An Experiment to Investigate the Use of Proteolytic Enzymes in Teeth Discoloration Reduction: The Effect of

Increasing Volumes of Bromelain Enzyme extracted from Ananas comosus on the Volume of Semi-Solid Gelatin
Liquified as an Indicator of the Rate of Catalysis Reaction of Denatured Collagen into Amino Acid Monomers
INTRODUCTION
Aim: This experiment aims to investigate the effect of different volumes of Ananas comosus containing bromelain (0 mL, 20
mL, 40 mL, 60 mL, 80 mL (± 5 mL)) on the volume of semi-solid gelatin liquified as an indicator of the rate of catalysis
reaction of collagen into amino acid monomers when the amount of substrate (collagen) (200 mL ± 5 mL), the source of
bromelain (Pineapple Bhulae), surface area of contact (42.43 cm2 ± 2.340 cm2), freshness of Ananas comosus, time given for
reaction (50.00 minutes ± 0.01 s), and temperature (30.0°C ± 0.1°C) are held constant. .
Research Question: How does different volumes of Ananas comosus containing bromelain enzyme (0 mL, 20 mL, 40 mL,
60 mL, 80 mL (± 5 mL)) affect the rate of enzyme activity which will be calculated by measuring the change in volume of
semi-solid gelatin dissolved into liquid in 50.00 minutes (± 0.01 s) as an indicator of the rate of catalysis reaction of denatured
collagen into amino acid monomers when amount of substrate (collagen) (200 mL ± 5 mL), source of bromelain (Pineapple
Bhulae), surface area of contact (42.43 cm2 ± 2.340 cm2), freshness of Ananas comosus, time given for reaction (50.00 minutes
± 0.01 s), and temperature (30.0°C ± 0.1°C) are held constant?
Background Information:
The brightness of a teeth is determined both by its intrinsic (internal) color and
the extrinsic (external) stains that accumulate when chromophores from food
and drinks embed to the protein pellicle layer on the enamel surface1. Any
macromolecule/stain embedded onto the tooth surface diminishes light
reflection, therefore reducing the white color of the tooth2.
Teeth whitening products treat this yellow appearance by bleaching the teeth or
using abrasives and chemical agents in toothpaste. The most popular technique
is hydrogen peroxide-based bleaching gels, which catalyze the macromolecules
embedded into smaller components through oxidation reaction, giving the tooth
Figure 1. Reduction in the dental pellicle over time with the a brighter appearance. However, hydrogen peroxide is associated with
addition of hydrogen peroxide – Image by Hannig et al. hypersensitivity and pulp inflammatory reactions in the tooth, damaging the
surface of the enamel and causing texture change, mineral loss, and decay in hardness3,4.
A recent alternative method in the literature is natural enzymes. As the pellicle layer is made up of proteins, proteolytic
enzymes were proposed to temporarily break down the dental pellicle and remove embedded stains. This method is more
favorable, as it averts creating tooth sensitivity. Studies by Münchow et al.3 and Chakravarthy & Acharya4 investigated the
clinical stain removal effects of dentifrice involving papain and bromelain extracts, which are cysteine
protease enzymes. In both experiments, the bromelain and papain-based gels removed extrinsic stains
from the tooth samples by catalyzing the stains that prevented light reflection from the tooth surface,
resulting in a whitening effect3,4.
Bromelain is a protease extracted from the fruit/stem of Ananas comosus, pineapple. It is a mixture of
thiol endopeptidases and other chemicals like phosphates, glucosidases, carbohydrates, glycoproteins,
cellulases, and protease inhibitors5. Bromelain has several therapeutic uses, such as treating
inflammation and swelling, relieving pain, decreasing platelet aggregation, and digestive aid. Literature
review has shown that the optimum temperature for Bromelain enzyme activity is 50°C while its
optimum pH is 76. The enzyme activity of bromelain is often determined with substrates like casein, Figure 2. Molecular
structure of Bromelain –
gelatin, or chromatographic tripeptides7. Image from National Center
for Biotechnology
Information.

1
Intertek Group. “Pellicle Cleaning Ratio (PCR) Oral Care Product Stain Removal Testing.” Intertek, 2018, www.intertek.com/consumer-healthcare-trials/oral-care-pellicle-cleaning.
2
Bhargava, Hansa D., MD. “Tooth Discoloration Causes and Treatments.” WebMD, 3 June 2003, www.webmd.com/oral-health/guide/tooth-discoloration.
3
Münchow, Eliseu, et al. “Stain Removal Effect of Novel Papain- and Bromelain-Containing Gels Applied to Enamel.” Clinical Oral Investigations, 2016, doi:10.1007/s00784-016-1840-1.
4
Chakravarthy, P. K., and S. Acharya. “Efficacy of Extrinsic Stain Removal by Novel Dentifrice Containing Papain and Bromelain Extracts.” Journal of Young Pharmacists, vol. 4, no. 4, 2012, pp.
245–49. Crossref, doi:10.4103/0975-1483.104368.
5
Renu, I. C., and Hari Kumaran Thampi. “Studies on Collagen Hydrolysis by Pineapple (Ananas Comosus) Stem Bromelain.” International Journal of Pharmacy and Biological Sciences, vol. 9, no. 1,
2019, pp. 118–26. IJPBS, doi:10.13140/RG.2.2.29822.13126.
6
Martins, Bianca C., et al. “Characterization of Bromelain from Ananas Comosus Agroindustrial Residues Purified by Ethanol Factional Precipitation.” The Italian Association of Chemical
Engineering, vol. 37, 2014, pp. 781–86, doi:10.3303/CET1437131.
7
Bhattacharyya, Barun K. “Bromelain: An Overview.” Natural Product Radiance, vol. 7, no. 4, 2008, pp. 359–63, nopr.niscair.res.in/handle/123456789/5694.
1
Gelatin is produced from mammalian collagen, a structural protein forming connective tissues in the skin, tendons, ligaments,
and bones. It has a rope-like helix structure and consists of three-polypeptides. As the most abundant protein in the human
body, collagen forms mesh of fibers in the skin and in the blood vessel walls, providing high tensile strength that prevents
structures from tearing apart. It also averts cracks and fractures in the bones and the teeth8,9
To obtain gelatin, collagen is extracted from animals’ hides and bones through boiling, drying, reacting with a strong acid or
base, and filtering. Next, the extracted collagen is treated with a powder and sifted to produce gelatin. When the powdered
gelatin is mixed with boiling water to make “jello”, the collagen protein denatures. The stabilized bonds between the R groups
of the amino acids that stabilize collagen’s 3D structure begin to vibrate more vigorously under extreme heat (over 100°C),
causing the bonds to break and the protein to lose its shape and function permanently. Once the long chains of proteins are
unwound, the denatured collagen reforms messy and tangled as it cools down and solidifies. As its original structure is
disturbed, the orientation of the amino acids is also altered, which exposes the hydrophilic R groups of the protein. This
process is called hydrogenation. The hydrogen atoms attached to the sides of these collagen chains form hydrogen bonds with
the partially negative oxygen atoms in the water molecules. Due to this hydrophilic property, this 3D maze traps water
molecules in the middle of the long-chain proteins. The initially liquid gelatin becomes a complex matrix of loosely bonded
water molecules and gelatin chains, all stuck together to create a semi-solid5,10,11,12. When pineapple is added to gelatin, the
catalytic properties of Bromelain accelerate the breakdown of peptide bonds in collagen. Water molecules trapped between
tangled collagen chains are able to gradually set free, returning the gelatin solution back to liquid from semi-solid form5,10,11,12.
Personal Engagement:
While I was researching the literature for my internship with a dentist, I came across the
recent use of proteolytic enzymes for less harmful and equally as effective teeth
whitening procedures3,4. Reading about these investigations immediately made me think
of the “Enzymes” and “Proteins” units we covered in class. I came across an exercise
question that involved the catalysis reaction between bromelain from Ananas comosus
and collagen in gelatin. Noticing that Bromelain was also employed in the studies about
teeth bleaching, I realized the mechanism behind this experiment was similar to how
Bromelain removed extrinsic stains from the surface of the tooth. However, the question
in the book included a very simplistic procedure that did not allow the collection of Figure 3. Production of gelatin in gel form from
quantitative data. I concluded that conducting this experiment with slight improvements collagen protein – At the sol-gel transition temperature,
could be a useful demonstration of the recent teeth bleaching methods. Examining water molecules in the gelatin solution become trapped
within the gelatin network, forming a semi-rigid gel.
whether Bromelain is effective in catalyzing proteins is an important question to answer, Image by Katie Fegan.
since it could provide teeth discoloration patients with a less harmful alternative treatment.
During this experiment, I also employed one-way analysis of variance (ANOVA) testing to examine the validity of my results.
This test is used to examine whether there are statistically significant differences between the means of independent
experimental groups. The degree of significance is determined by the p-value, which is a measure of the probability that a
recorded difference between the experimental groups has occurred by random chance. The p-value being smaller than 0.05
indicates there is a 5% probability or less that the differences between the groups were due to chance. Therefore, researchers
can reject the H0 and accept the HA. Likewise, a p-value greater than 0.05 indicates that the differences are "not statistically
significant," suggesting a lack of strong evidence to reject the null hypothesis and argue that the groups are different13.
Null Hypothesis (H0): If different volumes of pineapple containing bromelain enzyme (0 mL, 20 mL, 40 mL, 60 mL, 80 mL
(± 5 mL)) are applied to 200 mL (± 5 mL) of semi-solid gelatin when the amount of substrate (collagen) (200 mL ± 5 mL),
source of bromelain (Pineapple Bhulae Pineapple), surface area of contact (42.43 cm2 ± 2.340 cm2), freshness of Ananas
comosus, time given for reaction (50.00 minutes ± 0.01 s), and temperature (30.0°C ± 0.1°C) are held constant, there will be
no significant difference between experimental groups containing different volumes of bromelain, thus indicating no
significant difference between rates of catalysis of denatured collagen into amino acid monomers.

8
Nimni, Marcel E. “Collagen: Structure, Function, and Metabolism in Normal and Fibrotic Tissues.” Seminars in Arthritis and Rheumatism, vol. 13, no. 1, 1983, pp. 1–86. Crossref,
doi:10.1016/0049-0172(83)90024-0.
9
Allott, Andrew. “Molecular Biology.” IB Biology Course Book: 2014 Edition: Oxford IB Diploma Program, edited by David Mindorff, 2014th ed., Great Clarendon Street, Oxford,
OX2.
10
Baguley, Richard. “Appliance Science: The Firm Chemistry of Gelatin.” CNET, 10 Sept. 2015, www.cnet.com/home/kitchen-and-household/appliance-science-the-firm-chemistry-of-
gelatin.
11
Groves, Melissa. “Is Jello Good for You? Nutrition, Benefits, and Downsides.” Healthline, 19 Feb. 2019, www.healthline.com/nutrition/what-is-jello-made-of#what-it-is.
12
“Pineapple Enzymes and Gelatin - The Sci Guys: Science at Home.” YouTube, uploaded by The Sci Guys, 11 May 2016, www.youtube.com/watch?v=7t7v8w7EqTM&t=208s.
13
LaMorte, Wayne W. “P-Values.” Module 7 - Comparing Continuous Outcomes, 2019, sphweb.bumc.bu.edu/otlt/MPH-Modules/PH717-QuantCore/PH717-Module7-T-tests/Module7-
ttests3.html.
2
 Alternative Hypothesis (HA): If different volumes of pineapple containing bromelain enzyme (0 mL, 20 mL, 40 mL, 60 mL,
80 mL (± 5 mL)) are applied to 200 mL (± 5 mL) of semi-solid gelatin when the amount of substrate (collagen) (200 mL ± 5
mL), source of bromelain (Pineapple Bhulae Pineapple), surface area of contact (42.43 cm2 ± 2.340 cm2), freshness of Ananas
comosus, time given for reaction (50.00 minutes ± 0.01 s), and temperature (30.0°C ± 0.1°C) are held constant, experimental
groups with higher bromelain volumes will obtain greater amounts of liquified gelatin as a result of higher rates catalysis of
denatured collagen into amino acid monomers.
Independent Variable: Volume of bromelain enzyme determined by differing volumes of mashed Ananas comosus (0 mL,
20 mL, 40 mL, 60 mL, 80 mL (± 5 mL)). The volumes of pineapple will be manipulated by cutting the fruit into smaller
cubes via a knife and a ruler (3.00 cm ± 0.10 cm each side of the cube). The cubes will be mashed using a mortar-and-pester
and added into a plastic metered cup (± 5 mL) until the desired volumes (mL) are obtained. As the original procedure does
not specify the amount of Bromelain enzyme used, the range for the independent variable was determined to be between 0
mL and 80 mL (± 5 mL) for this replication to have a control group with no bromelain enzyme and significantly observe the
liquification of the gelatin according to the preliminary experiment.
Dependent Variable: The volume of semi-solid gelatin liquified as an indicator of the rate of catalysis reaction of collagen
after 50.00 minutes (± 0.01 s) when different volumes of bromelain enzyme (0 mL, 20 mL, 40 mL, 60 mL, 80 mL (± 5 mL))
are added. The dependent variable will be measured by a metered cup (± 5 mL) through eight consecutive trials in to minimize
the standard deviation and increase the validity. As bromelain concentration increases, the rate of catalysis is expected to
increase proportionally. An increase in the volume of the liquified gelatin would imply that the denatured collagens in the
gelatin are broken down into amino acid monomers more rapidly.
Controlled Variables:

Variables Why the Variable is Controlled How the Variable is Controlled

Amount of As increasing the substrate concentration increases the All groups will have 200 mL (± 5 mL) of semi-solid
substrate possibility of substrates’ collision with the active site, rate gelatin. This volume will be controlled through the use
of reaction increases proportionally. However, if there is of a glass metered cup with mL divisions on it (± 5 mL).
too much substrate present, there remains no available According to my preliminary experiment, when 200 mL
enzymes to catalyze the remaining substrates, since the of water is added to gelatin, 10-20 mL of the water
active sites are already occupied. Therefore, the rate of evaporates during the boiling and freezing processes.
reaction increases rather gradually after this point9,14,15. After this observation, 220 mL (± 5 mL) of water was
If some groups had too much substrate present, their added to crystal gelatin instead and 200 mL (± 5 mL) of
enzymes would be highly occupied, which would slow semi-solid gelatin was obtained in the end. Therefore,
down the rate of reaction. Therefore, it would be the modified procedure also states that 220 mL (± 0.5
impossible to conclude a cause-and-effect relationship. mL) of water should be used for each group.

Source of Different species of Ananas comosus have differing For all of the experimental conditions, the Ananas
Bromelain concentrations of Bromelain enzyme present16. Therefore, comosus species called “Pineapple Bhulae” will be used.
if different experimental groups had obtained the enzyme Thus, all of the pineapples were bought from the same
from different pineapple species, it would be very hard to market on the same day. Pineapple Bhulae was selected
manipulate the enzyme concentration precisely. For to be the source of Bromelain, since it is the most
example, even if a group was given the highest volume of abundant pineapple species in Turkey. It is a small
pineapple among the other four groups, the actual amount pineapple breed cultivated in the north of the country17.
of Bromelain it had could be less than the others, based on Plus, it is big enough to obtain sufficient amounts of the
the pineapple species it was given. Therefore, not enzyme. If a less abundant species was selected, the
controlling this variable would make it harder to amount of Bromelain obtained could be insufficient for
manipulate the independent variable and hence harm the some of the experimental groups, which would have
causality of the findings. created a disadvantageous difference.

14
Libretexts. “Enzyme Activity.” Chemistry LibreTexts, 17 Aug. 2020, https://chem.libretexts.org/@go/page/16022.
15
BBC. “Effect of Temperature, Substrate Concentration and PH on Reaction Rate - Enzymes - Edexcel - GCSE Combined Science Revision - Edexcel.” BBC Bitesize, 2021,
www.bbc.co.uk/bitesize/guides/zwxv6yc/revision/2.
16
Omotoyinbo, O. V., and D. M. Sanni. “Characterization of Bromelain from Parts of Three Different Pineapple Varieties in Nigeria.” American Journal of BioScience, vol. 5, no. 3,
2017, pp. 35-41. Crossref, doi: 10.11648/j.ajbio.20170503.11.
17
Jones, Chris. “Pineapple.” Abbay Trading Group, 2019, www.abbaytradinggroup.net/pineapple-3294618.h
3
Surface area The rate of enzymatic reaction depends on the substrate to Since all experimental groups will have 200 mL (± 5
of the properly collide with the active site of the enzyme9. mL) of semi-solid gelatin and be placed in identical
contact Hence, a greater region of contact would indicate a higher glass metered cups, the surface area of the contact
region probability of collision. If one of the experimental groups region of gelatin and Ananas comosus will also be equal
had a bigger surface area of contact with Bromelain, that for all groups. As 200 mL coincides with the exact half
group would have an unfair advantage to display a higher of the metered cup, the average of the areas of the open-
rate of reaction. end and the bottom of the cup would give the surface
area of the contact region.

Freshness of The Bromelain enzyme is extracted from the juices of the All pineapples will be stored in the refrigerator until
Ananas pineapple fruit18. If the pineapple is exposed to direct experimentation to maintain its Bromelain content.
comosus sunlight for a long time, the fruit may dry out, which Literature review has shown that the ideal temperature
would reduce the amount of Bromelain content inside and for the storage of pineapple is 7-12°C19. The temperature
slow the rate of reaction. of the refrigerator was measured to be 9°C (± 0.1°C).

Time for Giving more time for reaction increases the rate of The time for reaction will be controlled using a
reaction catalysis, since the enzyme has more time to break down chronometer (± 0.01 s), starting immediately once the
more substrates. Therefore, if an experimental group had groups are prepared and ending immediately after 50.00
a longer reaction time than the others, the volume of minutes (± 0.01 s). Time given for reaction was selected
liquified gelatin in that group would be higher. Likewise, based on the pre-experiment. To eliminate individual
if the time was cut short for one group only, that differences in starting time, the order in which the
experimental condition would have less volume of conditions are prepared and measured will be
liquified gelatin than anticipated, as the enzyme would
randomized between trials.
not be able to fully catalyze all of the substrates9.
Temperature As greater temperatures increase the kinetic energy of the A water-bath was created to control the temperature. All
molecules, substrates and enzymes move around faster, five groups were placed inside a deep tray filled with
increasing the probability of collision. After the optimum water. A digital thermometer was positioned inside the
temperature, the enzymatic activity decreases as the tray to monitor the temperature of the water. To ensure
enzyme denatures. The high energy causes the bonds that the water remained at room temperature (30.0°C ±
between the R groups of amino acids to vibrate faster and 0.1°C), cold or hot water was added whenever the
eventually break, which alters the shape of the active site temperature fluctuated. The design was adapted from
to permanently lose its function9, 20. “How to Set Up a Water Bath” video21.
Table 1. Controlled variables, possible effects on data, and method of control

Control Group: To deduce that the change in enzyme activity due to increasing volumes of Bromelain enzyme, a control
group with no enzymes will be used. This group will only have 200 mL (± 5 mL) gelatin.
PRELIMINARY EXPERIMENT:
Since I had found this experiment in an exercise question (Appendix 1), the procedure was not mentioned in detail.
Therefore, before I conducted the actual experiment, I wanted to test the success of my modifications and fill in missing
information from the original procedure.
Since the optimum temperature of bromelain was around 50°C according to the literature review, I set my water-bath to
50.0°C (± 0.1°C). I created a water-bath by filling a deep tray with hot water from the sink, positioning my thermometer
so that it did not touch the container. I held the temperature constant throughout the experiment, which proved my design
was functioning. To prepare the gelatin, I boiled 200 mL (± 5 mL) of water on the stove and poured the water into the
metered cup that contained 3 tablespoons of crystal gelatin. I stirred the mixture until all gelatin was dissolved, and placed
it in the refrigerator. After 40.00 minutes (± 0.01 s) in the refrigerator and 10.00 minutes (± 0.01 s) in the freezer, gelatin
was semi-solid. After I added 50 mL (± 5 mL) of mashed pineapple on top of the gelatin, I placed the metered cup inside
the water-bath and started the timer. I observed the change in the solution and stopped the chronometer when the gelatin
appeared to be significantly liquified. Through this method, I determined that the reaction needed 10.00 minutes to yield

18
Manzoor, Zoya, et al. “Bromelain: Methods of Extraction, Purification and Therapeutic Applications.” Brazilian Archives of Biology and Technology, vol. 59, no.0, 2016. Crossref, doi:
10.1590/1678-4324-2016150010.
19
Paull, Robert E., and Ching Cheng Chen. “Pineapple: Postharvest Quality-Maintenance Guidelines.” College of Tropical Agriculture and Human Resources, May 2014.
20
Solomon, Sally. Introduction to General, Organic, and Biological Chemistry (General Chemistry 103, Department of Che. Vol. 10, Wiley Custom Services, 2021.
21
“How to Set Up a Water Bath.” Flinn Scientific, uploaded by Flinn Scientific, 5 Nov. 2016, www.flinnsci.com/how-to-set-up-a-water-bath/vfm0384.
4
significant results. Out of the 190 mL of semi-solid gelatin (approximately 10 mL of it evaporated during the boiling and
freezing processes), 180 mL (± 5 mL) of it was dissolved.
However, 40 mL of 180 mL gelatin (± 5 mL) in the control group was also dissolved. To solve this, I firstly decreased the
temperature of my water-bath to 30.0 °C (± 0.1°C), added 4 tablespoons of crystal gelatin, poured 220 mL (± 5 mL) of
boiling water, and kept the solution in the freezer for 20.00 minutes (± 0.01 s) after 40.00 minutes (± 0.01 s) of
refrigeration. After these modifications, the gelatin became more stable, the control group did not dissolve, and the
experimental group with 50 mL (± 5 mL) of mashed pineapple experienced liquification of 130 mL (± 5 mL). Since gelatin
was stronger, the reaction required 50.00 minutes (± 0.01 s) to yield a significant result, which was modified in the
procedure.
MATERIALS:
- 4 x Pineapple (Pineapple Bhulae) - 1 x chopping board (Appendix 4)
- 3 x 150 g crystal gelatin (see Appendix 2) - 1 x mortar-and-pester (Appendix 4)
- Tap water - 1 x tablespoon (Appendix 5)
- 1 x digital thermometer (see Appendix 3) (± 0.1°C)
- 1 x plastic stirrer (Appendix 5)
- 1 x deep tray (see Appendix 3)
- 1 x pot - 1 x refrigerator and freezer
- 1 x stove - 10 x glass metered cups (± 5 mL)
- 1 x ruler (Appendix 4) (± 0.5 mm) - 1 x mask
- 1 x knife (Appendix 4) - 1 x lab coat
- 1 x chronometer (± 0.01 s) - 1 x gloves
PROCEDURE:
1) Wear a lab coat, gloves, and a mask
2) Via a ruler and a knife, cut the pineapples into cubes that are 3.00 cm (± 0.10 cm) on each side.
3) Put one of the cubes into a mortar-and-pester and mash it. Not included in the original procedure, this step aims
to soften the fruit so that Bromelain can leave the fruit easier. However, do not mash the pineapple to the point it
becomes more of a liquid, as this could be hard to distinguish from the liquified gelatin later on.
4) Place the smashed pineapple in a glass metered cup and keep adding smashed pieces until the desired volume is
reached.
5) Repeating the procedures 2-4, obtain 20 mL, 40 mL, 60 mL, and 80 mL (± 5 mL) of mashed pineapples.
6) Place the prepared pineapples in the refrigerator, so that they do not dry out while waiting.
7) Using a glass metered cup, pour 1100 mL (± 5 mL) of water into a pot and boil it on the stove.
8) While the water is boiling, add 4 tablespoons of crystal gelatin into each of the five metered cups.
9) Label the cups as “0”, “20”, “40”, “60”, and “80”.
10) Carefully pour 220 mL (± 5 mL) of boiling water into each of the metered cups.
11) Stir the gelatin solution until it is fully dissolved in all of the cups.
12) Place the metered cups in the refrigerator and set the chronometer to 40.00 minutes (± 0.01 s).
13) After 40.00 minutes (± 0.01 s), place the cups into the freezer for 20.00 minutes (± 0.01 s).
14) When 10.00 minutes (± 0.01 s) is left until the gelatin is ready to be taken out from the freezer, securely place the
thermometer on to the side of a deep tray. Make sure that the tip of the thermometer does not touch the bottom or
the sides of the container.
15) Turn on the thermometer and start pouring water into the container from the sink.
16) Adjust the temperature of the water via the faucet handle until the thermometer shows the temperature as 30.0 °C
(± 0.1°C). Make sure at least half of the water-bath is filled.
17) Once the timer turns off, remove the gelatin from the freezer and take the pineapples out from the refrigerator.
18) Place the cups inside the water-bath.
19) Put the pineapples on top of the semi-solid gelatin. Do not add any pineapples to the control group.
20) Once the pineapples are placed, immediately set the chronometer to 50.00 minutes (± 0.01 s).
21) While waiting, constantly monitor the thermometer for any fluctuations in temperature. If the temperature of the
water-bath changes, carefully pour more water from the sink into the container until the temperature is 30.0 °C (±
0.1°C) again.
22) After 50.00 minutes (± 0.01 s) have passed, remove the cups from the water bath.
5
 23) Note down the changes in the appearance of the gelatin to collect the qualitative data.
24) In the opposite order the pineapples were added, gently pour the liquified gelatin into separate metered cups.
25) Through the metered cups that the liquified gelatin were poured in, measure the volume of the products of the
catalysis reaction.
26) Once the results are obtained, pour the gelatin and pineapples into the bin.
27) Clean the metered cups, the spoons, and the stirrer using the water in the water-bath, so that it is repurposed.
28) Repeat the steps 2-27 7 more times, so that eight trials are conducted. Randomize the order in which you place
the pineapples into the cups, so that individual differences in starting time is eliminated between different trials.
29) In order to ensure that the results obtained are significant in terms of indicating a difference in catalysis rates
among experimental groups, employ ANOVA testing to make comparisons between groups.

SAFETY, ETHICS, AND ENVIRONMENTAL CONCERNS22,23:

During the preparation of gelatin, boiling water has to be prepared using the stove, which has to be handled with caution. It
is advised to use gloves and a lab coat. Although not caused by a dangerous chemical, the smell coming while preparing the
gelatin can cause a headache after hours of exposure. To prevent this, ventilate the room and wear a mask while working. In
terms of ethics, this experiment does not include any direct testing on any humans or animals. However, gelatin is made from
the bones and hides of cattle, which requires the slaughter of an animal for commercial uses. Although there are vegan options
for gelatin, such as agar-agar from red algae, this experiment could not utilize this ethically-just alternative, since agar-agar
does not contain collagen or any other form of protein. Regarding environmental concerns, due to the pandemic, a home-
made water-bath was used rather than a digital instrument. Although a successful alternative, a great amount of water is used
in the making of this water-bath. To prevent water going to waste, the water in the tray could be used for the cleaning of the
materials before it is refreshed. If conducted in a laboratory, it is strongly recommended that the experimenter uses a digital
water-bath. Moreover, the remaining products are pieces of pineapple, water, and small amounts of semi-solid gelatin. None
of these materials constitute a danger to the environment and can be thrown into a trash can. However, it is recommended
that all waste products are thrown into a chemical-waste container, if conducted in a laboratory.
DATA COLLECTION AND ANALYSIS:
Qualitative Data: (See Appendix 6 for bigger pictures)

Figure 4. The appearance of the experimental group with 80 mL of Figure 5. The volume of liquified gelation for the experimental group with
pineapple at different time intervals of trial one – The time intervals are a) 80 mL of pineapple at different time intervals of trial one – The time
Initial (t=0), b) 30 minutes after the pineapples are added, c) 50 minutes after intervals are a) Initial (t=0), b) 30 minutes after the pineapples are added, c)
the pineapples are added. 50 minutes after the pineapples are added. The liquid gelatin was separated
from the experimental group and was measured in a different glass metered
cup for observation purposes.
a) Initial: There was no liquified gelatin once pineapples were added.
b) 30.00 minutes later: The gelatin appeared to be softer, indicated by the pineapples sinking deeper. There were also small
amounts of liquification observed for the gelatin in the groups with 60 mL and 80 mL (± 5 mL) of pineapple.
c) 50.00 minutes later: The majority of the gelatin in all of the groups (except for the control condition) were in liquid state.
The fruit sample sank the deepest in the experimental group with 80 mL (± 5 mL) of pineapple, and the least in the group
with 20 mL (± 5 mL) of pineapple. There was very slight liquification of the gelatin in all of the control groups. No bubble,
odor, or color formation was observed in any of the trials.

22
Adams, Ashley. “Agar-Agar Is a Healthy Vegetarian Gelatin Substitute.” The Spruce Eats, 31 July 2020, www.thespruceeats.com/what-is-agar-agar-p2-1000960.
23
Song, E. H., et al. “9.08 - Polysaccharides.” Polymer Science: A Comprehensive Reference, vol. 9, Elsevier, 2012, pp. 137–55.
6
 Volume of Pineapple Containing Bromelain Enzyme (mL) (± 5 mL)

0 mL 20 mL 40 mL 60 mL 80 mL

Volume of Liquified Gelatin Trial #1 5 100 120 125 140

(mL) (± 5 mL)
Trial #2 5 113 123 150 155

Trial #3 10 120 140 135 160

Trial #4 7 117 125 140 143

Trial #5 10 125 140 143 147

Trial #6 3 95 133 140 150

Trial #7 3 100 125 145 157

Trial #8 5 120 137 147 150

Table 2. Raw data table – Volume of liquified gelatin (mL) vs volume of pineapple containing bromelain (mL) for eight
different trials | Uncertainty for the volumes were determined to be ± 5 mL as the smallest unit on the analogous glass
metered cup was 10 mL.

Data Processing: The results demonstrated in Table 2 were processed according to the series of calculations depicted under
the title “Sample Calculations”, and were summarized in Table 3 for further statistical investigation. The uncertainty for the
volume of pineapple containing bromelain enzyme was determined based on the constant uncertainty of the analogous
instrument used, while the uncertainty for the rate of enzyme reaction was obtained by taking the sum of the relative
uncertainties of the volume of liquified gelatin and time given for reaction. However, this uncertainty was rather treated as
an overall uncertainty for the rate of enzyme reaction, and separate standard deviations were calculated for each
experimental group to be more precise regarding uncertainties.

Volume of Pineapple Containing Bromelain Enzyme (mL) (± 5 mL)

0 mL 20 mL 40 mL 60 mL 80 mL

Rate of Reaction (mL/min) Trial #1 0.10 2.00 2.40 2.50 2.80

(± 1.25%)
Trial #2 0.10 2.26 2.46 3.00 3.10

Trial #3 0.20 2.40 2.80 2.70 3.20

Trial #4 0.14 2.34 2.50 2.80 2.86

Trial #5 0.20 2.50 2.80 2.86 2.94

Trial #6 0.06 1.90 2.66 2.80 3.00

Trial #7 0.06 2.00 2.50 2.90 3.14

Trial #8 0.10 2.40 2.74 2.94 3.00

Average 0.12 2.23 2.61 2.81 3.01

Standard Deviation 0.05 0.21 0.15 0.15 0.13

ANOVA Testing* P-value = 1.80 x 10-29

Table 3. Processed data table – Rate of enzyme reaction (mL/min) versus concentration of bromelain enzyme (mL) for
eight different trials, their average, standard deviations, and P-value | *P-value was calculated using the Excel tool “Anova:
Single Factor”. The ANOVA testing will be further investigated for significance between different pairs of experimental
groups, as demonstrated in Table 4.
7
Sample Calculations:
𝐴𝑏𝑠𝑜𝑙𝑢𝑡𝑒 𝑈𝑛𝑐𝑒𝑟𝑡𝑎𝑖𝑛𝑡𝑦
𝑃𝑒𝑟𝑐𝑒𝑛𝑡𝑎𝑔𝑒 𝑈𝑛𝑐𝑒𝑟𝑡𝑎𝑖𝑛𝑡𝑦 = × 100
𝑉𝑎𝑙𝑢𝑒
8 :.:B
For volume = 9:: ;< × 100 = ±1.25% For time = CD:: E × 100 = ±0.000278%

𝑉𝑜𝑙𝑢𝑚𝑒<KLMKNKOP QORSTKU (𝑚𝐿)

𝑅𝑎𝑡𝑒 𝑜𝑓 𝑅𝑒𝑎𝑐𝑡𝑖𝑜𝑛 = = 1.25 + 0.000278 = 1.25028% ≈ 1.25%
𝑇𝑖𝑚𝑒 (𝑚𝑖𝑛𝑢𝑡𝑒𝑠)
𝑉𝑜𝑙𝑢𝑚𝑒<KLMKNKOP QORSTKU (𝑚𝐿)
𝑅𝑎𝑡𝑒 𝑜𝑓 𝑅𝑒𝑎𝑐𝑡𝑖𝑜𝑛24 =
𝑇𝑖𝑚𝑒 (𝑚𝑖𝑛𝑢𝑡𝑒𝑠)
B:: ;<
For Trial 1, 20 mL of pineapple = 8:.:: ;KUMTOE = 2.00 𝑚𝐿/𝑚𝑖𝑛𝑢𝑡𝑒𝑠 (±1.25%)

∑e _`RM;O `N RKLMKNKOP aORSTKU N`b OScd TbKSR

Average of data for 20 mL of pineapple = f g
2.00 + 2.26 + 2.40 + 2.34 + 2.50 + 1.90 + 2.00 + 2.40
= = 2.23
8
Note: Average of trials was calculated through excel formula AVERAGE
Standard deviation for experimental group with 20 mL of pineapple:

(𝑥 − 𝑥̅ )t (𝑥 − uuuuuu
2.23)t
𝑆𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝐷𝑒𝑣𝑖𝑎𝑡𝑖𝑜𝑛 = op = op = 0.21
𝑛−1 8−1

Note: Standard deviation was determined via excel formula STDEV.P

Graph 1. Rate of Enzyme Reaction versus Volume of Bromelain Enzyme

Rate of Enzyme Reaction versus Volume of Bromelain Enzyme

4

3,5
Rate of Enzyme Reaction / mL/min

y = 0,0318x + 0,9016
3

2,5

1,5

0,5

0
0 10 20 30 40 50 60 70 80 90
Volume of Pineapple Containing Bromelain Enzyme / mL
Average Trendline

Interpretation of Processed Data:

Graph 1 was plotted according to the processed data demonstrated in Table 3. To draw the curved line, labelled as “Average”,
the volume of liquified gelatin for all of the experimental groups was divided to 50.00 (time given for reaction) to deduce the
rate of enzyme reaction, and the average of all eight trials were taken. The curved line appears to be rising throughout the
24
Chandran, Nithin. “Enzyme Kinetics.” SlideShare, 9 Jan. 2012, www.slideshare.net/nithinaneesh/enzyme-kinetics-10903597.
8
 graph, although the steepest increase is observed between 0mL-20 mL of pineapple containing bromelain enzyme. As the
volume of the enzyme increases, the slope of the curved line was found to become more gradual, although still increasing.
An additional trendline was constructed via Excel to see the overall linear trend in the rate of enzyme reaction with different
volumes of bromelain enzyme. The trendline displayed a slope of 0.0318, indicating a trend of increase.
The uncertainty of the volume of pineapple containing bromelain enzyme was represented via horizontal error bars as ± 5
mL due to the use of an analogous instrument. The horizontal error bars are not included in the control group due to the
absence of any pineapple. The uncertainty for the rate of reaction for each experimental group and the control were calculated
individually through standard deviation calculations as demonstrated in Table 3, and were represented in the graph through
vertical error bars. The graph shows notable overlaps between the vertical error bars for the pairs 40mL-60mL and 60mL-
80mL of pineapple containing bromelain enzyme.
Further Statistical Analysis:
In addition to the descriptive analysis of the processed data in Graph 1, further statistical analysis via one-way ANOVA
testing was applied to understand if there was a significant difference in the rate of enzyme reaction for different volumes
of pineapple containing bromelain enzyme. The results of the ANOVA testing were calculated via the website
atsatsa.com25, and are demonstrated in Table 4.
Treatment Pairs / Different Amounts of Enzyme p-value Significance level

0 mL versus 20 mL 0.0010053 p<0.01 - significant

0 mL versus 40 mL 0.0010053 p<0.01 - significant

0 mL versus 60 mL 0.0010053 p<0.01 - significant

0 mL versus 80 mL 0.0010053 p<0.01 - significant

20 mL versus 40 mL 0.0010053 p<0.01 - significant

20 mL versus 60 mL 0.0010053 p<0.01 - significant

20 mL versus 80 mL 0.0010053 p<0.01 - significant

40 mL versus 60 mL 0.0911005 p>0.05 - insignificant

40 mL versus 80 mL 0.0010053 p<0.01 - significant

60 mL versus 80 mL 0.1266441 p>0.05 - insignificant

Table 4. Results of the ANOVA testing – The p-values and levels of significance for different treatment pairs containing
different volumes of bromelain enzyme. | The p-values are calculated via the website atsatsa.com25.
According to the results of the ANOVA testing, it can be concluded that except for the treatment pairs 40mL-60mL and
60mL-80mL, the p-value for all treatment groups are smaller than 0.01, indicating a significant difference. Therefore, the
alternative hypothesis can be accepted while the null-hypothesis can be rejected for these experimental groups. However,
the p-value was found to be greater than 0.05 for 40mL-60mL and 60mL-80mL groups, meaning that their differences were
insignificant and could have occurred by chance, unable to reject the null-hypothesis.
CONCLUSION:
Inspired by the newest treatments to teeth discoloration in the field of dentistry, this investigation aimed to investigate whether
increasing volumes of Bromelain impacted the volume of dissolved gelatin as an indicator of the rate of hydrolysis reaction
of collagen polypeptide. It was anticipated for the rate of enzyme activity to increase as the volume of Bromelain increased,
simultaneously expecting this increase to become more gradual at higher enzyme concentrations. When the results of this
study are evaluated, the experimental data significantly supports the alternative hypothesis: If different bromelain enzyme
concentrations (0 mL, 20 mL, 40 mL, 60 mL, 80 mL (± 5 mL)) are applied to 200 mL (± 5 mL) of semi-solid gelatin when
the amount of substrate (denatured collagen in gelatin) (200 mL ± 5 mL), the source of bromelain (Pineapple Bhulae
Pineapple), surface area of contact region (42.43 cm2 ± 2.340 cm2), the freshness of Ananas comosus, time for reaction (50.00

25
Vasavada, Navendu. “One-Way ANOVA (ANalysis Of VAriance) with Post-Hoc Tukey HSD (Honestly Significant Difference) Test Calculator for Comparing Multiple Treatments.”
Astatsa.Com, 2016, astatsa.com/OneWay_Anova_with_TukeyHSD.
9
minutes ± 0.01 s), and temperature (30.0°C ± 0.1°C) are held constant, experimental groups with higher bromelain enzyme
concentrations will obtain greater amounts of liquified gelatin as a result of higher rates catalysis of denatured collagen into
amino acid monomers, thus indicating greater rate of enzyme reaction.
Table 2 shows the raw data of the change in liquified gelatin volume with increasing volumes of pineapple containing
bromelain enzyme in eight trials. A clear increasing trend can be seen from the raw data, since the volume of dissolved gelatin
was reported to become greater with the presence of greater volumes of bromelain enzyme. A corresponding observation was
also noted for the qualitative data, as experimental groups with more pineapples were observed to dissolve in greater amounts,
allowing more pineapples to sink in. There were more liquified gelatin in all of the groups when t=50.00 (± 0.01 s) minutes
compared to t=30.00 (± 0.01s) minutes, meaning that longer exposure to Bromelain corresponded to a greater rate of
hydrolysis. The qualitative data also supported the raw data in the aspect that the greatest amount of liquified gelatin was
seen in the experimental group with 80 mL (± 5 mL) of pineapple and the least was seen in the control group. This indicates
that greater amounts of enzyme increase the rate of enzymatic reaction. The raw data in Table 2 and the qualitative
observations combined provide evidence for the argument that more gelatin is liquified as a result of the catalysis of denatured
collagen fibers via the protease bromelain, thus allowing more water molecules to escape from the semi-solid gelatin matrix.
Table 3 summarizes the rate of enzyme reaction of each experimental group with increasing concentrations of bromelain
enzyme for eight different trials, their average, standard deviations, and overall p-value obtained from standardized ANOVA
testing. While the uncertainty for the volume of bromelain remained constant as ± 5 mL, the overall uncertainty for the rate
of reaction was calculated by adding the relative uncertainties of the volume of liquefied gelatin and time given for reaction.
However, the individual uncertainties for the rate of reaction of each experimental group was determined by calculating their
standard deviations, and these unique uncertainties for each group were taken into account for both Graph 1 and the discussion
of the uncertainties. The rate of enzyme reaction of bromelain was determined by dividing the volume of liquified gelatin to
the time given for reaction. This process led to a range of reaction rates from 0.06 mL/min to 3.14 mL/min. The distribution
of values within each trial was also calculated through an assessment of standard deviation, which resulted in a range of 0.05
to 0.21. In order to eliminate individual differences within trials, averages were calculated for the data of each experimental
group to obtain a singular, curved line as demonstrated in Graph 1.
Based on the processed data in Table 3, a rate of reaction versus enzyme concentration
trend was shown in Graph 1, which was created by taking the averages of the eight
trials for each experimental group. The uncertainties calculated for rate of reaction
and volume of pineapple containing bromelain were also displayed through error bars.
The resulting graph was consistent with the graph created by Sattler et al.26, who also
investigated the effect of increasing enzyme concentration on the rate of reaction.
Although the enzyme and substrate used in the study by Sattler et al.26 is different than
those employed in the current study, the trend in reaction rate appears to be
interchangeable between catalysts. Consistent with Figure 6, Graph 1 demonstrates a Figure 6. Effect of celluclast enzyme dosage on the extent
of hydrolysis of cellulose after 24 hours – Letters A, B,
significant increase (from 0.12 to 3.01 mL/min) in reaction rate with respect to and C represent different pretreatment temperatures. Image
increasing volumes of enzyme. In this investigation, the trendline generated according by Sattler et al. (1989).
to the resulting curve was found to have a gradient of 0.0381, which indicates a strong positive correlation, thus supporting
the hypothesis that increasing bromelain concentration will also increase the rate of hydrolysis of collagen. This is because
more active sites will be available, allowing more substrate molecules to be catalyzed, increasing the rate of enzyme reaction.
In comparison to the control condition, experimental groups appeared to have significantly greater reaction rates, which is
due to the presence of bromelain enzyme. Bromelain, as a protease, provides an alternative pathway with a lower activation
energy for the hydrolysis of collagen, thus accelerating the rate of the reaction. Likewise, since the control groups only had
substrate and no bromelain enzyme, there was close to no reaction taking place. Another observation that can be drawn from
Graph 1 is that the increase in reaction rate seems to be more gradual with increasing enzyme concentrations. While there is
only an increase of 0.20 mL/min between the experimental conditions with 60 and 80 mL of pineapple, an increase of 0.38
mL/min (almost the double) was observed between 20 and 40 mL. This reduction in pace can be explained as more collagen
polypeptides are catalyzed into amino acid monomers, the probability of the remaining substrates colliding with the active
site of Bromelain decreases at a more rapid pace. Therefore, while increasing the volume of pineapple from 20 to 40 mL
creates a significant increase in the reaction rate, transitioning from 60 to 80 mL results in a rather gradual difference.

26
Sattler, W., et al. “The Effect of Enzyme Concentration on the Rate of the Hydrolysis of Cellulose.” Biotechnology and Bioengineering, vol. 33, no. 10, 1989, pp. 1221–34. Crossref,
doi:10.1002/bit.260331002.
10
To conclude, the results clearly support the alternative hypothesis by demonstrating Bromelain enzyme concentration is
directly proportional to the rate of reaction. Furthermore, the results are also consistent with research by Münchow et al.3 and
Chakravarthy & Acharya4, highlighting that the enzyme Bromelain is an effective catalyst for proteins like collagen. Although
collagen is not found in the enamel of the tooth2, the results can still contribute to the discussion on whether natural proteolytic
enzymes can be employed in the treatment of teeth discoloration. As this investigation demonstrated that Bromelain is a
sufficient protease in breaking down peptide bonds, the results have strong implications for the use of Bromelain in dentistry,
considering the tooth enamel is predominantly composed of proteins2.
EVALUATION:
Uncertainty, Standard Deviation, and Error Bar Discussion:
The horizontal error bars presented in Graph 1 demonstrate the uncertainty in volume measurements for the volume of
pineapple containing bromelain enzyme, having a constant value of ± 5 mL. As ± 5 mL is a small value, the uncertainties for
volume of the bromelain enzyme do not seem to threaten the clear interpretation of the trend in the graph and the validity of
the results. Furthermore, the vertical error bars in Graph 1 were obtained via standard deviation calculations for each
experimental group, and appear to be somewhat consistently small among groups (2 groups having an uncertainty of 0.15
mL/min and 1 having that of 0.13 mL/min), suggesting that the experiment has strong precision.
However, the great degree of confidence of the results obtained is best demonstrated by the results of the standardized
ANOVA testing. The testing for the results overall yielded a p-value of 1.80 x 10-29, demonstrating that the results had high
reliability and were sufficient to reject the null hypothesis. Furthermore, except for two treatment pairs (40mL-60mL and
60mL-80 mL), all of the differences in the experimental groups were found to be highly significant, as their p-values were
smaller than 0.01. Therefore, the results of the ANOVA testing suggest a great degree of confidence in the results.
The curve depicted in Graph 2 also appears to be consistent with the trend found in the studies by Sattler et al.26 and Michaelis
& Menten27 and the observed effects of bromelain enzyme on proteins as reported by Münchow et al.3 and Chakravarthy &
Acharya4, which suggests that the results also possess high accuracy within the literature. Overall, the validity of the findings
is not greatly affected by the uncertainties of the materials and their standard deviations, remaining highly reliable.
Strengths, Limitations, and Improvements Discussion:
Strengths:
Although laboratory resources were unavailable due to the pandemic, a homemade water-bath was designed to keep the
temperature of the system constant. Temperature is an important extraneous variable to manipulate, as it can accelerate the
rate of reaction if close to the optimum temperature of the enzyme. This would, in return, limit the causality of the findings,
as it would be impossible to determine whether the increase in reaction rate for certain experimental groups were caused by
the independent variable or differences in temperature. The use of the homemade water-bath prevented this consideration,
ensuring high internal validity in the results.
By testing possible scenarios through a preliminary experiment, possible errors in the procedure were eliminated beforehand,
making the results more reliable. Important considerations such as the ideal temperature for the water-bath, appropriate
amount of crystal gelatin to use, refrigeration time, and the time given for reaction were determined through the results of the
preliminary experiment.
Lastly, the results of the one-way ANOVA testing yielded a very small p-value for almost all of the treatment pairs, indicating
a great level of confidence for the data. P-value being smaller than 0.05 indicates that there is a significant difference between
the results of the experimental groups, thus strongly depicting that the results did not occur by chance. Therefore, we can
confidently accept the alternative hypothesis while rejecting the null-hypothesis.
Limitations and Improvements:
In addition to its strengths, this experiment also had several weaknesses. For example, the vertical error bars in Graph 1
showed great standard deviation for the experimental groups with 20 and 40 mL of pineapple. Although the p-value of
ANOVA testing was very small, the large range of these standard deviations led to several overlaps in the vertical error bars,
suggesting the differences observed between each group could be less reliable. This weakness may be because bromelain was

27
Johnson, Kenneth A., and Roger S. Goody. “The Original Michaelis Constant: Translation of the 1913 Michaelis–Menten Paper.” Biochemistry, vol. 50, no. 39, 2011, pp. 8264–69.
Crossref, doi:10.1021/bi201284u.
11
obtained from pineapple fruit, rather than used as a purified extract. Although the volumes of pineapple were measured for
each experimental group, there was no way to determine the volume of enzyme present in the prepared sample. Hence, the
independent variable was measured indirectly. So, groups with small volumes of pineapple could have had a greater bromelain
concentration, portraying a greater reaction rate than expected. Thus, since bromelain was not in purified form, the pineapple
could have contained other enzymes that affected the rate of reaction. These considerations combined limit the internal
validity of the findings. The experiment could be replicated using purified bromelain rather than pineapple fruit. Bromelain
can be directly purchased in its purified form, or can be extracted using methods such as the reverse micellar system, aqueous
two-phase system (ATPS), membrane filtration, ion exchange chromatography, or salting out via ammonium sulfate20.
Another limitation was that the control groups also displayed liquification of gelatin. As no enzymes are present in these
groups, their rate of reaction would be expected to be zero. With regards to the preliminary experiment, a possible explanation
could be that the temperature of the water-bath has caused gelatin to melt, or the gelatin was not refrigerated enough. Although
the contribution of enzymes to the rate of reaction is apparent when the amounts of dissolved gelatin are compared, this
observation still limits the cause-and-effect relationship between the amount of enzyme and catalysis rate. A digital water-
baths could ensure a temperature that would prevent the gelatin in the control groups from melting on their own.
The procedure did not include any method to control the pH of the system, which is an important factor in the rate of reaction.
When varying greatly from the optimum pH of the enzyme, the peptide bonds of the protein can be permanently broken
down, changing the orientation of the R groups and ending the proper function of the active site. This denaturation would
jeopardize the reaction rate if not controlled. A pH probe can be used to monitor and regulate the changes in pH.
The volume of liquified gelatin was measured by first removing the pineapples from the cup and then pouring the dissolved
gelatin into a metered glass. However, a portion of gelatin may adhere to the surface of the pineapples and get thrown out, or
not all of the liquid may be successfully transferred into the cup. Therefore, the dependent variable may be measured
imprecisely, reducing the reliability of the findings. To eliminate this, the volume of the dissolved gelatin can be determined
via the measurements on its metered cup, subtracting the volume of the pineapple.
Lastly, the preliminary experiment showed that a portion of the water added to crystal gelatin evaporated during the boiling
and cooling processes. Hence, in the actual experiment, 220 mL of water was added to each group to eventually obtain 200
mL of gelatin. However, this uncertainty in how much water would be left in the end of the preparation of gelatin caused
some experimental groups to have excess substrate compared to others. Having more substrate present would increase the
probability of collision with the active site of the enzyme, which would also increase the rate of reaction, weakening the
causality of the investigation. To prevent this, readily made gelatin that is fixed in volume can be purchased from the market.
Extensions:
Further experimentation can compare the rate of catalysis of collagen when Bromelain enzyme is mobilized versus
immobilized. As immobilized enzymes are more frequently used in the industry due to their advantages, (i.e. stability and
higher concentrations), it would make the results more generalizable.
The changes in the rate of hydrolysis of collagen and Bromelain enzyme when competitive and non-competitive inhibitors
are present can be tested. According the study by Esti et al.28, non-competitive inhibitors for Bromelain include grape seed,
while sulfur dioxide acts as a strong mixed-type inhibitor. While non-competitive inhibitors would prevent the enzyme from
reaching its maximum rate, competitive inhibitors would have a similar effect only if the amount of substrate is limited.
Researchers could also compare the rate of reaction of Bromelain with similar enzymes, such Papain from the papaya fruit,
or Actinidin from kiwi. Moreover, the Bromelain enzyme used in this experiment was obtained from the fruit of pineapple.
However, research has shown that there are higher concentrations of Bromelain in the stem20. Therefore, using stem
Bromelain could yield more significant results.
Finally, literature review has shown that the dental pellicle does not contain collagen in its structure. To make the results
generalizable to Bromelain’s recent use in dentistry, the effect of this protease can be tested on proteins that are present in the
dental pellicle, such as albumin or cystatin29.

28
Esti, Marco, et al. “Effect of Wine Inhibitors on Free Pineapple Stem Bromelain Activity in a Model Wine System.” Journal of Agricultural and Food Chemistry, vol. 59, no. 7, 2011,
pp. 3391–97. Crossref, doi:10.1021/jf104919v.
29
Odanaka, Hibiki, et al. “Comparison of Protein Profiles of the Pellicle, Gingival Crevicular Fluid, and Saliva: Possible Origin of Pellicle Proteins.” Biological Research, vol. 53, no. 1,
2020. Crossref, doi:10.1186/s40659-020-0271-2.
12
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 APPENDICES:

Appendix 1: Original procedure in the book

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Appendix 2: Crystal gelatin

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Appendix 3: Water-bath setup
Thermometer

Water

Deep tray

Water

Deep
tray

Thermometer

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Appendix 4: Setup for the preparation of Bromelain

Mortar-and-pester

Chopping board

Ruler

Knife

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Appendix 5: Setup for the preparation of the gelatin

Chronometer

Tablespoon

Plastic stirrer

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Appendix 6: Bigger pictures of the qualitative data of the experimental group with 80 mL of pineapple

Figure 4. The appearance of the experimental group with 80 mL of pineapple at different time intervals of trial one – The time intervals are a) Initial (t=0), b) 30
minutes after the pineapples are added, c) 50 minutes after the pineapples are added.

Figure 5. The volume of liquified gelation for the experimental group with 80 mL of pineapple at different time intervals of trial one – The time intervals are a)
Initial (t=0), b) 30 minutes after the pineapples are added, c) 50 minutes after the pineapples are added. The liquid gelatin was separated from the experimental group and
was measured in a different glass metered cup for observation purposes.

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