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Effect of temperature on pancreatic lipase on lipid digestion measured using pH sensor. Please give proper reference to my IB student, Gina if you use this material.
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IB Biology HL
Name: Yoojin Lee
Candidate Number: 002213-067
1
Candidate Name :Yoojin Lee
Candidate Number :002213-067
Date of Practical :November 18, 2010
Internal Assessment – Effect on changing the temperature of pancreatic lipase
on lipid digestion
Research Question
How will changing the temperature of pancreatic lipase together with bile solution affect the
rate of digestion of milk into glycerol and fatty acids, measured using pH sensor?
Introduction
Pancreatic lipase1 is an enzyme that digests dietary lipids into glycerol and three fatty acids
in alkaline condition. In this investigation, milk that contains fats is used. The dietary lipid in
milk is insoluble while lipase is soluble in water. Thus, by nature, lipase cannot directly break
down the dietary lipid, because they will form two layers. Hence, an emulsifier called the bile
salts is essential. Bile salts are amphipathic, having both hydrophilic and hydrophobic
characteristics.2 By making the lipids soluble in water, bile salts enable lipase to successfully
digest.
Figure 1 shows the chemical process of lipase activity on a triglyceride3
1 “Pancreatic Lipase,” Wikipedia, the free
encyclopedia, http://en.wikipedia.org/wiki/Pancreatic_lipase (accessed January 8, 2011). 2 R. Bowen, “Absorption of Lipids,”
Colostate,http://arbl.cvmbs.colostate.edu/hbooks/pathphys/digestion/smallgut/absorb_lipids.html(accessed January 8,
2011). 3 “Fatty Acid Metabolism,”
IB Biology HL
Name: Yoojin Lee
Candidate Number: 002213-067
2
The dietary lipid itself is neutral in terms of acidity. However, as shown in Figure 1, when the
lipase breaks down the fats, producing glycerol and three fatty acids, the pH will decrease.
Thus, the pH sensor is used to measure the change in pH over time. Hence, the change in pH
over time represents the rate of digestion of milk.
All enzymes have optimal pH and temperature ranges. The optimal pH of the pancreatic
lipase activity is around 8.4 However, milk is slightly acidic, because it is a fermented
product of lactic acid. Thus, in order to make the condition suitable for lipase activity, sodium
bicarbonate, a weak base, is added to increase the pH. In this investigation, the pH of the milk
will be fixed and the temperature will be altered to test how different temperatures affect the
rate of enzyme activity. In extreme temperatures, the enzyme might denature, so only
reasonable temperatures ranging from 5℃ to 55℃ are tested.
Natuurlijkerwijs,http://www.natuurlijkerwijs.com/english/Fatty_acid_metabolism.htm (accessed January 8, 2011). 4 “Effects of pH (introduction to Enzymes),” Worthington Biochemical Corporation,http://www.worthington-
biochem.com/introbiochem/effectsph.html (accessed January 8, 2011).
IB Biology HL
Name: Yoojin Lee
Candidate Number: 002213-067
3
Hypothesis
The rate of lipase activity is represented by the change in pH over time. Since pancreatic
lipase can be found in human body, surely it works at temperatures around body temperature,
36.5℃ and the optimal temperature should be close to the body temperature as well. Hence,
the pH will drop constantly until the substrate, dietary lipid in milk, is completely used. In
high temperatures, the enzyme may denature and not function at all, thereby not changing the
pH. Likewise, in low temperatures, the enzyme may be inactive, if not denatured, and thus
the pH will not drop. Hence, the optimal temperature will produce the highest rate of enzyme
activity and as the temperatures deviate from the optimal temperature, the rate will decrease
and eventually reach 0.
ㅣ ㅣ
Figure 2 shows the predicted relationship between the rate of lipase activity and temperature
IB Biology HL
Name: Yoojin Lee
Candidate Number: 002213-067
4
Variables Variables Description Method of Measuring
Independent Temperature (℃) of the
lipase-bile solution
The lipase-bile solution were left in
different temperatures at 5℃, 25℃, 35℃,
45℃, and 55℃ using refrigerator, room
temperature, and water baths. The
solutions were incubated for 30 minutes
and were tested immediately after
incubation to limit changes in
temperature.
Dependant Rate of lipase activity
ㅣ ㅣ
Rate of lipase activity is represented by
the change in pH over time. Since the
lipase activity produces fatty acids, it is
directly proportional to the acid
formation. pH was measured using the
pH sensor. Also, to limit errors, the same
pH sensor was used throughout the
experiment.
Controlled Recording initial rate As soon as the lipase-bile solution was
released into the test tube, it was capped
with pH sensor to record the data
immediately.
Amount of lipase-bile
solution
The amount of lipase-bile mixture was
set to 5cm3. Micropipette was used to
accurately measure and transfer the
solution.
Temperature of the
surrounding
All experiments were conducted at room
temperature, approximately 25℃. Since
the independent variable is the
temperature of the lipase-bile solution, it
was vital to have the same temperature of
the surrounding.
Volume of milk For all trials, 5cm3 of milk was tested.
Micropipette was used to accurately
measure and transfer the solution.
Size and type of test tubes The size and type of test tubes were
constant, because they can alter the
surface area of milk, which is vital for
initial rate. The same size and type of test
tubes were used throughout.
Milk Different brands of milk contain different
amount of dietary lipids. Hence, milk
from the same package was used
throughout the experiment.
Table 1 shows the independent, dependent, and controlled variables and the methods of
measuring
IB Biology HL
Name: Yoojin Lee
Candidate Number: 002213-067
5
Procedure
1. 50cm3 of 2% lipase was prepared and mixed with 20cm
3 of bile solution.
2. 5cm3 of the lipase-bile prepared in Step 1 was transferred to a separate test tube and
mixed with 1cm3 of NaHCO3.
3. Step 2 was repeated 14 times to produce 15 of identical lipase-bile solution samples.
4. Three lipase-bile solutions were incubated at different temperatures for 30 minutes
for triplicate trials.
Table 2 shows the preparation methods for various temperatures
5. 5cm3 of milk was transferred to a separate test tube and an incubated lipase-bile
solution was added.
6. Immediately after, data was recorded using the pH sensor and Logger Pro.
(While collecting data, magnetic stirrer was used to mix the lipase-bile solution and
milk thoroughly for complete reaction.)
7. Steps 5 and 6 were repeated to obtain valid triplicate data for each temperature.
Apparatus Materials pH sensor
Micropipette (± 0.006cm3)
Test tubes
Water baths
Refrigerator
Temperature probe
Magnetic Stirrer
Milk Lipase Bile solution NaHCO3 solution
Temperature, ℃ Preparation Method
5 Incubated in a refrigerator
25 Incubated at room temperature
35
Each incubated in water bath 45
55
IB Biology HL
Name: Yoojin Lee
Candidate Number: 002213-067
6
Data Collection – Quantitative Data
Graph 1 shows the raw data for the effect of changing temperature on the rate of lipase activity
IB Biology HL
Name: Yoojin Lee
Candidate Number: 002213-067
7
Table 3 shows the raw data collected on Logger Pro (a)
– represents uncollected data
Time,
t/s
pH(±0.05)
5 25 35 45 55
Trial 1 Trial 2 Trial 3 Trial 1 Trial 2 Trial 3 Trial 1 Trial 2 Trial 3 Trial 1 Trial 2 Trial 3 Trial 1 Trial 2 Trial 3
0.00 7.63 7.58 7.60 7.80 8.01 7.80 7.68 7.64 7.62 7.75 7.68 7.68 7.70 7.66 7.66
50.0 7.70 7.59 7.52 7.81 7.98 7.81 7.68 7.63 7.63 7.67 7.65 7.67 7.64 7.41 7.67
100 7.65 7.58 7.58 7.81 7.97 7.81 7.67 7.61 7.76 7.67 7.61 7.62 7.66 7.57 7.61
150 7.65 7.57 7.57 7.80 7.96 7.80 7.65 7.58 7.58 7.66 7.59 7.61 7.66 7.58 7.62
200 7.63 7.56 7.58 7.79 7.94 7.78 7.62 7.57 7.58 7.65 7.58 7.59 7.64 7.59 7.63
250 7.62 7.55 7.55 7.77 7.93 7.76 7.60 7.55 7.56 7.64 7.56 7.58 7.66 7.57 7.62
300 7.61 7.53 7.53 7.76 7.91 7.75 7.58 7.53 7.52 7.62 7.56 7.56 7.65 7.57 7.63
350 7.60 7.52 7.51 7.74 7.89 7.73 7.56 7.51 7.51 7.60 7.55 7.57 7.65 7.57 7.62
400 7.58 7.51 7.52 7.73 7.88 7.72 7.54 7.49 7.50 7.59 7.54 7.56 7.65 7.57 7.63
450 7.57 7.50 7.50 7.72 7.85 7.71 7.54 7.48 7.48 7.59 7.53 7.55 7.65 7.59 7.61
500 7.56 7.49 7.50 7.70 7.85 7.69 7.52 7.46 7.46 7.58 7.51 7.53 7.66 7.58 7.61
550 7.55 7.48 7.47 7.69 7.83 7.68 7.50 7.45 7.45 7.57 7.52 7.52 7.66 7.57 7.61
600 7.55 7.46 7.47 7.68 7.82 7.67 7.49 7.43 7.44 7.56 7.51 7.50 7.65 7.58 7.63
650 7.53 7.46 7.45 -(a)
- - 7.48 7.43 7.40 7.56 7.50 7.49 7.65 7.58 7.62
700 7.52 7.45 7.44 - - - 7.47 7.41 7.41 7.55 7.49 7.48 7.65 7.58 7.63
750 7.51 7.44 7.43 - - - 7.44 7.40 7.41 7.54 7.48 7.48 7.66 7.59 7.62
800 7.51 7.43 7.42 - - - 7.44 7.39 7.39 7.54 7.46 7.47 7.66 7.60 7.62
IB Biology HL
Name: Yoojin Lee
Candidate Number: 002213-067
8
Data Collection – Qualitative Data
There were no visible changes for all different temperatures. The solutions remained opaque
because of milk and lipase solution and gave off bad odor. The solutions were yellowish-
white because small amount of bile solution, which was brown in color, was added.
Data Processing
The absolute value of the gradient of Graph 1 represents the change in pH over time. Thus, it
represents the rate of lipase activity. The processed data is shown in Table 3 below.
Rate of lipase activity, r/s
-1
Temperature,
T/℃
(± 0.05)
Trials
Mean(a)
Mean ±
SD(b)
1 3 3
5.00
25.0
35.0
45.0
55.0
Table 4 shows the rates of pressure increase for different hydrogen peroxide concentrations (a)
Mean: average of triplicate trials for each set. (b)
SD: standard deviation for triplicate trials.
IB Biology HL
Name: Yoojin Lee
Candidate Number: 002213-067
9
Sample Calculations
ㅣ ㅣ
Calculation of the mean rate of 5℃ lipase-bile solution from the triplicate trials.
Mean ( ) =
=
s-1
Calculation of the standard deviation of 5℃ lipase-bile solution from the triplicate trials
Standard deviation =
=
= s-1
IB Biology HL
Name: Yoojin Lee
Candidate Number: 002213-067
10
Data Presentation
Graph 2 shows the processed data of average rates of evaporation against the number of the carbon chain. (a) Vertical error bar shows the standard deviation of the triplicate trials for the rate of evaporation.
y = -3E-07x2 + 2E-05x + 0.0002
R² = 0.7577
0
0.0001
0.0002
0.0003
0.0004
0.0005
0.0006
0 5 10 15 20 25 30 35 40 45 50 55 60
Rate
of
Lip
ase
Act
ivity, r/
s-1
Temperature, T/℃
Effect of Changing Temperature on the Rate of Lipase Activity
(a)
IB Biology HL
Name: Yoojin Lee
Candidate Number: 002213-067
11
Conclusion
The data suggests that the optimal temperature for lipase activity is close to 35℃ and that my
hypothesis was valid as Graph 2 seems to be similar to Figure 2, which is my predicted
outcome. Also, the data shows that the rate of lipase activity decreases as the temperature
deviates, both decrease and increase, from the optimum, which the parabolic trend line
suggests as well. For instance, the maximum rate occurs at 35℃ while the rates at 5℃ and 55℃
are ostensibly lower than the rate at 35℃. The R2 value tells that there is a correlation.
However, the limitation of this investigation is that the exact optimal temperature cannot be
found out. Although 35℃ lipase-bile solution produced the highest rate of enzyme activity,
the exact optimum may not be 35℃. Hence, the optimum temperature range is between 25℃
and 45℃. According to online research, the exact optimal temperature is found out to be
37℃, which is very close to the empirical data in this investigation.
Evaluation
This experiment is justifiable because reliable triplicate trials were obtained. Yet, the data
consists of wide uncertainties, partly because the rate itself was too small ranging from
to . Since the fatty acids did not lower the acidity a lot, the change
in pH was very opaque. Because the change in pH was hard to detect, it naturally
accompanied a great uncertainty in measurement.
Moreover, in terms of procedure, it accompanied greater uncertainty, because temperature is
always changing. Even though the experiment was immediately performed after incubation at
IB Biology HL
Name: Yoojin Lee
Candidate Number: 002213-067
12
certain temperature, it will nevertheless assimilate into the room temperature as the
experiment proceeds. Hence, the greater uncertainty is present for the temperature, but the
extent is unknown.
Limitations and Improvements
Limitations Improvements
The temperature could not be fixed as the
experiment proceeded. Although the lipase-
bile solutions were incubated enough, as
soon as the incubation was over, the
temperature inevitably started to assimilate
into the room temperature. This would have
produced the major error, since temperature
itself is the independent variable.
In order to prevent temperature assimilation,
a more advanced method is needed. Perhaps,
if the whole experiment was done inside the
incubator, it could prevent the temperature
change.
Only certain range of temperatures could be
tested due to technical limitations. For
example, 15℃ could not be tested, because
the room temperature was around 25℃ and
the water bath temperature range starts from
the room temperature. Lacking diversity in
temperature is another major limitation. Due
to time constraints, specific temperatures
could not be tested.
Better equipment is needed to test a variety
of temperatures. If more time was given, this
research could be further investigated by
narrowing down the temperature ranges and
finding the empirical optimal temperature.
Then, the percent error could be calculated to
make the investigation more justifiable and
reliable.
Since the test tube had to be manually capped
with the pH sensor, it inevitably included
human error because of human reaction time.
Thus, the initial rate might not be accurate.
To reduce human errors, more advanced
apparatus has to be used. To obtain accurate
data, the pH has to be measured as soon as
the lipase-bile solution hits the surface of
milk, because the reaction starts
instantaneously, even if the rate of lipase
activity is low.
Table 5 shows the limitations and the improvements
IB Biology HL
Name: Yoojin Lee
Candidate Number: 002213-067
13
Bibliography
1
“Pancreatic Lipase.” Wikipedia, the free
encyclopedia. http://en.wikipedia.org/wiki/Pancreatic_lipase(accessed January 8, 2011).
2 Bowen, R. “Absorption of Lipids.”
Colostate.http://arbl.cvmbs.colostate.edu/hbooks/pathphys/digestion/smallgut/absorb_lipids.h
tml(accessed January 8, 2011).
3
“Fatty Acid Metabolism.”
Natuurlijkerwijs.http://www.natuurlijkerwijs.com/english/Fatty_acid_metabolism.htm (acces
sed January 8, 2011).
4 “Effects of pH (introduction to Enzymes).” Worthington Biochemical
Corporation.http://www.worthington-biochem.com/introbiochem/effectsph.html (accessed
January 8, 2011).