Comparative Evaluation of the Antimicrobial Effects of Xylitol, Aspartame, and Stevia and the Antibiofilm Activity of Stevia against Escherichia coli: An in vitro Study

Background

Artificial sweeteners are widely used as non-cariogenic alternatives to dietary sugars, and beyond their sweetening properties, certain sweeteners have demonstrated antimicrobial activity, suggesting potential pharmaceutical applications. Thus, they are extensively incorporated into pharmaceutical formulations and oral healthcare products as functional excipients. Escherichia coli, a common Gram-negative bacterium capable of forming resilient biofilms, contributes to persistent infections and increased antimicrobial resistance. Although emerging evidence indicates that artificial sweeteners may influence bacterial growth and biofilm formation, their comparative antimicrobial efficacy and antibiofilm potential remain insufficiently characterised.

Aim

To evaluate the in vitro antimicrobial activity of xylitol, aspartame, and stevia using the agar well diffusion assay and to further investigate the antimicrobial and antibiofilm activities of stevia against Escherichia coli using MIC, Crystal Violet (CV), MTT, and CFU assays.

Materials and Methods

Three sweetener-based formulations, xylitol (5 g/100 mL), stevia (0.2 g/100 mL), and aspartame (0.2 g/100 mL), were prepared in saline, stored at 4ºC, and used within seven days. All formulations were initially screened for antimicrobial activity using the agar well diffusion assay. Based on the screening results, the stevia formulation was further evaluated against Escherichia coli using Minimum Inhibitory Concentration (MIC), Crystal Violet (CV), 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and Colony-Forming Unit (CFU) assays. Data were expressed as mean±SD and analysed using one-way ANOVA.

Results

Stevia demonstrated significant antimicrobial activity, producing the largest Zones of Inhibition (ZOI) against Escherichia coli [32 mm] and Staphylococcus aureus [28 mm], moderate inhibition of Klebsiella pneumoniae [11 mm], and no activity against Streptococcus mutans. Xylitol and aspartame showed no detectable antimicrobial effects. Stevia exhibited concentration-dependent inhibition of E. coli, with a Minimum Inhibitory Concentration (MIC) of 175 µg/mL. Biofilm formation was reduced to approximately 10% at three times the minimum inhibitory concentration, while metabolic activity decreased by approximately 90%. Colony-Forming Unit (CFU) analysis confirmed complete bactericidal activity at the highest tested concentration. Data were analysed using IBM SPSS Statistics version 21.0. Results were expressed as mean±SD. One-way ANOVA was used to compare groups, with p<0.05 considered statistically significant.

Conclusion

Among the tested sweeteners, stevia exhibited potent and selective antimicrobial and antibiofilm activity. These findings highlight its potential as a functional sweetener with intrinsic antimicrobial properties and support its possible application as a bioactive ingredient in pharmaceutical and oral healthcare formulations. Further investigations are warranted to validate its therapeutic potential and facilitate the development of natural antimicrobial products.