Non-caloric artificial sweeteners are among the most commonly used food additives in the world, consumed by both healthy and obese individuals. People like the "no calorie" or "no guilt" aspect that comes along with this zero calorie sweetener when choosing between regular and diet soda or any other sweetened treat. What people do not realize is that these artificial sweeteners can actually lead to obesity indirectly by affecting the plethora of microbiota in the human gut and affecting glucose intolerance (Suez). While people are possibly choosing the no calorie option because they believe it is healthier and want to lose weight, they do not realize that they could actually be causing more damage and weight gain than they would just consuming regular sugar. Diabetes and obesity are huge problems, especially in America. About nine percent of the American population suffers from diabetes and about one third of the population is considered to be obese, so as much research as possible should be conducted in order to prevent this trend from growing (Fryar).

It has been previously proven in both mice and humans that changes in the gut microbiota after consuming high, yet FDA acceptable, amounts of sweetener leads to a greater likelihood of developing diabetes (Suez). Instead of testing on living subjects, this research uses an in-vitro approach in testing the direct effects of a realistic amount of sweetener on the microbiota. Using a standardized in-vitro digestion method, the sweeteners are digested and then exposed to three types of bacteria found in the microbiome, Escherichia coli, Staphylococcus Epidermidis, and Enterobacter Aerogenes (Minekus). The growth curves of these bacteria cultures are plotted using absorption spectroscopy, and the different curves are compared to a control curve.

In addition to NAS, caffeine is often consumed along with these additives. There have been previous studies showing the positive effects of caffeine on the human gut microbiome, but more research is needed to fully understand the effects. If NAS inhibits gut microbiome growth and caffeine promotes it, the two food additives could possibly cancel out the effects of each other, which is also a question being examined in this research.

This experiment consists of twenty-seven experimental groups, nine for each type of bacteria. The first group is a control group of bacteria growing in the digest medium, but no type of additive is present. The second group is the bacteria growing in a culture of digested sucrose, or normal sugar. The third, fourth, and fifth groups are bacteria cultures growing in saccharin, sucralose, and aspartame respectively. The sixth and seventh groups are the bacteria growing in two different concentrations of caffeine. The eighth group is the bacteria growing in a combination of aspartame and caffeine. The ninth group is the combination of Sucralose and caffeine. Each trial is done for each type of bacteria and repeated three times.

Aspartame increases bacteria growth while saccharin and Sucralose decrease it. Caffeine increases growth. When combined, aspartame and caffeine significantly increase growth and Sucralose and caffeine balance out bacteria growth towards the control.