Experiment 3

LABORATORY TESTING OF BIOLOGICAL MOLECULES FOR THE PRESENCE OF

STARCH, NON-REDUCING SUGARS, REDUCING SUGARS, LIPIDS AND PROTEINS
Aim

 To test for the presence of biological Molecules in food samples, non-reducing sugars,
reducing sugars and starches, lipids and proteins.
Introduction
All biological systems are made up of four major classes of macromolecules and these are the
carbohydrates, lipids, proteins, and nucleic acids. Carbohydrates are the most abundant
macromolecules on earth, and they are a source of immediate energy needs in living systems.
Carbohydrates also participate in defining the structure of cells and living systems. There are 3
general chemical grouping for carbohydrates and these are monosaccharides, disaccharides, and
polysaccharides. Monosaccharides, also known as simple sugars, are made up of a single sugar
molecule. The major example of monosaccharide is glucose (C6H12O6). Other monosaccharides
include isomers of glucose, such as fructose and galactose. Monosaccharides are transported in the
blood of animals, broken down to produce chemical energy inside the cell, and can be found within
other macromolecules, such as nucleic acids. Disaccharides are composed of two single monomers of
sugar linked together. Examples of disaccharides are maltose (glucose + glucose) and sucrose
(glucose + fructose). Disaccharides are broken down into their subunits for use inside living systems.
Polysaccharides are polymers, or long chains of sugar monomers linked together, and are stored
inside the cell for future energy use. In plants, the major storage polysaccharide is the starch. While in
animals it is the glycogen. Plants also contain cellulose, which is the most abundant of all
carbohydrates. Cellulose is found in the plant cell wall, where it provides structure and support to the
plant cell.
On the 0ther hand, lipids are nonpolar macromolecules and because of this, they are insoluble in
water. Lipids include oils and fats, phospholipids, and steroids. In this case, fats and oils are
triglycerides, composing of one glycerol and 3 fatty acids. A fatty acid is a long chain of carbon-
hydrogen (C-H) bonds, with a carboxyl group (-COOH) at one end. Fatty acids can be classified as
saturated or unsaturated. In this case, fatty acids are saturated when they do not contain any double
bonds between the carbons and unsaturated when they contain double bonds. An example of a
saturated fat is butter, while an example of an unsaturated fat is vegetable oil.
Phospholipids are found primarily in the cell membranes of living systems, of which they are the
major component. Structurally, a phospholipid contains a hydrophilic head and a hydrophobic tail.
The head of the phospholipid contains a phosphate group while the tail is typically a diglyceride. The
cell membrane is a double layer of phospholipids in which the tails are turned inwards and the heads
are exposed to the intracellular and extracellular environments. Steroids are lipids which are typically
made up of fused hydrocarbon rings. Each type of steroid is different in the type of chemically active
functional groups that it contains. Examples of steroids include cholesterol, estrogen, and testosterone.
Cholesterol found in the cell membrane of animals, where it provides structural support. Cholesterol
is also the precursor for other steroids, such as testosterone and estrogen. Some vitamins, such as
Vitamin D, are also classified as steroids.
When it comes to proteins, these are polymers of amino acids. Amino acids are small molecules that
contain an amine (-NH2), a carboxyl acid (-COOH), and a side chain (R). There are twenty naturally
occurring amino acids, and each amino acid has a unique side-chain or R-group. Amino acids are
connected by peptide bonds to form protein polymers. This gives rise to different levels of structure
for proteins. The primary (1°) structure of a protein is made up of a string of amino acids connected
via peptide bonds. The secondary (2°) structure of a protein is formed by the coiling and folding of
the 1° structure, due to hydrogen bonding. At this level, structures called alpha-helices and beta-sheets
are visible. The tertiary (3°) structure of a protein is formed by interactions between the components
of the 2° structure. Some proteins have quaternary (4°) structure, which includes the assembly of
multiple individual subunits to form the functional protein. Proteins have numerous biological
functions. The common types of proteins that are found in biological systems are enzymes, antibodies,
transport proteins, regulatory proteins, and structural proteins.
Apparatus/Materials
a. Egg albumen, groundnuts, cassava, table sugar, tomato, and sample of solution k
b. Biuret reagent, ethanol, distilled water, iodine solution, Benedict’s reagent, dilute
hydrochloric acid and sodium hydroxide solution.
c. Two Beakers 250 ml, 6 test tubes, pipettes/droppers, test-tube racks and hot water bath..
Procedure Part A- Preparation of Food Sample Solutions
1. If the food sample to be tested is in solid form (i.e. ground nuts or maize grains), grind crush a
small amount of that food sample using a mortar and pestle.
2. Then after that, put the crushed food sample into a 250 ml beaker to a depth of about 2cm and
fill the same beaker with distilled water up to a quarter full.
3. Shake the mixture gently and allow it to stand for few minutes to let the sediments settle at
the bottom of the beaker.
4. After sediments have settled, get a clear sample solution into a clean 250 ml beaker and label
it for that particular food sample name.
5. If the food sample is in fresh form (i.e. orange or tomato), cut the fruit and squeeze out all the
juice into a 250 ml beaker. Or alternatively grind crush the sample using the mortar and pestle
and then fill the mortar with water up to a quarter full to make a solution. Mix the solution
using a pestle and then pour into the250 ml beaker and shake further the mixture gently.
6. Allow the solution to stand for few minutes to let the fresh sediments settle and then after
that, pour out a clear solution into a clean 250 ml beaker and label the beaker for that
particular food sample name.
7. If the food is in solution form, test it directly, but if it is concentrated (i.e. egg albumen),
dilute it using distilled water.
Procedure Part B- Testing for Reducing Sugars
1. Familiarize yourself with all procedural steps and instructions for the testing of reducing
sugar and then set up the water bath at 80 °C to 100 °C.
2. Label the test tubes and each according to the solution of food sample that will be prepared in
that test tube for testing of reducing sugar.
3. Place 2 ml of each solution of food sample prepared into the respectively test tube labeled for
particular food sample solution and then place 2 ml of water into test tube C which will acts
as the control and then add 2 ml of Benedict’s reagent to each of the test tubes above.
4. Swirl each of the tubes and place the test tubes containing food solutions into the hot water
bath, which is set at 80 °C to 100 °C, and heat for 5 minutes.
 5. When heating test tubes in a water bath, keep on observing for initial colour change of testing
reagent (Benedict solution) and record each step of any colour change observed.
6. Using the test-tube holder, carefully remove test tubes from the water bath and place them in
the test-tube rack and then record the observed results on the table of results.
Technical Notice for Reducing Sugars

 If the reducing sugar test comes out negative or no change in the colour of benedict’s solution
from blue, to green, yellow, orange and brick red, then the test for non-reducing sugars can
then be done and if it comes positive then no need to test for non-reducing sugars.

Procedure Part C -Testing for Non-Reducing Sugars

1. Set up the water bath at (80 °C to 100 °C) and then label the test tubes each according to the
solution of food sample that will be prepared in it to be tested for the reducing sugar
2. Prepare a sample solution following the appropriate extraction procedure as indicated above
for steps of sample preparations.
3. Place 2 ml of each solution of food sample prepared into the respective test tube labeled for
that particular food sample solution.
4. Add 3 drops of 0.5M of hydrochloric acid to each of the sample solutions in the test tubes and
heat in water bath for 5 minutes to hydrolyze the non-reducing sugars into reducing.
5. Add 3 drops of 0.5M sodium hydroxide to the mixture. Shake the mixture safely and heat
gently to neutralize the acid from the sample that has become hydrolyzed.
6. Add an equal volume of Benedict’s solution to the test tube containing the food sample that
has become hydrolyzed and neutralized.
7. Heat the mixture in a water bath by boiling gently, while observing colour change from initial
testing reagent (Benedict solution) and record each step of any colour change observed.
8. When heating, observe safety by putting on safety goggles to protect your eyes, and then
using test-tube holder, carefully remove both tubes from a water bath and place them in a test-
tube rack and then record results a similar table like the one showing below.
Procedure Part D -Testing for Starch
1. Label test tubes each according to the solution of food sample that will be tested for starch.
2. Place 2 ml of each solution of food sample prepared into the respective test tube labeled for
the particular food sample solution and then place 2 ml of water into test tube C that will acts
as a control.
3. Add 2 to 5 drops of iodine solution to each of test tubes including tube C, and swirl each of
the tubes.
4. Observe colour change from initial colour and then reproduce a similar table of results like
the one showing below on a plain paper and record the results.

Procedure Part F - Testing for Proteins

1. Label the test tubes each according to the solution of food sample that will be prepared into it
to be tested for protein.
2. Add 2 ml of each of food sample solution prepared into the respective test tube containing the
particular food sample solution.
5. After that, add 2 to 10 drops (2 ml) of biuret reagent to each test tube containing food samples
and swirl to mix.
6. Observe the colour change after the addition and record results in the table of results.
7. Dispose of Biuret sample waste in the discard container.
8. Repeat steps 1 to 7 for the unknown solution V and record the results.

Procedure Part G - Testing for Lipids Using Ethanol and Water

1. If the food sample to be tested is in solid form (i.e. ground nuts or maize grains), grind crush a
small amount of that food sample using a mortar and pestle.
 2. Label test tubes each according to the solution of food sample that will be prepared into it for
testing of lipids and fats.
3. Put the crushed food sample into a respective labeled test tube to a depth of about 2cm and
then fill the test tube with 95 - 99 % ethanol up to a quarter full of test tubes. Shake the
content of each test tube gently to mix.
4. Allow the test tubes to stand for 15 to 20 minutes to let the sediments settle at the bottom of
the test tubes.
5. After sediments have settled, get a clear sample solution from each of the test tubes and
decant into clean test tubes whilst labeling them for the particular food sample name.
6. Then after that, using a pipette, add drops of water to the test tubes one at a time while
observing what is happening. Record the results in the table of results below.
7. Note: If the food sample to be tested is naturally in liquid form like an egg albumen dilute it
a little with water and then after that add drops of ethanol and observe the happenings. Record
also the results in the table of results.
8. Repeat the same steps of the procedure from 1 to 8 and test for the unknown solution V and
the control sample C which is water and record the results in the table of results below.

Task

 Write a full laboratory report and discuss it in details according to the results collected on all
biomolecules (food samples) tested in the lab.