Materials Included In Kit

Additional Materials Required

Prelab Preparation

Prepare the following solutions up to five days in advance of the lab.

• Use 100 mL of DI water to prepare the 2% albumin (protein) solution. Add 10 mL of the DI water to the 2 g of albumin in the bottle. Allow the albumin to soak for several hours, then add 90 mL of DI water. Gently shake the bottle to mix and refrigerate.
• Use 100 mL of DI water to prepare the 1% glucose solution. Add 100 mL DI water to the 1 g of glucose powder in the bottle and mix. Refrigerate.
• Use 100 mL of boiling DI water to prepare the 1% starch solution. Add a small amount of the boiling DI water to the 1 g of starch in the bottle. Cap the bottle and gently shake. Continue to add 10 mL of boiling water to the bottle until the entire 100 mL of boiling water has been added. Allow the solution to slowly cool to room temperature and refrigerate.

Prepare the following solutions up to two days in advance.

• Use 100 mL of DI water to prepare the 1% litmus–milk solution. Add 100 mL DI water to the 1 g of litmus–milk powder in the bottle and mix. May be prepared up to two days in advance and refrigerated.

Prepare the following solution the day of the lab.

• Prepare the 1% pepsin solution by adding the 100 mL of 0.01 M hydrochloric acid to the 1 g of pepsin in the bottle. The solution should have a pH of 1.5 to 2.5.
• Use 100 mL of DI water to prepare the 1% lipase solution and the 1% amylase solution. Add 100 mL DI water to the each bottle and mix.
• Prepare the boiling water bath for the Benedict’s test.
  1. Fill a 250-mL beaker about one-third full with hot tap water.
  2. Add several boiling stones.
  3. Heat the water on a hot plate to boiling.

Safety Precautions

Biuret test solution contains copper(II) sulfate and sodium hydroxide and is a corrosive liquid. It is moderately toxic by ingestion and is dangerous to skin and eyes. Benedict’s solution contains copper(II) sulfate, sodium citrate and calcium carbonate; it is moderately toxic by ingestion and a skin and body tissue irritant. Hydrochloric acid is severely toxic by ingestion and is corrosive to skin and eyes. Iodine solution contains iodine and potassium iodide and is an eye and skin irritant; it will stain skin and clothing. Avoid contact of all chemicals with eyes and skin. Wear chemical splash goggles and chemical-resistant gloves and apron. Remind students to wash their hands thoroughly with soap and water before leaving the laboratory. Please review current Safety Data Sheets for additional safety, handling and disposal information.

Disposal

Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. All of the waste solutions and excess Benedict’s reagent may be disposed of down the drain with plenty of excess water according to Flinn Suggested Disposal Method #26b. Excess biuret solution may be neutralized with acid and then disposed of according to Flinn Suggested Disposal Method #10. Excess hydrochloric acid may be disposed of by neutralizing with base and then disposing of down the drain with plenty of excess water according to Flinn Suggested Disposal Method #24b. Excess iodine solution may be reduced with sodium thiosulfate solution and then disposed of down the drain with water according to Flinn Suggested Disposal Method #12a.

Lab Hints

• Enough materials are provided in this kit for 30 students working in pairs or for 15 groups of students. This laboratory activity can reasonably be completed in one 50-minute class period. The prelaboratory assignment may be completed before coming to lab, and the data compilation and calculations may be completed the day after the lab.
• Table sugar (sucrose) is not a reducing sugar. It will give a negative Benedict’s test.
• Biuret test solution does not contain the compound biuret. Biuret is the simplest compound that gives a positive test result with Biuret test solution.

Teacher Tips

• Catalysts cause slow reactions to occur more quickly by lowering the activation energy necessary for the reaction to occur. A ski lift is an analogy for a catalyst. If the reaction is “skiing,” then the skier must first get to the top of the ski hill. One option is for skiers to climb to the top and once they reach the top, enjoy the potential energy they earned as they ski back down the hill. The ski lift allows many more skiers to reach the top of the hill very quickly without the skiers expending much energy. Once at the top, they still enjoy the same energy release as they ski down the ski hill. The reaction can occur many, many more times and the ski lift (the catalyst) is not changed during the process.
• For an advanced class you may want to use this activity to introduce the terms substrate, active site, co-factors, coenzymes and the induced fit theory.
• In order to help students understand that amylase is found in saliva, have each student chew on an unsalted, unsweetened saltine type cracker until the cracker tastes sweet. The sweetness is due to the amylase hydrolyzing the starch into glucose and other mono- and disaccharides. Complete this activity in a food-appropriate area.
• This experiment may be extended into an inquiry-based laboratory in which the students determine the optimal pH and temperature for each enzyme.

Further Extensions

This experiment may be extended into an inquiry-based laboratory in which the students determine the optimal pH and temperature for each enzyme.

Answers to Prelab Questions

1. Define the term enzyme.

   Enzymes are globular, three-dimensional proteins with specific characteristic shapes that act as catalysts in the body.

2. For each of the following enzymes, describe the type of food digested, the essential nutrient that results from the digestion, and the organ where digestion occurs: (a) amylase, (b) pepsin and (c) lipase.
   1. Amylase digests starch yielding glucose in the mouth.
   2. Pepsin digests protein into polypeptides in the stomach.
   3. Lipase digests fat into fatty acids and glycerol in the small intestine.

Sample Data

Answers to Questions

1. Compare and contrast the observations of the biuret test results for test tube 1 and test tube 2. Describe the evidence, if any, for the digestion of protein using pepsin.

   Test tube 1 contains albumin, a protein and biuret. The solution is a cloudy, blue-purple color. It is positive for polypeptides. Test tube 2 contains pepsin which digests the albumin protein. As the solution heats, solids can be seen in the solution. The biuret test solution is a clear, pink-purple color. It is positive for peptides.

2. The pepsin solution was prepared using 0.01 M hydrochloric acid in order to optimize the pepsin enzyme. Why was this necessary?

   Pepsin is active in the acidic environment of the stomach. A basic or neutral pH would inactivate the enzyme.

3. Compare and contrast the observations of the test results for test tube 3 and test tube 4. Describe the evidence, if any, for the digestion of butterfat using lipase.

   Test tube 3 contains litmus and buttermilk, a fat. The solution is blue because the buttermilk is basic. Test tube 4 contains lipase which hydrolyzes the buttermilk fat into fatty acids creating an acidic solution. The litmus appears pink in an acidic solution.

4. Compare and contrast the iodine test results for starch (test tube 5) and starch/amylase (test tube 6). Explain the test results based on the activity of amylase.

   Test tube 5 is the control sample yielding a positive iodine test for starch. Test tube 6 has a negative iodine test because the amylase has hydrolyzed the starch to glucose.

5. Explain the results of the Benetict’s test for the starch–amylase solution in test tube 7. What are the products of the digestion of starch by amylase?

   Test tube 7 yielded a positive Benedict’s test result matching the positive control for Benedict’s in test tube 8. The positive Benedict’s test indicates that the amylase has hydrolyzed the starch into a reducing sugar.

6. Summarize the digestion of a sausage pizza served with a tall glass of milk. Indicate the enzymes responsible for digestion in the mouth, stomach and small intestine.

   Amylase in the mouth starts digesting the starch of the pizza crust. Pepsin and acid begin digesting the sausage proteins in the stomach. The carbohydrates leave the stomach first and are digested into simple sugars in the small intestine by sucrase, maltase, lactase and pancreatic amylase. The protein digestion of the sausage continues in the small intestine when the three peptidases finish splitting the protein into amino acids for absorption into the bloodstream. Nucleases in the small intestine convert nucleic acids into nucleotides. Finally, fats are hydrolyzed into fatty acids and glycerol by lipases in the small intestine.

Introduction

People must eat to live but how does the body transform a pizza into the essential nutrients (peptides, amino acids, fatty acids and glucose) it needs to carry out cell processes and cell growth? This activity explores the biochemistry of digestion.

Concepts

• Catalysts
• Enzymes
• Digestion
• Gastrointestinal tract

Background

The human body is composed of millions of cells that need oxygen, water, and nutrients to survive. The amazing transformation of food into the simpler molecules that can be absorbed by the body for use by the cells is called digestion. Digestion occurs in the gastrointestinal (GI) tract, which is also called the alimentary canal (see Figure 1). The GI tract is a mucous membrane-lined tube that extends from the mouth to the anus. While in the GI tract, food is first mechanically broken down and then chemically treated with acids, bases and enzymes within the organs of the digestive system. Enzymes are biochemical catalysts. A catalyst is any substance that causes a chemical reaction to occur without being permanently altered in the process. A single molecule of catalyst can perform the same reaction thousands of times a second. Enzymes are globular, three-dimensional proteins with characteristic shapes that allow only a few specific substances called substrates, to temporarily bond with the enzyme. Because of the exclusive nature of enzyme/substrate binding, the human body contains thousands of different enzymes that are needed to catalyze all the different biochemical reactions that must occur.

Digestion begins in the mouth. The food mixes with saliva while the teeth grind the food. The tongue shapes the food and saliva mixture into a ball called a bolus (plural, boluses). Saliva provides the first chemical treatment of the food. Saliva is composed of a neutral pH mixture of water, mucus, proteins, mineral salts, and the enzyme amylase. Amylase begins the breakdown of starch, a carbohydrate, into glucose (see Figure 2). Glucose is the sugar used during cellular respiration as a source of cellular energy. The bolus is swallowed for further digestion in the stomach. Gastric juices in the stomach contain mucus, hydrochloric acid, pepsinogen and small amounts of other enzymes. Hydrochloric acid acts to denature (uncoil) the proteins in food and activates pepsinogen, the inactive precursor of the enzyme pepsin. Since pepsin would digest the muscular walls of the stomach along with the food proteins, the inner layer of stomach cells secrete viscous alkaline mucus that coats the inside of the stomach. The mucus protects the stomach walls from the action of pepsin. The gastric juices are mixed with the bolus by movements of the stomach wall, producing a very thick liquid called chyme. The amount of time that chyme spends in the stomach depends upon the type of food present. Foods that are predominantly carbohydrates pass through the stomach quickly, followed by high-protein foods, and finally by high-fat foods, which may spend several hours in the stomach. Glucose, alcohol, fat-soluble drugs, some salts, and small amounts of water are absorbed through the walls of the stomach directly into the bloodstream for transport to the liver, where they are metabolized or sent on to other cells in the body. Movements by the stomach wall muscles, called peristaltic waves, push the chyme toward the bottom of the stomach where the stomach connects to the small intestine. Once the first section of the small intestine is full, the chyme combines with excretions from the pancreas, liver, and the small intestine. Pancreatic juice from the pancreas, bile salts from the liver, and excretions from the epithelial cells of the small intestine contain enzymes that are capable of completing the digestion of carbohydrates, proteins, nucleic acids and fats.

The digestion of carbohydrates into glucose and other simple sugars is completed in the small intestine by the enzymes sucrase, maltase, lactase, and pancreatic amylase. The resulting sugars are absorbed through the mucous lining of the small intestine into the bloodstream for transport to the liver where they are converted to glucose, glycogen or fat. Glycogen is used for intermediate energy storage. The partially digested proteins from the stomach are still too large to be absorbed through the small intestine. Pancreatic juice contains three peptidases that complete the digestion of protein into amino acids for absorption into the bloodstream. Each peptidase in the pancreatic juice is very specific and splits the bonds only between particular combinations of amino acids. Chyme also contains the nucleic acids of the plant and animal cells that were ingested. Nucleases found in the pancreatic juice convert these nucleic acids into nucleotides, which are absorbed and transported to the liver.

Fats (lipids) are hydrolyzed into fatty acids and glycerol by intestinal and pancreatic lipase with help from bile salts secreted by the liver. Hepatic cells of the liver produce bile, which is stored in the gall bladder before being excreted into the small intestine. Bile salts help with the digestion of fat globules by acting like soap. The globules of fat are small clumps of lipids that stick together in the chyme. Bile salts break the globules into smaller drops, creating greater surface area for pancreatic lipase to break the lipids into fatty acids and glycerol.

Once the nutrients produced by the enzymes have been absorbed by the small intestine, they travel to the liver where they are metabolized, if necessary, before being transported by the blood and lymph to every cell in the body. The material remaining in the small intestine travels to the large intestine where more mucous is added and where water and electrolytes are absorbed before the “waste” is expelled from the body.

Experiment Overview

The purpose of this experiment is to visualize the products of enzyme digestion in the human gastrointestinal tract. Activity 1 highlights the digestion of protein in the stomach by the enzyme pepsin. Activity 2 illustrates the digestion of milk fat by intestinal lipase into fatty acids and the resulting change in pH. Activity 3 uses iodine and Benedict’s reagent to visualize the digestion of starch to glucose by amylase in the mouth.

Materials

Prelab Questions

1. Define the term enzyme.
2. For each of the following enzymes, describe the type of food digested, the essential nutrient that results from the digestion, and the organ where digestion occurs: (a) amylase, (b) pepsin and (c) lipase.

Safety Precautions

Biuret test solution contains copper(II) sulfate and sodium hydroxide and is a corrosive liquid. It is moderately toxic by ingestion and is dangerous to skin and eyes. Benedict’s solution contains copper(II) sulfate, sodium citrate and calcium carbonate; it is moderately toxic by ingestion and a body tissue irritant. Iodine solution contains iodine and potassium iodide and is an eye and skin irritant; it will stain skin and clothing. Avoid contact of all chemicals with eyes and skin. Wear chemical splash goggles and chemical-resistant gloves and apron. Wash hands thoroughly with soap and water before leaving the laboratory. Follow all laboratory safety guidelines.

Procedure

Part A. Protein Digestion

1. Use a marker to label two test tubes 1 and 2.
2. Use a clean, graduated pipet to add 3 mL of the 2% protein solution to test tube 1.
3. Add 1 mL of the 2% protein solution to test tube 2.
4. Use a clean, graduated pipet to add 2 mL of the 1% pepsin solution to test tube 2. Gently swirl the test tube to mix the contents.
5. Place both test tubes in a 40 °C water bath for 15 minutes.
6. Remove the test tubes from the water bath and use a clean, graduated pipet to add 1 mL of biuret test solution to each test tube.
7. Observe the color and appearance of the resulting solution in each test tube and record the observations in the data table on the Digestive Enzyme Worksheet. Note: Biuret test solution is bluish-purple in the presence of polypeptides and lavenderpink in the presence of amino acids.
8. Answer Questions 1 and 2 on the Digestive Enzyme Worksheet.

Part B. Fat Digestion

9. Use a marker to label two test tubes 3 and 4.
10. Use a clean, graduated pipet to add 2 mL of the 1% litmus–milk solution to test tube 3. This solution contains buttermilk, a fat.
11. Add 1 mL of the litmus–milk solution to test tube 4.
12. Use a clean, graduated pipet to add 1 mL of the 1% lipase solution to test tube 4. Gently swirl the test tube to mix the contents.
13. After 3 minutes, record the color of the solutions in the data table on the Digestive Enzyme Worksheet. Note: Litmus is a pH indicator. Litmus appears blue in basic solutions and pink in acidic solutions.
14. Answer Question 3 on the Digestive Enzyme Worksheet.

Part C. Carbohydrate Digestion

15. Use a marker to label four test tubes 5–8.
16. Use a clean, graduated pipet to add 1 mL of the 1% starch solution to each test tube 5, 6 and 7.
17. Use a clean, graduated pipet to add 1 mL of the 1% amylase solution to test tubes 6 and 7.
18. Use a clean graduated pipet to add 1 mL of the 1% glucose solution to test tube 8.
19. Gently swirl the test tubes to mix the contents.
20. Allow the test tubes to sit undisturbed for about 2 minutes.
21. Test the starch–amylase solution for starch.
    1. Use a clean, graduated pipet to add 4–6 drops of iodine solution to test tubes 5 and 6.
    2. Record the color of the resulting solutions in the data table on the Digestive Enzyme Worksheet. Note: Test tube 5 is a positive (control) sample for the iodine–starch test. Iodine forms a blue-black colored complex with starch.
22. Test the starch–amylase solution for glucose.
    1. Use a clean, graduated pipet to add 1 mL of Benedict’s reagent to test tubes 7 and 8.
    2. Place the test tubes in a boiling water bath using a test tube clamp.
    3. After 2–3 minutes, remove the test tubes from the hot water bath using a test tube clamp.
    4. Record the color of the resulting solutions in the data table on the Digestive Enzyme Worksheet. Note: Test tube 8 is a positive (control) sample for Benedict’s test. Benedict’s reagent contains Cu²⁺(aq) which, when heated, reacts with glucose to form a red, orange or mustard colored precipitate.
23. Answer Questions 4 and 5 on the Digestive Enzyme Worksheet.

Student Worksheet PDF

10818_Student1.pdf