1

The effect of varying temperatures (℃) on the digestion rate of egg albumin looked at
through wavelength transmittance (%) over the period of 8 minutes.
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1: Introduction
1.1: Introduction
Pepsin is an endopeptidase and is responsible for breaking down dietary proteins in the stomach
into amino acids. It functions by breaking down the peptide bonds in pepsin. Pepsin is the active
form of pepsinogen. When mixed with HCl, pepsinogen becomes active and turns into pepsin.
Proteins are broken downy by pepsin and are then either absorbed by the intestine or broken down
even further by pancreatic enzymes. My initial interest into enzymes and, more specifically pepsin,
is that in my family there is a history of chronic reflux. This means that there is a backflow of acid,
pepsin and other substances into the esophagus. When given the opportunity, I knew that I wanted
to investigate this topic for the reason above, but also because I have always been interested in
human physiology and hope to one day make a career related to it. I combined my two interest and
decided to investigate the digestion of protein, more specifically egg white, at varying
temperatures. Looking at this will hopefully allow me to gain a greater understating of how
enzymes function as well as broaden my knowledge of reflux.
1.2 Research Question
What is the effect of varying temperatures (℃) on the digestion rate of egg albumin looked at
through wavelength transmittance (%) over the period of 8 minutes?
1.3: Hypothesis
H1: The digestion rate will increase with the temperature until it has reached optimum temperature,
once this has happened the digestion rate will steadily decrease. As the average human temperature
is slightly lower than 40 degrees, the optimum digestion rate will be at 40 degrees.
1.4: Background Knowledge
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The experiment that is being done is meant to demonstrate how proteins are digested in the human
body by using the enzyme pepsin produced in the mucus-lining of the stomach. Proteins are
complex and large molecules that have a critical role in many processes in the body. They consist
of a carboxyl (–COOH) and an amino group (–
NH2) which are attached to the same carbon
atom, the ⍺-carbon. The identity of a protein is
determined by its variable R-group. Proteins
are present in every living organism and are
directly involved in the chemical processes
essential for life. Due to their irreplaceable
function in the human body, the word protein
Diagram 1. comes from the Greek word prōteios, which
means “holding first place’’(Protein Digestion
and Absorption). A protein molecule consists of many amino acids joined together by peptide
bonds which form a 3-dimensional structure (Protein Digestion and Absorption). The 3
dimensional structure can be seen in diagrams 1.
An enzyme is responsible for all chemical reactions that keep us alive, i.e. our metabolism.
Enzymes are biological catalysts and in some cases, they can make reaction rates a million times
faster (Protein Digestion and Absorption). An example of how enzymes work is shown through
this experiment. Pepsin is a type of enzyme that is used to break down proteins into smaller
peptides. The stomach contains gastric juice and pepsin which then initiate the breakdown of the
protein through which the protein is broken down. Then, pepsin dismantles the chains into smaller
fragments for easier digestion.
In this experiment, the digestion rate of egg whites will be measured using wavelength
transmittance. Transmittance was used as the solution should become clearer the higher the
digestion rate of the egg white. As the enzyme works, the egg mixture will become more and more
clear as it is being digested and so its wavelength transmittance will be measured at different time
intervals. The higher the transmittance, the higher the digestion rate of the egg.
1.5: Variables
Independent variables: Temperature of water bath that egg albumin solution was placed in (℃)
(±0.10℃). These temperatures are 20, 30, 40, 50 and 60 degrees Celsius.
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Dependent variables: Wavelength transmittance (100%) (±0.05%)

Controlled variables: The volume of water in each water bath was kept at 150 ml for each trial
and at each temperature. This was done to ensure that at each varying temperature an equal amount
of water needed to be heated up to get the desired temperatures.
The volume of egg suspension was kept constant throughout the experiment at 10 ml. This was
done to control the amount of egg that needed to be digested/ that needed to react.
The volume and concentration of hydrochloric acid (HCl) was kept the same throughout the
experiment. Each time 1 ml was used, and combined with the suspension as well as the pepsin to
eliminate the factor that pH concentrations change reaction times.
The concentration of pepsin placed into each test tube was consistent throughout. Each time 3 ml
of pepsin was placed into the test tube. This was once again done to eliminate the factor that
enzyme concentrations change reaction times.
The amount of time was kept the same; each test tube was placed into the water bath for 8 minutes.
This was repeated during each trial to eliminate any other variables that could affect the reaction
rate. If the time was not kept consistent, the enzyme could have had more or less time to react, and
so, the results couldn’t be comparable once the experiment was completed.
1.6: Preliminary Experiment
A preliminary experiment was conducted a week in advance to assesses if there needed to be any
alterations to the original procedure. The method that was used was almost identical to the one in
section 2.2, except for the fact that absorbance was looked at compared to transmittance. This was
changed since it seemed to be a more efficient method as it directly measures how much light
passes through compared to how much light the sample absorbs. Also, the scale, percentages, made
the data easier to compare and analyze.
2: Procedure
2.1: Apparatus

➢ 250ml egg white suspension ➢ Approximately 2L of tap water

➢ 60ml, 1% pepsin solution ➢ Labelling tape
➢ 50, 0.5mol Hydrochloric acid ➢ Pen and pencil
➢ 1 thermometer ➢ Lab Burner
➢ 5 test tubes, standard size ➢ Distilled water
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➢ 1, 500ml glass beaker ➢ Syringe

➢ 1, 10ml graduated cylinder ➢ Blender
➢ 2, 5ml graduated cylinders ➢ 1,1ml pipette

2.2: Procedure

1. Cook one egg on a lab burner until hard-boiled.

2. Prepare the egg white suspension by placing only the egg white into 100 ml of distilled
water and blend until the mixture is smooth and there are no chunks of egg white left.
3. Measure out 25 0.5 mol of HCl into a cylinder
4. Measure out 60 ml of 1% pepsin solution into a cylinder.
a. The 1% pepsin solution can be made by using 100 ml of distilled water and adding
1 mg of pepsin.
5. The next step is to set up the colorimeter. Connect the device to your laptop and start the
program. First, fill a cuvette with 3 ml of distilled water, cover it and place it into the
colorimeter covering the lid.
a. The green indicator light should be on and located below the 430 nm label. Press
collect on your device and a graph will appear on the screen, once it has shown the
wavelength transmittance of the water, take the sample out and clear it for the egg
white sample. The transmittance should be 100%.
6. In a large cylinder add 300 ml of regular tap water. Using a lab burner heat this up to 30
degrees.
a. Monitor the temperature using a thermometer.
7. In a test tube add 10 ml of egg white suspension. Label this tube 1 with labelling tape and
a pen.
a. Place the tube into the heated up water bath.
8. Pipette 3 ml of 1% pepsin solution as well as 1 ml of 0.5mol of HCl. At the same time
place both substances into the beaker with egg white solution that is in the water bath.
9. Pipette 2 ml of the solution after 0, 2, 4, 6 and 8 min out of water bath.
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a. Add these 2 ml to the sample cuvette and place into the colorimeter. Cover the
device and click collect on your device to record wavelength transmittance at that
specific time.
10. Repeat steps 4-8 five times for 20, 40, 50 and 60 ℃ and record any qualitative data as well
as the wavelength transmittance for each time interval and temperature. Sort the data using
tables as seen below in section 3.
11. Clean up the station making sure to be careful with the acid.

2.3: Justification

The presented independent variable values were used for specific reasons. Since temperature is
one of the key factors when looking at enyzme activity and rate of reaction, 20, 30, 40, 50 and 60
℃ were used to determine at which temperature the enzyme would have the highest digestion rate.

The experiment consisted of 5 trials at each varying temperature to improve the accuracy of the
data gained from the experiment. Both the concentration of pepsin and HCl were chosen based on
their approximate concentration in the body. This was done to hopefully replicate actual conditions
of the stomach.

2.4: Safety

Wear safety goggles and a lab apron. Acids are destructive and spills should be avoided. Along
with being cautious around the acid, be sure to be cautious around the lab burner as it will be hot.

3: Raw Data

3.1: Raw data table 1- A table showing the wavelength transmittance (±0.05) of the egg white
suspension at 20℃ (±0.10)during a period of 8 minutes.

Wavelength transmittance (%)(±0.05)

0 min (±0.01) 2 min (±0.01) 4 min (±0.01) 6 min (±0.01) 8 min (±0.01)

Trial 1 1.10 1.29 1.20 1.28 4

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Trial 2 1.11 1.25 1.52 1.72 1.87

Trail 3 1.07 1.30 1.54 1.83 1.92

Trial 4 1.05 1.29 1.50 1.74 1.79

Trial 5 2.15 1.35 1.56 1.78 1.80

3.2: Raw data table 2- A table showing the wavelength transmittance (±0.05) of the egg white
suspension at 30℃ (±0.10)during a period of 8 minutes.

Wavelength transmittance (%)(±0.05)

0 min (±0.01) 2 min (±0.01) 4 min (±0.01) 6 min (±0.01) 8 min (±0.01)

Trial 1 1.08 1.25 1.29 1.35 1.40

Trial 2 1.14 1.58 1.59 1.73 1.98

Trail 3 1.01 1.54 1.69 1.87 1.97

Trial 4 1.18 1.63 1.74 1.72 1.92

Trial 5 2.25 1.72 1.63 1.90 2.02

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3.3: Raw data table 3- A table showing the wavelength transmittance (±0.05) of the egg white
suspension at 40℃ (±0.10)during a period of 8 minutes.

Wavelength transmittance (%)(±0.05)

0 min (±0.01) 2 min (±0.01) 4 min (±0.01) 6 min (±0.01) 8 min (±0.01)

Trial 1 1.12 1.35 1.32 3.14 4.12

Trial 2 1.11 1.98 2.03 3.06 4.20

Trail 3 1.05 1.89 2.08 3.07 4.19

Trial 4 1.06 1.93 2.01 3.10 4.11

Trial 5 1.03 1.94 2.02 3.05 4.09

3.4: Raw data table 4- A table showing the wavelength transmittance (±0.05) of the egg white
suspension at 50℃ (±0.10)during a period of 8 minutes.

Wavelength transmittance (%) (±0.05)

0 min (±0.01) 2 min (±0.01) 4 min (±0.01) 6 min (±0.01) 8 min (±0.01)

Trial 1 1.19 1.37 1.46 1.50 1.63

Trial 2 1.18 1.43 1.53 1.70 1.82

Trail 3 1.17 1.45 1.52 1.73 1.83

Trial 4 1.16 1.42 1.63 1.74 1.79

Trial 5 1.18 1.41 1.67 1.76 1.90

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3.5: Raw data table 5- A table showing the wavelength transmittance (±0.05) of the egg white
suspension at 60℃ (±0.10)during a period of 8 minutes.

Wavelength transmittance (%) (±0.05)

0 min (±0.01) 2 min (±0.01) 4 min (±0.01) 6 min (±0.01) 8 min (±0.01)

Trial 1 1.20 1.29 1.41 1.67 1.70

Trial 2 1.19 1.35 1.56 1.72 1.83

Trail 3 1.09 1.28 1.60 1.63 1.82

Trial 4 1.11 1.37 1.61 1.78 1.79

Trial 5 1.12 1.33 1.59 1.75 1.78

3.6: Qualitative data

For 20 degrees
➢ The temp was a bit higher than 20 in the beginning, had to find a way to cool it down using
tap water
➢ It seems like 8 min is far too short
➢ No visible change, at the end it seems almost the same
For 30 degrees
➢ At minuet 6 the solution is clearing up a bit, however, still not translucent
➢ No visible change, at the end it seems almost the same
➢ There are clumps which make it hard to transmit
➢ The temp was a bit higher than 20 in the beginning, had to find a way to cool it down using
tap water
➢ It seems like 8 min is far too short
➢ Is a little more visible than 20, but not that big of a difference
For 40 degrees
➢ The reaction seems to be going at a much faster rate
➢ There are no chunks in this batch making the transmittance percentage much clearer
➢ While in the colorimeter, the solution became more and more clear
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➢ It was much harder to control this temperature compared to the others

For 50 and 60 degrees
➢ The reaction seems slower than for 40 degrees
➢ The first trial had some inconsistent data as I wasn’t able to control the temperature so
easily
➢ Notably slower than the 40 degree one, as suspected since enzymes peak at a certain temp
and then their reaction rate lowers
4: Processed Data
4.1: Processed Data Table 1: A table showing the average transmittance of egg white
suspension during the period of 8 minutes

Average wavelength transmittance (%) for each time interval

0 min 2 min 4 min 6 min 8 min

20 degrees 1.30 1.46 1.67 1.85 1.92

30 degrees 1.54 1.59 1.71 1.89 1.95

40 degrees 1.82 1.89 3.09 4.14 4.60

50 degrees 1.42 1.56 1.69 1.79 1.83

60 degrees 1.32 1.55 1.71 1.78 1.81

∑ 𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇 𝑜𝑜𝑜𝑜 𝑒𝑒𝑒𝑒𝑒𝑒ℎ 𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖

Calculation for average transmittance:
𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜 𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖

In order to establish a statistical difference between the varying temperature, an ANOVA test was
conducted on processed data table 1 using ‘’Social Science Statistics’’ online calculator. This test
allowed me to test for a statistical relationship across all my 5 different temperatures.

Note: RF [degrees of freedom (d.f) between groups, d.f total]= F value p (probability level)
The f-ratio value is 5.8618. The p-value is .002733. The result is significant at p < .05. This meant
there was a statistically significant relationship between temperature and the rate of digestion of
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an egg. However, when looking at the conditions compared to one another, an interesting note can
be made. Between almost every value that is not 40 ℃, there was no statistical significant
difference. Instead the only times when there was a statistical significant difference was when
other temperatures were compared to 40 ℃. This was then explored further using a bar graph.
5: Graphs
5.1: Bar graph showing average wavelength transmittance vs. varying temperatures

The values in the bar graph were achieved by averaging all the value for each temperature seen in
processed data table 1. What can be seen is that the standard deviation for conditions of 20℃, 30℃,
50℃ and 60℃ all overlap meaning they are insignificant. The only value that does not is 40℃.
This can be supported using the ANOVA results as well as the knowledge that enzymes have
optimum temperatures at which they work, and for pepsin it is around 40 since this is the average
temperature of the human body.
5.2: Line graph showing the wavelength transmittance of the of egg white suspension vs.
varying temperatures and time intervals
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From this line graph we have further data to conclude that temperature of 20, 30, 50 and 60 ℃
have no significant difference when it comes to the activation of an enzyme. Since the lines
overlap, so do the R2, making them insignificant. The only condition which has a statistical
difference is 40 ℃ which can be rationalized as this is the optimum temperature at which enzymes
work.

5: Evaluation
5.1: Conclusion
Firstly, I would like to define a few things in order to be able to analyze the data. The transmittance
is referring to how much light is able to pass through the sample in the colorimeter. 100%
transmittance would be a solution that is completely clear, example, water. Next, as the protein is
broken down, the solution will become more clear. And so, by looking at how much light is able
to pass through we are able to track the breakdown of the protein and the action of the enzyme;
pepsin.
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The results, as seen on the bar graph and scatter diagram, show that the optimum temperature as
predicted in the hypothesis seems to be 40°C, close to human body temperature. The results of the
experiment also show that temperature has a definite effect on the rate pepsin reacts to breakdown
the protein in the egg whites. As can be seen in table 4.1, 20°C and 30°C have the lowest
wavelength transmittance, meaning that the least amount of light was able to pass through, i.e. they
were the least clear and so they had the lowest digestion rate. This is because at lower temperature
pepsin does not react as readily and so the process is a lot slower. The same can be said for
temperatures above 40 degrees. At both 50 and 60 °c, we can see that the transmittance is once
again much lower compared to 40, and so the protein is not being broken down as readily. Since
the optimum temperature for pepsin to act is about 37°C, the solution at 40°C produces the highest
transmittance rate. Since the average change in transmittance was 2.78%, which is almost 4 times
the transmittance changes for the other temperatures. This verifies the theory that at 40°C,
optimum temperature, the enzyme is working the most effectively and so the rate is the highest
making the solution clearer much faster than the other conditions.

Next, the kinetic collision theory can be used to describe some of the findings of the experiment.
The theory states that temperature affects a system as a higher temperature will increase collision
between molecules and a lower temperature will have the opposite effect (“Factors Affecting
Enzyme Action). When these molecules collide, the kinetic energy is transformed into chemical
potential energy, and if this energy is enough to reach the activation energy, the enzyme can start
working and breaking down the molecules(“Factors Affecting Enzyme Action). The more
collisions there are, the more chemical potential energy molecules will have and so a greater
number of molecules will have the required activation energy to bind the enzyme's active site to
the protein which in turn results in a quicker rate of reaction(“Factors Affecting Enzyme Action).
In this experiment that would mean a higher transmittance meaning how clear the egg white
solution becomes. Furthermore, if the temperature gets too high some of the weak bonds that
determine the shape of a protein and its active site could be broken resulting in the enzyme
becoming denatured and decreasing the rate of reaction sometimes rendering the enzyme
inactive(“Factors Affecting Enzyme Action)e. Graph 2 shows that after 50°C the enzyme reaction
rate slows down considerably, the enzyme is denatured at a faster rate than it is below 30°C. Pepsin
is less active at lower temperatures until it reaches its activation energy around 30°C and anything
beyond around 50°C – 55°C will rapidly denature the pepsin so the molecules in the active site
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can no longer bind to the protein and produce a reaction, rendering the enzyme inactive
permanently. This can be seen from the line graph, as every temperature has the same trendline,
except for 40°C. This is supported by what is stated above as this is the optimum temperature and
so it will have the highest transmittance change and therefore, the highest digestion rate.

Lastly, from the standard deviation on the bar graph we can see that the spread of the data was
quite low for each trial, meaning that the results are not significant. We can also notice that the
40°C condition had the widest spread of data meaning it also had the greatest change. This is once
again supported by the theory mentioned above and the other results as 40°C is the optimum
temperature for the enzyme to work at and so it digests the most egg and creates the biggest change.
Hence, the widest spread of data is for 40°C.

I would also like to mention that no previous research could be found that used transmittance as a
way of measuring digestion rate. Therefore, the results could not be compared to identical results,
However, what was found was a journal labelled ‘’ Digestibility of Cooked and Raw Egg Protein
in Humans as Assessed by Stable Isotope Techniques-Pieter Evenepoel, Benny Geypens, Anja
Luypaerts, Martin Hiele, Yvo Ghoos, Paul Rutgeerts’’. The data from this journal confirms the
conclusion of my experiment, which is that pepsin works at an optimum temperature of 40°C,
which leads to the highest digestion rate of proteins.
5.2: Evaluation & Extension
The limitations of the experiment are the following: temperature control, acid concentration/
pepsin concentration and egg white suspension preparation. Firstly, the temperature was at times
quite inaccurate since it was difficult to control. I would heat up the water to the desired
temperature, however, the water starts to cool and so I would have to reheat which would cause
the temperature to rise, but sometimes too much. Because of this, it was difficult to keep it at a
constant temperature. As there is not real solution to this, only close attention to the temperature
and monitoring it. The other limitation could have possibly been the pepsin concentration as well
as the HCl concentration. Since the concentrations were quite low, the reaction was slow. By
possibly increasing the concentrations, the reaction could be sped up and with that more extensive
data could have been collected. Lastly, when making the egg white suspension, I didn’t blend the
egg whites with the water long enough and so the mixture was quite chunky. This caused many
issues, the first one was that when I would place a sample in the colorimeter, the chunks would
 15

completely block the wavelength and because of this I wasn't able to accurately measure the
transmittance in the first few trials. This could have affected the averages, however, the fix to this
was quite simple and I was able to simply blend the egg whites for a little bit longer for the other
trails and I did not have this problem anymore. Another issue with the chunks was that it would
have taken a lot more pepsin as well as acid to digest full egg whites. Because of this, there was
some inconsistency among a variable that was meant to be controlled and so the trials are in ways
not comparable. Again, the solution was quite simple, simply to blend the egg whites longer.
Additionally, when taking out the samples I had left them on the counter while the colorimeter
was measuring the previous sample. It was visible that during this time the enzyme was working
and I noticed the solutions become more clear as I was waiting to put them into the colorimeter.
Because of this, the time stamps that were used and written might be slightly inaccurate and
therefore, when comparing values, we must take this into account since one sample might have
had more time to react. Improvements to the experiment itself could be to allow more time between
intervals as this would eliminate the limitation mentioned in the previous sentence, as well as allow
more time for the enzyme to react. Another extension to the experiment would be to prepare a
stronger concentration of acid and pepsin as we would be able to get clearer and more extensive
results since none of the solution turned clear in this experiment by the end of the 8 minutes. Lastly,
in order to confirm that the protein was even digested at the end, a Biuret test could be performed
which helps in determining the presence of protein in a sample.
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Works Cited

Protein Digestion and Absorption, 2012books.lardbucket.org/books/an-introduction-to-

nutrition/s10-03-protein-digestion-and-absorpti.html.
“Factors Affecting Enzyme Action - Cell Metabolism - Eduqas - GCSE Biology (Single Science)
Revision - Eduqas - BBC Bitesize.” BBC News, BBC,
www.bbc.co.uk/bitesize/guides/zgp2v9q/revision/2.
“What Are Proteins and What Do They Do?: MedlinePlus Genetics.” MedlinePlus, U.S. National
Library of Medicine, 18 Sept. 2020,
medlineplus.gov/genetics/understanding/howgeneswork/protein/.