Work in Groups of 4

LAB EXERCISE 7

DIGESTION

(Martini p. 40,41, 45-57, 915-918)

Work in groups of 4

Objectives:

Identify the types of carbohydrates, fats and proteins present in the diet, state their general formulas, and outline the reactions by which they are digested to absorbable endproducts.
Identify the enzymes of the GI tract which act on each group of food substances, state their origin, activators (if present), substrates and endproducts.
State the pH in each part of the GI tract where digestion occurs, outline the mechanisms whereby it is established, and state its significance.
Explain the role of bile salts in digestion, and state their origin.

SAFETY (for Exercise C):

Do not burn yourself with boiling water or the reagent when you perform the Benedict test.
Use the test tube clamps to pick up test tubes from the beakers on the hot plates.
Put the test tubes inside a test tube holder before carrying them to your bench.

I. DIGESTION OF FATS

A. THE DIGESTION OF MILK FATS BY PANCREATIC LIPASE

EXERCISE A

Equipment:

bile salts (powder)
pancreatin solution (0.5% - contains pancreatic lipase)
cream
litmus solution (1%)
distilled water
pipettes
pipette suction pump
test tubes
water bath at 37 oC

Label four test tubes #1A 2A - 3A - 4A, and add solutions as designated in Table 1. Add the enzyme last!

Table 1. Fat Digestion Tests with Pancreatic Lipase and Bile Salts

Test
Tube / SUBSTRATE
(Cream) / BILE
SALTS / DISTILLED WATER / INDICATOR
(Litmus Solution) / ENZYME
(Pancreatic Lipase)

Add last

1A / 3.0 ml / pinch / - / 3.0 ml / 3.0 ml
2A / 3.0 ml / pinch / 3.0 ml / 3.0 ml / -
3A / 3.0 ml / - / - / 3.0 ml / 3.0 ml
4A / - / pinch / 3.0 ml / 3.0 ml / 3.0 ml

Notes:

i) Litmus is a pH indicator which changes colour from red (below pH 4.5) through various shades of violet to blue (above pH 8.3). Try it yourself: take a test tube, put approximately 1 ml of litmus solution. The colour in the test tube is blue. Add a few drops of 2N HCl. Is there a change of colour? If yes what is the colour?

ii) Enzymatic digestion of fat (triglyceride) releases fatty acids (e.g. butyric acid) which react with litmus and change its color to red.

iii) Pancreatin is a commercial preparation of dried pancreatic enzymes.

Shake the tubes to mix thoroughly, make note of the colour of each tube, and place in a water bath at 37oC. Incubate for 1 to 1 1/2 hours.

Shake tubes and record their colour in Table 2a every 5 minutes for the first 15, then for every 15 minutes until no colour change is observed between colour tests.

Choose terms to describe the colours such as: milky blue, milky violet, milky purple, pinkish purple, reddish purple, transparent blue, transparent red… Write your results in Table 2a.

Table 2a. Colour observations during digestion tests of milk fat by pancreatic lipase

Colours in test tubes during incubation
Test tube / 0min / 5min / 10min / 15min / 30min / 45min / 1hour / 1 hr 15 min / 1.5 hr
1A
2A
3A
4A

Complete the table below:

Table 2b: Summary of results of milk fat digestion tests

Test tube / Presence of fatty acids (yes/no) / Is the digestion of cream, fast, slow or is there no digestion at all? / Brief explanation of each result
1A
2A
3A
4A

B. DEMONSTRATION OF THE EMULSIFYING PROPERTIES OF BILE SALTS

EXERCISE B

Equipment:

bile salts (powder)
distilled water
oil

Prepare two test tubes: In the first one, put 3.0 ml. olive oil and 3.0 ml. distilled water. In the second test tube, put 3.0 ml water and add a pinch of bile salts. Shake vigorously. Then add 3.0 ml of oil.

When both test tubes are ready, take one in each hand and shake them at the same time. Put them back in your tray and keep both test tubes until the end of the laboratory period. Do NOT shake them again. Describe the appearance of each test tube after first shaking and changes in the appearance over time. Note generally how long it takes for the oil and water to separate.

II. DIGESTION OF CARBOHYDRATES

EXERCISE C

Equipment:

starch solution (1%)
distilled water
2N HCl
pancreatin solution (2.5% - contains pancreatic amylase)
test tubes
paraffin
pH paper
water bath at 37oC
iodine solution
glucose solution (1%)
maltose solution (1%)
Benedict's solution
hot plate
test tube clamp

THE DIGESTION OF STARCH BY AMYLASE

Label four test tubes #1C 2C - 3C - 4C and add solutions as described in Table 3. Add the enzyme last.

Table 3. Carbohydrate Digestion Tests with Pancreatic Amylase

Test
Tube / SUBSTRATE
(starch solution) / OTHER REAGENTS / ENZYME (Pancreatic Amylase)

Add last

1C / 5.O ml / --- / 5.0 ml
2C / 5.0 ml / 5.0 ml dist. Water / ---
3C / --- / 5.0 ml dist. Water / 5.0 ml
4C / 5.0 ml / 5 drops 2N HCl / 5.0 ml

Shake the tubes and determine and record the pH of each reaction mixture in table 4.

Place the test tubes in a water bath at 37oC and incubate for at least 45 minutes. Shake the tubes every 15 minutes. (Complete the iodine test and Benedict’s test control test tubes as instructed below while waiting for these tubes to incubate for the 45 minutes)

Divide each of the 4 samples by pouring half of it into a clean test tube (label).

Test one set of 4 test tubes for starch by adding 5 drops of iodine solution. Shake and record the colour of the iodine solution in table 4. See the control tests below to analyze results of iodine test.

Test the second set of 4 test tubes for reducing sugar by adding 5 ml. of Benedict's reagent to each, and placing in a boiling water bath for 5 min. Remove and rate the precipitate for colour according to the following scale:

Blue

Green +

Yellow ++

Orange +++

Red ++++

Record the colour of the precipitate in Table 4. See the control tests below to analyze results of Benedict’s test.

Iodine solution indicates the presence of starch by turning blue. Try it yourself!

- Prepare three test tubes. Place 2ml of 1% starch solution in one test tube, 2ml of 1% glucose solution in a second, and 2ml of 1% maltose in the third.

- Add 5 drops of iodine solution to each test tube and shake.

Iodine solution is yellow. Is there any change in colour after it is added to the starch solution? If yes, what colour is formed?

Is there any change in colour after it is added to the glucose solution? If yes, what colour is formed?

Is there any change in colour after it is added to the maltose solution? If yes, what colour is formed?

Keep these test tubes as controls to compare to the test tubes prepared for starch digestion.

Benedict’s reagent tests for the presence of glucose and other reducing sugars. Reducing sugars (glucose, fructose, maltose, lactose, etc.) react with Cu+2 ions (blue, soluble) in Benedict's reagent to form Cu+, which combines with O2 to form Cu2O (red, insoluble). Nonreducing sugars (starch, galactose, sucrose) do not react with Cu+2. Try it yourself!

- Prepare three test tubes by adding 5 ml of Benedict's reagent to each test tube

- Place 4ml of 1% starch solution in one test tube, 4ml of 1% glucose solution in a second, and 4ml of 1% maltose in the third.

- Heat in a boiling water bath for 5 min.

- Cool.

A green, yellow or red precipitate should form if glucose is present. The extent of the colour change is related to the amount of red Cu+ formed in relation to the amount of blue Cu++ remaining. This is determined by the amount of glucose present.

Record colour of the precipitate for starch:

Record colour of the precipitate for glucose:

Record colour of the precipitate for maltose:

Keep these test tubes as controls to compare to the test tubes prepared for starch digestion.

Table 4. Results of iodine and Benedicts tests for starch digestion by amylase.

Test tube / pH / Iodine test (colour) / Benedict test (colour) / Presence of starch (yes/no) / Presence of reducing sugar (yes/no) / Brief explanation of each result
1C
2C
3C
4C

III. DIGESTION OF PROTEINS

EXERCISE D

Equipment:

Capillary tubes which have been completely filled with coagulated egg white and incubated at 37 oC for 24 hours in the following solutions:

(1)pepsin in buffer, pH 2.0

(2)pepsin in buffer, pH 4.0

(3)pepsin in buffer, pH 8.0

(4)buffer, pH 2.0

(5)buffer, pH 4.0

(6)buffer, pH 8.0

(7)trypsin in buffer, pH 2.0

(8) trypsin in buffer, pH 4.0

(9) trypsin in buffer, pH 8.0

Ruler

Each of these tubes was totally filled with coagulated egg white. Determine the amount of egg white that has disappeared by measuring the length of empty tube at each end in mm, summing them and expressing them in percentage of the total length of the capillary tube (tubes were completely filled with egg white at the start of the experiment).

Tabulate results. Compare the amount of disappearance at each pH with and without enzyme. Record your results in Table 5 and on blackboard.

Table 5: Digestion of proteins by pepsin and trypsin.

Capillary tube / Solutions / Amount of egg white that has disappeared (in %)
1 / pepsin in buffer, pH 2.0
2 / pepsin in buffer, pH 4.0
3 / pepsin in buffer, pH 8.0
4 / buffer, pH 2.0
5 / buffer, pH 4.0
6 / buffer, pH 8.0
7 / trypsin in buffer, pH 2.0
8 / trypsin in buffer, pH 4.0
9 / trypsin in buffer, pH 8.0

Why did we bother to incubate egg white in buffer solutions without enzymes?

How would you calculate the amount of egg white digested by pepsin and trypsin at different pH values?

CLEAN UP:

Wash all glassware with soap and warm water,
Return equipment to where you found it.

NAME & section #:

Biology 153 LAB REPORT

EXERCISE 7: DIGESTION.

INTRODUCTION

State the purpose of this exercise

MATERIALS AND METHODS

See the lab manual.

RESULTS

I: DIGESTION OF FATS

1. Copy results in Table 2a to the table below.

Table A. Changes in colour observed during the digestion of milk fat by pancreatin.

Colours in test tubes during incubation
Test tube / 0min / 5min / 10min / 15min / 30min / 45min / 1hr / 1h15min / 1h30min
1A
2A
3A
4A

2. Briefly describe the distribution of oil with and without bile salts (Exercise B). Describe the appearance of each test tube after first shaking and changes in the appearance over time. Note generally how long it takes for the oil and water to separate.

II: DIGESTION OF CARBOHYDRATES

In the following table, copy results for the digestion of starch by amylase.

Table B. Benedict’s and iodine test results for starch digestion by amylase.

Test tube / pH / iodine test (colour) / Benedict test (colour) / Presence of starch (yes/no) / Presence of reducing sugar (yes/no)
1C
2C
3C
4C

III: DIGESTION OF PROTEINS

1. Complete the following table (Exercise D).

Table C. Digestion of egg white protein by pepsin and trypsin.

Capillary tubes / Solutions / Amount of egg white that has disappeared (in %) / Amount of egg white that has been digested (in %)
1 / pepsin in buffer, pH 2.0
2 / pepsin in buffer, pH 4.0
3 / pepsin in buffer, pH 8.0
4 / buffer, pH 2.0
5 / buffer, pH 4.0
6 / buffer, pH 8.0
7 / trypsin in buffer, pH 2.0
8 / trypsin in buffer, pH 4.0
9 / trypsin in buffer, pH 8.0

2. Prepare onegraph showing the percent digestion of egg white by pepsin and trypsin. Percent of digestion should be on the Yaxis and pH on the Xaxis. Describe your results in no more than 5 lines and explain the significance of the capillary tubes filled with egg white incubated in buffers pH 2, 4 and 8.

DISCUSSION

Answer in the spaces provided below. Do not use extra pages.

I: DIGESTION OF FATS

Explain what happened in each tube used in Exercise A by completing the following table.

Table D: Discussion of results of milk fat digestion by pancreatic lipase

Test tube # / Is the digestion of cream, fast, slow or is there no digestion at all? / Explain. Be sure to include: function of components of the test tube, products of digestion, explanation for colour change or no colour change, explanation for changes in rate of digestion, etc.
1A
2A
3A
4A / What is the significance of using this test tube?

II: DIGESTION OF CARBOHYDRATES

Explain what happened in each tube used in Exercise C by completing the following table.

Table E: Discussion of results of carbohydrate digestion by pancreatic amylase

Test tube / Presence of starch (yes/no) / Presence of reducing sugar (yes/no) / Explain your results.
1C
2C
3C
4C

III:DIGESTION OF PROTEINS

Explain the results you graphed for the digestion of proteins (Exercise D).

Explain how optimum pH is created for these enzymes in the gastrointestinal tract (consider the organs secreting these enzymes, the nonenzyme secretions at these locations, and the importance of pH in the activity of each enzyme).