*2.2. Effects of Sweeteners on the Gut Microbiota in Animal Models*

A summary of the "animal" studies analyzed is given in Table 4. Mainly murine species have been studied and the work focuses primarily on the number of total anaerobic and aerobic bacteria, bacterial diversity, the Bacteroidetes/Firmicutes ratio, fecal transplantation, and the effects of maternal intake of sweeteners on offspring in adulthood. In most studies, sweeteners were administered to the animals as part of the drinking water at different concentrations using the ADI for saccharin and sucralose as a reference (Table 4).

One of the first studies on saccharin and the intestinal microbiota was conducted in 1980 by Anderson and Kirkland in rats. They compared the total anaerobic and aerobic microbial populations of the cecum and the proportion of both in male rats fed 0 or 7.5% saccharin sodium, in Purina laboratory chow, for 10 days. After this period, the authors observed that the highest doses of saccharin in cecal content showed an increase in anaerobes and maintenance of aerobes, implying a downward shift in the anaerobic/aerobic ratio [33]. However, Serrano et al., showed that short-term saccharin supplementation with an equivalent dose to the highest acceptable level (JECFA) is insufficient to alter gut microbiota in apparently healthy mice [46].

Conversely, Falcon et al., found that chronic feeding of a commercial non-nutritive sweetened yogurt (0.3% sodium saccharin and sodium cyclamate, Zero-Cal, SP, Brazil) did not induce differences in the bacterial diversity of adult male Wistar rats, compared to animals fed a standard low-fat yogurt supplemented with 20% sucrose [47].




ADI: Acceptable daily intake; MS: maternal sucralose; d: day; wk: weeks, mo: months; HFD: high-fat diet; LS: low saccharin; HS: high saccharin.↔: unmodified;↑: increase;↓: decrease.

In addition, the study by Abou-Donie et al., (2008) found adverse effects of sucralose on the gut microbiota. Splenda was administered to male Sprague-Dawley rats by oral gavage at 100, 300, 500, or 1000 mg/kg for 12 week, to evaluate the concentration of sucralose administered to these experimental animals. In the current review, an estimation was carried out taking into account the sucralose consumption of an adult rat drinking between 30 and 50 mL of the substance prepared in the study by Abou-Donia et al., according to the concentrations shown above and compared with the ADI (EFSA, JECFA), observing that all the values used exceeded admissible limits for humans. These data show that the consumption of sucralose produces an imbalance in the gut microbiota, specifically in the total numbers of anaerobic and aerobic bacteria that are reduced, with a significant decrease in beneficial anaerobic bacteria such as *Bifidobacteria*, *Lactobacilli*, and *Bacteroides*. In this study, equivalent levels of sucralose (Splenda®) in a single drink sweetened with sucralose per day were used [32]. Likewise, another study by Uebanson et al., using different doses of sucralose, found alterations in the microbiota, specifically suggesting that sucralose intake affected in a dose-dependent manner the relative amount of *Clostridium* cluster XIVa [48].

Sánchez-Tapia et al., studied whether the type of sweetener and the presence of a high-fat diet differentially could regulate the gut microbiota. Sucralose was dissolved in water to a concentration of 1.5%. Sucralose increased the Firmicutes abundance showing a decreasing trend in Bacteroidetes, with lower alpha diversity [49]. In this respect, Wang et al., in 2018 performed an 8 week sucralose treatment in mice; they found no changes in alpha diversity, Actinobacteria, and Proteobacteria, but they did find an increase in the abundance of the Firmicutes group [39].

Recently, Zhang et al., in their study with different low doses of sucralose in obese rats, found that ~0.43 mg (0.11 mg/kg translated to human) sucralose increased the relative abundance of Firmicutes but decreased the relative abundance of Bacteroidetes, and that ~0.62 mg sucralose (0.16 mg/kg translated to human) decreased the relative abundance of Firmicutes but increased that of Bacteroidetes. Therefore, the dose of sucralose consumed influenced the Bacteroidetes/Firmicutes ratio. There were no changes in alpha diversity. The authors concluded that the two lower doses of sucralose used in the study might alter the compositions of fecal microbiota [50]. However, in this study, the authors did not use a normal weight control animal model to evaluate the extent to which the establishment of obesity in these rats could modify the results.

Li et al., in 2021, evaluated the bacterial composition at different taxonomic levels in guinea pigs that for 28 days had received saccharin in their drinking water (5 mM). The abundance of Firmicutes tended to decrease in the saccharin-consuming group compared to the control group, while the abundance of Bacteroidetes increased. Therefore, the Bacteroidetes/Firmicutes ratio was affected. In addition, at the family level, the relative abundances of Muribaculaceae and Lactobacillaceae increased in the saccharin group and at the genus level, the relative abundance of *Lactobacillus* increased, while at the family level, the relative abundance of Erysipelotrichaceae and Eubacteriaceae decreased as well as *Ileibacterium* at the genus level [51].

Bian et al., conducted studies in male C57BL/6 J mice with sucralose and saccharin at concentrations equivalent to the ADI for humans (FDA). In 2017, concentrations of sucralose of 0.1 mg/mL [52] and concentrations of saccharin of 0.3 mg/mL administered to male mice [53], in a long-term study for 6 months, were found to induce gut microbiome perturbations, exemplified by the alteration of inflammation-related bacterial pathways and metabolites [52,53].

In 2014, Suez et al., had already demonstrated that the administration of saccharin, sucralose, and aspartame to mice can modulate gut microbiota composition and function, which leads to a higher risk of glucose intolerance, and this is associated with an increase in *Bacteroides* spp. and Clostridiales when performing fecal transplants in germ-free mice from the animals treated with commercial sweeteners. The sweeteners were dissolved in mouse drinking water to obtain a 10% solution: Sucrazit (5% saccharin, 95% glucose), Sucralite (5% Sucralose), Sweet'n Low Gold (4% Aspartame). As controls, 10% glucose and 10% sucrose solutions were used [34].

In relation to the possible effect of sweeteners on the offspring, Dai et al., in 2020 investigated the effects of maternal sucralose (MS) intake on the offspring susceptibility to suffer from hepatic steatosis in adulthood. C57BL/6 pregnant mice were randomized into an MS group (MS during gestation and lactation) and a maternal control (MC) group (MC diet). MS group mice were given sucralose solution of 0.1 mg/mL, approximately 5–15 mg/kg BW/day, and equal to the upper limit of the FDA ADI. After weaning, all offspring were fed a control diet until 8 weeks of age, and then treated with a high-fat diet (HFD) for 4 weeks. The maternal intake of sucralose was found to inhibit intestinal development, induce intestinal dysbiosis, and decrease the production of butyrate-producing bacteria and butyrate in offspring through downregulation of G-protein-coupled receptor 43 (GPR43), and to exacerbate HFD-induced hepatic steatosis in adulthood. Likewise, at the phylum level, an increase in the relative abundance of Verrucomicrobia and Proteobacteria and a reduction in Bacteroidetes was observed in animals with MS. However, at the genus level, MS increased the abundance of *Akkermansia*, *Blautia*, *Corynebacterium*, and *Robinsoniella*, while, *Alistipes*, *Barnesiella*, *Paraprevotella*, *Saccharibacteria*\_genera\_inc\_ertaesedis, and *Streptococcus* were reduced, with a decrease in alpha diversity [54].

However, we would like to emphasize that after reviewing the studies included in this review, not only the dilution of the sweetener in the drinking water should be considered, but also the adjustment to the amount of water ingested by the animals, because the consumption can vary among different species and strains. For example, the average dose/day of liquid drunk by one mouse can range from 3.9 ± 0.2 mL/mouse to 8.2 ± 0.3 mL [36]. There are also physiological and metabolic differences between rodents and humans [55], and, depending on the type of study and the duration of treatment, inferring the results of investigations using rodent models to those in humans may lead to misleading scientific interpretations. In addition, the metabolism of the sweeteners reviewed in this study can be different between animals and humans, and also among different types of animal species. In fact, in relation to sucralose, there is variability within the types of animals used. However, regarding sucralose (organochlorine), when administered orally, similar results have been found among all species evaluated, showing very low absorption levels and light metabolism. For saccharin, being a water-soluble acid with a pKa of 1.8, absorption is increased in those animal species with lower stomach pH, such as rabbits and humans, compared to those with higher stomach pH, including rats [24]. Thus, studies in animal models are a proxy to studying the potential human effects but human evidence should be gathered at the widest possible extent that the ethics premises in biomedicine and clinical trials may allow.

The animal studies reviewed, except that by Serrano et al. [46], show that saccharin and sucralose produce time- and dose-dependent changes in the gut microbiota. Some studies highlight the modification of the amount of anaerobic and aerobic microbiota, while others emphasize the effect of sucralose on the Bacteroidetes/Firmicutes ratio and others are focused on how maternal consumption can affect the offspring.

However, the mechanisms that mediate the physiological effects of low- or noncalorie sweeteners remain unclear and are most likely diverse. According to the literature, sucralose and saccharin, since they are not absorbed, can influence the maintenance of the pH of the bolus in its trajectory through the intestine, which implies a change in the microenvironmental conditions. Thus, this outcome could be a factor influencing the selective proliferation of certain bacterial groups. In addition, the presence in greater or lesser quantity of cells expressing the T1R2/T1R3 taste heterodimer would be related to the inflammatory effect and possible adaptations of the microbiota [45].
