Abstract

This study aims to examine the effects of artificial and natural sweeteners on gut bacteria, modeled by Escherichia coli. In reality, the human gut microbiome is made up of many types of bacteria, responsible for digesting and extracting energy from consumed foods and maintaining human health. E. coli is found in the human intestine, but it does not have a major role in metabolism; the internal chemical reactions necessary to support life. Sweeteners are classified into two groups: natural and artificial, where artificial sweeteners cannot be digested by the human body. This study hypothesises that if sweeteners, both natural and artificial, have an effect on gut bacteria, model E. coli strains will not develop when exposed to agar with artificial sweeteners. Sweeteners including aspartame, cyclamate, dextrose, saccharin, sucralose, lactose, maltose, and sucrose were used to test the hypothesis. Nutrient agar served as a control plate. After four days, only the control petri dishes and petri dishes made with natural sweeteners had developed bacteria colonies. Thus it was found that artificial sweeteners inhibit the growth of E. coli colonies while natural sweeteners allow development to proceed, implying that artificial sweeteners have a negative impact on human gut bacteria.

Introduction

Sweeteners are classified into two groups; natural and artificial. natural, or natural sweeteners are disaccharides; two molecules of simple sugars linked by a glycosidic bond. On average, natural sweeteners contain around 2.6 to 4 calories per gram^[1]. Artificial sweeteners contain little to no calories and have a much higher relative sweetness than natural sweeteners. As the American diet grows sweeter, artificial sweeteners are incorporated in many foods to lower the number of calories consumed on a daily basis, whereas natural sweeteners are less commonly found. While artificial sweeteners are advertised as the better, healthier option, not all artificial sweeteners have been approved universally. Countries vary in the type of artificial sweeteners allowed for consumption, which is subject to change as new research is carried out. Artificial sweeteners are also popular because they can prevent a surplus or lack of glucose in diabetics’ diets^[1].

Israeli researchers carried out a study on the adverse side effects of artificial and natural sweeteners. The researchers studied two groups of mice; one group received artificial sweeteners while the other group received natural sugars, both through feeding the mice sugar solutions. The artificial sweeteners used included aspartame, sucralose and saccharin. Glucose and sucrose were used as natural sweeteners. The glucose levels of the mice were measured after eleven weeks of feeding. The mice consuming artificial sweeteners had abnormally high glucose levels in their blood, indicating a poor absorption of glucose into the tissue. This would lead to high glucose levels and associated health complications such as diabetes^[2].

The gut microbiome is an ecosystem of commensal and pathogenic microbes existing in the human digestive tract that plays an essential role in human health. The gut microbiome has one of the largest populations of microorganisms in the human body, where over 90% of the bacteria are classified as bacteroidetes and firmicutes.^[3] Firmicutes are commonly known as low GC% Gram-positive bacteria, referring to the lesser amount of guanine and cytosine bases in their DNA compared to that of adenine and thymine, and include clostridia, mollicutes, and bacilli^[4]. Bacteroidetes, commonly known as bacteroids, are Gram-negative bacteria which can only survive in low partial pressures of oxygen. The difference between Gram-negative and Gram-positive bacteria lie in their surface charges and outer membrane structure. Gram-positive bacteria are positively-charged bacteria with thin, easily permeable membranes, while gram-negative bacteria are negatively charged and have an extra periplasmic layer, making them less permeable. Chi et al. studied the effect of neotame, an artificial sweetener, on the gut microbiome of mice. The study separated eight week old mice into two groups; one group was given neotame-enriched water and the other was given unadulterated water for four weeks. The enriched water contained a concentration of neotame that was 2.5 times the daily intake recommended for human consumption. The researchers found that the development of gut bacteria was disrupted, especially that of bacteroidetes and firmicutes within the gut. The number of bacteroidetes increased and the number of firmicutes decreased in the microbiome of the mice given neotame compared to those given unadulterated water, specifically a decrease in lachnospiraceae and ruminococcaceae populations. Following the exposure to neotame, the natural alpha-diversity of the gut was disturbed, resulting in a skewed ratio of bacteroidetes to firmicutes. Firmicutes serve as short-chain fatty acid (SCFA) producers and plant degraders; they break down and ferment carbohydrates to create and absorb energy – the loss of these microbes can lead to reduced ability of the gut microbiome to harvest and break down energy, leading to weight gain and damage to host metabolism^[5].

The most commonly studied gut bacteria is Escherichia coli^[6]. E. coli is found in the human intestine, but does not play a major role in metabolism. However, E. coli is used to model gut bacteria and is described as the “guinea pig” of bacterial research due to its extensive use^[7]. Wang et al. examined the effect of artificial sweeteners on the gut microbiome of mice. The research was carried out using different strains of E. coli to model the gut microbiome. In the experiment, the researchers used LB broth plates and measured the number of E. coli colonies before and after the introduction of artificial sweeteners: sucralose, saccharin, acesulfame potassium and rebaudioside A or a mixture of sodium chloride or sucrose. The study found that the artificial sweeteners consistently decreased the number of E. coli colonies, though to varying degrees depending on the strain, demonstrating that artificial sweeteners have negative effects on all gut bacteria but not all species are affected equally^[8].

There are many factors that influence the gut microbiome. To begin with, flora can vary greatly between individuals, due to variations in genetics, lifestyle, weight, and age. Diet can also impact the microbiome by promoting environments that are suited to specific types of microbes, thus changing the bacterial makeup. A key limitation to longitudinal microbiome studies is that the types of microbes present in the subjects’ gut microbiomes may have changed over time, influencing the results presented^[9].

Drawing from the literature, the hypothesis of this research is based on the observed effect of both natural and artificial sweeteners on the proportions and types of bacteria present in the gut microbiome. Thus, this experiment is designed to expose E. coli bacteria to different sugars, with the hypothesis that the bacteria will not develop colonies in the agar containing artificial sweeteners.

Method

The E. coli K12 bacteria mixture was made according to package instructions^[10] and incubated for 24 hours. To create the agar base, 350 mL of deionised water was poured into three 500 mL Erlenmeyer flasks and 5.25 g of nutrient agar added into each flask. The flasks were then placed in an autoclave for 25 minutes at 121℃to sterilize the equipment^[11]. Three petri dishes were labeled for each of the eight sweeteners used, as well as the controls. C&H brand sucrose, Flinn brand lactose, maltose, and dextrose, NectaSweet brand saccharin, Acros Organics brand cyclamate, and Bulk Supplements brand sucralose and aspartame were used as sweeteners. All materials were moved to the hood and 3 control petri dishes were filled with nutrient agar solution.

For the experimental groups, 50mL of nutrient agar was poured into eight Erlenmeyer flasks. 5.25g of each sweetener was mixed into the corresponding Erlenmeyer flask. Each flask was microwaved for 45 seconds and the solutions poured into petri dishes to solidify.

To dilute the solution, 2.77mL LB broth and 30μL E. coli culture were extracted into a sterile tube, then inverted to disperse the E. coli. To colonize the petri dishes, 10 μL of the diluted E. coli solution was spread evenly onto each Petri dish using Drigalski spatulas. The colonized petri dishes were placed in an incubator set to 37℃. After 24 hours, the petri dishes were checked for visible colonies and photographed on a light box with graph paper.

The frequency of colonies was calculated on each petri dish. All petri dishes and materials were aseptically treated with a 10% bleach solution and soaked for 24 hours.

Results

After four days, none of the petri dishes containing artificial sweeteners had any observable colonies on the agar (Table 1 and Figure 4). The petri dishes containing natural sweeteners and the control petri dishes had nearly identical numbers of colonies present on the agar. As displayed in Figure 2 and 3, the petri dishes containing natural sweeteners and the control petri dishes had similar rates of E. coli colony growth while the petri dishes made with artificial sweeteners had no growth. To compare the rate of growth between plates, the percentage of agar covered by E. coli colonies was calculated for each day of observation and the difference between daily concentrations calculated. When the frequency of E. coli colonies on control and natural sweetener petri dishes are compared, their rate of growth and frequency of colonies is about equal (see Figure 2). In both the experimental and control group, there were no anomalies in colony frequency and a minimal deviation. The growth of E. coli colonies was inhibited by the presence of artificial sweeteners in the agar and growth was neither inhibited nor increased by the presence of natural sweeteners in agar.

Table 1: Average E. coli Colony Frequency

    The above table shows the average frequency and standard deviation of E. coli colonies observed on each petri dish.

Figure 1: Frequency of E. coli Colonies on Agar with Various Sweeteners

The above graph shows the average of how much of each petri dish was covered by E. coli growth on the fourth and final day of observation. The control and natural sweetener petri dishes reached similar levels of average frequency with minimal deviation between petri dishes; as shown by the error bars, this was consistent across samples. The artificial sweetener petri dishes had no growth on any of the petri dishes.

Table 2: Rate of E. coli Colony Growth

The above table contains the average frequencies of E. coli colonies from the three observed petri dishes for each type of sweetener and the control. The average for each of the four days is displayed.

Figure 2: Rate of E. coli Colony Growth with Natural Sweeteners

The above graph shows the rate of E. coli growth by comparing the frequency of E. coli colonies grown on natural sweetener and control petri dishes. The rate for all samples was consistent, with the most rapid rate observed from lactose sugar growth.

Figure 3: Comparison of E. coli Growth on Control versus Natural Sweetener

The above photographs compare bacterial growth between the control plate (on the left) and agar made with the natural sweetener sucrose (on the right) at the end of two weeks of observation. Qualitative observations made by comparing the photographs above support the claim of equal bacterial growth between agar made with natural sweetener and LB agar.

Figure 4: Comparison of E. coli Growth on Control versus Artificial Sweetener

The above photographs compare bacterial growth between the control plate (on the left) and agar made with the artificial sweetener cyclamate (on the right) following two weeks of observation. Qualitative observations made by comparing the above photographs show the absence of bacterial growth on agar made with artificial sweeteners.

Discussion

The purpose of the experiment was to find the effect of artificial and natural sweeteners on gut bacteria. E. coli was grown on petri dishes infused with sweeteners and observed for four days. Based on background research, it was hypothesized that if both natural and artificial sweeteners have an effect on gut bacteria, then E. coli colonies will only develop on agar with natural sweeteners. Dextrose, maltose, lactose, and sucrose were used as the natural sweeteners. The artificial sweeteners were aspartame, cyclamate, saccharin, and sucralose.

The collected data supported the hypothesis that none of the petri dishes with artificial sugar had any E. coli growth, as displayed in Figure 1. The petri dishes with natural sweeteners (dextrose, maltose, lactose, and sucrose) all developed E. coli colonies. As Figure 2 demonstrates, all petri dishes made with natural sweeteners had a nearly identical rate of growth. In the presence of artificial sweeteners, E. coli growth was inhibited. According to Table 2, there was no growth on any petri dishes made with artificial sweeteners. Since the artificial sweeteners are not metabolized, this prevented E. coli from using the nutrients in the agar to sustain growth. Thus, the bacteria available in the digestive tract for metabolism and energy homeostasis is lower, resulting in weight gain, obesity, and diabetes; consequences which artificial sweeteners were designed to prevent. Artificial sweeteners are rapidly becoming more common in foods marketed as low-calorie due to their non-caloric nature. Further knowledge regarding the risks of artificial sweeteners in the human diet is essential for ensuring healthy consumption in at-risk populations.

By working in a hood and using an autoclave to sterilize materials, the petri dishes did not become contaminated throughout the experiment, leading to unconfounded results. However, while carrying out the experiment, some sources of error may have originated from a high concentration of E. coli, disruptions while documenting bacterial growth, and reliance on visual counting of colonies. To improve future experiments, the E. coli mixture should be serially diluted prior to streak plating. By using a lower concentration, more separate colonies would form, allowing for more accurate observations of differences between petri dishes. Using the quadrant streaking method instead of simply spreading the E. coli across the petri dishes would also help show how E. coli growth differentiates across the petri dishes. As the experiment was carried out over a limited period of time during the academic year, bacterial growth could not be observed over the weekend and was only recorded once a day. In addition to observing the bacteria for a longer duration to allow for more detailed data, future experimentation would be improved by recording growth on an hourly basis to calculate a more reliable mean. Additionally, relying on visual observation to quantify bacterial growth could lead to inaccuracies in bacterial counts. In future experiments, bacteria should be grown in liquid media and its optical density measured using a spectrophotometer, which is proportional to the rate of bacterial growth, to more accurately record the rate of growth.

Conclusion

To conclude, E. coli is used to model bacteria in the gut microbiome of humans^[6]. By examining E. coli growth in the presence or absence of sweeteners, scientists can examine the effect food items have on human gut health. Running the experiment with other types of bacteria found in the gut microbiome would help determine whether the negative effects of artificial sweeteners can be extrapolated to different species. This research shows that artificial sweeteners inhibit E. coli growth, but further study is necessary to confirm the validity of these conclusions. Currently, the health effects of artificial sweeteners are unclear; scientific quantification of their interaction with the gut microbiome is essential in understanding the consequences of diet, as well as in preventing related diseases such as obesity and diabetes.

Acknowledgements:

Dr. Koltermann and Ms. Beck of Notre Dame High School were essential to this experiment with their assistance in planning and carrying out the experiment. An additional thanks and acknowledgement to the hood, E. coli , and all sweeteners used in the experiment.

References

1. Newton, David E. \”Synthetic Foods.\” In The New Chemistry . N.p.: Facts on File, 2015.
2. Shell, Ellen Rupell. \”Artificial Sweeteners May Change Our Gut Bacteria in Dangerous Ways.\” Scientific American , April 2015, 32-34. Accessed December 22, 2019. https;//doi.org/10.1038/scientificamerican0415-32.
3. Maczulak, Anne. \”Bacteria.\” In Science Encyclopedia . N.p.: Facts on File, 2015. http://online.infobase.com/Auth/Index?aid=17163&itemid=WE40&articleId=285176.
4. “Low G C Gram Positive Bacteria.” Low G C Gram Positive Bacteria | Department of Microbiology. Accessed April 2, 2020. https://micro.cornell.edu/research/epulopiscium/low-g-and-c-gram-positive-bacteria/.
5. Chi, Liang, Xiaoming Bian, Bei Gao, Pengcheng Tu, Yunjia Lai, Hongyu Ru, and Kun Lu. \”Effects of the Artificial Sweetener Neotame on the Gut Microbiome and Fecal Metabolites in Mice.\” Molecules , February 2018, 1-11. https://0-web-b-ebscohost-com.catalog.sjlibrary.org/ehost/command/detail?vid=1 3&sid=899de462-d4e5-4ffb-ad9a-f5ccbfd4f7d3%40sessionmgr102&bdata=JkF1d GhUeXBlPWNvb2tpZSxpcCx1cmwsY3BpZCZjdXN0aWQ9czU4NTU1Nzgmc2 l0ZT1laG9zdC1saXZl#AN=128281454&db=a9h.
6. Dobrijevic, Dragana, Anne-Laure Abraham, Alexandre Jamet, Emmanuelle Maguin, and Maarten Van de Gutche. \”Functional Comparison of Bacteria from the Human Gut and Closely Related Non-Gut Bacteria Reveals the Importance of Conjugation and a Paucity of Motility and Chemotaxis Functions in the Gut Environment.\” Public Library of Science , July 14, 2016, 1-24. https://0-web-a-ebscohost-com.catalog.sjlibrary.org/ehost/detail/detail?vid=0&sid=cbe73 238-e786-4f08-a216-59e22b627200%40sdc-v-sessmgr02&bdata=JkF1dGhUeXBlPWNv b2tpZSxpcCx1cmwsY3BpZCZjdXN0aWQ9czU4NTU1Nzgmc2l0ZT1laG9zdC1saXZl# AN=116815124&db=a9h.
7. Maczulak, Anne. \”Escherichia Coli.\” In Science Encyclopedia . N.p.: Facts on File, 2015. http://online.infobase.com/Auth/Index?aid=17163&itemid=WE40&articleId=285183.
8. Wang, Qiao-Ping, Duncan Browman, Herber Herzog, and Gregory G. Neely. \”Non-nutritive Sweeteners Possess a Bacteriostatic Effect and Alter Gut Microbiota in Mice.\” Public Library of Science , July 2018, 1-13. https://0-web-b-ebscohost-com.catalog.sjlibrary.org/ehost/command/detail?vid=11&sid= 899de462-d4e5-4ffb-ad9a-f5ccbfd4f7d3%40sessionmgr102&bdata=JkF1dGhUeXBlPWNvb2tpZSxpcCx1cmwsY3BpZCZjdXN0aWQ9czU4NTU1Nzgmc2l0ZT1laG9zdC1saXZ l#AN=130506285&db=a9h.
9. Yong, Ed. \”Why Are Your Gut Microbes Different From Mine?\” The Atlantic , April 28, 2016. Accessed December 22, 2019. https://www.theatlantic.com/science/archive/2016/04/why-are-your-gut-microbes-differe nt-from-mine/480207/.
10. \”Escherichia Coli, K-12 Strain, Living, Nutrient Broth, Tube.\” Carolina Biological Supply. Accessed July 07, 2020. https://www.carolina.com/bacteria/escherichia-coli-living-k-12-strain-nutrient-broth-tube/155068.pr.
11. Autoclave Overview, October 31, 2016. https://blink.ucsd.edu/safety/research-lab/biosafety/autoclave/index.html.

About the Author

Beginning in middle school, Jocelyn worked in biology and biotechnology research. Now in high school, Jocelyn’s experiments range from microbiology to mycology. Her experience includes group research as well as solo research projects. She is passionate about research and driven to contribute to the body of scientific knowledge. In addition, Jocelyn teaches community science classes.