Neotame (-[-(3,3-dimethylbutyl)-l-α aspartyl]--phenylalanine-methyl ester) is one of five FDA-approved artificial sweeteners that are 7000–13,000 times sweeter than sugar [ 16 ]. In human bodies, neotame can be metabolized by esterase into de-esterified neotame and methanol, and eliminated in the urine and feces within 72 h [ 16 17 ]. It has been demonstrated that neotame is well tolerated in many species including Sprague-Dawley CD rats, CD-1 mice, beagle dogs and New Zealand rabbits; similar to other approved artificial sweeteners, neotame is considered as a safe additive to human diets [ 16 ]. However, neotame safety studies found that long-term neotame consumption is associated with low body weight and low body weight gain, although this has long been considered the result of low food consumption [ 18 ]. In summary, the potential adverse effects of neotame on the gut microbiome, which serves as a key regulating factor to host metabolism, remains unclear and should be addressed.

It is known that gut microbiota plays a key regulating role in host metabolism, which is deeply involved in food digestion, energy supplement, and immune system development [ 9 10 ]. Environmental factors-induced dysbiosis of gut microbiome is associated with many human diseases, especially obesity, inflammatory bowel disease (IBD) and type 2 diabetes [ 11 13 ]. In recent years, the influence of artificial sweeteners on gut microbiome have raised concerns as it has been found that many types of artificial sweeteners could perturb the composition of gut bacteria and then affect host health. For example, saccharin could disturb the normal gut microbiota and cause glucose intolerance in rat and human [ 5 ]. Our recent study also showed that saccharin could modulate mouse gut microbiota as well as its metabolic functions and induce liver inflammation in mice [ 14 ]. Likewise, acesulfame-K (Ace-K) consumption could alter the profile of mouse gut bacteria that is associated with the increase of body weight gain [ 15 ].

Artificial sweeteners are important sugar substitutes which are widely used in food and drinks to enhance flavor while avoiding extra energy intake. Some studies indicated artificial sweeteners play a positive role in weight loss, suggesting that it can be employed as a potential dietary tool to assist in weight-loss plan adherence [ 1 4 ]. However, adverse health effects of artificial sweeteners, such as inducing glucose intolerance and causing metabolic syndrome, have been found in recent studies, which indicate that artificial sweeteners have an active metabolic role in the human body and could perturb human metabolism [ 5 8 ]. An epidemiologic study also spotted a positive association between artificial sweetener intake and body weight gain in children [ 6 ].

We further investigated whether neotame consumption could perturb the fecal metabolome of the mouse. As we predicted, along with the perturbed gut microbiota, fecal metabolite profiles were also largely altered, as shown in Figure 4 A,B. Most of the altered metabolites were decreased in neotame-treated mice, such as malic acid, mannose-6-phosphate, 5-aminovaleric acid and glyceric acid ( Figure 5 A). Interestingly, we found most of the identified lipids and fatty acids were significantly decreased in treated mice, including 1,3-dipalmitate, 1-monopalmitin, linoleic acid and stearic acid ( Figure 5 B). Moreover, the concentrations of cholesterol, campesterol and stigmastanol were also increased in the fecal samples of neotame consumption, as shown in Figure 5 C. More altered metabolites can be found in Table S2

The perturbation of gut bacteria composition generally alters the functional gene profile. Therefore, we further investigated whether neotame consumption altered functional pathways in gut microbiome. As shown in Figure 3 A, neotame-treated gut microbiota shows a different pattern of metabolic pathways compared to controls. Specifically, in the neotame-treated microbiome, amino acid metabolism, LPS biosynthesis, antibiotics biosynthesis and folate biosynthesis pathways were enriched. However, for the abundances of pathways, such as fatty acid metabolism, carbohydrate metabolism, lipid metabolism and ABC transporters, they are generally lower than in controls. Besides, we found multiple genes in two classical butyrate fermentation pathways have been significantly reduced ( Figure 3 B). Three genes, which encode 4-hydroxybutyryl-CoA dehydratase, butyryl-CoA dehydrogenase and acetate CoA-transferase, respectively while participating in the succinate fermentation to butyrate, were significantly decreased. For the other pathway of butyrate fermented from pyruvate, although the genes of phosphate butyryltransferase and butyrate kinase were increased, four upstream genes were significantly reduced, including acetyl-CoA C-acetyltransferase, 3-hydroxybutyryl-CoA dehydrogenase, 3-hydroxybutyryl-CoA dehydratase and butyryl-CoA dehydratase and butyryl-CoA dehydrogenase ( Figure 3 B).

We first investigated whether a four-week neotame consumption would affect the gut microbiome of CD-1 mouse. No significant difference of alpha-diversity was observed between two groups before neotame consumption. After the four-week experiment, alpha-diversity of gut microbiome in neotame-consuming group was much lower than the control group, as shown in Figure 1 A. PCoA analysis showed a separation of gut bacteria between control and neotame-consuming animals after the four-week treatment, compared to their clustering distribution before neotame consumption ( Figure 1 B). The results suggest that neotame consumption significantly altered both the alpha- and beta-diversities of gut bacteria of mice. Dysbiosis analysis found that neotame-treated mice had a significantly higher microbial dysbiosis index (MD-index) than controls ( Figure 2 A). Specifically, phylum Bacteroidetes was largely enriched, while Firmicutes was significantly decreased in neotame-treated animals ( Figure 2 B). Before neotame treatment, no such significant taxonomy difference was observed on the phylum level. On genus levels, we found thatand an undefined genus in family S24-7 mainly contributed to the increase of phylum Bacteroidetes, as shown in Figure 2 C. Over 12 genera have been significantly altered in Firmicutes ( Table S1 ). Notably, multiple components of family Lachnospiraceae and family Ruminococcaceae in neotame-treated animals were significantly lower than controls, such asand Figure 2 D,E). More altered genus can be found in Table S1 (Supplementary Materials) . Taken together, the results suggested that the four-week neotame consumption perturbed the diversities as well as the community compositions of gut microbiome in male CD-1 mice.

3. Discussion

Bacteroides and a genus in family S24-7 ( Bacteroides plays important roles in glycan digestion and polysaccharide fermentation [ S24-7 , also called Candidatus Homeothermaceae , is a substantial component of mouse and human gut microbiota, and metagenomics sequencing indicates the genome of S24-7 contains polysaccharide utilizing genes and multiple vitamin synthetic genes [ Bacteroides . The upregulation of some pathways like folate synthesis and LPS biosynthesis should mainly come from the improvements of Bacteroides and S24-7 ( Artificial sweeteners generally cannot be utilized by human bodies. For a long time, they have been considered as safe food additives with a negligible influence on the normal metabolism of human. However, recent studies indicated that some of artificial sweeteners could perturb gut microbiota in mammals and further affect the host health, such as inducing glucose intolerance [ 5 14 ]. The results of our current study clearly demonstrated that a four-week neotame consumption disturbed the mouse gut microbiome. Notably, we found Bacteroidetes was largely enriched, mainly due to the increases of genusand a genus in family Figure 2 B,C). According to previous studies, other artificial sweeteners such as saccharin and Ace-k also could induce a promoted growth of Bacteroides [ 5 15 ]. As one of the most abundant bacteria in mammal gut,plays important roles in glycan digestion and polysaccharide fermentation [ 19 20 ]., also called, is a substantial component of mouse and human gut microbiota, and metagenomics sequencing indicates the genome ofcontains polysaccharide utilizing genes and multiple vitamin synthetic genes [ 21 ], which is similar for. The upregulation of some pathways like folate synthesis and LPS biosynthesis should mainly come from the improvements ofand Figure 3 A).

On the other hand, however, neotame consumption extensively induced the decline of various components in Firmicutes, which corresponded to the reduced alpha-diversity ( Figure 1 and Figure 2 ). Notably, multiple genera in family Lachnospiraceae and Ruminococcaceae were significantly decreased ( Figure 2 D,E). As the important components of Firmicutes, Lachnospiraceae and Ruminococcaceae have been considered as two of major plant degraders and short-chain fatty acid (SCFA) producers, with critical roles in host nutrition supplement and energy homeostasis [ 22 23 ]. This is in alignment with the downregulation of carbohydrate metabolism pathways and multiple butyrate synthetic genes in the gut microbiome of neotame-consuming mice ( Figure 3 ). To conclude, the extensive decline of Lachnospiraceae and Ruminococcaceae as well as other components of Firmicutes may reduce the energy-harvesting capacity of gut microbiome and therefore, influence host energy homeostasis, which is correlated to the observed low body weight gain in previous chronic neotame safety studies [ 18 24 ].

27, Bacteroides (a Bacteroidetes) is known as an important plant polysaccharides degrader, as is Ruminococcus (a Firmicuties) [ The ratio of Firmicutes and Bacteroidetes (F/B) has been widely studied regarding their associations with obesity. However, previous studies have very inconsistent conclusions. For example, some studies found high F/B ratio in the microbiome of obese subjects [ 11 25 ], while other studies reported a reverse trend or even a negligible difference of F/B ratio between obese and lean subjects [ 26 28 ]. Nevertheless, it is clear that components in Firmicuties and Bacteroidetes may share some functions. For example,(a Bacteroidetes) is known as an important plant polysaccharides degrader, as is(a Firmicuties) [ 29 30 ]. For this study, the alterations in functional pathways and fecal metabolite profiles may reflect the overall functional effects of neotame on gut microbiome, as the functional damage caused by extensive decrease of Firmicuties could be partially complemented by the increase of Bacteroidetes.

35,41, Fecal metabolome data further confirmed the observed effects of neotame consumption on gut microbiota. As shown in Figure 4 , the concentrations of hundreds of metabolites have undergone a significant decrease in the feces of treated mice, including malic acid, mannose-6-phosphate and glyceric acid ( Figure 4 and Figure 5 A). The decreased concentrations of these metabolites may be caused by the decline of Firmicutes ( Figure 2 B). Conversely, we found that the concentrations of most of the identified lipids and fatty acids in feces of neotame-consuming mice were higher than controls, including linoleic acid, stearic acid, 1-monopalmitin and 1,3-dipalmitate ( Figure 5 B). It seems that the absorption of those lipids and fatty acids in neotame-treated mice are lower than controls, which may partially explain the observed neotame-induced low body weight gain in previous neotame safety studies [ 18 ]. It is established that gut microbiota plays an important role in host lipid and fatty acid absorption and metabolism [ 31 32 ]. A recent research has demonstrated that Firmicutes could promote the fatty acid absorption and increase epithelial lipid droplet in Zebrafish [ 33 ]. In this study, neotame consumption largely reduced the abundance of Firmicutes, which may result in a lowered absorbing efficiency of fatty acids and lipids and increased levels of them in feces. However, previous studies also proposed other potential explanations. First, it has been found that SCFAs could inhibit gastric motility by increasing peptide YY levels [ 34 36 ]. The decline in gastric motility allows for more intestinal epithelial contact time and therefore increase energy absorbing efficiency [ 37 ]. For our current study, the large decrease of Lachnospiraceae and Ruminococcaceae and the reduced butyrate synthetic genes upon neotame-induced perturbation of gut microbiome may indicate a decline in SCFA production, thereby reducing lipid and fatty acid absorption. Moreover, previous study indicated that SCFAs could promote the L-cell differentiation and increase the L-cell number, which increased the glucagon-like peptide 1 (GLP-1) release [ 38 ]. The decrease of SCFAs production might influence the GLP-1 release, which can deeply affect the lipid metabolism [ 39 ]. Besides, it is known that gut microbiota also influences the bile acids metabolism, which is essential in lipid and cholesterol metabolism [ 40 42 ]. In this regard, the perturbation of gut bacteria may lead to an altered bile acid metabolism and further influence the absorption of lipids and fatty acids. The increased levels of cholesterol, campesterol and stigmastanol may share similar mechanisms with the alterations of fatty acids and lipids; however, our current data cannot demonstrate such a causation. More work needs to be done in future studies to reveal the mechanism of how neotame affects the fecal profiles of fatty acids and lipids.

Metabolic effects of the artificial sweetener neotame are still poorly understood. This study for the first time investigates the effects of neotame consumption on mouse gut microbiome. The results indicate negative effects on gut microbiota in mice that the use of neotame can induce perturbation in gut bacteria, including bacterial community compositions, functional genes, and the metabolome. The results yielded in this study may provide insights towards an improved mechanistic understanding of the interaction of neotame, the gut microbiome and host metabolism, and may be useful to resolving the much controversial health impacts of artificial sweeteners.

The current research has several limitations. First, the sample size in this study is relative small, that future studies need to further validate the effects of neotame on gut microbiome using a larger number of animals or a human cohort. Second, our results are based on 16S sequencing, PICRUSt analysis, and non-target metabolomics. However, shotgun metagenomics sequencing and target analysis especially for metabolites like SCFAs might have yielded better results. Moreover, the current study is a four-week neotame exposure, while human exposure is frequently long term and at lower concentrations. Future studies are necessary to explore effects of long-term exposure in human.