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Mammalian and Physiology Laboratory

Chemical and Physical Processes of Digestion

Introduction: In this laboratory, we studied the chemical and physical processes of digestion. The purpose of this laboratory was to investigate and assess digestion of starch, proteins, and fats via salivary amylase, pepsin hydrolysis, and lipase enzymes, respectively. Furthermore, we explore the specificity of amylase action in starch breakdown, producing an increase in sugar such as maltose and glucose while starch decreases. The addition of water helps hydrolases breakdown larger molecules into smaller subunits. Amylase activity is affected widely by different temperatures and pH levels. Moreover, we identified the specificity of pepsin’s enzymatic activity to hydrolyze peptide bonds into smaller chains and free amino acids. Lipases work to hydrolyze triglyceride to monoglycerides and fatty acids. Insoluble fats are emulsified to lipids for bile salts to separate clumps in order to lipase to easily breakdown triglycerides. In these studies, the preparation of control groups provided a known standard for comparisons to be made and assess the validity of the experiments. Negative controls determined contamination of the substances in thee reagents. Finally, the learning objectives were placed in a physiological context by the different enzyme assays which allow us to detect the presence or absence of substances. Materials & Methods: The computer program we used to perform the activities was Pearson’s PhysioExTM 10.0 Laboratory Stimulations in Physiology, exercise 8: Chemical and Physical Processes of Digestion. In this experiment, we determined the presence of starch and sugars by enzyme assays such as IKI and Benedict’s assay test. These tests change the coloration of samples to indicate their presence or absence of substances. Furthermore, to assess pepsin activity, BAPNA was utilized as a substrate, which released a yellow dyed when hydrolyzed and in the presence of pepsin or active peptidases. To digest fat with lipases a bile salts were used to separate fats into smaller droplets. Lipase activity was measured via pH values. Some common laboratory equipment used across experiments were pH buffers, deionized waters, test tubes, incubators used for temperature treatments, pH meters, and spectrophotometer to measure optical density. Results: see attached pdf below for the Chemical and Physical Processes of Digestion results. Discussion & Conclusion: The chemical and physical processes of digestion take place via distinct mechanism that produce one common effect: the breakdown of a larger substance or molecule of interest into smaller substances or chemicals. These mechanisms can be altered by temperatures and pH values. First, the hydrolysis of starch to maltose takes place via enzymatic breakdown by salivary amylase. From our results, we found amylase activity was most active at pH levels of 7.0. This is significant because salivary amylase is an enzyme secreted into the mouth by salivary glands. The pH level of the mouth is 7.0, which results in higher peak activity. Furthermore, findings suggested body temperature allow the enzymatic activity of amylase to increase and consume less time to digest starch because 37*C is amylase’s optimal temperature. Other effects such as boiling reduced the activity due to the denaturation of chemicals bonds causing a change in the chemical structures and thereby inactivating amylase enzymes. Similarly, boiling denatured pepsin into free amino acids and reduced pepsin’ enzymatic activity as well as its optical density. In contrast to amylase, pepsin’s optimal activity was seen at pH levels of 2 because pepsin resides in the stomach, which contains high acidity. As noted in test tube 2, optical density was highest due to low pH and no boiling. In the other hand, lipase’s optimal activity was seen at pH levels of 6.21 (test tube 1) because lipases are secreted into the small intestines which contain pH levels similar to 6.21. Unlike pepsin, lipase cannot survive and work effectively in pH levels of 2. Lipase activity is improved when breakdown occurs on a physical basis, rather than chemically. In conclusion, the mechanism of action of these digestive enzymes work independently from one another and their exposure to different pH levels, temperatures, or environmental conditions can hinder or promote their enzymatic activity.

11/9/2020

PhysioEx Exercise 8 Activity 1

PhysioEx Lab Report Exercise 8: Chemical and Physical Processes of Digestion Activity 1: Assessing Starch Digestion by Salivary Amylase Nam Dat

er 2020

Session ID: sessio

Pre-lab Quiz Results You scored 100% by answering 6 out of 6 questions correctly. 1 The substrate for amylase is You correctly answered: starch and carbohydrate. 2 Which of the following is true of enzymes? You correctly answered: Their activity can be affected by temperature and pH. 3 The reagent IKI tests for the presence of You correctly answered: starch. 4 Which of the following is not true of controls? You correctly answered: A negative result with a positive control is required to validate the test. 5 Which of the following is an end product of starch digestion? You correctly answered: maltose and glucose. 6 Hydrolases are enzymes that break down large molecules into smaller subunits through the addition of

.

You correctly answered: water.

Experiment Results Predict Question 1 Predict Question: What effect do you think boiling and freezing will have on the activity of the amylase enzyme? Your answer: Boiling will decrease amylase activity and freezing will have no effect. Stop & Think Question https://media.pearsoncmg.com/bc/bc_0media_ap/physioex/10/ex8/act1/

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11/9/2020

PhysioEx Exercise 8 Activity 1

1 Tube 2 (amylase, starch, pH 7.0 buffer) appears to have the same amount of starch digested as tube3(amylase, starch, pH 7.0 buffer) because You correctly answered: freezing had no effect on the enzyme. Experiment Data Tube №

Reagent Reagent Reagent Treatment 1 2 3

Time

Temp

IKI

Benedict's

1

Amylase

Starch

pH 7.0 Buffer

Boiled

60

37

+

-

2

Amylase

Starch

pH 7.0 Buffer

Frozen

60

37

-

++

3

Amylase

Starch

pH 7.0 Buffer

None

60

37

-

++

4

Amylase

Deionized Water

pH 7.0 Buffer

None

60

37

-

-

5

Deionized Water

Starch

pH 7.0 Buffer

None

60

37

+

-

6

Deionized Maltose Water

pH 7.0 Buffer

None

60

37

-

++

7

Amylase

Starch

pH 2.0 Buffer

None

60

37

+

+

8

Amylase

Starch

pH 9.0 Buffer

None

60

37

+

+

Post-lab Quiz Results You scored 100% by answering 4 out of 4 questions correctly. 1 Tubes 3 (amylase, starch, pH 7.0 buffer), 7(amylase, starch, pH 2.0 buffer), and 8(amylase, starch, pH 9.0 buffer) reveal that You correctly answered: amylase activity was highest at pH 7. 2 This activity includes a number of negative controls. Which tube indicates that the amylase solution was not contaminated with maltose? You correctly answered: tube 4 (amylase, deionized water, pH 7.0 buffer). 3 Which tubes indicate that the deionized water did not contain contaminating starch or maltose? You correctly answered: tubes 4 (amylase, deionized water, pH 7.0 buffer), 5(deionized water, starch, pH 7.0 buffer), and 6(deionized water, maltose, pH 7.0 buffer). i https://media.pearsoncmg.com/bc/bc_0media_ap/physioex/10/ex8/act1/

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11/9/2020

PhysioEx Exercise 8 Activity 1

4 Explain where and why salivary amylase would be most active. You correctly answered: Salivary amylase would be most active in the mouth because pH 7 is where its peak activity is.

Review Sheet Results 1 List the substrate and the subunit product of amylase. Your answer: starches and carbohydrates substrates for amylase. 2 What effect did boiling have on enzyme activity? Why? How well did the results this compare with your prediction? Your answer: Boiling decreased amylase activity, while freezing had no effect because boiling denatured the chemical-bonds of enzymes which causes the lost of chemical structures, thereby the enzymes will no longer bind with its target molecule. My prediction matched my results. 3 At what pH was the amylase activity the most active? Describe the significance of this result. Your answer: Amylase activity was the most active at pH = 7.0, which is the same pH as the mouth, thereby signifying its significance. 4 Briefly describe the need for controls and give an example used in this activity. Your answer: Control groups allow for comparisons to validate the experiments' results. An example is test tube 5, in which the enzyme that tests for the contaminating glucose in the starch or buffer is not present. 5 Describe the significance of using a 37°C incubation temperature to test salivary amylase activity. Your answer: The 37°C incubation is significant because it is the same temperature as body temperature, which is the ideal temperature for enzymatic activity.

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11/9/2020

PhysioEx Exercise 8 Activity 3

PhysioEx Lab Report Exercise 8: Chemical and Physical Processes of Digestion Activity 3: Assessing Pepsin Digestion of Protein Nam Date: 9 Nove

r 2020

Session ID: se

Pre-lab Quiz Results You scored 100% by answering 5 out of 5 questions correctly. 1 Where in the body does protein digestion begin? You correctly answered: the stomach. 2 The substrate for pepsin is You correctly answered: protein and peptides. 3 In this activity the substrate you will be using to detect protein digestion is You correctly answered: BAPNA. 4 Negative results with the negative controls You correctly answered: are expected and validate the experiment. 5 A spectrophometer measures You correctly answered: optical density.

Experiment Results Predict Question 1 Predict Question: At which pH do you think pepsin will have the highest activity? Your answer: pH 9.0. Stop & Think Questions 1 Which of the following tubes are negative control tubes? You correctly answered: tubes 3 (pepsin, deionized water, pH 2.0 buffer) and 4 (deionized water, BAPNA, pH 2.0 buffer). 2 An optical density greater than zero indicates that https://media.pearsoncmg.com/bc/bc_0media_ap/physioex/10/ex8/act3/

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PhysioEx Exercise 8 Activity 3

You correctly answered: BAPNA digestion has occurred and pepsin is active. Experiment Data Tube No.

Reagent 1

Reagent 2

Reagent 3

Boiled

Time (min)

Temp (°C)

Optical Density

1

Pepsin

BAPNA

pH 2.0 Buffer

+

60

37

0.00

2

Pepsin

BAPNA

pH 2.0 Buffer

-

60

37

0.40

3

Pepsin

Deionized Water

pH 2.0 Buffer

-

60

37

0.00

4

Deionized Water

BAPNA

pH 2.0 Buffer

-

60

37

0.00

5

Pepsin

BAPNA

pH 7.0 Buffer

-

60

37

0.03

6

Pepsin

BAPNA

pH 9.0 Buffer

-

60

37

0.00

Post-lab Quiz Results You scored 100% by answering 4 out of 4 questions correctly. 1 Pepsin would be most active You correctly answered: in the stomach. 2 Which two tubes validated the results of the experiment? You correctly answered: tubes 3 (pepsin, deionized water, pH 2.0 buffer) and 4 (deionized water, BAPNA, pH 2.0 buffer). 3 With more enzyme activity the optical density You correctly answered: increased. 4 If pepsin were digesting an actual protein substrate, the product would be You correctly answered: peptides and amino acids.

Review Sheet Results 1 Describe the effect that boiling had on pepsin and how you could tell that it had that effect. Your answer: Boiling breakup or denatured pepsin, which is noticeable as pepsin is broken down into peptide chains and free amino acids. https://media.pearsoncmg.com/bc/bc_0media_ap/physioex/10/ex8/act3/

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PhysioEx Exercise 8 Activity 3

2 Was your prediction correct about the optimal pH for pepsin activity? Discuss the physiological correlation behind your results. Your answer: The optimal pH of pepsin activity was at pH 2. pH values influence the enzymatic activity of enzymes such as pepsin, the stomach has high acidity and that is why pepsin works best at acidic environment. 3 What do you think would happen if you reduce the incubation time to 30 minutes for tube 5 (pepsin, BAPNA, pH 7.0 buffer)? Your answer: If the incubation time was shorter then, enzymatic activity increases and optical density will decrease closer to 0.

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PhysioEx Exercise 8 Activity 4

PhysioEx Lab Report Exercise 8: Chemical and Physical Processes of Digestion Activity 4 Name Date: 9 Nove

r 2020

Session ID: s

Pre-lab Quiz Results You scored 100% by answering 4 out of 4 questions correctly. 1 Which of the following is/are true of bile? You correctly answered: It works by a physical process. 2 The substrate used in this simulation is You correctly answered: vegetable oil. 3 When fatty acids are liberated by lipase, the pH You correctly answered: decreases. 4 One of the products of the chemical digestion of lipids is You correctly answered: fatty acids.

Experiment Results Predict Question 1 Predict Question: Which tube do you think will have the highest lipase activity? Your answer: tube 3 (lipase, deionized water, bile salts, pH 9.0 buffer). Stop & Think Questions 1 What does the pH measure? You correctly answered: both lipase activity and fatty acid release. 2 Why is it difficult to detect whether lipase is active in tube5(lipase, vegetable oil, bile salts, pH 2.0 buffer)? You correctly answered: The pH is already very low, so a decrease in pH might be difficult to detect. Experiment Data https://media.pearsoncmg.com/bc/bc_0media_ap/physioex/10/ex8/act4/

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PhysioEx Exercise 8 Activity 4

Tube No

Reagent 1

1

Lipase

2 3 4

Reagent 2 Vegetable Oil

Reagent 3

Reagent 4

Time (min)

Temp (°C)

pH

Bile salts

pH 7.0 Buffer

60

37

6.21

Lipase

Vegetable Deionized Oil Water

pH 7.0 Buffer

60

37

7.00

Lipase

Deionized Water

Bile salts

pH 9.0 Buffer

60

37

9.00

Deionized Vegetable Water Oil

Bile salts

pH 7.0 Buffer

60

37

7.00

5

Lipase

Vegetable Oil

Bile salts

pH 2.0 Buffer

60

37

2.00

6

Lipase

Vegetable Oil

Bile salts

pH 9.0 Buffer

60

37

9.00

Post-lab Quiz Results You scored 100% by answering 4 out of 4 questions correctly. 1 What is the product of lipase hydrolysis? You correctly answered: fatty acids. 2 From your results, which pH is ideal for pancreatic lipase digestion? You correctly answered: pH 7.0. 3 Which tube confirms that there is no lipase in bile salts or vegetable oil? You correctly answered: tube 4 (deionized water, vegetable oil, bile salts, pH 7.0 buffer). 4 Where in the body does pancreatic lipase hydrolyze triglycerides? You correctly answered: small intestine.

Review Sheet Results 1 Explain why you can't fully test the lipase activity in tube5(lipase, vegetable oil, bile salts, pH 2.0 buffer). Your answer: It's hard to test the lipase activity in test tube #5 due to low levels of pH. 2 Which tube had the highest lipase activity? How well did the results compare with your prediction? Discuss possible reasons why it may or may not have matched. Your answer: https://media.pearsoncmg.com/bc/bc_0media_ap/physioex/10/ex8/act4/

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PhysioEx Exercise 8 Activity 4

The test tube with the highest lipase activity was test tube #1 because it had the closest pH value to that of the intestines. Results did not match my predicted answer because I thought higher lipase activity would be present on higher pH values. 3 Explain why pancreatic lipase would be active in both the mouth and the pancreas. Your answer: Pancreatic lipase would be active in both the mouth and pancreas because it has the highest enzymatic activity in areas with a pH of 7.0. 4 Describe the process of bile emulsification of lipids and how it improves lipase activity. Your answer: The process of bile emulsification of lipids occurs due to physical breaking of biles to fat globules. Lipase activity is improved because the breakdown occurs on a physical basis, not chemically.

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