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Process Of Digestion

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Autor:  back_up  22 December 2009
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Introduction
Since digestive enzymes actually function outside of the body cells in the digestive tract, their hydrolytic activity can be studied in a test tube. We studied starch digestion by salivary amylase, trypsin digestion of protein and pancreatic lipase digestion of fats. The goal of these tests was to understand the optimal environmental conditions needed for digestive enzymes to function. Additionally, it was the goal of the above experiments to learn the role of temperature and pH in the regulation of enzyme activity.
Starch Digestion by Salivary Amylase--Relevant Observations:
Salivary amylase and pancreatic amylase break down starch.
Boiling the mixtures denatures the enzymes and therefore starch is not reduced to sugar.
The pH of 7 is optimal for reducing starch to sugar.
If the pH is too high or too low starch and sugars will remain present.
Protein Digestion by Trypsin--Relevant Observations:
Trypsin breaks down protein.
When the substrate, BAPNA, was added to the test tubes containing Trypsin, we learned that hydrolysis had occurred.
Emulsification of Bile and Fat Digestion by Lipase--Relevant Observations:
Bile emulsifies fats. Emulsified fats provide a larger surface area for enzymatic activity.
When bile salt is added to the test tube with vegetable oil, the bile salt acts as a detergent and emulsifies the fat so that the surface area was greater, allowing the greater enzymatic activity.
In the case of the Pancreatic Lipase, we learned that fats are digested by fatty acids during hydrolysis, they will lower the pH of the sample they are in. Litmus cream turns from blue to pink if the solution is acidic.
Hypotheses: Hydrolytic enzymes hydrolyze only a small group of substrate molecules, and specific environmental conditions are necessary for it to function optimally.

Materials and Methods:
Hot plates
250-ml beakers
Boiling chips
Test tubes and test tube rack
Wax markers
Water bath set at 37 degrees C
Ice water bath
Chart to record results
Dropper bottle of distilled water
Dropper bottles of: 1% amylase, 1% boiled starch solution, 1% maltose solution, Lugol’s iodine solution (IKI), Benedict’s solution.
Spot plate
Dropper bottles of 1% trypsin and .01% BAPNA solution
Dropper bottles of: 1% pancreatic solution, litmus cream, .1 N HCL, vegetable oil
Bile salts
Parafilm


Directions for conducting the experiment:
Activity 1:
1. Obtain a test tube rack, 10 test tubes, and a wax marking pencil. Obtain a dropper bottle of distilled water and dropper bottles of maltose, amylase and starch solutions.
2. To test tube 1A; add 3 drops of starch and 3 drops of water.
3. To test tube 2A; add 3 drops of amylase and 3 drops of water.
4. To test tube 3A; add 3 drops of maltose and 3 drops of water.
5. To test tube 4A; add 3 drops of amylase then boil for 4 minutes then add 3 drops of starch.
6. To test tube 5A; add 3 drops of amylase and 3 drops of starch.
7. Incubate the 5 test tubes for 1 hour at 37 degrees C. Shake test tubes periodically.
8. Obtain a spot plate and dropper bottles of Lugol’s solution and Benedict’s solution. Set up the boiling water bath using a hot plate, boiling chips and a 250 ml beacker.
9. Mark 5 depressions of the spot plate 1A-5A for sample identification.
10. Pour about a drop of the sample from each of the tubes 1A-5A into the appropriately numbered spot. Into each sample drop, place a drop of Lugol’s IKI solution.
11. Into the remaining mixture in each tube, place 3 drops of Benedict’s solution. Put each tube into the beaker of boiling water for about 5 minutes.
12. Record data.
Activity 2:
1. Obtain 5 test tubes and a test tube rack. Get a dropper bottle of trypsin and BAPNA.
2. Mark each tube with a wax pencil; 1T-5T.
3. To test tube 1T; add 3 drops of trypsin and 3 drops of distilled water.
4. To test tube 2T; add 3 drops of BAPNA and 3 drops of distilled water.
5. To test tube 3T; add 3 drops of Trypsin, boil 4 min, then add 3 drops of BAPNA.
6. To test tube 4T; add 3 drops of trypsin and 3 drops of BAPNA.
7. To test tube 5T; add 3 drops of trypsin and 3 drops of BAPNA.
8. Incubate tubes 1T-4T at 37 degrees C for one hour. Put tube 5T in an ice bath for 1 hour. Shake rack occasionally.
9. At the end of 1 hour, examine the tubes for the trypsin assay.
10. Record data.
Activity 3:
1. Obtain 9 test tubes and a test tube rack.
2. To tube 1E; add 10 drops of water and 2 drops of vegetable oil.
3. To tube 2E; add 10 drops of water, 2 drops of vegetable oil and a pinch of bile salts.
4. Cover each tube with a small square of parafilm, shake, and allow the tubes to stand at room temperature.
5. After 15 minutes, observe both tubes .
6. Record emulsification data.
8. To tube 1L; add 3 drops of lipase and 3 drops of distilled water.
9. To tube 2L; add 3 drops of litmus cream and 3 drops of distilled water.
10. To tube 3L; add 3 drops of lipase, boil for 4 minutes then add 3 drops of litmus cream.
11. To tube 4L; add 3 drops of lipase and 3 drops of litmus cream.
12. To tube 5L; add 3 drops of lipase and 3 drops of litmus cream--put in ice bath for 1 hour.
13. To tube 4B; add 3 drops of Lipase, 3 drops of litmus cream and a pinch of bile salts.
14. To tube 5B; ad 3 drops of Lipase, 3 drops of litmus cream and a pinch of bile salts. Cover with parafilm, shake and put in ice bath for 1 hour.
15. Incubate tubes 1L-4L and tube 4B at 37 degrees C for 1 hour.
16. Prepare a color control by adding 0.1 N HCL drop by drop to tubes 1L and 2L until the cream turns pink.
17. Record data.
Results:
Salivary amylase digestion of starch
Tube no. 1a 2a 3a 4a 5a
Additives
3 drops ea. Starch
Water Amylase
Water Maltose
Water Boil amylase 4 min then add starch Amylase
Starch
Incubation
Condition 37 C 37 C 37 C 37 C 37 C
IKI test No change Yellow Slight orange Slightly cloudy, hint of blue orange
+ or - + - - - -
Benedict’s Blue Blue Blue Blue Blue
+ or - - + - + +

Starch when exposed to amylase is broken down to simple sugars.


Trypsin digestion of protein:

Tube no. 1T 2T 3T 4T 5T
Additives
3 drops ea. Trypsin
Water BAPNA
Water Trypsin boil 4 min.
BAPNA Trypsin BAPNA Trypsin
BAPNA
Incubation
condition 37 C 37 C 37 C 37 C 0 C
Color Change Slight yellow No change Cloudy yellow Yellow yellow
+ or - + - + + +


Pancreatic Lipase digestion of fats:
Tube # 1L 2L 3L 4L 5L 4B 5B
Adds.
3 drops Lipase
Water Litmus cream water Boil lipase 4 min.
Litmus cream Lipase litmus cream Lipase litmus cream Lipase litmus cream
Bile salts Lipase litmus cream bile salts
Incuba-
tion 37 C 37 C 37 C 37 C 0 C 37 C 0 C
Color change Cloudy Pink More pink Pink Pink Pink Very pink
+ or - - + + + + + +



Discussion and Conclusion:
Salivary amylase and pancreatic amylase in the mouth and small intestine respectively break down starch to its’ simpler sugars.
Trypsin, a pancreatic enzyme breaks down proteins.
Fats are emulsified by the detergent action of bile salts ducted in from the liver. Pancreatic lipase further breaks fats down to become fatty acids and monoglycerides and glycerol.
I conclude that the data did support my hypothesis.
A major error of the testing was the time factor. There simply was not enough time to accurately run the tests and properly record the data. It would have been beneficial to have each group run one of the tests and record their data on a common class chart in order to facilitate group discussion regarding the results.








Source:

Marieb, Elaine N., and Susan J. Mitchell. Human Anatomy and Physiology Laboratory Manual. 9th ed. San Francisco: Pearson Education, 2008. 597-608.



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