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

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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

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Name: Yoojin Lee

Candidate Number: 002213-067

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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

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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

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Data Collection – Quantitative Data

Graph 1 shows the raw data for the effect of changing temperature on the rate of lipase activity

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Name: Yoojin Lee

Candidate Number: 002213-067

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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

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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

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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

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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)

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Name: Yoojin Lee

Candidate Number: 002213-067

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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

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Name: Yoojin Lee

Candidate Number: 002213-067

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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

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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).