D I G ES T I O N

Extractionof GastricEnzyme:

Turn apig’sstomachinsideout, washit with

water, and strip off the mucousmembrane.
Mincethe membrane, placeit in aclean bottle
and completely submergeit in glycerol. Stir
frequently and allow to stand at room
temperaturefor two(2) days.
Decant theglycerolportion into a smallf lask
and set aside.
Extractionof PancreaticEnzyme:

Removethefatsfrom apig’s pancreasand

grind them in ameat grinder. Placethe
pancreatictissue in af laskand add 100 ml. of
water and 50 ml. of 95%alcohol. Shake well
and allow to stand for two(2) days. Strain the
alcoholicextract through cheesecloth.Testthe
pH of theextract,and neutralized it.
Preparation of IntestinalExtract:

Scrapethemucousmembrane of the
washedduodenum and jejunum of apig’s
intestine. Grind thescrapingswith washed
sand in amortar, transfer it to af lask,and
add 50 ml. of 1%NaCland 5 ml. of toluene.
Allow to stand for two(2) daysat room
temperature. Shake themixturefrequently.
Then strain themixturewith acheeseclothand
storetheextractin a refrigerator.
Salivary Amylase
Reagents:
Iodine solution, Benedict’s solution.
 Procedure:
1. Place 5 ml. of 1% starch solution in a test tube.
2. Warm in a water bath at 40C, maintaining the temperature throughout the
experiment.
3. Add 1 ml. of saliva to the mixture and mix thoroughly.
4. At one- minute interval, transfer f ive (5) drops to one depression on the spot
plate, and 5 drops of Benedict’s solution. Continue the test until the starch solution
no longer gives a color reaction with iodine.
5. Treat the remaining mixture in the test tube with 10 drops of Benedict’s solution.
6. Place the test tube in a water bath for three (3) minutes. 7.Observe.
RESULTS:

Positive color for starch = deep blue color

Negativecolor for starch= yellow
Positive color f or sugar=green, yellow,
orange and red Negative color for sugar= blue
color
PICTURE RESULT:
Pancreatic Amylase
Reagents:
Iodine solution, Benedict’s solution.
 Procedure:
1.Place5 ml. of starch solution in 2 ml. of pancreaticextract in a test tube.
2.Shakewell and placein a water bath at 40C for 30 minutes. 3.Divide the mixture into
two (2) equal portions.
4.To the f irst test tube add a drop of iodine solution and to the second test tube add
f ive (5) drops of Benedict’s solution.
5.Observe.
RESULTS:

Positive color for starch = deep blue color

Negativecolor for starch= yellow
Positive color f or sugar=green, yellow,
orange and red Negative color for sugar= blue
color
PICTURE RESULT:
DIGESTION OF PROTEINS
GASTRIC ENZYME:
Reagent:
0.4%HCI,1%Na2CO3, 1%CuSO4, toluene
.
 Procedure:
1. Place the following in four (4) separate test tubes.
A. 5 ml of gastric extract
B.5 ml of 0.4% HCI
1. 5 ml of gastric extract + 3 ml of 0.4%HCI
2. 5 ml of gastric extract + 3 ml of 1%Na2CO3

Add equal slices of coagulated egg white to each tube and place the test tubes in a water
bath at 400C for two (2) hours. Add four (4) drops of toluene to each test tube and store
until next laboratory period. Determine the extent of protein digestion by noting the size
of the coagulated egg white.
RESULTS:

Test tube 1 did not work becauseit wasacontrolthatreplaced amylasewithwater.Thelackof

pepsin resultedin no protein being broken down, and sothe solution wasadark purple.This
indicatedthe presenceof proteins.
Test tube 2, did not work becausebyboiling the pepsin, wedenature it.Therefore, it cannot
breakdownthe starchand soit remainedin solution.
In test tube 3, the results werenegativeNaOHwasadded.Thisisnot the desiredpH, pepsin
favors amore acidic environment.
Test tubes 4 and 5 had apositive result, indicatedbyalightpink color.Thismeantthatthat
smallpeptides werepresentin the solution.
PICTURE RESULT:

Tube 1 Tube 2

Tube 3 Tube 4&5

PANCREATIC ENZYME:
Reagent:
0.4%HCI,1% Na2CO3
.
 Procedure:
1. Place the following in four (4) different testtubes.
a. 5 ml of gastricextract
b. 5 ml of0.4% HCI
c.5 ml of gastric extract + 3 ml of 0.4% HCI
d. 5 ml of gastric extract + 3 ml of 1%Na2CO3

Place equal sizes of coagulated egg white in each test tube and place them in a water
bath at 400C for one and a half hours. Add three (3) drops of toluene to all test tubes
and set aside until the next laboratory period. Then add 1 ml of CUSO4 to all mixtures.
RESULTS:

Test tube 1 -Blue/Purple

Test tube 2, Blue/Purple
In test tube 3, Light Purple Test
tubes 4 Light Purple Test Tube
5- LightPurple
PICTURE RESULT:

Tube 1 Tube 2

Tube 3 Tube 4&5

DIGESTION OF LIPID
Reagent:
0.05N NaOH, phenolphthalein.
Procedure:
1. Place the following in eachof the four (4) test tubes
2. 1 ml coconut oil + 4 ml pancreatic extract + 5 ml of H2O
3. 1 ml coconut oil + 9 ml of water
4. 1 ml coconut oil + 2 ml bile + 7 ml of H2O
5. 1 ml coconut oil + 2 ml bile + 4 ml pancreatic extract + 3 ml of H2O
1. Shake the test tubes well and place in a water bath at 400Cfor one and a half hours.
2. Add a drop of phenolphthalein to eachtest tube.
3. Add drop by drop of 0.05 N NaOH using a syringe until the solution turns to a faint
pink color.
4. Record the amount of 0.05 N NaOH use for eachtest tube.
TESTTUBE CONTENTS Initial FINAL CONCLUSION

2mllipase+ 4mlbile (37C) negative

A Result

2mllipase+ 4ml
distilled water
positive
B (37C) Result

4ml bile + 2ml negative

C distilled water (37C)
result

2mllipase+4ml bile negative

(room temperature)
D Result
 IntestinalDigestion for proteases:

Obtain 5 test tubes and labelthem with a wax pen asfollows:

#1
#2 (positivecontrol
#3
#4 (negativecontrol)
#5
1) In test tube #1:
• add 2mL of albumin solution
• add 3mL pepsin solution
• add 1 drop of DI water
• Stretch paraf ilm over the opening and mix well.
•Placetest tube the 37 ºC water bath for 60 minutes while mixing the
test tube every 10 minutes.
2) In test tube #2:
o add 2mL of albumin solution
o add 3mL pepsin solution
o add and 1 drop of 10N HCl
o Stretch paraf ilm over the opening and mix well.
oPlace test tube the 37 ºC water bath for 60 minutes while mixing the
test tube every 10 minutes.
3) In test tube #3:
• add 2mL of albumin solution
• add 3mL pepsin solution
• add 1 drop of 10NNaOH
•Stretch paraf ilm over the opening and mix well.
•Placetest tube the 37 ºC water bath for 60 minutes while
mixing the testtube every 10 minutes.
4) In test tube #4:
• add 2mL of albumin solution
• add 5mL of DI water
• add 1 drop of 10N HCl.
•Stretch paraf ilm over the opening and mix well.
Placetesttube the 37 ºC waterbath for 60 minutes
while mixing the testtube every 10 minutes.
5)In testtube #5:
• add 2mLof albumin solution
• add 3mLpepsin solution
• add 1drop of 10N HCl.
•Stretchparaf ilm over the opening and mix well.
• Placetesttube the ICEBATHfor 60
minutes while mixing the testtube every 10 minutes.
1) Remove all the testtubes from the 37ºCwaterbath and the ice bath.
2) Add 10drops of Biuret reagent into eachtest.Stretch paraf ilm over the
opening. Mix well.
3) Determine the Biuret reagent results Compare eachtest tube to the positive
control and negative control. Think about the ideal environment found in the
stomach. Determine how pepsin affects protein digestion.Determine how pH
affects protein digestion.Determine how temperature affects protein digestion.
 9). Clean-up: Dispose of paraf ilm in regular trash.
Dispose of allliquids in the chemical wastecontainer
near asink. Placethe test tubesin the testtube collection
bucket.
Rinse and washallserologicalpipets and graduated
cylinders.
Intestinal Enzymes:
Reagents:
Procedure:
1. Prepare four (3) testtubes as follows:
A. 5 ml of water
B. 5 ml of intestinal extract+ 1 ml of 1% Na2CO3
C.5 ml of boiled intestinal extract+ 1ml of 1%Na2CO3
Add 1 ml of milk to eachtest tube and mix thoroughly.
Divide the contents of the four
(3) test tubes equally and set aside the remaining three
test tubes as control
tubes. Placethe test solutions in awater bath 40C for
one hour. Add 1 ml of CUSO4 for both incubated tubes
and the control
tubes.
1.Give the effect of the following:

Boiling the enzyme on its activity

Enzymes are large proteins that catalyze chemical reactions. That means they assist in the formation or
disruption of atomic bonds. Enzymes, like other proteins, get their properties from their shapes.
Anything that disrupts the shape of an enzyme -- including boiling and freezing -- will make it
inactive.The higher the temperature, the more an enzyme will vibrate. If it vibrates enough it will distort
out of shape, or denature. If the temperature goes up even more, the different regions of the protein
will swingso far apart from each other that they can't come back .When enzymes boil they irreversibly
denature and become inactive.
Bile on fat digestion

When digesting fats, bile actsas an emulsif ier to break the large fat globules into smaller emulsion
droplets. The use of this is that it gives the fat a much larger surface area on which the lipase enzymes
(fat digesting) canact on, which in turn makes it a much quicker and ef ficient process.
Bile also helps to make fat more "soluble". On it's own, fat will separate from water which makes it hard
to transport in the human body. This is because it is hydrophobic. However, the bile bonds to the fat on
it's hydrophobic (water- hating) tail and the water on the hydrophilic (water-loving) head. This allows it
to be carried by water.
HCIon gastric digestion

Hydrochloric acidor HCLis an acidthat forms when hydrogen and chloride combine in your stomach.
Your body uses HCLin the early stages of digestion. It has a low pH of about 2, which means it is actually
strong enough to dissolve metal. Along with water and other stomach secretions, HCLmakes up the
gastric juice that f ills your stomach when you eat. Your stomach is coated with a form of mucus that
protects the lining against such a strong acid. Otherwise, HCLwould digest your stomach along with its
contents.
1.Proteolytic enzymes are secreted as proenzymes. What is the significanceof this?
Proteolytic enzymes are essential for many important processes in your body. They’re also called
peptidases, proteases or proteinases.In the human body, they are produced by the pancreas and
stomach.While proteolytic enzymes are most commonly known for their role in the digestion of dietary
protein, they perform many other critical jobs as well. (For example, they are essential for cell division,
blood clotting, immune function and protein recycling,among other vital processes)
1.Proteolytic enzymes are secreted as proenzymes. What is the significanceof this?
Proteolytic enzymes are essential for many important processes in your body. They’re also called
peptidases, proteases or proteinases.In the human body, they are produced by the pancreas and
stomach.While proteolytic enzymes are most commonly known for their role in the digestion of dietary
protein, they perform many other critical jobs as well. (For example, they are essential for cell division,
blood clotting, immune function and protein recycling,among other vital processes)
1. Discuss brief ly the activation of:

A.Pepsinogen

Pepsinogens are synthesized and secreted primarily by the gastric chief cells of the human stomach
before being converted into the proteolytic enzyme pepsin, which is crucial for digestive processes in
the stomach. Furthermore, pepsin can activate additional pepsinogen autocatalytically.

In the digestive tract pepsin effects only partial degradation of proteins into smaller units called
peptides, which then either are absorbed from the intestine into the bloodstream or are broken down
further by pancreatic enzymes
A.Trypsinogen

Trypsinogen is an enzyme released by the pancreas that is used to break down proteins found in
foods. Before it reaches the digestive tract, the enzyme is inactive. Once it reaches the intestines,
trypsinogen is converted to its active form, trypsin. Levels of this enzyme present in the bloodstream
canbe monitored to determine if there is a problem with pancreas function.
.Chymotrypsinogen
an inactive precursor (zymogen) of chymotrypsin, a digestive enzyme which breaks proteins down into
smaller peptides. Chymotrypsinogen is a single polypeptide chain consisting of 245 amino acid
residues.[1] It is synthesized in the acinar cells of the pancreas and stored inside membrane-bounded
granules at the apexof the acinar cell. Release of the granules from the cell is stimulated by either a
hormonal signal or a nerve impulse, and the granules spill into a duct leading into the duodenum
1.In these experiments on digestive enzymes, what factors inf luenced the rate of enzyme-catalyzed
reaction?

There are several factors that caninf luence the rate of enzyme reactions. The most common include
changes to pH, temperature, or substrate concentration. The substrate is the compound an enzyme
bonds with.

All enzymes have an optimal pH they work best in. The pH scaleruns from 1 to 14 and is a measure
of how acidic or basic a substance is. This image demonstrates how pH inf luences enzyme activity.
Here wehave three different enzymes (pepsin, salivary amylase, and arginase). Seehow pepsin works
very well at a pH of 2, yet both salivary amylase and arginase fail to function?
Thisisbecausepepsin's optimal pH is around 2,
whilesalivary amylasehas an optimum of
around 7,and arginaseat roughly 9.5. Placing
an enzyme in an environment with its optimal
pH will increasethe rate of reaction. If the
environment is outside itsoptimal range, the
enzyme rate of reaction will decrease.
Changing thetemperaturecanalso cause f luctuationsin ratesof
reaction. Again,as with pH, allenzymeshavean optimum
temperature. However,when explaining how temperature
inf luencesrateof reaction wecanusegeneraltrends. Thisis
because increasing temperatures willincreasethe rateof reaction
for nearly allenzymeswhile decreasing temperaturewillhavean
inverse effect.