The effect of allulose, sucralose, and xylitol on Streptococcus mutans acid production

Abstract

Background

Caries is a bacterial infection driven by a dysbiotic biofilm enriched and fueled by sugar. Frequent consumption of sugar can impart negative health effects, including caries; thus, there is increased interest in identifying less cariogenic sugar substitutes. In this study, the authors investigated the in vitro pH shift by Streptococcus mutans planktonic cells in the presence of various sugars and sugar substitutes.

Methods

S mutans UA159 cells were suspended in a potassium chloride and magnesium chloride salt solution, and acidogenesis was determined by pH drop assay after the additions of sugars (sucrose, glucose, fructose) or sugar substitutes (allulose, sucralose, xylitol). The pH of S mutans cell suspensions was recorded at 10-minute intervals for 1 hour. Planktonic cells were then challenged with glucose and pH was monitored for an additional hour.

Results

All sugars resulted in an initial pH drop to 3.5. For xylitol and sucralose, the pH drop was minimal. Allulose initially dropped to a pH of 5.4 before leveling to 5.7. In subsequent glucose challenges, all S mutans cell suspensions with sugar substitutes dropped to pH 3.5.

Conclusions

Allulose, sucralose, and xylitol did not cause a pH drop as low as sugars, and were not considered cariogenic, except for allulose potentially causing root caries. Sugar substitutes were not able to prevent a pH drop to near 3.5 after the glucose challenge indicating these substitutes are not anticariogenic in the presence of glucose.

Graphical abstract

Why Is This Important?

Acid production by the cariogenic bacterium Streptococcus mutans is critical for cariogenicity. S mutans UA159 acidogenesis using a pH drop assay has not been evaluated for allulose, sucralose, and xylitol with a clinically relevant 1-hour postexposure timeframe. Furthermore, limited data are available on the impact of allulose on S mutans acid production. Allulose, sucralose, and xylitol do not produce a sustained acid drop below the critical pH of 5.5 and are not considered cariogenic compared with sugars such as sucrose, glucose, and fructose, although allulose may contribute to root caries. These sugar substitutes do not inhibit an S mutans pH drop in the presence of glucose and should not be considered anticariogenic in the presence of other sugars.

Introduction

Caries may be considered a noncommunicable disease that afflicts human populations worldwide.1, 2, 3, 4 Caries lesions have been observed in human dentition since antiquity, with Aristotle first associating sweets (consumption of soft figs) with caries.⁵ Caries persists today, with a 1995-2019 estimate of worldwide caries in pediatric populations of 46.2% (primary teeth) and 53.8% (permanent teeth).⁶ The consequences of caries can lead to painful pupal infections, potentially resulting in head and neck infections that increase morbidity and mortality.⁷

Sugar in the human diet is an essential substrate for caries pathogenesis.8, 9, 10 Cariogenic bacteria within the dental plaque biofilm metabolize sugars through fermentation into organic acids that result in the demineralization of tooth structure.^11,12 If a person reduces the dietary intake of sugar, caries can be diminished.^13,14 Historically, this approach has been problematic because humans have a sweet tooth that must be satisfied.¹⁵ The frequency of sugar consumption determines the intensity and prevalence of tooth destruction in humans, ranging from acute early childhood caries in children through chronic root caries in older adults.16, 17, 18

The quintessential cariogenic bacterium of the oral cavity is Streptococcus mutans.^11,16,19, 20, 21 S mutans, along with other acidogenic and aciduric bacteria such as lactobacilli and bifidobacteria, Actinomyces, and Scardovia species, cause caries by rapidly metabolizing fermentable carbohydrate-sugar that lowers the pH below the critical pH of 5.5 within the dental plaque biofilm, beginning the process of tooth demineralization.^12,22, 23, 24 Therefore, an acidic milieu with a pH of less than 5.5 may be considered cariogenic.

There are several sugars dental plaque bacteria can metabolize to produce lactic acid and cause caries.²⁵ These sugars include but are not limited to sucrose (eg, table sugar), glucose, and fructose (eg, high fructose corn syrup). Sweetener substitutes have been used to avoid the negative health effects (including caries) of sugars while providing the desired sweetness of sugars. Those alternatives include sucralose^26,27 and the sugar alcohol xylitol.28, 29, 30 These sweeteners have shown benefits such as caries reduction.^13,31 However, these alternatives are not without their health risks. For example, xylitol has been associated with side effects such as gastrointestinal disturbances,³² and sucralose has been shown to affect gut microbiota negatively.³³ Therefore, the search continues for sweetener alternatives that may be free of adverse health consequences.34, 35, 36, 37

Xylitol has been reported to have antibacterial properties that are, in part, responsible for their ability to prevent caries.^28,38,39 Xylitol is indeed a viable sugar substitute for caries prevention, but its antimetabolic effect on S mutans remains controversial.40, 41, 42, 43, 44, 45

Another sugar alternative is allulose (d-psicose), which may provide health benefits such as weight loss, improved glycemic control, and reduced insulin resistance.⁴⁶ Allulose is a naturally occurring sugar found in wheat, raisins, and other foods and is a C-3 epimer of fructose. This minor configurational change gives allulose different properties than fructose.⁴⁷ Rigorous studies investigating the impact of allulose and its cariogenic properties, such as its ability to lower pH, are lacking.

We investigated the ability of allulose, sucralose, and xylitol to cause a pH drop below the critical cariogenic pH of 5.5 in planktonic S mutans UA159 in vitro and examined their antimetabolic properties after a glucose challenge, using a pH drop assay to determine acidogenesis.^48,49

Methods

pH Drop Assay

S mutans UA159 is recognized as a representative cariogenic strain of human origin, and its genome was sequenced in 2002.⁵⁰ The pH drop assay was performed as previously described with modifications.^48,49 S mutans cells were grown in Todd Hewitt broth (Beckton Dickenson) with 5% carbon dioxide at 37 °C. A 12-hour culture (500 mL) in late log phase was pelleted at 4,500 revolutions per minute for 20 minutes at 4 °C and resuspended in 400 mL of cold 50 mM potassium chloride (3.75 g/L) and 1 mM magnesium chloride (KMg) salt solution. This step was repeated, and the washed cells were resuspended in 200 mL of KMg to an optical density (at a wavelength of 600 nm) of 2.0 with a cell density of 2 × 10⁹ cells/mL. The cell suspension was incubated in a 37 °C water bath for 1 hour, and the final pH was adjusted to 7.0 with 0.1 N potassium hydroxide. Aliquots of 10 mL cell suspensions were transferred into 18 × 150-mm tubes, and an equal volume (10 mL) of 100 mM sugar (glucose, sucrose, fructose) or 100 mM sugar substitute (xylitol, sucralose, allulose) was added to each reaction suspension, bringing the final bacterial density to 10⁹/mL with a final sugar or sugar substitute concentration of 50 mM. A cell suspension with only 10 mL KMg added was used as a negative control. After 1 hour, 1 mL was removed from the 20 mL reaction mixtures, and 1 mL of 20% glucose/KMg was added to bring the final concentration to 1% glucose. After the addition of sugars or sugar substitutes, all tubes were immediately inverted several times to allow uniform mixing, and pH readings were obtained every 10 minutes with a Fisher Accumet AR 15 pH meter (Fisher Scientific). Results are reported as mean (SD) using a minimum of 3 independent experiments.

The KMg salt solution was adjusted to a pH of 7.0, autoclaved, and stored at 4 °C. The sugars (glucose [Sigma Aldrich], sucrose [Fisher Scientific], and fructose [Thermo Scientific]) and sugar substitutes (xylitol [Now Foods], sucralose [Pure Organic Ingredients], and allulose [Wholesome Sweeteners]) were prepared in KMg at 100 mM concentrations, filter sterilized, and stored at room temperature. Subsequent analysis with xylitol included 50 mM, 100 mM, and 1 M concentrations.

The pH is an indicator of acidity (hydrogen ion concentration) in aqueous solutions, and the pH drop assay permits the accurate assessment of acid production by planktonic bacterial cells in a nonbuffered environment.^48,49 We used this experimental approach to determine the magnitude of S mutans acidogenesis when planktonic cell suspensions are exposed to sugars and sugar substitutes. We determined mean planktonic pH at 10-minute intervals for each sugar and sugar substitute over 120 minutes, as shown in Figure 1, Figure 2, Figure 3. The baseline (B in Figures 1 and 2) was the pH of cell suspensions before the addition of sugar or sugar substitutes, and then they were added to the S mutans cell suspensions, followed by pH measurements at 10-minute intervals.

Figure 1.

Results of pH drop assay for Streptococcus mutans planktonic cells in potassium chloride and magnesium chloride salt solution with sugars. A. Control S mutans cells only (no sugar added initially, with glucose added at 60 minutes to a final concentration of 1%). B. 50 mM glucose. C. 50 mM sucrose. D. 50 mM fructose. The results are from 5 independent experiments. B: Baseline.

Figure 2.

Results of pH drop assay for Streptococcus mutans planktonic cells in potassium chloride and magnesium chloride salt solution with sugar substitutes. A. 50 mM allulose. B. 50mM xylitol. C. 50 mM sucralose. D.S mutans cells only control. Glucose was added after 60 minutes to a final concentration of 1%. The results are from 5 independent experiments. B: Baseline.

Figure 3.

Results of pH drop assay for Streptococcus mutans planktonic cells in potassium chloride and magnesium chloride salt solution with 50 mM, 100 mM, or 1 M (15.2%) xylitol. Glucose was added after 60 minutes to a final concentration of 1%. The results are from 3 independent experiments.

Results

When glucose, sucrose, and fructose were added to the cell suspensions, an immediate pH drop was observed that leveled off to pH 3.5 within 10 minutes (Figure 1). All the sugars resulted in a drop below the critical cariogenic pH for tooth enamel of 5.5. The control cell suspension in KMg salt solution maintained a neutral pH between 7.0 and 6.5 for 60 minutes until glucose was added to a final concentration of 1%, resulting in an immediate drop in pH (Figure 1A)

The sugar substitute allulose showed an initial pH drop to 5.4 but leveled off at a pH of 5.7, above the critical cariogenic pH of 5.5 (Figure 2A). Xylitol and sucralose addition to cell suspensions did not result in a pH drop and remained at approximately pH 6.5 for 60 minutes (Figures 2B and C). The addition of glucose to 1% after 60 minutes resulted in an immediate drop in pH and remained at a pH of approximately 3.5 over 60 minutes (Figure 2).

Xylitol was added at time 0 to a final concentration of 50 mM, 100 mM, and 1 M (15.2%) (Figure 3). Increasing concentrations of xylitol to cell suspensions did not result in a pH drop and remained at pH 6.0 through 6.5 for 60 minutes. The addition of glucose to 1% after 60 minutes resulted in a pH drop to less than 4.0 and remained at that level for at least 40 minutes. These results are comparable to Figure 2B using xylitol at 50 mM.

These pH drop experiments indicated that S mutans UA159 required sugars for acidogenesis and did not produce glycolytic fermentative acid from the sugar substitutes xylitol and sucralose. However, allulose did cause an initial drop to pH 5.4 before leveling at 5.7. Moreover, the sugar substitutes used in our study did not prevent the production of lactic acid from 1% glucose. Xylitol at a higher concentration of 1 M or 15.2% did not inhibit acidogenesis in S mutans UA159.

Discussion

Our study used a simple in vitro pH drop assay to evaluate the effects of various sugars and sugar substitutes on the glycolytic fermentative pH profiles for planktonic S mutans UA159 cells. When glucose, sucrose, and fructose were added to cell suspensions there was an immediate drop in pH to 3.5 which then leveled (Figures 1B-D). The control pH remained at 6.5 for 60 minutes and then dropped to a pH of 3.5 when 1% glucose was added (Figure 1A). These results are consistent with the acidogenic nature of lactic acid bacteria that includes S mutans.⁵¹ S mutans, a facultative anaerobe, has been reported to produce sufficient lactic acid to achieve a pH less than 5 via a homolactic glycolytic pathway in the presence of glucose, sucrose, and fructose.^11,52, 53, 54 The low pH of 3.5 after S mutans sugar fermentation has been substantiated by previous studies and validates the pH drop assay as a sensitive methodology for measuring bacterial acidogenesis.^48,49

Sucralose, a trichlorinated derivative of sucrose 600 times as sweet as a 10% sucrose solution, was approved by the US Food and Drug Administration (FDA) in 1999, later followed by the Academy of Nutrition and Dietetics in 2012 declaring it a safe, nonnutritive sweetener for human consumption.^55,56 Previous studies have indicated that sucralose is not metabolized by S mutans and is nonacidogenic^26,53 Our results in Figure 2C are consistent with these observations. Animal models have also revealed that sucralose is noncariogenic in rats infected with S mutans,³¹ and an experimental biofilm caries model suggested that S mutans UA159 has minimal cariogenic potential in the presence of sucralose.^57,58 Taken together, sucralose may be considered a noncariogenic sugar substitute.²⁷ Furthermore, sucralose did not inhibit acidogenesis when 1% glucose was added to the S mutans cell suspension (Figure 2C), and similar experimental conditions support this observation.^26,53

Allulose is a rare sugar of interest because of its potential use as a natural sugar substitute. Although it is not metabolized by humans, Klebsiella pneumonia is able to use allulose as a substrate^47,55 We found that S mutans metabolized allulose to a pH of 5.4 and then leveled off at pH 5.7 (Figure 2A) within 60 minutes of exposure. This finding is similar to other investigations that reported S mutans strains GS5 (at 12 hours incubation) and MTCC890 (after 48 hours incubation) produced pH reads of 5.5 and 5.6, respectively.^59,60 On the basis of our observations, we did not consider allulose a cariogenic substrate because the pH was transiently below the critical pH of 5.5 for the demineralization of hydroxyapatite. This position is also taken by the FDA, stating that allulose does not promote caries.⁶¹ However, dentin demineralization associated with root caries occurs at a critical pH of 6.7,⁶² and the fermentation of allulose to a pH of 5.7 may contribute to the pathogenesis of root caries in older adults with exposed roots because of gingival recession. Nevertheless, the initial drop to pH 5.4 would suggest that S mutans does use allulose as a substrate, or the commercial allulose (Wholesome Sweeteners) might contain trace amounts of fermentable sugars that could also contribute to this drop in pH. The addition of 1% glucose after 60 minutes results in an immediate drop to pH 3.5 (Figure 2A), clearly indicating that allulose does not exert an inhibitory effect on glucose metabolism by S mutans.

Xylitol, a 5-carbon sugar alcohol, is derived from birch tree xylan, corn cobs, and sugar cane waste.^28,63 It has the equivalent sweetness of sucrose and was approved by the FDA in 1963 for human use.^63,64 The dental benefits of xylitol as a sugar substitute were published in the 1976 Turku Sugar Studies from Finland.¹³ Numerous studies since then have indicated that cariogenic bacteria in dental plaque do not metabolize xylitol to lactic acid, resulting in pH levels remaining above the caries zone of less than 5.5.^40,41,44,53 Although the clinical S mutans strains used and suspension substrates differ among the previous studies and our study, our pH drop assay results (Figures 2B and 3) are in agreement with these observations because the pH remained from 6.0 through 6.5 for 60 minutes at xylitol concentrations of 50 mM, 100 mM, and 1 M (15.2%). Xylitol is in use clinically and as a dietary adjunct for the prevention of caries; therefore, higher concentrations compared with clinical and dietary concentrations were used in the pH drop assay to assess S mutans acidogenesis (Figure 3).⁶⁵

The addition of 1% glucose to S mutans cells after a 60 min incubation in 50 mM, 100 mM, and 1M xylitol resulted in an immediate drop in pH to less than 4.0 (Figures 2B and 3), indicating xylitol did not inhibit the S mutans glycolytic fermentative pathway and the acidogenic production of lactic acid. Earlier studies have reported similar results with S mutans OMZ 176,⁴⁰ S mutans NCTC 10832 and 10449,⁵³ and S mutans GS5-2.⁶⁶ Additional experiments with human dental plaque bacteria⁴¹ and in vivo plaque biofilm metabolic analysis⁴⁴ also support our observations. Takahashi and Washio⁴⁴ concluded that “xylitol is not an inhibitor of plaque acid production but rather a non-fermentative sugar alcohol ... [and] that the role of xylitol in caries prevention is as a non-fermentative sugar substitute.”

The intracellular accumulation of xylitol 5-phosphate in S mutans cells exposed to xylitol results in metabolic inhibition.^28,67 However, this inhibitory effect is strain-specific, and variations exist among different S mutans clinical isolates.⁶⁸ S mutans UA159 would be considered xylitol-resistant by not forming high levels of intracellular xylitol 5-phosphate, resulting in acidogenesis in the presence of 1% glucose (Figures 2B and 3). The presence of xylitol within the oral cavity would select xylitol-resistant S mutans and favor their dominance in dental plaque biofilms.^65,69,70 The emergence of xylitol-resistant ancestral strains of S mutans⁶⁹ could account for the questionable efficacy of xylitol as an anticariogenic agent, indicating the need to investigate their prevalence in human populations.^43,64,71, 72, 73, 74

Conclusions

The pH drop assay was used to assess S mutans UA159 acidogenesis in the presence of sugar and sugar substitutes. Glucose, fructose, and sucrose were metabolized to a pH of 3.5 and are considered cariogenic. The addition of xylitol and sucralose to S mutans cells did not result in a pH drop below 6.5; allulose initially caused a pH drop to 5.4 before leveling to 5.7. None of the sugar substitutes should be considered cariogenic, except that allulose potentially causes root caries. Sugar substitutes did not inhibit an S mutans pH drop after the addition of glucose. Moreover, xylitol in concentrations as high as 1 M or 15.2% also did not inhibit S mutans acid production in the presence of glucose. Xylitol, sucralose, and allulose can be considered a sugar replacement intervention in moderating the dietary prevention of caries but should not be regarded as anticariogenic in the presence of glucose.

Disclosure

None of the authors reported any disclosures.

Disclaimer

Hui Wu serves as an editorial board member for JADA-FS. Dr Wu was not involved in any decisions about the article he wrote, and peer review was handled independently.