HUMAN PHYSIOLOGY

LAB REPORT

ABCT2326

Digestion Lab Report

Name: Cheung Chi Ming

Student Number: 23086215D
Programme: BSc (Hons) in Physiotherapy
Team member’s names:
Chau Cho Yin, Chen Hao, Cheung Chak Lam, Cheung Chi Kit
 A. Digestion in mouth: The action of salivary amylase(α-amylase)

RESULTS
1. Iodine test

Time 0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0

(min)
Tube A Dark Dark Dark Dark Pale Pale Brown Brown Brown
blue blue blue blue blue blue
Tube B Dark Dark Dark Dark Dark Dark Dark Dark Dark
blue blue blue blue blue blue blue blue blue
Tube C Dark Dark Dark Dark Dark Dark Dark Dark Dark
blue blue blue blue blue blue blue blue blue
2. pH test and Benedict’s test

Tube A B C
pH 7 7 2
Benedict’s test Red precipitate (+++ Blue solution (-) Blue solution (-)
+)

DISCUSSION
Experiment A aims to test the activity of α-amylase on the digestion of starch. Amylase is a kind of
enzyme present in our saliva, and it can hydrolyze starch into mainly maltotriose and maltose
(Dhital, Warren, Butterworth, Ellis & Gidley, 2017). Regarding the iodine test results, a color
change in test tube A is observed at the 3-minute time mark. The color of content in tube A,
consisting of α-amylase and starch only, turns from dark blue to dark brown, and further decolorizes
to orange brown by the 4-minute mark. On the other hand, there is no color change in tube B
containing boiled amylase and starch only, and in test tube C containing α-amylase, HCl and starch.
The results imply that starch is digested in test tube A but not in test tube B and C at the end of the
experiment.

In test tube A, the digestion of starch occurred due to the activity of α-amylase, which is an enzyme
produced by the salivary glands and the pancreas. Hydrolysis of starch molecules into maltose and
glucose is catalyzed by α-amylase. Tube A has a color change from dark blue to dark brown to
orange brown, indicating the starch in test tube A is gradually being digested. By the fourth minute,
the brown color of the content in test tube A shows that all starch has been digested. Iodine solution
turns dark blue in the presence of starch because of an intense blue-black complex formed by the
amylose in starch and triiodide anion (Pesek et al., 2022). Without dark blue color, test tube A hence
contains no starch at the end of the experiment. However, tubes B and C remains dark blue
throughout the experiment, so starch is still present in tubes B and C. The reason for the inability to
digest starch is the denaturation of α-amylase in both test tubes B and C. The α-amylase is
denatured by high temperature in tube B and extreme pH in tube C, the configuration of the active
site is altered so the active site of α-amylase can no longer bind to the substrate starch to form
enzyme-substrate complexes. Hence, the hydrolysis of starch cannot be catalyzed by α-amylase,
and the starch remains undigested in tubes B and C.

In the pH test, tubes A and B both have a pH of 7 but tube C has a pH of 2. Due to hydrochloric
acid in tube C, its content is highly acidic as it is a strong acid. Nonetheless, the starch and amylase
in tubes A and B are all neutral so the pH value of contents in tube A and B are both 7.

In Benedict’s test, red precipitate is observed in tube A but not in the other two tubes. Red
precipitate, as known as the positive result in Benedict’s test, indicates the presence of reducing
sugar in tube A. This further proves that the α-amylase is tube A is functional and able to digest
starch into the reducing sugar maltose and glucose. For tubes B and C, the solutions inside remain
blue in color, which is a negative result showing the absence of reducing sugar and inability of
starch digestion by denatured α-amylase in test tube B and C.

All three test tubes are incubated at 37°C to mimic the digestion process in the human body. Test
tube has demonstrated how starch is digested in oral cavity by the α-amylase, secreted by salivary
glands during ingestion. On the other hand, tube B has demonstrated the digestion of starch in the
stomach, where α-amylase is denatured by strong hydrochloric acid. The digestion environment is
highly acidic in stomach so the denatured α-amylase can no longer digest starch.

B. Digestion in the stomach: the action of pepsin

RESULTS
Time Tube D E F G
Beginning pH 7 2 7 2
After 1 hour Precipitate Present Absent Present Present
 incubation Cloudiness +++ + +++ +++
Coagulation test Insoluble No Insoluble Insoluble
by saturated precipitate precipitate precipitate precipitate
(NH4)2SO4

DISCUSSION
The pH test at the start of the experiment shows that tubes D and F have a pH of 7, since the
distilled water, egg albumin and pepsin inside the tubes are all neutral. Tubes E and G both have a
pH value of 2 due to the presence of hydrochloric acid.

Among the 4 test tubes, precipitate is found in tubes D, F and G, but not in tube E. The precipitate is
the egg albumin, and the presence of precipitate indicates that egg albumin is not broken down in
tubes D, F and G. In tube D, the pH is not optimal for the action of pepsin, and at neutral pH the
pepsin is denatured (Compos & Sandro, 2003), the egg albumin remains undigested. In tubes F and
G, the pepsin used is boiled. The high temperature has denatured the pepsin, and the configuration
of the active site of the boiled pepsin is altered, pepsin can no longer bind to the substrate to form
the enzyme-substrate complex. Hence, the breakdown of albumin cannot be catalyzed by the boiled
pepsin in tubes F and G. In tube E, hydrochloric acid provides the optimum pH for pepsin to digest
the egg albumin, since the optimum pH of pepsin is around 2 (Compos & Sandro, 2003).
Hydrochloric acid is added to tube E to mimic the digestion environment in stomach, which is the
optimum pH for the action of pepsin. The active pepsin catalyzes the breakdown of albumin to
smaller and soluble peptides, so the turbidity of content in tube E is smaller.

The coagulation test aims to determine the presence of albumin. Saturated ammonium sulphate is
added to each test tube. Ammonium sulphate will react with egg albumin to form an insoluble
precipitate. The precipitates formed in tubes D, F and G indicate that the albumin remains
undigested. Nonetheless, in tube E, the absence of precipitate shows that the albumin is digested by
pepsin. Without albumin, there is no reaction between albumin and ammonium sulphate and
therefore no precipitate.

Tube E mimics the digestion of proteins by the action of pepsin in the stomach. We can note that
pepsin is most active in a highly acidic environment, in terms of digestion of proteins. Chief cells in
the stomach secrete pepsinogen, the inactive proenzyme, while parietal cells secrete hydrochloric
acid (HCl) to provide the optimum pH for pepsin activity. Pepsinogen is converted to pepsin, the
active proteolytic enzyme, by HCl in the gastric lumen. Pepsin then catalyzes the breakdown of
protein into small and soluble peptides, which can be further digested into amino acids in the
duodenum. Moreover, the peptides in chyme and nervous stimulation will stimulate the secretion of
gastrin by G cells. Gastrin then further stimulates the parietal and chief cells to secrete gastric juice,
so the digestion of proteins in stomach can be facilitated.

C. Digestion in the duodenum: the action of pancreatic enzymes

RESULTS

i) Test tubes H and H’

At the beginning

Time/ pH 0 10 20 30 40 50 60 70 80
s
H 8 Dark Dark Dark Pale Pale Pale Pale Brown Brown
blue blue blue blue blue brow brow
H’ 3 Dark Dark Dark Dark Dark Dark Dark Dark Dark
blue blue blue blue blue blue blue blue blue
At the end

Tube H H’
pH 8 3
Benedict’s test Red precipitate (++++) Blue (-)

ii) Test tubes J and J’

Time Tube J Tube J’

Beginning pH 8 3
After 1 hour Precipitate Absent Present
incubation in water Turbidity + ++
bath at 37°C Coagulation test by No precipitate Insoluble precipitate
(NH4)2SO4
 iii) Test tubes K and K’

Tube K Tube K’
pH at the beginning 8 3
pH after 1 hour of incubation 4 3
in water bath at 37°C

DISCUSSION
i) Test tubes H and H’

According to the results of the iodine test, only tube H has color change. The color of content in
tube H changes from dark blue to pale blue by 30 seconds, and then changes from pale blue to
brown by 70 seconds. After 70 seconds, the color remains brown since starch is completely broken
down by pancreatic amylase. However, tube H’ remains dark blue throughout the entire experiment,
as hydrochloric acid added to tube H’ has denatured the pancreatic amylase under extreme pH,
starch therefore remains undigested. As a result, H shows a positive result in Benedict’s test due to
the presence of maltose and glucose, the reducing sugar coming from the digestion of starch. On the
other hand, tube H’ shows a negative result in Benedict’s test due to the absence of maltose and
glucose from the digestion of starch. Tube H has a pH value of 8 due to the alkaline pancreatic juice
with a pH range of 8.3 to 8.6 (Itoyama et al., 2022), while tube H’ has a pH value of 3 due to the
hydrochloric acid added.

ii) Test tube J and J’

Precipitate is present in tube J’ but absent in tube J, and the content in tube J is more turbid than that
in tube J’. Proteolytic enzymes like proteases and peptidases in pancreatic juice will break down the
albumin into smaller and soluble peptides and amino acids in tube J, so there is no precipitate
formed in tube J. While the hydrochloric acid added in tube J’ provides a low pH which denatures
the proteolytic enzymes in the pancreatic juice, so the albumin in tube J’ remains undigested. In the
coagulation test, precipitate is absent in tube J but present in tube J’, because the albumin is
digested by proteolytic enzymes in tube J but not in tube J’.

iii) Test tubes K and K’

In tube K, the pH experiences a drop from 8 to 4 after incubation in the water bath at 37°C. During
incubation at 37°C, lipase in pancreatic juice catalyzes the breakdown of lipids to glycerol and fatty
acids, which are acidic. Due to the fatty acids formed in the digestion of lipids in tube K, the pH in
tube K drops from 8 to 4. On the other hand, the pH value in tube K’ remains unchanged at 3 after
incubation. At a low pH value of 3, the lipases are denatured. Hence, the lipids in the olive oil
cannot be digested by the denatured lipases in pancreatic juice in tube K’, and the pH remains
unchanged.

Experiment K mimics the digestion by the pancreatic enzymes in pancreatic in the duodenum.
When chyme arrives at the duodenum, the hormone secretin and cholecystokinin stimulate the
secretion of pancreatic juice. Pancreatic acini are stimulated to secrete pancreatic enzymes,
including lipases, α-amylase and proteolytic enzymes. These pancreatic enzymes break down the
ingested food into smaller and simpler molecules so that they can be absorbed in ileum.
Chyme from the stomach is acidic, the acidic nature of chyme is simulated by the addition of HCl
in tubes H’, J’ and K’. The acidic chyme will be neutralized by the bile juice containing sodium
bicarbonate when the chyme enters the duodenum. A slightly alkaline environment can therefore
facilitate the maximal action of the pancreatic enzymes.

Reference
Dhital, S., Warren, F. J., Butterworth, P. J., Ellis, P. R., & Gidley, M. J. (2017). Mechanisms of starch digestion by α -
amylase—Structural basis for kinetic properties. Critical Reviews in Food Science and Nutrition, 57(5), 875–892.
https://doi.org/10.1080/10408398.2014.922043
Pesek, S., Lehene, M., Brânzanic, A. M. V., & Silaghi-Dumitrescu, R. (2022). On the origin of the blue color in the
iodine/iodide/starch supramolecular complex. Molecules, 27(24), 8974. https://doi.org/10.3390/molecules27248974
Campos, L. A., & Sancho, J. (2003). The active site of pepsin is formed in the intermediate conformation dominant at
mildly acidic pH. FEBS Letters, 538(1–3), 89–95. https://doi.org/10.1016/S0014-5793(03)00152-2
Itoyama, S., Noda, E., Takamatsu, S., Kondo, J., Kawaguchi, R., Shimosaka, M., Fukuoka, T., Motooka, D., Nakamura,
S., Tanemura, M., Mitsufuji, S., Iwagami, Y., Akita, H., Tobe, T., Kamada, Y., Eguchi, H., & Miyoshi, E. (2022).
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bacterial flora. JGH Open, 6(1), 85–90. https://doi.org/10.1002/jgh3.12703