Experiment 1: Enzymes in Food;Amylase is used by humans to facilitate digestion. Specifically, it is an enzyme;which breaks down starch molecules into sugar 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 IodinePotassium 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).;Materials;(1) 2 oz. Bottle (Empty);(1) 100 mL Graduated;Cylinder;30 mL Iodine-Potassium;Iodide, IKI;Permanent Marker;Ruler;2 Spray Lids;30 mL Starch (liquid);*Cutting Board;*2 Food Products (e.g., ginger;root, apple, potato, etc.);*Kitchen Knife;*Paper Towel;*Saliva Sample;*Tap Water;*You Must Provide;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.;Set up a positive control for this experiment by spraying a paper towel;with the starch solution. Allow the starch to dry for approximately one;hour (this time interval may vary by location).;4.;In the mean time, set up a negative control for this experiment. Use your;knowledge of the scientific method and experimental controls to;establish this component (hint: what should happen when IKI solution;contacts something that does not contain starch?) Identify your negative;control in Table 1.;Note: Be sure to space the positive and negative controls apart from;each other to prevent cross-contamination.;5.;When the starch solution has dried, test your positive and negative;controls. This step establishes a baseline color scale for you to evaluate;the starch concentration of the food products you will test in Steps 7 11. Record your results in Table 1.;6.;Select two food items from your kitchen cabinet or refrigerator.;7.;Obtain a kitchen knife and a cutting board. Carefullycut your selected;food items to create a fresh surface.;Figure 3: Sample set-up.;8.;Gently rub the fresh/exposed area of the food items on the dry, starchsprayed paper towel back and forth 10 - 15 times. Label where each;specimen was rubbed on the paper towel with a permanent marker;(Figure 3).;9.;Provide a saliva sample by spitting in a separate bowl and rubbing the;paper towel in the saliva. Be sure not to spit on the paper towel directly;as you may unintentionally cross-contaminate your samples. Repeat;this step until you are able to adequately moisten the paper towel.;10.;Wait five minutes.;11.;Hold the IKI spray bottle 25 - 30 cm away from the paper towel, and;mist with the IKI solution.;12.;The reaction will be complete after approximately 60 seconds. Observe;where color develops, and consider what these results indicate. Record;your results in Table 1.;Table 1: Substance vs. Starch Presence;Substance;Resulting Color;Presence of;Starch?;Positive Control: Starch;Negative Control: Student Must Select;Food Product;Food Product;Saliva;experiment 2: Effect of Temperature on Enzyme Activity;Figure 4: Catalase catalyzes;the decomposition of;hydrogen peroxide to water;and oxygen.;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 lab 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.;Materials;(2) 250 mL Beakers;3 Balloons;30 mL 3% Hydrogen;Peroxide, H2O2;Measuring Spoon;Permanent Marker;Ruler;3 Test Tubes (Glass);Test Tube Rack;Thermometer;Yeast Packet;*Hot Water Bath;*Stopwatch;20 cm String;*You Must Provide;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 10 mL hydrogen peroxide. Then, keep one of the test;tubes 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;a.;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).;b.;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.;c.;If using a stove, place a small pot on the stove and turn the stove;on to a medium heat setting.;d.;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.;e.;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 100 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 hydrogen peroxide.;6.;Record the temperatures of each condition in Table 2. Be sure to;provide the thermometer with sufficient time in between each;environment to avoid obscuring the temperature readings.;7.;Let the tubes sit for 15 minutes.;8.;During the 15 minutes prepare the balloons with yeast by adding tsp.;of yeast each balloon. 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.;9.;Carefully stretch the neck of the balloon to help ensure it does not rip;when stretched over the opening of the test tube.;10.;Attach the neck of a balloon you prepared in step 8 to the top of Tube 2;(the room temperature test tube) making sure to not let the yeast spill;into the test tube yet. Once the balloon is securely attached to the test;tube lift the balloon and allow the yeast to enter the test tube. Tap the;bulb of the balloon to ensure all the yeast falls into the tube.;11.;As quickly and carefully as possible remove the Tube 1 (cold) from the;refrigerator and repeat steps 9 - 10 with Tube 1 using a balloon you;prepared in step 8.;12.;As quickly and carefully as possible remove Tube 3 (hot) from the hot;water bath and repeat steps 9 - 10 with Tube 3 using a balloon you;prepared in step 8.;13.;Swirl each tube to mix, and wait 30 seconds.;14.;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.;Table 2: Balloon Circumference vs. Temperature;Tube;1 - (Cold);2 - (RT);Temperature;(C);Balloon;Circumference;(Uninflated, cm);Balloon;Circumference;(Final, cm);3 - (Hot);To conduct your laboratory exercises, use the Laboratory Manual located under;Course Content. Read the introduction and the directions for each exercise/experiment;carefully before completing the exercises/experiments and answering the questions.;Save your Lab 4 Answer Sheet in the following format: LastName_Lab4 (e.g.;Smith_Lab4).;You should submit your document as a Word (.doc or.docx) or Rich Text Format;(.rtf) file for best compatibility.;Pre-Lab Questions;1. How could you test to see if an enzyme was completely saturated during an experiment?;2. List three conditions that would alter the activity of an enzyme. Be specific with your;explanation.;3. Take a look around your house and identify household products that work by means of an;enzyme. Name the products, and indicate how you know they work with an enzyme.;Experiment 1: Enzymes In Food;Data Tables and Post-Lab Assessment;Table 1: Substance vs. Starch Presence;Substance;Resulting Color;Presence of Starch?;Positive Control: Starch;Negative Control: Student Must;Select;Food Product;Food Product;Saliva;Post-Lab Questions;1. What were your controls for this experiment? What did they demonstrate? Why was;saliva included in this experiment?;2. What is the function of amylase? What does amylase do to starch?;3. Which of the foods that you tested contained amylase? Which did not? What;experimental evidence supports your claim?;4. Saliva does not contain amylase until babies are two months old. How could this affect;an infants digestive requirements?;5. There is another digestive enzyme (other than salivary amylase) that is secreted by the;salivary glands. Research to determine what this enzyme is called. What substrate does it;act on? Where in the body does it become activated, and why?;6. Digestive enzymes in the gut include proteases, which digest proteins. Why dont these;enzymes digest the stomach and small intestine, which are partially composed of protein?;Experiment 2: Effect of Temperature on Enzyme Activity;Data Tables and Post-Lab Assessment;Table 2: Balloon Circumference vs. Temperature;Tube;Temperature (C);Balloon;Circumference;(Uninflated, cm);1 - (Cold);2 - (RT);3 - (Hot);Post-Lab Questions;1. What reaction is being catalyzed in this experiment?;2. What is the enzyme in this experiment? What is the substrate?;Balloon Circumference;(Inflated, cm);3. What is the independent variable in this experiment? What is the dependent variable?;4. How does the temperature affect enzyme function? Use evidence from your data to support;your answer.;5. Draw a graph of balloon diameter vs. temperature. What is the correlation?;6. Is there a negative control in this experiment? If yes, identify the control. If no, suggest how;you could revise the experiment to include a negative control.;7. In general, how would an increase in substrate alter enzyme activity? Draw a graph to;illustrate this relationship.;8. Design an experiment to determine the optimal temperature for enzyme function, complete;with controls. Where would you find the enzymes for this experiment? What substrate would;you use?
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