THE EFFECTS OF SUCRALOSE AND SUCROSE ON STAPHYLOCOCCUS AUREUS
Layla Fijany and Sahar Meshkat
Department of Biological Sciences
Saddleback College
Mission Viejo, CA, 92692
The objective of this study is was to analyze and determine the effect of the sucrose and
sucralose on the growth inhibition of the bacteria, Staphylococcus aureus. Three trials were
conducted with nutrient agar plates of bacteria and different concentrations (5%, 10%,
20%, 30%, 40%, 50%, 80%, 90% and 100%) of the two types of sugars were prepared and
placed for an incubation period of 18-48 hours at 37ºC. No zones of inhibition were visible
on any of the plates and there was no significant difference between the types of growth
seen. The experimental results indicate that sucrose and sucralose do not inhibit the growth
of bacteria. A fourth trial was performed with non-nutrient agar mixed with sucrose and
sucralose at varying concentrations (25%, 50%, 75% and 100%) into separate Petri dishes
with Staphylococcus, using a streaking technique. No bacterial growth was observed after
an 18-hour incubation period at 37ºC. From these results, it can be concluded that a
sucrose or sucralose environment does not enable Staphylococcus aureus to grow, but that
the nutrient agar provided enough nutrients that the bacteria could withstand the high
concentrations of sucrose and sucralose. These results might also suggest that
Staphylococcus aureus is able to metabolize sucralose and sucrose, in an environment of low
water activity (high osmotic pressure), in which they are still able to reproduce.
Introduction
Bacteria are classified mostly as unicellular organisms that are able to multiply rapidly
under favorable conditions when variables such as temperature, oxygen content, pH, osmotic
pressure and nutrients available are ideal in their environment (Chirife et al., 1983). Bacteria,
like all other forms of life, require water for growth, and their water requirements are best
defined in term of water activity of the substrate (Chirife et al., 1983). When the aqueous
solutions in the environment of the microorganism are concentrated by the addition of a solute
such as sugar (sucrose), this creates a decrease in the water activity level, which causes
microorganisms to inhibit growth (Chirife et al., 1983).
Bacterial growth may either help or hinder human life. Therefore, it is important to
understand the environment in which growth may be controlled or inhibited. Food-borne
staphylococcal poisoning, caused by the ingestion of one or more preformed toxins in food
contaminated with Staphylococcus aureus, is one of the most prevalent causes of gastroenteritis
worldwide (Cole et al., 2002; Anas et al., 2008; Jamshidi et al., 2008). Knowing the precise
boundary for the growth/no growth interface of S. aureus and determining the conditions needed
for bacterial growth inhibition is necessary for food safety risk assessment (Jamshidi et al.,
2008).
Sucrose is very effective at inhibiting bacterial growth, as it is routinely used as a natural
food preservative and wound remedy. Sugar's success as an antimicrobial agent in wound
remedy and food preservation lies primarily in its ability to deprive bacteria of water that is
essential for growth. The explanation of the role of sugar in the treatment of infected wounds is
complex and perhaps impossible to reduce to a single mechanism, such as its antibacterial action.
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The Chirife et al. (1983) study has proposed that a function of sugar is to create an environment
of low water activity (meaning a high osmotic pressure), which inhibits bacterial growth. The
study concluded that sugar may might have played a role in the control of infection by
diminishing bacterial virulence in patients with lowered defense mechanisms (Chirife et al.,
1983).
The global population is presenting rises in overweight, obesity, diabetes, hypertension,
cardiovascular diseases, hyperlipidemia and hypercholesterolemia, a situation that has created
concern about changes in lifestyle and balanced diet (Azoubel et al., 2009; Astrup et al., 2011).
As a result, there is acceleration in the use of light or diet products, and in the consequent
consumption of sweeteners. Given that the use of sucralose, one of the newest sweeteners has
been gradually increasing, new studies are necessary to attest its affects on bacterial cells and the
human body. Sucralose is poorly absorbed in the intestinal tract, and is almost entirely excreted
in unaltered forms through the feces (Azoubel et al., 2009). Recent studies have proven an
association between ingestion of sweeteners and nephrotoxicity, hepatoxicity, or retardation of
placental and fetal development (Azoubel et al., 2009).
Microbial growth models are typically developed when the objective is to understand the
responses of microorganisms when part of the range of conditions studied permits growth to
occur (Cole et al., 2002). Such models can describe the increase in numbers with time, the
conditions allowing growth or no growth to occur, or the chance of growth. In the past several
years, there has been a growing interest in the effects of artificial sweeteners, but the effects of
whether sucralose exhibits similar growth inhibition as sucrose on Staphylococcus aureus,
however, are still unclear.
Therefore, it can be concluded that if sucralose exhibits similar properties on bacterial cells
as sucrose, then this type of artificial sweetener may be effective at treating wounds and acting as
a defense at such sites against invading bacteria and other foreign materials. A better
understanding of the effects of sucralose on Staphylococcus may contribute to the development
of treating solutions dealing with topical medications. This current study represents a further
attempt to better understand the growth inhibition of sucralose and sucrose on Staphylococcus
aureus by focusing on varying concentrations of sweetener solutions in agar plates. We will
measure the diameter of the zones of inhibition on the agar to further determine if there is a
similarity in the antimicrobial properties of sucralose and sucrose.
Materials and Methods
The aim of this in vitro study was to determine the efficacy of artificial sugar, sucralose,
and granulated cane sugar, sucrose, against Staphylococcus aureus. All sugars used were a
commercially available pure granulated white cane sugar (table sugar) and a Ralph’s brand
artificial sweetener, made of sucralose. All sugar stock solutions were made with deionized
water.
Three trials were performed in this study to examine zones of growth inhibition of
Staphylococcus aureus in a nutrient agar in response to sucrose and sucralose solutions. All
measurements were conducted at Saddleback College in Mission Viejo, CA from 2 November to
10 November 2011. Trial one consisted of 5 five agar plates of 5%, 10% and 20% solutions of
sucrose and sucralose (separately) made with DI water, for a total of 30 experimental plates and
an additional 10 control plates made with DI water (no sugar). Trial two tested 30%, 40% and
50% solutions of sucrose and sucralose, as well as a water control group. Trial three tested 80%,
90% and 100% solutions of sucrose and sucralose, with a water water as the control group. Each
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trial consisted of the same number of nutrient agar plates and were conducted using the same
procedure. The nutrient agar solution was prepared with 23 g of agar and 1000 mL of DI water
and was heated to boil until clear. The dry mass of the sucrose and sucralose was first measured,
then placed in a 10 mL-graduated cylinder and was filled up to 10 mL with DI water. Percent
solutions and agar (liquid) were placed in the autoclave to be sterilized. Forty 40 Petri dishes
were filled with agar and once hardened, 0.3 mL of Staphylococcus aureus was transferred onto
each plate using aseptic technique. Three chads were placed on each agar plate, with 5 µL of
corresponding solution on each chad, that were placed equidistance from each other in a triangle
formation. After all the agar plates were completely prepared, they were placed in an incubator
set at 37 ˚C. Trial 1 one was incubated for 45 hours; trial 2 two and 3 three were incubated for 18
hours. After the results of trial 1 one were obtained, the principal investigators decided to
decrease incubation to see if the zones of inhibition were present in earlier bacterial growth and
in order to conduct more trials within a given time period. After the incubation period, zones of
inhibition were measured (or lack thereof).
Due to the lack of growth inhibition, a fourth trial was conducted using 3 three nonnutrient agar plates of 25%, 50%, 75% and 100% solutions of sucrose and sucralose (separately)
for a total of 24 experimental plates. The agar solution was prepared by mixing 2.3 g of nonnutrient agar, 100 mL of DI water and sucrose or sucralose, of 25 g, 50 g, 75 g and 100 g. The
agar solution was boiled until clear and then placed into the autoclave for sterilization.
Staphylococcus aureus was added to the plates using a streaking technique, in order to isolate the
bacterial colonies. Once the agar plates were prepared, they were incubated for 18 hours at 37 ºC.
After 18 hours of incubation, isolated colonies were counted and measured to determine the
average size of the colonies present.
Results
There were no zones of inhibition present. Therefore, the mean diameter of the growth
inhibition was 0.0 cm. This result was observed for trials 1 one, 2 two and 3 three, which
included 5%, 10%, 20%, 30%, 40%, 50%, 80%, 90%, 100% sucrose and sucralose solutions and
for the water control as well. After the fourth trial was complete, no bacterial growth was visible
on the non-nutrient agar plates (mixed with the corresponding amount of sugar). As a result, no
isolated bacterial colonies were present.
Discussion
The first three trials resulted in no zones of growth inhibition present on any of the
nutrient agar plates with the sucrose or sucralose solutions when tested. Staphylococcus aureus
displayed the same growth in water and in the two types of sugar solutions. This result may
suggest a multitude of conclusions: that Staphylococcus aureus is able to withstand environments
of low water activity, or high osmotic pressure, and still be able to replicate; or that the nutrient
present in the agar powder provided a sufficient enough environment that the Staphylococcus
was able to survive in. To test whether the latter hold true, we conducted the fourth trial with a
non-nutrient agar to remove the variable of a nutrient enriched environment. The result of the
fourth trial suggested that Staphylococcus aureus is not able to survive in the sugar and DI water
agar alone, thus supporting that the nutrients in the agar led to the successful growth of the
bacteria and the lack of zones of inhibition.
Sugar possesses inherent microbial properties, some of which are due to high osmotic
pressure and low water activity, which is inhibitory to the growth of the majority of bacteria
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(Naama, 2009). Water activity is a measure of the consequential effect of the average
intermolecular forces between water molecules, being increased when water molecules become
oriented on the surface of the solute molecules (Cooper et al., 1999). When numerous molecules
are tied up in this way, the water molecules are on average less free to act, e.g. to hydrate
something, so the ‘activity’ is lower (Cooper et al., 1999). When applied topically to wounds,
osmosis would be expected to draw water from the wound into the sugar, helping to dry the
infected tissue and reduce bacterial growth (Dealey et al., 2011). Even when diluted with water,
sugar would be likely to retain a water activity sufficiently low enough to inhibit most bacteria.
The ability of topical disaccharides, in the form of sugar, to cleanse infected wounds and
facilitate healing has been recognized for many years (Efkors et al., 1999; Naama, 2009; Dealey
et al., 2011). Surgeons have applied sugar dressings to contaminated wounds on animals. There
is also support that sugar therapy has been used on infected surgical wounding, pressure ulcers,
deep tissue infections and for other skin defects that need a healthy granulated bed (Efkors et al.,
1999; Naama, 2009). In some developing countries, where modern advanced wound products are
unavailable or unaffordable, granulated sugar as a contact dressing for open wounds is seen as
the product of choice – its ability to reduce surface contamination and promote granulation is
well recognized (Dealey et al., 2011). A study done by Naama (2009) found that Staphylococcus
aureus displayed inhibition of 20 mm and 11 mm at 100% and 75%, respectively, using honey
solutions in a disc diffusion test. Also, according to this study, no effect was observed at 25% or
50% (Naama, 2009). The high antimicrobial effect of the honey sample in Naama’s 2009 study
may be attributable to the presence of glucose oxidase, which is activated by dilutions in water
resulting in the production of hydrogen peroxide, which is toxic to bacteria.
The expected results were not obtained because there were no zones of inhibition for any
of the concentrations of sucrose or sucralose. These results suggest that the nutrient agar supplied
an adequate environment for Staphylococcus, or that this strain of bacteria has a metabolic
pathway that utilizes sucrose and sucralose at high concentrations. Although our intentions of
discovering antimicrobial properties of sucralose and sucrose were not achieved, we believe
future studies may further determine new treatments pertaining to Staphylococcus inhibition.
Future studies regarding the effects of sucralose and sucrose against a variety of bacteria may
present new breakthroughs in clinical treatments of infected wounds. The next step of this study
would be to focus on the metabolic pathway of Staphylococcus aureus and how this
microorganism processes the uptake of nutrients in its environment and how this affects survival
and reproduction of the bacteria.
Literature Cited
Anas, M., Eddine, H., Mebrouk, K. 2008. Antimicrobial Activity of Lactobacillus Species
Isolated from Algerian Raw Goat’s Milk Against Staphylococcus aureus. World Journal of Dairy
& Food Sciences. 3(2): 39-49
Astrup, A., Flint, A., Holst, J., Moller, A., Moller, B., Raben, A., Vasilaras, T. 2011. Increased
Postprandial Glycaemia, Insulinemia, and Lipidemia after 10 weeks’ Sucrose-rich Diet
Compared to an Artificially Sweetened Diet: a Randomized Controlled Trial. Food and Nutrition
Research. 55: 5961.
Azoubel, R., Rodero, A., Rodero, L. 2009. Toxicity of Sucralose in Humans: A Review.
International Journal of Morphology. 27(1): 239-244.
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Chirife, J., Herszage, L., Joseph, A., Kohn, E. 1983. In Vitro Study of Bacterial Growth
Inhibition in Concentrated Sugar Solutions: Microbiological Basis for the Use of Sugar in
Treating Infected Wounds. Antimicrobial Agents and Chemotherapy. 23(5): 766-773.
Cole, M., Legan, D., Schaffnew, D., Slade, L., Stewart, C., Vendeven, M. 2002. Staphylococcus
aureus Growth Boundaries: Moving towards Mechanistic Predictive Models Based on SoluteSpecific Effects. Applied and Environmental Microbiology. 68(4): 1864-1871.
Cooper, R., Harding, K., Molan, P. Antibacterial activity of honey against strains of
Staphylococcus aureus from infected wounds. Journal of The Royal Society of Medicine. 1999.
92: 283-285.
Dealey, C., Murandu, M., Simms, M.H, Webber, M.A. 2011. Use of Granulated Sugar Therapy
in the Management of Sloughy or Necrotic Wounds: a Pilot Study. Journal of Wound Care.
20(5): 206-216.
Efkors, T., Kossi, J., Laato, M., Niinikoski, J., Peltonen, J. 1999. Effects of Hexose Sugars:
Glucose, Fructose, Galactose and Mannose on Wound Healing in the Rat. European Surgical
Research. 31:74-82.
Jamshidi, A., Kazerani, H., Seifi, H., Moghaddas, E. 2008. Growth Limits of Staphylococcus
aureus as a Function of Temperature, Acetic Acid, NaCl Concentration, and Inoculum Level.
Iranian Journal of Veterinary Research. 9(4): 353-359.
Naama, R. 2009. Evaluation of Inhibitory Effect of Honey on Some Bacterial Isolates. Iraqi
Journal of Medical Sciences. 7(4): 67-72.
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Review Form
Department of Biological Sciences
Saddleback College, Mission Viejo, CA 92692
Author (s): Layla Fijany and Sahar Meshkat
Title: The Effects of Sucralose and Sucrose on Staphylococcus Aureus
Summary
Summarize the paper succinctly and dispassionately. Do not criticize here, just show that you
understood the paper.
This paper was a study to determine the effect of sucrose and sucralose on the inhibition
growth of the bacteria Staphylococcus Aureus. The experimenters made several Petri dishes with
different concentrations of the sucralose and sucrose and bacteria and incubated the Petri dishes
over a certain amount of time to examine the inhibition. Since sucralose is one of the newest
light sweeteners, experimenting with its ability to inhibit bacterial growth is important because if
sucralose can inhibit the growth of bacteria in certain cells, it can be beneficial in treating certain
diseases. Their experiment didn’t have any zones of inhibition present.
General Comments
Generally explain the paper’s strengths and weaknesses and whether they are serious, or important to our current
state of knowledge.
This paper was well written and generally easy to follow. There were only a couple parts
such as, discussing the different concentrations that I was mildly confused on. There were some
parts in the introduction and discussion that seemed a little bit jumpy and out of place. In the
introduction, there was a paragraph that started out talking about obesity and ended with
discussing retardation of fetal growth development. They are both relevant to the paper, there
just needs to be a better transition between the two.
The in-text citation was accurate throughout the entire paper and I didn’t find any
spelling errors as well as minimal grammatical errors. The strengths definitely outweighed the
weaknesses but overall they didn’t take away from the state of knowledge.
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Technical Criticism
Review technical issues, organization and clarity. Provide a table of typographical errors, grammatical errors, and
minor textual problems. It's not the reviewer's job to copy Edit the paper, mark the manuscript.
This paper was a final version
This paper was a rough draft
The comments containing grammar are in red throughout the paper and the clarity issues
are outlined in the notes attached to the paper.
Recommendation
 This paper should be published as is
 This paper should be published with revision
 This paper should not be published
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