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LAB EXERCISE DIGESTION

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 end-products.

Identify the enzymes of the GI tract which act on each group of food substances, state their origin, activators (if present), substrates and end-products.

State the pH in each part of the GI tract where digestion occurs, explain how it is established, and state its importance.

Explain the role of bile salts in digestion.

SAFETY (for Exercise C):

● Use extreme caution around hot plate. Wear Protective eyewear!!!! 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.

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I. DIGESTION OF FATS

A. THE DIGESTION OF MILK FATS BY PANCREATIC LIPASE

1. Label four test tubes with the sharpie and colored tape provided, #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

TestTube

SUBSTRATE(Cream)

BILESALTS

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 color 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 color in the test tube is blue. Add a few drops of 2N HCl. Is there a change of color? If yes what is the color?

.ii) Enzymatic digestion of fat (triglyceride) releases fatty acids that react with litmus and change its color to red.iii) Pancreatin is a commercial preparation of dried pancreatic enzymes.

2. Cover the tubes with parafilm and shake the tubes to mix thoroughly or use vortex mixer, make note of the color of each tube, and place in a water bath at 37oC. Incubate for 1 to 1 1/2 hours.

3. Shake tubes and record their color in Table 2a every 10 minutes until no color change is observed between color tests. At the end of the incubation period measure the pH of each tube by taking a sample with disposable pipet and placing on probe sensor as shown in pictures. Don’t forget to rinse the probe in the beaker marked “ph Rinse” and blot dry with Kimwipe.

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

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

Colors in test tubes during incubation

Test tube

0min 10min 20min 30min 40min 50min 60min 70min 80min 90min

1A

2A

3A

4A

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4. 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

1. 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. Mix with vortex mixer. Then add 3.0 ml of oil.

2. When both test tubes are ready, mix using the vortex mixer. Try to mix one after another within 15 seconds. 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.

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II. DIGESTION OF CARBOHYDRATES

THE DIGESTION OF STARCH BY AMYLASE

1. 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 AmylaseTestTube

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 10 drops .2N HCl 5.0 ml

2. Shake the tubes and determine and record the pH of each reaction mixture in table 4. Since the pH probe is too big for the mouth of the test tubes, pour the contents of each test tube into a clean 25 ml beaker. The probe fits into these small beakers. After measuring the pH pour the contents of the beaker back into the test tube for incubation. After each pH measurement, clean the probe according to standard protocol.

3. Place the test tubes in a water bath at 37oC and incubate for at least 45 – 60 minutes. Shake the tubes every 15 minutes. Perform an iodine test and Benedict’s test at the end of the incubation period.

4. At the end of incubation, divide each of the 4 samples by pouring half of it into a clean test tube (label).

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

6. 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 color according to the following scale:

Blue -Green +Yellow ++Orange +++Red ++++

Record the color of the precipitate in Table 4.

Perform the following control tests while waiting for the test tubes to incubate:

Lugol’s (IKI)

Iodine solution (Lugol’s) indicates the presence of starch by turning blue or black. Try it yourself!- Prepare three test tubes. Place 2ml of 1% starch solution in one test tube, 2ml of 1% glucose solution in a

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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 color after it is added to the starch solution? If yes, what color is formed?

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

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

Benedict’s

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). Non-reducing 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. - 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 color 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 color of the precipitate for starch:

Record color of the precipitate for glucose:

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.6

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Test tube

pH Iodine test (color)

Benedict test (color)

Presence of starch (yes/no)

Presence of reducing sugar (yes/no)

Brief explanation of each result

1C

2C

3C

4C

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III. DIGESTION OF PROTEINS

EXERCISE DEquipment:

● Egg white, scalpel, balance, weighing boats and spatula

(1) pepsin in buffer, pH 2.0 2% pepsin, 2g/100 ml water

(2) pepsin in buffer, pH 8.0 2% pepsin, 2g/100 ml water

(3) trypsin in buffer, pH 2.0 2% trypsin, 2 g/100 ml water

(4) trypsin in buffer, pH 8.0 2% pepsin, 2g/100 ml water

1. Obtain 4 test tubes and them as shown in table 5.

2. Fill the test tubes with 10 ml of the solutions listed in table 5.

3. Cut and weigh 4 small pieces of egg white and carefully add 1 piece to each tube using a small spatula. Don’t forget to tare the balance with the weighing boat to make weighing easy.

4. Record the weight that was used in each tube in table 5.

5. Incubate at 37oC for 60 – 90 minutes. Try for 90 minutes

6. Pour out most of the liquid from each tube.

7. Remove each the egg white from each tube by tilting the tube and sliding the spatula under the egg white.

8. Remove most of the water by gently placing each side of the egg white on a paper towel, then transfer to a tarred weighing boat and weigh. Record the weights in table 5.

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Table 5: Digestion of proteins by pepsin and trypsin.

Test Tube

Solutions Weight of egg white at start

Weight of egg white after incubation

Weight change+/-

1D pepsin in buffer, pH 2.0

2D pepsin in buffer, pH 8.0

3D trypsin in buffer, pH 2.0

4D trypsin in buffer, pH 8.0

Why was the egg white in buffer solutions without enzymes?

Is there any way that the egg white could be transferred to improve the measurement at the end of the incubation period?

CLEAN UP:● Clean each table top with Lysol or Envirocide● Return equipment to the location in the prep room. Ask the lab technician or teacher for the return location.

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LAB REPORT EXERCISE DIGESTION.

INTRODUCTIONState the purpose of this exercise

MATERIALS AND METHODS See the lab manual.

RESULTS I: DIGESTION OF FATS1. Copy results in Table 2a to the table below.

Table A. Changes in color observed during the digestion of milk fat by pancreatin. Colors in test tubes during incubation

Test tube

0min 10min 20min 30min 40min 50min 60min 70min 80min 90min

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.

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II: DIGESTION OF CARBOHYDRATESIn 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

(color)Benedict test (color)

Presence of starch (yes/no)

Presence of reducing sugar (yes/no)

1C

2C

3C

4C

III: DIGESTION OF PROTEINS1. Complete the following table (Exercise D).

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

Solutions Weight of egg white (+ or -)

Amount of egg white that has been digested (in %)

1 pepsin in buffer, pH 2.0

2 pepsin in buffer, pH 8.0

3 trypsin in buffer, pH 2.0

4 trypsin in buffer, pH 8.0

2. Prepare one graph showing the percent digestion of egg white by pepsin and trypsin in different buffers.

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Percent of digestion should be on the Y-axis and pH on the X-axis. Paste or Insert graph below.

Describe your results and explain the significance of the tubes filled with egg white incubated in buffers pH 2, and 8.

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

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I: DIGESTION OF FATSExplain 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 lipaseTest 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 color change or no color 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 CARBOHYDRATES13

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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 amylaseTest tube

Presence of starch (yes/no)

Presence of reducing sugar (yes/no)

Explain your results.

1C

2C

3C

4C

III: DIGESTION OF PROTEINSExplain 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 non-enzyme secretions at these locations, and the importance of pH in the activity of each enzyme).

Lab Materials

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EXERCISE AEquipment:● bile salts (powder)● pancreatin solution (2% - contains pancreatic lipase) 2g/100ml water, equally divided into 6 small clear

glass dropping bottles with caps.● cream (half & half)● litmus solution (1%) 1g/100ml water, equally divided into 6 small clear glass dropping bottles with caps.● distilled water● 5 ml pipettes ● pipette suction pump● test tubes (20 x 100mm)● water bath with test tube racks at 37 oC● (6) 5ml, 2N HCl in small clear dropping bottle with attached eyedropper.

EXERCISE BEquipment:● bile salts (powder)● distilled water● olive oil

EXERCISE CEquipment:● starch solution (1%)● distilled water● .2N HCl (dropper bottles)● pancreatin solution (2% - contains pancreatic amylase) 2g/100ml water● test tubes● paraffin to cover test tubes● pH meter (Hach probe type)● water bath at 37oC● iodine solution (Lugol’s)● glucose solution (1%) - 1g/100 ml water● Benedict's solution● hot plate● test tube clamps

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