*2.3. Effects of Sweeteners on the Gut Microbiota in Human Trials*

Non-caloric sweeteners (sucralose and saccharin), as food additives, have been evaluated and approved for use in humans by the European Food Safety Authority and

subsequently authorized by the European Commission, the Parliament, and the Council of the European Union. Currently, their consumption, as we have already mentioned, is very widespread in the population, especially in hypocaloric foods and diets as an adjuvant for weight loss or in diabetic patients. The fact that their industrial use in a great variety of products has increased favors the non-adverted consumption.

The human studies reviewed, described in Table 5, studied microbial diversity and metabolites, specifically changes in SCFAs, the main metabolites produced by the microbiota in the large intestine [56]. The SCFAs are bacterial metabolites produced during the colonic fermentation of undigested carbohydrates, such as dietary fiber and prebiotics, and can mediate the interaction between the diet, the microbiota, and the host [57]. SCFA levels are influenced by the proportion of intestinal bacteria, whose alteration (dysbiosis) can lead to an unbalanced composition of the gut SCFAs and therefore it has been concluded that supplementation with pure saccharin did not alter microbial diversity or composition [58].

**Table 5.** Summary of the analyzed in vivo studies. Humans.


ADI: Acceptable daily intake; SCFA: short-chain fatty acid; d: day; wk: weeks. ↔: unmodified

The following are the results of human studies, with a sweetener concentration not exceeding the ADI and short-term intake. Among the intervention studies carried out with saccharin, Serrano et al., performed a double-blind, placebo-controlled, parallelarm study to explore the effects of pure saccharin compound on gut microbiota and glucose tolerance in healthy men and women (46 subjects completed the study; IMC ≤ 25). Participants were randomized into four treatment groups (placebo, saccharin, lactisole, or saccharin with lactisole) and consumed capsules containing pulp filler/placebo (1000 mg/d) sodium saccharin (400 mg/d), lactisole (670 mg/d), or sodium saccharin (400 mg/d) + lactisole (670 mg/d) twice daily for 2 weeks. The authors concluded that in these conditions, microbial diversity or composition at any taxonomic level were not changed by pure saccharin supplementation in humans. According to these results, short-term saccharin consumption at maximum acceptable levels (JECFA) is not sufficient to alter the gut microbiota or induce glucose intolerance in supposedly healthy humans [46]. However, Suez et al., did find some modifications in the gut microbiota in 4 of 7 healthy volunteers (5 men and 2 women, aged 28–36 years) from an ongoing clinical nutritional study who were selected as non-habitual sweetener consumers. A saccharin intervention was conducted for one week in which they consumed, on days 2 to 7, the FDA maximum acceptable daily intake (ADI) of commercial saccharin, in three daily doses (equivalent to 120 mg). Changes in the microbiota of only 4 participants, who had developed significantly worse glycemic responses in the study, were observed, and they suggest that humans exhibit a personalized response to non-caloric artificial sweeteners, possibly derived from differences in their basal microbiota [34].

In relation to sucralose, Thomson et al., (2019) conducted a randomized, double-blind study in 34 healthy men (18–50 years) with BMI 20–30 kg/m2. Sixteen subjects were administered for one week a dose of 780 mg of sucralose per day that was divided into three-260 mg intakes; the control group received a placebo (*n* = 17). In this study, at the phylum level, the gut microbiome was not modified in healthy individuals [60].

Similar results were obtained in a randomized, double-blind, crossover, controlled clinical trial involving the follow-up of 17 healthy participants. They performed a crossover design for 12 weeks (two 14 day treatment periods separated by a 4 week washout period). In weeks 5 and 6, the volunteers consumed aspartame (*n* = 9) or sucralose (*n* = 8). Prior to the washout period, in which no artificial sweeteners were consumed in weeks 11 and 12, all participants consumed the sweetener that they had not previously consumed. The participants were administered 14% (0.425 g) of the ADI for aspartame and 20% of the ADI for sucralose (0.136 g) (approximately 10.5 packets of sucralose with beverages). To define the ADI, they used Health Canada data (sucralose as 9 mg/kg body weight and 40 mg/kg/bw for aspartame). The relative abundance of the five most abundant genuslevel taxa within the four most dominant phyla (Actinobacteria, Bacteroidetes, Firmicutes, and Verrucomicrobia) before and after treatment were analyzed at the following days: 1, 28, 42, and 84. Alpha diversity estimation was performed with the Shannon index on the raw operational taxonomic unit. No changes were found for aspartame and sucralose in the gut microbiota composition or SCFAs after 14 days of a daily intake in healthy participants [59].

In relation to long-term studies with saccharin and sucralose in humans, there are not any studies to our knowledge. In the study conducted by Suez et al., in 2014 on the relation between artificial sweetener consumption and gut microbiota, the effect of long-term consumption of non-caloric artificial sweeteners was evaluated. To this end, a validated food frequency questionnaire comprising data collected from 381 non-diabetic individuals from an ongoing clinical nutritional study was used. The results show that artificial sweetener consumption increases the risk of glucose intolerance, these adverse metabolic effects being mediated by modulation of the composition, metabolic function, and the basal microbiota. In this regard, Aldrete-Velasco et al., pointed out in a review that under this design, eliminating completely the confounding variables was not possible, so changes in the microbiota and their metabolic characteristics could also be different due to other factors beyond the consumption of non-caloric sweeteners [61].

Considering the results mentioned above and according to other authors, by using high doses of saccharin and sucralose both in in vitro studies and in animal models, gut microbiota can be modified, whereas in human studies performed using amounts below the ADI and in short-term studies, no effects on gut microbiota are found [2,10,16,47–49]. Contrary to this outcome, Schiffman et al., in 2019 stated in an editorial regarding in vivo animal models, involving data on low- and non-caloric sweeteners and gut microbiota, that sucralose can unequivocally and irrefutably alter the gut microbiome at those levels approved by regulatory agencies, associated with human use. These authors also highlight that it is not appropriate to draw generalized conclusions about effects on the gut microbiota [62].

According to several studies, the explanation for these results may be due to the different doses used in in vitro and in animal model studies versus in human studies, where the doses are lower than the ADI [16,48]. In addition, in human clinical studies, the sample sizes are small, as well as the duration of the interventions. In addition, there is a relevant point to bear in mind like the failure in considering the knowledge regarding the basal gut microbiota of volunteers.

#### **3. Conclusions**

In conclusion, it is necessary to broaden the concept of food safety for sucralose and saccharin by re-evaluating toxicity referring to the effect on the gut microbiota and the possible consequences on health maintenance and disease amelioration in humans. Indeed, the mechanisms by which low-calorie and non-calorie sweeteners may alter the gut microbiota remain unclear, and it is not possible to conclude at present whether their effect is direct on the microbiota or mediated by the metabolic situation of the host, for which there are still no conclusive studies. In fact, the scientific literature in both health

and disease sometimes refers to beneficial strains and other studies focus on pathogenic strains, which may be due to the lack of clarity regarding what defines dysbiosis or eubiosis. In order to obtain sufficient evidence in these types of studies, clinical trials should be conducted bearing in mind an adequate number of subjects, as well as considering their baseline gut microbiota, dietary habits, and lifestyles. Although the preferred population is healthy adults due to its easy accessibility, more studies must be conducted taking vulnerable population groups into account, such as children, the elderly, pregnant women, lactating women, or subjects with intestinal pathologies, obesity, diabetes, cardiovascular diseases, etc. and chronic and/or excessive consumers of low- and non-calorie sweeteners.

**Author Contributions:** Conceptualization, S.d.P., S.G.-M. and L.E.D.; data curation, S.d.P., S.G.-M. and L.E.D.; methodology, S.d.P., S.G.-M. and L.E.D.; writing—original draft, S.d.P., S.G.-M. and L.E.D.; writing—review and editing, S.d.P., S.G.-M., L.E.D., E.N., R.U. and A.M. All authors will be informed about each step of manuscript processing including submission, revision, etc. via emails from our system or assigned Assistant Editor. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.
