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Processes of Digestion Simulation Lab
Activity 1:
Salivary Amylase Digestion of Animal Starch
Tube no. 1 2 3 4 5 6 7
Additives Amylase
Starch
pH 7.0
buffer Amylase
Starch
pH 7.0
buffer Amylase
Deionized
water
pH 7.0
buffer Deionized
water
Starch
pH 7.0
buffer Deionized
water
Maltose
pH 7.0
buffer Amylase
Starch
pH 2.0
buffer Amylase
Starch
pH 9.0
buffer
Incubation
condition Boil first,
incubate at 37oC 37oC 37oC 37oC 37oC 37oC 37oC
Benedict's
test (-) (+++)
A lot of Sugar (-) (-) (+++)
A lot of Sugar (++) (+)
IKI test (+) (-) (- ) (+) (-) (+) (+)
What do tubes 2, 6, and 7 reveal about pH and amylase activity?
- The results show that the activity max of amylase is @ 7.0, where pH 2.0 and 9.0 show very little activity.
Which pH buffer allowed the highest amylase activity?
- 7.0 showed highest amylase activity
Which tube indicates that the amylase did not contain maltose?
- Tube 3
Which tubes indicate that the deionized water did not contain starch or maltose?
-Tubes: 3, 4 , and 5
If we left out control tubes 3, 4, or 5, what objections could be raised to the statement: "Amylase digests starch to maltose"?
- Tube 3: shows that water didn’t have maltose or starch contamination.
- Tube 4: starch control, with same water, and showed NO maltose
- Tube 5:was a maltose control, which also had water and showed NO starch
- IF these three control tubes ( 3, 4, and 5) hadn’t been done, then whats’ perceived as digestion, might actually be maltose or starch contamination.
Would the amylase present in saliva be active in the stomach? Explain.
-No, because the pH in the stomach is too LOW.
What effect does boiling have on enzyme activity?
- Boiling deactivates, or denatures enzymes
Activity 2:
Enzyme Digestion of Animal Starch and Plant Starch
Tube no. 1 2 3 4 5 6 7
Additives Amylase
Animal
Starch
pH 7.0
buffer Amylase
Starch
pH 7.0
buffer Amylase
Glucose
pH 7.0
buffer Amylase
Cellulose
pH 7.0
buffer Amylase
Cellulose
Deionized
water
Peptidase
Starch
pH 7.0
buffer Bacteria
Cellulose
pH 7.0
buffer
Incubation
condition 37oC 37oC 37oC 37oC 37oC 37oC 37oC
Benedict's
test + + + -- -- -- +
IKI test -- -- -- + + + --
Which tubes showed a positive test for the IKI reagent?
- Tubes: 4,5, and 6
Which tubes showed a positive test for the Benedict's reagent?
- Tubes: 1,2,3, and 7
What was the effect of freezing tube 1?
- There was no effect.
How does the effect of freezing differ from the effect of boiling?
- Freezing doesn’t restrict enzyme activity, whereas boiling does.
What was the effect of amylase on glucose in tube 3? Explain.
- There was no effect.
-Glucose cannot be broken down further by amylase.
What was the effect of amylase on cellulose in tube 4?
? Amylase has no effect on the cellulose in tube 4
Popcorn and celery are nearly pure plant starch or cellulose. What can you conclude about the digestion of cellulose, judging from the results of test tubes 4, 5, and 7?
- That Cellulose is digestible by bacteria
What was the effect of the different enzyme, peptidase, used in tube 6? Explain based on what you know about peptidase.
-Peptidase doesn’t work on carbohydrate substrates, so it has no effect on digestion of these molecules
Activity 3:
Pepsin Digestion of Protein
Tube no. 1 2 3 4 5 6
Additives Pepsin
BAPNA
pH 2.0
buffer Pepsin
BAPNA
pH 2.0
buffer Pepsin
Deionized
water
pH 2.0
buffer Deionized
water
BAPNA
pH 2.0
buffer Pepsin
BAPNA
pH 7.0
buffer Pepsin
BAPNA
pH 9.0
buffer
Incubation
condition Boil first,
incubate at 37oC 37oC 37oC 37oC 37oC 37oC
Optical
density 0.00 0.40 0.00 0.00 0.03 0.00
Which pH provided the highest pepsin activity?
- pH 2.0
Would pepsin be active in the mouth? Explain.
- No, because the pH in the mouth is around 6.0
How did the results of tube 1 compare with those of tube 2?
- Boiling denatured the pepsin, which results in not being able to digest the protein.
Tubes 1 and 2 contained the same substances. Explain why their optical density measurements were different.
- There was no breakdown of the protein by the boiled pepsin. Because of that, there was no color change and no change in optical density in tube 1.
If you had not run the tube 2 and 3 samples, what argument could be made against the statement "Pepsin digests BAPNA"?
- Using water with pepsin shows that :
- without BAPNA there is no substrate. (The color change isn’t due just to the pepsin.)
What do you think would happen if you reduced the incubation time to 30 minutes? Use the simulation to help you answer if you are not sure.
- There will be less digestion in Tube 2
What do you think would happen if you decreased the temperature of the incubation to 10 oC ? Use the simulation to help you answer.
- There should be less digestion at a lower temperature, due to the face that the enzyme is most efficient at 37 * C ( which is body temp. )
Activity 4:
Pancreatic Lipase Digestion of Fats and the Action of Bile
Tube no. 1 2 3 4 5 6
Additives Lipase
Veg. oil
Bile salts
pH 7.0
buffer Lipase
Veg. oil
Deionized
water
pH 7.0
buffer Lipase
Deionized
water
Bile salts
pH 9.0
buffer Deionized
water
Veg. oil
Bile salts
pH 7.0
buffer Lipase
Veg. oil
Bile salts
pH 2.0
buffer Lipase
Veg. oil
Bile salts
pH 9.0
buffer
Incubation
condition 37oC 37oC 37oC 37oC 37oC 37oC
pH 6.21 6.72 9.00 7.0 2.00 8.97
Explain the difference in activity between tubes 1 and 2.
- The contents of tube 1 were boiled, which inactivated the pepsin; therefore there was no activity in tube 1
- Boiling inactivates, or denatures enzymes.
Can we determine if fat hydrolysis has occurred in tube 6? Explain.
- No because there isn’t much of a difference in tube 3
Which pH resulted in maximum lipase activity?
- 7.0
Is this method of assay sufficient to determine if the optimum activity of lipase is at pH 2.0?
- NO
-The buffer is 2.0 in tube 5, therefore the pH is 2.0
-The pH isn’t low because there was fat digestion.
- It’s low due to the buffer-? not optimum activity.
In theory, would lipase be active in the mouth?
-Pancreatic lipase would because the pH is close to 7.0
BUT:
- Lingual lipase works best in acidic environments
Would it be active in the stomach?
- Lingual Lipase would be
- Pancreatic would NOT
Explain your answers.
- The pH in your mouth is 6.5- 7.0
- Lingual lipase is produced by the salivary glands and isn’t active until there is an acidic environment.
- Pancreatic lipase likes a more neutral - basic environment. Therefore, it would be a little active in the mouth but would still work better in the small intestine.
- Bile salts in the small intestine break up fat globules into smaller molecules so lipase can work.
Based on the enzyme pH optima you determined, where in the body would we expect to find the enzymes in these experiments?
Amylase -- *Mouth
*Small Intestine
Pepsin -- *Stomach
Lipase -- * A small amount in the stomach- due to lingual lipase and no bile.
• A lot of digestion in small intestine by pancreatic lipase and presence of bile.
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