BC 102: Biochemistry (α) Activity 03: Hydrolysis of Starch

Hydrolysis of Starch
ACTIVITY 03

INTRODUCTION

Hydrolysis, by definition, is a common chemical reaction where water is mostly used to

break down the chemical bonds that exist between a particular substance including large ones
like polymers. This process is mostly the reverse process of a condensation reaction.

During hydrolysis, an —OH group from the surrounding water molecules attaches to one
monomer and an —H attaches to the other. Essentially, the water (hydro) will be used to break
(lyse) a bond.

This experiment illustrates the conversion of starch to a reducing sugar by the action of
hydrochloric acid at boiling point. The longer the starch is exposed to the acid the further
hydrolysis proceeds. The experiment is intended to show the contrast with enzymes, which do
not need high temperatures and prolonged exposure to reagents and give a quick reaction.

OBJECTIVE:

In this activity, perform hydrolysis on starch using an acid and an enzyme.

SAFETY PRECAUTIONS!

When dealing with acid and bases avoid direct contact with your skin and eyes. If any gets on
your skin, wash thoroughly with water.
Wear eye protection! If any gets in your eye, rinse immediately and thoroughly with water.
Never add water to acid. Always add acid to water.
Be careful when using heating water baths.

MATERIALS CHEMICALS
1 Beaker (250 mL) Concentrated HCl
3 Dropper Corn Starch solution
1 Graduated Cylinder (10.0 mL) Distilled water
1 Spot plate Flour solution
4 Test tube (15.0 mL) Iodine solution
1 Tongs Potato Starch solution
1 Tripod/iron stand w/ clamp Sodium carbonate
1 Wash bottle
1 Wire gauze
1 Beaker (250 mL)

Additional PPE Human Saliva

1 Bring pot holder
1 Gloves (Nitrile or latex)

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BC 102: Biochemistry (α) Activity 03: Hydrolysis of Starch

PROCEDURE

1. Place 15.0 mL of 1% corn starch solution into a test tube.

2. Carefully add about 10 drops of concentrated HCl in the test tube.
3. Put the test tube in a boiling water bath and start performing iodine test every minute in a
spot plate.
 Put 1 drop of corn starch solution from the boiled test tube in a spot plate.
 Add 1 drop of iodine solution to the drop of corn starch solution.
 Repeat this (use another spot on the plate) until the iodine test gives a negative result
Review the Iodine test from Activity 02.
4. Heat the test tube for two minutes more.
5. Take out the test tube from boiling water bath, then cool them at room temperature.
6. Add a very small quantity of solid sodium carbonate in the test tube.
7. Continue to add sodium carbonate until the effervescence stops.
8. Repeat the same process for the other two starch solutions with the following
concentrations: 1%, 5%, and 15%. Or, if you can manage, you can perform all three
solutions at once. Just add more spot plate on your list.
9. Repeat the same process but instead of adding several drops of HCl, use fresh saliva instead.
Make sure you can have 10 drops per tube. Assign one student saliva per group.

LABORATORY STATION AFTERCARE

1. Clean your glassware with a diluted dish washing soap solution. Cleaning materials like
test-tube brush will be provided to you from the stockroom.
2. Sponge, soap, tissues, matches, and wiping cloth are items you should personally
bring/buy for lab use.

DISPOSAL
1. CONFIRM FIRST WITH THE LABORATORY IN CHARGE IF DIRECT FLUSHING IS ALLOWED.
2. Broken Glassware: If your test tube broke while heating, put the broken pieces aside to
cool. After cooling, carefully collect with and wrap the broken pieces in a paper (about 2
to 3 sheets), label it as broken test tube, and hand it over to the SA together with the
rest of your borrowed materials. You will replace this broken item before the semester
ends as part of your clearance requirement.

REFERENCES:
1. Libretexts. (2022, July 4). Starch and iodine. Chemistry LibreTexts. Retrieved June 22, 2022,
from
https://chem.libretexts.org/Bookshelves/Biological_Chemistry/Supplemental_Modules_(Bi
ological_Chemistry)/Carbohydrates/Case_Studies/Starch_and_Iodine
2. Biochemistry lab Manual - Komar University of Science and Technology.

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BC 102: Biochemistry (α) Activity 03: Hydrolysis of Starch

Name: Jeanen Grace C. Cuadrado Score:

Section: N14 Date Performed:
Group #: 3 Date Submitted:

OBSERVATIONS
A.
I. 1% Corn Starch Solution
Stage of Colour Observed Time
Theoretical Explanation
Hydrolysis HCl Saliva HCl Saliva

1st Drop Yellow Dark Green 1 minute 1 minute Iodine detected more starches in
the solution.
(POSITIVE)

2nd Drop Yellow Dark Green 3 minutes 3 minutes Iodine detected more starches in
the solution.
(POSITIVE)

3RD Drop Yellow Royal Blue 6 minutes 6 minutes Iodine detected more starches in
the solution.
(POSITIVE)

4th Drop Yellow Green 9 minutes 9 minutes Iodine detected more starches in
the solution.
(POSITIVE)

5th Drop Yellow Green 12 minutes 12 minutes Iodine detected more starches in
the solution.
(POSITIVE)

6th Drop Pale Brown 40 minutes 40 minutes Iodine detected less starches in
Yellow Yellow the solution.
(NEGATIVE)

II. 5% Corn Starch Solution

Stage of Colour Observed Time
Theoretical Explanation
Hydrolysis HCl Saliva HCl Saliva

1ST Drop Yellow Dark Blue 1 minute 1 minute Iodine detected starch in the
solution. (POSITIVE)

2ND Drop Black Dark Blue 5 minutes 5 minutes Iodine detected starch in the
solution. (POSITIVE)

3RD Drop Dull Red Dark Blue 15minutes 15 minutes Iodine detected starch in the
solution. (POSITIVE)

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BC 102: Biochemistry (α) Activity 03: Hydrolysis of Starch

III. 1% Potato Starch Solution

Stage of Colour Observed Time
Theoretical Explanation
Hydrolysis HCl Saliva HCl Saliva

1st Drop Dark Dark Green 1 minute 1 minute Iodine detected more starches in
Yellow the solution.
(POSITIVE)

2nd Drop Dark Dark Green 3 minutes 3 minutes Iodine detected more starches in
Yellow the solution.
(POSITIVE)

3RD Drop Pale Green 6 minutes 6 minutes Iodine detected more starches in
Yellow the solution.
(POSITIVE)

4th Drop Pale Light 9 minutes 9 minutes Iodine detected more starches in
Yellow Green the solution.
(POSITIVE)

5th Drop Pale Light 12 minutes 12 minutes Iodine detected more starches in
Yellow Green the solution.
(POSITIVE)

6th Drop Clear Pale 40 minutes 40 minutes Iodine detected no starches in the
Yellow solution. Starches has been
hydrolyzed into glucose.
(NEGATIVE)

B.
I. 5% Potato Starch Solution
Stage of Colour Observed Time
Theoretical Explanation
Hydrolysis HCl Saliva HCl Saliva
Iodine detected starch in the
1ST Drop Yellow Gray 1 minute 1 minute solution. (POSITIVE)

Iodine detected starch in the

2nd Drop Dark Red Dark Blue 9 minutes 9 minutes solution. (POSITIVE)

Iodine detected starch in the

3rd Drop Gold- Dark Blue 15 minutes 15 minutes solution. (POSITIVE)
brown

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BC 102: Biochemistry (α) Activity 03: Hydrolysis of Starch

II. 1% Flour Starch Solution

Stage of Colour Observed Time
Theoretical Explanation
Hydrolysis HCl Saliva HCl Saliva

1st Drop Yellow Dark 1 minute 1 minute Iodine detected more starches in
Brown Yellow the solution.
(POSITIVE)

2nd Drop Yellow Dark 3 minutes 3 minutes Iodine detected more starches in
Brown Yellow the solution.
Green (POSITIVE)

3RD Drop Yellow Green Blue 6 minutes 6 minutes Iodine detected more starches in
the solution.
(POSITIVE)

4th Drop Yellow Light 9 minutes 9 minutes Iodine detected more starches in
Green the solution.
(POSITIVE)

5th Drop Yellow Green 12 minutes 12 minutes Iodine detected more starches in
the solution.
(POSITIVE)

6th Drop Pale Yellow 40 minutes 40 minutes Iodine less starches in the
Yellow solution.
(TURNING NEGATIVE-
SALIVA, NEGATIVE FOR
HCI)

III. 5% Fluor Starch Solution

Stage of Colour Observed Time
Theoretical Explanation
Hydrolysis HCl Saliva HCl Saliva

1st Drop Mustard Gray 1 minute 1 minute Iodine detected starch in the
solution. (POSITIVE)

2ND Drop Black Dark Blue 9 minutes 9 minutes Iodine detected starch in the
solution. (POSITIVE)

3RD Drop Dark Red Dark Blue 15 minutes 15 minutes Iodine detected starch in the
solution. (POSITIVE)

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 BC 102: Biochemistry (α) Activity 03: Hydrolysis of Starch

C.

RESULTS AND DISCUSSIONS: (You can write at the back or add a bond paper if you need
more space.)

Starting with 1 % concentrated starches (CS, PS, AP) boiled for 1 minute. We have gathered data
that the appearance of all the solutions gives positive results for polysaccharide detection. The same
appearance was found until after 6 drops and 40 minutes of boiling. The starches appeared to have
a light color or almost no color in each solution. We therefore concluded that during the last stage
of hydrolysis, in 40 minutes of boiling, the starches have been completely hydrolyzed, meaning that
to show that there are only glucose molecules governing the solution and that the glycosidic
linkages of starches have been hydrolyzed, or in other words, broken by the acid and enzyme.

Furthermore, for the case of starches with a 5% concentration filled with HCI AND Amylase,
separately (CS, PS, AP). Due to time conflict, we have only gathered data up to 3 stages of hydrolysis.
However, despite the conflicts encountered, we still gathered important data, like, during the first
drop, basically, the solution appears to have a darker color shade. This indicates that after 1 minute
of boiling, the starches are still far from being hydrolyzed. After our second drop (9 minutes), we
then discover that the temperature given by the boiling process is still not enough to completely
break the glycosidic linkages that starches have. And thereafter, in the last third drop, the solution is
starting to show lighter shades than the first and second drops. Therefore, after 15 minutes of
boiling of 5 % concentrated starches, the starches have finally shown an obvious indicator that the
starches have been slowly broken into their monosaccharide glucose by the enzyme and acid. Note
that, during the last drop, the starches have not been completely hydrolyzed but are close to being
hydrolyzed.

And for the comparison of data between the HCI and Amylase found in saliva. Through our
experiments, we have found that HCI gives the fastest rate of hydrolysis than amylase. And we
found that HCI appears to like being exposed to prolonged heat more than Amylase. As a result, as
the HCI is exposed to heat for a longer period of time, the rate of hydrolysis increases based on the
time that the solution begins to show a lighter shade. Unlike saliva being heated, it gives a slower
rate of hydrolysis. And we have connected this to the idea that the amylase enzyme is a protein.
Therefore, when amylase is exposed to extreme heat, the amylase gets denatured. And this idea
has happened during our experiment. There, we have concluded that it's not recommendable to
have the solution with amylase exposed to heat for a longer period of time as this may cause the
rate of hydrolysis to get slower.

REFERENCES: At least 3. Follow proper citation. (You can use this site:
https://www.citationmachine.net/)

https://www.ucl.ac.uk/~ucbcdab/enzass/temperature.htm

https://laney.edu/cheli-fossum/wp-content/uploads/sites/210/2012/01/10-Enzymes.pdf

https://www.youtube.com/watch?v=kquaf6GPhpc
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BC 102: Biochemistry (α) Activity 03: Hydrolysis of Starch

REPORT QUESTIONS:

1. What is the final product of hydrolysis for all of the three solutions?

The intended final product for all the two solutions is to have a clear color solution to indicate
that starches have been completely hydrolyzed by amylase and HCI. However, due to time
conflict, we are only able to discover that during the 6 th drop, with 40 minutes of boiling, the
solution finally produces a pale-yellow color, meaning to show that the starches have
already/almost been completely hydrolyzed into their monosaccharide glucose. While in the
other solution, with a 5% concentration, we are only able to do 3 stages hydrolysis. And during
our last stage of 30 minutes boiling, the solution appeared to give a slight light color, especially in
the case of HCI, while the solution with saliva appeared to give a blue-red color, indicating there
were still more starches in the solution.

2. What is the active enzyme present in a human saliva? Which bonds are in the carbohydrate
structure are attacked by this enzyme?

α-Amylase is the major digestive enzyme present in saliva. And it hydrolyses α-1,4 glycosidic
linkages in starch carbohydrates.

3. Which between the concentrated HCl and the Saliva has the fastest hydrolysis reaction?
How is this so?

The HCI with 5% concentration appeared to be the fastest solution to be hydrolyzed

compared to the solution with 1%. For this case, we have concluded that it might be caused
by the increased concentration that the 5% have. Thus, emphasizing the idea that the
higher the concentration, the faster the rate of enzyme activity. Furthermore, we also
consider the idea that boiling time/temperature also influenced the acid activity- the
higher the temperature, the easy-faster it is to be hydrolyzed.