Experiment 1: Enzymes in Food

Amylase is used by humans to facilitate digestion. Specifically, it is an enzyme that breaks down starch molecules into glucose molecules. This is why people sometimes observe a sweet taste after sucking on a starch-containing food for an extended period of time. Amylase is found naturally in human saliva and the pancreas. However, it is also present in some of the common plant foods consumed by humans.

This experiment tests for the presence of amylase in food by using Iodine-Potassium Iodide, IKI. IKI is a color indicator used to detect starch. This indicator turns dark purple or black in color when in the presence of starch. Therefore, if the IKI solution turns to a dark purple or black color during the experiment, one can determine that amylase is not present (because presence of amylase would break down the starch molecules, and the IKI would not change color).

Procedure:

1. Remove the cap from the starch solution. Attach the spray lid to the starch solution.
2. Rinse out the empty two ounce bottle with tap water. Use the 100 mL graduated cylinder to measure and pour 30 mL of IKI into the empty two ounce bottle. Attach the remaining spray lid to the bottle.
3. Use a permanent marker to create four quadrants of equal size by drawing two perpendicular lines that intersect in the center of the paper towel (Figure 3).

1. Spray the sectioned paper towel and another blank paper towel with the starch solution. Allow the starch to dry for approximately one hour (this time interval may vary by location).
2. Once the paper towels are completely dry, label the blank paper towel “saliva” and set it aside to be used in Step 14.
3. Use the permanent marker to label the top left quadrant of the sectioned paper towel “positive control” and the top right quadrant “negative control”.
4. Select two food items from your kitchen cabinet or refrigerator, and obtain a fresh ginger root sample.

Note: If ginger root is not available, use banana as a positive control. Be sure to adjust data tables and labels accordingly.

1. Obtain a kitchen knife and a cutting board. Carefully cut your selected food items and the ginger root to expose the internal surfaces.
2. Set up a positive control for this experiment. Use the permanent marker to add the label “ginger root” under the positive control label. Carefully rub an area in the positive control quadrant with the fresh surface of the ginger root (which is known to contain amylase). Ensure a good application by firmly pressing the exposed surface of the root onto the paper towel. Let the area rest.
3. Set-up a negative control for this experiment in the “negative control” quadrant. Use your knowledge of the scientific method and experimental controls to establish this component. Identify your negative control in Table 1.

Note: What result would you expect if amylase was not present when tested with IKI solution?

1. Use the permanent marker to label the bottom two quadrants with the name of the two food items you chose to test for amylase.
2. Carefully, but firmly, rub the fresh/exposed area of the food items on the paper towel in their designated quadrants back and forth 10 – 15 times.
3. Allow the areas to dry for at least five minutes.
4. While you wait, provide a saliva sample by spitting into the center of the paper towel labeled “saliva” that you prepared with starch in Step 5. Spread the saliva out by gently rubbing the halves of the paper towel together.
5. After your food samples and controls have rested for at least five minutes, test them with IKI solution.

Note: Use caution when spraying the IKI solution. Hold the IKI spray bottle 25 – 30 cm away from the paper towel, and mist the IKI solution over each quadrant.

1. Allow the IKI solution to dry. Observe where and what color develops, and consider what these results indicate. Record your results in Table 1.
2. Take a picture of your results. Include a note with your name and date on an index card in the picture. Insert picture here.

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Experiment 2: Effect of Temperature on Enzyme Activity

Yeast cells contain catalase, an enzyme which helps convert hydrogen peroxide to water and oxygen. This enzyme is very significant as hydrogen peroxide can be toxic to cells if allowed to accumulate. The effect of catalase can be seen when yeast is combined with hydrogen peroxide (Catalase: 2 H₂O₂ → 2 H₂O + O₂).

In this experiment, you will examine the effects of temperature on enzyme (catalase) activity based on the amount of oxygen produced. Note, be sure to remain observant for effervescence when analyzing your results.

Procedure

1. Use a permanent marker to label test tubes 1, 2, and 3. Place them in the test tube rack.
2. Fill each tube with 9 mL hydrogen peroxide. Then, keep Test Tube 2 in the test tube rack, but transfer the two additional test tubes to two separate 250 mL beakers.
3. Find one of the balloons, and the piece of string. Wrap the string around the uninflated balloon and measure the length of the string with the ruler. Record the measurement in Table 2.
4. Create a hot water bath by performing the following steps:
   1. Determine if you will use a stovetop or microwave to heat the water. Use the 100 mL graduated cylinder to measure and pour approximately 200 mL of water into a small pot or microwave-safe bowl (you will have to measure this volume in two separate allocations).
   2. If using a stovetop, obtain a small pot and proceed to Step 4c. If using a microwave, obtain a microwave-safe bowl and proceed to Step 4e.
   3. If using a stove, place a small pot on the stove and turn the stove on to a medium heat setting.
   4. Carefully monitor the water in the pot until it comes to a soft boil (approximately 100 °C). Use the thermometer provided in your lab kit to verify the water temperature. Turn the stove off when the water begins to boil. Immediately proceed to Step 5.

      CAUTION: Be sure to turn the stove off after creating the hot water bath. Monitor the heating water at all times, and never handle a hot pan without appropriate pot holders.

   5. If using a microwave, place the microwave-safe bowl in the microwave and heat the water in 30 second increments until the temperature of the water is approximately °C. Use the thermometer provided in your lab kit to verify the water temperature. Wait approximately one minute before proceeding to Step 5.
5. Place Tube 1 in the refrigerator. Leave Tube 2 at room temperature, and place Tube 3 in the hot water bath.

Important Note: The water should be at approximately 85 °C when you place Tube 3 in it. Verify the temperature with the thermometer to ensure the water is not too hot! Temperatures which exceed approximately 85 °C may denature the enzymes.

1. Let the tubes sit for 15 minutes in their respective condition.
2. During the 15 minutes, prepare three balloons by using a sheet of paper to create a funnel by wrapping a sheet of paper into a cone shape with an opening at the point of the cone. Place the point of the funnel into the mouth of the balloon. One at a time, funnel ¼ tsp. of yeast into each of the three balloons. Make sure all the yeast gets settled to the bulb of the balloon and not caught in the neck. Be sure not spill yeast while handling the balloons.
3. After 15 minutes, use the thermometer to record the temperature of the hydrogen peroxide in Test Tube 2 in Table 2. Remove the thermometer after a reading has been recorded and set it aside.
4. Carefully stretch the neck of the balloon to help ensure it does not rip when stretched over the opening of the test tube. Remember, there is yeast inside of the balloons.
5. Attach the neck of a balloon you prepared in Step 7 to the top of Tube 2 (the room temperature test tube) making sure to not let the yeast spill into the test tube yet (Figure 5). Once the neck of the balloon is securely attached to the test tube lift the balloon so that is in a vertical position in line with the test tube and allow the yeast to drop from the bulb and enter the test tube. Tap or rub the sides of the balloon together to ensure all the yeast enters the test tube. The balloon should remain attached to the test tube.

Note: To ensure the balloon does not pop off of the test tube during the reaction, ensure the neck of the balloon extends a least two centimeters down the neck of the test tube.

1. As quickly and carefully as possible remove Tube 1 (cold) from the refrigerator and repeat Steps 8 – 10 with Tube 1 using a balloon you prepared in Step 7.
2. As quickly and carefully as possible remove Tube 3 (hot) from the hot water bath and repeat Steps 8 – 10 with Tube 3 using a balloon you prepared in Step 7.
3. Swirl each tube to mix the yeast and hydrogen peroxide. Observe the test tubes for at least two minutes.
4. Wrap the string around the center of each balloon to measure the circumference. Measure the length of string with a ruler. Record your measurements in Table 2.
5. Take a picture of your results. Include a note with your name and date on an index card in the picture. Insert picture here.

© 2014 eScience Labs, LLC.
All Rights Reserved