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The Effect of Sucrose and Sucralose on the Growth of Gut Microbiota

Gabrielle Bonifacio gabrielle.bonifacio@ocvts.org
Marine Academy of Technology and Environmental Science, NJ

Introduction:
The human gut microbiome is composed of trillions of bacteria, viruses, fungi, and other
microorganisms. This influences the body by regulating food digestion, the central nervous
system, the immune system, and various other processes. There are up to 1,000 species of
bacteria living in the gastrointestinal tract (Robertson, 2017). Most are probiotic bacteria and are
vital for one’s well-being, although some are pathogenic bacteria and may cause illness. The
balance of these bacteria is essential for a healthy gut. For example, probiotic bacteria multiply
frequently so pathogenic bacteria do not have an area to grow. Yet when too many unhealthy
bacteria are in the gut, it can result in the disease dysbiosis, a microbial imbalance in the gut that
can lead to bloating, diarrhea, and constipation. Normal or disturbed populations of bacteria have
been connected to diseases such as asthma, cancer, celiac disease, diabetes, eczema, heart
disease, multiple sclerosis, and more (Sethi, 2018).
Diet impacts the composition of the gut microbiome. A diet made up of different food
types can lead to a diverse microbiota, which is deemed healthy since more species of bacteria
increase the number of health benefits they can provide. Fermented foods, prebiotic foods, and
foods rich in probiotics and fiber promote healthy gut bacteria (Robertson, 2016). However,
foods that use artificial sweeteners can do the opposite of that and cause harmful conditions like
dysbiosis (West, 2017).
According to the 2015-2020 Dietary Guidelines for Americans, the average American
intakes 71.14 grams of added sugar daily (U.S. Department of Health and Human Services &
U.S. Department of Agriculture, 2015). The American Heart Association recommends that men
should consume a maximum of 36 grams per day, while women should intake no more than 25
grams daily (American Heart Association, 2021).
Sucralose is a synthetic artificial sweetener made from C12H22O11 (sucrose) but is sweeter
and essentially calorie-free. The sucralose molecule appears to be identical to the sucrose
molecule on the surface. However, sucralose differs from sucrose in that three of the
oxygen-hydrogen groups on the sucrose molecule are replaced by chlorine atoms. Research
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conducted by Corder and Knobbe found that sucralose is a powerful inhibitor for the gut bacteria
Escherichia coli, but also that E. coli can gradually adapt to sucralose inhibition (2018).
Table sugar, scientifically named sucrose, is a naturally occurring sugar made up of
glucose and fructose. When digesting sucrose, its fructose and glucose units are freed. A study
on mice found that large doses of fructose and glucose prevent the production of proteins that
nurture the growth of bacteria often seen in those who are lean and healthy (Townsend et al.,
2019).
This research was directed at identifying the effect of different amounts of sucrose and
sucralose on the growth of human gut bacteria. E. coli is a bacteria that will model bacteria in the
human gut microbiome, and its growth will be recorded after being exposed to the sugar and
artificial sweetener. A majority of E. coli are safe and an integral bacteria in a healthy intestinal
tract and help to digest food, while some E. coli are pathogenic and can cause illnesses like
diarrhea or diseases outside of the intestinal tract (Escherichia coli (E. coli), 2021). It was
hypothesized that by analyzing the mean gray value of concentrations of E. coli, the variations in
the growth of E. coli that would be found were caused by sucrose and sucralose, causing sucrose
to decrease the amount of E. coli. Furthermore, since the human body absorbs 15% of consumed
sucralose in the digestive tract, the bacteria that absorbs the sucralose would kill itself off
(especially more so when consumed in large amounts) and the sucralose would decrease the
amount of E. coli (Food Insight, 2018). This study's results may help individuals to better
regulate their sucrose and sucralose intake after becoming more informed of their effects on gut
bacteria.

Methodology:
Bacteria Quantity and Growth
1. E. coli was grown with sucrose and sucralose five times in two different amounts:
a. There were five trials with two grams of sucrose added to the E. coli growth
medium, five trials with five grams of sucrose, five trials with two grams of
sucralose, and five trials with five grams of sucralose.
b. Two grams was used to mimic the effects of the average recommended intake of
added sugar.
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c. Five grams was used to mimic the effects of the average intake of added sugar for
an American adult.
2. A plate of E. coli was grown once with no additives to act as the control.
3. E. coli was grown in Coliscan™ Easygel Media.
4. Bacteria was grown in petri dishes for 24 hours in an incubator set at 37ºC.
Preparing Additives
1. The appropriate amount of grams of sucrose or sucralose was added to a beaker on a
balance, making sure that the mass of the beaker and additive is two or five more grams
than the mass of the beaker.
2. The sucrose or sucralose was poured into a Coliscan™ Easygel bottle using a funnel
(Figure 1).
3. The funnel was removed and swirled the bottle to mix its contents well.
Plating E. coli with Additives and Incubation

1. The incubator was set to 37°C and was allowed to cycle empty for at least 20 minutes to
reach a thermal equilibrium.
2. The bottom of the petri dish was labeled appropriately according to the type of additive
and amount of grams with a marker.
a. For example, if two grams of sucrose were added to the E. coli growth medium,
the plate would be labeled “Sucrose 2.”
3. The contents of the bottle were poured onto the smaller side of the Easygel pretreated
petri dish, ensuring that the medium covered the entire surface of the bottom and that
there were minimal bubbles.
4. The lid was put back on the plate and was given about five hours for the medium to fully
solidify in order to successfully apply the E. coli:
a. For the control, nothing was added to the Coliscan™ Easygel bottle and it needed
only 30 minutes to solidify.
5. After the medium fully solidified, the lid was removed and a cotton swab was used to
absorb the E. coli sample liquid. The bacteria was applied to the plate by making streaks
in a Z-pattern over the medium with the swab, making sure to turn the swab as it is
moved across the plate (Figure 2).
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6. The lid was put back on the plate and the plate was placed upside down in the incubator
for 24 hours.
7. After 24 hours, the plate was retrieved from the incubator and the lid was removed to
take a picture of the plate against a white background (Figure 3).
8. Steps 1-7 were repeated for a total of five trials for each additive and amount.
Quantifying the Data
1. ImageJ, an open-source image processing software for multidimensional scientific image
data, was used to convert an image of one of the plates of E. coli tested from RGB color
to 8-bit grayscale.
2. The polygon selection tool was used to outline the concentrated streak of E. coli (Figure
4).
3. The mean gray value that was given for the selected area of the image was recorded:
a. The mean gray value is the sum of the gray values of all the pixels in the selection
divided by the number of pixels. The pixel value in a grayscale image is a number
that reflects the pixel's brightness. This number is stored as an 8-bit integer with a
range of 0 to 255 potential values. 0 is considered black and 255 is considered
white, with shades of gray comprising the values in between.
4. Steps 1-3 were repeated for the five trials of each additive and amount.

Statistical Analysis
A one-way ANOVA test applied with Tukey’s Honest Significant Difference (HSD) test
was performed to determine if there was a significant difference between the concentration of E.
coli for the control and each additive and amount, as well as between the additives and amounts
themselves (α = 0.05).
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Figure 2: The black arrow

Figure 1: Two grams of shows the Z-pattern that was
sucralose were poured into a used to apply the E. coli
funnel placed on a Coliscan™ using a cotton swab for each
Easygel bottle. plate.

Figure 3: (from left to right, top to bottom) Images of the control plate, a plate with 2 g of
sucralose, a plate with 5 g of sucralose, a plate with 2 g of sucrose, and a plate with 5 g of
sucrose against a white piece of paper after 24 hours of incubation.
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Figure 4: (from left to right, top to bottom) Images of the control plate, a plate with 2 g of
sucralose, a plate with 5 g of sucralose, a plate with 2 g of sucrose, and a plate with 5 g of
sucrose after being converted to 8-bit grayscale and the streak E. coli on each plate was outlined
using the polygon selection tool. The mean gray values ranged from 107.248 to 161.341 and a
significant difference was calculated.

Results:
Among all 20 trials, the mean gray values ranged from 107.248 to 161.341, while the
mean gray value of the control was 101.146 (Table 1; Figure 5). A one-way ANOVA test
applied with Tukey’s HSD test calculated that the control versus the five trials of plates with two
grams of sucralose added was significantly different. This was the same result when the control
was paired with the plates with five grams of sucralose, two grams of sucrose, and five grams of
sucrose. From the Tukey’s HSD test, it was also found that when the four different types of
plates were paired with each other, they were all significantly different, except for the plates with
two grams of sucralose versus five grams of sucralose and two grams of sucrose versus five
grams of sucrose.
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Table 1: The mean gray values of the concentration of E. coli of each trial for each additive and
amount, where the possible values range from 0 to 255. 0 is black and 255 is white (N=20).
Mean Gray Value
Plate
Trial #1 Trial #2 Trial #3 Trial #4 Trial #5 Average

Sucralose - 2 g 144.158 159.819 141.412 136.268 137.920 143.915

Sucralose - 5 g 161.341 137.303 142.795 152.555 154.214 149.642

Sucrose - 2 g 124.103 107.248 114.825 123.358 122.808 118.468

Sucrose - 5 g 124.043 125.769 131.747 133.868 125.585 128.202

Control 101.146

Figure 5: The averages of the mean gray values of the five trials conducted
for each additive and amount added to the E. coli growth medium with ± 5%
error bars, as well as the mean gray value of the control (N=20, p<0.05).

Discussion:
Both additives and amounts were statistically different when paired with the control. The
plates with a higher mean gray value had a greater effect on reducing E. coli. The mean gray
value of the concentrations of E. coli on the plates of each sweetener and amount was higher than
the concentration of E. coli grown with no additive. It can also be gathered that sucralose
decreased bacteria growth more efficiently than sucrose for both amounts. This indicates that
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adding sucrose and sucralose to the bacteria’s growth medium decreased the amount of E. coli
that would have normally grown. This correlation in terms of sucralose can be accounted for due
to E. coli’s inability to metabolize sucralose, thus inhibiting E. coli from utilizing nutrients in the
medium to maintain growth. Bacteria available in the gastrointestinal tract for energy
homeostasis and metabolism are then reduced, leading to weight gain, obesity, and diabetes, all
of which are health risks artificial sweeteners were meant to prevent (Swift, 2020). However,
unlike sucralose, sucrose is metabolized by the body. The similarity in molecular formulas may
account for the decrease in sucrose, but an exact reasoning behind this decrease was not
determined. Further studies on the impact of sucrose on the growth of gut bacteria must be
conducted to come to a strong evidence-based conclusion.

Conclusion:
My hypothesis was correct. Adding sucralose to the growth medium of E. coli decreased
the number of bacteria that would have normally grown due to E. coli’s inability to metabolize
sucralose, thus hindering E. coli from using nutrients in the medium to support growth. Adding
sucrose to E. coli’s growth medium also impeded E. coli growth, but not as effectively as
sucralose. Ultimately, it is best for people to consume sucrose over sucralose, although both
should be consumed in moderation. This research shows that sucrose and sucralose inhibit the
growth of E. coli, although additional research is needed to verify the reasoning behind these
deductions. The results acquired from the research are relevant because it can help bring
attention to the importance of taking care of the gut microbiome and improve people’s health all
around, since gut health is connected to many bodily cycles. Overall, this research can help
inform the public of the importance of monitoring one’s diet and sweetener intake in regards to
one’s gut microbiome.

Acknowledgments:
I would like to acknowledge my teachers for supplying me with my materials for this
project, giving me guidance on culturing E. coli, answering all my questions, and giving helpful
feedback, as well as the upperclassmen who took time to review my project and provide
suggestions and/or comments. I would also like to thank Adrian Perono for providing emotional
support throughout this entire project.
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