Biology 13A Lab #13: Nutrition and Digestion

Biology 13A Lab #13: Nutrition and Digestion Lab #13 Table of Contents: Expected Learning Outcomes.... 102 Introduction...... 103 Activity 1: Food Chemistry & Nutrition.. 104 Activity 2: Parts of the Digestive System.. 109 Activity 3: Chemical Digestion.... 109 Expected Learning Outcomes At the end of this lab, you will be able to list the essential nutrients found in food; describe the basic chemical composition of carbohydrates, proteins, fats, and vitamins; identify nutrient content in foods and test for nutrients in unknown samples; learn the parts of the digestive system; demonstrate how nutrients are broken down in the presence of digestive enzymes; and explain functions of major nutrients in the body. Figure 13.1 Digestive System Lab #13 Nutrition and Digestion 102

Introduction Food, glorious food! Movement, processing information and responding to the environment, and maintenance of homeostasis all require energy. Ultimately, energy is derived from food. In addition, food provides building material for cells and tissues. The job of the digestive system is to break down food and absorb nutrients: carbohydrates, proteins, and lipids and smaller quantities of vitamins and minerals. Most of the water we need also comes from food. Few foods combine all six nutrients. As primates, we are omnivores, adapted to eat a wide variety of foods to obtain a full complement of necessary nutrients. Our digestive system anatomy and physiology reflects the eclectic diet for which we are adapted. Releasing nutrients from food requires mechanical digestion where large pieces are crushed and ground primarily by teeth, with the aid of tongue and saliva. This increases the surface area for chemical digestion in which digestive enzymes break down complex large molecules such as proteins and carbohydrates to their basic components (e.g. amino acids and simple sugars). Chemical digestion is performed by many organs: for example, salivary glands produce amylase, an enzyme that breaks down starches (polysaccharides) to disaccharides; the pancreas and small intestine produce numerous enzymes that complete the breakdown of proteins, lipids, and carbohydrates to forms usable by cells. The nutrients are absorbed through the lining of the small intestine and are then transported throughout the body. Today we will examine nutrients in food, review the structures of the digestive system, and investigate enzymes responsible for chemical digestion. Testing Your Comprehension: Answer the following questions based on your reading of the introduction. 1. List the five nutrients found in food. 2. What is an omnivore? How does the fact that humans are omnivores influence our anatomy and behavior? 3. Where and how does mechanical digestion occur? 4. What molecules are necessary for chemical digestion? Give examples of organs that perform chemical digestion. Lab #13 Nutrition and Digestion 103

Activity 1: Food Chemistry & Nutrition Carbohydrates, proteins, lipids, and vitamins and minerals are the nutrients in food. Carbohydrates are either simple sugars (monosaccharides) consisting of a single sugar molecule such as glucose, or disaccharides, two monosaccharides joined together (e.g. sucrose, or table sugar), or polysaccharides, large chains of monosaccharides. Starch and glycogen are polysaccharides. They are major sources of energy for cellular work. Common sources of carbohydrates in the diet are breads, cereals, fruits and vegetables. Proteins have numerous functions. They are the basis for tissue and organ structure; some are capable of movement (socalled motor proteins ) while others act as enzymes. All proteins are chains of amino acids. Twenty amino acids combine to form thousands of different proteins. Twelve amino acids can be assembled in the body but eight must be obtained directly from the diet. Dietary sources of proteins include fish, soybeans, meat, and dairy products. Lipids are hydrophobic (insoluble in water). They include fats, oils, waxes, phospholipids, and steroids. Concentrated sources of energy, each gram of lipid has more calories than a gram of protein or carbohydrate. In addition to energy storage, lipids form the basic structure of plasma membranes and provide support for joints, tendons, and internal organs. Dietary sources of lipids include nuts, meat, butter and cheese, and vegetable oils. Although only minute quantities of vitamins and minerals are required, a deficiency can have devastating effects. Vitamins help control chemical reactions, often facilitating the actions of enzymes. They are necessary for normal growth and metabolism. Thirteen vitamins are essential for health four of those are fat soluble and are stored for months at a time in adipose tissue; nine are water soluble and must be regularly replaced. Minerals such as calcium and phosphorus are also derived from the diet and perform vital functions. Vitamins are obtained from a wide variety of foods. For example, vitamin C is obtained from citrus fruits and tomatoes whereas vitamin B is found in nuts, whole grains, and beans. Vitamin pills may supplement the diet. Each vitamin has specific functions in the body, leading to particular symptoms if there is a lack. The first symptom of vitamin C deficiency is fatigue, followed by anemia, back and joint pain, bleeding of the gums, and poor wound healing. With time, death ensues, as was the fate of numerous sailors who succumbed to scurvy. Lab #13 Nutrition and Digestion 104

Testing Your Comprehension: Answer the following questions based on your reading. 1. Define monosachharide, disaccharide, and polysaccharide, and give examples of each. 2. What is the basic building block of proteins? What are dietary sources of proteins? 3. List several functions of lipids. 4. What causes scurvy and what are some of its symptoms? Simple chemical tests using indicators can be used to determine the presence of nutrients in food. A color change of an indicator is usually a positive test for the presence of a certain nutrient. In this experiment, you will use several indicators to test for the presence of nutrients in solutions. This purpose of this lab is to demonstrate how different foods can contain one, some, or all four of these organic compounds that are important to cells. Summary of Activities 1. Make a hypothesis about the content of food samples you and your lab partners have brought from home. 2. Test each food, along with the appropriate positive and negative controls, for protein, monosaccharide, and complex carbohydrate. We will be using three different tests to identify protein, monosaccharide (glucose and/or fructose), and complex carbohydrates (starch) in various foods. Monosaccharides and starch are both carbohydrates. Monosaccharides are the small organic molecules that are the subunits used to build large starch macromolecules. Bring one food or drink to lab that you think contains one of these compounds as an experimental sample. Some examples of what to bring: juice or milk fruit or vegetables flour, or tofu, or bread anything that can mix with water NO: oily foods uncooked beans or pasta, or other dry unhydrated food dark-colored foods or drinks acidic, or sour foods like orange juice These items will not react well with the test chemicals. Foods will contain at least one type of organic compound. Before beginning, make a prediction about which type of compound or compounds you think your experimental sample contains. Record your prediction on your RESULTS page. You will perform three different tests on the same four experimental samples (share with your lab partners). Each test will use a different indicator reagent that will change color in the presence of the particular organic compound that is being tested for. As part of the experimental method, you must include control samples to insure the validity of your results. A control is a test sample with a known result. If your control samples do not give you the expected result, then your experimental results are not valid and you must reevaluate your experimental set-up (maybe your test chemicals are no good). A negative control will result in no change in color. It will either contain no sample at all or it will contain a nonreactive sample like water. For example, if you are testing for the presence of monosaccharides, the test chemical, called Benedict's solution, will remain the original color blue when mixed with water. A positive control will result in a color change indicating the presence of the compound you are testing for. For example, a 5% glucose solution will react with Benedict's solution and change it from blue to rust (brown-red). You can compare your experimental results to your control results to determine if you obtained a positive reaction. To Prepare Your Samples: 1. If your experimental sample is solid, chop or break it up into the smallest pieces possible using a razor blade or glass rod, whichever is Lab #13 Nutrition and Digestion 104

appropriate. Use a pinch of sample small enough so that the material can be easily suspended in 1.5 ml of water and does not fill the bottom of the tube. Lab #13 Nutrition and Digestion 105

Add 1.5 ml distilled water to your sample and mix it well. 2. If your sample is a liquid, add 1.5 ml of the liquid to the appropriate tube. 3. Each tube should have the same volume of fluid, regardless of the type of sample. I. Identification of Protein Materials: test tube rack test tubes tape for labeling your tubes 0.5% CuSO4 10% NaOH albumin protein solution (egg white) distilled water four different experimental samples (share with the people at your table) The test chemicals used in this experiment react with the covalent bonds that link amino acids together in protein chains. In the presence of protein, the chemicals will turn varying shades of purple. NOTE: NaOH (sodium hydroxide) is very caustic and will burn your skin and damage your clothes. Handle it with caution. If you do come into contact with it, notify the instructor and flush the area thoroughly with running water. NaOH is the ingredient in hair removal products like Nair. It works by dissolving protein, which is what hair is made of. Procedure: 1. Predict which organic compounds your experimental samples might contain. 2. Label your tubes 1 through 6. 3. Prepare your sample following the instructions from above. 4. CAUTIOUSLY add 20 drops of 10% NaOH solution to each tube. Agitate the tube gently. See instructor for demonstration. 5. Add 4 drops of 0.5% CuSO4 to each tube. Agitate the tubes again. 6. Let the tubes sit at room temperature a few minutes, until you see a color change in your positive control. 7. Record the results. II. Identification of Monosaccharides Materials: hot plate 400 ml beaker half filled with boiling water test tube clamp for picking hot test tubes Benedict's solution 5% glucose solution 5% fructose solution 5% sucrose solution distilled water 4 experimental samples Benedict's solution is a blue solution that will change color in the presence of monosaccharides, such as glucose or fructose. Benedict's changes from blue --> green --> orange --> brown, depending on the amount of monosaccharide present. Any change in color indicates the presence of monosaccharides. Procedure: 1. Set up a boiling water bath using a 400 ml beaker filled with approximately 150 ml of tap water on a hot plate. Add 2 or 3 boiling chips to the water in the beaker. These will prevent boiling water from slashing up when you add your test tubes. Set the hot plate on high to get the water boiling, then reduce the temperature to a setting of "2" to keep the water simmering. The water should be boiling BEFORE you place your tubes in it. 2. Label your tubes 1 through 8. If you are using tape, make sure the tape is high enough on the tube so that it does not get wet in the water bath. Otherwise, the tape will fall off and you will not be able to identify your samples. 3. Prepare your samples as before. 4. Add 1.5 ml of Benedict's solution to each test tube, for a total of 3 ml. 5. Agitate your tubes so that the sample and Benedict's is well mixed. See instructor for demonstration. Lab #13 Nutrition and Digestion 106

6. Place the all the test tubes at the same time in the boiling water bath for 5 minutes. 7. Remove the tubes using the test tube clamp and record the resulting color. Do not mix the tubes again once they have been boiled. If your sample was a solid, note the color change in the immediate vicinity of your sample. The rest of the Benedict's may stay blue since a solid cannot mix well. III. Identification of Starch (Complex Carbohydrate) Materials: Iodine solution (IKI) Starch suspension distilled water 4 experimental samples Iodine will react with starch and turn from a yellow/brown color to a purple/black color. Only the purple/black color is an indication of starch. Procedure: 1. Number your tubes. 2. Prepare your samples as before. Remember, each tube should have 1.5 ml of liquid. 3. Add 2-3 drops IKI to each tube. Agitate your tubes. 4. Record your results. 5 Lab #13 Nutrition and Digestion 105

Data Sheets: Turn in with lab report Before you begin, predict which organic compounds may be found in each of your samples. Experimental Samples #1: #2: #3: #4: Color of Sample Predicted Organic Compound(s) Part 1: Testing Samples Where Nutrient Content is Known In the first part of the experiment, you will determine the presence of nutrients in prepared samples where the contents are known, at least to the teacher! You won t know what s in them until you run the tests, then you can check to determine whether your results are accurate. We will then proceed to testing nutrient contents of a variety of foods. Procedure 1. Obtain 4 small plastic medicine cups. Use a wax pencil to label them from #1 through #4, for the four samples. 2. Pour approximately 5 ml of each of the samples into the corresponding labeled cups. Use the ml marks on the cup to measure the amount. 3. Each group should obtain 1 white spot plate. Each plate has 4 columns with 3 rows (figure 13.2). Use the wax pencil to number the wells 1 through 12 from left to right (e.g. 1, 5, and 9 will be in Col. 1). Use a plastic pipette to transfer 1 ml of Nutrient Sample #1 to each of the four wells across Row 1 of the spot plate. Rinse the pipette. Add 1 ml of Sample #2 to the four wells in Row #2 then rinse. Add 1 ml of Sample #3 to Row 3. Figure 13.2 Spot Plate Lab #13 Nutrition and Digestion 106

4. Use a clean pipette to add 1-2 drops of indophenol solution to Wells #1, 5, and 9 in column 1. Indophenol is an indicator for ascorbic acid (vitamin C). If ascorbic acid is present, it will bleach the indophenol and change the color from blue to colorless. Record observations in Table 13.1. If the color changed, put a + in the appropriate column. 5. Use a clean pipette and add 1-2 drops of iodine solution to Wells #2, 6, and 10 in Column 2. Iodine is an indicator for starch. If starch is present, the mixture will change color from yellow-brown to blue-black. Record observations in Table 13.1. 6. Use a clean pipette to add 1-2 drops of Biuret reagent to Wells # 3, 7, and 11 in Column 3 of the spot plate. In the presence of protein, the mixture will change color from light blue to pinkish or purplish violet. Record observations in Table 13.1. 7. Use a clean pipette to add 1-2 drops of Sudan III to Wells #4, 8, and 12 in Column 4. Sudan III is an indicator for lipids and if they are present, the mixture will stain red. Record observations in Table 13.1. Table 13.1 Results for Nutrient Sample Tests Sample1 Sample2 Sample3 Sample4 Color (+/-) Color (+/-) Color (+/-) Color (+/-) Indophenol Iodine Solution Biuret Reagent Sudan III Lab #13 Nutrition and Digestion 107

Part 2: Testing Samples Where Nutrient Content is Not Known Now that you know how to test for carbohydrates, proteins, Vitamin C, and lipids, we can apply the tests to some common foods to determine what nutrients they contain. 1. Thoroughly rinse the spot plate and dry it. Use a clean pipette to transfer 1 ml of Nutrient Sample #4 to each of the four wells across Row 1 of the spot plate. 2. There are food samples prepared for you to test. Each group will be assigned 1 to 2 samples. Predict what nutrients you will find in: Potato Chips Orange Juice Strawberries Rice Test the food samples as you did the unknown nutrient samples in steps #3-7 on pages 105-106. Record results in Table 13.2. 3. Clean Up!!! Lab #13 Nutrition and Digestion 108

Indophenol Table 13.2 Results for Known Food Items Potato Chip Orange Juice Strawberries Color (+/-) Color (+/-) Color (+/-) Rice Color (+/-) Iodine Solution Biuret Reagent Sudan III Testing Your Comprehension: Answer the following questions based on the experiment. 1. Which nutrients were identified in the unknown nutrient samples? What were the samples? Record answers in the following table. Nutrient(s) Sample 1 Sample 2 Sample 3 Sample 4 Sample I.D. 2. Which indicators are used to detect lipids, starch, vitamin C, and proteins? 3. Which of the following do you think might test positive for the presence of all the nutrients: carbohydrates, proteins, fats, minerals, and vitamins? Circle your choice. Corn, potatoes, olive oil, red meat, dairy products Lab #13 Nutrition and Digestion 109

4. What nutrients did you find when you tested potato chips, orange juice, strawberries, and rice? Did the results match your predictions? 5. Why is it important to eat a variety of foods? In your answer, refer to the results from tests of different food items. Activity 2: Parts of the Digestive System The previous experiment explored some of the nutrients in food. How are nutrients extracted from the food we eat? In this activity, we will follow a bite of food through the digestive system and identify the structures that it passes through. 1. Get a hand-out with the diagram of the digestive system from the instructor. 2. Use the torso model to examine the parts of the system. Beginning at the mouth, follow an imaginary bite of food through the system. 3. On the diagram, place an arrow to the place where mechanical digestion occurs. What structures are involved in mechanical digestion? 4. On the diagram, place an arrow and label the place where absorption of nutrients occurs. What structures are involved? 5. On the diagram, place an arrow and label the location where most water is reabsorbed. Why is reabsorption of water important? Activity 3: Chemical Digestion Chemical digestion is due to enzymes that break down large food molecules to their basic components. Several organs produce enzymes that break down specific food molecules. The mouth contains amylase, an enzyme from saliva that breaks starches (polysaccharides) into disaccharides. Cells in the lining of the stomach produce pepsin, which in the presence of hydrochloric acid serves to break proteins into peptide chains. Chemical digestion is completed in the first part of the small intestine. Enzymes such as lactase, maltase, and sucrase break disaccharides into simple sugars. Polypeptide chains are further broken down to single amino acids by trypsin and peptidase. Lipids are broken into fatty acids and glycerol by lipases. The smaller molecules can then be absorbed by the villi of the small intestine. Procedure Lab #13 Nutrition and Digestion 110

Each group needs the following: 1 spot plate ph test strips toothpicks 3 ml Albumin 2% solution 3 ml Biuret solution plastic pipettes Starch solution Amylase solution Corn oil Lipase solution 10% Liquid soap Protein Digestion 1. Label the wells of a spot plate 1 through 12 with #1 to 4 going from left to right across the top row. 2. Place 5 drops of the albumin solution in wells 1 to 4, across the top. 3. To wells #2, #4 and #5, add 5 drops of hydrochloric acid. To wells #3 and #4 add 5 drops of pepsin. 4. Stir the mixture in each well using a clean toothpick. Allow the mixture in each well to remain undisturbed for 5 10 minutes. 5. Determine the ph in each of the wells. Dip a separate ph strip in each well and compare the color change to the ph chart provided on the package. Record results in Table 13.3. 6. Test for the presence of protein by adding 2 drops of Biuret reagent to each well. A positive test for protein is indicated by a pinkish color. In the observations column, write down whether the sample is a control, whether the protein is completely digested, etc. Lab #13 Nutrition and Digestion 111

Table 13.3 Protein Digestion Test Results Well # Contents PH Protein Test (+/-) Observations 1 Albumin 2 Albumin + HCl 3 Albumin + Pepsin 4 Albumin + Pepsin + Hydrochloric Acid 5 Hydrochloric Acid Questions: 1. How could you tell that protein digestion took place? 2. Explain what may have happened in well #2 after the addition of hydrochloric acid. 3. Explained what happened after addition of pepsin in #3. 4. Explain what happened in well #4 after adding pepsin and HCl. 5. Which well showed the greatest degree of protein digestion and why? 6. Does the effect of pepsin depend on ph? Explain your answer with reference to human physiology. Lab #13 Nutrition and Digestion 110

Carbohydrate Digestion 1. Use a clean spot plate. Label wells 1 and 2. Place 15 drops of starch solution in the wells of the spot plate that are numbered 1 and 2. 2. To well #2, add 5 drops of amylase solution. Stir thoroughly with a toothpick and leave the mixture undisturbed for 5 to 10 minutes. 3. Test for the presence of starch (a complex carbohydrate) in the two wells. Using a clean pipette, add 1 drop IKI (potassium iodine) solution to each of the two wells. In the presence of starch, the mixture will change color from yellow-brown to blue-black. 4. Observe any color changes and record results in Table 13.4. Table 13.4 Carbohydrate Digestion Test Results Well # Contents Starch Test Observations 1 Starch 2 Starch & Amylase Questions: 1. How do you know that carbohydrate digestion has taken place? Explain the process. 2. Explain what happened in well #2 after the addition of amylase. 3. What is the purpose of well #1? Fat Digestion 1. Clean and dry the spot plate thoroughly. Use a wax pencil to number the first three wells in the top row #1-3. 2. Place 10 drops of water and 3 drops of corn oil in the wells of the spot plate numbered 1-3. 3. Stir each well thoroughly using a toothpick. Lab #13 Nutrition and Digestion 111

4. Test the ph of each of the wells by dipping a ph strip in the well and comparing the color change to the ph chart provided on the package. Record the ph of each well in data table 13.5. 5. Add 5 drops of lipase solution to well #2, then add 5 drops of lipase solution and 2 drops of soap solution to well #3. 6. Stir the mixture in each well thoroughly with a toothpick and allow the spot plate to remain undisturbed for about 20 minutes. 7. After 20 minutes, test the ph in each well and record results and observations in table 13.5. Table 13.5 Fat Digestion Test Results Well # Contents ph Observations 1 Oil and water 2 Oil, water, and lipase 3 Oil, water, lipase, and liquid soap 1. What are the end products of digestion of fats? How does this relate to ph? 2. In this experiment, what data were collected? How were the data used to show that fat digestion occurred? 3. What is the role of the soap in the experiment? What substance in the body performs a similar function? 4. Which well showed the greatest amount of fat digestion? Explain why. Lab #13 Nutrition and Digestion 112