PART 2: PANCREATIC LIPASE ACTIVITY

INTRODUCTION
In order for the nutrients in food to be absorbed, they must first be broken down into particles
that are small enough to be transported through carrier proteins into the epithelial cells that
form the mucosal lining of the digestive tract. This process of breaking down food is called
digestion, and occurs primarily within three particular segments of the digestive tract: the
mouth, the stomach, and the small intestine. There are many different substances that are
secreted into the different segments of the digestive tract like bile salts, bilirubin,
hydrochloric acid (HCl), and sodium bicarbonate (NaHCO3). It just some of the substances
mixed with the food as it passes through the digestive tract, and many of these substances
facilitate the breakdown of food. However, the most important substances secreted for the
purpose of digestion are the digestive enzymes. Digestive enzymes greatly enhance the rate at
which the covalent bonds that link subunits together to form polymeric biomolecules are
broken. Indeed, without the presence of these enzymes, chemical digestion would essentially
not occur (Bengt, 2007).

Lipase is a type of enzyme known as a hydrolase and is responsible for catalysing the
hydrolysis of triglycerides (the substrate) into fatty acids and glycerol. It is referred to as a
hydrolase because the reaction that it catalyses is a hydrolysis reaction – a reaction in which
large molecules are broken down into smaller ones with the addition of water (Johnson,
2010). Fat digestion in the human body takes place mostly in the duodenum of the small
intestine. Fat digestion requires enzymes called Lipase, bile salt and also with the present of
sodium bicarbonate. This sodium bicarbonate that buffer stomach acid, will maintaining a
chemical environment which pancreactic enzyme can function. While for the bile salt that
secrete by liver will make fat digestion more efficient. Bile salt are like a detergent they
emulsify, or break up larger unit of fat into smaller one (Cecie Star, 2014).In this experiment
we going to investigate the influence of bile salt and temperature on lipase digestion activity
in full fat milk. Milk is a white liquid composed mostly of water (87.3%), with small amounts
of fats (3.9%), and non-fat solids such as proteins and lactose (8.8%).Milk contains more fat
than most liquids and a majority of these lipids are classed as triglycerides which therefore
makes milk a suitable liquid to be used for this experiment. The globules of fat found in the
milk gives the lipids a larger surface area and provides more ‘surfaces’ that the lipase enzyme
can bind to.
OBJECTIVE
1. To determine the absent or present of bile salt and different temperature in different
solution of the test tube for the lipase digestion activity.

MATERIAL

37 º C water bath

Ice water

Beaker full of water heated to 70 º C on hot plate

Tap water

250ml beaker

Test tubes in holder

Marker

Ph paper

Phenol red solution in dropper

Milk

2% sodium bicarbonate solution

1% bile salt solution

1% pancreatin solution

METHOD

1. 5 test tube was labelled with number 1, 2, 3, 4 and 5.

2. 5 ml of milk was added into each test tube.
3. 3 ml of sodium bicarbonate was added into each test tube.
4. 3 drop of phenol red was added into each test tube.
5. Litmus paper was used to determined pH level in each test tube must be in basidic
condition around 7.5 to 8.0.
6. 3 drop of phenol red was added into each test tube.
7. 15 drop of pancreatin solution was added into test tube 2, 3, 4 and 5.
8. 10 drop of bile salt was added into test tube 3.
 9. All the test tube was swirl.
10. Test tube 1, 2 and 3 was placed in the 37o C water.
11. Test tube 4 was placed in the beaker with 70o water.
12. Test tube 5 was placed in the ice bath.
13. 10 minutes over the next hour the test tube was swirl.

RESULT

Figure 1 shown the result of the mix between milk, sodium bicarbonate and phenol red
solution in room temperature after 1 hour.

Figure 2 shown the result of the mix between milk, sodium bicarbonate, phenol red and
pancreatin solution in room temperature after 1 hour.
Figure 3 shown the result of the mix between milk, sodium bicarbonate, phenol red and
pancreatin solution in room temperature after 1 hour.

Figure 4 shown the result of the mix between milk, sodium bicarbonate, phenol red and
pancreatin solution in 70o C temperature after 1 hour.

Figure 5 shown the result of the mix between milk, sodium bicarbonate, phenol red and
pancreatin solution in ice bath after 1 hour.
 Test tube Color before Color after Color change
1 Pink Pink Insignificant
2 Light pink Pale pink Moderate
3 Pale pink-green Orange cream Substantial
4 Light pink Pale pink Moderate
5 Light pink Baby pink Slight
Table 1 shown the result of color change in all test tube after 1 hour

DISCUSSION
In this experiment shown that test tube 3 was the best condition for lipase enzyme activity to
working and take action in it. It is because test tube 3 was change from pale pink green into
orange cream. To follow the reaction, we will make use of the fact that fats are neutral, while
fatty acids are acidic. The release of fatty acids from fats by hydrolysis will increase the
acidity (lower the pH) of the reaction mixture. This change can be observed by using the
indicator dye, phenol red, which is above pH 8.2, phenol red turns a bright pink (fuchsia)
color. This solution contains a pH indicator which helps in monitoring of the pH changes in
the solution. It will change from pink to orange when the pH value decreases. At pH 6.8 the
indicator is orange and at pH 8.4, the indicator turns pink. That mean it is useful for
measuring pH values between 6.8 and 8.4 (Reginald,1998).
Pancreatin solution is an enzyme that breaks down dietary fats into smaller molecules called
fatty acids and glycerol (Cecie Star,2014). The sensitivity of the enzyme’s means that any
changes in physical and chemical conditions will lead to a denatured enzyme that will be
unable to break down the specific substrate that matches the shape of the active sites cleft. If
this occurs during the procedure then the lipids will no longer be hydrolysed to fatty acids
and no readings will be able to be taken as there will no longer be a change in pH. If the
solution of lipase experiences too high temperatures then the atoms making up the lipase will
have more kinetic energy causing them to vibrate vigorously, tearing apart the hydrogen
bonds and other bonds holding the protein structure together. Low temperatures however
would cause the enzyme to hibernate, as it would have a too minimal amount of kinetic
energy to function efficiently.

Despite denaturing enzymes being useful to prevent food spoilage by various enzymes found
in food materials, the denaturing of the lipase enzyme would mean that no results could be
harvested, and so the experiment would lose its purpose. Many enzymes in the human body
have an optimum temperature of approximately 37˚C. We anticipate that the lipase enzyme
will hydrolyse fats most efficiently at a temperature of about 37 – 40˚C, as this is close to our
human body temperature (37˚C) and matches the temperature of the digestive organs in
which lipase acts. It is possible however that the lipase may have an optimum temperature
higher than this, as the human body uses 37˚C as additional energy (food) would be needed to
maintain a higher temperature. The solution of milk, lipase, bile salts, sodium carbonate and
phenol red will be mixed at a range of higher and lower temperatures to test the optimum
temperature of the lipase enzyme. The preliminary experiment will be used to verify the
optimum temperature in which lipase works best.

If one test tube were to contain bile salts than another, then the fat present in the full fat milk
may be emulsified to a greater extent than a test tube without bile salt. For example, test tube
3 contain of lipase was mixed with equal amounts of phenol red, full fat milk, bile salt and
sodium carbonate compare to the test tube without bile salt, then the fats in the full fat milk
could be dispersed over a small surface area, making it easier for the test tube 3 solution to
act on the lipids. This in turn could allow the test tube 3 solution of lipase to hydrolyse the
lipids in the milk faster than the other test tube because bile salt act as detergent it is
emulsify, it can break large unit into smaller unit and give fat digesting enzyme much greater
surface to act on (Cecie Star,2014). These results would insinuate that the test tube 3 can
hydrolyses lipids than other test tube when realistically the hydrolyse the lipids faster because
there is a greater enzyme concentration (Johnson,2010).

CONCLUSION

In this experiment we can conclude that lipase enzyme activity can take action in the present
of bile salt with the condition of room temperature which is 37 o C. That happen because of
bile salt was helping to make fat digestion more efficient, bile salt was like detergent it
emulsify, or break up large unit of fat into smaller one. Temperature 37 oC is the best
temperature because this condition was same with our body or stomach condition optimum
temperature.
REFERENCE

Cecie S. & Beverly M. (2014). Human biology. Canada: Nelson Education.

Reginald M. A. (1998). Determination of Lipase Activity. Retrieved May 2, 2019, from

http://www.jbc.org/content/165/2/443.full.pdf

Johnson. (2010). The volume of lipase affects the rate of the hydrolysis of lipids. Retrieved
May 2,2019, from https://www.ucl.ac.uk/~zcapf71/lipase_littlelaptop%5B1%5D.pdf

Bengt B. (2007). Influence of bile salt, pH, and time on the action of pancreatic lipase;
physiological implications. Retrieved May 2, 2019, from
http://www.jlr.org/content/5/4/522.full.pdf