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CHAPTER 3

MATERIALS AND METHODS

The study on various aspects and objectives related to the present research

work was carried out during the year 2009-2013 at Department of Food Processing

Technology, A. D. Patel Institute of Technology, New Vidya Nagar, Anand and

Sophisticated Instrumentation Centre for Applied Research and Testing, Vidyanagar,

Anand.

In the present investigation, selected variety of flaxseed was microwave

roasted to optimize the reduction of cyanogenic glycosides. Roasted flaxseed powder was

used to develop the process technology of extruded product and cookies. Optimized

flaxseed fortified food products were analyzed for their proximate composition and

sensory evaluation. Moreover, the current investigation studied storage effect on flaxseed

fortified products packed in two different packaging films. This chapter deals with the

description of materials, experimental set ups, analytical techniques and processing

techniques used in various experiments.

3.1 Materials

3.1.1 Flaxseed

Flaxseed Variety NL-260 was collected from AICRP Project on Linseed,

College of Agriculture, Punjabrao Krushi Vidyapeeth, Nagpur. The procured flaxseed

were cleaned and stored properly at room temperature (28 to 350C) in air tight container

prior to its use in actual experiment. The cleaned flaxseeds were ground in domestic

mixer for 3 minutes and sieved through 30 mesh sieve. The undersize ground powder of

flaxseed was used in the further experimental work.

3.1.2 Raw materials

For the manufacturing of extruded product, rice flour (Brand Gaay), Salt

(Brand Tata), Black pepper powder (Brand Everest) and corn flour were procured from

local market. For the manufacturing of cookies, commercial refined wheat flour (Uttam

Brand), hydrogenated fat (Raag Brand), Sugar, skim milk powder (Sagar Brand), custard

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powder (Wiekfield Brand), baking powder (Blue Bird Brand) and keshar pista essence

were procured from the local market. Sugar was ground in the domestic mixer and sieved

powder was used.

3.1.3 Chemicals and Reagents

Chemicals used in the analysis were of analytical grade from Merck, S.D.

Fine and Loba Chem companies.

3.2 Methods

3.2.1 Proximate composition of flaxseed

3.2.1.1 Determination of moisture

The sample was weighed in an aluminium dish and allowed to dry in a hot

air oven maintained at 1100C for 4 hours until constant weight achieved. It was cooled in

a desiccators to room temperature. Difference in percentage weight was reported as

moisture (AOAC, 2000).

Moisture % , 𝑤𝑏 = A − B X 100

A − C

............. (3.1)

Where,

A = Initial weight of sample + Aluminium dish before drying

B = Final weight of sample + Aluminium dish after drying

C = Aluminium dish weight

3.2.1.2 Determination of ash content

The sample was weighed into silica crucible and placed in muffle furnace

at 5500C for 4 hours. It was cooled in desiccators and weighed. This was repeated until

two consecutive weights were constant. The percent ash content was calculated by on the

basis of initial sample (AOAC, 2000).

Ash % =(W3−W1) X 100

W2−W1

……….. (3.2)

Where,

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W1 = Weight of silica crucible

W2 = Weight of silica crucible + sample

W3 = Weight of silica crucible + ash

3.2.1.3 Determination of fat

The ground sample was weighed accurately in thimble and defatted with

petroleum ether in Soxhlet apparatus for 6 hours at 700C. The resultant ether extract was

evaporated to remove traces of ether and lipid content was calculated (AOAC, 2000).

3.2.1.4 Determination of protein

It was determined by Micro-kjeldahl method (AOAC, 2000). Weighed

sample was transferred to a digestion flask followed by the addition of 3 g of catalyst

mixture (K2SO4:CuSO4:SeO2 in 100:20:2.5) and 20 ml of concentrated sulphuric acid.

The content was then digested till transparent liquid was obtained. The volume of

digested material was made up to 100 ml with distilled water. Carry out a blank digestion

without the sample and make the digest to 100 ml. Measured aliquot of digested material

was distilled with excess of 40% NaOH solution and the liberated ammonia was collected

in 20 ml of 2% boric acid solution containing 2-3 drops of mixed indicator (10 ml of 0.1

percent bromo cresol green + 2 ml of 0.1 percent methyl red indicator in 95 percent

alcohol). The entrapped ammonia was titrated against 0.01 N hydrochloric acid. A

reagent blank was similarly digested and distilled. Nitrogen content in the samlple was

calculated as follows and a factor of 6.25 was used to convert nitrogen to protein.

% Nitrogen content

= Sample titre – Blank titre X Normality of HCl X 14 X vol. made of digest X 100

Aliquot of the digest taken X Weight of sample X 1000

……… (3.3)

% Protein content = % Nitrogen X 6.25 ……… (3.4)

3.2.1.5 Determination of crude fiber

The sample was weighed into 500 ml beaker and 200 ml of boiling 0.255

N sulphuric acid (1.25 percent w/v) was added. The mixture was boiled for 30 min

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keeping the volume constant by the addition of hot water at frequent intervals (a glass rod

stirred in the beaker helps smooth boiling).

At the end of this period, the mixture was filtered through a muslin cloth

and the residue washed with hot water till free from acid. The material was then

transferred to the same beaker and 200ml of boiling 0.313 N (1.25 percent w/v) NaOH

was added. After boiling for 30 min., the mixture was filtered to a crucible, dried

overnight at 80-1000C and weighed. The crucible was kept at in a muffle furnace at

5500C for 3 hours. Then it was cooled in desiccators and weighed again. The difference

in residue weights and ash represents the weight of crude fiber (Ranganna, 2000).

3.2.2 Fatty acid profile of extracted lipids/oil

Fatty acid profile analysis was carried out by Gas Chromatography (GC)

instrument (Make – Perkin Elmer, Model – Auto System XL) according to the method

suggested by Petrovic et al (2010). Homogenized sample was weighed and dried for 1 h

at 1050C. The sample was cooled in desiccators. Lipids were extracted with petroleum

ether by soxhlet apparatus for 4 h on heating mentle. After extraction, petroleum ether

was evaporated and the flask was dried at 1050C, cooled in desiccators and then weighed.

Lipids were converted to fatty acid methyl esters by trans-esterification. Sample (60 mg)

was dissolved in 4 ml of isooctane in test tube and 200 µl of methanolic potassium

hydroxide solution (2 mol/l) was added. Solution was shaken vigorously for about 30 sec.

The solution was neutralized by addition of 1 g of sodium hydrogen sulphate

monohydrate. After the salt has settled, 1ml of upper phase was transferred into 5 ml vial

and analyzed. Analyses were performed on GC instrument equipped with flame

ionozation detector and spilt/splitless injector. Injection temperature was 1800C and

samples were injected manually (1 µl). Column was packed with 12.5% diethylene glycol

solution. Column temperature 2200C, detector temperature 230

0C and carrier gas nitrogen

at 15 ml/min were maintained. 30 min run time in every analysis was maintained. The

peaks were identified by comparing retention time with those of authentic standards and

represented as relative percentage.

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3.2.3 Estimation of cyanogenic glycosides (CG) content

CG was determined by AOAC official method 915.03 (part B, alkaline

titration method). CG level in food or feed is expressed through content of HCN

equivalents. Sample (20 g) was weighed in a kjeldahl flask. The flask was given rest for

two hours after adding 200 ml water into it. Then, distillation was carried out by the

distillation assembly as shown in Plate 1. The distillate was collected in a flask

containing 20 ml of 2.5% NaOH solution until distilled to a definite volume. Ammonium

hydroxide solution (8 ml of 6M) and 2mL of 5% potassium iodide solution were added to

the distillate. Then the whole contents were titrated against 0.02 M AgNO3 solution.

HCN contents of sample were calculated according to the formula given below.

HCN (mg) = mL of 0.02 M AgNO3 solution used x 1.08 ….. (3.5)

3.2.4 Methodology for optimization of microwave roasting of flaxseed

Response Surface Methodology (RSM) was used to generate the

experimental designs, statistical analysis and regression model with the help of Design

Expert Software Version 8 (Statease Inc.). Kenstar microwave oven (Model OM-34

ECR) was used for treatment of flaxseed samples. Samples were placed in a thin layer on

glass bowl (Inner diameter 20 cm) uniformly distributed. The Central Composite

Rotatable Design (CCRD) with quadratic model (Box and Draper, 1987) was employed.

Two independent variables namely microwave power percent and time of treatment in

seconds were considered. Each independent variable had three levels which were -1, 0

and +1 (Cochran and Cox, 1957). The α-values in the design outside the ranges were

selected for design rotatability (Thompson et al., 1982). A total 13 combinations

(including five replicates of central point each assigned coded value 0) were chosen.

Operating frequency was 2450 mHZ and working power of 40%, 60% and 80%

microwave power corresponding to output of 370W, 580W and 790W respectively were

used. CG content (HCN equivalents) was taken as the response. Numerical optimization

was carried out to derive specific microwave power percent and time of treatment. The

optimized conditions of microwave roasting adopted for reduction in CG. The

experimental design was shown in Table 3.1 in actual and coded levels of variables.

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The coefficients of the polynomial were represented by b0 (Constant

terms), b1, b2 (linear effects), b11, b22 (quadratic effects) and b12 (interaction effects). The

analysis of variance (ANOVA) tables were generated and the effect and regression

coefficients of individual linear, quadratic and interactions terms were determined. The

significances of all terms in the polynomial were judged statistically by computing the F-

value and compared with standard significance level of 0.1%, 1% and 5%. The regression

coefficients were then used to make statistical calculation to generate contour maps from

the regression models.

Table 3.1 Central Composite Experimental Design employed for microwave

roasting of flaxseed

Run No. Microwave Power (%)

X1 (x1)

Time of heating

(seconds)

X2 (x2)

1 40 (-1) 360 (+1)

2 60 (0) 410 (+α)

3 60 (0) 240 (0)

4 60 (0) 70 (-α)

5 60 (0) 240 (0)

6 60 (0) 240 (0)

7 90 (+α) 240 (0)

8 60 (0) 240 (0)

9 30 (-α) 240 (0)

10 60 (0) 240 (0)

11 80 (+1) 360 (+1)

12 40 (-1) 120 (-1)

13 80 (+1) 120 (-1)

The values in bracket i.e. x1 and x2 represent the coded values for respective factors

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Plate 1. Distillation assembly for cyanogenic glycosides (HCN equivalents)

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3.2.5 Methodology for process development of flaxseed fortified rice flour based

extruded product

3.2.5.1 Preparation of extrusion feed

Rice flour was mixed with distilled water to attain the desirable moisture

levels. Corn flour was mixed and sieved the mixture. Flaxseed flour, salt and black

pepper powder were added as per formulations and then again sieved for uniform blend.

Each flour blend was conditioned for 1 h to attain the uniform moisture throughout. Sieve

analysis of extrusion feed was carried out according to Sahay and Singh (2001).

3.2.5.2 Determination of moisture for flours and extrusion feed

Moisture for flours and extrusion feed was analyzed by using Infra red

moisture analyzer (Advance equipment). The desired moisture content levels in the blend

(extrusion blend) were obtained by adding distilled water as calculated using equation

below (Oluwale and Olapade, 2011).

𝑄 =𝐴 𝑀𝑓−𝑀𝑖

(1−𝑀𝑓) ------------ (3.5)

where A = Initial mass of the blend

Mi = Initial fraction of moisture content of the blend (wet basis)

Mf = Desired fraction of moisture content of the blend (wet basis)

Q = Mass of water to be added (kg)

3.2.5.3 Extrusion

Laboratory scale twin screw extruder (M/s Basic Technology Pvt. Ltd.,

Kolkata, India) as shown in Plate 6 was employed for this study . The extruder has two

heating zones with electrical resistance heaters and thermocouple sensor to monitor the

temperature. The extruder barrel is consisting of two co-rotating screws intermeshing

each other. Two screws were 30 mm in diameter and 350 mm long. Thus, the length to

diameter ratio of the screws was 11.66:1. The screw had constant pitch of 20 and 3.5 mm

flight. Double screw volumetric feeding system was used to feed the blend through the

extruder. Parameters i.e. Feeder rpm –25, Second heater temperature - 600C, Cutter RPM

– 55 and Extruder die diameter - 4 mm are kept constant throughout the study. During

each extruder run, the machine was allowed to stabilize for 5-10 min until a stable torque

was achieved. Extrudates were collected on stainless steel plate to allow excess steam

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flash off. The samples were packed in polyethylene bags after cooling and stored at cool

and dry place. Extruded product preparation is shown in Flowchart 1.

3.2.5.4 Experimental Design and Statistical Analysis

Response Surface Methodology (RSM) was used to generate the

experimental designs, statistical analysis and regression model with the help of Design

Expert Software Version 8 (Statease Inc.). The Central Composite Rotatable Design

(CCRD) with a quadratic model (Box and Draper, 1987) was employed. Four

independent variables namely Flaxseed flour replacement (%), Moisture content of

extruder feed (%), Temperature of extruder barrel (0C) and Screw speed were chosen.

Each independent variable had 3 levels which were -1, 0 and +1. A total of 30 different

combinations (including six replicates of the centre point each signed the coded value (0)

were chosen in random order according to a CCRD configuration for three factors

divided in three blocks (Cochran and Cox, 1957). The α- values in the design outside the

ranges were selected for rotability of the design (Thompson, 1982). The experimental

design in the coded (x) and actual (X) levels of variables is shown in Table 3.2.

The responses function (y) measured were Expansion ratio, Breaking

strength, Bulk density and Overall acceptability of extruded product. These values were

related to the coded variables (xi, i = 1, 2, 3 and 4) by a second degree polynomial using

the equation below.

y = b0 + b1x1 + b2x2 + b3x3 + b4x4 + b12x1x2 + b13x1x3 + b14x1x4 + b23x2x3 + b24x2x4 +

b34x3x4 + b11x12 + b22x2

2 + b33x3

2 + b44x4

2 ……. (3.6)

The coefficients of the polynomial were represented by b0 (Constant

terms), b1, b2, b3 and b4 (linear effects), b11, b22, b33 and b44 (quadratic effects) and b12,

b13, b14, b23, b24 and b34 (interaction effects). The analysis of variance (ANOVA) tables

were generated and the effect and regression coefficients of individual linear, quadratic

and interactions terms were determined. The significances of all terms in the polynomial

were judged statistically by computing the F- value and compared with standard

significance level of 0.1%, 1% and 5%. The regression coefficients were then used to

make statistical calculation to generate contour maps from the regression models.

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Table 3.2. Central Composite Experimental Design employed for development of

flaxseed fortified extruded product

Sr.

No.

Flaxseed flour

(%)

Moisture

content (%)

Temperature

(0C)

RPM

X1 (x1) X2 (x2) X3 (x3) X4 (x4)

1. 20(0) 14(0) 130(0) 315(0)

2. 20(0) 14(0) 130(0) 315(0)

3. 20(0) 14(0) 130(0) 315(0)

4. 20(0) 14(0) 130(0) 315(0)

5. 20(0) 14(0) 130(0) 315(0)

6. 20(0) 14(0) 130(0) 315(0)

7. 20(0) 10(-α) 130(0) 315(0)

8. 10(-α) 14(0) 130(0) 315(0)

9. 20(0) 14(0) 110(-α) 315(0)

10. 20(0) 18(+α) 130(0) 315(0)

11. 20(0) 14(0) 130(0) 345(+α)

12. 30(+α) 14(0) 130(0) 315(0)

13. 20(0) 14(0) 150(+α) 315(0)

14. 20(0) 14(0) 130(0) 285(-α)

15. 15(-1) 12(-1) 140(+1) 330(+1)

16. 25(+1) 12(-1) 140(+1) 300(-1)

17. 15(-1) 16(+1) 120(-1) 300(-1)

18. 15(-1) 12(-1) 120(-1) 330(+1)

19. 25(+1) 12(-1) 140(+1) 330(+1)

20. 15(-1) 16(+1) 140(+1) 330(+1)

21. 25(+1) 12(-1) 120(-1) 330(+1)

22. 15(-1) 12(-1) 140(+1) 300(-1)

23. 15(-1) 12(-1) 120(-1) 300(-1)

24. 25(+1) 16(+1) 120(-1) 330(+1)

25. 15(-1) 16(+1) 120(-1) 330(+1)

26. 25(+1) 16(+1) 120(-1) 300(-1)

27. 25(+1) 16(+1) 140(+1) 330(+1)

28. 25(+1) 16(+1) 140(+1) 300(-1)

29. 25(+1) 12(-1) 120(-1) 300(-1)

30. 15(-1) 16(+1) 140(+1) 300(-1)

Values in bracket i.e. x1, x2 x3 and x4 represents the coded values for respective factors

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Flowchart 1. Extruded product process chart

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Plate 2. Twin screw extruder

Plate 3. Extruder die holder and 4 mm Die

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3.2.5.5 Extruded product characteristics

a) Expansion ratio (ER)

ER was measured as the ratio of cross sectional area of the cylindrical

extrudate to that of the die (4 mm) (Chakraborty et al., 2009). The diameter of the

extrudate was the average of ten random measurements. This index describes the degree

of puffing undergone by the sample as it exits the extruder.

b) Bulk density (BD)

Cylindrical section was weighed and the diameter measured using vernier

caliper. The bulk density was then calculated as the ratio of the extrudate to the volume

of extrudate (Barret and Peleg, 1992).

𝐵𝐷 =4𝑤

𝜋𝑑2𝐿

…………. (3.7)

Where w is weight in gram, d is diameter of the extrudate (cm) and L is length (cm). Ten

pieces of extruded product were randomly selected and average was reported.

c) Breaking strength (BS)

BS was measured by using TA Plus Lloyd Texture analyzer as shown in

Plate 6. Warner – Bratzler cutting blade (Plate 7) in snap test setup was used. Probe speed

of 2 mm/sec, trigger force of 0.1 N and limit of 15 mm were set parameters in snap test.

Breaking strength can be well correlated with textural attributes of the product. Breaking

strength in kgf was then calculated as mean of ten random determinations (Ilo et al.,

1999).

d) Overall acceptability score (OAA score)

To evaluate sensory attributes, OAA score was given on 9-point hedonic

scale ranging from extremely dislike (1) to extremely like (9). (Ranganna, 2000). Ten

panelist evaluated each extruder run product. Mean value of each sample score was

reported. Evaluation card is given on Annexure – I.

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Plate 4. Lloyd Texture analyzer

Plate 5. Warner – bratzler cutting blade (probe) used for determination of breaking

strength

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3.2.6 Methodology for the development of flaxseed fortified cookies

3.2.6.1 Preparation of cookies

Cookies were prepared as per the formulations given in Table 3.3. Roasted

flaxseed flour (RFF) was fortified by substituting refined wheat flour at 5%, 10%, 15%,

20%, 25% and 30%. Table 3.4 represents the details of treatment and their respective

coding.

Dry ingredients i.e. refined wheat flour, custard powder, baking powder

and RFF were thoroughly mixed and sieved twice for uniform blend. Hydrogenated fat

and shortening were creamed together manually for 10 min. to get bright and fluffy mass.

Milk and essence was added in the mass and mixed for another 5 min. Finally, dry

ingredient blend flour was slowly added to the above creamed mass and mixed for 2 min.

The cookie dough was sheeted to 8 mm thickness and cut into circular shape using 42

mm diameter cutter. The cookies were then baked at 1800C for 15 min. The cookies were

cooled for 10 minutes, then wrapped in aluminum foil and packed in polyethylene bag.

3.2.6.2 Evaluation of cookies

a) Physical characteristics:

Diameter (D), thickness (T) and spread factor were analyzed as physical

characteristics for control and cookies with RFF. For the determination of cookies

diameter (D), six cookies were placed edge to edge. The total diameter of the six

randomly selected cookies was measured in mm by using a ruler. The cookies were

rotated at an angle of 900 for duplicate reading. The average diameter was reported in

mm. To determine the thickness (T), six cookies were placed on top of another. The total

height was measured in mm with the help of ruler. This was repeated thrice to get an

average value and results are reported in mm. Spread ratio was determined with the help

of following formula:

Spread ratio =Average diameter mm X 10

Average thickness mm ............ (3.8)

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b) Sensory evaluation of cookies:

Sensory evaluation was carried out by a panel of ten judges. Hedonic

rating test was employed using 9-point hedonic scale (from like extremely -9 to dislike

extremely -1). Sensory parameters such as color, taste, texture and overall acceptability

were evaluated. (Ranganna, 2000). Sensory score card used in this study was attached as

appendix – II.

c) Texture analysis:

Prepared cookies were analyzed for the effect of refined wheat flour

replacement with roasted flaxseed flour for its hardness and fracturability using Texture

analyzer (Stable Microsystems, UK, Model TA-HDi) as shown in Plate 8. Hardness and

fracturability of the cookies was measured by following Three point bending rig setup

(Gaines, 1991). The sample was placed on two supporting beams aparted by 3 cm

distance. Another beam connected to moving part was brought down to break down the

biscuit (Plate 9). Precaution was taken that the point of contact was uniform from the

both supporting beam. Different parameters for textural attributes analysis were set as

followings. Mean value of three determinations are reported.

Parameter Setup values

Load cell 5 kg

Pretest speed 1.0 mm/s

Test speed 3 mm/s

Trigger force 50 g (Auto)

Distance to travel 5 mm

Data Acquisition rate 400 pps

d) Statistical Analysis:

Data was analyzed with Daniel‘s XL Toolbox Version 5.08 using one –

way analysis of variances (ANOVA) with tukey comparision of means values.

Differences between mean values with probability p < 0.05 were recognized as

statistically significant differences.

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Table 3.3 Formulation of cookies

(Reference: Kamaliya and Kamaliya, 2001)

Table 3.4 Treatment levels of roasted flaxseed powder in cookies formulation

Sr. No. Ingredient Quantity

1. Refined Wheat flour 100 g

2. Sugar 50 g

3 Hydrogenated fat 60 g

4 Custard powder 2 g

5 Milk powder 2 g

6 Milk 5 to 6 ml

7 Baking powder 0.5 g

Treatments Refined wheat flour (%)

Roasted

flaxseed

flour (%)

Control 100 0

A 95 5

B 90 10

C 85 15

D 80 20

E 75 25

F 70 30

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Plate 6. Texture analyzer (Stable Microsystems, UK, Model TA-HDi)

Plate 7. Three point bending rig setup

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3.2.7 Proximate composition of developed flaxseed fortified extruded food product

and cookies

Moisture content (%), ash (%), protein content (%), crude fiber (%), fat

content (%) was estimated according to the methods mentioned in 3.2.1. Carbohydrate

content (%) was estimated by difference method. Fatty acid profile of extracted lipids

from the respective developed product to confirm the enhancement of omega-3 fatty acid,

were analyzed as mentioned in 3.2.2.

3.2.8 Storage stability study of optimized products in different packaging material

To estimate shelf life of product, storage stability of developed optimized

food products in the present investigation were studied in different packaging material for

90 days. Two laminates i.e. PET/Al/LD (Metalized polyester with low density

polyethylene) and PP/LD (Polypropylene with low density polyethylene) were taken for

this study. At 15 days interval, each sample was analyzed for their responses.

3.2.8.1 Analysis of packaging material laminates

a) Thickness :

Thickness of packaging laminate was determined by using dial gage

micrometer (Make – Mitutoyo Manufacturing Co. Ltd., Tokyo, Japan, Model – 7327 P,

Least count – 1 µ). Laminate was cleaned with carbon tetrachloride to remove oil

residues and dust particles. Again it was cleaned with tissue paper. Five readings on one

specimen (four corners and central position) were taken by using dial gage micrometer.

Average of these five readings was reported as thickness of laminate in micron.

b) Water vapour transmission rate (WVTR) :

WVTR was measured by according to ASTM D1653/E96 using Labthink

instrument model No. TSY-T1. The preconditioned sample was used for measurement.

The specimen size was 100 mm and actual test area was 63.58 cm2. The test parameters

used were temperature of 380C and relative humidity of 90 per cent.

Principle: Saturated water vapour transmits through specimen in a unit time under

specified conditions of temperature and humidity. Determine transmitted mass by testing

the decreasing weight of distilled water with time going.

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c) Oxygen transmission rate (OTR) :

The oxygen transmission rate was measured by using Labthink instrument

model VAC-VBS according to standard method ASTM D1434. The specimen size was

80 mm and actual test area was 28.3 cm2. The test was conducted under proportional

mode. The gas pressure used was between 4-6 kg/h and vacuum used was less than 26 Pa.

Principle: Place the sample between the upper and lower chambers, clamp tightly, open

the valve of the whole system. When the vacuum reaches to the certain level, close the

valve of the lower chamber. Feed certain pressure of testing gas into the upper chamber

to generate a constant pressure difference between the two chambers (adjustable). The

gas will penetrate the film from the higher pressure side to the lower pressure side under

the function of the pressure gradation.

3.2.8.2 Storage study of developed extruded product in different packaging material

In case of extruded product, moisture content, breaking strength and

overall acceptability score were taken as response during storage stability study of 90

days.

a) Moisture content :

It was determined by the procedure mentioned in 3.2.1.1.

b) Breaking strength :

It was determined by the procedure mentioned in 3.2.5.5 section c.

c) Overall acceptability score :

It was determined by the procedure mentioned in 3.2.5.5 section d.

d) Statistical Analysis :

It was carried out by performing the analysis of variances (ANOVA). Two

way ANOVA without replicate was adopted on the data collected for the above

mentioned response parameters. Critical difference (CD) was calculated by using two

tailed t-test value at 0.05 probability.

3.2.8.3 Storage study of developed cookies in different packaging material

In case of cookies, moisture content, peroxide value and sensory score

were taken as responses during storage stability study of 90 days.

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a) Moisture content :

It was determined by the procedure mentioned in 3.2.1.1.

b) Peroxide value of lipids extracted from cookies :

It was determined by the standard procedure of AOAC (2000). Weigh oil

sample and mixed with 30 ml of solvent (glacial acetic acid: chloroform in the ratio

3:1,v/v) in 500 ml conical flask. Add 0.5ml of saturated KI solution, mix well and allow

standing for 1 min. Add 430 ml of water and 3-4 drops of starch indicator and mixing

well. Titrate against standard 0.01N sodium thio-sulphate with vigorous shaking to

liberate all from chloroform layer until the blue colour just disappears. Treat the blank

similarly in the absence of oil.

Peroxide value (meq / kg of oil) = A x N x 1000 ……… (3.9)

Weight of oil

Where A = ml Na2 S2 O3 (Test - blank)

N = Normality of Na2S2O3 solution

c) Overall acceptability score :

It was determined by the procedure mentioned in 3.2.5.5 section d.

d) Statistical analysis :

It was performed as mentioned in 3.2.8.2 section d.