**2. Materials and Methods**

A descriptive review was conducted to investigate whether there are potential effects of saccharin and sucralose consumption on gut microbiota composition.

The PubMed, Scopus, Google Scholar, ScienceDirect, and Scielo databases were used for the search. The terms entered in this search were as follows: sweeteners, non-calorie sweeteners, sucralose, splenda, saccharin, sugar-win, sweet'n low, microbiota, gut microbiota, human, animal model, mice, rat, and in vitro studies.

Using the term "sweeteners", for the last 5 years, 1573 clinical trials, meta-analyses, and randomized controlled trials, together with 2984 reviews and systematic reviews, were found. When narrowing the search also including the term "microbiota", we found 41 clinical trials, meta-analyses, and randomized controlled trials, plus 144 reviews and systematic reviews.

The following exclusion criteria were used: studies that focused on microbiota other than the gut microbiota, studies that did not include the effect of saccharin and sucralose on the gut microbiota, studies that included supplements and/or prebiotics and/or probiotics that affect the gut microbiota, and studies carried out in populations with diseases.

All these studies were divided into in vitro and in vivo studies, differentiating in the latter between studies in animal models and in humans. Finally, for the present review, 6 in vitro studies were evaluated, plus 14 in vivo studies in animal models and 4 in vivo studies in humans (Figure 1). Of the studies included in this publication, 10 were not present in previous reviews, 2 were studies in humans, 6 were studies in animal models, and 2 were in vitro studies.

The following formula was used to estimate the concentrations of saccharin and sucralose used in the animal studies with respect to the ADI in humans when the work did not indicate this, when it was possible with the published data.

ADI (EFSA/JECFA) (mg/kg/day) × Average animal weight (kg)/Average daily liquid intake (mL) (modified from Suez et al.) [34].

The amount of water consumed by the experimental animals was estimated according to the data indicated by Bachmanov et al. [36,37] and the animal care and use committee of the Johns Hopkins University [37].

**Figure 1.** Flow chart regarding selection method.
