Cuyamaca College Chemical and Physical Processes of Digestion Biology Lab

Cuyamaca College Chemical and Physical Processes of Digestion Biology Lab Cuyamaca College Chemical and Physical Processes of Digestion Biology Lab T Chemical and Physical Processes of Digestion he digestive system, also called the gastrointestinal system, consists of the digestive tract (also gastrointestinal tract or GI tract ) and accessory glands that secrete enzymes and fluids needed for digestion. The digestive tract includes the mouth, pharynx, esophagus, stomach, small intestine, colon, rectum, and anus. The major functions of the digestive system are to ingest food, to break food down to its simplest components, to extract nutrients from these components for absorption into the body, and to expel wastes. Most of the food we consume cannot be absorbed into our bloodstream with- out first being broken down into smaller subunits. Digestion is the process of breaking down food molecules into smaller molecules with the aid of enzymes in the digestive tract. See Figure 8.1b for an overview of chemical digestion sites in the body. Digestive enzymes are hydrolases: They catalyze (accelerate) the ad- dition of water to food molecules in order to break them down into smaller sub- units. For example, when two amino acids join together to form a protein, an — O H – group is removed from the carboxyl end of one amino acid, and an —H + is removed from the amino group of the second amino acid to form a dipeptide bond between the two amino acids plus w a t e r . T o break down such a protein, a digestive enzyme catalyzes the addition of water ( — OH – plus —H + ) to . Cuyamaca College Chemical and Physical Processes of Digestion Biology Lab the dipeptide bond, cleaving the bond to restore the carboxyl group of the first amino acid and the amino group of the second amino acid, and effectively breaks the protein down into two amino acid subunits. Once a food molecule is broken down into its simplest components, the components are absorbed through the ep- ithelial cells that line the intestinal tract and then enter the bloodstream. In addition to being hydrolases, digestive enzymes are substrate specific — they work on some substances but not others. For example, salivary amylase is an enzyme in the saliva that breaks down starch (found in foods like corn, pota- toes, bread, and pasta) and glycogen (animal starch), but not cellulose (found in the cell walls of plants), even though cellulose is made up of glucose, just like starch and glycogen. Two factors that play key roles in the efficacy of digestive enzymes are tem- perature and pH level. An increase in temperature may cause a reaction to speed up, as it causes molecules to move faster and thus increases contact with an en- zyme; however, too high a temperature will disrupt molecular bonding that sta- bilizes enzyme configuration, causing the enzyme to denature (i.e., undergo a 103 E X E R C I S E 8 O B J E C T I V E S 1 . To define digestive tract, accessory glands, digestion, hydrolases, sali- vary amylase, carbohydrates, proteins, lipids, bile salts, pepsin, and li- pase 2 . To understand the main functions and processes of the digestive system 3 . To understand the specificity of enzyme action 4 . To explain the impact of temperature and pH levels on enzyme activity 5 . To identify the three main categories of food molecules 6 . To explain how enzyme activity can be assessed with enzyme assays 7 . To identify the main enzymes, substrates, and products of carbohydrate, protein, and fat digestion T Chemical and Physical Processes of Digestion he digestive system, also called the gastrointestinal system, consists of the digestive tract (also gastrointestinal tract or GI tract ) and accessory glands that secrete enzymes and fluids needed for digestion. The digestive tract includes the mouth, pharynx, esophagus, stomach, small intestine, colon, rectum, and anus. The major functions of the digestive system are to ingest food, to break food down to its simplest components, to extract nutrients from these components for absorption into the body, and to expel wastes. Most of the food we consume cannot be absorbed into our bloodstream with- out first being broken down into smaller subunits. Digestion is the process of breaking down food molecules into smaller molecules with the aid of enzymes in the digestive tract. See Figure 8.1b for an overview of chemical digestion sites in the body. Digestive enzymes are hydrolases: They catalyze (accelerate) the ad- dition of water to food molecules in order to break them down into smaller sub- units. For example, when two amino acids join together to form a protein, an — O H – group is removed from the carboxyl end of one amino acid, and an —H + is removed from the amino group of the second amino acid to form a dipeptide bond between the two amino acids plus w a t e r . T o break down such a protein, a digestive enzyme catalyzes the addition of water ( — OH – plus —H + ) to Cuyamaca College Chemical and Physical Processes of Digestion Biology Lab the dipeptide bond, cleaving the bond to restore the carboxyl group of the first amino acid and the amino group of the second amino acid, and effectively breaks the protein down into two amino acid subunits. Once a food molecule is broken down into its simplest components, the components are absorbed through the ep- ithelial cells that line the intestinal tract and then enter the bloodstream. In addition to being hydrolases, digestive enzymes are substrate specific — they work on some substances but not others. For example, salivary amylase is an enzyme in the saliva that breaks down starch (found in foods like corn, pota- toes, bread, and pasta) and glycogen (animal starch), but not cellulose (found in the cell walls of plants), even though cellulose is made up of glucose, just like starch and glycogen. Two factors that play key roles in the efficacy of digestive enzymes are tem- perature and pH level. An increase in temperature may cause a reaction to speed up, as it causes molecules to move faster and thus increases contact with an en- zyme; however, too high a temperature will disrupt molecular bonding that sta- bilizes enzyme configuration, causing the enzyme to denature (i.e., undergo a 103 E X E R C I S E 8 O B J E C T I V E S 1 . To define digestive tract, accessory glands, digestion, hydrolases, sali- vary amylase, carbohydrates, proteins, lipids, bile salts, pepsin, and li- pase 2 . To understand the main functions and processes of the digestive system 3 . To understand the specificity of enzyme action 4 . To explain the impact of temperature and pH levels on enzyme activity 5 . To identify the three main categories of food molecules 6 . To explain how enzyme activity can be assessed with enzyme assays 7 . To identify the main enzymes, substrates, and products of carbohydrate, protein, and fat digestion. Cuyamaca College Chemical and Physical Processes of Digestion Biology Lab ORDER NOW FOR CUSTOMIZED AND ORIGINAL NURSING PAPERS 104 Exercise 8 (a) F I G U R E 8 . 1 Chemical digestion. (a) Opening screen of the Amylase experiment. Part (b) follows. structural change that renders it functionless). In addition, each enzyme has an optimal pH at which it is most active. Within range of this optimal pH, the enzyme will work as ex- pected; beyond the optimal pH, the enzyme may have no effect. Most food molecules fall into one of the following cate- gories: carbohydrates, proteins, or lipids. Carbohydrates are the principal source of calories for most people and include glucose, sugars, and starch. Larger carbohydrates are broken down into monosaccharides (simple sugars, such as glucose) before being absorbed into the blood. Proteins are very im- portant for growth, especially among young people. Proteins are broken down into amino acids before being absorbed into the body to build new proteins. Lipids, most of which are triglycerides (the major constituents of fats and oils), are not water soluble and thus pose special problems for digestion. Lipase, the enzyme that acts on lipids, is hydrolytic (like all digestive enzymes) and can only work on the surfaces of lipid droplets because the lipids are water insoluble. To increase the rate of digestion by lipase, lipids are first emulsified (bro- ken down into smaller droplets) with the aid of bile salts, a cholesterol derivative. Emulsification results in smaller droplets with larger surface areas, making it easier for lipase to bind to substrates and digest lipids. Bile salts also form micelles, which aid in the absorption of products of lipid di- gestion: fatty acids and monoglycerides. In the following experiments you will be examining the effects of different digestive enzymes on carbohydates, pro- teins, and lipids. To begin, follow the instructions for starting up PhysioEx 8.0 in the Getting Started section at the begin- ning of this lab manual. From the Main Menu, choose Exer- cise 8: Chemical and Physical Processes of Digestion from the drop-down menu and click GO. Then click Amylase. The opening screen will appear (see Figure 8.1). This screen will be used for the first two activities, in which you will be test- ing the effects of salivary amylase on starch and cellulose. Amylase Notice the 11 dropper bottles in the top right quadrant of the screen. You will be preparing test tubes containing various Chemical and Physical Processes of Digestion 105 Salivary glands: produce salivary amylase for the digestion of starch. Small intestine: produces enzymes for digestion of starch and proteins. Pancreas: produces pancreatic lipase for the digestion of fats, and pancreatic amylase for the digestion of starch (enzymes are ducted to the small intestine). Liver: produces bile for the emulsification of fats (ducted to the small intestine). Stomach glands: produce pepsin (in the presence of HCl) for the digestion of proteins. (b) F I G U R E 8 . 1 (continued) Chemical digestion. (b) A few sites of chemical digestion and the organs that produce the enzymes of chemical digestion. combinations of the dropper bottle contents. Below the drop- per bottles is an incubation unit that will enable you to boil, freeze, and incubate the test tubes. The closed cabinet in the upper left quadrant of the screen is an assay cabinet, contain- ing chemicals that you will add to your experimental test tubes in order to interpret your test results. Below the assay cabinet is a test tube washer where you will deposit used test tubes. Next to this is a test tube dispenser from which you will click test tubes and drag them to the holders in the incu- bation unit, where you will prepare them for experimenta- tion. Along the bottom of the screen is the data recording box, where you will be recording your experimental data. A C T I V I T Y 1 Salivary Amylase and Starch Starch digestion begins in the mouth with the action of sali- vary amylase, which is secreted by salivary glands. Salivary amylase breaks starch down into maltose, a disaccharide formed from two molecules of glucose. Thus the presence of maltose in an experimental sample would indicate that starch digestion has occurred. Your goals in this experiment are to test the effects of amylase on starch, determine the optimal pH level at which. Cuyamaca College Chemical and Physical Processes of Digestion Biology Lab 106 Exercise 8 amylase works, and observe the effects of temperature on en- zyme activity. 1 . Click the test tube hanging nearest to the incubator, drag it to slot number 1 on top of the incubator, and release the mouse button. The test tube will click into place. Repeat this action, filling slot number 2, slot number 3, and so on, until all seven slots on top of the incubator contain test tubes. 2 . Fill your seven test tubes with three substances each, as follows: Test tube #1: starch, deionized water, pH 7.0 buffer Test tube #2: amylase, deionized water, pH 7.0 buffer Test tube #3: starch, amylase, pH 7.0 buffer Test tube #4: starch, amylase, pH 7.0 buffer Test tube #5: maltose, deionized water, pH 7.0 buffer Test tube #6: starch, amylase, pH 2.0 buffer Test tube #7: starch, amylase, pH 9.0 buffer To do this, click the dropper cap of the desired substance, drag it to the top of the desired test tube, and release the mouse button. 3 . Click the number “3” under tube #3. The tube will lower into the incubation unit. Then click Boil. The tube will boil and then resurface. Note that the only difference between tube #3 and tube #4 is that tube #3 is being boiled. 4 . Be sure the incubation temperature is set for 37°C and that the timer is set for 60 minutes. If not, click the ( + ) o r ( – ) buttons accordingly. 5 . Click Incubate. All seven test tubes will be lowered into the incubation unit and gently agitated while incubated. At the end of the incubation period, the tubes will resurface and the door of the assay cabinet will open. You will notice that there are seven empty test tubes and two dropper bottles in the assay cabinet. The two dropper bottles contain IKI and Benedict’s reagents. IKI tests for the pres- ence of starch, while Benedict’s tests for the presence of sug- ars such as glucose or maltose (which, you will recall, is the product of starch digestion). You will be adding these reagents to your seven experimental test tubes to determine whether or not digestion has taken place. 6 . Click test tube #1 in slot 1 of the incubator. You will see your cursor change into a miniature test tube. Drag this miniature test tube to the rim of the first tube in the assay cab- inet, then release the mouse button. The contents of the miniature test tube will empty into the assay tube. 7 . Repeat step 6 for the remaining test tubes. Be sure to do this in sequence; that is, do test tube #2, then test tube #3, and so on. 8 . Once solutions from each of the tubes on the incubator have been transferred to the tubes in the assay cabinet, click the top of the IKI dropper bottle, drag the dropper to the opening of the first tube in the assay cabinet, and release the mouse button. Drops of IKI will be dispensed. The dropper will automatically move across and dispense IKI to the re- maining tubes. Note any color changes. A blue-black or gray color indicates a positive test for starch; a yellow color indi- cates a negative test. Record your IKI results in the following chart. 9 . Next, click the dropper top for Benedict’s, drag it to the opening of test tube #1, (in slot 1 of the incubator), and re- lease the mouse button. Drops of Benedict’s will be added to the test tube. Repeat this for the remaining test tubes on top of the incubator. 1 0 . After Benedict’s reagent has been added to each test tube on top of the incubator, click Boil. All the test tubes will de- scend into the incubator unit, boil, and resurface. Examine the tubes for color changes. A green, orange, or reddish color indicates the presence of maltose, considered a positive CHAR T 1 Results of Activity 1 Tube No. 1 2 3 4 5 6 7 Additives starch amylase starch starch maltose starch starch deionized water deionized water amylase amylase deionized water amylase amylase pH 7.0 buffer pH 7.0 buffer pH 7.0 buffer pH 7.0 buffer pH 7.0 buffer pH 2.0 buffer pH 9.0 buffer Incubation condition 37°C 37°C boiled, then incubated at 37°C 37°C 37°C 37°C 37°C IKI test Benedict’s test Chemical and Physical Processes of Digestion 107 Benedict’s result. A blue color indicates that no maltose is present, and is considered a negative Benedict’s result. Record your data in Chart 1. Use a ( + ) for a green sample, a ( + + ) f o r a reddish-brown sample, and a ( – ) f o r a blue sample. 1 1 . Click Record Data to record your data onscreen. You may also click Tools and select Print Data for a hard-copy print out of your results. 1 2 . Click and drag each test tube to the opening of the test tube washer and release the mouse button. The tubes will dis- appear. What was the purpose of tubes #1 and #2? What can you conclude from tubes #3 and #4? What do tubes #4, #6, and #7 tell you about amylase activity and pH levels? (Hint: What variable was changed in the procedure?) What is the optimal pH for amylase activity? Does amylase work at pH levels other than the optimal pH? What is the end-product of starch digestion? In which tubes did you detect the presence of maltose at the end of the experiment? Why wasn’t maltose present in the other tubes? Salivary amylase would be greatly deactivated in the stom- ach. Suggest a reason why, based on what you have learned in this activity. ? A C T I V I T Y 2 Salivary Amylase and Cellulose If there are any test tubes remaining in the incubator, click and drag them to the test tube washer before beginning this activity. In the previous activity we learned that salivary amylase can digest starch. In this activity we will test to see whether or not salivary amylase digests cellulose, a substance found in the cell walls of plants. We will also investigate whether bacteria (such as that found in the stomachs of cows and other rumi- nants) will digest cellulose, and whether peptidase (a pancre- atic enzyme that breaks down peptides) will digest starch. 1 . Click the test tube hanging nearest to the incubator, drag it to slot number 1 on top of the incubator, and release the mouse button. The test tube will click into place. Repeat this action, filling slot number 2, slot number 3, and so on, until all seven slots on top of the incubator contain test tubes. 2 . Fill your seven test tubes with three substances each, as follows: Test tube #1: amylase, starch, pH 7.0 buffer Test tube #2: amylase, starch, pH 7.0 buffer Test tube #3: amylase, glucose, pH 7.0 buffer Test tube #4: amylase, cellulose, pH 7.0 buffer Test tube #5: amylase, cellulose, deionized water Test tube #6: peptidase, starch, pH 7.0 buffer Test tube #7: bacteria, cellulose, pH 7.0 buffer To do this, click the dropper cap of the desired substance, drag it to the top of the desired test tube, and release the mouse button. 3 . Click the number “1” under tube #1. The tube will lower into the incubation unit. Then click Freeze. The tube will be frozen and then resurface. 4 . Be sure the incubation temperature is set for 37°C and that the timer is set for 60 minutes. If not, click the ( + ) o r ( – ) buttons accordingly. 5 . Click Incubate. All seven test tubes will be lowered into the incubation unit and gently agitated while incubated. At the end of the incubation period, the tubes will resurface and the door of the assay cabinet will open. Again, you will notice that there are seven empty test tubes and the IKI and Benedict’s dropper bottles in the assay cabi- net. Recall that IKI tests for the presence of starch, while Benedict’s tests for the presence of glucose or maltose. IKI turns blue-black in the presence of cellulose as well as starch. You will be adding these reagents to your seven experimental test tubes to determine whether or not digestion has taken place. 6 . Click test tube #1 in slot 1 of the incubator. You will see your cursor change into a miniature test tube. Drag this miniature test tube to the rim of the first tube in the assay cab- inet, then release the mouse button. The contents of the miniature test tube will empty into the assay tube. 108 Exercise 8 CHAR T 2 Results of Activity 2 Tube No. 1 2 3 4 5 6 7 Additives amylase starch pH 7.0 buffer amylase starch pH 7.0 buffer amylase glucose pH 7.0 buffer amylase cellulose pH 7.0 buffer amylase cellulose deionized water peptidase starch pH 7.0 buffer bacteria cellulose pH 7.0 buffer Incubation condition frozen, then incubated 37°C 37°C 37°C 37°C 37°C 37°C 37°C IKI test Benedict’s test 7 . Repeat step 6 for the remaining test tubes. Be sure to do Which tubes showed that starch or cellulose was still present? this in sequence; that is, do test tube #2, then test tube #3, and so on. 8 . Once solutions from each of the tubes on the incubator have been transferred to the tubes in the assay cabinet, click the top of the IKI dropper bottle, drag the dropper to the Which tubes tested positive for the presence of reducing sugars? opening of the first tube in the assay cabinet, and release the mouse button. Drops of IKI will be dispensed. The dropper will automatically move across and dispense IKI to the re- maining tubes. Note any color changes. A blue-black or gray What was the effect of freezing tube #1? color indicates a positive test for starch; a yellow color indi- cates a negative test. Record your IKI results in the following chart. 9 . Next, click dropper top for Benedict’s, drag it to the opening of test tube #1 (in slot 1 of the incubator), and release the mouse button. Drops of Benedict’s will be added to the How does the effect of freezing differ from the effect of boiling? test tube. Repeat this step for the remaining test tubes on top of the incubator. 1 0 . After Benedict’s reagent has been added to each test tube on top of the incubator, click Boil. All the test tubes will de- scend into the incubator unit, boil, and resurface. Examine the tubes for color changes. A green, orange, or reddish color What is the smallest subunit into which starch can be broken down? indicates the presence of glucose or maltose, considered a positive Benedict’s result. A blue color indicates that no glu- cose or maltose is present, and is considered a negative Bene- d i c t ’ s result. Record your data in Chart 2. Use a ( + ) for a green sample, a ( + + ) f o r a reddish-brown sample, and a ( – ) for a blue sample. Does amylase use cellulose as a substrate? Hint: Look at the results for tube 4. 1 1 . Click Record Data to record your data onscreen. You may also click Tools and select Print Data for a hard copy print out of your results. 1 2 . Click and drag each test tube to the opening of the test tube washer and release the mouse button. The tubes will dis- appear. Chemical and Physical Processes of Digestion 109 F I G U R E 8 . 2 Opening screen of the Pepsin experiment. Popcorn and celery are nearly pure cellulose. What effect did the addition of bacteria have on the digestion of cellulose? the acidic (low pH) environment of the stomach. The extent to which protein in the stomach is hydrolyzed or digested is significant but variable. It is estimated that 15% of dietary protein is reduced to amino acids by pepsin. Most protein di- gestion occurs in the duodenum of the small intestine. What was the effect of the enzyme peptidase, used in tube #6? Explain your answer, based on what you know about peptidase and its substrate. ? Pepsin Proteins are composed of subunits known as amino acids. When subjected to enzyme activity, proteins are broken down to their amino acid components. Pepsin is an example of an enzyme that breaks down protein. Pepsin is secreted by stom- ach glands as an inactive proenzyme, pepsinogen, which is converted to pepsin by the cleavage of acid-labile linkages in A C T I Get a 10 % discount on an order above $ 100 Use the following coupon code : NURSING10

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