CHEMICAL AND PHYSICAL PROCESSES OF DIGESTION

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Activit y 3: Protein digestion by tryp sin Trypsin, an enzyme produced by the pancreas, hydrolyzes proteins to small fragments (proteoses, peptones, and peptides). BAPNA (N-alpha-benzoyl-L-arginine-p-nitroanilide) is a synthetic trypsin substrate consisting of a dye covalently bound to an amino acid. Trypsin hydrolysis of BAPNA cleaves the dye molecule from the amino acid, causing the solution to change from colourless to bright yellow. Since the covalent bond between the dye molecule and the amino acid is the same as the peptide bonds that link amino acids together, the appearance of a yellow colour indicates the presence and activity of an enzyme that is capable of peptide bond hydrolysis. Because the colour change from clear to yellow is direct evidence of hydrolysis, additional tests are not required when determining trypsin activity using BAPNA. Table 3: Trypsin Digestion of Protein Assessing Protein Digestion by Trypsin 1. Obtain 6 test tubes and a test tube rack, trypsin, BAPNA, distilled water and the appropriate labelled pipettes and bring them to your bench. 2. Prepare the controls (tubes 1T and 2T) and the experimental samples (tubes 3T- to 6T), as shown in Table 3. 3. Mark each tube with a wax pencil and load the tubes as indicated in the Table 3, using 3 ml of each indicated substance. 4. Place all tubes in a rack in the cold water bath or incubator for approximately 1 hour. Shake the rack occasionally to keep the contents well mixed. 5. While these tubes are incubating, proceed to Activity 6: Physical Processes: Mechanisms of Food Propulsion and Mixing. • Once the incubation period is over, you are ready to carry out the trypsin assay. Trypsin Assay Since BAPNA is a synthetic colourigenic (colour-producing substrate), the presence of yellow colour indicates a positive hydrolysis test; the dye molecule has been cleaved from the amino acid. If the sample mixture remains clear, no detectable hydrolysis has occurred. Record the negative hydrolysis test results in Table 3 and on the chalkboard. 6
Activit y 4: Pancreatic Lipase Digestion of Fats and th e A ct ion of Bile The treatment that fats and oils go through during digestion in the small intestine is a bit more complicated than that of carbohydrates or proteins – pre-treatment with bile to physically emulsify the fats is required. Hence, two sets of reactions occur. First: Bile Fats/Oils � (Emulsification) Then: Lipase Fat/oil droplets � (Digestion) Minute fat/oil droplets Monoglycerides and fatty acids The term pancreatin describes the enzymatic product of the pancreas, which includes enzymes that digest proteins, carbohydrates, nucleic acids, and fats. It is used here to investigate the properties of pancreatic lipase, which hydrolyzes fats and oils to their component monoglycerides and two fatty acids (and occasionally to glycerol and three fatty acids). The fact that some of the end products of fat digestion (fatty acids) are organic acids that decrease the pH provides an easy way to recognize that digestion is ongoing or completed. You will be using a pH indicator called litmus blue to follow these changes; it changes from blue to pink as the test tube contents become acid. Fig 1: Emulsification action of bile Demonstrating the Emulsification Action of Bile Although bile, a secretory product of the liver is not an enzyme, it is important to fat digestion because of its emulsifying action (the physical breakdown of larger particles into smaller ones) on fats. Emulsified fats provide a larger surface area for enzymatic activity. NOTE: use disposable tubes with caps for this experiment. 1. To demonstrate the action of bile on fats, prepare two test tubes and mark them 1E and 2E (fig.1). 2. To tube 1E add 5 ml of water and 1 ml of vegetable oil. 3. To tube 2E add 5 ml of water, 1 ml of vegetable, and a pinch of bile salts. 4. Cap each tube, shake vigorously, and allow the tubes to stand at room temperature. 5. After 10-15 minutes, observe both tubes. If emulsification has not occurred, the oil will be floating on the surface of the water. If emulsification has occurred, the fat droplets will be suspended throughout the water, forming an emulsion. In which tube has emulsification occurred?______________ 7
Page 1 and 2: LABORATORY - CHEMICAL AND PHYSICAL
Page 3 and 4: Materials Part 1: Enzyme Action •
Page 5: Activity 2: Assessing Starch Digest
Page 9 and 10: Activit y 5: Reporting Results an d

CHEMICAL AND PHYSICAL PROCESSES OF DIGESTION

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