IB Biology HL Name: Yoojin Lee Candidate Number: 002213-067
Candidate Name Candidate Number Date of Practical
:Yoojin Lee :002213-067 :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,”
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IB Biology HL Name: Yoojin Lee Candidate Number: 002213-067
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.worthingtonbiochem.com/introbiochem/effectsph.html (accessed January 8, 2011). 2
IB Biology HL Name: Yoojin Lee Candidate Number: 002213-067
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. ă…Ł
ă…Ł
Figure 2 shows the predicted relationship between the rate of lipase activity and temperature
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IB Biology HL Name: Yoojin Lee Candidate Number: 002213-067
Variables Variables Independent
Dependant
Description Temperature (℃) of the lipase-bile solution
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. 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. As soon as the lipase-bile solution was released into the test tube, it was capped with pH sensor to record the data immediately. The amount of lipase-bile mixture was set to 5cm3. Micropipette was used to accurately measure and transfer the solution. All experiments were conducted at room
Rate of lipase activity ㅣ
Controlled
Method of Measuring The lipase-bile solution were left in
ㅣ
Recording initial rate
Amount of lipase-bile solution
Temperature of the surrounding
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 4
IB Biology HL Name: Yoojin Lee Candidate Number: 002213-067
Apparatus
Materials Milk Lipase Bile solution NaHCO3 solution
pH sensor Micropipette (± 0.006cm3) Test tubes Water baths Refrigerator Temperature probe Magnetic Stirrer
Procedure 1. 50cm3 of 2% lipase was prepared and mixed with 20cm3 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. Temperature, ℃
Preparation Method
5
Incubated in a refrigerator
25
Incubated at room temperature
35 45
Each incubated in water bath
55 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.
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IB Biology HL Name: Yoojin Lee Candidate Number: 002213-067
Data Collection – Quantitative Data
Graph 1 shows the raw data for the effect of changing temperature on the rate of lipase activity 6
IB Biology HL Name: Yoojin Lee Candidate Number: 002213-067
pH(Âą0.05) Time, t/s
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 Table 3 shows the raw data collected on Logger Pro
7.47
7.66
7.60
7.62
(a)
– represents uncollected data 7
IB Biology HL Name: Yoojin Lee Candidate Number: 002213-067
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 yellowishwhite 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 ± Mean(a)
1
3
3
SD(b)
5.00 25.0 35.0 45.0 55.0 Table 4 shows the rates of pressure increase for different hydrogen peroxide concentrations Mean: average of triplicate trials for each set. (b) SD: standard deviation for triplicate trials. (a)
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IB Biology HL Name: Yoojin Lee Candidate Number: 002213-067
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
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IB Biology HL Name: Yoojin Lee Candidate Number: 002213-067
Data Presentation y = -3E-07x2 + 2E-05x + 0.0002
Effect of Changing Temperature on the Rate of Lipase Activity
R² = 0.7577
0.0006
Rate of Lipase Activity, r/s-1
0.0005
0.0004
0.0003
0.0002
(a)
0.0001
0 0
5
10
15
20
25
30
35
40
45
50
Temperature, T/℃
(a)
Graph 2 shows the processed data of average rates of evaporation against the number of the carbon chain. Vertical error bar shows the standard deviation of the triplicate trials for the rate of evaporation. 10
55
60
IB Biology HL Name: Yoojin Lee Candidate Number: 002213-067
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 11
IB Biology HL Name: Yoojin Lee Candidate Number: 002213-067
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 lipasebile 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 Better equipment is needed to test a variety example, 15℃ could not be tested, because of temperatures. If more time was given, this research could be further investigated by the room temperature was around 25℃ and narrowing down the temperature ranges and the water bath temperature range starts from finding the empirical optimal temperature. the room temperature. Lacking diversity in Then, the percent error could be calculated to temperature is another major limitation. Due make the investigation more justifiable and to time constraints, specific temperatures reliable. could not be tested.
To reduce human errors, more advanced apparatus has to be used. To obtain accurate Since the test tube had to be manually capped data, the pH has to be measured as soon as with the pH sensor, it inevitably included the lipase-bile solution hits the surface of human error because of human reaction time. milk, because the reaction starts Thus, the initial rate might not be accurate. instantaneously, even if the rate of lipase activity is low. Table 5 shows the limitations and the improvements
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IB Biology HL Name: Yoojin Lee Candidate Number: 002213-067
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).
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