Oscar Mora Snchez

304
Biology Higher Level
Laboratory report | Lipase

Enzymatic Reaction Of Lipase In Different Types Of Milk

Introduction

Lipase is an enzyme the body uses to break down fats in food so they can be absorbed in
the intestines. Lipase is produced in the
Diagram 1: Obtained from
(Ummedu, 2015)
pancreas, mouth, and stomach. (Ummedu,
2015)

Milk may be defined various ways.

Chemically speaking, milk is a complex fluid in
which more than 100 separate chemical compounds have been found. Its major components are
water, fat, lactose, casein, whey proteins, and minerals (or ash) in amounts varying with the milk of
various species of animals. (Ummedu, 2015).

There are different types of milk, and each type of milk has different composition and
nutritional content. And each type of milk has a different quantity of lipids. In the
experiment that is going to be done, there were used 5 types of milk: whole, siluette, light,
developing, and semi-skim. Here are shown the quantities of lipids that the milk Lala has in
its products of this types of milks:

Whole milk: 7.5 grams per 250 mL

Siluette milk: 0.5 grams per 250 mL
Light milk: 2.5 grams per 250 mL
Developing milk: 6.4 grams per 250 mL
Semi-skim milk: 4 grams per 250 mL

Through the realization of the experiment, it is going to be shown how does the lipase
affects in each of the milk types, by measuring its pH at the initial time, 15 minutes and 30
minutes after the lipase is added, and seeing how does the pH becomes more acid; so, by
the pH it is shown how does it affect.
Research Question and hypothesis
In what way does the different types of milk (whole, light, semi-skim, siluette and
developing) affect in the work of the enzyme lipase in it (the milk) at optimum temperature
of the enzyme (45C) in a period of 30 minutes? If the type of milk has more lipids, then it
will have a higher change in the ph and will become more acid, because the enzyme lipase
is going to have more substrates to interact with.

Variables
Type of Variable
Independent

Dependent

Controlled

Variable
Type of milk

Control
5 different types of milk were used in the
experiment (whole, siluette, light, developing,
and semi-skim). Each type of milk is contained
in a different beaker with its proper mark.
Ph
The ph is the indicator of the reaction taking
place in the experiment. It is measured 3 times,
after a period of time. It is measured with a ph
meter.
Control group
The control milk shows the ph that a control
group of each type of milk has.
Temperature
The temperature is the same for all the trials of
the experiment (45C), the best temperature
for the work of the enzyme.
Time
Time is measured with a chronometer. After
the tube test is in the incubator, it is set for 15
minutes. When is collocated in the incubator
again, it is set again for 15 minutes.
Quantity of milk All the trials with all types of milk use 10mL of
and lipase
the milk. All the trials with all types of milk use
3mL of lipase.

Materials
The materials needed for the realization of the experiment were the following:

Two test tube container per table

Two beakers of 100 mL per table
Four test tubes of 10 mL per table
Lipase (5 mL at 3%)
Incubator (at 45C)
Chronometer
pH meter
Lab coat
Siluette, light, whole, developing, and semi-skim milks (40 mL of each one)
Temperature bath

Marker
Tape
Test tube container
Graduated cylinder

Procedure
For the realization of the experiment there must be prepared the materials of the
laboratory first. Each beaker of 100mL has 40 mL of milk. Different beakers for each type of
milk are used. The test tubes are in the test tube container.
1. When you are in your work table, identify and mark the substances you have in your
table, by pasting a piece of tape with the name of the substance written with the
marker.
2. In a graduated cylinder, empty 10 mL of the beaker that contains 100 mL of the milk
tested in your table. Be careful and try to pour this quantity exactly by seeing the
measures in the graduated cylinder.
3. Empty the milk contained in the graduated cylinder into a test tube. Try to empty all
the substance and to do not let it in the graduated cylinder.
4. Put the test tube in the test tube container.
5. Do the same three times more with the same milk type, because each milk type has
4 repetitions of the experiment. As you are going to collocate the 4 test tubes in the
same test tube container, it is important to put them in order so you now which is
the trial 1, 2, 3, and 4 when taking notes.
6. Add 5 mL of lipase 3% to each test tube containing milk (4 test tubes). Be careful
and try to add the exact quantity of lipase.
7. Measure the ph of each substance with the ph meter, and take notes, according to
how they are arranged in the test tube (trial 1, 2, 3, 4). It is important to do not let
pass much time after the lipase was added to measure the ph.
8. Take the test tube container into the incubator at 45C (degrees Celsius). At the
same time start the chronometer. The incubator must be preheated before, for it to
be at 45 at the moment when the test tube container is taken in.
9. When 14:40 minutes have passed, prepare to take the test tube container out of
the incubator. At 15:00 minutes, take the test tube container out of the incubator.
10. Measure the ph of each test tube of the experiment and take notes of it. It is
important to have identified each test tube for them to acquiesce with the notes
taken at 0:00 minutes.
11. Reset the chronometer. Start it and get the test tube container into the incubator
again, for 15 minutes more.

12. Repeat steps 9 and 10 of this section.

13. The experiment with one type of milk is finished. Repeat steps 1 to 12 of this
section for each type of milk, excepting the one done in the first time.
Now the experiment finished. There must be 4 trials of each type of milk, 4 test tubes. And
each trial has 3 measures of ph; one at 0 minutes, one at 15:00 minutes and one at 30:00
minutes.
Raw data
This section shows the raw data obtained in the realization of the experiment. There are
shown the pHs of the milk in 0, 15 and 30 minutes after the lipase is added. These results
are shown for each milk type (whole, silouette, light, developing, and semi-skim). The
control group shows the pH of the milk without the lipase being added.
Whole milk pHs obtained after each period of
time
pHs at each time
Trial
0min
15min
30min
1
6.49
6.29
6.16
2
6.48
6.29
6.15
3
6.48
6.28
6.14
4
6.49
6.32
6.2
control
6.46
6.46
6.46

Table 2: This table shows

the pH of whole milk at 0,
15 and 30 minutes of the
experiment
since
its
beginning. Also the control
group is shown.

Light milk pHs obtained after each period of

time
pHs at each time
Trial
0min
15min
30min
1
6.5
6.25
6.04
2
6.51
6.27
6.15
3
6.53
6.28
6.09
4
6.54
6.29
6.11
control
6.42
6.42
6.42

Table 3: This table shows

the pH of light milk at 0, 15
and 30 minutes of the
experiment
since
its
beginning. Also the control
group is shown.

Semi-skim milk pHs obtained after each period

of time
pHs at each time

Table 4: This table shows

the pH of semi-skim milk
at 0, 15 and 30 minutes of

Trial
1
2
3
4
control

0min
6.54
6.56
6.57
6.59
6.47

15min
6.24
6.22
6.24
6.26
6.47

30min
6.01
6
6.01
6.07
6.47

the experiment since its

beginning. Also the control
group is shown.

Siluette milk pHs obtained after each period of

time
pHs at each time
Trial
0min
15min
30min
1
6.56
6.35
6.22
2
6.57
6.39
6.25
3
6.58
6.39
6.24
4
6.59
6.41
6.28
control
6.41
6.41
6.41

Table 5: This table shows

the pH of siluette milk at 0,
15 and 30 minutes of the
experiment
since
its
beginning. Also the control
group is shown.

Developing milk pHs obtained after each period

of time
pHs at each time
Trial
0min
15min
30min
1
6.55
6.21
6.01
2
6.57
6.2
6.02
3
6.6
6.25
6
4
6.61
6.28
6.03
control
6.48
6.48
6.47

Table 6: This table shows

the pH of developing milk
at 0, 15 and 30 minutes of
the experiment since its
beginning. Also the control
group is shown.

Processing data
In this section it is shown the operations done with the raw data to analyze and compare
the data obtained. The first thing done, to reduce the number of data was to get a mean of
the pHs obtained in the four trials of the experiment. To get the mean, the pHs of the four
trials of a milk type in a specific time are summed, and then it is divided by 4 because there
are 4 data being added. For example to get the mean of the developing milk at 0 minutes
the operations done will be:
6.55 + 6.57 + 6.6 + 6.61 = 26.33

26.33 / 4 = 6.5825
pHs average of each milk type in each time
Milk type
0min
15min
30min
Whole
6.485
6.295
6.1625
Light
6.52
6.2725 6.0975
Semi-skim
6.565
6.24
6.0225
Siluette
6.575
6.385
6.2475
Developing
6.5825 6.235
6.015

Table 7: Shows the

average pH of each
milk type in after each
lapse of time.

And for each mean, the standard deviation was obtained. The standard deviation is a
measurement of the dispersion of the data. The standard deviation is obtained by the
following general formula:

(Where n is the sample size). Formula from : (Bbccouk, 2015)

The standard deviations obtained for each average are the following:
Table 8: Shows the standard
Standard deviations of each average
deviations of each pH average
Milk type
0min
15min
30min
Whole
0.0057735 0.01732051 0.02629956 of table 7.
Light
0.01825742 0.01707825 0.04573474
semi-skim 0.02081666 0.01632993 0.03201562
Siluette
0.01290994 0.02516611 0.025
Developing 0.02753785 0.03696846 0.01290994

The next thing done for processing data was to obtain the differences in each type of milk
between 0 and 15 minutes and between 15 and 30 minutes. For this, the 0 minute average
pH is subtracted by the 15 minutes average pH. And also the 15 minutes average pH is
subtracted by 30 minutes average pH. Also a difference between the 0 and 30 minutes
average pH was obtained, by subtracting the 0 minutes pH average by the 30 minutes
average pH. For example, to obtain this data in the whole milks ph averages, the
operations done were the following:
0 15 minutes = 6.485 6.295 = 0.19
15 30 minutes = 6.295 6.1625 = 0.1325
0 30 minutes = 6.485 6.295 = 0.19

Differences between lapses of time

Milk type
0-15
15-30
0-30 min
min
min
Whole
0.19
0.1325
0.3225
Light
0.2475
0.175
0.4225
semi-skim
0.325
0.2175
0.5425
Siluette
0.19
0.1375
0.3275
Developing 0.3475
0.22
0.5675

Table 9: Shows the

differences
between
lapses of time gotten with
the pHs average.

Processed data presentation

Graphs below show the differences obtained between the lapses of time, shown in the
section of processing.
The graph below shows the absolute change in pH of milk in all the different milk types in
two intervals of time, from 0 to 15 and from 15 to 30 minutes. With this graph it is
compared the change in the first and in the second lapse of time of each milk, and it also
can be compared between different milks.

Absolute Change in pH of milk types in

lapses of time
0.6
0.5

Change in pH

0.4
0.3

0-15 min
15-30 min

0.2
0.1
0
Whole

Light

semi-skimm

Siluette

Developing

Milk type

Graph 1: This graph shows the absolute change in pH of each milk type in the two different lapses of time.

The graph below shows the absolute changes in pH in all the time of the experiment, from 0 to 30
minutes. This graphs main purpose is to compare between the change between each milk and see
which milk has a higher or lower change.

Absolute Change in pH of milk types from 0 to

30 minutes
0.6
0.5

Change in pH

0.4
0.3
0-30 min
0.2
0.1
0
Whole

Light

semi-skimm

Siluette

Developing

Milk type

Graph 2: This graph shows the absolute changes from 0 to 30 minutes in each milk type.

Conclusion

By seeing the graphs it is possible to infer some things. In graph 1 are shown the absolute
changes in the two lapses of time of 15 minutes. Here is shown that no matter the milk
type, the first 15 minutes show through the pH change that there were more enzymatic
reaction that in the second lapse of 15 minutes. The second lapse shows an smaller change
in the pH, thus, lower enzymatic reaction. It can be also seen that the difference in the
changes of pH between the lapses of time is bigger in some milk types than in others. For
example, the developing milk shows a high change in the first lapse of 15 minutes, and the
change in the next lapse of time is significantly smaller. And in the other hand, in the
siluette milk the change in the first lapse does not have a difference with the second lapse
as the developing milk has.

And in graph 2 it is shown the absolute change in the total time of the experiment. In this
graph it is observed which milk type lead to a bigger change in the pH, so have more
enzymatic reaction in that period of time. The milk with more enzymatic reaction was the
developing milk, then the semi-skim, then the light, then the whole and the siluette, having
this last two a very similar change.
According to the nutritional content of Lala, the milk with more lipids is the whole milk,
then the developing milk, then the semi-skim, then the light, and at the end the siluette
milk.
My hypothesis was: If the type of milk has more lipids, then it will have a higher change in
the pH and will become more acid, because the enzyme lipase is going to have more
substrates to interact with.
For my hypothesis to be correct, the whole milk should have the bigger change in the pH,
because is the milk with more lipids to be broken down by the lipase. However, the milk
that had a bigger change in the pH was the developing milk.
And the milk with less lipids, the siluette, should be the milk with less change in the pH, and
it is, so here the hypothesis is correct, but the whole milk has a very similar absolute
change in the pH, so the enzymatic reaction took place more or less in the same way in the
milk with more lipids and with less lipids. The reaction might not occurred so fast in the
whole milk because, as there are many lipids, many substrates, they are very tight together
so it is harder for the substrate to bind to the active site of the lipase. And in the siluette,
with very few lipids, the molecules are far apart from each other, so it is harder for the
substrates to find to the active site of the lipase.
But in the developing milk, the second milk with more lipids, there is observed the bigger
change in pH, so the more enzymatic reaction taking place. This can mean that there is the
optimum number of substrates for the enzymes to work properly. And also it can be seen
in graph 1 that the first lapse of time is where was observed the major part of the change,
so this means that the enzymes were working at full capacity and then the number of
substrates decreased much so the enzymatic reaction took place in a lower rate.
So, it cannot be said that the hypothesis was correct, because the whole milk is not the one
with a major change in pH and major enzymatic reaction; however, the idea of the
hypothesis is correct; that with more substrates, there is more enzymatic reaction, but
there was not taking in account that if the number of substrates is bigger than the
optimum, the reaction will go slower.

The experiment was done as it was planned, and the results obtained are satisfactory. The
procedure was done according to the times set before and as it was wanted to do. May be
at the way of processing data there could be more things done in order to understand in a
better way the behavior of the reaction. If the differences were gotten first and then the
average, may be the data processed will be more accurate.
To improve the experiment, somethings can be done. First, as all the milk beakers at the
moment of measuring its pH there is just one pH meter, so it is going one by one, and while
measuring one the time happens and the other beakers are still reacting, so the measure it
is not in 15 and 30 minutes exactly for each one. So there could be used more pH meters to
get better in this aspect.
Something else that could be improved is the number of measurements of the pH. To
understand how the enzymatic reaction takes place in a better way, the measurements
could be done in shorter lapses of time and for a longer time; for example, each 8 minutes
for 40 minutes. In this way it will be understood in a better way how the reaction does
behaves with the pass of the time.
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