely; Maha, Wonder King, Sky Red and P-6 will
be purchased from local seed market of Faisalabad and will be sown under plastic tunnels
in Vegetable Area, Institute of Horticultural Sciences, University of Agriculture,
Faisalabad. All standard practices of crops will be performed.
3.2 Methods
The freshly produced fruits of chillies will be picked at horticultural maturity stage; full
green color with hue intensity of (nm) and with uniform size and shape of the fruits. The
pickings will perform at the early in the morning because at noon temperature will
increase sporadically which deteriorates the quality of the fruits. Picking will be
performed manually and fruits will collected in plastic trays. After picking the fruits will
be shifted to vegetable seed lab, Institute of Horticultural Sciences, University of
Agriculture, Faisalabad. The fruits will be cooled to remove the field heat and then
dipped in 50 ppm sodium hypochlorite solution for 5 minutes. After surface sterilization
of fruits will be dried under hot wind dry by fans. Properly drying will be done because
water droplets stayed at the calyx or fruit skin will facilitate the deterioration and decay.
Fully dried fruits will be packed in various polyethylene bags with different thickness or
gauge of the sheets. The gauge was 7, 15 and 25 microns and sized of bags was 20 x 30
cm. Each bag will be contained 250 grams of fruits and after packaging these bags will be
sealed and placed under the surveillance of low temperature of 7, 14 and 21 ˚C. The data
will be collected on following parameters; weight loss, color, internal temperature, pH,
vitamin C and SSC.

3.3 Treatments
1. 7 ˚C
2. 14 ˚C
3. 21 ˚C
3.4 Hybrids
1. Maha
2. Wonder King
3. Sky Red
4. P-6
3.5 Packaging Materials
1. Open or unpackaged one (control)
2. Polyethylene packaging bag with7 μ thickness
3. Polyethylene packaging bag with 15 μ thickness
4. Polyethylene packaging bag with 21 μ thickness

Treatments combinations

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T1 Maha at 7˚C in 21 μ thickness bag

T2 Maha at 7˚C in 15 μ thickness bag
T3 Maha at 7˚C in 7 μ thickness bag
T4 Maha at 7˚C open (control)
T5 Wonder King at 7˚C in 21 μ thickness bag
T6 Wonder King at 7˚C in 15 μ thickness bag
T7 Wonder King at 7˚C in 7 μ thickness bag
T8 Wonder King at 7˚C open (control)
T9 Sky Red at 7˚C in 21 μ thickness bag
T10 Sky Red at 7˚C in 15 μ thickness bag
T11 Sky Red at 7˚C in 7 μ thickness bag
T12 Sky Red at 7˚C open (control)
T13 P-6 at 7˚C in 21 μ thickness bag
T14 P-6 at 7˚C in 15 μ thickness bag
T15 P-6 at 7˚C in 7 μ thickness bag
T16 P-6 at 7˚C open (control)
T17 Maha at 14˚C in 21 μ thickness bag
T18 Maha at 14˚C in 15 μ thickness bag
T19 Maha at 14˚C in 7 μ thickness bag
T20 Maha at 14˚C open (control)
T21 Wonder King at 14˚C in 21 μ thickness bag
T22 Wonder King at 14˚C in 15 μ thickness bag
T23 Wonder King at 14˚C in 7 μ thickness bag
T24 Wonder King at 14˚C open (control)
T25 Sky Red at 14˚C in 21 μ thickness bag
T26 Sky Red at 14˚C in 15 μ thickness bag
T27 Sky Red at 14˚C in 7 μ thickness bag
T28 Sky Red at 14˚C open (control)
T29 P-6 at 14˚C in 21 μ thickness bag
T30 P-6 at 14˚C in 15 μ thickness bag
T31 P-6 at 14˚C in 7 μ thickness bag
T32 P-6 at 14˚C open (control)
T33 Maha at 21˚C in 21 μ thickness bag
T34 Maha at 21˚C in 15 μ thickness bag
T35 Maha at 21˚C in 7 μ thickness bag
T36 Maha at 21˚C open (control)
T37 Wonder King at 21˚C in 21 μ thickness bag
T38 Wonder King at 21˚C in 15 μ thickness bag
T39 Wonder King at 21˚C in 7 μ thickness bag
T40 Wonder King at 21˚C open (control)
T41 Sky Red at 21˚C in 21 μ thickness bag
T42 Sky Red at 21˚C in 15 μ thickness bag
T43 Sky Red at 21˚C in 7 μ thickness bag
T44 Sky Red at 21˚C open (control)
T45 P-6 at 21˚C in 21 μ thickness bag
T46 P-6 at 21˚C in 15 μ thickness bag

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CHAPTER 3 MATERIALS AND METHODS

T47 P-6 at 21˚C in 7 μ thickness bag

T48 P-6 at 21˚C open (control)

Experiment 1. Sky Red evaluation

Experiment 2. Maha evaluation
Experiment 3. Wonder King Evaluation
Experiment 4. P-6 Evaluation

Parameters
Physical Parameters
1. Length of fruits (cm)
2. Diameter of fruits (cm)
3. Size of fruits (cm2)

Analytical Parameter
1. Weight loss (%age)
2. Color (nm, wavelength)
3. Internal temperature (˚C)
4. pH
5. Vitamin-C (mg/ 100g)
6. SSC (brix)
7. Titratable acidity (%)
8. Total sugar
9. Reducing Sugars
10. Non-Reducing Sugars
11. Specific gravity (mm3)
12. Total phenolic compounds (mg/ g)
13. Total carotenoids (mg/ 100 g)

STATISTICAL ANALYSIS:
The treatments will be arranged in (4 x 3) packaging materials and temperature
Factorial Experiment under Completely Randomized Design (CRD). The mean values of
treatments will be calculated and analyzed statistically by using DMR test analysis of
variance was obtained at 5 % level of significance (Steel et al., 1997).
PHYSICAL PARAMETERS
3.1 LENGTH OF FRUITS
In each treatment ten fruits randomly selected and length of fruits was recorded with the
help of vernier caliper. Length calculated in (cm) and mean of the length calculated with
the help of software Microsoft Excel (Microsoft Office, 2004).

3.2 DIAMETER OF FRUITS

In each treatment ten fruits randomly selected and diameter of fruits was calculated with
the help of vernier caliper. Diameter will be recorded with three days interval in (cm) and
mean of the diameter calculated with the help of software Microsoft Excel (Microsoft
Office, 2004).

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3.3 SIZE OF THE FUITS

Size of fruits was calculated by the formula as written under. The diameter was calculated
at three days interval in the entire period of storage life span. Mean of ten fruits was
calculated in software Microsoft Excel (Microsoft Office, 2004).
Size (cm2) = Length (cm) x Width (cm)

ANALYTICAL PARAMETERS
3.4 WEIGHT LOSS
Percent weight loss of fruits packs was calculated on fresh basis by the formula

Percent weight loss ( % ) = W2 – W1 x 100

W1
Where as:
W1 is the initial weight of the packs stored in cold storage
W2 is the weight of packs after loss of water through evapo-tranpiration from the fruits.

3.5 COLOR OF FRUITS

The color of green hot pepper fruits was calculated use to determine the color changes for
the surface of green hot pepper fruits samples. Color changes were quantified by the
tristimulus color values (L*, a*, b*). L* refers to the lightness of the hot pepper pericarp,
and ranges from black = 0 to white = 100. A negative values of a* indicates green, while
a positive number indicates red-purple color. Positive b* indicates yellow and negative
blue color (McGuire, 1992). The hue angle [h˚ = tan-1(b*/a*)] was calculated from a*
and b* values (Lancaster et al., 1997). The deviation from the raw material color was
represented as ∆E*, and calculated as (L*- L0)2 + (a* - a0)2 + (b* - b0)2, where L0, a0 and b0
were the color parameters of fresh hot pepper fruit. The meter was calibrated without any
treatment. The meter was calibrated using the manufacture’s standard white plate (L* =
95.73; a* = -0.68 and b* = 3.25). With a decrease in L* and hue angle values as well as
the increase of a* and ∆E* values, the color of the hot pepper pericarp became less and
less greening or more and more yellowing. On each fruit pericarp, three readings were
taken at 1, 2 and 3 cm from the tip (Siomos, Dogras and Sfakiotakis, 2001), and the final
presented data is the mean of the three readings.

3.6 INTERNAL TEMPERATURE

Internal temperature was of ten randomly selected fruits was calculated by inserting the
probe of the digital thermometer and reading recorded directly by the LCD of the meter.
Mean of internal temperature of ten fruits was calculated and same process is repeated
after every third day of storage of hot pepper fruits.

3.7 pH
pH of ten randomly selected fruits was calculated by sqeezing a drop of juice of the green
fruit on the digital pH meter sensor. Mean of the fruits was recorded and the same process
is repeated after every third day of the storage of fruits.

3.8 ASCORBIC ACID

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The method described by Ruck (1969) was used for estimation of vitamin C in
juice. For this purpose extracted juice of green hot pepper fruit from each sample was
filtered through Watman No. 1 filter paper. 10ml of filtered aliquot was taken in 100ml
round bottom flask, then volume was made up to the mark by adding 0.4 % oxalic acid.
Out of 100ml aliquot, 5 ml was taken in a beaker and titrated against 2, 6-dichlorophenol
indophenol dye to a light pink color which persisted for 10-15 seconds.

PREPARATION OF DYE
Dye was prepared by adding 42 mg NaHCO3 and 52 mg 2, 6-dichlorophenol
indophenol in a 200 ml volumetric flask. Volume was made up to the mark by adding
distilled water. It was filtered and used as freshly prepared dye.
Vitamin C was calculated as ascorbic acid by using formula:

Ascorbic Acid (mg/100g) = 1 x R1 x V x 100

R x W x V1
Where as
R1 = ml dye used in titration of aliquot.
R = ml of dye used in titration of 1 ml standard ascorbic acid solution prepared
by adding 1 ml of 0.1 % ascorbic acid + 1.5 ml of 0.4 % oxalic acid.
V1 = ml of juice used.
V = volume of aliquot made by addition of 0.4 % oxalic acid.
W = ml of aliquot used for titration.

3.9 SOLUBLE SOLID CONTENTS

Soluble Solids Contents (SSC) was determined using Atago RX 5000 Digital
Refractometer (Atago, Japan). A drop of juice of green hot pepper fruit was extracted and
placed on clean prism of Refract meter and the lid was closed after placing for each
sample. Reading was taken directly from the scale at room temperature.

3.10 TITRATABLE ACIDITY

Acidity was determined by taking 5ml juice in conical flask and adding few drops of
phenolphthalein as an indicator and some distilled water to make end point clear. The
samples were titrated against N/ 10 NaOH solutions (Hortwiz, 1960). The results were
expressed as percent citric acid.

T.A. ( % ) = N/10 NaOH used x 0.0064 x 100

Volume of sample

3.11 SUGARS
To estimate the sugar in juice of each treated sample, the method of Lane and
Eynon (1923) as described by Hortwitz (1960) was used, according to which 10 ml of
juice was taken in 250 ml flask in which 100 ml distilled water, 25 ml lead acetate
solution (430 g/1000 ml) and 10 ml of 20 % potassium oxalate solution added. Volume
was made up to the mark with distilled water and filtered. Then the filtered was used for
the estimation of sugars as follows.
3.12 REDUCING SUGARS.

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The above mentioned filtrate was taken in burette and titrated against 10 ml
Fehling’s solution using 2-3 drops of Methylene blue with continuous boiling till brick
red end point appeared. Reducing sugars were calculated by:
% reducing sugars = 6.25 (X/Y)
Where
X = ml of standard sugar solution used against 10 ml Fehling‘s solution.
Y = ml of sample aliquot used against 10 ml Fehling’s solution.

3.13 NON-REDUCING SUGARS

Non-reducing sugars for juice were estimated with formula:-
= (Total sugars % - Reducing sugars %) x 0.95
3.14 TOTAL SUGARS
For estimation of total sugars 25 ml of aliquot already prepared for reducing
sugars was taken in a 100 ml volumetric flask in which 250 ml distilled water and 5 ml
concentrated HCl was added and solution was kept overnight for converting the non-
reducing sugars into reducing sugars. Then it was neutralized with 50 % concentrated
NaOH solution and volume was made 100 ml with distilled water. That prepared solution
was taken in burette and titrated against 10 ml Fehling’s solution to brick red end point
using Methylene blue as an indicator. Total sugars were calculated by:
% Total sugars = 25 x (X/Z)
where
X= ml of standard sugar solution used against 10 ml Fehling’s solution.
Z = ml of sample aliquot used against 10 ml Fehling’s solution.
PREPARATION OF STANDARD INVERT SUGAR SOLUTION
Dissolve 23.75 g pure sucrose in about 120 ml water in a 250 ml volumetric flask,
add 9 ml concentrated hydrochloric acid and keep it for 8 days at room temperature.
Make volume up to the mark with water. (When inversion is complete, rotation in a 200
mm tube = 11.80° + 0.05° S). Transfer 200 ml into a 2 L volumetric flask, add 200 ml
water and with shaking add 71.4 ml of Sodium hydroxide solution (40 g/L) containing 4
g benzoic acid. Add 1 L water mix and check with indicator paper that the solution is
approximately pH 3. Adjust if necessary and make up to the mark. This produces a stable
1 % m/v stock solution when required. Alternatively add 4 g benzoic acid dissolve in 400
ml of hot water to the 200 ml acid solution containing the inverted sucrose and make up
to the mark. When 0.25 % standard inverted sugar solution is required, neutralize 50 ml
of the acidic stock solution in a 200 ml flask with 1 N NaOH using litmus paper and
make up to the mark. Neutral or alkaline solution of sugar do not keep and should be
prepared when required (Ronald and Sawyer, 1981).

THE LANE AND EYNON TITRATION

Fehling’s 1:- Dissolve 69.3 g Copper sulphate pentahydrate (CuSO4.5H2O) in water and
make up to 1 L.
Fehling’s 2:- Dissolve 100 g Sodium hydroxide and 345 g Sodium potassium tartrate
(KNaC4O6.4H2O) in water and make up to 1 L.

3.15 SPECIFIC GRAVITY

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The specific gravity of hot pepper fruits was determined by the method as followed
(Talburt and Smith, 1959). The weight of 10 cleans fruits of hot pepper from four hybrids
used in the study calculated in air (W1) and completely immersed in a container of water
(W2) was recorded. The specific gravity was calculated as formula:
Specific Gravity = W1 / (W1 – W2)

3.16 TOTAL PHENOLIC CONTENT

The total phenolic content (TPC) was determined by a Folin-Ciocalteu assay (Singleton
& Rossi, 1965) with slight modifications (Xu & Chang, 2007) using gallic acid (GA) as
the standard. The original unprocessed or processed legume extract or processing water
(50 lL), distilled water (3 mL), 250 lL of Folin-Ciocalteu’s reagents solution, and 7%
NaCO3 (750 lL) were mixed in a tube and incubated for 8 min at the room temperature.
Then a dose of 950 lL of distilled water was added. The mixture was allowed to stand for
2 h at the room temperature. The absorbance was measured at 765 nm against distilled
water as a blank. The total phenolic content was expressed as gallic acid equivalents (mg
of GAE/g sample) through the calibration curve of gallic acid. Linearity range of the
calibration curve was 50–1000 lg/mL (r = 0.99).

3.17 TOTAL CAROTENOIDS

Two g of ripe hot pepper fruit pericarp with 0.05 g of (MgCO 3) magnesium
carbonate was grounded and extracted two times with a 20 ml of acetone:n-hexane
(75:60, v/v). The pool extract was washed with a 40 ml of 10 % NaCl and 2 x 40 ml of
distilled water to remove acetone. The hexane extract was measured for its absorbance at
436 nm using spectrophotometer model 640 UV/VIS (Jenway Ltd., Essex, England).
Total carotenoids were expressed as mg/g-1 of β-carotene equivalent from a standard
curve of β-carotene (Lale, 2003).

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REFERENCE:
Hortwitz, W. 1960. Official and tentative methods of analysis. Association of the Official
Agriculture Chemist. Washington, D.C. Ed. 9: 320-341.
Lancater, J.E., C.E. Lister, P.F. Reay and C.M. Triggs. 1997. Influence of pigment
composition on skin color in a wide range of fruit and vegetables. Journal of the
American Society for Horticultural Sciences. 122: 594-598.
Lane, J.H. and L. Eyanon. 1923. Determination of reducing sugars by Fehling’s solution
with methylene blue indicator. J. Soc. Chem. Ind., 42, 32T, 663T.
Ronald, S. and K.R. Sawyer. 1981. Pearson’s chemical analysis of foods. Longman
Scientific an Technical, England, p. 211.
Singleton, V.L. and J.A.Rossi. 1965. Colorimetry of total phenolic with
phosphomolybdic–phosphotungstic acid reagents. American Journal of Enology
and Viticulture. 16: 144–158.
Siomos, A.S., C.C. Dograa and E.M. Sfakiotakis. 2001. Color development in harvested
white asparagus spears in relation to carbon dioxide and oxygen concentration.
Postharvest Biology and Technology. 23: 209-2014.
Talburt, W.F. and O. Smith. 1959. Potato processing. Westport C.T: The AVI Publishing
Company, Inc.
Xu, B.J. and S.K.C. Chang. 2007. A comparative study on phenolic profiles and
antioxidant activities of legumes as affected by extraction solvents. Journal of
Food Science. 72: 159–166.

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