Sweeteners are a versatile food ingredient because of their low caloric content. In recent years, several population groups have begun to use these products, even if they have normal blood sugar levels. Sweetness perception is crucial for an individual’s acceptance of food, and the physiological process depends upon the sweetener (maximum 4 mM concentration) and receptor interactions [1
]. Two of the most important steps post intake are represented by the absorption and interaction with the physiological processes in the human body. Sweeteners have even been found in breast milk, and they directly impact the child’s responses to sweet taste during the growth period. Over the long term, this high acceptance of sweet taste determines the incidence of diabetes at very young age [2
The effect of sweeteners on human health has been extensively explored because of the incidence of obesity and diabetes [3
]. The biological effect on the microbiota is significant because the impact of regular consumption helps to explain the progression of degenerative pathologies or cancer [4
]. From recent studies, it is evident that a direct relationship exists between sweetener consumption, the establishment of dysbiosis, and the development of neurodegenerative diseases [5
]. Setting up pre-diabetes is favored by the interaction of the microbiota with different types of sweeteners, which are increasingly being used in food [6
]. Understanding the initiation of dysbiosis and pre-diabetic prognosis necessitates a metabolomic approach, as a modern preclinical study method [7
]. The physiological mechanism is a reduction in the time of insulin sensitivity, which once initiated has a linear progression until the pathology is established and manifested simultaneously with an increase in body weight [8
Colonic pH variation could be associated with a reduction in insulin sensitivity—an important feature in people diagnosed with type 2 diabetes. The regular consumption of sweeteners causes the increased incidence of this pathology even at an early age, with the establishment of dysbiosis. A biomarker was considered to be the Bacteroides
strain level as an indicator of the ability of the microbiota to control insulin resistance. The level of sweetener consumption is an essential factor that is capable of disrupting the functional plasticity of the microbiota, and reflects a clinical risk factor [9
In vitro studies in simulation systems are a viable alternative to in vivo assays. Dynamic simulation aims at involving the entire physiological system in the digestion of the samples, as well as the absorption of the essential nutrients. The static GIS1—Phase 2 system was adapted for the in vitro dynamic transit, SHIME (Simulator of the Human Intestinal Microbial Ecosystem) being an accepted example in the scientific community [10
]. Previous studies [11
] have reported that the modulating response of the microbiota in this case was similar to that in vivo. The results revealed the modulation ability of both the microbial and the metabolomic patterns [11
]. This study intended to establish the effect of the sweeteners on the microbiota pattern of healthy individuals, associated with alterations in the metabolomic response, through the production of organic acids and ammonium. Untreated healthy microbiota were used as the control to compare the affected pattern that was altered by the in vitro treatment of different sweeteners.
3.1. Alteration in the Metabolomic Pattern Post Sweetener In Vitro Treatment
The quantity of ammonia synthesized is the crucial factor in microbiota modulation. A significant drop (p
< 0.05) in the ammonia was noted after the in vitro treatment with steviol and oligofructose from chicory-containing sweeteners (Figure 1
). In all the other cases, a minimum 10% increase was recorded for all samples, particularly for sucralose and sodium saccharin, with their passage through the descending colon (data not shown).
In vitro sweetener treatment, including those chemically synthesized, exerted a stimulating effect on the patterns. The rise in the SCFAs post sweetener in vitro treatment has been linked to the stimulation of the in vivo transit [20
]. Cyclamate and sucralose caused the same metabolomic response, altering the ratio of the butyric and propionic acids when compared to the control sample. A direct relationship was identified between steviol in vitro treatment and the butyrate quantity determined. A significant butyrate value (p
< 0.001) was recorded after steviol powder was added (Table 3
). For this sample, the exception made was the propionic acid synthesis, with about 100 μg/mL less than that of the steviol capsule. These differences can be explained by the presentation of the samples, their dissolution, as well as the presence of other compounds. From the results it is clear that the in vitro treatment of a particular type of sweetener controls the metabolomic response of the microbiota.
The molar ratios of the chief SCFAs are shown in Figure 2
and Figure S1
. The microbiological response was the one that generated significant differences in the in vitro treatment of all the sweeteners. The oligofructose from chicory was the exception, where it seemed like a balance was struck compared to the control sample. Steviol led to the most altered values of the molar ratio, while steviol powder led to variations of about three times. These results indicate a change in the metabolomic response even with steviol [21
]. There was also an almost 10-fold decrease in the propionate after in vitro steviol treatment. The data revealed a rise in the acetate/butyrate quantities, depending on the type of sample presentation (Table 3
Another microbiological response to the in vitro treatment of sweeteners was the raised benzoic acid, PLA concentrations, and the HO-PLA in the culture media containing steviol powder, steviol and brown sugar, and white sugar.
Bifidobacteria and lactobacilli produced significant quantities of PLA and HO-PLA in vitro, using phenylalanine and ά-ketoglutarate [22
]. These compounds appear to play a crucial part as antimicrobial and antioxidant compounds [23
]. Apart from these metabolic compounds, benzoic acid released by the Serratia marcescens
has been found to play a role in inhibiting the formation of reactive oxygen species in the neutrophils [22
The higher concentrations of these compounds in the samples post sweetener treatment may have been caused by the multiplication of the bacterial populations that synthesize them or by the presence in the environment of certain compounds that stimulate the synthesis of these metabolic compounds. Other organic acids (e.g., oxalic, succinic, malic, tartaric, and citric acids) were not determined after in vitro tests.
3.2. Alterations in the Microbiota Pattern Post Sweetener In Vitro Treatment
From the findings shown in Figure 3
, significant differences were evident between the samples containing different sweeteners. Considering the general bacterial cells, the highest values were obtained for the sodium cyclamate, sucralose, sodium saccharin, and steviol powder samples (109
genomes/mL), and the lowest value for the oligofructose from chicory sample (107
genomes/mL). For the steviol and brown sugar, steviol capsule, and white sugar samples, however, the number of bacterial cells achieved 108
In most samples, the enterobacteria revealed about 108 genomes/mL, barring the steviol capsule and oligofructose from chicory samples, where they decreased, respectively, to 105 genomes/mL and to 106 genomes/mL. In addition, the Bacteroides–Prevotella–Porphyromonas groups revealed lower values in these two samples, achieving the minimum detection threshold (101 genomes/mL).
Species included within phylum Firmicutes occurred in large numbers, particularly for sodium saccharin, steviol powder, steviol and brown sugar, and steviol capsule, while their numbers decreased by one unit in samples sodium cyclamate, sucralose, white sugar, and oligofructose from chicory. In the case of the oligofructose from chicory sample, the large number of species within phylum Firmicutes could be linked to the development of the Lactobacillus–Leuconostoc–Pediococcus species, where their numbers were roughly equal, achieving 107 genomes/mL. Bifidobacterium sp. were found to be low in number in all the samples analyzed, registering below 102 genomes/mL.
The main goal of this study was accomplished by demonstrating the effects of different sweeteners on the human microbiota pattern. One of the most significant findings was the dramatic drop in the number of bifidobacteria after adding the steviol capsule, white sugar, and oligofructose from chicory (Figure 3
). The results showed similarity to the data drawn from colorectal cancer patients, and revealed a direct link between the synthesis of SCFAs and modulation of the microbial pattern [25
]. When the steviol samples were added in powder form alone or combined with brown sugar (steviol powder and steviol and brown sugar), the number of bifidobacteria was higher than in the control or in the other samples. These results suggest that steviol products could be used as a carbon source by these strains. Thus, when steviol and brown sugar were consumed, the pH of the medium declined (pH < 5). This behavior was characteristic of the descending colon segments, which contained a high number of lactic bacteria. The pH drop was accompanied by the presence of different organic acids (e.g., acetic and lactic acids) in higher amounts (Table 3
) upon the administration of steviol powder plus brown sugar (p
≤ 0.05) and white sugar. Sweeteners produced by chemical synthesis caused the pH values to increase (>7.5) for saccharin and sucralose (p
≤ 0.05; Figure S2
). Reports revealed an increase in the number of Gram-negative bacteria—coliforms in particular—which negatively affected the microbiota balance.
Sucralose and sodium saccharin caused a decrease in the number of genomes belonging to Firmicutes, which had a direct correlation with the SCFA level. This behavior was reported in an earlier study, which established the impact of various antibiotics on the SCFAs and precursors of biomarkers [26
]. The in vivo effect bears similarity to sucralose in vitro treatment, a compound that can induce the development of inflammatory processes [27
]. This finding has been linked to disturbances in the normal microbial pattern, which caused dysbiosis and some alterations in a few physiological functions. This behavior precedes the development of degenerative pathologies [28
]. Changes in the microbiota patterns trigger glucose intolerance—one of the stimuli causing type 2 diabetes [29
]. Modification of the microbiota pattern corresponds to alterations in the glucose tolerance because the Bacteroides
species are significant in metabolism regulation [9
]. The establishment of dysbiosis was attributed to the rise in the number of the coliform strains that induced the pH to increase and high resistance to long-term modulation of the microbiota [9
By using an improved in vitro static model, the steviol-based sweeteners were shown to have a similar effect to that resulting from prebiotic in vitro treatment. However, no negative physiological changes were observed [30
]. From these data (Figure 2
and Figure S1
), it is obvious that the in vitro treatment with the steviol-containing products mirrored that of fiber consumption (oligofructose from chicory contains approximately 60% fiber). For the in vitro steviol capsule treatment, the propionic acid/butyric acid ratio [31
] was balanced, and similar was seen with oligofructose from chicory. For the other samples, an increase in the butyrate concentration was identified as a biomarker to maintain colon health. The resulting values (Figure S1
) offer an explanation for a decline in health status after the sweetener in vitro treatment and the occurrence of dysbiosis.
Modification of the metabolomic pattern after in vitro sweetener treatment was the cause of the microbiota modulation. Sweeteners are generally unaffected by the gastrointestinal environment (neither in low pH nor in bile salts) and do not undergo biotransformation [32
]. In addition, some sweeteners (e.g., saccharin) are mostly absorbed in the stomach, and our system does not allow us to highlight this process [32
]. A reduced metabolic activity of the microbiota was noted. The results of this study did not confirm an earlier in vivo study, which stated that in vitro sucralose treatment induced an alteration in the number of Enterobacteriaceae
]. The differences were understood to be a result of the types of experiments conducted, as well as of the specificity of the tested microbiota.
The study showed that the microbial load was reduced, even with in vitro steviol treatment (e.g., steviol capsule). This limits the plasticity of the microbial pattern to exogenous factors. In our case, the behavior was due to the selective consumption (use as carbon source) of the sweetener by the lactic bacteria strains [6
]. Also, it was supported by acetic acid (Table 3
) and lactic acid synthesis, for steviol and brown sugar and white sugar [34
Microbiota control demonstrated that in vitro sweetener treatment caused the fermentation processes to escalate, due to the selective use of these compounds. This behavior was evident for oligofructose from chicory, and the steviol action on the metabolomic profile was confirmed by an earlier study on the effect of fiber on healthy donors [35
]. According to earlier studies [36
], steviol was shown to be able to enhance the glucose uptake, revealing an effect similar to human insulin. The partially contradictory in vitro data can also be explained by the limitations of using a static simulator. Further, the final pattern revealed a specific signature, affected directly by the carbon source and selective modulation of the microbiota. Steviol significantly raised the quantity of the SCFAs (Table 1
), and the phenomenon mirrored the in vivo behavior of obese individuals, where the assimilation of the SCFAs increases the daily calorie intake [37
]. The increased quantity of the SCFAs was also related to supporting the physiological function of the colon by promoting an anti-inflammatory response. In vitro steviol treatment data lend support, via the SCFAs content, to the fact that it does not exert any negative effect on the body [38
This study is relevant when considering large-scale sweetener consumption, by demonstrating their impact on the colon microbiota. The metabolomic modulation by the steviol was demonstrated by the complete metabolism compared to the rest of the samples [6
]. Some differences were noted between the samples containing steviol, which can be explained as one of the effects of product presentation (powder, tablet, or combination with other compounds). Steviol capsule along with oligofructose from chicory determined a significant decrease in the Gram-negative strains, and also in bifidobacteria. The sodium cyclamate, sodium saccharin, steviol powder, and steviol with brown sugar induced an increase in bifidobacteria. The possible presence of other compounds (e.g., carrier ingredients; Table 1
) may represent one of the limitations of this study. In our study, one example is sodium bicarbonate, which is present in small quantities in steviol capsules. Though the presence of this ingredient does not have a negative effect, and it is considered to prevent type 2 diabetes incidence, in our research the quantities were too small to express an effect and to have an influence on microbiota activities [40
]. The results of the study refer to the effects of these sweeteners on the microbiota starting from the action of the major active compound in the sweetener composition. Other minor compounds (e.g., excipients, carrier ingredients, or the presence of other minor sweeteners) may have a synergistic role, amplifying the effect of the principal active compound [41