OBJECTIVES:

- To analyze the presence or absence of biological macromolecules in various foods.

- To test the food samples for the presence of lipids, proteins, and simple and complex
sugars.
- To understand the tests and procedures that used to identify the presence of
macromolecules in various foods.
- To recognize both the positive and the negative results from each of the
macromolecule
tests.
- To understand the use of a positive and a negative control in this experiment.
- To identify the functional groups for each of the biomolecules that react in the following
biochemical tests: Benedicts test, Iodine test, Brown Bag test, Sudan III/IV test, and the
Biuret's
Test.
- To understand the mechanism of reaction for: Benedicts test, Iodine test and the
Biuret's
Test.

INTRODUCTION:
All living things contain organic macromolecules: Lipids, proteins, carbohydrates and
nucleic acids. Characteristic for these organic molecules is that they are made up of only
a small number of elements: carbon, hydrogen, oxygen, and to smaller amounts
nitrogen, phosphorus and sulfur. They are called "macromolecules" because they are
very large, containing long chains of carbon and hydrogen atoms and often consist of
repeating smaller molecules bonded together in a repeating pattern (polymers).
Macromolecules have a very big impact on our diet. Therefore, in this experiment, three
macromolecules that are important energy sources for biological organisms:
carbohydrates, proteins and fats will be focused to figure out the right balance that we
need to sustain a healthy body. By using the chemical tests and comparing the solutions
to the control, the macromolecules in the solution can be identified.
A carbohydrate is an organic compound that consists only of carbon, hydrogen, and
oxygen. Body uses carbohydrates to make glucose. It is critical for diet since it provides
a source of energy, and it is the simplest form of carbohydrates which is the easiest to
store in the body. Body can use glucose immediately or store it in liver and muscles
when it is needed. In general, the smaller carbohydrate compounds are commonly
referred to as sugars, which are found naturally in foods such as fruits, vegetables, milk,
and milk products. Simple carbohydrates also include sugars added during food
processing and refining. However, starch and dietary fiber are the two types of complex
carbohydrates. Starch is basically a long chain of sugar (glucose) molecules. Therefore,
eating starch enables us to take in lots of glucose efficiently, which is used for energy. It
must be broken down through digestion before body can use it as a glucose source.
Proteins are essential for our diet because proteins help build new cells, maintain
tissues, and synthesize new proteins that make it possible to perform basic body
functions. For instance, the results prove that milk contains proteins, and our bones are
1

mainly made up of proteins with calcium for strength, as well as magnesium and
phosphate. Protein is mainly found in the following foods: meats, poultry, and fish,
legumes (dry beans and peas), tofu, eggs, nuts and seeds, milk and milk products, and
grains. The protein in the foods is digested into amino acids that are later used to
replace these proteins in bodies. Actually, there are 20 different amino acids that join
together to make all types of protein. Our bodies cannot make some of these amino
acids so these are known as essential amino acids it is essential that our diet provide
these. A complete protein source is one that provides all of the essential amino acids, for
example, meat, poultry, fish, milk, eggs, and cheese are considered complete protein
sources. An incomplete protein source is one that is low in one or more of the essential
amino acids. Complementary proteins are two or more incomplete protein sources that
together provide adequate amounts of all the essential amino acids (e.g. rice and
beans).
Lipids are molecules that contain hydrocarbons and make up the building blocks of
the structure and function of living cells. For examples, fats, oils, waxes, certain vitamins,
hormone and most of the non-protein membrane of cells. Besides that, lipids are a large
and diverse group of naturally occurring organic compounds that are related by their
solubility in non-polar organic solvents and general insolubility in water. It had a bad
press, to the extent that some foods are designed and promoted as fat-free. However,
fat should not be completely banned from body because obtaining some fat from our diet
is vital as lipid provide long-term energy storage in cells and are very diverse. It is
primarily digested in the small intestine where the bile produced by the liver breaks lipid
globules into smaller droplets and then the pancreatic enzymes break large lipid
molecules into smaller components for absorption. Nowadays, almost all foods contain
some fat because almost all living organisms in this world use fat to store energy.
Moreover, lipid is needed for growth, development, and function. And so, we are much
like other animals, therefore we do need some fat in our diet for survival.
MATERIALS AND METHODS:
Apparatus and materials:
- Benedicts reagent
- Starch solution
- Iodine reagent
- Concentrated KOH
- 0.5% CuSO4
- Brown paper
- Test tubes
- Test tube holder
- Water bath
- Mortal and pestle
- Droppers

- Distilled water
- Sucrose solution
- Apple Juice
- Potatoes
- Soft drinks
- Onions
- Instant noodles
- Soya beans
- Peanut butter
- Bread
- Cheesecloth

Methods:
Testing for carbohydrates
1. The solid foods and semi-solid foods were grinded with mortal and pestle by adding
some distilled water.
2. The sample foods are strained into each test tube.
3. About 1 ml of each food sample was placed into different test tubes.
4. A control which is a tap water was prepared.
5. 5ml of Benedicts solution were added into each of all test tubes.
6. Then, all the test tubes were heated in the water bath for 2-3 minutes.
7. The colour of the solutions were observed and noted down whether a precipitate has
formed.
Testing for starch
1. About 1 ml of each food sample was placed into different test tubes.
2. A control was prepared by adding about 1ml of tap water.
3. A few drops of iodine reagent were added into each test tube.
4. The colour of the solutions were observed and noted down.
Testing for protein
1. About 1 ml of each food sample was placed into different test tubes.
2. A control was prepared by adding about 1ml of tap water.
3. 2ml of concentrated KOH (~20%) were added to all test tubes and mixed thoroughly.
4. Then. 1ml of 0.5% CuSO4 was added slowly and mixed well.
5. The colour of the solutions of each test tube were observed and noted down after 2
minutes.
Testing for lipid
1. 2 small rectangular of brown papers were taken.
2. 4 food samples were written on one piece and another 4 were on the other piece.
3. A tiny drop of each food sample was put and allowed the spots to dry.
4. As a negative control, put a tiny drop of water on the half of the paper labeled water.
5. The paper was holded up to the light when the spots are dry.
6. The observations were recorded.

RESULTS:
Food
samples/
Macromolecules
Apple juices

Potato

Soft drinks

Onions

Instant noodles

Soya milk

Peanut butter

Bread

Tap water as
negative control

Carbohydrates

Starch

Protein

Lipid

Orange precipitate
is formed. The
blue solution turns
to orange colour.
Orange precipitate
is formed. The
blue solution turns
to orange colour.
Orange precipitate
is formed. The
blue solution turns
to orange colour.
Orange precipitate
is formed. The
blue solution turns
to orange colour.
The blue solution
remains
unchanged.

The
brown
iodine solution
remains
unchanged.
The
brown
iodine solution
turns
blueblack.
The
brown
iodine solution
remains
unchanged.
The
brown
iodine solution
remains
unchanged.
The
brown
iodine solution
turns
blueblack.
The
brown
iodine solution
remains
unchanged.
The
brown
iodine solution
turns
blueblack.
The
brown
iodine solution
turns
blueblack.
The
brown
iodine solution
remains

The
greenishblue solution is
formed.

A
translucent
spot
is
observed.

The blue solution

remains
unchanged.
Orange precipitate
is formed. The
blue solution turns
to orange colour.
Orange precipitate
is formed. The
blue solution turns
to orange colour.
The blue solution
remains unchange.

The blue solution

with precipitate is
formed.
The blue solution
remains
unchanged.

No
change.
A
translucent
spot
is
observed.

The blue solution

remains
unchange.

No
change.

The blue solution

turns to light blue
with precipitate.

No
change.

The blue solution

turns to purple
colour.

No
change.

The blue solution

remains
unchanged.

A
translucent
spot
is
observed

The bluish-purple
solution
is
formed.

No
change.

The blue solution

remains
unchange.

No
change.

unchange.

DISCUSSION:
A control is a test sample with a known result. If the control samples do not give the
expected result, then the experimental results are not valid. The purpose of preparing
controls is to compare the results. During the experiment, tap water which serves as a
negative control sample was prepared for each part of the experiment to insure the
validity of your results. A negative control will result in no change in color. It will either
contain no sample at all or it will contain a nonreactive sample like water. For example,
for testing the presence of monosaccharides, the test chemical, called Benedict's
solution, will remain the original color blue when mixed with water. However, a positive
control will result in a color change indicating the presence of the compound you are
testing for. For example, a starch solution will react with iodine reagent and change it
from brown to blue-black colour.
During the first part of the experiment, Benedict's reagent is used to test all of the
food samples. This is a test for testing the presence of simple carbohydrates. Benedict's
reagent is a blue colored liquid that contains copper (II) ions. When Benedict's reagent
and simple carbohydrates are mixed and heated for 2-3 minutes, the color change
sequence will changes from blue to green or to yellow or to orange or to brick red
depending on the amount of monosaccharide present in the food samples. This reaction
is caused by the reducing property of simple carbohydrates. The copper (II) ions in the
Benedict's reagent are reduced to Copper (I) ions, which causes the color change.
Sometimes a brick red solid, copper oxide, precipitates out of the solution and collects at
the bottom of the test tube. In the experiment, it is found that all the 8 food samples
except instant noodles and soya milk show a positive result in this test. An orange
precipitate is formed but not brick red precipitate. This means that simple carbohydrates
are slightly present in these food samples. However, instant noodles and soya milk show
negative result in this test. The blue colour solution remains unchanged. This means that
there is no simple carbohydrates present in these two food samples. For the test tube
which contains tap water that serves as a negative control, the blue solution remained
unchanged.
In part 2, iodine reagent is used to test for the presence of starch. Since iodine on its
own is a small non-polar molecule and is insoluble in water. Therefore, potassium
triiodide solution which is iodine dissolved in an aqueous solution of potassium iodide is
used as a reagent. In this test, the starch-iodide complex is formed as charge is
transferred between the starch and iodide ion. The transfer of charge between the starch
and the iodide ion changes the spacing between the energy levels. This change results
in the starch-iodide complex absorbing light at a different wavelength than any other

species, resulting in an intense purple colour; Biologists call this colour as blue-black
colour. In this experiment, it is found that potatoes, instant noodles, peanut butter and
bread show a positive result. Each of them reacts with the iodine reagent, producing a
blue-black colour solution. This proves that starch is present in these 4 food samples.
However, for apple juices, soft drinks, onions and soya milk, when each of them is tested
with iodine reagent, there is no colour change. This indicates that there is no starch in
these food samples and that is why a negative result is obtained.

REFERENCES:
Hadikusumo, M. , 2014. Testing in Macromolecules in Food. [Online] Available at:
https://prezi.com/eeyvfxmf0-f7/testing-macromolecules-in-food/ [Accessed 31 July
2015].
n.d. Testing for Lipids, Proteins and Carbohydrates. [Online]
http://www.seplessons.org/node/362 [Accessed 1 August 2015].

Available

at:

Sone, H., 2013. ~ Experiment 2: Protein Experiment ~. [Blog] Available at:

http://biochemistrygirls.blogspot.com/2013_04_01_archive.html [Accessed: 1 August
2015].