METHODOLOGY

The present investigation was carried out to find glycemic index of the RTE
extruded snack food prepared using maize flour, pearl millet flour and morigna leaf
powder. The research procedure carried to achieve the objectives described below
under the following headings and subheadings:
1. Selection and nutritional analysis of the snack product
1.1 Selection and details of the selected product
1.2 Analysis of recipe for proximate principles
2. Assessment of glycemic index
2.1 Selection of the subject
2.2 Development of tool and collection of data
a) Profile of the subject and family
b) Anthropometric measurement –Weight, Height Waist and Hip
circumference
c) Blood pressure
2.3 Determination of glycemic index of snack product
a) Oral glucose tolerance test
b) Feeding of test recipe
c) Blood glucose test

3. Statistical analysis

1.1 Selection

1.1.1 Details of the selected product: Snack food product developed and
standardized at Center of Excellence (COE) on Maize Processing and Value Addition,
CDFST was taken for the study. The product comprised of combination of QPM
flour, pearl millet and morigna leaf powder.QPM was selected as a source of good
quality protein, pearl millet due to its high dietary fiber and mineral content. Moringa
leaf powder was selected due to its high dietary fiber and protein content. Besides
these, secondary raw material such as chilli, salt, maggie masala (spices), baking
powder and benzoic acid were added for taste and flavor.

Grains of QPM were procured from KVK Farm, Banswara (Rajasthan) and
milled to make flour in the laboratory while pearl millet were purchased from local

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market of Udaipur and milled to make flour. Fresh mature Moringa oleifera leaves
were carefully harvested from a Moringa tree in the outskirts of Udaipur region. The
leaves were washed with clean water at 1:5 leaves and water ratio to remove dusts,
pests, and subsequently drained on draining table for about one hour to before taking
them for drying inside a tunnel dryer at ambient temperature of 50 ± 5°C and relative
humidity of 75 ± 2 % for 2 to 3 days to reach 11 to 12% moisture content. Dried
leaves were milled using electric grinder. All the flour was passed through sieve of
300 μm.

2. Formulation

The selected snack food product comprised of 12 per cent protein 6 per cent
carbohydrates and 75 per cent dietary fibre. The tabulated nutrient content of the
product (tabulated values) is given in Table 1

Table 1 Nutrient content of the selected flour (g/100g)

Raw materials Protein Carbohydrates Dietary fiber
QPM 9 74 2
Pearl millet 11 73 8
Moringa powder 27 38 19

(Source: NIN, Hyderabad; Jones et al. 1970)

Process flow chart

Fig. 1 Process flow chart for preparation of QPM based extruded product

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Proximate Analysis:

One serving of the recipe was homogenized and used for analyzing the following
proximates in triplicate. The analysis was conducted in the laboratory of Agricultural
University, Junagadh, Gujarat in the academic year of 2016 by the researcher. The
methods used as follows.

Moisture (NIN, 1983):

Moisture is an important constituent of all foods. It is essential to determine the

moisture constituents to express the nutrients estimated on moisture free basis.

Method: Five gram fresh sample of the product was weighed in a previously weighed
petridish in triplicate and the again dried in oven (Yorcko hot air oven) at 60c and
cooled in a dessicator and weighed. The process of heating and cooling was repeated
till a constant weighed was achieved. The moisture was calculated as under:

Initial Weight – Final Weight

Moisture ( g % )= x 100
Weight of the sample taken

Plate 1.1 Universal oven for determination of moisture content

Crude Protein

The protein assay was performed by using Folin-Lowry method (Lowry et al.,
1951). 0.1 g of sample was weighed and aliquot was prepared by centrifugation.
Standard curve was prepared by using following procedure Bovine Serum Albumin of
concentration one mg/0.1 ml was prepared. A series of dilutions (0.2, 0.4, 0.6, 0.8 and
1.0) were made in duplicates with final volume of one ml as shown in plate .color
developed after 15 minutes at room temperature and dark was then compared with

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standard readings at 470 nm in absorption type Spectrophotometer as shown in plate .
Standard graph was then prepared to estimate the protein content.

Graph factor × Optical density × Total volume of buffer (ml)

Protein ( g %)= ×100
Volume taken from extract (ml) × weight of sample (gm)

Fat (NIN, 1983): Fat content of the product was analyzed by Soxhlet’s extraction
method which is based on siphon principle.

Method: Five gram moisture free sample was wrapped in Whatman No.41 filter
paper of 10x10 cm in such a way that during extraction the sample does not come out.
The packet was placed in the extracting tube of Soxhlet’s apparatus. An empty round
bottom flask was weighed and was filled to three fourth with petroleum ether (40-60
c). The flask was kept on heating mantle (Khera Instrument Pvt. Ltd.) and then
connected with extractor. The flask was heated for about 16 hours which ensured
complete extraction of fat then the flask was disconnected and ether was evaporated.
The flask was cooled and weighed. The difference in initial and final weight of the
flask was calculated to find out the fat content. The fat content was calculated as
under.

Weight of fat
Crude fat ( g % )= X 100
Weight of sample

Plate 1.2 Soxhlet apparatus for estimation of oil content

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Ash: The ash content of food stuff is the inorganic residue remaining after the organic
matter has been burnt away. It is a measure of the total mineral content of the food.

Method: Five grams moisture free sample in triplicate was placed in previously
weighed crucibles (after heating to about 600c & cooling). The crucible was then
heated over low flame till the material was completely charred, followed by heating
in a muffle furnace at 600c (Yorcko sales Pvt. Ltd.) for about 3-5 hrs. It was then
cooled in a desiccator and weighed. The process of heating &cooling was repeated till
constant weight were obtained and the ash was almost white. The ash content was
calculated as under:

Weight of ash
Ash ( g % )= x 100
Weight of the sample

Plate 1.3 Muffle furnace for determination of ash content

Crude Fibre (NIN, 1983): Fibre is an insoluble vegetable matter which is

indigestible by proteolytic and diastic enzyme and cannot be utilized by the human
body. It is usually composed of cellulose hemicelluloses and lignin.

Method: Two gram of moisture and fat free sample in triplicate was weighed into
500ml beaker. The 200ml of boiling 0.25 N sulphuric acid was added to it. The
mixture was boiled for 30 minutes and the volume was kept constant by adding water
at frequent intervals. The mixture was then filtered through a muslin cloth and the
residue was washed with hot water till free from acid. The residue was then
transferred to the same beaker and 200 ml of 1.25 per cent NaOH was added. After
boiling for 30 minutes as earlier, the mixture was filtered again through muslin cloth.

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The residue was washed with hot water till free of alkali followed by washing with
50ml alcohol and 50ml ether. It was then transferred to a crucible of known weight
(dried overnight at 80-100c). The crucible was heated in a muffle furnace at 600°c for
2-3 hours, cool and weighed. The difference in weight represents the weight of the
crude fibre . The fibre content was calculated as under :

Weight of residue−Weight of ash

Crude fibre ( g % )= x 100
Weight of sample

Carbohydrates (Gopalan et at., 1994): The total carbohydrates was calculated by

the different method as : sum of moisture, crude protein, crude fat, ash, crude fibre
being subtracted from 100.

Energy: It was calculated by using physiological fuel values i.e. 4,9,4 kcal per gram
for protein, fat and carbohydrates respectively.

2. Assessment of glycemic index

2.1 Selection of the subject: A total of 30 female subjects residing in Udaipur city
were selected randomly on the basis of following criteria.

 The age group between 30-50 year.

 No biochemical evidence of any other known disease.

Each subject was explained in detail about the experiment and objectives of the study.
A informal written consent was taken from each participant for prior to the study.

2.2 Development of tool and collection of data: An interview schedule was

developed and pretested to collect the following information about the subject and
their family. Appointment was taken telephonically and each subject was described
in details about the experiment.

A Profile of family and subject

Family: At the beginning of the study, information about the family of the subject
was enquired. The details are discussed below.

Caste and religion: It was recalled by subjects.

Family type: It was assessed in terms of nuclear and joint on the basis of the
following definition.

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Nuclear family : A family consisting of husband, wife and their children or any other
member of the family, who is/are dependent on this unit and living with them.

Joint Families: The collection of more than one primary unit on the basis of close
blood ties and common residence.

Family size: It was operationally defined as total number of members living in the
family and sharing the same kitchen.

Family income: It was calculated by assessing the income of all the earning members
of the family. Income from sources like agriculture, house rent and interest was also
considered. The total family income per month was divided by number of family
members to get the per capita income of the family.

Subject: It included information related to food habits the educational level,

designation, work place, type, pay scale and marital status.

Food habits: The information regarding food habits as vegetarian, non vegetarian and
ova vegetarian was recalled from each subject.

Educational level: The educational level of the subject was evaluated in term of
literate, secondary, higher secondary, graduation, post graduation and professional
degree /certificate.

Occupation Type: Designation at work place and pay were recalled for each of the
subject.

Marital status: It was recalled and recorded as marital, unmarried, widow or divorce.

Age: It was recorded by the date of birth of each subject.

B Anthropometric Measurements It provides information on gross body size,

skeletal form, configuration, on skeletal and soft tissue development. These method
are noninvasive, inexpensive, universally applicable and can reflect nutritional and
health status. Following measurements were taken of each subject to assess Body
Mass Index, and Waist Hip Ratio.

Weight - (Jelliffe, 1966): It is the measurement of body mass and is most in use. It
also provides a wide evaluation of overall fat and muscle stores. For healthy adults,
increase in body weight usually but not always indicates an increase in body fatness
(Robinson et.al 1987)

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 Plate 1.4 Anthropometric measurements

Technique: A platform spring balance was used for measuring weight. The subject
was asked to stand in the center of the platform bare feet, the minimum clothes and
without touching anything else, weight was recorded to the nearest of 0.25kg.

Height : It is a linear measurement made-up of sum of components legs, pelvis, spinal

and skull. While, for detailed studies of body proportions all these measurements are
required (Jelliffe1966).

Technique: Height of a subject was measured using a vertical anthropometric rod.

After removing shoes and socks the subject was asked to stand on horizontal platform
with heels together and hands hanging by the side. The anthropometric rod was
positioned behind the subject, so that the lower and stands between the heels and the
beam passes vertically between the buttocks touching the back of the head. The
subject was made to look straight and it was ensured that the inferior orbital margin
and the tragus of the ear fall in the same horizontal plane parallel to the ground. The
head piece of the rod which consisted of a mental bar was gently lowered touching the
hair and making its contact with the top of the head. Height was recorded to the
nearest of 1mm.

Body Mass Index (BMI): It accounts for difference in body composition by defining
the level of adiposity according to the relationship of weight and height, thus
eliminating dependence on form size (Stensland and Margolis, 1990).

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Technique: The body weight and height were taken as described above to obtain the
body mass weight was divided by the sources of height in meters square as follow

weight ( Kg)
BMI = 2
Height (m )

The value obtained was interrupted as per the classes of BMI with their presumptive
diagnosis suggested by James et.al, (1988) and recommended in Nutritive News
(1998).

Waist and Hip circumference (Depress et al. 1997): The recognition of fat
distribution as an indicator of risk, circumferential or girth measurement have been
ascribed importance. The most frequently used measure of adiposity is the waist to
hip ratio.

Plate 1.5 Waist and Hip circumference

Technique: The circumference of waist and hip was measured using a non stretchable
and durable plastic tape. The waist circumference was measured at the navel point.
The hip circumference was measured at the waist over the greater trochanters. The
subjects were asked to stand erect with the weight distributed equally on both the legs.
The readings were made with an accuracy of 0.1cm.

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Waist hip ratio: It differentiates between android and gynoid obesity. A WHR of
greater than 0.85 in women and 1.0 in man is indicative of android obesity and
increased risk of obesity related disease (WHO, 2000). It was calculated as follows.

Waist circumference( cm)

WHR=
Hip circumference(cm)

C Blood pressure (mm Hg):   Blood pressure is defined as the lateral pressure
exerted by blood on vessel walls while flowing through it (Chatterjee, 1976). Levels
of blood pressure were analyzed for each subject.

Plate 1.6 Measurement of Blood pressure

Technique: The blood pressure was measured by instrument called mercury

sphygmomanometer which consists of rubber bag connected by rubber tubing, bag
supported by a cloth cuff to be placed around the upper arm, bulb, manometer and
stethoscope. The cloth cuff was wrapped around the arm and then inflated with air to
compress the brachial artery. Then with the stethoscope placed on the skin above the
bend in the elbow, the air pressure in the cuff was slowly released. The first sound
was heard as the brachial artery begins to open, it was caused by the blood coming
into the forearm in spurts and hitting the stationary column of the blood. The level of

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the mercury column on manometer gauge at that point was recorded as systolic
pressure. The level of mercury column at which the sound becomes faint and die
away was recorded as diastolic pressure. The normal blood pressure recommended for
an adult are: Systolic 120-130 mm Hg and Diastolic 80-90 mm Hg.

2.3 Determination of glycemic index of snack product

Oral glucose tolerance test

On the first day after overnight fast the blood glucose fasting (0 hour) was estimated,
than 30 gm of glucose diluted in 100ml of water was given orally to the subjects.
Blood glucose levels were estimated at 30, 60, 90, 120 minutes using by glucometer
(Alera GI ) based on glucose – oxidase mediated reaction.

Plate 1.7 : Glucometer

Feeding of test recipe

Next day 30gm carbohydrate selected snack product was served to each member after
an overnight fast taking fasting (0 hour) blood sample. The subject was asked to
consume the snack product sitting comfortably within a time span of 15-20 minutes.
The blood glucose levels were estimated at 30, 60, 90, 120 minutes.

Blood glucose test

Serial estimation of blood glucose used for deriving area under the 2 hour glucose
curve (AUC-G) as per the given formula.

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 Areaunder blood glucose response curve for
30 gm of test carbohydrate food
GI =
Area under blood glucose response for 30 gm of glucose

Plate 1.8 : Blood glucose test

The glycemic load was also calculated for the selected snack product .It was
calculated in the study by using the formula of GL Food = (GIFood x amount (g) of
available carbohydrateFood per serving)/100. Glycemic loads below 10 to be "low," and
Glycemic Loads above 20 to be "high." Because Glycemic load is related to the food's
effect on blood sugar, low Glycemic load meals are recommended for diabetic
control. Monro et. al (2008).

3. Data analysis: The data on religion, caste family type size, food habits, educational
information, marital status, family history, blood pressure and food consumption
pattern was expressed as frequencies and percentages. Height, BMI, Waist
circumference, hip circumference, WHR was calculated. Area Under Curve (AUC)
formula was used to calculated glycemic index and Mean + SD were also calculated
of glycemic values of the subjects.

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