*2.1. Effects of Sweeteners on the Gut Microbiota: In Vitro Trials*

In vitro models can be used to study the potential effects of sweeteners, specifically saccharin and sucralose, in humans. Data obtained from in vitro studies can serve as hypothesis generators and as indicators of possible interactions between these sweeteners and the gut microbiota.

In vitro studies focus on the changes in the main microbial groups and selected species together with their metabolites, analyzing the diversity, richness, and abundance in the community over time. The in vitro studies included in this review (Table 3) have commonly addressed the interactions between bacteria, intestinal epithelium, and simulated transit.




#### **Table 3.** *Cont.*

ADI: acceptable daily intake; SCFA: short-chain fatty acid. ↔: unmodified; ↑: increase; ↓: decrease.

In 2018, Harpaz et al., evaluated the relative toxicity for the bacteria of artificial sweeteners, approved by the FDA and in a range of concentrations based on acceptable daily intake (ADI). Genetically modified bacteria (*E. coli*) showing luminescence after exposure to certain stresses were used. Both the induced luminescent signals and bacterial growth were measured. The dose-dependent toxicity effect on *E. coli* in vitro was demonstrated [38]. In addition, Wang et al., (2018) evaluated the bacteriostatic effect of sucralose and saccharin on the growth of *E. coli* in liquid and solid media, finding that the ability to selectively inhibit the growth of enteric bacterial species may be due to inhibition of metabolic enzymes or alterations in nutrient transport [39,44,45].

According to Markus et al., using concentration ranges of non-calorie sweeteners, with comparable concentrations within FDA-approved acceptable daily intake (ADI), aspartame, sucralose, and saccharin are not bactericidal but may affect the bacterial communication system via a molecular system termed quorum sensing (QS)-inhibition and by extension may also affect the host metabolism. According to these authors, this outcome may be due to the significant inhibitory actions of these sweeteners on the Gram-negative bacteria N-acyl homoserine lactone-based (AHL) communication system. However, there is a need to continue to elucidate the mechanisms of action involved in the effects of these sweeteners and other related products on gut microbiota [40].

Gerasimidis et al., in 2020 investigated the effect of artificial sweeteners on the gut microbiome and fiber fermentation capacity. To conduct their study, they fermented fecal samples from 13 healthy volunteers in cultures with sweeteners (aspartame, sucralose, stevia-based sweetener). They measured short-chain fatty acid (SCFA) production by gas chromatography and characterized the composition of the microbiome with 16S rRNA sequencing and quantitative polymerase chain reaction (qPCR). Among their results they found that compared to the control, sucralose (*p* = 0.025) significantly increased valeric

acid production and induced significant changes in microbiome community structure (β-diversity); using the Bray–Curtis dissimilarity index, it also increased the relative abundance of *Escherichia/Shigella* species as well as *Bilophila* [41].

However, Shil et al., conducted a study using gut microbiota and epithelial models on the role of commonly consumed sweeteners in the pathogenicity of gut bacteria. The effect of non-calorie sweeteners on *E. coli* and *E. faecalis* growth in planktonic culture was measured in vitro after exposure for 4 days to varying concentrations of non-calorie sweeteners (saccharin, sucralose, and aspartame). All these sweeteners increased the ability of model gut bacteria to adhere to and invade intestinal epithelial cells except for saccharin, which had no significant effect on *E. coli* invasion. Furthermore, a negative effect of these artificial sweeteners has been shown on intestinal epithelial cell apoptosis and permeability, thus further increasing the opportunity for bacteria to traverse the gut epithelium and cause septicemia [42].

Some authors (Vamanu et al., 2019), with the aim of establishing the effect of sweeteners on the microbiota pattern of healthy individuals, used a static in vitro system to simulate the transit through the three segments of the human colon. Under these conditions, both the fermentative response and microbial diversity were found to be altered after treatment with in vitro sweeteners, specifically sucralose and saccharin (equivalent to 9 g of sugar), also showing that non-nutritional sweeteners can induce toxicity, expressed by the establishment of dysbiosis [43].

All the reviewed in vitro studies allow us to hypothesize that in one way or another the consumption of artificial sweeteners can affect the bacteria present in the gut microbiota. We must be careful when interpreting the results and consider different aspects, such as the fact that the in vitro conditions may not correspond to the in vivo conditions of the organism. In addition, the different methodologies used in these studies may make it difficult to interpret the results.
