RESEARCH PROPOSAL

Development of low glycemic index (GI) bun from cornlettes and

cornsilks powder (CFCP)

NAME: NURUL ALI’IM BT ZAINAL ABIDIN

Table of Content

Chapter 11.0 Background of Study

1.1Justification of Study1.2Research Objective1.3Research Questions1.4Research Hypothesis

4 - 6

Chapter 22.0 Literature Review

2.1Non Communicable Diseases (NCDs)2.2Dietary fiber2.3Cornlettes2.4Bakery Products2.5Glycemic Index (GI)

7 – 10

Chapter 33.0 Methodology

3.1Research design3.2Sample collection3.3Sample preparation3.4Formulation and preparation of burger bun3.5Determination of moisture content3.6Determination of ash3.7Determination of fat3.8Determination of protein3.9Determination of total carbohydrate.

3.10 Determination of total dietary fibre3.11 Thermal Profiles using Differential

Scanning Calorimetry (DSC)Starch crystallinity using X – ray Diffraction

3.12 Colour profile determination3.13 Morphological Characterization Using

Scanning Electron Microscope (SEM)3.14 Physical analyses

I. Determination of diameter, thickness and weight

II. Determination of spread ratio and density

III. Determination of hardness and fracturability

3.15 Texture profile analysis (TPA) of burger bunI. Analyses of hardness, springiness,

resilience, cohesiveness and

11 - 26

2

chewiness

3.16 Sensory evaluation3.17 Glycemic index (GI) determination

I. Selection of subjectII. Preparation by subjectIII. Serving size of test foodsIV. Preparation of reference foodV. Study protocol for GI determinationVI. Capillary blood samplingVII. Glucose measurementVIII. Calculation of glycemic index

3.18 Data Analysis4.0 References 27 – 305.0 Appendixes

5.1Flow chart5.2Gantt chart of research activities 31 & 32

3

CHAPTER 1

1.0 Background of Study

There are varieties of foods available from plant source as many plants or plants part

are eaten as food. One of plants main part can be eaten is seed and grain. Seed contains

nutrients necessary for human body thus classify as a good source, in fact, majority of foods

consumed by human beings are seed based foods. Edible seeds include cereals (maize, wheat

and rice), legumes (beans, peas and lentils) and nuts.

Young corn or cornlettes (Zea mays) is one of the commonly consumed vegetables

especially in Asian regions. Cornlettes (Zea mays) is a type of corn harvested in the early

stages. During harvesting, young corn ears are small, immature, silk either do not appear or

appear and fertilisation not occurred (Hooda et al., 2013). There are different varieties of

cornlettes in the market, results from natural hybrids and mutation which cause a very large

numbers of cultivars (Adejumo et al., 2013). Properties of each variety differentiate type of

cornlettes, especially physical appearance, texture, and structure – function of the starch.

Cornlettes can be eaten either raw or used as ingredients in food preparation as pickled, soup

and others. As cornlettes taste delicious and very nutritious, people tend to add them in their

prepared foods. Moreover, cornlettes is popular at the national and international stage even

though the nutritional has not been known clearly. On the other hand, it can grow four times a

year; with some amount of irrigation thus it has proven to be a good alternative to rice

(Chutkaew & Paroda, 1994). Presently, many researchers are interested in investigating

functional properties of young corn.

1.1 Justification of Study

Cornlettes or young corn is one of the local vegetables that have been neglected despite of the

nutritional value and content. Food – based crop is one of the major food sources, thus the

4

demand and production getting higher day by day, and to fulfil those demands, something has

to be done.

This study is done in order to partially replace imported wheat flour as main ingredients by

reducing its usage in food products. Wheat is a cereal grain grown all over the world. It is the

third most-produced cereal after maize and rice and is the staple food of millions of people.

Furthermore, wheat flour is the main ingredients in all flour – based food products such as

cakes, pastries, cookies, noodles and breads. China is the world’s largest producer of wheat

followed by India, Russian Federation, United States of America and France.

Apparently, unavailability of local based locally produced dietary fibres resources cause

limitation in the bakery based products. Generally, Malaysian bakery entrepreneurs rely on

dietary fibres such as oat, whole grain wheat flour and wheat meal in their processed products

(Wan Rosli et al., 2014). As most of wheat is cultivated and produce worldwide, Malaysia

has to import them thus increase production of baked – based products. Tharise et al. (2014)

reported that, Indonesia and other tropical countries have been dependent on imported wheat

to full fill their requirement for the manufacture of various food products based on wheat

flours. Besides that, wheat and some cereals can causes gluten intolerance in some

individuals, which can impairs intestinal absorption and lead to severe malnutrition (Ciclitira

& Ellis, 1987).

Dependency on the imported raw material cause instability of the price, thus it may trigger

food manufactures to find an alternative resource in sustaining their income and productions

of the food products. However, there is no such local vegetable can be used as an alternative

raw material. Therefore, there is a need to explore and apply new alternative from locally

available vegetable.

1.2 Research Objectives

1.2.1 General Objective

The present study aimed to investigate changes in physiochemical properties, pasting profiles

and sensory acceptability of burger bun utilize with cornlettes and cornsilk powder in

reducing the glycemic index (GI) values.

1.2.2 Specific Objective

5

1. To formulate bakery products utilize with cornlettes and cornsilk powder.

2. To analyse gelatinization and pasting profiles of bakery products utilize with

cornlettes and cornsilk powder.

3. To examine the physical and textural properties of bakery products utilize with

cornlettes and cornsilk powder.

4. To evaluate the sensory acceptability of bakery products utilize with cornlettes

powder.

5. To determine the glycemic index (GI) of bakery products utilize with cornlettes

powder.

1.3 Research Questions

1) Are there any qualities differences in bakery products utilize with cornlettes powder?

2) Is bakery products utilize with cornlettes powder can be accepted by the consumer?

3) Can glycemic index (GI) values reduce in bakery products utilize with cornlettes

powder?

1.4 Research Hypothesis

I. Null hypothesis, Ho :

There are no significant differences in pasting profiles of composite flours blends with

different percentage of cornlettes.

II. Alternative hypothesis, HA:

There are significant differences in pasting profiles of composite flours blends with different

percentage of cornlettes.

6

CHAPTER 2

2.0 Literature Review

2.1 Non Communicable Diseases (NCDs)

Nowadays, the number of non-communicable diseases (NCDs) around the world and

Malaysia especially have been increasing. NCDs is defined as a diseases of long duration

and generally have a slow progression. Non-communicable diseases such as hypertension,

diabetic, cancer, heart disease as well as chronic respiratory diseases now become the major

morbidity and mortality factors around the worlds. 63% of deaths around the worlds caused

by NCDs. More than 36 million people die each year (Merten, 2013); and 28 million or 80%

of the deaths occurred in low and middle income countries (WHO, 2015). According to

World Health Organization (WHO), non-communicable diseases have shorten and torn

down the life of too many people not only in higher income countries but also in low and

middle income countries. Moreover, people from developing countries suffered the most as

the lifestyle are changes and becomes more similar to the developed countries. Besides that,

NCDs also can be caused by the unhealthy diet, tobacco consumption and alcohol abused

and physical inactivity (WHO, 2013). Thus, prevention need to be taken from earlier to

reduce the number of NCDs from developing any further.

The most important things to do to reduce NCDs is by changing unhealthy diet intake.

Dietary fiber intake may prevent the increase number of chronic diseases. Dietary fiber main

sources such as vegetables, fruits and whole grain should be taken regularly as it contains

carotenoids, flavonoids, vitamins and minerals as well as dietary fiber (Shikany and White

(2000); Ng and Wan Rosli (2013). Gomez et al. (2003) mentioned that dietary fiber is a

common and important ingredients of a healthy food products that have been highly

requested by the new generation consumers.

The recommendation daily intake (DRIs) of dietary fiber is 25g for women and 38g

for men with the suggestion of 14g/1000kcal dietary fiber (Timm and Slavin, 2008). For

children under 8 years old, the recommended intake 25g and under 13 years old, the value is

31g (Dietaryfiberfood.com, 2005). However, the usual dietary intake of dietary fiber is low

with the amount of 16g per day. Therefore, the intake of dietary fiber should be increased.

7

2.2 Dietary Fiber

Fruits, vegetables and wholegrain are the major sources of fiber. There are two types

of fiber which is soluble fiber and insoluble fiber. Soluble fiber or fermentable fiber can be

found in grain (oats and barley), fruits (bananas and apples), beans and pulses (baked beans

and chick peas), and root vegetables (carrot and potatoes), while insoluble fiber can be found

in cereal foods, wholemeal breads, pasta, brown rice, vegetables, potatoes with skin, nuts,

and seeds. Soluble fiber can dissolves in water and forms a gel in the gut where it helps to

prevent or treat constipation.

Dietary fiber is defined as the edible parts of plant that are resistant to digestion and

absorption during the digestion processes in the human body. The edible parts include

polysaccharides, lignin, oligosaccharides (inulin) and resistant starches (Gomez et al. 2003);

Anderson et al. (2009). Dietary fiber is known to have several health benefits including

lowering prevalence rates for chronic heart disease (CHD), stroke, hypertension, diabetes

and cancer. Besides that, it’s also important to maintain a good health and prevent diseases

as it reducing cholesterol levels, controlling blood sugar and promoting bowel regularity

(whfoods.org, 2001). Furthermore, with the presence of dietary fiber, gastric emptying can

be delayed thus slowing down carbohydrates digestion and absorption. Previous study by

Aune et al. (2011) showed that high intake of dietary fiber is associated with a reduced risk

of colorectal cancer. Their finding reveal that for every 10g/day of total dietary fiber intake

may reduce 10% of colorectal cancer risks and it further reduced with higher intake.

Furthermore, high level of dietary intake also associated with significantly lower prevalence

rates for CHD, stroke and peripheral vascular disease (Anderson et al. 2009). In addition,

previous studies showed that the young corn powder is believed to reduce the postprandial

glycemic index when added to the chiffon cakes (Wan Rosli &, Che Anis, 2014). According

to Ho et al. (2012), the substitution of dietary flour into food may also contribute to the

reduction of prevalence of malnutrition.

2.3 Cornlettes

Cornlettes is simply a regular corn plants that is harvested at early age while the ears

are still very immature. According to Miles & Zenz (1997), cornlettes is harvested at early

age of ear development because the ear can grow very quickly and tend to grow larger than it

need to be in one or two days. Cornlettes is harvested when it reaches the maturity stag,e

which occurs one to three days after the corn silk is emerging. During harvesting cornlettes

8

optimum size should be reach where cornlettes is generally two – four inches long and 1/3 –

2/3 inch in diameter. Upon harvesting, cornlettes immediately stored and cooled, perhaps

cornlettes is sold in the husk in order to maintain ear moisture and quality. Furthermore,

cornlettes is popular in Asian cuisine and often is eaten as either cooked or raw vegetable

due to its delicate flavor and crispiness. Most of cornlettes production widely produces in

East Asia countries such as Thailand and is exported all over the world including United

State and Europe.

Cornlettes is a good source of essential nutrients as it contains an abundance of

nutritional content that make it closer to a non – starchy vegetable. Moreover, sugar in

immature cornlettes is not yet developed during harvesting, thus is make cornlettes as a good

low – carb foods. Dried cornlettes powder has showed to has shown to have high fiber

contents where it contains 30.4% of dietary fiber (Wan Rosli, & Che Anis, 2012) and it can

be used to improve functional properties of food products (Wan Rosli et al., 2014).

2.4 Bakery Products

Bakery products such as bread, biscuits, cakes, pastries and pies are historically one of

mankind’s oldest food staples where both can give nourishment and enjoyment at the same

time. Baked products are referred as all food products which are based on the use of wheat

flour (Cauvin, 2016). Wheat flour is used together with various ingredients such as sugar,

fats and many; in order to produce a tasty and delicate bakery products. According to

Vitaglione et al. (2015), bakery product quality can be measured based on the texture,

aroma, colour, taste and appearance. Based on the research done by Malaysia Adult

Nutrition Survey, the consumption of bakery products in Malaysia especially bread and

biscuit appeared to be in the top ten list of the most consumed foods daily (Norimah et al,

2008; Anis Jauharah et al. 2014). Hence the addition of cornlettes in bakery products might

increase the health value of the products. This is because cornlettes contains high dietary

fiber and other nutrients as Hooda et al. (2013) reported that the nutritive value of cornlettes

is comparable to several high-priced vegetables like cauliflowers, cabbage; french beans,

spinach, lady finger, brinjal, radish and potato. Thus, cornlettes may potentially be used to

incorporate and partially replace wheat flour in bakery products.

2.5 Glycemic Index (GI)

9

The glycemic index (GI) is a numerical index that ranks carbohydrate based on their

rate of glycemic response such as the conversion of glucose within the human body. The

value is used to quantify the differences in blood postprandial glucose response to a food

after finding that different carbohydrate foods draw out different glycemic response each

(Jenkins et al. 1981; Kendall et al. (2010). It is measured using a glycemic index scale with

the value of 0 to 100; pure glucose is used as the reference point with the GI value of 100.

Higher values are given to the foods that cause the most rapid rise in blood sugar upon

ingestion. Meanwhile carbohydrate with a low GI value are more slowly digested, absorbed

and metabolised in the human body thus cause a lower blood glucose levels. As most

vegetables are rich in dietary fiber, the glycemic index value may be lower. Study done by

Weickert and Pfeiffer (2008) reported that, gastric emptying and macronutrient absorption

from the gut can be slow down by the soluble dietary fiber while insulin sensitivity can be

increased by insoluble fiber, whereby the elevation of postprandial glycemic response can be

controlled by both dietary fiber.

10

CHAPTER 3

3.0 METHODOLOGY

3.1 Research Design

This study is based on experimental study design. The experimental samples are

physiochemical, pasting and thermal properties of cornlettes and cornsilk powder in the

development of burger bun with low glycemic index (GI). Analysis will be done by

comparing three substitutions of cornlettes and cornsilk powder in which is mixed with wheat

flour. The 4%, 6%, 8% and 10% of cornlettes and cornsilk powder will be blended with

wheat flour accordingly. Cornlettes and cornsilk powder will partially replace wheat flour.

Previous study shows that replacement of 10% cornlettes powder in wheat flour results in

reduction of postprandial blood glucose response thus reducing the glycaemic index of the

food products (Wan Rosli WI et al., 2014).

3.2 Sample Collection

In this study, young corn/cornlettes samples will be collected and purchased from

Kampung Salor, Pasir Mas district in Kelantan, Malaysia. Cornlettes should be free from

pesticide, harvest at early age (1 -3 days after the silks become visible) and size requirements

should be 2 – 4 inches long and 1/3 – 2/3 inch in diameter. After harvested, cornlettes is

placed in refrigerated storage with the hush intact to conserve ear moisture and preserve their

quality. Besides that, wheat flour, corn flour and rice flour will be purchased from the local

supermarket in Kubang Kerian, Kelantan.

3.3 Sample Preparation

To prepare cornlettes and cornsilk powder (CLCP), several procedures should be done.

First, cornlettes ears and cornsilks will be detached and separate from silk, tassel and husk.

Next, cornlettes will be sliced and chopped into small size while cornsilk also will be cut into

smaller size in order to increase the total surface area. Then, air dried for three days at room

temperature and two days in oven – dried (Memmert GmbH & Co. KG, Germany) at 55°C

until its turns brownish in colour. Dried young corn will be ground using electric grinder

(National MX – 895M) to obtain fine cornlettes and cornsilk powder. Then cornlettes and

11

cornsilk powder will be sifted through 125 microns mesh – sieve (Endecotts Ltd. England) to

acquire fine powder. Finally, cornlettes and cornsilk powder (CLCP) will be keep in screw

cap bottle (Scoot Duran) and store in refrigerator at 4°C.

3.4 Formulations and preparation of burger bun

Formulations of burger bun for this study were listed in Table 3.1. All ingredients were

carefully weighed on a portable digital scale (OHAUS Scout, USA). In this study, four

formulations of young corn biscuits were prepared. YCP was used to partially replace the

wheat flour. Each formulation differed from one another by various level of YCP

incorporation.

Ingredients (g) Formulation (% YCP used to replace wheat flour)

0 4 6 8 10

Wheat flour 1000 960 940 920 900

CLCP 0 10 10 20 30

Fat 10 10 10 10 10

Yeast 15 15 15 15 15

Sucrose 200 200 200 200 200

Salt 3 3 3 3 3

Bread Improver 1 1 1 1 1

Water 11 11 11 11 11

First, yeast will be dissolved in warm water. Fat (shortening) and sugar will be added

and mixed together. Then, bread improver and salt will be added into the mixture and mix

thoroughly. After that, wheat flour blended with cornlettes and cornsilk powder will be added

to form soft dough. The dough will be left stand for five minutes. Turn onto a floured surface;

knead until smooth and elastic, and will be rested for 30 to 60 minutes and covered with

warm towel/plastic wrap. Next, the dough will be kneaded again and divided into small

pieces weighing 50 grams each and shaped into a ball. The dough will be rested again for 30 -

60 minutes and covered with warm towel. The dough will be arranged on a flat pan and

baked in an oven (Zanussi ZCG841W, England) at 150° for 10-15 minutes or until golden

brown. Burger bun will be stored rein four different air-tight plastic containers according to

their formulations. The containers will be labelled with the type of formulation and date of

preparation.

12

3.5 Determination of Moisture Content

In determination of moisture content, air-oven method or drying method (AOAC,

1996) will be followed. An empty aluminium dishes will be dried for 3 hours in an oven

(Memmert GmbH & Co. KG, Germany) at 105oC. They will be placed in a desiccator

(NORMAX, Portugal) to cool down to reach room temperature. It will be weighed using an

analytical balance (Mettler-Toledo Dragon 204, Switzerland). Readings will be taken at four

decimal places. The weight of dried empty dish was named as ‘W1’.

Next, dishes with samples will be put in the oven at 105oC overnight. Then, they will

be taken out from oven and immediately will be transferred into a desiccator to reach room

temperature and absorb any remaining moisture. The final weight (W3), which will be

referred as the weight of the dish and dried sample was then repeatedly measured until

constant weight achieved.

The following formula will be used to calculate moisture content of sample:

Moisture (%) =Loss of weight of the sample (g)

x 100Weight of sample (g)

=W2 – W3 – W1

x 100W2

Samples will be grounded in a laboratory blender (Waring Commercial 8010S, USA)

until fine. The homogenized sample will be weighed at 5.0000 ± 0.001 g into the aluminium

dish. The initial weight of the sample was noted as ‘W2’. There will be three replicates for

each sample.

3.6 Determination of ash

Ash determination will be performed according to method 942.05 (AOAC, 1996).

Initially, crucibles will be dried in an oven (Memmert GmbH & Co. KG, Germany) at 105 oC

for 3 hours. Once taken out, they immediately will be cooled in a desiccator (NORMAX,

Portugal). The dried empty crucibles will be weighed (W1) on an analytical balance (Mettler-

Toledo Dragon 204, Switzerland). Bakery product samples will be homogenized by grinding

in a laboratory blender (Waring Commercial 8010S, USA). The sample will be weighed at

0.500 ± 0.01 g into the crucible. The weight will be noted as ‘W2’. Each sample will be made

in three replicates.

13

The samples in the crucibles will be charred on a hotplate (ERLA EM2-V7070,

Malaysia) until smoke performed. Later, the crucibles will be transferred into the cold muffle

furnace (Barnstead Thermolyne F6020C-33, USA) which will be brought to a temperature of

550oC. The furnace system will be pre-set to at 550oC, for 3 hours and to cool down

automatically. As the samples in the crucibles turn into whitish or greyish, they will be

removed from the furnace, cooled in a desiccator and weighed. They will be replaced in

muffle furnace and reheated until constant weight obtained. The weight will be noted as

‘W3’.

Total ash content of bakery products will be calculated using formula as follow

Ash (%) =Weight of ash (g)

x 100Weight of sample (g)

=(W3 – W1)

x 100W2

3.7 Determination of fat

Crude fat content will be analysed based on method 960.39 (AOAC, 1996).

Extraction cups (VELP Scientifica, Italy) will be dried in advance and weighed as ‘W1’ on

analytical balance (Mettler-Toledo Dragon 204, Switzerland).

Homogenized samples will be acquired by grinding in laboratory blender (Waring

Commercial 8010S, USA). Samples will be weighed to 3.000 ± 0.01 g in every extraction

thimble and noted as ‘W2’. Then, the thimbles with samples will be introduced to the solvent

extractor (VELP Scientifica SER148/6, Italy). Next, 80 ml of petroleum ether will be added

into each of the extraction cup. The cups then will be brought into the extractor. After that,

the unit will be closed to prevent solvent in the extraction cup from evaporating into the air

and later cooling water flow and heating will be started. The slider of the extractor will be

changed into ‘Immersion’ position in order to immerse the thimbles.

After 30 min, the thimbles will be took out from solvent by pushing the slider into

‘Washing’ position. This step allows reflux washing which took about 25 min. Then, in

‘Recovery’ position, the stopcock located under the water cooled condenser will beclosed.

The final step of extraction took 30 min in ‘Recovery’ position. Then, the unit will be

switched off. All extraction cups will be then took off from the extraction unit and transferred

14

into an oven (Memmert GmbH & Co. KG, Germany) at temperature 100oC for 30 min to let

the remaining petroleum ether dry and only fat will be left in the extraction cup. After that,

they will be cooled in a desiccator (NORMAX, Portugal) prior to weighing the cups

containing fat of the samples (W3).

Fat content of the bakery products will be determined using the following formula:

Fat (%) =Weight of fat (g)

x 100Weight of sample (g)

=(W3 – W1)

x 100W2

3.8 Determination of protein

I. Preparation of reagents

40% sodium hydroxide (NaOH) solution will be prepared by dissolving 400 g of

NaOH crystals in distilled water in a 1000 ml volumetric flask and making up to volume with

distilled water. 4% boric acid (H3BO3) solution will be obtained by dissolving 40 g H3BO3

powder in distilled water in a 1000 ml volumetric flask. Then, distilled water will be used to

make up to volume. Indicator for titration will be prepared by dissolving 80 mg methyl red

and 20 mg methylene blue in 95% ethanol which were made up to 100 ml with 95% ethanol.

Hydrochloric acid (HCl), 0.1 N will be obtained with dilution of 8.9 ml 37% HCl in 700 ml

distilled water in a 1000 ml volumetric flask and making up to volume using distilled water.

II. Analysis of protein

Protein analysis will be performed using Kjeldahl method 991.20 AOAC (1996).

Homogenized sample of each bakery products will be obtained by grinding in a laboratory

blender (Waring Commercial 8010S, USA). The samples will be weighed to obtain 1000 ±

10 mg into each digestion tubes. Following that, one catalyst tablet (Gerhardt 1000 Kjeltabs

ST, Germany) and 20 ml sulfuric acid will be added into each digestion tube. As for blank,

the digestion tube will be filled with sulfuric acid and the catalyst.

The tubes will be brought to Kjeldal herm block digestion unit (Gerhardt GmbH &

Co. KG, Germany) and placed in inclined position on the electric coil heating rack. The

Turbosog scrubber unit (Gerhardt GmbH & Co. KG, Germany) will be turned on to remove

15

and neutralize acid fumes. The digestion unit will be programmed to achieve 400oC by

gradual heating. Heating will be started and when the mixture in tubes became clear and

colourless, heating will be stopped. The unit will be left to cool.

Once the room temperature is achieved, the tubes will be transferred to Vapodest

distillation unit (Gerhardt GmbH & Co. KG, Germany). A conical flask containing a few

drops of indicator will be placed on the receiver platform. Solutions for distillation, i.e. 40%

NaOH, 4% H3BO3 which is prepared earlier will be added into their respective tanks connect

to the distillation unit. The unit will be switched on to run the distillation process. After

distillation took place, the content of the conical flask will be took out from the platform and

titrated to light purplish colour by the addition of 0.1 N HCl delivered from a burette.

Amount of HCl required for titration will be noted.

The following formula shows the calculation of protein content in the bakery products:

Protein (%) =(ml HCl – ml HCl blank) x 14.008 x 0.1 N HCl x 6.25

x 100Weight of sample (mg)

3.9 Determination of total carbohydrate

Total carbohydrate content will be calculated by difference (Southgate, 1991). The

calculation was as follow:

Total carbohydrate (%) = 100 – [% moisture + % fat + % ash + % protein]

3.10 Determination of total dietary fibre

Determination of dietary fibre in bakery products sample will be carried out according

to enzymatic-gravimetric method 991.43 (AOAC, 1996). In this study, Fibertec E system

(FOSS Analytical, Sweden) which applies the enzymatic-gravimetric principle will be used to

perform the analysis. The system comprises of Fibertec 1023 Filtration Module and FOSS

1024 Thermostatic Shaking Water Bath. Samples will be homogenized by grinding in

laboratory blender (Waring Commercial 8010S, USA) and defatted with petroleum ether by

Soxhlet method 960.32 (AOAC, 1996). The defatted sample of each product formulation will

be weighed 1000 ± 5 mg on lid of incubation flask where it will be performed on analytical

16

balance (Mettler-Toledo Ross Series ML204/02, Switzerland). The lid is properly attached to

the flask.

Step 1 incubation will be started by adding 50 ml phosphate buffer solution into each

flask. To ensure that the samples are completely dispersed, they will be stirred on magnetic

stirrer. The pH will be checked using HANNA pH 211 microprocessor pH meter (USA). The

pH 6.0 ± 0.2 was required. If necessary, either 0.275 N NaOH or 0.325 M HCl will be added

for adjustment. 50 µl α-amylase (Sigma-Aldrich A3306, USA) then will be added as the

required pH is achieved. Flask will be stirred slightly and covered with aluminium foil. Next,

the samples will be took for incubation in boiling water bath (Protech Model 830, Malaysia)

at 95100oC. After 30 min of incubation, the flasks will be removed from water bath and

brought to cool to room temperature.

In step 2 incubation, pH will be adjusted to 7.5 ± 0.2 using 0.275 N NaOH. Later, 100

µl of freshly prepared protease enzyme solution (50 mg/ml) (Sigma-Aldrich P3910, USA)

will be added to each flask when the target pH is obtained. Incubation period at 60oC in the

shaking water bath will take about 30 minutes at speed 2.5. Once taken out from the water

bath, flasks will be cooled down to attain room temperature.

Next incubation step will be carried out by adjusting pH between 4.04.6 with 0.325 M

HCL. Later, 200 µl amyloglucosidase (Sigma-Aldrich A9913, USA) will be added after

acquired pH is achieved. After that, incubation will be started again at 60oC at 2.5 speed in

shaking water bath for 30 minutes. The flasks will be transferred out after 30 min. The

digested samples containing 280 ml or 4 volumes of 95% ethanol is immediately added. They

were let to precipitate for 1 hour at room temperature. Zero point five gram celite (Sigma-

Aldrich C8656, USA) will be weighed in fritted crucibles (FOSS Analytical, Sweden), then

will be dried by heating at 105oC overnight.

Once taken out and cooled down, they will be weighed. The crucibles will be brought

to filtration unit. The beds of celite in the crucibles will be wet and redistributed using a

stream of 78% ethanol. Suction will be applied to draw the celite onto the fritted glass in

order to get an even mat. The crucibles will be removed from the unit and mounted upside

down on top of the incubation flask containing the sample. The flasks will be attached to the

bayonet fittings and they will be folded up. The bottom lids of each flask will be removed.

17

Residues on the lid and seal will be washed off into the flask with a small portion of 78%

ethanol. In each flask, washing procedure will be carried out using ethanol and acetone.

The procedure will be repeated 3 times using 20 ml 78% ethanol, 2 times using 10 ml

95% ethanol and 2 times using 10 ml acetone. Water aspiration pump will be started for

filtration. The control valves will be turned to ‘V’-position for suction and ‘P’-position for

breaking up clogged residue, whichever necessary. After filtration, residues in crucibles will

be dried at 105oC overnight and weighed. The dried residues will be divided into two groups

of replicates.

The first replicates will be continued with determination of ash in furnace (Carbolite

CWF1100, UK). Weight of residue ash will be took. The second replicates will be took for

determination of protein. As to continue with protein analysis, the residue in each crucible

will be scraped into digestion flask and protein analysis will be ran. Protein content of

residual will be calculated.

Residue weight, blank value and dietary fibre will be calculated as follow:

Residue weight = (Residue + Celite + Crucibles) – (Celite + Crucibles)

Blank = ( B1 + B2

) – mg protein – mg ash 2

Dietary

fibre

(%)

= {

[(R1 + R2)

] – mg protein – mg ash – Blank } x 100 2

M1 + M2

2

References for the formulas are as follow:

B1/B2 = Residue weight (mg) of blank duplicates

R1/R2 = Residue weight (mg) of sample duplicates

M1/M2 = Weight (mg) of sample duplicates

mg protein = protein (mg) in blank/sample residue

mg ash = ash (mg) in blank/sample residue

18

3.11 Thermal Profiles using Differential Scanning Calorimetry (DSC)

The thermal properties of the samples will be analysed using the Perkin-Elmer DSC-7

(Norwalk, CT, USA) equipped with an intra - cooler I and Thermal Analysis Controller TAC

7/DX. An empty pan will be used as the reference and calorimeter will be calibrated with

indium (melting point = 156.6°C, ΔH = 28.5J/g). The operation of DSC analyser is runs

under nitrogen gas atmosphere (30 mL/min). Burger bun thermal transitions define as T˳

(onset temperature), Tp (peak of gelatinization temperature) and Tᴄ (conclusion temperature)

and ΔHgel (enthalpy gelatinization) where enthalpies are calculated on samples dry weight

basis. DSC analysis will be used approximately 3 mg of flours samples and every sample will

be carried out in triplicate.

Approximately 3 mg of samples (dry basis) will be weighted in an aluminium pan.

Distilled water will be added into the samples subsequently using 1:2 ratio (sample: water,

w/w). Control the weight of the sample continuously until the desired moisture content is

attained. The pan will be sealed hermitically and is allowed to stabilize temperature at 4°C for

overnight to reach equilibrium before heating in the calorimeter. Water amount will be

adjusted accordingly in order to achieve sample– water suspension. Sample pans will be

heated at a rate of 10°C/min from 20 – 950°C.

3.12 Colour profile determination

Determination of bread crust and crumb profile colour will be determined using

colorimeter (Minolta Chroma Meter 300, Japan). There are three parameters will be recorded

which are lightness (L), redness (a) and yellowness (b). All results will be recorded.

3.13 Morphological Characterization Using Scanning Electron Microscope (SEM)

SEM test will be done to analyse the morphology of cornlettes granules by using

scanning electron microscope (SEM) (QUANTA FEG 250 ESEM). Starch samples will be

suspended in 95% ethanol and mount on circular aluminium stubs with double-sided sticky

tape. Starch granules will be dispersed evenly on the surface of the tape, and ethanol will be

allowed to evaporate. Sample will be dispersed with 12 nm gold, then examine and

photograph at an accelerating voltage of 5 kv with a magnification of x5000 and x10000

19

3.14 Physical analyses

I. Determination of diameter, thickness and weight

Measurements of physical characteristics (Saha et al., 2011; Tiwari et al., 2011) will

be carried out using a 15 cm scale. A piece of burger bun from each formulation will be

weighed (OHAUS Scout, USA) simultaneously and the average weight of each piece will be

noted. It will be placed in the centre to measure the diameter and thickness, respectively.

Measurement will be repeatedly taken and the mean value was then calculated.

II. Determination of spread ratio and density

Spread ratio and density will be derived from the diameter (D), weight (W) and

thickness (T) measurements by simple calculation. Formulas for spread ratio and density

were;

Spread ratio = D/T

Density (kg/m2) = W/D2

III. Determination of hardness and fracturability

A common technique known as the three-point break (Gaines, 1991) will be followed

to measure breaking strength and fracturability of biscuits. Texture Profile Analyzer; Texture

Analyser TA XT PLUS (Stable Micro System, Surrey, UK) powered by Exponent software

package will be used to conduct the test. Accessories needed in the test were three-point

bending rig (HDP/3PB) and Heavy Duty Platform (HDP/90). The two adjustable supports of

the rig base will be adjusted to separate 20 mm apart as to support the biscuits. The gap

distance will be kept in a constant rate for comparison purposes. The base plate will be fixed

onto the Heavy Duty Platform. The platform will be moved and locked in a position that

allowed equal distance between the upper blade and the two lower supports. Bun sample will

be placed centrally over the supports.

As to run the test, the following settings will be applied: test mode: compression, pre-

test speed: 1 mm/s, test speed: 3 mm/s, target mode: distance, distance: 5 mm, trigger force:

50 g. When the trigger force is achieved, the force will be increased until the bun broke into

20

two pieces. The maximum force will be observed to break the bun into two pieces where it is

referred as ‘hardness’ and mean distance compressed before breaking value will be known as

‘fracturability’. When the test is performed, a curve and values of interest, i.e. hardness and

fracturability will be obtained from the software. The peak force (kg) and mean distance at

point break (mm) will be referred as hardness and fracturability of the biscuits.

3.15 Texture profile analysis (TPA) of burger bun

I. Analyses of hardness, springiness, resilience, cohesiveness and chewiness

Instrumental analysis of bun textural properties will be performed to record hardness,

springiness, cohesiveness, resilience and chewiness (Baixauli et al., 2008a; Sanz et al., 2009).

According to instrumental definition, hardness is ‘the peak force during the first compression

cycle’, springiness is ‘the height that the food sample recovers during the time that elapses

between the end of the first bite and the start of the second bite’, cohesiveness is ‘the ratio of

the positive force during the second compression to that during first compression’ and

resilience is ‘the area during withdrawal of the first compression divided by the area of first

compression’. The values of each parameter will be acquired from the TPA curve analysis.

The test will be conducted using TA XT PLUS Texture Analyser (Stable Micro Systems Ltd.,

Surrey, UK) which is driven by Exponent software package. The soft inner portion of bun

will be evaluated. Each bun will be cut into 2.5 cm sided cube, where the upper and lower

crusts is eliminated. A 75-mm diameter aluminium plate (P/75) will be used for compression.

Probe height is initially calibrated to ensure that the travel distance of the probe can be

recorded.

The test will be performed under the following states: test speed: 1 mm/s, strain 50%,

trigger force 5 g. Muffin cube is compressed twice to obtain TPA curve which consists of

double cycles. The curve will be analysed to obtain values of interest, i.e. peak force of first

compression cycle, areas of first and second cycles, positive peak area of first cycle and

distance travelled during the first and second compressions. The peak force of first

compression is noted as hardness and expressed in Newton (N) unit. Ratio of distance

travelled during second compression to that during the first compression is taken as

springiness. Cohesiveness will be acquired by dividing the positive area of first compression

by positive area of second compression. Resilience will be obtained by calculating the ratio

21

of first decompression (withdrawal) area to the first compression area. Springiness,

cohesiveness and resilience were dimensionless. Chewiness will be calculated as the product

of hardness x cohesiveness x springiness (Pons and Fiszman, 1996) and is expressed in

Newton (N).

3.16 Sensory evaluation

Seven-point hedonic scale

Sensory evaluation session will be conducted based on seven-point hedonic scale

(Aminah, 2000) where higher score indicates better quality attributes (1- dislike very much

and 7 - like very much).

Sensory attributes such as crumb and crust color, aroma, texture, mouth feel, taste,

overall acceptability (Menon et al., 2015) will be evaluated.

Each product is independently judged by 60 volunteers based on their likenesses.

Volunteers will be randomly selected and they consist of staffs and students of School of

Health Sciences, Universiti Sains Malaysia. They need to be voluntarily agreed to participate

in the sensory evaluation session.

Short briefing will be given before the sensory evaluation session started. In order to

minimize bias, volunteers will be provided with drinking water for rinsing before testing the

next sample. Four samples of burger bun will be served to every volunteer.

Every sample will be assigned with three-digit code. The code is generated from a table

of random numbers; three digits numbers will be used as a code. The random numbers will be

recorded on a master sheet, one code for each sample for each volunteer.

Serving order will be determined according to random permutation principle. Using the

random permutation table, numbers will be read from top to bottom within a column. Only

number 1, 2, 3 and 4 will be read since in total there were four samples to be evaluated at one

time. The set of numbers will be wrote in the presentation order column of the master sheet.

The four-digit number will be assigned for each volunteer and it shows the order in which

each sample is presented to the respective volunteer.

22

3.17 Glycemic index (GI) determination

I. Selection of subject

Twelve healthy human subjects (five males and seven females) will be randomly

selected from the School of Health Sciences. Inclusion criteria are: age between 18 to 75

years, BMI of 18.5 to 24.9 kg/m2, non-pregnant, non-lactating, non-smoker, having no history

of acute or chronic illnesses and did not undergo any surgical procedures during past 6

months. After obtaining a written informed consent, a clinical examination will be done by a

physician. The clinical examination form will be attached as Appendix 2. Ethical approval for

the study will be acquired from the Research Ethics Committee (Human) of Universiti Sains

Malaysia.

Body weight will be measured on an electronic scale (Seca 767, UK) with subject

wearing light clothing and without shoes. Reading is recorded to the nearest 0.1 kg. Height

measurement will be carried out using an electronic stadiometer (Seca 242, UK) with subject

standing straight and shoes taken off. Measurement of height is recorded to the nearest

centimetre. Body mass index (BMI) will be obtained by calculation using standard formula:

weight (kg)/height2 (m2).

II. Preparation by subject

Subject will be instructed to fast overnight for about 1014 hours prior to attending the

test session in the morning. They will be advised to avoid any unusually vigorous activity

on the day before test was conducted (Brouns et al., 2005). As to minimize carry-over

effects, two test sessions will be separated for at least 48 hours as washout period.

III. Serving size of test foods

All formulations of burger bun will be choose for GI determination. Subjects were served

with 25 g available carbohydrate portions of the control food and the test food to avoid

unrealistically oversized serving. Calculation of available carbohydrate will be measured

using this formula:

23

Available carbohydrate = Total carbohydrate dietary fibre

IV. Preparation of reference food

The reference food that will be used in this study is glucose. Glucose drink will be

prepared by dissolving 25 g of original-flavoured Glucolin (Reckitt Benckiser, Malaysia) in

250 ml drinking water. 25 g Glucolin contains 25 g available carbohydrate (dextrose

monohydrate).

V. Study protocol for GI determination

The protocol will be applied according to WHO/FAO (1998) recommendation. Each

subject will be tested with glucose drink (reference food) for three times at different occasion

in order to improve the precision of measurement, thus reduce variation of mean GI values

(Brouns et al., 2005). Meanwhile, every formulation of test food will be tested once by each

subject. There will be four formulations of test foods involved in this study. Thus, in total

there will be seven test sessions conducted in random order. A standard drink of water (250

ml) is given with each test meal. During each session, fingertip capillary blood samples will

be collected at fasting, then repeatedly at 15, 30, 45, 60, 90, and 120 minutes after consuming

the test food. Subjects is encouraged to consume the test foods served within 1015 minutes

and remain sedentary during the test session.

VI. Capillary blood sampling

Capillary blood will be recommended over the venous blood because of easier access,

better sensitivity and avoiding potentially large variations in measured glucose concentration

(FAO/WHO, 1998; Brouns et al., 2005; Venter et al., 2005). Guidelines of capillary blood

sampling by WHO (2010) will be followed. Subjects are encouraged to warm their hands

before finger prick to increase blood flow. Alcohol pad containing 70% v/v isoprophyl

alcohol (BD Alcohol Swabs, USA) will be gently applied to the entry site and let to air dry.

Skin will be deliberately punctured with a quick and continuous stroke using a lancing device

(Accu-Chek Multiclix, USA). The first drop of blood will be wiped away due to possible

contamination with tissue fluid or debris. Whole blood of approximately 4 µl will be drew

from fingertip capillary into cavity of disposable plastic microcuvette (HemoCue Glucose

201 RT Microcuvette, Sweden) by capillary action. During fingertip blood extraction, hard

finger squeezing will be avoided to minimize dilution of plasma.

24

VII. Glucose measurement

Fill microcuvette will be inserted without delay into glucometer (HemoCue Glucose

201 RT, Sweden) which applies modified glucose dehydrogenase method to measure

concentration of glucose in the blood. The HemoCue Glucose testing systems use whole

blood sample for measurement and automatically transformed the result to plasma equivalent

reading. HemoCue system has a very good correlation with classic gold standard analyzer

Yellow Spring Instrument (YSI) system in glucose determination (r=0.9787) (Stork et al.,

2005). Unlike glucose meters, HemoCue is reported to have negligible hematocrit and protein

bias and is free from operator influences (Rajadhyaksha et al., 2007). According to the

manufacturer’s note, external quality control for calibration before test is optional since the

instrument would automatically perform self-test each time it is turned on. Internal self-test

will verifies that the instrument’s optronic unit is working properly.

VIII. Calculation of glycemic index

Calculation of incremental area under curve (iAUC) will be performed using Microsoft

Excel (Version 2007, USA), in which the trapezoid rule will be applied. If the blood glucose

response value falls below the baseline, only the area above the fasting level will be included.

Glycemic index (GI) of the test foods was calculated using the following equation

(FAO/WHO, 1998)

GI =Area under curve of test food

x 100Area under curve of reference food

The iAUC for each control and test food will be expressed as a percentage of the mean iAUC

for glucose taken by the same subject and the resulting values averaged to give the food GI

values.

3.18 Data Analysis

25

Data obtain will be analyse using ANOVA procedure in IBM SPSS 20.0 (USA)

software. Results are express as mean ± standard deviation. All measurements will carry out

in triplicate (n = 3). Significant level establish at P ≤0.05.

4.0 References

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5.0 Appendixes

5.1 Flow Chart

\

31

Tittle selection

Sample and method selection

Purchasing of sample

Sample preparation

Preparation of burger bun using cornlettes cornsilk powder (CLCS).

Thesis writing, presentation and publication

Interpretation of analyse data.

Statistical analysis of the obtain results by SPSS statistical programme.

CLCS and product analysis.

Sensory evaluation

5.2 Gantt Charts of Research Activities

PROJECTACTIVITIES

2016 2017

Research Activities

A M J J A S O N D J F M A M J J A S O

Writing proposal

Presentation of proposal

Collection of samples

Bun formula using CLCS Powder

Analysis of samples

Data Analysis/Interpretation

Presentation and Report Writing

Submission of Research Paper

32