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Cariogenic potential of commercial sweeteners in an experimental biofilm caries model on enamel
archives of oral biology 58 (2013) 1116–1122
Available online at www.sciencedirect.com
journal homepage: http://www.elsevier.com/locate/aob
Cariogenic potential of commercial sweeteners in an experimental biofilm caries model on enamel Rodrigo A. Giacaman , Pı´a Campos, Cecilia Mun˜oz-Sandoval, Ramiro J. Castro Cariology Unit, Department of Oral Rehabilitation, University of Talca, Talca, Chile
article info
abstract
Article history:
Objective: Scarce evidence is available on the cariogenic potential of the widely used
Accepted 10 March 2013
commercial sweeteners. The aim of this study was to evaluate the effect of several sweeteners on enamel demineralisation and on the cariogenic properties of Streptococcus mutans
Keywords:
biofilms in an artificial caries model.
Dental caries
Methods: S. mutans-UA159 biofilms were cultured on bovine enamel slabs and exposed to
Sweetener
one of the following commercial sweeteners in tablet or powder form: stevia, sucralose,
Streptococcus mutans
saccharin, aspartame or fructose. Ten percent sucrose and 0.9% NaCl were used as caries-
Oral biofilm
positive and caries-negative controls, respectively. Slabs/biofilms were exposed to the
Sucralose
sweeteners three times per day for 5 min each time. After 5 days, biofilms were recovered
Stevia
to determine: biomass, bacterial counts and intra- and extracellular polysaccharides.
Aspartame
Surface microhardness was measured before and after the experiment to assess enamel
Fructose
demineralisation, expressed as percentage of surface hardness loss (%SHL). Data were
Saccharine
analysed using analysis of variance (ANOVA) and Bonferroni ( p < 0.05). Results: All tested commercial sweeteners, except fructose, showed less enamel demineralisation than sucrose ( p < 0.05). Only saccharine showed less biomass and intracellular polysaccharides than the rest of the groups ( p < 0.05). Stevia, sucralose and saccharine reduced the number of viable cells when compared with sucrose ( p < 0.05). All sugar alternatives reduced extracellular polysaccharide formation when compared with sucrose ( p < 0.05). Conclusions: Most commercial sweeteners appear to be less cariogenic than sucrose, but still retaining some enamel demineralisation potential. Products containing stevia, sucralose and saccharine showed antibacterial properties and seem to interfere with bacterial metabolism. Further studies are necessary to deepen these findings. # 2013 Elsevier Ltd. All rights reserved.
1.
Introduction
The detrimental role of sugars, particularly in dental caries is still a matter of controversy.1 Sucrose has been traditionally considered a highly cariogenic substrate for the oral biofilm. Upon fermentation by oral bacteria, sucrose molecules are transformed into energy and large amounts of acids.2 Thus,
frequent exposures to this carbohydrate create conditions for caries onset by promoting demineralisation. As an additional virulent mechanism, cariogenic bacteria populating the dental biofilm generate exopolysaccharides to create a protective environment against physiological antibacterial mechanisms of the mouth.3 Artificial sweeteners are becoming increasingly used to sweeten beverages, such as soda, juice, coffee and tea. In an
Corresponding author at: Escuela de Odontologı´a, Universidad de Talca, 2 Norte 685, Talca, Chile. Tel.: +56 71 201546; fax: +56 71 201761. E-mail address: [email protected] (R.A. Giacaman). 0003–9969/$ – see front matter # 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.archoralbio.2013.03.005
archives of oral biology 58 (2013) 1116–1122
era where obesity and overweight have become a serious health problem with large populations affected,4 sweeteners arise as a way to replace sucrose consumption to deal with this public-health matter. These products have been considered as safe in a recently published position article from the Academy of Nutrition and Dietetics.5 Several research articles have been published claiming a non-cariogenic or an apparent anti-caries potential of sweeteners.6 Sugar alcohols (polyols), that is, xylitol and sorbitol have been intensely tested for caries prevention, especially in chewing gums. Despite the multiple investigations into a putative anti-caries effect of xylitol or sorbitol as caries-safe sugar substitutes, these products are not the most frequently used by the food industry. Saccharine, a sulphamide, was the first and most used sweetener. From there, several other artificial sweeteners have been introduced over the time. Currently, five non- or low-caloric sweeteners have been approved by the Food and Drug Administration (FDA): aspartame, saccharine, acesulfame potassium, sucralose and neotame. Moreover, five non- or low-caloric sweeteners are generally recognised as safe by the same institution: sorbitol, xylitol, erythritol, tagatose and stevia.7 Generally assumed as caries-safe, sweetening beverages with sugar substitutes is becoming increasingly popular. One of the most investigated artificial sweeteners is sucralose. In its pure form, sucralose has been regarded as non-cariogenic8 and when combined with bulking ingredients is less cariogenic than sucrose. Likewise, scarce evidence, mostly in rats, suggests that aspartame would be non-cariogenic.9 Stevia is a highly used commercial sweetener derived from a plant, but only one study in rats with a controlled diet reports a non-cariogenic effect of the sweetener.10 Research on the effect on caries of the currently available commercial sweeteners is rather insufficient. Importantly, most of the available research on a presumptive anticariogenic or non-cariogenic effect of sweeteners comes from the pure chemical compound. Information on the cariogenicity of the sweeteners when in combination with bulking carbohydrates is more limited and may be of importance for enamel and dentine caries. Given the fact that evidence on the caries effect of sweeteners is still inconclusive11 and that limited data on the cariogenic potential of carbohydrate-containing products have been reported, the aim of this investigation was to test the cariogenic potential on enamel and the effect on Streptococcus mutans biofilms of several commercial sweeteners.
2.
Materials and methods
2.1.
Experimental design
(5) aspartame, (6) fructose and (7) 0.9% NaCl (caries-negative control). Biofilms were exposed to the different treatments for 5 min three times a day, simulating what can be considered a typical snack-consumption pattern. The culture medium was changed twice a day. Biofilms were separated from the slabs for analysis of biomass, viable bacteria, polysaccharide production and biofilm protein content. Final surface hardness was measured from the enamel slabs and the demineralisation produced throughout the experiment was estimated by the percentage of surface hardness loss (%SHL). Acidogenicity of the biofilms was estimated through medium pH, measured twice a day at each medium change. Samples were coded to allow blind measurements of the treatment groups. The whole experiment was repeated twice with each condition in triplicate (n = 6, per treatment).
2.2.
Enamel slabs
Bovine incisors were obtained, disinfected with a 5% NaOCl solution and stored in 0.9% NaCl (w/v) until use for no longer than 30 days. Slabs (4 mm 7 mm 1 mm) were prepared using diamond discs with a low-speed hand piece and Soflex polishing discs (3M, St. Paul, MN, USA). Initial SH was determined by three indentations 100 mm apart from each other, performed with a Knoop microindenter with a microhardness tester (402 MVD, Wolpert Wilson Instruments, Norwood, MA, USA) at 50 g for 5 s. Only those slabs of SH 340.87 24.4 kg mm 2 (n = 42) were included to avoid bias derived from using enamel with different initial SH values. Slabs were sterilised with ethylene oxide13 and covered with ultrafiltered (0.22 mm) pooled human saliva treated for 30 min with a protease inhibitor cocktail, to emulate the acquired pellicle on the enamel that further facilitates S. mutans adhesion.14 Slabs were suspended into the wells of a 24-well plate by means of a specially designed device made of orthodontic wire.
2.3.
S. mutans biofilms
Frozen stocks of S. mutans UA159 (kindly provided by Prof. J.A. Cury, UNICAMP, SP, Brazil) were reactivated in 1% glucosecontaining brain heart infusion (BHI; Merck, Darmstadt, Germany) at 37 8C and 10% CO2 for 18 h. Slabs were inoculated with S. mutans culture (optical density (OD) 0.8 at 600 nm) and 1% sucrose-containing medium to form the adherent biofilm14 and incubated for 8 h. Slabs were then maintained in BHI supplemented with 0.1 mM glucose for 24 h, which simulates glucose basal concentration in saliva.12
2.4.
S. mutans UA159 biofilms were grown using a previously described in vitro caries model,12 with modifications; three exposures per day for 5 min, instead of eight exposures for 1 min. Bovine enamel slabs served as substrates for S. mutans anaerobic biofilm formation for 5 days. Initial surface microhardness (SH) was assessed and the slabs were randomly sorted into seven treatment groups: (1) 10% sucrose (caries-positive control), (2) sucralose, (3) saccharine, (4) stevia,
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Sweetener application to the biofilms
Sweeteners in tablets or powder available in the Chilean market were used in this study. Although not in their pure state, the type of sweetener (groups 2–6, as indicated above) was that informed by the manufacturer on the label of the product. Besides the sweetener of interest, all the products contained additional bulking components. Manufacturer information on the composition of the products is presented in Table 1. Treatment solutions were prepared according to the product labelling to a concentration equivalent to 2 teaspoons
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archives of oral biology 58 (2013) 1116–1122
Table 1 – Commercial sweetener used and composition as informed by the manufacturers. Identification
Commercial name
Presentation
Active sweetener
Composition per portion (%)
Other components
Total carbohydrate (%)
14.9 6.18 14.5
Lactose L-Leucine Lactose L-Leucin Lactose L-Leucin Sodium bicarbonate Lactose Leucin Carboximetilcelulose N/A
69.8
Stevia
Cero K Stevia, Iansa1
Tablets
Sucralose
Cero K Sucralosa, Iansa1
Tablets
Stevia Sucralose Sucralose
Saccharine
Sacarina, Lider1
Tablets
Sodium saccharine
40.0
Aspartame
Naturalist, Laboratorios Prater1 Tablets
Aspartame
40.0
Fructose
Regimel, Fructosa1
Fructose
99.0
Powder
of sugar (8 g) in 100 ml of distilled water, considered as a common amount used to sweeten beverages. A confirmation of the sweetness of the solutions was performed by blind sips of the solution. Enamel was treated with the different treatments for 5 min three times per day at defined time points (8:30, 12:30 and 16:30). After treatment, the biofilms were washed three times with 0.9% NaCl and repositioned in the same position in the plate. Culture medium was replenished twice a day, before the first and after the last exposure. The exposure cycles were repeated to complete 5 days, which has been reported as sufficient to induce enamel demineralisation.12
2.5.
Biofilm acidogenicity
This study considered a pH-cycling model, obtained by exposing the biofilms three times during the day to the experimental sweeteners and leaving them unexposed overnight only with a basal glucose concentration. The latter produced a cyclic pH drop in the system, similar to the clinical situation. As a way to verify acid production by the biofilms formed on enamel, culture medium pH was measured inside each well twice per day before each medium change during the entire experiment12 by a microelectrode (HI 1083B, Hanna Instruments, Rumania) coupled to a portable pH metre (HI 9126-02, Hanna Instruments, Rumania).
2.6.
Enamel demineralisation
SH has been extensively used as a reliable methodology to evaluate demineralisation15 and it has been validated for enamel caries.16 Briefly, a second enamel SH (kg mm 2) reading was obtained after the experimental phase by a row of three indentations separated by 100 mm from those performed before the experimental phase. Mean values from the initial and final measurements were used to obtain %SHL calculated as follows: (mean initial SH mean final SH) 100/ initial SH.
2.7.
Biofilm analysis
Once the 5 days of the experimental phase were completed, enamel slabs were washed three times with 0.9% NaCl and sonicated for 30 s at 7 W to separate the biofilms from the slabs,12 which were stored for further analysis. The resulting
82.0 7.82
50.0
99.0
suspension containing the biofilm was further divided into aliquots to assess biomass, viable microorganisms,12 intraand extracellular polysaccharides (EPSs)17 and total proteins18 as previously described, but a brief explanation of each dependent variable follows below: (a) Biomass. To estimate biomass, dry weight was used.14 A volume of 200 ml of the biofilm suspension was transferred to a pre-weighted tube and incubated with 100% ethanol at 20 8C for 15 min. The resulting suspension was centrifuged (10 min at 5000 g and 4 8C) and the resulting pellet washed with 500 ml of 75% ethanol, centrifuged again and the pellet dried for 24 h in a desiccator. Biomass was obtained by subtracting the final weight from the initial weight of the empty tube and expressed in milligrammes. (b) Number of live bacteria from the biofilms. Using serial dilutions of the biofilm suspension in 0.09% NaCl (v/v), 10 ml was drop-plated on BHI agar plates in duplicate, incubated anaerobically for 24 h at 37 8C and colonies counted from the dilution that allowed visualisation of distinctively isolated colonies. Counting was corrected by the dilution factor and expressed as colony forming unit (CFU)/mg of biofilm dry weight.17 (c) Intra- and extracellular polysaccharides. Three different polysaccharide fractions were assessed: soluble (SEPS) and insoluble (IEPS) extracellular (EPS) and intracellular (IPS) polysaccharides from aliquots (200 ml) of the S. mutans biofilm suspension.17 The suspension was centrifuged (10,000 g for 5 min at 4 8C) to obtain SEPS from the supernatant. The resulting pellet was treated with 200 ml of 1 M NaOH, homogenised and centrifuged to obtain IEPS from the supernatant.19 The resulting pellet from the previous extraction containing the IPS was incubated with 200 ml 1 M NaOH for 15 min at 100 8C, centrifuged (10,000 g for 5 min at 4 8C) and the IPS concentration was measured from the supernatant. Each fraction from the supernatants was treated with three volumes of cold 100% ethanol and incubated for 30 min at 20 8C. Samples were immediately centrifuged and the resulting pellet was washed with cold 70% ethanol and centrifuged again. The new pellet was resuspended in 1 M NaOH and total carbohydrate concentration contained in each fraction was estimated by the sulphuric phenol method.20 Results were standardised by biofilm dry weight and expressed as
archives of oral biology 58 (2013) 1116–1122
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percentage of the different fractions of polysaccharides by milligramme of biomass.
2.8.
Statistical analysis
Normal distribution of the data was verified and values obtained for the different treatment groups were compared by analysis of variance (ANOVA) followed by the Tukey test, using the Statistical Package for Social Sciences (SPSS) 15.0 statistical software. Differences were considered significant at a 95% confidence level.
3.
Results
Biofilm acidogenicity is illustrated in Fig. 1. At 32 h from the beginning of the experiments, samples exposed to sucrose, fructose and aspartame had already decreased pH below 5.5 (enamel demineralisation critical pH), significantly lower than the remaining sweeteners. At 56 h, however, the sucrosetreated group showed the lowest pH drop ( p < 0.05). Although lower than sucrose, fructose induced more acidogenicity than the other sweeteners ( p < 0.05). At 80 and 104 h, the acidogenicity elicited by sucrose continued to be the highest ( p < 0.05), followed by fructose and aspartame ( p < 0.05). Saccharine, stevia and sucralose, on the other hand, induced significantly lower acidogenicity throughout the experiment. Regarding enamel demineralisation, all the tested sweeteners showed lower %SHL ( p < 0.05) as compared with the caries-positive control (Fig. 2). Fructose, however, induced more demineralisation than the other sweeteners ( p < 0.05). When S. mutans biofilm properties were analysed, only saccharine-treated samples showed significantly lower biomass than the rest of the experimental products, albeit a trend to induce less biomass for stevia, and sucralose (Fig. 3). Stevia, sucralose and saccharine retrieved significantly less viable cells from the biofilms when compared with the other sweeteners, with similar counts to the negative control (Fig. 4). Likewise, polysaccharide analysis indicated that, except for fructose, all the sweeteners induced lower IEPS production than sucrose ( p < 0.05) (Fig. 5). A similar trend was observed for SEPS (data not shown). Finally, all treatments,
Sucrose
Medium pH
7.0
Stevia
Sucralose
Saccharine
Aspartame
Fructose
NaCl
6.5 6.0 5.5 5.0 4.5
0
8
16
24
32
40
48
56
64
72
80
88
96
104 112 120
Time Points
Fig. 1 – Acidogenicity of S. mutans biofilms formed on enamel an exposed to the sweeteners. Plot shows mean pH of the culture medium of each group (as indicated). Measurements were performed after 24 h of biofilm formation, twice per day, at defined times (as indicated).
Fig. 2 – Enamel surface hardness loss (%SHL) by sweetener tested. Bars represent mean of the %SHL of the slabs (n = 6). Error bars indicate SD. Different letters represent significant differences among treatments ( p < 0.05).
Fig. 3 – Biomass induced by each experimental condition. Plot depicts mean biomass (mg) for each condition W SD (n = 6). Different letters represent significant differences among treatments ( p < 0.05).
including the negative control, showed significantly lower IPS formation (Fig. 6).
4.
Discussion
Although not new, sweeteners are increasingly consumed worldwide. The cariogenic potential of these nutritive and non-nutritive products in their commercial form has not been elucidated. Results from the present study suggest a less cariogenic effect for all tested products when compared with sucrose. Indeed, most of the sweeteners tested, only fructose and aspartame excluded, induced lower pH drop than sucrose (Fig. 1). Similar results have been previously published.21,22 As we found here, fructose had been reported to be as acidogenic as sucrose, in vitro.23 The latter is important as diabetic patients are generally advised to replace sucrose by fructose. As expected and in consistency with the acidogenicity data, sweeteners induced lower enamel demineralisation than sucrose, but still higher than the negative control (Fig. 2). Since no sweetener was capable of demineralising the enamel at levels comparable to those induced by sucrose, these findings confirm the higher cariogenic potential of sucrose
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archives of oral biology 58 (2013) 1116–1122
Fig. 4 – Viable microorganisms from S. mutans biofilms. Bacterial cells retrieved from each sweetener-exposed biofilm were counted and expressed as CFU/biofilm dry weight (mg). Bars show mean counting for each condition (n = 6, triplicate of two independent experiments). Error bars indicate SD. Different letters represent significant differences among treatments ( p < 0.05).
Fig. 5 – Insoluble extracellular polysaccharides (IEPS) produced by the biofilms. IEPS produced by the different conditions were measured and expressed as mg/mg of biofilm. Plot shows mean of each treatment in triplicate of two independent experiments W SD (n = 6). Different letters represent significant differences among treatments ( p < 0.05).
when compared with any other carbohydrate.16,17,24 Although reduced with respect to sucrose, the products tested did induce enamel demineralisation. Hence, commercial sweeteners appear to induce less demineralisation than sucrose, but preserving some demineralising potential. This is particularly important since the sweeteners used for the experiments, and commonly consumed by the population, contain other potentially fermentable carbohydrates, including lactose (Table 1). In a previous study, we have shown that although it does have a demineralising potential, lactose seems to be less cariogenic than sucrose, when tested in a similar in vitro caries model.25 S. mutans biofilms treated with sweeteners containing stevia, sucralose and saccharine showed less biomass (Fig. 3) and viable cells (Fig. 4) than sucrose, at comparable levels with
Fig. 6 – Intracellular polysaccharide (IPS) production by each treatment. Sweetener-exposed biofilms were retrieved and the IPS measured as mg/mg of biofilm. Bars indicate mean values for each treatment W SD (n = 6). Different letters represent significant differences among treatments ( p < 0.05).
the negative control. The latter suggests that the sweeteners most likely do not act as bactericidal agents, but they do not promote cell proliferation as sucrose. Furthermore, sweeteners, except fructose, showed a general trend to decrease polysaccharide production (Fig. 5). EPSs are responsible for 40% of the composition of the dental biofilm, and they are one of the main virulence factors of the bacterial consortium, as they allow bacterial cell adhesion to the acquired pellicle, serve as scaffolds for biofilm maturation and increase the porosity of the structure allowing sugar diffusion within the biofilm.26 Mechanisms to explain the lower demineralising potential of the commercial sweeteners tested here may derive from a lack of metabolisation of the products by S. mutans. For example, sucralose has been deemed to be metabolically inert and thus non-cariogenic, at least in animal models.27 Since sucralose has been shown not to interfere with bacterial metabolism, a decrease in polysaccharide production is not expected to be observed by an inhibitory effect of the sweetener.28 IEPS production, however, did show a decrease when compared with sucrose. This may derive from a highly increased sucrose-induced polysaccharide production rather than from an inhibition caused by the sweeteners. As a matter of fact, an increased metabolic activity upon sucrose exposure has been previously reported.29 Yet, exposure to treatments was performed three times per day for 5 min for each exposure. Although this regime of sweetener exposure intended to mimic a moderate and probably common consumption, cariogenicity could increase upon an increased frequency of exposure. By itself and as the sole source of carbon, sucralose does not support bacterial growth28 and could be, therefore, considered as antibacterial. Although our data do show a decrease in bacterial count levels comparable with the caries-negative control, stevia, sucralose and saccharine still allow bacterial growth. The apparent contradiction may be explained by the other components contained in the commercial form of the products that support bacterial growth throughout the experiment (Table 1). Artificial sweeteners are in general
archives of oral biology 58 (2013) 1116–1122
hundreds of times sweeter than sucrose.7 Products sold in the market, for that reason, cannot be commercialised in their pure form. For instance, a tablet of a commercial product advertised as sucralose usually contains only about 10% of sucralose with higher proportions of other carbohydrates, usually lactose, starch or starch hydrolysates. Like lactose, starches alone30 or in combination with simple carbohydrates31–33 are potentially cariogenic. Indeed, we have shown that the combination of starch and sucrose causes more demineralisation than sucrose alone on dentine.34 In that regard, an interesting finding was that although lower, biomass was not dramatically reduced by the sweeteners (Fig. 3). Since bacterial counts and EPS were lower than sucrose, it could have been expected to find lower biomass in the biofilms exposed to the sweeteners. Lactose, contained in the commercial products, may be used by the S. mutans biofilm increasing biomass without affecting bacterial counts and EPS production. Regarding IPS, S. mutans biofilms exposed to the sweeteners showed a similar low production as the caries-negative control (Fig. 6). IPS may have been used by the bacteria treated with the artificial sweeteners to provide energy, as energy availability was not as abundant for the sweetener-exposed biofilms as it was for those treated with sucrose. Glucose contained in the culture medium and the exposure to the sweeteners three times a day to an alternative source of nutrient may have not been enough to supply the energy demands of the biofilm. It is important to highlight that the results of this study were obtained using an in vitro approach. Despite the fact that we used a relevant caries model that intends to resemble the oral environment, for example; by using a S. mutans biofilm intermittently exposed to a metobolisable substrate, this model only can serve as proof of principle and its limitations must be acknowledged. Indeed, the oral biofilm comprises a metabolically active and organised consortium of hundreds of bacterial species.35 Likewise, we used bovine enamel slabs for the experiments. This substrate has been reported to be quite similar to human enamel,36 but some differences still exist that may generate different results, had human tissues been used. Moreover, the model used here does not include saliva. Saliva has important anti-caries properties that may modulate the mere effect of the nutrients presented to the oral biofilm.37 Further in vivo studies must be conducted to actually replicate the complexity of the oral environment, including the complete dental biofilm and the presence of saliva. Hence, the results from clinical studies with sweeteners might be different from those reported here. Taken together, these results suggest that commercial sweeteners have lower cariogenic potential than sucrose. Fructose appears to maintain a comparatively higher demineralising potential than the other tested products. The latter seems to derive from the incapacity of S. mutans biofilms to metabolise the products with the same efficiency the bacterium ferments sucrose, despite the presence of other fermentable carbohydrates. Further studies must be performed to confirm these findings, but the results indicate that the use of artificial sweeteners is not as caries-safe as it is widely assumed and they should be carefully recommended.
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Contribution All authors have made substantive contribution to this study and/or manuscript, and all have reviewed the final paper prior to its submission.
Funding These investigations have been funded by the Chilean Government Grant Fondecyt Number: 11100005 to Rodrigo A. Giacaman.
Competing interests Authors declare no competing interests.
Ethical approval Not required.
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Available online at www.sciencedirect.com
journal homepage: http://www.elsevier.com/locate/aob
Cariogenic potential of commercial sweeteners in an experimental biofilm caries model on enamel Rodrigo A. Giacaman , Pı´a Campos, Cecilia Mun˜oz-Sandoval, Ramiro J. Castro Cariology Unit, Department of Oral Rehabilitation, University of Talca, Talca, Chile
article info
abstract
Article history:
Objective: Scarce evidence is available on the cariogenic potential of the widely used
Accepted 10 March 2013
commercial sweeteners. The aim of this study was to evaluate the effect of several sweeteners on enamel demineralisation and on the cariogenic properties of Streptococcus mutans
Keywords:
biofilms in an artificial caries model.
Dental caries
Methods: S. mutans-UA159 biofilms were cultured on bovine enamel slabs and exposed to
Sweetener
one of the following commercial sweeteners in tablet or powder form: stevia, sucralose,
Streptococcus mutans
saccharin, aspartame or fructose. Ten percent sucrose and 0.9% NaCl were used as caries-
Oral biofilm
positive and caries-negative controls, respectively. Slabs/biofilms were exposed to the
Sucralose
sweeteners three times per day for 5 min each time. After 5 days, biofilms were recovered
Stevia
to determine: biomass, bacterial counts and intra- and extracellular polysaccharides.
Aspartame
Surface microhardness was measured before and after the experiment to assess enamel
Fructose
demineralisation, expressed as percentage of surface hardness loss (%SHL). Data were
Saccharine
analysed using analysis of variance (ANOVA) and Bonferroni ( p < 0.05). Results: All tested commercial sweeteners, except fructose, showed less enamel demineralisation than sucrose ( p < 0.05). Only saccharine showed less biomass and intracellular polysaccharides than the rest of the groups ( p < 0.05). Stevia, sucralose and saccharine reduced the number of viable cells when compared with sucrose ( p < 0.05). All sugar alternatives reduced extracellular polysaccharide formation when compared with sucrose ( p < 0.05). Conclusions: Most commercial sweeteners appear to be less cariogenic than sucrose, but still retaining some enamel demineralisation potential. Products containing stevia, sucralose and saccharine showed antibacterial properties and seem to interfere with bacterial metabolism. Further studies are necessary to deepen these findings. # 2013 Elsevier Ltd. All rights reserved.
1.
Introduction
The detrimental role of sugars, particularly in dental caries is still a matter of controversy.1 Sucrose has been traditionally considered a highly cariogenic substrate for the oral biofilm. Upon fermentation by oral bacteria, sucrose molecules are transformed into energy and large amounts of acids.2 Thus,
frequent exposures to this carbohydrate create conditions for caries onset by promoting demineralisation. As an additional virulent mechanism, cariogenic bacteria populating the dental biofilm generate exopolysaccharides to create a protective environment against physiological antibacterial mechanisms of the mouth.3 Artificial sweeteners are becoming increasingly used to sweeten beverages, such as soda, juice, coffee and tea. In an
Corresponding author at: Escuela de Odontologı´a, Universidad de Talca, 2 Norte 685, Talca, Chile. Tel.: +56 71 201546; fax: +56 71 201761. E-mail address: [email protected] (R.A. Giacaman). 0003–9969/$ – see front matter # 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.archoralbio.2013.03.005
archives of oral biology 58 (2013) 1116–1122
era where obesity and overweight have become a serious health problem with large populations affected,4 sweeteners arise as a way to replace sucrose consumption to deal with this public-health matter. These products have been considered as safe in a recently published position article from the Academy of Nutrition and Dietetics.5 Several research articles have been published claiming a non-cariogenic or an apparent anti-caries potential of sweeteners.6 Sugar alcohols (polyols), that is, xylitol and sorbitol have been intensely tested for caries prevention, especially in chewing gums. Despite the multiple investigations into a putative anti-caries effect of xylitol or sorbitol as caries-safe sugar substitutes, these products are not the most frequently used by the food industry. Saccharine, a sulphamide, was the first and most used sweetener. From there, several other artificial sweeteners have been introduced over the time. Currently, five non- or low-caloric sweeteners have been approved by the Food and Drug Administration (FDA): aspartame, saccharine, acesulfame potassium, sucralose and neotame. Moreover, five non- or low-caloric sweeteners are generally recognised as safe by the same institution: sorbitol, xylitol, erythritol, tagatose and stevia.7 Generally assumed as caries-safe, sweetening beverages with sugar substitutes is becoming increasingly popular. One of the most investigated artificial sweeteners is sucralose. In its pure form, sucralose has been regarded as non-cariogenic8 and when combined with bulking ingredients is less cariogenic than sucrose. Likewise, scarce evidence, mostly in rats, suggests that aspartame would be non-cariogenic.9 Stevia is a highly used commercial sweetener derived from a plant, but only one study in rats with a controlled diet reports a non-cariogenic effect of the sweetener.10 Research on the effect on caries of the currently available commercial sweeteners is rather insufficient. Importantly, most of the available research on a presumptive anticariogenic or non-cariogenic effect of sweeteners comes from the pure chemical compound. Information on the cariogenicity of the sweeteners when in combination with bulking carbohydrates is more limited and may be of importance for enamel and dentine caries. Given the fact that evidence on the caries effect of sweeteners is still inconclusive11 and that limited data on the cariogenic potential of carbohydrate-containing products have been reported, the aim of this investigation was to test the cariogenic potential on enamel and the effect on Streptococcus mutans biofilms of several commercial sweeteners.
2.
Materials and methods
2.1.
Experimental design
(5) aspartame, (6) fructose and (7) 0.9% NaCl (caries-negative control). Biofilms were exposed to the different treatments for 5 min three times a day, simulating what can be considered a typical snack-consumption pattern. The culture medium was changed twice a day. Biofilms were separated from the slabs for analysis of biomass, viable bacteria, polysaccharide production and biofilm protein content. Final surface hardness was measured from the enamel slabs and the demineralisation produced throughout the experiment was estimated by the percentage of surface hardness loss (%SHL). Acidogenicity of the biofilms was estimated through medium pH, measured twice a day at each medium change. Samples were coded to allow blind measurements of the treatment groups. The whole experiment was repeated twice with each condition in triplicate (n = 6, per treatment).
2.2.
Enamel slabs
Bovine incisors were obtained, disinfected with a 5% NaOCl solution and stored in 0.9% NaCl (w/v) until use for no longer than 30 days. Slabs (4 mm 7 mm 1 mm) were prepared using diamond discs with a low-speed hand piece and Soflex polishing discs (3M, St. Paul, MN, USA). Initial SH was determined by three indentations 100 mm apart from each other, performed with a Knoop microindenter with a microhardness tester (402 MVD, Wolpert Wilson Instruments, Norwood, MA, USA) at 50 g for 5 s. Only those slabs of SH 340.87 24.4 kg mm 2 (n = 42) were included to avoid bias derived from using enamel with different initial SH values. Slabs were sterilised with ethylene oxide13 and covered with ultrafiltered (0.22 mm) pooled human saliva treated for 30 min with a protease inhibitor cocktail, to emulate the acquired pellicle on the enamel that further facilitates S. mutans adhesion.14 Slabs were suspended into the wells of a 24-well plate by means of a specially designed device made of orthodontic wire.
2.3.
S. mutans biofilms
Frozen stocks of S. mutans UA159 (kindly provided by Prof. J.A. Cury, UNICAMP, SP, Brazil) were reactivated in 1% glucosecontaining brain heart infusion (BHI; Merck, Darmstadt, Germany) at 37 8C and 10% CO2 for 18 h. Slabs were inoculated with S. mutans culture (optical density (OD) 0.8 at 600 nm) and 1% sucrose-containing medium to form the adherent biofilm14 and incubated for 8 h. Slabs were then maintained in BHI supplemented with 0.1 mM glucose for 24 h, which simulates glucose basal concentration in saliva.12
2.4.
S. mutans UA159 biofilms were grown using a previously described in vitro caries model,12 with modifications; three exposures per day for 5 min, instead of eight exposures for 1 min. Bovine enamel slabs served as substrates for S. mutans anaerobic biofilm formation for 5 days. Initial surface microhardness (SH) was assessed and the slabs were randomly sorted into seven treatment groups: (1) 10% sucrose (caries-positive control), (2) sucralose, (3) saccharine, (4) stevia,
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Sweetener application to the biofilms
Sweeteners in tablets or powder available in the Chilean market were used in this study. Although not in their pure state, the type of sweetener (groups 2–6, as indicated above) was that informed by the manufacturer on the label of the product. Besides the sweetener of interest, all the products contained additional bulking components. Manufacturer information on the composition of the products is presented in Table 1. Treatment solutions were prepared according to the product labelling to a concentration equivalent to 2 teaspoons
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archives of oral biology 58 (2013) 1116–1122
Table 1 – Commercial sweetener used and composition as informed by the manufacturers. Identification
Commercial name
Presentation
Active sweetener
Composition per portion (%)
Other components
Total carbohydrate (%)
14.9 6.18 14.5
Lactose L-Leucine Lactose L-Leucin Lactose L-Leucin Sodium bicarbonate Lactose Leucin Carboximetilcelulose N/A
69.8
Stevia
Cero K Stevia, Iansa1
Tablets
Sucralose
Cero K Sucralosa, Iansa1
Tablets
Stevia Sucralose Sucralose
Saccharine
Sacarina, Lider1
Tablets
Sodium saccharine
40.0
Aspartame
Naturalist, Laboratorios Prater1 Tablets
Aspartame
40.0
Fructose
Regimel, Fructosa1
Fructose
99.0
Powder
of sugar (8 g) in 100 ml of distilled water, considered as a common amount used to sweeten beverages. A confirmation of the sweetness of the solutions was performed by blind sips of the solution. Enamel was treated with the different treatments for 5 min three times per day at defined time points (8:30, 12:30 and 16:30). After treatment, the biofilms were washed three times with 0.9% NaCl and repositioned in the same position in the plate. Culture medium was replenished twice a day, before the first and after the last exposure. The exposure cycles were repeated to complete 5 days, which has been reported as sufficient to induce enamel demineralisation.12
2.5.
Biofilm acidogenicity
This study considered a pH-cycling model, obtained by exposing the biofilms three times during the day to the experimental sweeteners and leaving them unexposed overnight only with a basal glucose concentration. The latter produced a cyclic pH drop in the system, similar to the clinical situation. As a way to verify acid production by the biofilms formed on enamel, culture medium pH was measured inside each well twice per day before each medium change during the entire experiment12 by a microelectrode (HI 1083B, Hanna Instruments, Rumania) coupled to a portable pH metre (HI 9126-02, Hanna Instruments, Rumania).
2.6.
Enamel demineralisation
SH has been extensively used as a reliable methodology to evaluate demineralisation15 and it has been validated for enamel caries.16 Briefly, a second enamel SH (kg mm 2) reading was obtained after the experimental phase by a row of three indentations separated by 100 mm from those performed before the experimental phase. Mean values from the initial and final measurements were used to obtain %SHL calculated as follows: (mean initial SH mean final SH) 100/ initial SH.
2.7.
Biofilm analysis
Once the 5 days of the experimental phase were completed, enamel slabs were washed three times with 0.9% NaCl and sonicated for 30 s at 7 W to separate the biofilms from the slabs,12 which were stored for further analysis. The resulting
82.0 7.82
50.0
99.0
suspension containing the biofilm was further divided into aliquots to assess biomass, viable microorganisms,12 intraand extracellular polysaccharides (EPSs)17 and total proteins18 as previously described, but a brief explanation of each dependent variable follows below: (a) Biomass. To estimate biomass, dry weight was used.14 A volume of 200 ml of the biofilm suspension was transferred to a pre-weighted tube and incubated with 100% ethanol at 20 8C for 15 min. The resulting suspension was centrifuged (10 min at 5000 g and 4 8C) and the resulting pellet washed with 500 ml of 75% ethanol, centrifuged again and the pellet dried for 24 h in a desiccator. Biomass was obtained by subtracting the final weight from the initial weight of the empty tube and expressed in milligrammes. (b) Number of live bacteria from the biofilms. Using serial dilutions of the biofilm suspension in 0.09% NaCl (v/v), 10 ml was drop-plated on BHI agar plates in duplicate, incubated anaerobically for 24 h at 37 8C and colonies counted from the dilution that allowed visualisation of distinctively isolated colonies. Counting was corrected by the dilution factor and expressed as colony forming unit (CFU)/mg of biofilm dry weight.17 (c) Intra- and extracellular polysaccharides. Three different polysaccharide fractions were assessed: soluble (SEPS) and insoluble (IEPS) extracellular (EPS) and intracellular (IPS) polysaccharides from aliquots (200 ml) of the S. mutans biofilm suspension.17 The suspension was centrifuged (10,000 g for 5 min at 4 8C) to obtain SEPS from the supernatant. The resulting pellet was treated with 200 ml of 1 M NaOH, homogenised and centrifuged to obtain IEPS from the supernatant.19 The resulting pellet from the previous extraction containing the IPS was incubated with 200 ml 1 M NaOH for 15 min at 100 8C, centrifuged (10,000 g for 5 min at 4 8C) and the IPS concentration was measured from the supernatant. Each fraction from the supernatants was treated with three volumes of cold 100% ethanol and incubated for 30 min at 20 8C. Samples were immediately centrifuged and the resulting pellet was washed with cold 70% ethanol and centrifuged again. The new pellet was resuspended in 1 M NaOH and total carbohydrate concentration contained in each fraction was estimated by the sulphuric phenol method.20 Results were standardised by biofilm dry weight and expressed as
archives of oral biology 58 (2013) 1116–1122
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percentage of the different fractions of polysaccharides by milligramme of biomass.
2.8.
Statistical analysis
Normal distribution of the data was verified and values obtained for the different treatment groups were compared by analysis of variance (ANOVA) followed by the Tukey test, using the Statistical Package for Social Sciences (SPSS) 15.0 statistical software. Differences were considered significant at a 95% confidence level.
3.
Results
Biofilm acidogenicity is illustrated in Fig. 1. At 32 h from the beginning of the experiments, samples exposed to sucrose, fructose and aspartame had already decreased pH below 5.5 (enamel demineralisation critical pH), significantly lower than the remaining sweeteners. At 56 h, however, the sucrosetreated group showed the lowest pH drop ( p < 0.05). Although lower than sucrose, fructose induced more acidogenicity than the other sweeteners ( p < 0.05). At 80 and 104 h, the acidogenicity elicited by sucrose continued to be the highest ( p < 0.05), followed by fructose and aspartame ( p < 0.05). Saccharine, stevia and sucralose, on the other hand, induced significantly lower acidogenicity throughout the experiment. Regarding enamel demineralisation, all the tested sweeteners showed lower %SHL ( p < 0.05) as compared with the caries-positive control (Fig. 2). Fructose, however, induced more demineralisation than the other sweeteners ( p < 0.05). When S. mutans biofilm properties were analysed, only saccharine-treated samples showed significantly lower biomass than the rest of the experimental products, albeit a trend to induce less biomass for stevia, and sucralose (Fig. 3). Stevia, sucralose and saccharine retrieved significantly less viable cells from the biofilms when compared with the other sweeteners, with similar counts to the negative control (Fig. 4). Likewise, polysaccharide analysis indicated that, except for fructose, all the sweeteners induced lower IEPS production than sucrose ( p < 0.05) (Fig. 5). A similar trend was observed for SEPS (data not shown). Finally, all treatments,
Sucrose
Medium pH
7.0
Stevia
Sucralose
Saccharine
Aspartame
Fructose
NaCl
6.5 6.0 5.5 5.0 4.5
0
8
16
24
32
40
48
56
64
72
80
88
96
104 112 120
Time Points
Fig. 1 – Acidogenicity of S. mutans biofilms formed on enamel an exposed to the sweeteners. Plot shows mean pH of the culture medium of each group (as indicated). Measurements were performed after 24 h of biofilm formation, twice per day, at defined times (as indicated).
Fig. 2 – Enamel surface hardness loss (%SHL) by sweetener tested. Bars represent mean of the %SHL of the slabs (n = 6). Error bars indicate SD. Different letters represent significant differences among treatments ( p < 0.05).
Fig. 3 – Biomass induced by each experimental condition. Plot depicts mean biomass (mg) for each condition W SD (n = 6). Different letters represent significant differences among treatments ( p < 0.05).
including the negative control, showed significantly lower IPS formation (Fig. 6).
4.
Discussion
Although not new, sweeteners are increasingly consumed worldwide. The cariogenic potential of these nutritive and non-nutritive products in their commercial form has not been elucidated. Results from the present study suggest a less cariogenic effect for all tested products when compared with sucrose. Indeed, most of the sweeteners tested, only fructose and aspartame excluded, induced lower pH drop than sucrose (Fig. 1). Similar results have been previously published.21,22 As we found here, fructose had been reported to be as acidogenic as sucrose, in vitro.23 The latter is important as diabetic patients are generally advised to replace sucrose by fructose. As expected and in consistency with the acidogenicity data, sweeteners induced lower enamel demineralisation than sucrose, but still higher than the negative control (Fig. 2). Since no sweetener was capable of demineralising the enamel at levels comparable to those induced by sucrose, these findings confirm the higher cariogenic potential of sucrose
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Fig. 4 – Viable microorganisms from S. mutans biofilms. Bacterial cells retrieved from each sweetener-exposed biofilm were counted and expressed as CFU/biofilm dry weight (mg). Bars show mean counting for each condition (n = 6, triplicate of two independent experiments). Error bars indicate SD. Different letters represent significant differences among treatments ( p < 0.05).
Fig. 5 – Insoluble extracellular polysaccharides (IEPS) produced by the biofilms. IEPS produced by the different conditions were measured and expressed as mg/mg of biofilm. Plot shows mean of each treatment in triplicate of two independent experiments W SD (n = 6). Different letters represent significant differences among treatments ( p < 0.05).
when compared with any other carbohydrate.16,17,24 Although reduced with respect to sucrose, the products tested did induce enamel demineralisation. Hence, commercial sweeteners appear to induce less demineralisation than sucrose, but preserving some demineralising potential. This is particularly important since the sweeteners used for the experiments, and commonly consumed by the population, contain other potentially fermentable carbohydrates, including lactose (Table 1). In a previous study, we have shown that although it does have a demineralising potential, lactose seems to be less cariogenic than sucrose, when tested in a similar in vitro caries model.25 S. mutans biofilms treated with sweeteners containing stevia, sucralose and saccharine showed less biomass (Fig. 3) and viable cells (Fig. 4) than sucrose, at comparable levels with
Fig. 6 – Intracellular polysaccharide (IPS) production by each treatment. Sweetener-exposed biofilms were retrieved and the IPS measured as mg/mg of biofilm. Bars indicate mean values for each treatment W SD (n = 6). Different letters represent significant differences among treatments ( p < 0.05).
the negative control. The latter suggests that the sweeteners most likely do not act as bactericidal agents, but they do not promote cell proliferation as sucrose. Furthermore, sweeteners, except fructose, showed a general trend to decrease polysaccharide production (Fig. 5). EPSs are responsible for 40% of the composition of the dental biofilm, and they are one of the main virulence factors of the bacterial consortium, as they allow bacterial cell adhesion to the acquired pellicle, serve as scaffolds for biofilm maturation and increase the porosity of the structure allowing sugar diffusion within the biofilm.26 Mechanisms to explain the lower demineralising potential of the commercial sweeteners tested here may derive from a lack of metabolisation of the products by S. mutans. For example, sucralose has been deemed to be metabolically inert and thus non-cariogenic, at least in animal models.27 Since sucralose has been shown not to interfere with bacterial metabolism, a decrease in polysaccharide production is not expected to be observed by an inhibitory effect of the sweetener.28 IEPS production, however, did show a decrease when compared with sucrose. This may derive from a highly increased sucrose-induced polysaccharide production rather than from an inhibition caused by the sweeteners. As a matter of fact, an increased metabolic activity upon sucrose exposure has been previously reported.29 Yet, exposure to treatments was performed three times per day for 5 min for each exposure. Although this regime of sweetener exposure intended to mimic a moderate and probably common consumption, cariogenicity could increase upon an increased frequency of exposure. By itself and as the sole source of carbon, sucralose does not support bacterial growth28 and could be, therefore, considered as antibacterial. Although our data do show a decrease in bacterial count levels comparable with the caries-negative control, stevia, sucralose and saccharine still allow bacterial growth. The apparent contradiction may be explained by the other components contained in the commercial form of the products that support bacterial growth throughout the experiment (Table 1). Artificial sweeteners are in general
archives of oral biology 58 (2013) 1116–1122
hundreds of times sweeter than sucrose.7 Products sold in the market, for that reason, cannot be commercialised in their pure form. For instance, a tablet of a commercial product advertised as sucralose usually contains only about 10% of sucralose with higher proportions of other carbohydrates, usually lactose, starch or starch hydrolysates. Like lactose, starches alone30 or in combination with simple carbohydrates31–33 are potentially cariogenic. Indeed, we have shown that the combination of starch and sucrose causes more demineralisation than sucrose alone on dentine.34 In that regard, an interesting finding was that although lower, biomass was not dramatically reduced by the sweeteners (Fig. 3). Since bacterial counts and EPS were lower than sucrose, it could have been expected to find lower biomass in the biofilms exposed to the sweeteners. Lactose, contained in the commercial products, may be used by the S. mutans biofilm increasing biomass without affecting bacterial counts and EPS production. Regarding IPS, S. mutans biofilms exposed to the sweeteners showed a similar low production as the caries-negative control (Fig. 6). IPS may have been used by the bacteria treated with the artificial sweeteners to provide energy, as energy availability was not as abundant for the sweetener-exposed biofilms as it was for those treated with sucrose. Glucose contained in the culture medium and the exposure to the sweeteners three times a day to an alternative source of nutrient may have not been enough to supply the energy demands of the biofilm. It is important to highlight that the results of this study were obtained using an in vitro approach. Despite the fact that we used a relevant caries model that intends to resemble the oral environment, for example; by using a S. mutans biofilm intermittently exposed to a metobolisable substrate, this model only can serve as proof of principle and its limitations must be acknowledged. Indeed, the oral biofilm comprises a metabolically active and organised consortium of hundreds of bacterial species.35 Likewise, we used bovine enamel slabs for the experiments. This substrate has been reported to be quite similar to human enamel,36 but some differences still exist that may generate different results, had human tissues been used. Moreover, the model used here does not include saliva. Saliva has important anti-caries properties that may modulate the mere effect of the nutrients presented to the oral biofilm.37 Further in vivo studies must be conducted to actually replicate the complexity of the oral environment, including the complete dental biofilm and the presence of saliva. Hence, the results from clinical studies with sweeteners might be different from those reported here. Taken together, these results suggest that commercial sweeteners have lower cariogenic potential than sucrose. Fructose appears to maintain a comparatively higher demineralising potential than the other tested products. The latter seems to derive from the incapacity of S. mutans biofilms to metabolise the products with the same efficiency the bacterium ferments sucrose, despite the presence of other fermentable carbohydrates. Further studies must be performed to confirm these findings, but the results indicate that the use of artificial sweeteners is not as caries-safe as it is widely assumed and they should be carefully recommended.
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Contribution All authors have made substantive contribution to this study and/or manuscript, and all have reviewed the final paper prior to its submission.
Funding These investigations have been funded by the Chilean Government Grant Fondecyt Number: 11100005 to Rodrigo A. Giacaman.
Competing interests Authors declare no competing interests.
Ethical approval Not required.
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