Sucralose or rebaudioside A at recommended doses did not alter the gut microbiota composition in rats under two dietary conditions

doi:10.21203/rs.3.rs-2802760/v2

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Sucralose or rebaudioside A at recommended doses did not alter the gut microbiota composition in rats under two dietary conditions

https://doi.org/10.21203/rs.3.rs-2802760/v2

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Research reported that non-nutritive sweeteners (NNS), including sucralose, elicit metabolic changes through the gut microbiota (GM) modulation, nonetheless, this topic remains controversial. Furthermore, the impact of rebaudioside A (reb A) on GM has received limited scrutiny. Consequently, we aim to investigate the response of GM composition to both sucralose and reb A in rats, considering two distinct dietary conditions. Male Wistar rats (150–200 g) fed either a normal diet (ND) or a high-fat diet (HFD) were randomly assigned to receive sucralose (SCL), reb A (REB), glucose (GLU, control), or sucrose (SUC). The NNS were administered in water at doses equivalent to the human acceptable daily intake (ADI). Following eight weeks, the GM composition in fecal samples was analyzed through 16S ribosomal RNA gene sequencing. The NNS did not modify the diversity, the structure, the composition at the phylum level, and the Firmicutes/Bacteroidetes (F/B) ratio of the GM. At the class level, REB with HFD decreased Bacilli and increased Faecalibacterium abundance. SCL and REB in combination with ND reduced the genera Romboutsia and Lactobacillus. On the other hand, the analysis of the effect of the diet without NNS showed that HFD increased the proportions of Bacilli and Coriobacteriia, despite not observing changes in the F/B ratio. Our study indicates that when sucralose or reb A is consumed at recommended doses, there is no observed alteration in the diversity and composition of the GM at the phylum level. Consequently, our data suggest that these NNS do not substantially impact the GM. We encourage further research to delve into the potential modifications of the GM at the level of specific bacterial taxa, as this could offer valuable insights for clinical interventions.

Nutrition & Dietetics

non-nutritive sweeteners

sucralose

rebaudioside A

high-fat diet

gut microbiota

Non-nutritive sweeteners (NNS) are food additives that provide high potency in sweetness but few or null caloric content [1]. Sucralose and rebaudioside A (reb A) are two widely NNS consumed [2, 3]. Sucralose is a disaccharide derived from sucrose and exists in over 4500 products which represent 62% of the global market for artificial sweeteners [4]. Food and Drug Administration (FDA) has established the acceptable daily intake (ADI) of sucralose as 5 mg/kg body weight (BW) and of steviol glycosides as 4 mg/kg BW [5].

Originally, the NNS were introduced as an alternative to help control caloric intake, body weight, or glycemia in individuals with obesity or diabetes [6]. Nevertheless, there is an ongoing controversy about the possible effects of these substances on metabolic health [7]. Diet has emerged as a pivotal determinant in shaping microbial communities [8]. Likewise, the disruption of the normal balance of gut microbiota (GM) promotes the development of chronic diseases including obesity, type 2 diabetes, or cognitive disorders, and NNS exposure can contribute to the GM alterations [9].

A study in lean and obese mice exhibited that sucralose for 11 weeks modified the gut bacteria belonging to the phylum Bacteroidetes and Actinobacteria, such as Bacteroides uniformis and Bifidobacterium longum [10]. Increased proinflammatory metabolites and expression of hepatic pro-inflammatory markers by disrupting the GM have been observed in lean C57BL/6J mice after sucralose consumption for 6 weeks [3, 11]. Low-dose (0.0003 mg/mL) of sucralose for 16 weeks increased Tenacibaculum, Ruegeria, Staphylococcus and Allobaculum proportions in mice jejunum, ileum, and colon [12]. Reb A for 9 weeks reduced the abundance of health-promoting microbiota in lean Sprague-Dawley rats [2]. Stevia supplementation for 10 weeks did not prevent high fat diet-induced changes in glucose tolerance or the microbiota in obese C57BL/6 J mice [13]. In contrast, other findings founded that artificial NNS have minimal or null effects on GM in animal models and humans [14, 15].

In this line, the scientific community still has no unanimous consensus on the effects of sucralose on GM, and the reb A impact on microbiota has barely been evaluated. Besides, since the high-fat content affects the GM community, the aim of the present study was to examine the effects of sucralose or reb A, at doses equivalent to the ADI, on GM composition in rats with a ND or a HFD.

Animals

Male Wistar rats weighing 150–200 g (6 to 8 weeks old) were provided by the Faculty of Medicine of the National Autonomous University of Mexico (UNAM) and they were housed in polycarbonate cages (four per cage) at a 12-h light/dark cycle (7:00 h to 19:00 h), controlled temperature (18 °C to 26 °C), ventilation cycles (12 to 15 air changes per hour), and relative humidity (45% to 60%). All procedures were performed according to the Official Mexican Standard NOM-062-ZOO-1999 and the guidelines established by the Research Committee for the Care and Use of Laboratory Animals of the UNAM. The study protocol was approved by the Institutional Review Board of the Academic Division of Health Sciences, Juarez Autonomous University of Tabasco (002/CIP/DACS).

Diets

Animals were randomly allocated into two groups to receive either a normal diet (ND) or a hypercaloric diet (HCD) for 8 weeks. ND was comprised of standardized LabDiet Rodent® 5001 chow (28.67% protein, 57.94% carbohydrate, 13.38% fat; 3.36 kcal/g) and pure water, whereas the HCD was comprised of High-fat diet (HFD) (4.6 kcal/g of energy, 26.7% carbohydrates, 13.2% proteins and 60.0% fat) along with a 30% sucrose solution. Diets were administered in pellet form. Before intervention phase, rats underwent a one-week acclimation period receiving ND and pure water. All animals had ad libitum access to food and fluid over the experimental period.

Treatments and study design

The following sweeteners were employed in this study: 1.3% sucralose (Sweeny® plus), and pure reb A (Anhui Minmetals, Hefei, China), glucose (Roquette corporation®), and sucrose (Zulka®). Each dietary group was divided into four subgroups to receive 4% glucose (GLU), 4% sucrose (SUC), sucralose (SCL) (5 mg/kg BW/d), or reb A (REB) (4 mg/kg BW/d) (n = 8 for each group) for 8 weeks. Sweeteners were administered in drinking water. The sucralose and reb A dose were equivalent to the ADI established by the FDA. To control the high glucose content in the commercial sweeteners, a 4% glucose solution was introduced as a primary control. Glucose concentrations were adjusted to 4% in NNS groups. The SUC groups served as a secondary comparator. Food and fluid consumption were measured daily, and energy intake was calculated weekly, while BW was determined twice a week using an electronic precision balance (Precision BJ 2200C). After eight weeks, animals did not change weekly body weight evolution, total weight gain, and energy intake among NNS treatments in both ND and HFD rats. A detailed description of the metabolic outcomes presented in the original study is reported in Ramos-García et al. (2021) [16]. For the secondary outcome, each animal was isolated for handling, and fresh fecal samples in duplicate were collected. All samples were immediately stored at -80 °C for further analysis.

We decided to examine the sucralose and reb A sweeteners due to their widespread popular consumption. In addition, reb A is one of the most abundant GE in the stevia leaf, with greater sweetness power (200-400x) and the one that leaves the least bitter aftertaste when ingested.

DNA extraction and 16S rRNA PCR

The GM composition was assessed through the sequencing pf the16S rRNA gene in fecal samples. Total DNA was isolated from feces of rats using the QIAamp® Fast DNA Stool Mini Kit (QIAGEN, Hilden, GE) (Cat No. 51604) following the manufacturer’s instructions. Purified DNA was quantified by duplicate in a Nanodrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA). All samples were stored at −80 °C until further analysis. The V3−V4 hypervariable regions of the bacterial 16S rRNA gene were targeted using the following specific universal bacterial primer pairs:

515F	5′–TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG –3′
806R	5’–GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC–3

For each sample, 5 ng/ µL of the purified DNA, 5 µL of forward primer (1µM), 5 µL of reverse primer (1 µM), 12.5 µL of 2X Platinum SuperFi PCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA), and 10 µL of water PCR grade for a total volume of 25 µL were used for the initial PCR reaction. The PCR reaction was performed on an Applied Biosystems GeneAmp 9700 96-well Thermal Cycler (Thermo Fisher Scientific, Waltham, MA, USA) using the following parameters: 98 °C for 3 min predenaturation, followed by 25 cycles of amplification consisting of denaturation (98 °C for 30 sec), alignment (55 °C for 30 sec), elongation (72 °C for 30 sec) and a final elongation step (72 °C for 5 min). Amplicons were generated, cleaned, indexed, and sequenced according to the Ilumina MiSeq 16S Metagenomic Sequencing Library Preparation Protocol [17].

16S rRNA gene sequencing

Libraries were demultiplexed using a dual-index approach with the Nextera XT Index kit (Illumina Inc., San Diego, CA, USA). Paired end 2 × 250 (MiSeq Reagent kit V2, 500 cycles) reads were generated. Trimmomatic v. 0.36 software was used to process the raw fastQ files [18] and the mate-paired files were trimmed to dispose of bases prior to estimating the sequencing error. The aligned data were analyzed using quantitative insights into microbial ecology (QIIME) [19]. Dada2 and deblur denoising packages were used to match operational taxonomic units (OTUs) with 97% sequence similarity against Silva database release 138. Alpha diversity measurements (Shannon) were calculated. Unweighted UniFrac distances were used to perform the principal coordinate analysis (PCoA) for beta diversity (Bray-Curtis). All sequencing reactions were performed at Sequencing Unit from the National Institute of Genomic Medicine in Mexico.

Data analysis

Data were analyzed and plotted using Graphpad Prism Software version 7.0 (San Diego, CA, USA) or QIIME Software version 1.9.1 (San Diego, CA, USA). Kruskal-Wallis test was employed to compare the differences in the ɑ-diversity (Shannon index) across different treatments. Permutational multivariate analysis of variance (PERMANOVA) with Bray-Curtis dissimilarity and principal component analysis (PCoA) was used to analyze β-diversity. The ratio of Firmicutes/Bacteroidetes (F/B ratio) was calculated by dividing the relative abundance of Firmicutes by that of Bacteroidetes. The differences in the relative abundances of bacterial taxa and F/B ratio between sweeteners were compared using one-way analysis of variance (ANOVA) for multiple comparisons or the Kruskal-Wallis nonparametric equivalent test with Tukey or Dunns post hoc tests, respectively. A two-tailed Student’s t or Mann Whitney test was used to analyze the differences between diets. Data were expressed as media ± standard error of the mean (SEM) unless otherwise specified. Differences were considered statistically significant at p <0.05.

Effect of sucralose or reb A on GM diversity

PCoA distances displayed that neither SCL nor REB altered the microbe structure in ND and HFD-fed rats. The SUC consumption with ND and the SCL with the HFD tended to cluster distinctly from the GLU treatment, however, the p adjust does not support these differences (PERMANOVA, p = 0.02 and p adjust= 0.16). The variation in the dataset was explained by two principal components, with a cumulatively variance of 25.29%. Fig. 1, panels A, B, and C shows the comparisons between NNS and between diets. No significant differences were found in the α-diversity (Shannon index) between NNS treatments in ND or HFD group (p> 0.05) (Fig. 2, panels A and B).

Effect of sucralose or reb A on GM composition

Fig. 3 shows the distribution of the four dominant phyla between treatments in ND and HFD groups. The dominant phylum across all treatments in both ND and HFD-fed rats were Firmicutes (ND: 67.44% ± 5.06%; HFD: 75.59% ± 4.72%), and Bacteroidetes (ND: 31.49% ± 5.07%; HFD: 19.26% ± 4.60%), with a lower proportion of Actinobacteria (ND: 0.38% ± 0.06%; HFD: 1.38% ± 0.40%) and Proteobacteria (ND: 0.105% ± 0.04%; HFD: 0.47% ± 0.21%).

In ND-fed rats, SUC (52.08% ± 9.40%, p = 0.02) decreased Firmicutes and increased Bacteroidota (47.05% ± 9.59%, p = 0.02) relative to GLU (90.16% ± 4.40%, 9.09% ± 4.34%, respectively) (FIGURE 3A). In HFD-fed rats, the presence of Actinobacteria and Proteobacteria was observed in smaller proportions, but without reaching statistical significance between treatments (FIGURE 3B).

The ratio between the Firmicutes and Bacteroidetes (F/B) has been reported to influence the maintenance of gut homeostasis and the onset of several pathologies [13]. Our analysis showed that in ND-fed rats, the SUC group lowered the F/B ratio [0.88 (0.54,3.77)] compared to GLU [17.52 (5.17,94.87)] (p = 0.038) (Fig. 4A). In the HFD fed rats, no significant differences were observed between NNS treatments (Fig. 4B).

Fig. 5 display the distribution of the GM at class level between NNS treatments in ND and HFD groups. We observed that in ND-fed rats, SUC consumption (47.05% ± 9.59%) induced an increase in the abundance of Bacteroidia compared to GLU (9.09 % ± 4.34%, p = 0.024) (Fig. 5A). In HFD-fed rats, REB (16.45% ± 7.11%) decreased the media proportions of Bacilli in comparison with GLU (63.72% ± 10.74%, p = 0.003) (Fig. 5B). No significant changes were identified with the consumption of NNS in other bacterial classes (p > 0.05).

The major genera were found in Firmicutes, Bacteroidetes, and Actinobacteria phylum. In rats fed with a ND, SCL (3.60% ± 1.41%), REB (3.71% ± 1.61%), and SUC (1.78% ± 1.29%) significantly reduced the relative abundance of genus Romboutsia (Phylum: Firmicutes; Family: Peptostreptococcaceae) compared to GLU (21.40% ± 8.23%, p <0.05) (Fig. 6A). In HFD rats, SCL (42.67% ± 6.98%) and REB (6.85% ± 6.67%) reduced Lactobacillus (Phylum: Firmicutes; Family: Lactobacillaceae) with respect to GLU (59.69% ± 11.51%). We noted that REB (11.53% ± 4.22%) increased Faecalibacterium (Phylum: Firmicutes; Family: Ruminococcaceae) compared to SUC (1.99% ± 0.68%, p <0.01) (Fig. 6 B). In other genera, we did not observe statistical significance.

Effect of the HFD on diversity, composition and F/B ratio

The effect of HFD on GM was evaluated regardless of the type of sweetener. Fig. 7 shows the effects of HFD on GM diversity, composition, and F/B ratio. With the consumption of the HFD, no significant effects were identified in the β-diversity (Table S3, Fig. 7A), α-diversity (Shannon index, Fig. 7B), composition at the phylum level (Fig. 7C) or F/B index (p > 0.05) (Fig. 7D). At the class level, HFD increased the proportions of Bacilli [40.30% (21.24, 74.54)] (p = 0.03) and Coriobacteriia [0.62% (0.24, 1.80)] (p = 0.02)] relative to ND [16.68 % (8.14, 28.29); 0.29% (0.15, 0.50), respectively] (Fig. 7E). A potential reduction of Clostridia was observed; however, no statistical significance was reach. At the genus level, HFD did not induce significant modifications on Lactobacillus, Romboutsia and Faecalibacterium (p > 0.05)

In the present study, neither the relative abundance of dominant phyla in the GM nor the F/B ratio was altered after 8 weeks of consuming recommended doses of sucralose or reb A, which suggests that these NNS did not cause substantial changes in GM composition. In addition, no changes were observed in the GM α- and β-diversity. Collectively, our results agree with previous results that evaluated sucralose or reb A in animal models that ingested similar diets [20]. Many of the acute studies on the biological fate of sucralose have shown that this substance is not absorbed but it is eliminated unchanged in faeces, which makes it unlikely to be a substrate for gut microbiota [21, 22]. Reb A is known to resist hydrolysis by pancreatic or brush border enzymes but is converted to the aglycone steviol and glucose by gut microbes expressing β-glucosidases in the gastrointestinal tract [1, 23]. After degradation, steviol is absorbed and transformed into steviol glucuronide [20], while, glucose is mainly absorbed by the enterocyte and have limited effects on glucose homeostasis [24]. The residual glucose remaining in the gut is not considered important to induce GM diversity and composition changes [23].

The alterations in the F/B ratio have been extensively examined in relation to obesity. Nevertheless, there is currently no consensus on this matter, as prior research has yielded conflicting conclusions [25, 26]. For instance, certain studies have identified an elevated F/B ratio in the microbiota of individuals with obesity [27], while others have reported the opposite outcome or even a minimal distinction in the F/B ratio between obese and lean animals or humans [28, 29]. Here, we did not find effects of NNS on this outcome. Since it is known that the F/B ratio can be influenced by the availability of specific nutrients to different bacterial populations, we think that the lack of an energetic content in these sweeteners may have contributed to the minimal impact on this variable even in animals fed HFD.

At class level, exposure to reb A decreased the abundance of Bacilli in HFD, which could be considered beneficial since the increase in this genus has been associated with alterations in metabolism [30]. Sucralose and reb A induced a decrease in the genus Romboutsia in rats with ND. These bacteria can utilize carbohydrates, ferment various amino acids, and modulate lipid metabolism [31]. Moreover, reb A and sucralose decreased Lactobacillus in HFD rats. It has previously been reported that both genera are associated with beneficial functions by participating in the regulation of lipid metabolism and in the maintenance of intestinal epithelial barrier function [32]. Our results agree with other investigations where stevia glycosides showed an inhibitory influence on Lactobacilli [23] or even reb A inhibited the growth of probiotic Lactobacillus strains in vitro [33]. Further studies should be carried out to evaluate the influence of these NNS on widely utilized probiotic strains in humans. The focus should be put on the influence of sucralose or stevia glycosides on the metabolism of probiotic bacteria to elucidate observed effects.

In the present study, the HFD regimen showed modifications in the GM at the class level, increasing Bacilli but no significant effects on the F/B ratio was observed. The absence of significant effects of HFD and NNS on the F/B ratio could be mainly explained by the reduction in Clostridia. In addition, it has been proposed that certain bacterial groups as Bacilli can metabolize lipids, which could result in a main mechanism for the observed changes in our findings [34, 35]. The excessive consumption of fats in Western diets is related to alterations in the microbiome that contribute to inflammatory processes and obesity-induced insulin resistance [36, 37].

Our microbiota results are in line with the absence of effects of NNS on glycemic response, body weight, or energy intake in healthy rats and with glucose intolerance reported in our previous research [16]. On the contrary, other studies in male rodents have shown GM alterations. Acesulfame K and saccharin, administered at 2.5 or 650 times the ADI, respectively, induced GM changes, including the over-representation of Bacteroides, Anaerostipes, and Sutterella, or the under-representation of Clostridiales [10, 38]. Notably, these interventions utilized doses surpassing admissible limits for humans, and while they elicited significant changes, they are not reflective of real daily consumption. In a recent investigation, a low dose (0.0003 mg/mL) of sucralose over 16 weeks led to an increase in Tenacibaculum, Ruegeria, Staphylococcus, and Allobaculum at the genus level in the jejunum, ileum, and colon of mice [12]. Similarly, alterations in bacterial genera were observed in C57BL/6 mice after 6 months of sucralose at the human ADI, including an increase in Ruminococcaceae and Ruminococcus, and a reduction in Dehalobacterium, Lachnospiraceae, Anaerostipes, and Lachnospiraceae Ruminococcus [3]. These results suggest that at low doses, sucralose has a selective impact on specific types of microorganisms.

Our study exhibits some advantages: a model of glucose intolerance induced by HFD in rats; natural and artificial NNS; doses equivalent to the ADI; and the use of glucose and sucrose as controls. Also, the use of 16S rRNA gene sequencing constitutes a reliable method to study the diversity and composition of the microbiota. The design of this study was based on the primary outcome, which was the postprandial glucose response during an oral glucose tolerance test and this study was focused on microbiota composition as an exploratory secondary outcome. Even though we could not evaluate the mechanisms by which dietary patterns acts on GM, our research compares the effects of NNS under two dietary conditions and contributes to the knowledge about the effects of consuming foods containing NNS in combination with foods with a high-fat content.

Collectively, our study indicates that when sucralose or reb A is consumed at recommended doses, there is no observed alteration in the diversity and composition of the GM at the phylum level. Consequently, our data suggest that these NNS do not have a substantial impact on the GM. We encourage further research to delve into the potential modifications of the GM at the level of specific bacterial taxa, as this could offer valuable insights for clinical interventions.

Acknowledgment

We acknowledge to Consejo Nacional de Ciencia y Tecnología (CONACYT), México, for the grant awarded to MR-G. We thank Rodrigo Zamora, Araceli Gutiérrez, and Paola Mejía for their technical assistance.

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The authors declare no competing interests.

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Sucralose or rebaudioside A at recommended doses did not alter the gut microbiota composition in rats under two dietary conditions

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