1
Research Question: What is the effect of exposure to different ratios of ethanol and soju
mixtures (1:9, 3:7, 5:5, 7:3, 9:1) and thus the increase in concentration of ethanol (25.37%,
42.01%, 58.55%, 75.09%, 91.63%) on the permeability of blueberry (Vaccinium
corymbosum) cell membrane measured by absorbance of green light (absorbance)?
1: Introduction
Often times in Korea, soju, which is a traditional Korean liquor, is infused with flavor by putting different
types of fruits inside a sealed container of soju and letting it sit for a couple of months. This allows for the
liquor to be flavored by the fruit. As I began gaining interest in ethanol’s effect on biological systems, I
began to notice that the blueberry infused soju that my parents had prepared during Lunar New Year had
changed from colorless to dark blue. Pigments present inside the fruit could not have passed through the
phospholipid bilayer by simple diffusion or facilitated diffusion in a rate that could noticeably change the
color of the liquor. I wanted to see whether the ethanol in soju compromised the integrity of the berries’
membrane, thus increasing the permeability. I chose to test this using varying concentrations ethanol in
soju and blueberries or Vaccinium corymbosum. Soju, a common Korean liquor, has a varying alcohol
percentage of 16.8% to 53.0% and is made by fermenting rice with yeast. Blueberry contains more than
ten different derivatives of anthocyanin pigment, highly concentrated in its skin cells (Lee et al., 2016).
More significantly, unlike other plants, the pigments in blueberry skin cells are not bound in any specific
vacuoles inside the skin cell, but are rather contained freely inside the cytoplasm. Hence, I came up with
the research question: What is the effect of exposure to different ratios of ethanol and soju mixtures
(1:9, 3:7, 5:5, 7:3, 9:1) and thus the increase in concentration of ethanol (25.37%, 42.01%, 58.55%,
75.09%, 91.63%) on the permeability of blueberry (Vaccinium corymbosum) cell membrane
measured by absorbance of green light (absorbance)?
2: Investigation
2.1: Background
Anthocyanins, found most commonly within the Vaccinium genus, are water soluble pigments that are
responsible for red, purple and blue colors in fruits and vegetables. These pigments are often vacuole
bound and are situated in the fruit’s skin. In exception to this, in blueberry skin cells, the pigments are not
vacuole bound. Thus, if the outer plasma membrane is compromised, pigments are able to freely escape
out to the surrounding liquid by diffusion (Sun et al., 2019). Fruits and other botanical structures often
have a cuticular protective layer on the surface formed by epicuticular wax. This forms a thin, white wax
layer on top of the structure’s outermost surface (Percy, & Baker, 1990). Ethanol is an organic solvent
that has sterilizing properties due to its ability to interact with and denature proteins, killing small
organisms like bacteria. Thus, when in contact with the plasma membrane, ethanol denatures membrane
proteins, causing punctures and disturbances in the structure of the cell membrane (Haimi et al., 2008).
2.2: Hypothesis
Ha: Rise in ethanol concentration in soju increases the permeability of blueberry skin cell membranes,
thus increases the final absorbance of green light in ethanol solution after 5 days.
H0: Rise in ethanol concentration in soju has no effect on the permeability of blueberry skin cell
membranes, thus has no effect on the final absorbance of green light in ethanol solution after 5 days.
, 2
2.3: Variables
Independent Variable: Ethanol concentration in soju was manipulated by changing the ratio of ethanol
and soju in solution. The range of independent variable chosen for experimental conditions was the
following ratios (ethanol:soju), in addition to a control condition conducted with distilled water:
1:9, 3:7, 5:5, 7:3, 9:1.
These ratios translate to the following concentrations of ethanol respectively:
25.37%, 42.01%, 58.55%, 75.09%, 91.63%
This range was chosen to show the change in permeability of blueberry cell membrane at various ethanol
concentration, from low to high. This was done to highlight the effect of ethanol concentration on cell
membrane, specifically. Manipulation of ratios was done rather than direct manipulation of ethanol
concentration to simplify the preparation process of ethanol-soju solutions.
Dependent Variable: Concentration of anthocyanin pigment in solution after 5 days was measured by
absorbance of green light. More light would be absorbed as concentration of pigment in solution increases,
thus absorbance was chosen to measure dependent variable. Green light (565 nm) was selected because
anthocyanin has its peak absorbance at around 520~570 nm wavelength (Qin et al., 2010; Sims & Gamon,
2002). There was no unit of measurement for the dependent variable as “absorbance is a unitless measure
of the amount of light of a particular wavelength that passes through a volume of liquid, relative to the
maximum possible amount of light available at that wavelength” (Vernier, 2011).
Table 1: Controlled Variables, Their Possible Impacts on the Investigation, and Methods of Control
Variable Impact Method of Control
Type of soju Different types of soju may have different The same brand and type of soju was used which
used concentrations of ethanol and other is Chamisul Fresh. (17.2% ethanol)
characteristics which may be a source of
confounding variables.
Size and Higher surface area would mean that there All blueberries were measured to be greater than
condition of would be greater amount of pigment in the 16 mm and less than 17 mm in diameter, and
blueberries blueberry skin. This could affect the final were made sure to have no prior damages to the
amount of pigment present in the solution. skin. Whole blueberries were used to best mimic
the procedure of making infused soju.
Infusion Greater lengths of exposure to ethanol will All trials started at the same time (January 10,
period lead to greater disruption of the membrane. 2019; 3:30 PM, UTC + 9) and ended at the same
time (January 14, 2019; 3:32 PM, UTC + 9).
Quantity of Different quantities of ethanol solution may Each trial involved exactly 20 mL of solution.
solutions result in different amounts (mole) of ethanol
involved in each trial, which may impact
final results.
Cuvette Any damages, such as scratches and Same cuvette was used for all data collections.
staining, on the cuvette may hinder the Between trials, cuvette was cleaned thoroughly
absorbance readings. with water and cotton swabs.
Research Question: What is the effect of exposure to different ratios of ethanol and soju
mixtures (1:9, 3:7, 5:5, 7:3, 9:1) and thus the increase in concentration of ethanol (25.37%,
42.01%, 58.55%, 75.09%, 91.63%) on the permeability of blueberry (Vaccinium
corymbosum) cell membrane measured by absorbance of green light (absorbance)?
1: Introduction
Often times in Korea, soju, which is a traditional Korean liquor, is infused with flavor by putting different
types of fruits inside a sealed container of soju and letting it sit for a couple of months. This allows for the
liquor to be flavored by the fruit. As I began gaining interest in ethanol’s effect on biological systems, I
began to notice that the blueberry infused soju that my parents had prepared during Lunar New Year had
changed from colorless to dark blue. Pigments present inside the fruit could not have passed through the
phospholipid bilayer by simple diffusion or facilitated diffusion in a rate that could noticeably change the
color of the liquor. I wanted to see whether the ethanol in soju compromised the integrity of the berries’
membrane, thus increasing the permeability. I chose to test this using varying concentrations ethanol in
soju and blueberries or Vaccinium corymbosum. Soju, a common Korean liquor, has a varying alcohol
percentage of 16.8% to 53.0% and is made by fermenting rice with yeast. Blueberry contains more than
ten different derivatives of anthocyanin pigment, highly concentrated in its skin cells (Lee et al., 2016).
More significantly, unlike other plants, the pigments in blueberry skin cells are not bound in any specific
vacuoles inside the skin cell, but are rather contained freely inside the cytoplasm. Hence, I came up with
the research question: What is the effect of exposure to different ratios of ethanol and soju mixtures
(1:9, 3:7, 5:5, 7:3, 9:1) and thus the increase in concentration of ethanol (25.37%, 42.01%, 58.55%,
75.09%, 91.63%) on the permeability of blueberry (Vaccinium corymbosum) cell membrane
measured by absorbance of green light (absorbance)?
2: Investigation
2.1: Background
Anthocyanins, found most commonly within the Vaccinium genus, are water soluble pigments that are
responsible for red, purple and blue colors in fruits and vegetables. These pigments are often vacuole
bound and are situated in the fruit’s skin. In exception to this, in blueberry skin cells, the pigments are not
vacuole bound. Thus, if the outer plasma membrane is compromised, pigments are able to freely escape
out to the surrounding liquid by diffusion (Sun et al., 2019). Fruits and other botanical structures often
have a cuticular protective layer on the surface formed by epicuticular wax. This forms a thin, white wax
layer on top of the structure’s outermost surface (Percy, & Baker, 1990). Ethanol is an organic solvent
that has sterilizing properties due to its ability to interact with and denature proteins, killing small
organisms like bacteria. Thus, when in contact with the plasma membrane, ethanol denatures membrane
proteins, causing punctures and disturbances in the structure of the cell membrane (Haimi et al., 2008).
2.2: Hypothesis
Ha: Rise in ethanol concentration in soju increases the permeability of blueberry skin cell membranes,
thus increases the final absorbance of green light in ethanol solution after 5 days.
H0: Rise in ethanol concentration in soju has no effect on the permeability of blueberry skin cell
membranes, thus has no effect on the final absorbance of green light in ethanol solution after 5 days.
, 2
2.3: Variables
Independent Variable: Ethanol concentration in soju was manipulated by changing the ratio of ethanol
and soju in solution. The range of independent variable chosen for experimental conditions was the
following ratios (ethanol:soju), in addition to a control condition conducted with distilled water:
1:9, 3:7, 5:5, 7:3, 9:1.
These ratios translate to the following concentrations of ethanol respectively:
25.37%, 42.01%, 58.55%, 75.09%, 91.63%
This range was chosen to show the change in permeability of blueberry cell membrane at various ethanol
concentration, from low to high. This was done to highlight the effect of ethanol concentration on cell
membrane, specifically. Manipulation of ratios was done rather than direct manipulation of ethanol
concentration to simplify the preparation process of ethanol-soju solutions.
Dependent Variable: Concentration of anthocyanin pigment in solution after 5 days was measured by
absorbance of green light. More light would be absorbed as concentration of pigment in solution increases,
thus absorbance was chosen to measure dependent variable. Green light (565 nm) was selected because
anthocyanin has its peak absorbance at around 520~570 nm wavelength (Qin et al., 2010; Sims & Gamon,
2002). There was no unit of measurement for the dependent variable as “absorbance is a unitless measure
of the amount of light of a particular wavelength that passes through a volume of liquid, relative to the
maximum possible amount of light available at that wavelength” (Vernier, 2011).
Table 1: Controlled Variables, Their Possible Impacts on the Investigation, and Methods of Control
Variable Impact Method of Control
Type of soju Different types of soju may have different The same brand and type of soju was used which
used concentrations of ethanol and other is Chamisul Fresh. (17.2% ethanol)
characteristics which may be a source of
confounding variables.
Size and Higher surface area would mean that there All blueberries were measured to be greater than
condition of would be greater amount of pigment in the 16 mm and less than 17 mm in diameter, and
blueberries blueberry skin. This could affect the final were made sure to have no prior damages to the
amount of pigment present in the solution. skin. Whole blueberries were used to best mimic
the procedure of making infused soju.
Infusion Greater lengths of exposure to ethanol will All trials started at the same time (January 10,
period lead to greater disruption of the membrane. 2019; 3:30 PM, UTC + 9) and ended at the same
time (January 14, 2019; 3:32 PM, UTC + 9).
Quantity of Different quantities of ethanol solution may Each trial involved exactly 20 mL of solution.
solutions result in different amounts (mole) of ethanol
involved in each trial, which may impact
final results.
Cuvette Any damages, such as scratches and Same cuvette was used for all data collections.
staining, on the cuvette may hinder the Between trials, cuvette was cleaned thoroughly
absorbance readings. with water and cotton swabs.
