FA2 Student Experiment Assignment

Effect of fat percentage on lipase activity

Hayley Westman
Due date: 14-06-2023
Teacher: Miss Taylor
Table of Contents
Rationale...............................................................................................................................................2
Research Question.................................................................................................................................2
Original Experiment...............................................................................................................................2
Modifications.........................................................................................................................................3
Safety and ethical considerations..........................................................................................................4
Raw data................................................................................................................................................4
Processed Data......................................................................................................................................5
Sample calculations...........................................................................................................................6
Interpretation:.......................................................................................................................................7
Analysis..............................................................................................................................................7
...............................................................................................................................................................8
Evaluation..............................................................................................................................................8
Suggested improvements and extensions.............................................................................................9
Conclusion.............................................................................................................................................9
Reference List......................................................................................................................................10

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Rationale
Enzymes are large globular proteins commonly located in the stomach, small intestine and pancreas,
that act as biological catalysts. Throughout this report, the lipase enzyme will be focused on and to
what extent the independent variable (IV) of differing fat percentages ( <0.01%, 0.018%, and 0.034%
per 10mL ) in milk affects its activity. Lipase is a catabolic digestive enzyme that catalyses the
breakdown of lipids and fats into fatty acids and glycerol. In this experiment, the fats in milk are the
substrates for lipase to bind to which allows for the breakdown catalysation to occur. While the ‘lock
and key’ theory was the original belief on how enzymes and substrates interact, the induced fit
model is the updated understanding. This states despite enzymes structure being different and
highly specific they can change shape of the active site to accommodate the substrate. Sodium
carbonate (Na2CO3) was also an essential aspect of the experiment as it was added to the milk to
make the solution alkaline. By making the solution more alkaline it mimics the
duodenum where lipase normally works. After lipase activity takes place catalysing the breakdown
of fats into fatty acids, the sodium carbonate is to some degree neutralised thus reducing pH which
will be seen in the data recorded.

When enzymes and substrates interact there are certain environmental conditions that must be
met, either by being in the tolerance range or at the optimal point for that specific enzyme. The rate
of reaction will be very slow or simply not take place as the enzyme will denature if these conditions
are not met. Some of these conditions include pH level, temperature, and concentration of reactants
and products. The original experiment focused on the condition of temperature and how it affects
enzyme function, whereas this experiment focuses on the condition of concentration of reactants
and products as the aim is to investigate the effect of varying fat percentages in milk on enzyme
activity.

Research Question
To what extent does varying fat percentages of <0.1%, 0.18%, and 0.34% per 10mL of milk in a
solution made alkaline by 0.5 mol sodium carbonate, affect Lipase enzyme activity on pH levels?

Original Experiment
The original experiment methodology was from the Education Perfect experiment “The Effect of
Temperature on Enzyme Activity” (2020 Education Perfect) which aimed to “investigate the effect of
temperature on the activity of lipase.” (2020 Education Perfect) Over the course of three trials for
the 5 different temperatures (20℃ , 30℃ , 40℃ or 50℃ ) the change in pH was measured by
observation of colour evaporation as a result of the indicator, phenolphthalein. A stopwatch was
used in conjunction with this observation to time how long it took for the solution to become
colourless, to the naked eye, completely.

2
Modifications
Original experiment
3 trials for each independent
variable
7mL of Sodium carbonate
(Na2CO3)
Experiment materials at
temperatures of either 20℃ ,
30℃ , 40℃ or 50℃ .
Fat percentage of milk was 3.4
g/100mL
The ratio of sodium carbonate
to milk to lipase was 7:5:1
5mL of milk was used
Stopwatch used to measure
the dependent variable
Data was recorded for 13
minutes
Phenolphthalein was used as
an indicator
One independent variable
(temperature) was tested at a
time
Modifications to experiment
6 trials for each independent
variable
5mL of Sodium carbonate
(Na2CO3)
Experiment materials at 21℃
constantly
Fat percentage of milk was
either 3.4 g/100Ml (full
cream), less than 1 g/100mL
(skim) or 1.8 g/100mL
(almond)
The ratio of sodium carbonate
to milk to lipase was 2:1:2
10mL of milk was used
Data probes/ loggers were
used to measure and record
the dependent variable
Data was recorded for 180
seconds (1 minute 20 seconds)
Phenolphthalein was not
required for this experiment
All three independent
variables (3 different fat %)
from one trial were tested at
the same time
3
Justification of modifications
more trials promotes
increased accuracy more
precision within results
Only 5mL of Na2CO3 was
needed for the substance to
still fulfil its purpose
Temperature was no longer
the independent variable
therefore had to remain the
same/ didn’t need to change
The fat percentage of milk was
the independent variable in
this experiment therefore
there must be multiple
different amounts
This ratio had to adapt as
there was a change in amounts
of each substance being used
There was a change in the
quantity of milk as the
increased amount allowed for
complete coverage of a section
of the data probes as well as
easier observations
The method of recording and
measuring results was
changed to be more accurate
and record changes in pH
rather than just human
observation
The decreased time of
recording was done to ensure
the completion of all trials as
well as considering the length
of time taken for enzymes to
denature and adapted
accordingly
Phenolphthalein was no longer
needed as change in pH was
recorded through the data
probes rather than human
observation
Change in circumstances (e.g.
the people/ number of people
in the group, equipment set up
and aim) were different thus
requiring a different method of
experiment execution
Safety and ethical considerations
Risk Safety considerations
Dropping/ breaking of glass equipment (e.g. If glass is broken it may cause cuts and injure
stirring rod, beakers measuring pipettes etc.) people, handle glass with care and caution
Spilt chemicals If chemicals are spilt it may cause burns,
contamination or slipping hazards, handle all
chemical and materials with care and caution
Improper disposal of waste If chemicals are not disposed of correctly there
may be negative environmental repercussions,
ensure appropriate disposal by asking lab techs
and teachers
Inhalation of toxic fumes Inhalation of toxic fumes may cause bodily
harm, refrain from breathing in fumes and keep
face a safe distance away from chemicals
Mercury thermometer breaking If the mercury thermometer breaks the glass
could cause cuts and injuries or the mercury
could be released and could cause bodily harm,
handle glass thermometer with care and
caution
Chemical burns If chemicals are spilt it may chemically burn the
skin and be corrosive, handle all chemicals with
care and caution
Allergies An allergic reaction may take place from
substances/chemicals being used, ensure all
allergies have been noted and considered, and
have allergic reaction protocols in place in case
of emergency

Raw data
Skim
Time Trial 1 Trial 2 Trial 3 Trial 4 Trial 5 Trial 6
0 10.93 10.89 10.6 10.62 8.9 10.69
20 10.81 10.88 10.6 10.59 8.33 10.69
40 10.83 10.89 10.6 10.6 8.24 10.71
60 10.84 10.9 10.6 10.85 8.21 10.71
80 10.85 10.9 10.6 10.65 8.22 10.72
100 10.85 10.9 10.6 10.96 8.21 10.72
120 10.85 10.91 10.6 10.6 8.21 10.73
140 10.86 10.91 10.6 10.6 8.22 10.73
160 10.86 10.91 10.6 10.6 8.25 10.73
Figure180
1 (skim10.86
milk) 10.91 10.6 10.6 8.27 10.73

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Full cream
Time Trial 1 Trial 2 Trial 3 Trial 4 Trial 5 Trial 6
0 8.96 9 9.07 8.92 7.28 8.65
20 8.97 9.08 9.07 8.8 6.82 8.25
40 8.98 9.09 8.99 8.65 6.84 8.15
60 8.99 9.09 8.99 8.63 6.84 8.12
80 9.01 9.09 8.99 8.63 6.83 8.12
100 9.01 9.09 8.99 8.62 6.7 8.14
120 9.02 9.1 9 8.6 6.68 8.16
140
Figure 9.02
2 (full cream) 9.1 9.01 8.6 6.67 8.19
160 9.02 9.1 9.01 8.58 6.66 8.23
180 9.04 9.1 9.01 8.57 6.65 8.25

Almond
seconds trial 1 Trial 2 Trial 3 Trial 4 Trial 5 Trial 6
0 10.6 10.8 9.41 9.03 7.28 9.1
20 10.59 10.6 9.41 9.02 7.24 9.06
40 10.59 10.6 9.37 9.03 7.24 9.03
60 10.59 10.61 9.3 9.02 7.24 9.01
80 10.59 10.61 9.3 9.02 7.24 9
100 10.59 10.61 9.28 9.02 7.23 8.99
120 10.59 10.61 9.27 9.02 7.21 8.98
140 10.59 10.62 9.27 9.03 7.19 8.98
160 10.59 10.62 9.26 9.03 7.16 8.98
180 10.59 10.62 9.25 9.03 7.15 8.99
Figure 3 (almond)

Processed Data
Skim
Averag Uncertaint Uncertaint Standard
Time e y y% Deviation
10.438
0 3 1.015 9.72377 0.76612
10.316
20 7 1.275 12.3586 0.97999
10.311
40 7 1.325 12.8495 1.02175
10.351
60 7 1.345 12.9931 1.05492
10.323
80 3 1.34 12.9803 1.03678
100 10.373 1.375 13.2551 1.06774

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 3
10.316
120 7 1.35 13.0856 1.0398
140 10.32 1.345 13.0329 1.03678
160 10.325 1.33 12.8814 1.02463
10.328
180 3 1.32 12.7804 1.01653

Figure 4 (skim)
Full cream
Averag Uncertaint Uncertaint Standard
Time e y y% Deviation
8.6466
0 7 0.895 10.3508 0.6848
8.4983
20 3 1.13 13.2967 0.8783
40 8.45 1.125 13.3136 0.86072
8.4433
60 3 1.125 13.3241 0.86324
80 8.445 1.13 13.3807 0.86952
100 8.425 1.195 14.184 0.91636
8.4266
120 7 1.21 14.3592 0.92587
8.4316
140 7 1.215 14.41 0.92925
8.4333
160 3 1.22 14.4664 0.93046
8.4366
180 7 1.225 14.52 0.93569
Figure 5 (full cream)

Almond
Averag Uncertaint Uncertaint Standard
Time e y y% Deviation
0 9.37 1.76 18.7834 1.27405
20 9.32 1.68 18.0258 1.24446
40 9.31 1.68 18.0451 1.24481
60 9.295 1.685 18.128 1.24789
9.2933
80 3 1.685 18.1313 1.24835
9.2866
100 7 1.69 18.1981 1.25211
120 9.28 1.7 18.319 1.25917
140 9.28 1.715 18.4806 1.26747
160 9.2733 1.73 18.6556 1.2774

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 3
9.2716
180 7 1.735 18.7129 1.28029
Figure 6 (almond)

Sample calculations
Full cream milk Skim milk Almond milk
Standard Shown in graph through Shown in graph through error Shown in graph through error
error: indicates how error bars bars bars
different the o o o
SE= SE= SE=
population mean is √n √n √n
likely to be from a
sample mean 0.88 1.00 1.26
(to calculate standard SE= SE= SE=
√6 √6 √6
error excel was used SE=0.359258496 SE=0.40824829 SE=¿ 0.514392846
as well as the formula SE=0.36 SE=0.41 SE=0.51
o
SE= )
√n

√ √ √
Standard deviation: a Σ(x i−µ)² Σ(x i−µ)² Σ(x i−µ)²
measure of how o= o= o=
N N N
dispersed the data is
o=0.879419324 o=1.004504728 o=1.259600869
in relation to the
o=0.88 o=1.00 o=1.26
mean
(to calculate standard
deviation the STDEV
function in excel was
used

o=
√ Σ(x i−µ)²
N
Averages: the mean of ∑ of observations ∑ of observations ∑ of observations
the results from each total number of observationstotal number of observations total number of observations
trial
(to calculate the
averages excel was
used as well as
∑ of observations
total number of observations
)
Range: the area of Range = max – min Range = max – min Range = max – min
variation between
highest and lowest
value results
(max – min)

Interpretation:
Literature suggests that the increased pH from the addition of sodium carbonate in the solution
would begin to decrease as the fats present in milk get broken down. From Figure 7 it can be
interpreted that while the average pH change over the 180 seconds for all fat percentage trials was

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limited, each varying milk remained relatively consistent after a small decrease in pH in the
beginning of the experiment. As shown on the graph (figure 7) skim milk with the fat percentage of
<0.1% dependably remained with the highest pH, just below 10.5 pH, out of the three milks tested.
Almond milk with the fat percentage of 0.18% followed suit remaining, on average, just below the
9.5 pH mark. Full cream milk with the highest fat percentage of 0.34% consistently remained around
8.5 pH. The consistency of these results for each milk type directly reflects the effect of fat
percentage on pH levels, regardless, it doesn’t show significant change over the 180 seconds of data
recording as there were instant effects instead. This is seen as full cream milk unswervingly has the
lowest pH due to its higher fat percentage (0.34%) while skim milk (<0.1%) constantly has the
highest pH levels as a result of obtaining the lowest fat percentage out of the three samples.

Analysis
This relationship between pH levels and fat percentage in the milks tested is illustrated in figure 7.
The linear trendline on the graph below depicts the consistency of the rate of change in pH for each
fat percentage. As the line is relatively straight horizontally and close to the plotted data this shows
the limited yet consistent rate of reaction occurring. While standard deviation is present the average
standard deviation for all milks tested was less than two which indicates the data values plotted are
more likely to be true and reliable thus showing the data collected has a relatively high level of
precision with less variability. Furthermore, the pH levels recorded are a direct reflection of the
consequences of reliability and validity errors to the results. This data suggests that despite possible
human error, the results from the six trials remained somewhat close to the mean which upholds
the reliability and shows immediate effect of fat percentages on lipase activity and consequently pH
levels.

Average Change in pH for Skim, Full Cream and Almond Milk

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10.5

10

9.5
pH scale

8.5

7.5
0 20 40 60 80 100 120 140 160 180
time (s)

Skim (<0.1%) Linear (Skim (<0.1%))

Full Cream (0.34%) Linear (Full Cream (0.34%))
Almond (0.18%) Linear (Almond (0.18%))
Figure 7

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Evaluation
The validity and reliability of the results were affected by the numerous limitations encountered
over the course of the experiment. Through standard error and standard deviation, these limitations
and the influence they have had on the data are shown.

Limitation and sources of error affecting reliability

 Technology error
- Data loggers repeatedly disconnecting from data recording device (Ipad)
- Data loggers change label names thus making the data unidentified when originally
presented in results.
 Human error
- Milk measurements were all done by different people thus slight variation may arise.
- One milk (skim) was refrigerated therefore changing the temperature of the milk in the
first experiment.
- There was slight variation in the time the recording of data began for each experiment
after the combination of lipase, milk, and sodium carbonate mixture.
- Trial 3 onwards the mixtures began getting stirred to fully disperse/mix the solution for
better results (didn’t mix trials 1 and 2)

Limitations and sources of error affecting validity

 Confounding or extraneous variables were not controlled (the relationship between IV and
DV is subject to undesired external effects)
 Human error:
- Lipase spillage
- Measuring of liquids (Milk measurements were all done by different people thus slight
variation may arise)
- There was a minor amount of lipase left in the test tubes after pouring it into the milk
and sodium carbonate mixture.
- There was a difference in pour in time of the lipase combining with the sodium
carbonate mixture.
- There was a slight variation in the time the recording of data began for each experiment
after the combination of lipase, milk, and sodium carbonate mixture.

Suggested improvements and extensions

Improvements:

- More accurate and detailed methodology fully finished before the experiment.
- More accuracy and precision practised throughout the experiment in measuring and
pouring substances.
- Reducing random error
- Control more extraneous variables
- Ensure all data loggers are labelled so data can be easily identified.
- Ensuring consistency is practised and detailed throughout the methodology.

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Extensions:

- Increase the variety of fat percentages in milks being tested (e.g. 6 different fat
percentage milks trialled and compared)
- Trial fat percentage in a variety of other liquids/substances such as different fat
percentages in sauces, kinds of butters or types of yoghurts
- Trial the entire experiment at the optimal temperature for lipase activity (in a controlled
room purposely remaining at 35℃ ) (Hiol et al., 2000)

Conclusion
The findings over the course of this experiment have been analysed to answer the following research
question. “To what extent does varying fat percentages of <0.1%, 0.18%, and 0.34% per 10mL of milk
in a solution made alkaline by 0.5 mol sodium carbonate, affect Lipase enzyme activity on pH
levels?” As shown on the graph above and represented through the findings calculated in figures 3
to 6, regardless of there being limited change in pH levels over the course of the 180 seconds there
were immediate effects instead. The sample of milk (full cream) with the highest fat percentage
constantly remained with the lowest average pH over the course of the experiment. This trend was
corroborated throughout the rest of the data as the pH levels continued to increase the fat
percentage decreased. This demonstrates that varying fat percentages will have immediate effects
on the lipase enzyme activity as revealed throughout this report highlighted through varying pH
levels in each sample. However, the reliability and validity of this study could be argued against as
there were numerous limitations, sources of error and extraneous variables effecting the
experiment. Thus, further research, statistical analysis and multitudes of improvements to the
methodology would be required to confirm these findings and to answer the research question
definitively.

Reference List
Hiol, A., Jonzo, M.D., Rugani, N., Druet, D., Sarda, L. and Comeau, L.C. (2000).
Purification and characterization of an extracellular lipase from a thermophilic Rhizopus
oryzae strain isolated from palm fruit. Enzyme and Microbial Technology, 26(5-6), pp.421–
430. doi:https://doi.org/10.1016/s0141-0229(99)00173-8.

Loreto Sixth Form College A level Biology. (2023). Investigating effects of temperature on
enzymes. [online] Available at: http://loretocollegebiology.weebly.com/investigating-effects-
of-temperature-on-enzymes.html [Accessed 2023].

National Library of Medicine (2006). Standard Deviation. [online] NLM. Available at:
https://www.nlm.nih.gov/nichsr/stats_tutorial/section2/mod8_sd.html [Accessed 2023].

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Niaki, A. (2018). Low R-squared? [online] ResearchGate. Available at:
https://www.researchgate.net/post/Low_R-squared#:~:text=A%20low%20R%2Dsquared
%20value [Accessed 2023].

www.vedantu.com. (2023). A milkman adds a very small amount of baking soda to class 10
chemistry CBSE. [online] Available at: https://www.vedantu.com/question-answer/a-
milkman-adds-a-very-small-amount-of-baking-class-10-chemistry-cbse-
5f62645001faef2daa4e25d5 [Accessed 2023].

Yasaman Pirahanchi and Sandeep Sharma (2022). Biochemistry, Lipase. [online] Nih.gov.
Available at: https://www.ncbi.nlm.nih.gov/books/NBK537346/ [Accessed 2023].

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