The effect of Alcohol on the cellular membrane of dragonfruit (Selenicereus
undatus) as observed by absorbance on pigment .
Research Question: How does increasing concentration of alcohols; methanol, and ethanol impact
the damage incurred by the cellular membrane of Dragon fruit (Selenicereus undatus) cell pigment
(betanin), evaluated by the absorbance of red pigment diffused in solution determined via a
colorimeter at 470 nm?
This Internal Assessment investigates the disruptive effects of two forms of alcohol on the cellular
membrane of the Dragon Fruit (Selenicereus undatus) using methanol, ethanol. Initially starting
my IA, I wanted to investigate pigments; as I came across the experiment of the testing effects of
alcohol on cell membranes of beetroot was commonly seen, I wanted to test out with a similar
high in betalin fruit, which is the red dragon fruit. Red-fleshed dragon fruit also known as
Selenicereus undatus, it has high concentrations of betalains, which are natural red colour
pigments (Hylocereus polyrhizus) (Cheok et al.) Though both fruits hold different properties and
qualities, I set out to investigate by holding a pilot trial experimenting the lengths to which the
dragon fruit will work for this experiment. This will be further explored in the pilot study below but
in considering this, the result proved to be feasible therefore I was able to continue with this
investigation. The aim of this lab was to determine the stress that various concentrations of
alcohols have on biological membranes. This was demonstrated by experimenting with dragon
fruits and observing how the change in alcohol concentrations affected the colour in which it was
released.
The essence of this investigation lies in the detrimental effect of alcohol on cells, particularly in
relation to the effect that continuous use of alcohol may have on humans. It is important to study
the impacts of natural solvents such as alcohols because they have significant impact on our body
and more specifically, the structure of the plasma membrane therefore understanding it and being
able to maintain its integrity is necessary for the efficiency of numerous metabolic activities.
Dragonfruit’s relevance as a topic may also be imputed to the rising consumer scepticism of
synthetic food colourants, as they are becoming increasingly wary of the harmful effects of
artificial food colourants due to research linking them to the development of ADHD symptoms and
other cognitive issues, including hyperactivity, which has prompted food manufacturers to employ
more natural pigments like betalains (“Is Food Colouring Safe for Kids?”). In addition, Betalains are
now extensively employed in commercial and confectionery food colours, and even more so in
some cultures. Furthermore, beetroot is the only source of betalain that has been approved for
use as a natural red colourant in food and pharmaceutical applications. Consequently, I became
interested in researching more about the potential uses for dragon fruit as a substitute, particularly
in the cooking (Devadiga and Ahipa). Thus, not only is this investigation exploring the damage of
alcohol on membranes but also how this phenomenon might be exploited to extract pigment from
cells for use in cooking and other applications.
Background information:
Dragon fruit is a species rich in bioactive phytochemicals such as betalains and contains high levels
of fibre which proves to be beneficial properties for improving health. It has long been used as a
traditional folk remedy in many cultures and is now grown as a crop vegetable, making it an
important part of our modern diet used in various ways even for colouring. Red-violet betacyanins
and yellow-orange betaxanthins are the two structural groups that make up the water-soluble
nitrogen complex containing vacuolar pigments known as betalains. (Amaya)
Figure 1.1: structure of betanin (“Betanin” wikipedia)
The effects of alcohol on the cellular physiology
Alcoholism remains to be one of the most prevalent health, social, and economic crises in the
world today since it has an adverse effect on all human tissues. Ethanol is known to have a
multitude of cytotoxic effects, regardless of cell type. These detrimental outcomes include the
hyperfluidization of membranes, denaturation of proteins, and the accumulation of an abundance
of reactive oxygen species (ROS), which can interact with different cell components and result in
lipid and protein peroxidation as well as DNA
damage.
Ethanol
has
antiproliferative
properties and can affect how mitochondria
work and how they expend energy. Figure 1.2
illustrates how the lipid bilayers are affected by
the alcohol through lipid peroxidation, the
damages include both chemical disruptions and
physical breaches. The plasma membrane has
biophysical properties that can be altered by
chemical instabilities, which can ultimately lead
to a permeability gap. Then, lipid peroxidation
of polyunsaturated fatty acids is induced by the
oxidative stress resulting in the elimination of
defective areas and instability of the plasma
membrane. The breakdown of enzymes in membrane lipids occur when phospholipases attack,
altering membrane fluidity. However, depending on the magnitude and frequency of the breaches,
both lytic and non lytic damages are incurred and necessary repairs need to be made in order to
replenish the membrane integrity otherwise pore formation may occur. (Ammendolia et al.)
Study has shown that alcohol affects biological membranes by impacting the chemical structure
which includes the plasma membrane, liposomes and organelle membranes such as the
endoplasmic reticulum, and the mitochondria (Goldstein 1986). It promotes membrane fluidity
which contributes directly to the release of toxicants. In addition, other biological processes such
as membrane transportation, enzymatic reactions, and the signalling of pathways are affected by
the changes in the lipid environment of membranes, which in turn can affect the functioning of
proteins receptors, ion channels, and enzymes (Escribá et al., 2008). Additionally, this direct
reaction with the membrane proteins induces conformational changes, ultimately altering its
functions.
The area of the cell membrane that has less cholesterol is where alcohols have their greatest
effect. This will cause a tear in the membrane, allowing substances inside the bilayer to escape.
Given this, the degree of damage in a cellular membrane may be deduced from the intensity of the
colour in a solution. The effects of alcohol causes fluid membranes to become disorientated. Even
though the membrane impact is minimal, pharmacological, temporal, and genetic evidence reveal
its significance. Goldstein observed that the membranes of animals that are resistant to ethanol
intoxication are a result of their genetic background or constant exposure to ethanol. When
membranes are repeatedly exposed to ethanol, their structure stiffens, which can be considered as
a long-term adaptive response. Nevertheless, certain alcohols may promote the absorption of
cholesterol or saturated fatty acids into membranes, thereby decreasing its own effect. (Goldstein).
Alcohols are relatively small molecules hence, they don't specifically bind to a site. With this in
mind, mechanisms in place allow them to absorb into the membrane and this results in the
alteration of its structure. Variations in membrane structure can affect the conformation of
proteins and other structures contained in the membrane. This establishes a rear channel through
which alcohol and other tiny amphiphilic chemicals can enter the cell and thus, affect its cellular
activity. Alcohols have important chemical properties that increase membrane permeability. In
cases of increasing concentration, alcohols as polar substances, also react with other polar
substances, hence reducing bilayer stability (Patra et al.) According to Helgi Ingolfsson and Ole
Andersen's study, the efficacy of alcohol is reliant on the chain length of that alcohol, despite the
fact that rising alcohol concentration is expected to cause greater damage to the cell membrane.
This is because certain alcoholic products have the so-called ‘cut off effect’. This phrase refers to
the specific level of the alcohol's chain length at which it is most effective and beyond which it
vigors and becomes less strong, hence its plateaus. ‘Modulators’ are Alcohols that are known to
change the properties of lipid bilayers. Its biological properties have traditionally been linked to
their ability to influence the bilayer by affecting the protein function directly through their
interactions. Alcohols with shorter chain lengths, such as methanol and ethanol, would be more
potent than alcohols with longer chain lengths. Alcohols with 1-16 carbons in their chains were
evaluated for their ability to affect the bilayer, and results showed that all of them changed
characteristics; Short-chained alcohols relationship between their bilayer-modifying potency and
their bilayer partitioning reveal to be linear, while the effect weakens with increasing chain length
and eventually plateaus. This is seen to be reversed for the longest-chain alcohols, demonstrating
an alcohol cutoff effect in a system with no alcohol-binding pocket. (Ingólfsson and Andersen).
Pilot Study
It is necessary to address that a pilot research was undertaken in order to test and build the most
effective approach for the experiment; therefore, this paragraph is dedicated to documenting my
trial and error and explaining why I made certain decisions.
To begin my comparison of dragon fruit and beetroot, I had to determine the
range in which the absorbance and diffusion period would be most effective
and relevant. To do so, experimentation with both fruits was required. I
discovered that at a concentration range of 0 to 20%, the diffusion that
occurred was insufficient to produce an accurate comparable value, however
at a concentration range of 60 to 100%, the concentration would overtake a
clear result and there would be no good middle ground. As a result, I decided
to conduct a 40% concentration trial to assess the permeability and real
influence on cell membrane in both fruits in order to compare the quantitative
and qualitative properties that occur throughout the reaction. Upon
completing my trials, I noticed that the pigment released in the beetroot trial was overall a more
concentrated set than the red dragon fruit given that the diffuse period was only 10 minutes which
I later found that 15 minutes would allow for sufficient diffusion evident in figure 2.
As part of my pilot, I also researched various alcohols to see how they
would respond to the experiment. At first, my study with ethanol was
straightforward; everything went according to the methodology , and I
gathered evidence that seemed to confirm my hypothesis. Then I tried
experimenting with methanol, which proved to be increasingly more
challenging but ultimately successful as well. Upon further investigation,
I found that propanol, hexanol, and 2-methylpropan-2-d all appeared to
share the same appearance while I was searching for my third alcohol
source. Because diffusion occurred mainly in the lower part, where the
distilled water was, the alcohol was able to gradually separate from the
liquid and have only a minor impact on the process overall. In addition
this may be due to the products being expired that can render them ineffective in disrupting the
cell membrane as it can no longer form bonds with water, hence the reason why it is separating
with water. Due to time constraints, I have decided to only compare the effects of only two
alcohols.
The relevance of controlled experimental duration varies depending on the type of experiment.
During the pilot, I carefully observed the diffusion and the time for diffusion from 5 minutes up to
25 minutes. This allowed the result in Controls to be required for the objective, unbiased
observation of the measurement of the dependent variable in response to the experiment.
Whereas experimental duration addresses biologically diverse aspects and demonstrates different
responses. Therefore, the period of an experiment is intrinsically connected to the subject of
discussion: short duration tests often examine the acute responses of organisms to abrupt
environmental change, whereas long-term investigations examine the adapted and possibly even
evolved responses. Thus, It is necessary to weigh the benefits of collecting long-term responses
against the gradual loss of realism as the experiment progresses. With longer exposure to alcohol,
the more damaged the membrane becomes and hence, more pigment is leaked out. In
establishing this, the pilot trial found that 10 minutes was not sufficient and data did not vary
therefore it was appropriate to extend the duration of this period to 15 minutes.
Methodology: Method of Analysis
Why 470nm:
A key point in this step of data collection is the absorbance. Plant pigments, likewise, the common
betaxanthin found in red beet root with λ max = 482 nm (Corneliu, ResearchGate), absorb light in
the wavelength range of 400nm - 700nm. This wavelength range, known as
photosynthetically-active radiation, is the only one in which plant pigment molecules can absorb
light. Chlorophyll and other pigments tend to absorb most effectively at 470 nm.
Absorbance was tested using a colorimeter since its results are more reliable than those of
qualitative techniques and in addition, colorimetric analyses require the use of absorbance units,
which may be expressed to three decimal places. And the colorimeter has to be calibrated before
each experiment to minimise device uncertainty.
Why 5 trials:
It is important to conduct numerous trials in order to maximise accuracy. Numerous trials are
conducted in order to minimise the impact that errors have and, as a consequence, enhance the
dependability of the outcomes of the experiment. When determining whether or not the
hypothesis was supported, the amount of confidence you can have in your data increases in
proportion to the number of times the experiment was performed. Hence, the more trials there
are, the more results there are to create a range making it easier to identify definite outliers and to
reduce the effect it has on the actual results. Which is the following reason why outliers are usually
excluded from most data processing to produce a more accurate and supportive conclusion.
Why only 2 alcoholic drinks?
During the pilot investigation which is discussed below, time constraint and lack of materials led to
only being able to compare 2 alcohols that reacted with the subject of the experiment well enough
within the constraints of the methodology.
Why 5x5x5mm cubes of dragon fruit from the middle?
The shape and size of the dragon fruit cubes must remain the same because larger surface areas
will enable for faster and more leakage of pigment which makes the comparison of each and every
trial inaccurate. In addition, cutting the dragon fruit in small cubes increases surface area and
expedites the diffusion of pigment. This would essentially shorten the time needed to diffuse as
there are several leakage points producing more obvious results in order to compare between the
treatments. Moreover, the betalains within the vacuoles of the differing dragon fruit cells may
contain varying amounts of pigments which enables different amounts of pigments to leak. This
variation can be condensed by using the same fruit and cutting from the most concentrated area
which is the middle of the fruit as well as randomly placing the beetroot cubes in different test
tubes to disperse the pieces.
Concentration range and volume :
Since there is a discernible variation in diffusion between the various alcohol concentrations, the
range was selected using 20% increments—0%, 20%, 40%, 60%, 80%— this also enables the
variance in data as the concentration is spread out. To eliminate errors in anticipated
concentrations of pre-prepared solutions, the alcohols were diluted manually before each trial.
While the volume of the alcohol would not affect the amount of damage sustained by the
membrane, it would affect the colour intensity observed resulting in differing absorbance results
which would essentially affect the overall results.
Data processing:
The data was processed by computing the average and standard deviation in order to produce the
graphs, one for each alcohol and a comparison graph that illustrate the trend predicted by the
hypothesis and include error bars reflecting the margin of sampling error and to maximise the
accuracy of data.
Hypothesis:
One major assumption that can be made from this experiment is that membrane permeability is
proportional to the amount of pigment that is released from the cell membrane vacuoles. Thus, if
the concentration of alcohol increases, the rate of absorption will also increase relative to the
change. This is the result of the damaged cell membranes enabling more pigment to be released
and hence, while observing quantitatively, we should see a gradual change in pigment coloration
as the pinkish-red colour intensities and the absorbance of pigment measured by colorimeter
would increase accordingly. Relative to the Beer-Lambert law, increasing absorbance corresponds
to increasing pigment concentration, which should be directly proportionate to the concentration
of each alcohol solution. Furthermore, it is expected that these short-chain alcohols would exhibit
a tendency similar to the "cutoff effect": as chain length increases, so does the membrane lipid
disordering potency, and so the alcohol with the longest chain-length will cause the most damage
to the cellular membrane. Based on the explored background information, there should be a
positive correlation between the two variables. I predict that the trend would be logarithmic based
on my understanding of limiting factors and the alcohols cut off potential.
Methodology:
Part A: Preparing Alcohol Concentrations:
1. Acquire 5 test tubes and a rack, labelling each tube
1-5/A-E consecutively and label the rack like shown in
figure 4 (labelled in ratios of water and alcohol)
2. Using a graduated pipette, carefully add the
corresponding amount of water as shown in table 1
below.
3. Similarly, using another pipette, add the corresponding
amount of alcohol as shown on the table.
4. Use the table to prepare the alcohol concentrations for
the remaining alcohol (ethanol or propanol)
`
Table 1: Ratio of water and alcohol for dilution for each concentration
Test-tube
number
Volume H2O using pipette
#1 (cm3) ±0.05
Volume of alcohol (ethanol,
propanol) using pipette #2
(cm3) ±0.05
Alcohol concentration (%)
1
5.00
0.00
0
2
4.00
1.00
20
3
3.00
2.00
40
4
2.00
3.00
60
5
1.00
4.00
80
Part B: Dragon Fruit preparation
5. Put on your gloves and gather your dragonfruit.
6. Measure and cut out 5 x 5 x 5 mm using a ruler, knife and a cutting board, making sure you
use the same dragonfruit from the same area, close to the centre of the fruit.
7. Rinse the cubes in water to wash off the pigment released during cutting.
8. Using the tweezers, place 2 dragon fruit cubes into each test tube for 15 minutes with the
stopwatch and gently shake the test tube to homogenise the solution.
9. Immediately remove the dragon fruit bits with tweezers, once the 15 minutes have passed
and throw them into the waste bin/beaker.
10. Using the graduated pipette, transfer the 3cm3 of each solution from the test tubes into
separate cuvettes and securely seal it with the lid.
Part C: Measuring Absorbance in Colorimeter
11. Then Connect the colorimeter to the labquest and connect that to the computer and run
the LoggerPro app connected via an adapter.
12. On logger pro make sure to label ‘concentration’ and units ‘%’.
13. On the colorimeter, make sure the light corresponds to 470 nm to get the correct
measuring point.
14. To calibrate the colorimeter, insert a ‘blank’ cuvette filled with distilled water and press
‘CAL’....wait for the absorbance to reach ‘0’ and start data collection once ready.
15. Starting data collection: Replace the ‘blank’ cuvette with the first cuvette (0%).
a. Wait for the absorbance value to stabilise and press “save”.
b. Enter concentration of alcohol in Test-tube 1 and save the absorbance and
concentration values.
16. Replace cuvette with the next cuvette (20% alcohol). Repeat Step 9 with test tubes 3-6 for
the remaining concentrations.
17. Opening a new file, start the second run on LoggerPro and repeat Step 2-15 for Trials 2-5.
18. Then repeat step 16 for the next alcohol.
a. Make sure to rinse a pipette with water and allow it to dry
b. Then repeat the steps 1-4 with a different alcohol.
c. Stop data collection and evaluate data.
Data Collection:
Quantitative processed data:
Table 2: The absorbance of the dragon fruit diffused in different ethanol concentrations
Trials (#) - Absorbance (AU ± 0.001)
Ethanol
concentration (%)
Trial 1
Trial 2
Trial 3
Trial 4
Trial 5
AVG
St. Dev
0 (0:5)
0.199
0.203
0.197
0.193
0.210*
0.200
0.006
20 (1:4)
0.253*
0.249
0.245
0.248
0.249
0.249
0.003
40 (2:3)
0.250
0.260
0.255
0.257
0.261
0.257
0.004
60 (3:2)
0.283
0.286
0.290
0.283
0.281
0.285
0.003
80 (4:1)
0.317
0.323*
0.315
0.319
0.318
0.318
0.003
100 (5:0)
0.418
0.420
0.416
0.417
0.419
0.418
0.001
Table 3: The absorbance of the dragon fruit diffused in different methanol concentrations
Absorbance (AU ± 0.001)
Methanol
concentration (%)
Trial 1
Trial 2
Trial 3
Trial 4
Trial 5
AVG
St. Dev
0 (0:5)
0.111
0.116
0.120
0.113
0.114
0.115
0.003
20 (1:4)
0.162
0.167
0.150*
0.163
0.164
0.161
0.007
40 (2:3)
0.179
0.178
0.185
0.181
0.185
0.182
0.003
60 (3:2)
0.204
0.198
0.201
0.199
0.203
0.201
0.003
80 (4:1)
0.213
0.197*
0.218
0.210
0.207
0.209
0.008
100 (5:0)
0.278
0.276
0.285
0.282
0.279
0.280
0.004
In order to maximise the precision of data, it is necessary to identify potential outliers prior to data
processing that may be caused by systematic or just a random error. It is plausible to conclude that
the numbers shown in red* and an asterisk are outliers, as they deviate significantly from the
mean of each data set and will be omitted from further analyses. The sources of error are
discussed below however it is also important to recognize the uncertainties that partake in the
results, majority derived from the methodology and data collection processes.
Qualitative data:
Subsequent to the third trial, the alcohols followed a similar trend in pigment coloration as it
gradually intensifies as the concentration is increased. Though the pigment remained relatively
transparent, the diffused pigment was minimal. To facilitate the diffusion, we gave the test tubes a
good vigorous mix every two to three minutes. Within the first five minutes, there was hardly any
colour. However, by the 12th or 13th minute, the solutions had become sufficiently colored and
suitable for experimentation.
Figure 5: pigment change with increasing concentrations of alcohol
Conforming to figure 5 above it is apparent that the two alcohols followed a similar pattern when it
came to pigment release and solution colouring. In the alcohol solutions, the red pigment
significantly rose relative to the alcohol concentration; at 0%, there was minimal red pigment and
the solution largely remained transparent, while at 80% concentration the solution was heavily
concentrated with red-violet pigment. This was visibly evident in the steady increase of pigment
gradient from the alcohol concentrations of 20% to 40% to 60%.
Although the distinction in colour between the three alcohols was not immediately noticeable, it
appeared that the colour intensity of solution was greatest with propanol and least with ethanol.
Data processing:
Graphing:
Risk assessment (along with environmental and ethical considerations)
As this experiment deals with alcohol, it is important to address the potential hazards that are
involved. In reference to an official MSDS alcohol safety consideration sheet (Liebenberg and
Mudaly, 2018)
● Flammability: Ethanol and Methanol are both highly flammable substances both in liquid
and vapour form. Therefore some precautionary measures must be taken in order to
prevent this from occurring such as but not limited to:
○ Keeping liquid away from sparks, heat and open fire and hot surfaces.
○ Keeping the container tightly closed after use.
● Ingestion: These alcohols are harmful both swallowed and inhaled so there must be strictly
no consumption around the experimental area. Additionally, a mask can be worn in order
to avoid inhalation.
● Skin and eye irritation: When working with alcohol, prolonged contact may cause the skin
and eyes to be prone to irritation if not carefully handled. Protective gloves and clothing
must be worn as well as eye protection such as goggles. Proceed to wash skin and clothes
thoroughly after handling.
● Disposing considerations: When disposing of the alcohols, prevent liquid entering sewers
as this may cause future environmental consequences. Other disposals must be under
conditions approved by local authorization.
General risk assessments
Dragon Fruit are harmless to the environment as they are organic products. However it is
important to consider the responsibility of using them. Only purchase and prepare enough
amounts sufficient for the duration of the experiment and store the remaining for other use.
The data is recorded digitally rather than on paper to minimise waste production as well as
considering the impacts on the environment.
It is enough to say that there aren't any OTHER ethical/environmental issues.
Conclusion
To adequately respond to the question as to whether increasing concentrations of the alcohols
have an effect on the damage sustained by the cellular membrane of Dragon fruit (Selenicereus
undatus) cell pigment (betanin), it must be recognized that the pigment released during diffusion,
as measured by the absorbance of the colorimeter, is evidence of this statement. The alcohol has a
direct effect on the cell membrane cohesion, allowing pigment to escape through the phospholipid
bilayer and resulting in the breakdown of membrane structure and protein, as as demonstrated by
the fact that the absorbance value increased with rising alcohol concentration and that each
solution became more pigmented. However, it is indisputable that the permeability of the cell
membrane is affected by the concentration of alcohol, lending credence to the notion. Increased
pigment leakage at higher concentrations of ethanol indicates that irreversible changes were made
to the cellular membrane, which is parallel to literary values and scientific knowledge regarding the
effect of increased concentration on disordering of lipid acyl chains that maintain the integrity of
the cellular membrane. Based on the Beer-Lambert rule, it can be deduced that the concentration
of the pigment in the solution increases as a function of the concentration of the alcohol solution.
In regards to the graph, the blue set of data follows the mean absorption of Ethanol whereas the
red follows the mean absorption of Methanol with an logarithmic trend line. As a measure of how
well the regression model fits the data, the R2 value closest to 1 assures accuracy when
constructing a graph. According to "The basic practice of statistics, sixth edition," if the R2 value is
[0 r 0.70], this result indicates a small to moderate effect size. In contrast, if the R2 value is more
than 0.70, this is regarded as a strong impact size (basic practice of statistics, Moore, D. S.). In
establishing this, ethanol’s R2 values ranged from a linear R2 of 0.895, exponential: 0.931,
logarithmic: 0.672 and polynomial: 0.948. Whereas the methanol’s R2 values ranged from linear:
0.923, exponential: 0929, logarithmic: 0.737 and finally polynomial: 0.928. Though the differences
between each value is relatively small, they may be significantly important in a larger context. In
seeing this, based off of the information, the graph trends would be most accurate with ethanol
following a polynomial trend whereas methanol following an exponential trend. However, during
the hypothesis, it was predicted that a logarithmic trend would appear as it would demonstrates
how the alcohol concentration impacts the diffusion efficiency and essentially how membranes are
affected. Regarding the cut off affect and the limiting factor, the graph should be a logarithmic
trend. Though it does not correlate fully with the data, we can see that there is still a positive
corelation and that uncertainty is an inevitable factor that causes defective results leading to this
trend.
Statistical analysis:
Upon investigation in regards to an academic context, established literature values were not found,
however a relevant study regarding betacyanin pigment contained in a taiwanese fruit called djulis
(Chenopodium formosanum) explored the relative effect of ethanol from 10-80% concentration
testing its stability. This fruit, a staple food for Taiwan's indigenous people, is appreciated for its
rich nutrient and physiochemical content. It's high amylase activity makes them ideal for use as a
wine starter. The betacyanin that provides it's striking complexion was found to be unstable in the
hull, and thus were discarded. However, due to customer demand for more natural food products,
the practice toward replacing synthetic colourants with natural alternatives has emerged. In this
experiment, the samples were thermally controlled at 60°C and stored at room temperature of
25°C for approximately 21 days to allow the pigment to regenerate (4°C, 24h). The data revealed
that the ethanol promoted pigment degradation and that isomerization of pigment was more
conspicuous in high ethanol concentrated environments. Based off the analysis, no statistically
significant variations in pigment regeneration were observed in the first trial. For both pH 3.5 and
5.5, an increase in ethanol concentration resulted in a decrease in pigment retention and a grayer
red colour for all samples appeared. For instance, after heating samples with 60% ethanol at pH
3.5, only 51.73 percent of the pigment remained, and after 21 days in storage, only 23.6 percent of
the pigment remained. This indicated that ethanol at high concentrations would result in
significant degradation of pigment. The graph fit was an exponential graph, given that their factors
and axis values were different. Observing closely we are able to understand that alcohol in this
case as it increased in concentration, degraded the pigment potential which is a common
understanding towards the exploration currently being studied. The research revealed the
importance of controlling the alcohol content and the beverage's pH when the aim is to allow
betacyanin pigments to remain stable, as is the case with Djulis wine. Though this statistical study
aimed to distinguish the effects of alcohol and pH against pigment degradation, it revealed that
betalains were more as influenced by ethanol concentrations than by pH. Demonstrating that in
similar ways, how alcohol exhibits detrimental effects on pigments is undeniably parallel to its
destructive effects on cell membranes and pigments and other cellular processes and organisms.
(Narkprasom et al.) Finally, this study sheds light on the relevance of pigments in the food industry
and how it is important to understand and maintain pigment integrity.
Evaluation:
Time consuming
Although the experiment was moderately successful, the length of time it required to get a single
trial significantly reduced the students' productivity. Completing a trial would take 1-2 hours
including the preparations, the waiting time for the diffusion, and most importantly data collection
and due to this, the experimental period dragged over a course of 3-5 days and because the aim is
to use the same fruit so that it is more controlled, the exposure of oxygen and other atmospheric
molecules can affect the fruit’s ability to diffuse to it;s greatest potential enabling for a successful
investigation.
Absorbance is not an indicator for the overall molecular damage of membranes
A significant weakness of the investigation is the measure of absorbance as an indicator for the
damage sustained by the cell membrane as the release of pigment does not necessarily account
for the overall disruption and changes incurred to the phospholipid bilayer. Sources of error may
be identified within the diffusion period for each alcohol as it was inconsistent. Once 15 minutes
ended, test tubes did not receive equal time to diffuse as some may have additional time due to
the time required to transfer the dragon fruit out of each test tube. Though the interval times may
be small given it;s uncertainty of 1 second, it may have a significant impact on the experiment as
some trials may have more time to diffuse than others. However the large time frame and
conducting of numerous trials can potentially reduce the effects of this problem. Therefore
Increasing the number of trials can reduce the random error. As well as choosing equipment with
the lower absolute uncertainty. In addition, the duration causes the dragon fruit cube to become
frail and hence become more difficult to remove from the test tube leaving remnants in the
solution.
Human error
In addition, using a ruler to measure each piece of dragon fruit introduces the possibility of human
error. The uncertainty of the ruler was 0.5mm, which is essentially a small proportion.
Nevertheless, as subjective human error plays a substantial part in these measurements, the
uncertainty will inevitably grow. This indicates that certain solutions had larger/smaller bits of
dragon fruit, resulting in increased/decreased diffusion and thus different absorbance values.
0% still induced by the diffusion of the dragon fruit.
The migration of pigment into the 0% alcohol solutions is another recognized source of inaccuracy.
Since the 0% solutions purely contained distilled water, and didn't carry any alcohol, it was
assumed that no pigment would be produced because the membrane shouldn't have been
damaged. The absorbance of pigment at 0% alcohol was 0.200, 0.115 (AU) for ethanol and
methanol. This can be explained by the methodological approach to cut the dragon fruit into
cubes. Since cutting it with a knife disrupted the cell membranes, some of the initially leaked
pigment may have persisted even after the dragon fruit cubes were rinsed and drained,
contaminating the 0% solutions and making them unable to accurately reflect the diffusion of
pigment in pure distilled water.
The experiment can be extended in numerous of was such as exploring how pH is related to how
membrane is affected by alcohol as I found it interesting how the study I explored, looked at how
both alcohol and pH affected the membrane simultaneously on a fruit and thus could be a possible
extension to mu experiment. In addition there are other ways to explore how membranes are
affected also through experiments such as electron paramagnetic resonance, spectroscopy,
fluorescence anisotropy, gas chromatography and many more involving alcohol. The complexity of
biological membranes allows us to explore many different factors and many different areas which
sparked my curiosity in the first place. Thus, we are able to see how such intricate subjects can be
viewed under a broad range of conditions, allowing us to better understand our world.
Works cited:
Amaya, Rodriguez. “Betalain - an Overview | ScienceDirect Topics.” Www.sciencedirect.com, 2019,
www.sciencedirect.com/topics/agricultural-and-biological-sciences/betalain.
Ammendolia, Dustin A., et al. “Plasma Membrane Integrity: Implications for Health and Disease.”
BMC Biology, vol. 19, no. 1, 13 Apr. 2021, 10.1186/s12915-021-00972-y.
“Betanin.” Wikipedia, 8 Mar. 2020, en.wikipedia.org/wiki/Betanin.
Cheok, Alex, et al. “Betalain-Rich Dragon Fruit (Pitaya) Consumption Improves Vascular Function in
Adult Men and Women: A Double-Blind, Randomised Controlled Crossover Trial.” The
American Journal of Clinical Nutrition, 10 Mar. 2022, 10.1093/ajcn/nqab410. Accessed 12
Mar. 2022.
Devadiga, Deepak, and T. N. Ahipa. Betanin: A Red-Violet Pigment - Chemistry and Applications.
Www.intechopen.com, IntechOpen, 2 Jan. 2020, www.intechopen.com/chapters/70710.
Goldstein, Dora B. “Effect of Alcohol on Cellular Membranes.” Annals of Emergency Medicine, vol.
15,
no.
9,
Sept.
1986,
pp.
1013–1018,
www.sciencedirect.com/science/article/pii/S0196064486801202,
10.1016/s0196-0644(86)80120-2.
Ingólfsson, Helgi I., and Olaf S. Andersen. “Alcohol’s Effects on Lipid Bilayer Properties.” Biophysical
Journal, vol. 101, no. 4, 17 Aug. 2011, pp. 847–855, 10.1016/j.bpj.2011.07.013.
“Is Food Colouring Safe for Kids?” Health Essentials from Cleveland Clinic, 26 Dec. 2019,
health.clevelandclinic.org/is-food-coloring-safe-for-kids/#:~:text=A%3A%20Studies%20have
%20linked%20 artificial.
Liebenberg, D.D., and L. Mudaly. MATERIALS SAFETY DATA SHEET (MSDS) Ethanol (c 2 H 5 OH)
COMPANY DETAILS. NCP Alcohols, 20 Aug. 2018.
Nark Prasom, Kanjana, et al. ``Relative Effects of Alcohol and PH on Betacyanin Stability in Aqueous
Djulis Extracts and Their Colour Qualities after Organic Acid Addition.” Journal of Food
Quality,
vol.
35,
no.
4,
11
July
2012,
pp.
283–291,
onlinelibrary.wiley.com/doi/full/10.1111/j.1745-4557.2012.00453.x,
10.1111/j.1745-4557.2012.00453.x.
Patra, Michael, et al. “Under the Influence of Alcohol: The Effect of Ethanol and Methanol on Lipid
Bilayers.” Biophysical Journal, vol. 90, no. 4, Feb. 2006, pp. 1121–1135,
www.ncbi.nlm.nih.gov/pmc/articles/PMC1367264/, 10.1529/biophysj.105.062364.