OBJECTIVE

To examine the action of some of the key digestive enzymes and the factors which alter their activities.

MATERIALS
Amylase solution (400 units per 100 ml)
Test tubes (15 – 20 ml)
Test tube clamps
Measuring cylinders (10 ml)
Watch glass pH paper
Water baths with test tube racks
Hard-boiled egg whites
1% acetic acid
1% pancreatin solution in 0.2% Na2CO3
0.5% starch paste
Lugol’s solution
Benedict’s solution
5% pepsin solution
0.5% HCl
Concentrated HCl
0.5% NaOH
Ice
Powdered litmus
Dairy cream
Bile salts

METHODS AND PROCEDURE

1. Collect 10 ml of your own saliva in a graduated cylinder. Dilute the saliva with an equal amount of
water if you are unable to collect 10 ml.

2. A commercial Amylase solution (400 units per 100 ml) may be used as a substitute for saliva.
3. Place a small amount of saliva in a watch glass and add a few drops of 1% acetic acid. A precipitate
indicated that mucin (a glycoprotein) is present.

4. Prepare and label four test tubes as follows:

Tube 1 Tube 2 Tube 3 Tube 4

3 ml starch
3 ml starch 3 ml starch 3 ml starch (cooled) +
+ + 3 ml saliva + 3 ml saliva
3 ml water 3 ml saliva (cooled) +
↓ in ↓ in ↓ in 5 drops conc. HCl
37˚C water bath 37˚C water bath Ice bath ↓ in
37˚C water bath

5. After the tubes have incubated for d1 hour, pour half of each tube’s contents into a new test tube.
Test one set of tubes for starch using Lugol’s solution and the other set for maltose using Benedict’s
solution.

a. Starch Test: Add 3 drops of Lugol’s solution to each tube. A dark purple colour indicates
the presence of starch. Shades of reddish brown indicate lesser amounts of starch. Rate
the amount of starch (+++), (++), (+), or (-).

b. Maltose Test: Add 4 ml of Benedict’s solution to each tube and place in a boiling water bath
for 2 minutes. Remove the tubes using a clamp and compare the concentration of maltose
using the following scale: (+++) red, (++) orange-yellow, (+) green, (-) blue.

ACTIVITY 2

1. Place thin slices of cooked egg white in four test tubes. It is important to make these slices the same
size (about 0.5 cm2) and as thin as possible.

2. Add the following solutions to the tubes and determine the ph of each tube:

Tube 1 Tube 2 Tube 3 Tube 4

5 ml pepsin 5 ml pepsin
5 ml pepsin 5 ml HCl
(5% solution) (5% solution)
(5% solution) (0.5%)
+ +
+ +
5 ml HCl 5 ml NaOH
5 ml water 5 ml water
(0.5%) (0.5%)
3. Allow the tubes to incubate in a 37˚C water bath for 1 hour. Test the final pH of the solutions and
estimate the amount of protein digestion using a scale of (+++), (++), (+), and (-) to compare the four
tubes.

ACTIVITY 3

1. In each of two test tubes (A and B) place 3 ml of distilled water and 3 ml of vegetable oil. To
tube B add a small pinch of bile salts. Shake each tube for 30 seconds and observe it for several
minutes.

2. Add litmus powder to dairy cream until a blue colour is produced. Preincubate the litmus
cream and a 1% pancreatin solution at 37˚C for 5 minutes. Prepare a series of test tubes as
follows:

Tube 1 Tube 2 Tube 3 Tube 4

3 ml cream 3 ml cream
3 ml cream 3 ml cream + +
+ + 3 ml pancreatin 3 ml water
3 ml pancreatin 3 ml water + +
Pinch bile salts Pinch bile salts

3. Incubate all tubes in a 37˚C water bath for 1 hour, or until colour change occurs in one tube.
Blue litmus will turn pink in an acid environment. Test the pH using pH paper, and note the
colour of each tube.

RESULTS

Tube Initial Final Estimated Explanation

DISCUSSION

Based on this experiment, we had observed the effect of digestion of protein in different environment.
It is known that protein is digested by the enzyme pepsin that existed in our stomach. For this
experiment, we have prepared four test tubes with thin slice of eggs submerged in four different
environment. In the first test tube, the egg slice was submerged in the mixture of pepsin and
hydrochloric acid. The initial pH of the solution was 1 as the HCl is acidic. After incubated for an hour in a
37 degree water bath, the final pH is 2. This result is actually opposed to the theory. Because
theoretically, pepsin need HCl to be activated and the presence of HCl along with pepsin accelerate the
process of digestion. Supposedly, the pH must be lower than the initial pH because when the protein is
digested it will produce amino acid making the pH of the solution to actually decrease. We had
concluded that there are errors in determining the exact pH value of the solution using only the pH
paper.
In the second test tube, the egg slice was submerged in the mixture of pepsin and water. The
initial pH of the solution was 4 which was higher than the first test tube due to lack of acidic solution.
The initial pH and final pH is the same which is 4. This is because test tube 2 does not have acidic
environment to activate the pepsin enzyme. So the pepsin enzyme cannot work properly and there is no
digestion occur for the egg slice in test tube 2.
In the third test tube, the egg slice was submerged in the mixture of HCl and water. The initial
pH of the solution was 1. The initial pH and final pH is the same which is 1. Theoretically, HCl can actually
hydrolyze and digest protein but the rate of reaction is very slow. It would take too long for the protein
to be digested with only HCl without the presence of pepsin because pepsin is the protein catalyst. And
a catalyst speeds up the digestion. Thus, that’s why the pH of the solution in the test tube remain
unchanged because the reaction is too slow and does not occur much for the amino acid to change the
pH of the solution.
In the fourth test tube, the egg slice was submerged in the mixture of pepsin and NaOH. The
initial pH of the solution was 12. The initial pH and final pH is the same which is 12. This is because the
alkaline mixture prevents the enzyme to be activated. Hence the protein digestion cannot occur because
pepsin is not activated. Thus, the pH of the solution remained unchanged because of the absence of
amino acid as there are no digestion of the protein occur in the test tube.

Conclusion

Test tube