PhysioEx - Digestive
PhysioEx - Digestive
PhysioEx - Digestive
Processes of Digestion
Lu, Mangaba, Nieveras, Ortiz, Ramos, Yatco
Activity 1
Assessing Starch Digestion by Salivary Amylase
Objectives
1. Explain how enzyme activity can be assessed with enzyme assays
a. IKI assay
b. Benedict’s assay
2. Define enzyme, catalyst, hydrolase, substrate and control
3. Discuss specificity of amylase action
4. Discuss the possible effect of changes in temperature and pH on amylase
activity
Overview of Enzymes
● Enzymes have an optimal temperature and an optimal pH for maximum
activity.
● Human digestive enzymes have an optimal temperature equal to body
temperature (37°C) and most have an optimal pH near neutral.
● If the temperature or pH is too high, or if the pH is too low, enzymes will be
denatured
● Digestive enzyme are hydrolytic enzymes (hydrolases), which aids in the
breaking down of organic food molecules
○ By adding water to the molecular bonds, cleaving it between subunits
Introduction
● Focus of this activity is to investigate the hydrolysis of starch to maltose by salivary
amylase
● Starch digestion by amylase:
Amylase
Starch Water Maltose
● Salivary amylase: enzyme produced by salivary glands and is secreted in the mouth
● Enzyme assay: laboratory method of measuring enzymatic activity
○ IKI assay: test for the presence of starch
■ Blue or black: positive starch test
■ Diluted IKI color: negative starch test
○ Benedict’s assay test: for the presence of reducing sugars (glucose or maltose)
■ Green to reddish-brown: positive sugar test
■ No color changes: negative sugar test
Methodology
Methodology
Amylase
Starch Water Maltose Maltose Maltose
● Salivary amylase: enzyme produced by salivary glands and is secreted in the mouth
● Enzyme assay: laboratory method of measuring enzymatic activity
○ IKI assay: test for the presence of starch
■ Blue or black: positive starch test
■ Diluted IKI color: negative starch test
○ Benedict’s assay test: for the presence of reducing sugars (glucose or maltose)
■ Green to reddish-brown: positive sugar test
■ No color changes: negative sugar test
Starch & Cellulose
● Both are found in plants
○ Starch serves as energy storage
○ Cellulose provides rigidity to their cell walls
● Both are polymers of glucose
○ Starch: α-1,4-glycosidic bonds
○ Cellulose: β(1→4) glycosidic bonds
Methodology
Methodology
- The results in tube 1 and tube 2 are the same because amylase hydrolyzes
starch to maltose.
- Benedict’s test is a test used to detect the presence of reducing sugars
Review Questions
2. Describe the result in tube 3. How well did the results compare with your
prediction?
- As predicted, it will test positive in Iodine test and will test negative in
Benedict’s test.
- Amylase cannot hydrolyse cellulose
Review Questions
3. Describe the usual substrate for peptidase.
What is a peptide?
The stomach contains chief cells which secretes a protein digesting enzyme
called pepsin. The pepsin hydrolyzes peptide bonds to form simpler and free
amino acids.
In this activity the students will use BAPNA as a substrate that asses pepsin
activity. BAPNA is a synthetic “peptide” that releases a yellow dye product when
hydrolyzed. It will turn yellow in the presence of an active peptidase such as
pepsin.
Methodology
1. We place 1 test tube in each slot(until 6) in the incubator.
2. We filled the test tubes with the ff:
to measure how much yellow dye was released when BAPNA was “digested.”The
greater the optical density, the more BAPNA digestion by pepsin occurred.
Tube # 2 turns yellow because of the presence of pepsin, BAPNA and a pH of 2.0
which has an acidic environment
Tube # 5 turns slightly yellow since pepsin and BAPNA is added but with the pH of
which is 7.0 is neutral
Review Questions
1. Describe the effect that boiling had on pepsin and how you could tell that it
had that effect.
At boiling temperature, the enzyme will denaturate and then it will not have any
activity against the BAPNA, as shown in in tube 1 with optical density of 0.00 and
no color change.
Review Questions
2. Was your prediction correct about the optimal pH for pepsin activity? Discuss
the physiologic correlation behind your results.
As seen in the test tube 2 the optimal pH for pepsin activity is 2.0, compared to
tube 5 and 6 the optical density was higher in tube 2.
Review Questions
3. What do you think would happen if you reduce the incubation time to 30
minutes for tube 5?
Basically it won't have enough time to break down as much BAPNA as it would
have with longer time.
Conclusion
An optical density greater than zero indicates that BAPNA digestion has occurred
and pepsin is active. It also indicates that more hydrolysis occurs.
The optimal pH of pepsin is at 2.0 which indicates that the environment is acidic.
This shows that pepsin is the most active in an acidic environment especially in
the stomach.
3. Tubes were then placed in a pH meter to measure the final pH of the test
solutions.
Methodology
Experiment Data
Results & Discussion
Tube 1 investigated the action of bile on enzyme activity, and tube 2 examined
lipase activity without bile,
In test tube 5, even with the presence of bile salts and lipase, fat digestion does
not occur because the buffer (pH of 2.0) is already quite acidic.
Review Questions
1. Explain why lipase activity can’t be fully tested in tube 5.
Since the pH in test tube #5 is already very low, it is difficult to tell if fatty acids are
released.
Review Questions
2. Which tube has the highest lipase activity?
The correct prediction is tube #1, 7.0, which approximates the pH of the small
intestine
Review Questions
3. Explain why pancreatic lipase would be active in both the mouth and pancreas.
Since the activity of pancreatic lipase is highest at pH 7.0, the enzyme should be
active in the mouth and the pancreas.
Review Questions
4. Explain the process of bile emulsification of lipids and how it improves lipase
activity.