5 - Spring 2022 Digestion & Enzymes_ Fat Emulsion & Lipids - Fillable Form

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Santa Ana College *

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Feb 20, 2024

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LAB EXERCISE: Digestion & Enzymes: Fat Emulsification & Fat Digestion Summary In today’s lab we will continue learning about biological macromolecules and how the GI functions to break them down. We will learn about two main enzymatic concepts: how the presence of enzymes allows us to digest foods at a faster rate and how increased surface area increases chemical digestion. We will test for factors that affect fat digestion. We will also run tests to learn how surface area affects the rate of digestion. We will use chemical indicators to test for changes in pH. We will also learn how bile, an amphipathic molecule, mechanically breaks down fats to increase surface area to help us chemically digest fats. Triglyceride (fat) digestion mainly occurs in the mouth and in the small intestine. The enzymes responsible for breaking down triglycerides into monoglycerides and free fatty acids are called lipases (for example salivary lipase and pancreatic lipase). The amphipathic chemical that mechanically breaks down fats in order to increase the surface area for lipase to act is called bile. The higher the surface area of fat, the faster chemical digestion will occur. The liver makes bile salts and stores them in the gallbladder. The gall bladder then secretes bile into the small intestine to aid in increasing the rate of fat digestion. Today you will determine how quickly lipase acts with and without the presence of the emulsifier bile. You will determine how pancreatic lipase breaks down fats (we will use heavy cream). You will then use pancreatic lipase and bile to determine how the rate of fat digestion is influenced by both bile and pancreatic lipase. You will use litmus (a pH indicator) to determine how the fats are chemically digested; alkaline solutions are blue, neutral solutions are lavender, and acidic solutions are pink. 1

Goals Physiology students will learn about chemical digestion, mechanical digestion and various factors that affect their enzymatic rates. Physiology students will work safely and learn to use graduated cylinders, serological pipet, transfer pipets, enzymes, and chemical indicators. Physiology students will properly handle all waste materials, equipment, and chemicals. ● Understand the role of mechanical digestion and how this affects the rate of chemical digestion. ● Understand the basics of enzymes by using the digestive system as a model. In other words, understand the role that environmental factors have on the rate of enzymatic digestion. ● Understand how bile affects the rate of fat digestion by lipase. ● Learn to interpret the results of all chemical indicators. ● Learn how to use a transfer pipet properly. ● Learn how to use a serological pipet & pipetman properly. ● Learn how to use a graduated cylinder properly. ● Prepare for the lab, work safely and properly handle all waste materials, equipment, and chemicals. 2

Lab Exercises: Orientation to Lab Equipment: You should have a good understanding of how to use a graduated cylinder and serological pipette. Your instructor will show you how to use the equipment correctly if you need support. Ask for help if needed at any point during this lab. HINT: Before you begin, wash all your glassware and serological pipettes to prevent contamination. HINT: Begin the Bile Promotes Fat Emulsification Lab Activity & Fat Digestion in the Small Intestine Lab Activity at the same time. Bile Promotes Fat Emulsification (Mechanical Digestion) Equipment: ● test tubes ● wax pen ● graduated cylinder ● serological pipettes ● transfer pipettes ● test tube rack ● parafilm ● heavy cream ● DI water ● pancreatic lipase solution ● Litmus indicator ● bile solution ● soap ● 37 ºC water bath ● ice bath ● glass rod ● 2% HCl ● 2% NaOH Protocol: 1. Understand amphipathic molecule structure: a. An amphipathic molecule has a region that is polar and a region that is nonpolar. b. Examples are bile, soap, phospholipids, and surfactant. 3

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2. Understand micelle structure and function: a. A general rule for solubility is that “like dissolves like.” b. Micelles are structures made of amphipathic molecules. i. In a micelle, nonpolar molecules will arrange themselves next to the nonpolar region found ____________________ [inside / outside] the micelle. ii. In a micelle, polar molecules will arrange themselves next to the polar region found ____________________ [inside / outside] the micelle. iii. Micelles like to be suspended in _____________________ [nonpolar / polar] solutions. c. Micelles help to carry _____________________ [nonpolar / polar] molecules safely through water. 3. Understand surface area to volume ratio (SA/vol): a. Surface area is the total area of all the exposed surfaces of an object. It is the ______________________ [inside / outside] area of an object. b. Volume is the amount of space occupied by the object. It is the space ____________________ [inside / outside] of the object. c. Surface area to volume ratio is the amount of surface area of an item relative to its own volume. d. When the surface area to volume ratio is small, then the item is exposing very little surface area relative to its volume. e. When the surface area to volume ratio is large, then the item is exposing a lot of surface area relative to its volume. f. Pretend you have two identical blocks of ice and label them Ice A and Ice B. Both ice blocks have the same volume. i. You do not change Ice A and place it in a bucket of warm water. It takes a long time to melt. ii. You take Ice B and crush it into small pieces. The volume is the same as Ice Block A because you did not remove any ice. However by crushing the ice block, you increased the surface area of Ice B. You finally put Ice B in a bucket of warm water and it melts much faster than Ice A. iii. Ice A has a ____________________ [lower / higher] surface area to volume ratio compared to Ice B. iv. Ice B has a ____________________ [lower / higher] surface area to volume ratio compared to Ice A. 4

4. Make your predictions: a. Soap is amphipathic and will facilitate fat emulsification by creating micelles. If we emulsify fat with soap, then we will ____________________ [decrease / increase] the surface area to volume ratio of that fat. b. If Bile is also amphipathic, then it will facilitate fat emulsification by creating micelles. If we emulsify fat with bile, then we will ____________________ [decrease / increase] the surface area to volume ratio of that fat. 5. Obtain 2 test tubes and label them with a waxpen to indicate if soap or bile will be used as an emulsifier: i. Label one test tubes as: S (soap) ii. Label one test tubes as: B (bile) 6. Add 20 drops of vegetable oil and 20 drops of DI water into one of the test tubes labeled “ S ”. Stretch parafilm over the opening. Mix well using a vortex . a. Water is a(n) _________________________ [amphipathic / nonpolar / polar] molecule. b. Vegetable oil is a(n) _________________________ [amphipathic / nonpolar / polar] molecule. c. Water and vegetable oil do not mix. Vegetable oil is _________________________ [insoluble / soluble] in water. d. Water is found on the _______________________ [upper / lower] layer. e. Vegetable oil is found on the _______________________ [upper / lower] layer. 7. Add 2 drops of soap to the test tube labeled “ S ”. Stretch parafilm over the opening. Mix well using a vortex. a. Soap is a(n) _________________________ [amphipathic / nonpolar / polar] molecule and creates micelles. b. These micelles carry ____________________ [vegetable oil / water] safely inside of them. c. Micelles are found throughout the ____________________ [vegetable oil / water] layer. This is an example of emulsification. d. As emulsification increases, __________________________ [surface area / volume] increases. e. The “ S ” test tube now looks like a solution with ___________ [1 / 2 / 3] layers. 5

8. Add 20 drops of vegetable oil and 20 drops of DI water into one of the test tubes labeled “ B ”. Stretch parafilm over the opening. Mix well. a. Water is a(n) _________________________ [amphipathic / nonpolar / polar] molecule. b. Vegetable oil is a(n) _________________________ [amphipathic / nonpolar / polar] molecule. c. Water and vegetable oil do not mix. Vegetable oil is _________________________ [insoluble / soluble] in water. d. Water is found on the _______________________ [upper / lower] layer. e. Vegetable oil is found on the _______________________ [upper / lower] layer. 9. Add 2 drops of bile to the test tube labeled “ B ”. Stretch parafilm over the opening. Mix well by transferring the solution back and forth with a clean test tube. a. Bile is a(n) _________________________ [amphipathic / nonpolar / polar] molecule and creates micelles. b. These micelles carry ____________________ [vegetable oil / water] safely inside of them. c. Micelles are found throughout the ____________________ [vegetable oil / water] layer. This is an example of emulsification. d. As emulsification increases, __________________________ [surface area / volume] increases. e. The “ B ” test tube now looks like a solution with __________ [1 / 2 / 3] layers. 10.Compare your results in test tube “ S ” and test tube “B ”. a. Soap and bile are both _________________________ [amphipathic / nonpolar / polar] molecules and create micelles. b. Soap and bile are both emulsifiers of _________________________ [vegetable oil / water] resulting in a(n) _____________________ [decreased / increased] surface area to volume ratio of vegetable oil. 11. Clean-up: a. Dispose of parafilm in Regular Trash. b. Dispose of all liquids in the sink while running water. Then clean the sink with soap and water. c. Place the test tubes in the Test Tube Collection Bucket. d. Rinse and wash all serological pipettes and graduated cylinders. e. Clean your lab bench and common areas. Check and clean the sinks. Pick up trash around your lab bench and common areas. Organize your lab bench materials. f. Dispose of gloves in the Regular Trash. 6

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Fat Chemical Digestion in the Small Intestine 1. Understand the role of bile in fat emulsification: a. Micelles made with bile are responsible for _____________________ [chemical / mechanical] digest fats resulting in a(n) ______________________ [increased / decreased] surface area to volume ratio. 2. Understand the role of pancreatic lipase: a. Pancreatic lipase is an enzyme that chemically breaks down ____________________ [carbohydrates / proteins / fats]. b. Pancreatic lipase is responsible for _____________________ [chemical / mechanical] digestion of fats. c. Pancreatic lipase will function without the presence of bile. _____________________ [true / false] d. The fastest rate of digestion will occur in the presence of ____________________________ [both bile and pancreatic lipase / bile only / pancreatic lipase only]. 3. Understand how your reagents function: a. Litmus Indicator: i. Litmus Indicator will show a _______________________ [blue / lavender / red] color if the solution is acidic . 1. This indicates that there was triglyceride chemical digestion . ii. Litmus Indicator will show a _______________________ [blue / lavender / red] color if the solution is neutral . 1. This indicates that there was no triglyceride chemical digestion . iii. Litmus Indicator will show a _______________________ [blue / lavender / red] color if the solution is basic . 4. Make your predictions: a. If water (no pancreatic lipase, no bile) is added to heavy cream (triglyceride), then: i. The surface area to volume ratio of heavy cream (triglyceride) will ______________________ [decrease / increase / not change]. ii. Chemical digestion of triglyceride ______________________ [will / will not] occur. iii. The amount of free fatty acid in the solution will ______________________ [decrease / increase / not change]. iv. The pH of the solution will ______________________ [decrease / increase / not change]. 7

b. If pancreatic lipase and bile are added to heavy cream (triglyceride), then: i. The surface area to volume ratio of heavy cream (triglyceride) will ______________________ [decrease / increase / not change]. ii. Chemical digestion of triglyceride ______________________ [will / will not] occur. 1. If chemical digestion does occur, then the rate of chemical digestion will be ____________________ [fast / slow]. iii. The amount of free fatty acid in the solution will ______________________ [decrease / increase / not change]. iv. The pH of the solution will ______________________ [decrease / increase / not change]. c. If only pancreatic lipase is added to heavy cream (triglyceride), then: i. The surface area to volume ratio of heavy cream (triglyceride) will ______________________ [decrease / increase / not change]. ii. Chemical digestion of triglyceride ______________________ [will / will not] occur. 1. If chemical digestion does occur, then the rate of chemical digestion will be ____________________ [fast / slow]. iii. The amount of free fatty acid in the solution will ______________________ [decrease / increase / not change]. iv. The pH of the solution will ______________________ [decrease / increase / not change]. d. If only bile is added to heavy cream (triglyceride), then: i. The surface area to volume ratio of heavy cream (triglyceride) will ______________________ [decrease / increase / not change]. ii. Chemical digestion of triglyceride ______________________ [will / will not] occur. iii. The amount of free fatty acid in the solution will ______________________ [decrease / increase / not change]. iv. The pH of the solution will ______________________ [decrease / increase / not change]. 8

5. Obtain 4 test tubes and label them with a waxpen: a. #1 (triglycerides, no lipase, no bile) (negative control) b. #2 (triglycerides, lipase, bile) (positive control) c. #3 (triglycerides, lipase, no bile) d. #4 (triglycerides, no lipase, bile) 6. Prepare your test tubes at the same time: a. In test tube #1 (triglycerides, no lipase, no bile): add 1.5 mL of Heavy Cream, 3 mL DI water and 1.5 mL Litmus Indicator. Mix well using a glass rod or by transferring back and forth with a clean test tube. b. In test tube #2 (triglycerides, lipase, bile): add 1.5 mL of Heavy Cream, 2.5 mL pancreatic lipase, 0.5 mL bile, and 1.5 mL Litmus Indicator. Mix well using a glass rod or by transferring back and forth with a clean test tube. c. In test tube #3 (triglycerides, lipase, no bile): add 1.5 mL of Heavy Cream, 2.5 mL pancreatic lipase, 0.5 mL DI water, and 1.5 mL Litmus Indicator. Mix well using a glass rod or by transferring back and forth with a clean test tube. d. In test tube #4 (triglycerides, no lipase, bile): add 1.5 mL of Heavy Cream, 2.5 mL DI water, 0.5 mL bile, and 1.5mL Litmus Indicator. Mix well using a glass rod or by transferring back and forth with a clean test tube. e. Test tubes #1-4 should all have a neutral pH (pH 7) and should show the same lavender color. Show your instructor if you want to double check that all the colors match. Your instructor may suggest that you adjust the pH of your solutions by adding 2% HCl or 2% NaOH as needed to get all the test tubes to be the same color. 9

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7. Cover all test tubes with parafilm. Incubate all the test tubes in the 37 ºC water bath. Observe how the pH of each test tube changes every 3 minutes for 15 minutes by looking at the color change. Is the heavy cream solution becoming more red (acidic), more blue (basic), or is there no change in color? Litmus Indicator Color Test Tube #1 (triglycerides) Test Tube #2 (triglycerides, lipase, bile) Test Tube #3 (triglycerides, lipase) Test Tube #4 (triglycerides, bile) Time 0 min Lavender, neutral pH Lavender, neutral pH Lavender, neutral pH Lavender, neutral pH Time 3 min Time 6 min Time 9 min Time 12 min Time 15 min 8. Remove the test tubes from the 37 ºC water bath after the incubation period. 10

9. Compare your test tube results to the negative control and positive control: i. #1 (triglycerides, no lipase, no bile) (negative control) ii. #2 (triglycerides, lipase, bile) (positive control) iii. #3 (triglycerides, lipase, no bile) iv. #4 (triglycerides, no lipase, bile) b. The surface area to volume ratio of heavy cream (triglyceride) increased in test tubes #______________________ [1 / 2 / 3 / 4] because bile was present. c. Chemical digestion of triglyceride occurred in test tubes # ______________________ [1 / 2 / 3 / 4] because pancreatic lipase was present. d. Chemical digestion of triglyceride was the fastest in test tubes # ______________________ [1 / 2 / 3 / 4] because it had the lowest pH (most acidic). e. When the rate of triglyceride chemical digestion increases, the amount of free fatty acid _____________________ [decreases / increases]. f. The amount of free fatty acid in the solution was the highest in test tube #______________________ [1 / 2 / 3 / 4] because it had the lowest pH (most acidic). g. When the amount of free fatty acid increases, then the pH of the solution _______________________ [decreases / increases]. h. The pH of the solution was the lowest in test tube #______________________ [1 / 2 / 3 / 4] because it had the most free fatty acids. 10.Clean-up: a. Dispose of parafilm in Regular Trash. b. Dispose of all liquids in the Chemical Waste Container. c. Place the test tubes in the Test Tube Collection Bucket. d. Rinse and wash all serological pipets, graduated cylinders, and glass rods. e. Clean your lab bench and common areas. Check and clean the sinks. Pick up trash around your lab bench and common areas. Organize your lab bench materials. f. Dispose of gloves in the Regular Trash. 11

5 - Spring 2022 Digestion & Enzymes_ Fat Emulsion & Lipids - Fillable Form

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