Exercise Lab Enzymatic Breakdown of Food Adopted from Mr. Bob Nabors, Professor of Biology, retired, TCC, South Campus Introduction Food must be broken down into substances that the body can absorb and use. Digestive enzymes are proteins which speed up chemical digestion. Each enzyme has a set of optimal conditions of temperature and pH under which it performs best. A particular enzyme is very specific about the substrate with which it will bind. For example, maltose, a disaccharide sugar can bind with the enzyme maltase, but not sucrase. There are three major classes of digestive enzymes based on the types of food they affect. A series of carbohydrases act on complex carbohydrates to convert them to simple sugars (monosaccharides). For example, salivary amylase in the mouth breaks starches which are large polysaccharides into smaller ones. If food stays in the mouth long enough, this enzyme can break the polysaccharides all the way down to maltose, a disaccharide. Once the food is in the stomach, the acid soon stops the action of amylase, and carbohydrate digestion doesn't start again until the food reaches the duodenum, where pancreatic bicarbonate ions neutralize the acid, and pancreatic amylase continues the digestive process. Here the complex carbohydrates become maltose. From intestinal glands, three enzymes maltase, sucrase, lactase complete the conversion of all digestible carbohydrates to the monosaccharides glucose, fructose and galactose. Maltose becomes two glucose molecules. Sucrase action changes sucrose to glucose and fructose. Lactase action results in one glucose and one galactose from lactose. The small intestine absorbs these monosaccharides, and they are transported to the liver via the hepatic portal system. The liver stores much of the sugar as glycogen but some of it goes throughout the body as a source of fuel. If excess sugar is available, it is converted to fat. Proteases act upon proteins to release amino acids. In the stomach, pepsin breaks the bonds between some of the amino acids, resulting in shorter chains. In the small intestine, pancreatic trypsin, chymotrypsin, and some peptidases break more bonds, which results in a mixture of short chains and some individual amino acids. Peptidases from intestinal glands help finish the job of protein digestion with the result being individual amino acids. Amino acids are absorbed along with the sugars, and are taken to the liver and all parts of the body to be used for protein synthesis, or for energy. Lipases act upon fats (lipids) to result in fatty acids and glycerol. Most fat digestion occurs in the small intestine with the action of bile and lipase. Bile is not an enzyme; it emulsifies the fat, but does not cause a chemical change. Lipase is the enzyme which breaks the bonds connecting the fatty acids to the glycerol. Once these bonds are broken, the end products are absorbed, but most of the products do not go via the portal system to the liver. They are absorbed into the lymph vessels via lacteals in the intestinal villi, and go throughout the body to be used for energy or to be stored. This exercise is designed as a series of experiments to demonstrate the enzymatic breakdown of carbohydrates, fats and proteins.
Objectives After completing this exercise, the student should be able to: 1. Define and describe the function of an enzyme. 2. Give the action of each enzyme described in the introduction. 3. Describe the experiments in the laboratory exercise. 4. Describe the results of the experiments in the laboratory exercise. 5. Explain the significance of the control in a scientific experiment. 6. Answer all questions included in the exercise. Materials and Information Materials: Several racks of test tubes, with test tube holders 3 squeeze bottles of blue litmus solution 1 Liter of 5% pancreatin solution (dissolve 50g powdered pancreatin 5 and g Na 2CO3 In a liter of distilled water). 3 liters of distilled water 3 squeeze bottles of Benedict's solution 1 liter of 1% starch solution 1 quart of and , or cream 4 hard boiled eggs Water bath (1 large one for 40 degrees C.); hot plates (3) Wax marking pencils 24 10ml. graduated cylinders Beakers of various sizes Popcorn Information: A. Pancreatin contains trypsin, amylase and lipase. (!!) B. Egg white is a protein. C. Benedict's solution, which is blue, turns green or varying shades of brown when heated in the presence of increasing levels of sugar. D. Saliva contains salivary amylase. E. Cream contains butter fat. F. Blue litmus turns pink in the presence of an acid. Experimental Procedures 1. Protein Digestion (a demo) A whole boiled egg, shelled and allowed to stay 24 hours in a 5% pancreatin solution, shows evidence of digestion. Look carefully at this experimental egg, and compare it to another egg, the control, which has been soaked in distilled water for 24 hours. A. what evidence is there that the experimental egg has undergone some degree of digestion? B. Describe the surface appearance of the control egg. C. What is the purpose of a control?
D. Which pancreatic enzymes are responsible for protein digestion?
2. Carbohydrate Digestion (Starch) A. In each of 2 test tubes, labeled #1 and #2, pour 5 ml. of a 1% starch solution. Swirl container of starch solution, because starch has settled to the bottom of the container. B. Add 2 ml. of 5% pancreatin solution to test tube #1. C. Add 2 ml. of distilled water to test tube #2. D. Place both test tubes in a 40 C water bath for one hour. E. Remove both tubes from the water bath. F. Add 5 ml. of Benedict's solution to each tube. G. Use test tube holders to put tubes in a boiling water bath for 2 minutes. Answer the following questions: 1. What was the color of the solutions in both tubes before boiling?
2. What was the color of the solution in tube #1 after boiling?
3. What was the color of the solution in tube #2 after boiling?
4. Does this experiment indicate that something in the pancreatin facilitated the digestion of starch?
5. Explain your conclusion.
6. Which pancreatic enzyme is responsible for this digestion?
3. A. B. C. D. E. F. G. H. I.
Carbohydrate Digestion (using popcorn) Chew a natural size bite of popcorn until it has a watery consistency. Spit (carefully, and over a sink) the food and saliva into a test tube labeled #3. Crumble the same kind and amount of popcorn into small pieces, and put with 2 ml. of distilled water in a test tube labeled #4. Place these 2 tubes in a 40 C water bath for one hour. Remove both tubes and add 5 ml. of Benedict's solution to each. Using test tube holders, place both tubes in a boiling water bath for 2 minutes. Color change (if any) in tube #3____________________________________ Color change (if any) in tube #4 ___________________________________ If there was a noticeable difference in the 2 tubes after heating, what enzyme was responsible?_______________________________________________________
4. Fat Digestion A. Pour 10 ml. of cream into a test tube labeled #5. B. Finish filling the tube with blue litmus solution. Mix thoroughly by putting your thumb over the opening, and rocking the tube back and forth. C. Pour of the cream-litmus mixture into a test tube labeled #6. D. Add 2 ml. of 5% pancreatin solution to tube #5. E. Add 2 ml. of distilled water to tube #6. F. Place both tubes in the 40 C water bath for one and a half hours. G. Answer the following questions: (1) What was the color of the mixture in tube #5 after one hour? (2) What was the color of the mixture in tube #6 after one hour? (3) Which pancreatic enzyme facilitated the digestion of the fat? (4) The color change indicates the presence of which end product of fat digestion? (5) What is the other end product of fat digestion?