Investigating the Digestion of Starch, Protein and Lipid by Enzymes

Aim

This experiment investigates whether digestive enzymes break down starch, protein and lipid in food samples, using food tests to detect the substrate before and after the enzyme acts on it.

Variables

• Manipulated variable: presence of an active digestive enzyme (amylase for starch, pepsin or trypsin for protein, lipase with bile salts for lipid) compared with a boiled (denatured) enzyme used as a control.
• Responding variable: result of the relevant food test, the iodine test for starch, the Biuret test for protein, and the emulsion test for lipid, recorded before and after incubation.
• Controlled variables: temperature of incubation (such as a 37°C water bath), incubation time, pH of the mixture, and the volume and concentration of the substrate and the enzyme.

Materials and apparatus

• Starch solution, egg albumin (protein) solution, and cooking oil
• Amylase solution
• Pepsin or trypsin solution
• Lipase solution and bile salts
• Iodine solution
• Biuret reagent (or copper sulfate solution and sodium hydroxide solution)
• Ethanol (for the emulsion test)
• Water bath set at a constant temperature
• Test tubes, a test tube rack and a stopwatch
• Pipettes or droppers

Procedure

1. For starch: mix starch solution with amylase solution in a test tube, and incubate at a constant temperature such as 37°C.
2. At regular intervals, test a sample of the mixture with iodine solution and record whether it still turns blue-black.
3. Continue testing until the iodine solution no longer changes colour, showing that the starch has been fully digested.
4. For protein: mix egg albumin (protein) solution with pepsin or trypsin solution in a separate test tube, and incubate at the same temperature.
5. At regular intervals, test a sample with Biuret reagent and record whether the purple colour is still produced.
6. Continue testing until the Biuret test no longer gives a purple colour, showing that the protein has been digested.
7. For lipid: mix cooking oil with lipase solution and a few drops of bile salts in a test tube, and incubate at the same temperature.
8. At regular intervals, carry out the emulsion test on a sample and note whether a cloudy white emulsion still forms.
9. For each substrate, set up a control tube using the same enzyme that has first been boiled (denatured), and carry out the same food tests on it throughout the experiment.
10. Compare the results from the active-enzyme tubes with the boiled-enzyme control tubes.

Expected results

Active enzymes break down their substrate over time, so the positive food test fades or disappears, while the boiled-enzyme control still gives a positive result throughout. The table below summarises the typical pattern observed.

Conclusion

Digestive enzymes break down their specific substrates: amylase breaks starch into reducing sugars, pepsin or trypsin breaks protein into peptides and amino acids, and lipase (helped by bile salts, which emulsify the fat) breaks lipids into fatty acids and glycerol. This is shown by the loss of the positive food-test result in each active-enzyme tube over time.

The boiled-enzyme control stays positive throughout, because boiling denatures the enzyme so it can no longer catalyse digestion, confirming that the changes in the active-enzyme tubes are due to enzyme action, not some other factor.

Safety

Common mistakes

Paper 3-style questions

Question 1 (hypothesis). A student mixes starch solution with amylase and keeps it at 37°C. State a hypothesis for what the iodine test will show over time, and the result that would support it.

Model answer. Hypothesis: amylase digests the starch, so the iodine test stops giving a blue-black colour over time. It is supported if samples taken at intervals eventually give a brown iodine colour, showing the starch has been broken down.

Question 2 (variables and fair test). State the manipulated and responding variables when comparing the active-amylase tube with a boiled-amylase control, and one variable that must be controlled.

Model answer. The manipulated variable is whether the amylase is active or boiled (denatured); the responding variable is the iodine-test result (blue-black or brown). A controlled variable is the temperature and incubation time, kept the same for both tubes so the comparison is fair.

Question 3 (tabulation). The student tests a sample with iodine every two minutes. Suggest how the results should be recorded.

Model answer. Results are recorded in a table with the columns time / min and iodine-test colour (or 'starch present / absent'). This shows at which time the blue-black colour is first lost, marking when the starch has been fully digested.

Question 4 (inference). The boiled-amylase control still turns iodine blue-black after 20 minutes, but the active-amylase tube does not. What inference can the student make?

Model answer. The inference is that digestion of the starch was caused by the amylase acting as a catalyst, because boiling denatured the enzyme in the control so it could not break down the starch. The change is due to enzyme action, not to time or temperature alone.

Source:SRC-DSKP-EN

Frequently asked questions