Odontology DOI 10.1007/s10266-014-0154-5
ORIGINAL ARTICLE
Fatty acid effect on sucrose-induced enamel demineralization and cariogenicity of an experimental biofilm–caries model Rodrigo A. Giacaman • Pascale Jobet-Vila Cecilia Mun˜oz-Sandoval
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Received: 24 October 2013 / Accepted: 17 March 2014 Ó The Society of The Nippon Dental University 2014
Abstract Based on scarce evidence, fatty acids have been described as anticariogenic. The aim was to evaluate the effect of different types of fatty acids on enamel demineralization and on the cariogenic properties of Streptococcus mutans biofilms on a biofilm/caries model. Mature biofilms of S. mutans UA159 growing on bovine enamel slabs were exposed to 10 % sucrose for 5 min, 3 times per day followed by exposure to a panel of free fatty acids, including monounsaturated (oleic), polyunsaturated (linoleic) and saturated (stearic) fatty acids, in concentrations of 0.1, 1 and 10 mM for five additional minutes. Enamel demineralization was determined before and after the experiments by microhardness. Slabs were retrieved to analyze biofilm biomass, viable bacterial counts and polysaccharide production. Biofilms exposed to sucrose, followed by oleic and linoleic acids, showed less demineralization than sucrose alone (p \ 0.05). Biomass, S. mutans colonies and insoluble extracellular polysaccharide production were reduced from the biofilms treated with oleic and linoleic fatty acids (p \ 0.05). No differences with the positive control were observed with the saturated stearic acid. Poly and monounsaturated fatty acids presented to S. mutans biofilms after a cariogenic challenge appear to reduce demineralization on enamel and to interfere with cariogenicity of S. mutans biofilms. Keywords Caries Streptococcus mutans Fatty acids Biofilm Antibacterial agents
R. A. Giacaman (&) P. Jobet-Vila C. Mun˜oz-Sandoval Cariology Unit, Department of Oral Rehabilitation and Interdisciplinary Excellence Research Program on Healthy Aging (PIEI-ES), University of Talca, 2 Norte 685, Escuela de Odontologı´a, Talca, Chile e-mail: [email protected]
Introduction Caries, in its current conception, is considered as a biofilmand sucrose-dependent disease [1]. It has been clearly established that fermentable carbohydrates from the diet play a key role in the onset of the lesions [2, 3]. Sucrose, in particular, has been blamed as the most cariogenic of these molecules. This simple sugar is rapidly metabolized by the oral biofilm leading to high amount of demineralizing acids and to the synthesis of extracellular polysaccharides (EPS) [4], which in turn favors bacterial adhesion, aggregation and co-aggregation with other bacterial species [5]. Yet, a linear association between consumption of sucrose-containing foods and caries has been challenged by several studies [2, 6]. Reasons for this rather weak association may derive from multiple factors, including biological, behavioral and genetic factors, technical issues from the studies and the multifactorial etiology of the disease. In that context, we speculated that the modulatory role of other dietary nutrients, ubiquitous to normal diets and usually not considered in sugar-based studies, might be important to explain this apparently inconsistent association between sucrose intake and caries. In the same line of thoughts, an anticariogenic effect of fatty acids and lipids has been claimed for a long time [7, 8]. Mechanisms associated to the putative anticaries activity of fatty acids are diverse and include: antimicrobial activity [9], bacteriostatic properties [10, 11] and the lack of metabolization of fatty acids by the bacteria from the oral biofilm [12]. Most of the limited studies about the role of fatty acids on caries derive from experimental animal studies. Thus, animals fed with fatrich diets reduce caries incidence when compared with those treated with sucrose-rich diets [11, 13]. In the case of fatty acids, lauric acid has shown to be highly bacteriostatic on Gram-positive bacteria [9]. More recently, an
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antibacterial activity against S. mutans of several free fatty acids in low concentrations, including linoleic acid and oleic acid, has been confirmed by several reports [14–16]. An inhibition of nutrient transport through the cell membrane and cell adhesion by fatty acids has been proposed [11]. Other alternative mechanisms to explain the antibacterial activity of fatty acids include inhibition of enzyme activity, interference with oxidative phosphorylation, leakage of intracellular products, generation of peroxidation and auto-oxidation of degradation products and direct cell lysis [17]. Yet, insufficient evidence on this topic precludes from drawing more definitive conclusions and more research appears to be needed. Normal human diet contains all the macronutrients combined with some of them having anticaries properties. With that conception in mind, we conducted this study as a first scrutiny to model the potential anticaries effect of free fatty acids in a highly cariogenic environment provided by sucrose and modulated by the subsequent exposure to free fatty acids. Given the limited evidence available and the potential for a protective role in caries, we aimed to investigate whether immediate exposure to different types of free fatty acids after cariogenic challenges with sucrose reduces enamel demineralization and affects S. mutans biofilm properties on an in vitro pH-cycling biofilm caries model.
Materials and methods Enamel slabs Bovine incisors were obtained, initially disinfected with a 5 % NaOCl solution and stored in 0.9 % NaCl (w/v) until use for up to 30 days. Slabs (4 9 7 9 1 mm) were prepared using diamond disks and a low-speed handpiece and Soflex polishing disks (3M, St. Paul, MN, USA). Initial surface microhardness (SH) was determined by three Knoop indentations placed in one of the lateral aspects of the slab, apart 100 lm from each other (402 MVD, Wolpert Wilson Instruments, USA) at 50 g for 5 s and they were digitally assessed by the built-in reader in the microindenter. To maintain similar initial SH across the slabs, only those with SH 340.87 ± 24.4 kg/mm2 (n = 66) were included. Slabs were sterilized with ethylene oxide [18] and covered with ultrafiltered (0.22 lm) pooled human saliva for 30 min. Stimulated whole saliva was obtained from two healthy donors. To conserve its proteins, whole saliva was mixed with a buffer containing phenylmethylsulfonyl fluoride (PMSF) 1:100 (v/v), a protease inhibitor cocktail. Saliva coating of the slabs allows formation of an acquired pellicle on the enamel for S. mutans initial adherence [19]. Slabs were suspended into the wells of a
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24-well plate by means of a specially designed device made of stainless steel wire. Streptococcus mutans biofilms Frozen stocks of S. mutans UA159 (kindly provided by Prof. J.A. Cury, UNICAMP, SP, Brazil) were reactivated in BHI medium supplemented with 1 % glucose (Merck, Darmstadt, Germany) at 37 °C and 10 % CO2 for 18 h. Once they reached OD 0.8 (600 nm) [20], slabs were inoculated with the bacterial culture and grown for 8 h in 1 % sucrose-supplemented BHI to create an initial adhered biofilm [19]. To achieve maturity before treatment, biofilms on the slabs were allowed to grow for additional 16 h with 0.1 mM glucose, which is similar to the glucose basal concentration in saliva [21]. Fatty acid exposure to the sucrose-challenged biofilms A modified and previously validated in vitro caries model of S. mutans biofilms was used [21]. We carried out three sucrose exposures per day of 5 min each; instead of eight of 1 min each. With the biofilms mature and visibly thick after 48 h of growth, slabs were randomly allocated to one of the eleven groups and treated three times per day, at defined times (8:30, 12:30 and 16:30). Each slab with the mature biofilm was exposed to 10 % sucrose for 5 min followed by five additional minutes with treatment suspensions in 0.9 % NaCl of three different 18-carbon fatty acids; Oleic (monounsaturated) (Calbiochem, San Diego, CA, USA), linoleic (polyunsaturated) (Calbiochem) or stearic (saturated) (AppliChem, Darmstadt, Germany) fatty acids, at three different concentrations; 0.1, 1 and 10. One group exposed to 10 % sucrose followed by 0.9 % NaCl, but not to fatty acids, and another exposed only to 0.9 % NaCl, in two cycles of 5 min each, served as positive and negative control, respectively. After treatment, biofilms were washed three times with 0.9 % NaCl and returned to the same position in the plate. Culture medium was changed twice per day (9:00 a.m. and 5:00 p.m.). 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). Biofilm acidogenicity As a way to verify acid production by the biofilms formed on enamel, culture medium pH was measured inside each well by a microelectrode (HI 1083B, Hanna instruments, Rumania) coupled to a portable pH meter (HI 9126-02, Hanna instruments, Rumania). Measurements were carried out twice per day in the spent medium, before each medium change and during the entire length of the experiment [21].
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Enamel demineralization
Intra- and extracellular polysaccharides
After the experimental period of 5 days, biofilms were sonicated for 30 s to separate biofilms from the dental substrate and to release adhered bacterial cells. Slab’s SH was measured to estimate demineralization produced throughout the 5 days of the experiment, length reported as sufficient to induce enamel demineralization [22]. SH has been extensively used as a reliable methodology to evaluate demineralization [23] and it has been validated for enamel caries [24]. Briefly, a new enamel SH reading (kg/mm2) was obtained after the experimental phase by a row of three indentations separated by 100 lm from those performed before the experimental phase, using the microindenter. Mean values from the initial and final measurements were used to obtain the percentage of SH loss (%SHL) calculated as: (mean initial SH - mean final SH) 9 100/initial SH.
To assess polysaccharide formation by the S. mutans biofilms, three different fractions were analyzed: soluble (SEPS) and insoluble (IEPS) extracellular and intracellular (IPS) polysaccharides [25]. An aliquot of 200 lL from the biofilm suspension was centrifuged (10,000g for 5 min at 4 °C) to obtain SEPS from the supernatant. For IEPS, the pellet obtained from the previous step was treated with 200 lL of 1 M NaOH, homogenized, centrifuged and stored to extract the polysaccharides from the supernatant [26]. Finally, the pellet from the previous step containing the IPS was incubated with 200 lL 1 M NaOH for 15 min at 100 °C and centrifuged (10,000g for 5 min at 4 °C). Supernatant was used to measure the concentration of IPS. The three supernatants from each extraction step were separately treated with 3 volumes of cold 100 % ethanol and incubated for 30 min at -20 °C. Samples were immediately centrifuged and the resulting pellet was washed with cold 70 % ethanol and centrifuged again (10,000g for 5 min at 4 °C). The resulting pellet of each fraction was resuspended in 1 M NaOH and total carbohydrate concentration was estimated by the sulfuric phenol method [27]. Results were normalized by biofilm dry weight and expressed as percentage of polysaccharides by mg of biomass.
Biofilm analysis After the experimental phase, slabs were washed three times with 0.9 % NaCl and biofilms were detached by shaking the slabs for 30 s with a vortex mixer. A biofilm suspension was formed and aliquoted to evaluate the following biofilm properties: biomass, viable microorganisms [21] and intra and extracellular polysaccharides [25]. A description of each biofilm-dependent variable is briefly provided. Biomass Dry weight of the biofilm eluted from the slab was used to estimate biomass content [19]. Thus, 200 lL of the biofilm suspension was transferred to a pre-weighted tube and incubated with 100 % ethanol at -20 °C for 15 min, centrifuged (10 min at 5,000g and 4 °C) and the resulting pellet was washed with 500 lL of 75 % ethanol. After a second centrifugation, the pellet was dried for 24 h in a desiccator. Biomass was calculated by subtracting the final weight from the initial weight of the empty tube. Biomass content was expressed as mg per mL of biofilm suspension. Number of live bacteria from the biofilms From the biofilm suspension, serial dilutions of the biofilm suspension in 0.9 % NaCl (v/v) were drop-plated on BHI agar plates in duplicate, incubated anaerobically for 24 h at 37 °C and colonies were counted from the dilution that allowed visualization of distinctively isolated colonies. Data of counting was calibrated by the dilution factor and expressed as CFU/mg of biofilm dry weight [25].
Statistical analysis To determine whether the data had parametric distribution, the Kolmogorov–Smirnov test was performed. Means obtained from the different groups in each variable were compared by ANOVA followed by Bonferroni test, using SPSS 15.0 statistical software. Differences were considered significant if p \ 0.05.
Results Biofilms exposed to sucrose alone showed a general trend to significantly decrease medium pH more than biofilms exposed to sucrose first followed by the different fatty acids (Fig. 1a–c). Higher fatty acid concentration led to higher pH values. Higher pH values at an early stage of the experiment were observed for the oleic (Fig. 1a) and linoleic (Fig. 1b) acids in a dose-dependent manner, but not for the saturated fatty acid (Fig. 1c). Regarding demineralization, 1 and 10 mM oleic and linoleic acids exposed to S. mutans biofilms significantly reduced sucrose demineralization as compared to the positive control (Fig. 2). Conversely, stearic acid exposure after the cariogenic challenge failed to show changes in the %SHL with respect to 10 % sucrose only (p [ 0.05).
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Odontology Fig. 1 S. mutans biofilm acidogenicity. Biofilms on the slabs were exposed to sucrose followed by oleic (a), linoleic (b) or stearic acid (c) for 5 min, 39/day. Medium pH was measured 29/day throughout the experiment. Each point in the plot represent mean of 2 independent experiments in triplicate slabs. Medium changes are represented by the arrows. Error bars show SD. Different letters at each time point represent statistical significant differences (p \ 0.05)
Table 1 illustrates the effect of the tested free fatty acids on S. mutans biofilms. Biofilms exposed to 10 mM oleic and linoleic acids showed less biomass than the cariespositive control and the rest of the groups (p \ 0.05). Despite a trend for some antibacterial activity of linoleic acid, only 10 mM oleic acid reduced S. mutans counts when compared with sucrose exposure without fatty acids (p \ 0.05). When compared to the positive control, the highest concentration (10 mM) of oleic and linoleic acid decreased IEPS formation. Saturated stearic acid exposure did not affect any of the S. mutans biofilm properties. None
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of the fatty acids tested appeared to interfere with the effect of sucrose on SEPS and IPS formation by the biofilm.
Discussion It is unquestionable that lipids, triglycerides and fatty acids are non-cariogenic when compared with carbohydrates, particularly sucrose. The question of whether they decrease cariogenicity of sugars if they are presented to a cariogenic biofilm is yet to be clarified. Although several
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Fig. 2 Enamel demineralization in response to fatty acid exposure. Biofilms and slabs were exposed to 10 % sucrose for 5 min followed by either fatty acid for 5 min 39/day. Medium was changed 29/day. Surface microhardness (SH) was measure before and after the experiment to assess percentage of SH loss (%SHL) for slabs exposed
to oleic, linoleic and stearic acid, as indicated each in concentrations of 0.1, 1 and 10. 10 % sucrose was used as (?) control and 0.9 % as NaCl(-) control. Bars represent mean of 2 independent experiments in triplicate (n = 6). Error bars show SD. *p \ 0.05; **p \ 0.001 with respect to the positive control (10 % sucrose ? 0.9 % NaCl)
Table 1 Biofilm properties due to sucrose and free fatty acid exposure Biomass (mg)
Bacteria (CFU/mg biomass)
IEPS (%/mg biomass)
SEPS (%/mg biomass)
IPS (%/mg biomass)
10 % Sucrose ? 0.9 % NaCl
2.50 (0.44)
2.91E?10 (5.24E?09)
5.50 (0.95)
2.50 (0.44)
3.91 (0.72)
10 % Sucrose ? 0.1 mM oleic acid
2.17 (0.29)
2.12E?10 (2.80E?09)
5.18 (1.05)
2.17 (0.29)
3.74 (1.46)
10 % Sucrose ? 1 mM oleic acid
1.92 (0.40)
2.03E?10 (4.16E?09)
5.07 (1.51)
1.92 (0.40)
3.73 (0.58)
10 % Sucrose ? 10 mM oleic acid
1.29 (0.44)**
1.18E110 (6.80E109)*
3.30 (0.34)*
1.29 (0.44)
2.96 (0.91)
10 % Sucrose ? 0.1 mM linoleic acid
2.00 (0.58)
2.79E?10 (9.66E?09)
5.30 (2.62)
2.00 (0.58)
3.46 (1.36)
10 % Sucrose ? 1 mM linoleic acid
1.84 (0.15)
2.05E?10 (6.73E?09)
4.75 (0.99)
1.84 (0.15)
3.51 (0.92)
10 % Sucrose ? 10 mM linoleic acid
1.25 (0.44)**
2.06E?10 (1.06E?10)
2.57 (1.13)*
1.25 (0.44)
2.60 (1.53) 3.85 (1.01)
10 % Sucrose ? 0.1 mM stearic acid
2.17 (0.53)
3.21E?10 (7.93E?09)
5.12 (1.53)
2.17 (0.53)
10 % Sucrose ? 1 mM stearic acid
1.84 (0.72)
3.09E?10 (8.45E?09)
5.40 (0.99)
1.84 (0.72)
4.07 (0.82)
10 % Sucrose ?10 mM stearic acid
1.92 (0.29)
3.36E?10 (7.12E?09)
4.92 (0.83)
1.92 (0.29)
3.86 (0.86)
0.9 % NaCl ? 0.9 % NaCl
0.50 (0.40)**
7.90E109 (4.25E109)*
1.01 (0.53)**
0.93 (0.60)*
0.95 (0.34)*
Mean (SD), n = 6 SEPS soluble extracellular polysaccharides, IEPS insoluble extracellular polysaccharides, IPS intracellular polysaccharides * p \ 0.05; ** p \ 0.001 with respect to the positive control (10 % sucrose ? 0.9 % NaCl)
investigations have suggested an anticaries effect of free fatty acids through an antibacterial mechanism, to the best of our knowledge, this is the first time this fact has been tested using a relevant biofilm–caries model. The chief finding from this study was that S. mutans biofilms’ exposure to sucrose first and then to unsaturated free fatty acids reduces the cariogenicity induced by sucrose. Remarkably, the effect was not only on the acidogenicity (Fig. 1) and demineralization of the enamel (Fig. 2), but also on the biofilm properties (Table 1). Although the drop in pH may seem low, it was enough to lead to significant variations in surface microhardness (Fig. 2). It is important to mention that the reduction in acidogenicity and in
demineralization may not be of clinical relevance and definitive conclusions must be obtained only after clinical studies are conducted. The observed anticaries effect of the free fatty acids might be overcome, once the variability of the oral environment is present. Early investigations from the beginning of the past century on the role of diet on caries stated that although necessary, sucrose is not the sole dietary factor involved in the etiology of caries and dietary lipids may result in an anticariogenic nutrient when combined with carbohydrates (revised in Kabara JJ, 1986 [8]). The demineralization inhibition induced by the presence of fatty acids, as observed here is consistent with the results from classic studies using animal models [28, 29].
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When fatty acids are included in a sugar-containing diet in rats, caries scores significantly decrease when compared with the same diet without the fatty acids [11]. Fatty acids are carboxylic acids with a long aliphatic tail. Most naturally occurring fatty acids have a chain of an even number of carbon atoms, from 4 to 28, which may be either saturated or unsaturated [30]. An antibacterial activity of free fatty acids has been long recognized [31]. A higher antibacterial effect of these substances has been described on Gram-positive than Gram-negative microorganism and when the fatty acid chain is longer [31] and more unsaturated [9]. Likewise, although the monounsaturated (oleic) and the polyunsaturated (linoleic, an omega-6 fatty acid) acids reduced demineralization at two of the concentrations tested, linoleic acid showed lower demineralization at a lower concentration (Fig. 2). Yet, a broad range of fatty acids have shown antibacterial properties against many different Gram-positive and Gram-negative microorganisms [17]. Hence, to compare the fatty acids, we used same carbon chain length fatty acids (C18), but with different saturation. As expected, polyunsaturated and monounsaturated fatty acids resulted in higher antibacterial activity than the saturated stearic acid (Table 1). Despite the reported higher antibacterial effect of unsaturated fatty acids, an in vitro study showed activity of saturated medium chain fatty acids and their esters against the growth and metabolic activities of S. mutans [10]. The study showed that C8 to C15 fatty acids were more effective in inhibiting S. mutans than those with longer carbon chains. The saturated fatty acid used here, stearic acid, is a long chain (C18) fatty acid, which could explain why it showed neither reduction in acidogenicity (Fig. 1), demineralization (Fig. 2), nor an effect on the biofilm properties (Table 1). Importantly, the acidogenicity data were obtained by monitoring pH only twice per day. Thus, there might be several hours in which pH curve from some of the concentrations tested was below the critical pH for enamel demineralization (5.5) and the lower concentrations were above this threshold (Fig. 1a, b). This differential time of exposure to a demineralizing medium might have explained the changes observed in %SHL (Fig. 2). In the case of stearic acid, none of the concentrations tested was capable to maintain pH values above the critical pH (Fig. 1c), which is consistent with the %SHL data (Fig. 2). Our results are also consistent with more recent investigations showing that n-6, n-7 and n-9 fatty acids and their esters have inhibitory activity against several oral pathogenic bacteria [15]. Like in our study (Table 1), oleic and linoleic unsaturated fatty acids had a strong antibacterial effect on S. mutans and stearic saturated free fatty acid failed to show S. mutans inhibitory activity, confirming our findings [15]. In addition to their cardiovascular and systemic benefits when
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consumed in the diet [32], n-3 fatty acids have been also described as having antibacterial activity against oral microorganisms, including S. mutans [14]. We decided to test fatty acids of a similar chain length among them, but further studies should incorporate a broader range of fatty acids with this experimental approach. Oleic and linoleic fatty acid exposure showed less biomass than sucrose alone (Table 1). Since biofilm biomass comprises bacterial cells and polysaccharides [33], we speculate that these unsaturated fatty acids act as both, bacterial killers (Table 1) and metabolic inhibitor substances, resulting in lower EPS production (Table 1). Indeed, growth inhibition or the direct killing of bacteria has been described as the major antibacterial mechanisms of fatty acids [17]. The exact mechanisms of either bactericidal or bacteriostatic activity of fatty acids remain unclear. It has been proposed that fatty acids act on the bacterial cell membrane by detergent properties due to their amphipathic structure. This interaction creates pores that can solubilize the membrane disrupting its structural integrity and interfering with the electron transport chain and disrupting oxidative phosphorylation [34, 35]. Moreover, other processes may take place to contribute to bacterial growth inhibition or death, such as lysis, inhibition of enzyme activity, impairment of nutrient uptake and generation of toxic by-products [17]. In the case of oral microorganisms, it has been suggested that fatty acids would form micelles around the individual bacteria interfering with the adhesion of the cell to the enamel and also with transport across the cell membrane affecting metabolism and, therefore, acid production [11]. Furthermore, oleic and linoleic acids, but not stearic, contained in cocoa bean husk showed two mechanism to act against S. mutans [36]. On the one hand, the fatty acids kill bacteria and secondly, they exhibit an antiglucosyltransferase (GTF) activity, which also decrease caries in infected rats. The enzyme GTF is crucial in the pathogenesis of caries, as it takes part in the synthesis of adhesive glucans [4], one of the most relevant virulence factors of S. mutans [33]. Our data support the hypothesis that free unsaturated fatty acids affect bacterial metabolism, mainly on GTFs activity, with an added antimicrobial activity on S. mutans biofilms, but only at higher doses (Table 1). This design may be considered as a pH-cycling model. By exposing biofilms three times per day to a cariogenic challenge, represented by sucrose, and then keeping them overnight exposed only to a basal glucose concentration in fresh medium, this approach intends to mimic the pH variations to which the dental biofilm is exposed in the oral environment. The fatty acids used here are found in various commonly consumed foods. Oleic acid represents more than 63 and 69 % of the total constituents of olive oil and avocado, respectively. Linoleic acid represents 59 % of corn oil. The amount of fatty acids in foods is highly variable. For example, while a commercial cereal bar contains 0.5 and
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0.2 g of monounsaturated and polyunsaturated fatty acids, respectively, other bar has 2.1 and 0.1 g of monounsaturated and polyunsaturated fatty acids, respectively. The most efficient fatty acids to antagonize demineralization induced by sucrose and to have antagonist activity against the biofilm were oleic and linoleic at 10 mM concentration, which is equivalent to 2.8 g of mono and 2.7 g of polyunsaturated fatty acids. The concentrations of fatty acids used here, therefore, appear reasonable for a potential use of the fatty acids as anticaries agents. It has been reported that the structure of the fatty acids determines their antibacterial activity. Thus, free fatty acids are apparently more active than the fatty acids bound to their esters [17]. This assertion has been challenged in recent studies showing an equally effective activity of fatty acids and their esters against S. mutans in vitro [15]. In this study, we used free fatty acids, knowing that dietary lipids are not mainly found as free fatty acids. Oral bacteria, specifically S. mutans, are endowed with enzymes that have been reported to have lipolytic activity [37], nonetheless. Hence, potential nutritional and clinical applications may take both approaches; as the use of purified unsaturated free fatty acids or as dietary lipids. The state of the research on this field precludes the immediate use of this evidence in the clinic, albeit it certainly supports further investigation. Additional in vivo studies would be required to show a clinically relevant anticaries effect of unsaturated free fatty acids.
Conclusion Linoleic and oleic free fatty acids seem to interfere with the cariogenicity of S. mutans biofilms, when they are presented after highly cariogenic challenges with sucrose. Saturated stearic fatty acid shows no effect in inhibiting sucrose cariogenicity. The effect of the unsaturated free fatty acids appears to be metabolic, rather than antibacterial, albeit more studies are necessary to elucidate the mechanism. Acknowledgments Part of this work was presented at the 59th ORCA Congress, Cabo Frio, Brazil, 2012. Authors thank Ms. Gloria Correa-Beltra´n for statistical assistance. This manuscript was submitted in partial fulfillment of the requirements for the DDS degree by PJ. Conflict of interest These investigations were funded by a Chilean government grant Fondecyt #11100005 to Rodrigo A: Giacaman. The authors declare that they have no conflict of interest.
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31. Nieman C. Influence of trace amounts of fatty acids on the growth of microorganisms. Bacteriol Rev. 1954;18(2):147–63. 32. Hunter JE, Zhang J, Kris-Etherton PM. Cardiovascular disease risk of dietary stearic acid compared with trans, other saturated, and unsaturated fatty acids: a systematic review. Am J Clin Nutr. 2010;91(1):46–63. 33. Paes Leme AF, Koo H, Bellato CM, Bedi G, Cury JA. The role of sucrose in cariogenic dental biofilm formation—new insight. J Dent Res. 2006;85(10):878–87. 34. Sheu CW, Freese E. Effects of fatty acids on growth and envelope proteins of Bacillus subtilis. J Bacteriol. 1972;111(2): 516–24. 35. Wojtczak L, Wieckowski MR. The mechanisms of fatty acidinduced proton permeability of the inner mitochondrial membrane. J Bioenerg Biomembr. 1999;31(5):447–55. 36. Osawa K, Miyazaki K, Shimura S, Okuda J, Matsumoto M, Ooshima T. Identification of cariostatic substances in the cacao bean husk: their anti-glucosyltransferase and antibacterial activities. J Dent Res. 2001;80(11):2000–4. 37. Shah DS, Russell RR. A novel glucan-binding protein with lipase activity from the oral pathogen Streptococcus mutans. Microbiology. 2004;150(Pt 6):1947–56.
ORIGINAL ARTICLE
Fatty acid effect on sucrose-induced enamel demineralization and cariogenicity of an experimental biofilm–caries model Rodrigo A. Giacaman • Pascale Jobet-Vila Cecilia Mun˜oz-Sandoval
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Received: 24 October 2013 / Accepted: 17 March 2014 Ó The Society of The Nippon Dental University 2014
Abstract Based on scarce evidence, fatty acids have been described as anticariogenic. The aim was to evaluate the effect of different types of fatty acids on enamel demineralization and on the cariogenic properties of Streptococcus mutans biofilms on a biofilm/caries model. Mature biofilms of S. mutans UA159 growing on bovine enamel slabs were exposed to 10 % sucrose for 5 min, 3 times per day followed by exposure to a panel of free fatty acids, including monounsaturated (oleic), polyunsaturated (linoleic) and saturated (stearic) fatty acids, in concentrations of 0.1, 1 and 10 mM for five additional minutes. Enamel demineralization was determined before and after the experiments by microhardness. Slabs were retrieved to analyze biofilm biomass, viable bacterial counts and polysaccharide production. Biofilms exposed to sucrose, followed by oleic and linoleic acids, showed less demineralization than sucrose alone (p \ 0.05). Biomass, S. mutans colonies and insoluble extracellular polysaccharide production were reduced from the biofilms treated with oleic and linoleic fatty acids (p \ 0.05). No differences with the positive control were observed with the saturated stearic acid. Poly and monounsaturated fatty acids presented to S. mutans biofilms after a cariogenic challenge appear to reduce demineralization on enamel and to interfere with cariogenicity of S. mutans biofilms. Keywords Caries Streptococcus mutans Fatty acids Biofilm Antibacterial agents
R. A. Giacaman (&) P. Jobet-Vila C. Mun˜oz-Sandoval Cariology Unit, Department of Oral Rehabilitation and Interdisciplinary Excellence Research Program on Healthy Aging (PIEI-ES), University of Talca, 2 Norte 685, Escuela de Odontologı´a, Talca, Chile e-mail: [email protected]
Introduction Caries, in its current conception, is considered as a biofilmand sucrose-dependent disease [1]. It has been clearly established that fermentable carbohydrates from the diet play a key role in the onset of the lesions [2, 3]. Sucrose, in particular, has been blamed as the most cariogenic of these molecules. This simple sugar is rapidly metabolized by the oral biofilm leading to high amount of demineralizing acids and to the synthesis of extracellular polysaccharides (EPS) [4], which in turn favors bacterial adhesion, aggregation and co-aggregation with other bacterial species [5]. Yet, a linear association between consumption of sucrose-containing foods and caries has been challenged by several studies [2, 6]. Reasons for this rather weak association may derive from multiple factors, including biological, behavioral and genetic factors, technical issues from the studies and the multifactorial etiology of the disease. In that context, we speculated that the modulatory role of other dietary nutrients, ubiquitous to normal diets and usually not considered in sugar-based studies, might be important to explain this apparently inconsistent association between sucrose intake and caries. In the same line of thoughts, an anticariogenic effect of fatty acids and lipids has been claimed for a long time [7, 8]. Mechanisms associated to the putative anticaries activity of fatty acids are diverse and include: antimicrobial activity [9], bacteriostatic properties [10, 11] and the lack of metabolization of fatty acids by the bacteria from the oral biofilm [12]. Most of the limited studies about the role of fatty acids on caries derive from experimental animal studies. Thus, animals fed with fatrich diets reduce caries incidence when compared with those treated with sucrose-rich diets [11, 13]. In the case of fatty acids, lauric acid has shown to be highly bacteriostatic on Gram-positive bacteria [9]. More recently, an
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antibacterial activity against S. mutans of several free fatty acids in low concentrations, including linoleic acid and oleic acid, has been confirmed by several reports [14–16]. An inhibition of nutrient transport through the cell membrane and cell adhesion by fatty acids has been proposed [11]. Other alternative mechanisms to explain the antibacterial activity of fatty acids include inhibition of enzyme activity, interference with oxidative phosphorylation, leakage of intracellular products, generation of peroxidation and auto-oxidation of degradation products and direct cell lysis [17]. Yet, insufficient evidence on this topic precludes from drawing more definitive conclusions and more research appears to be needed. Normal human diet contains all the macronutrients combined with some of them having anticaries properties. With that conception in mind, we conducted this study as a first scrutiny to model the potential anticaries effect of free fatty acids in a highly cariogenic environment provided by sucrose and modulated by the subsequent exposure to free fatty acids. Given the limited evidence available and the potential for a protective role in caries, we aimed to investigate whether immediate exposure to different types of free fatty acids after cariogenic challenges with sucrose reduces enamel demineralization and affects S. mutans biofilm properties on an in vitro pH-cycling biofilm caries model.
Materials and methods Enamel slabs Bovine incisors were obtained, initially disinfected with a 5 % NaOCl solution and stored in 0.9 % NaCl (w/v) until use for up to 30 days. Slabs (4 9 7 9 1 mm) were prepared using diamond disks and a low-speed handpiece and Soflex polishing disks (3M, St. Paul, MN, USA). Initial surface microhardness (SH) was determined by three Knoop indentations placed in one of the lateral aspects of the slab, apart 100 lm from each other (402 MVD, Wolpert Wilson Instruments, USA) at 50 g for 5 s and they were digitally assessed by the built-in reader in the microindenter. To maintain similar initial SH across the slabs, only those with SH 340.87 ± 24.4 kg/mm2 (n = 66) were included. Slabs were sterilized with ethylene oxide [18] and covered with ultrafiltered (0.22 lm) pooled human saliva for 30 min. Stimulated whole saliva was obtained from two healthy donors. To conserve its proteins, whole saliva was mixed with a buffer containing phenylmethylsulfonyl fluoride (PMSF) 1:100 (v/v), a protease inhibitor cocktail. Saliva coating of the slabs allows formation of an acquired pellicle on the enamel for S. mutans initial adherence [19]. Slabs were suspended into the wells of a
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24-well plate by means of a specially designed device made of stainless steel wire. Streptococcus mutans biofilms Frozen stocks of S. mutans UA159 (kindly provided by Prof. J.A. Cury, UNICAMP, SP, Brazil) were reactivated in BHI medium supplemented with 1 % glucose (Merck, Darmstadt, Germany) at 37 °C and 10 % CO2 for 18 h. Once they reached OD 0.8 (600 nm) [20], slabs were inoculated with the bacterial culture and grown for 8 h in 1 % sucrose-supplemented BHI to create an initial adhered biofilm [19]. To achieve maturity before treatment, biofilms on the slabs were allowed to grow for additional 16 h with 0.1 mM glucose, which is similar to the glucose basal concentration in saliva [21]. Fatty acid exposure to the sucrose-challenged biofilms A modified and previously validated in vitro caries model of S. mutans biofilms was used [21]. We carried out three sucrose exposures per day of 5 min each; instead of eight of 1 min each. With the biofilms mature and visibly thick after 48 h of growth, slabs were randomly allocated to one of the eleven groups and treated three times per day, at defined times (8:30, 12:30 and 16:30). Each slab with the mature biofilm was exposed to 10 % sucrose for 5 min followed by five additional minutes with treatment suspensions in 0.9 % NaCl of three different 18-carbon fatty acids; Oleic (monounsaturated) (Calbiochem, San Diego, CA, USA), linoleic (polyunsaturated) (Calbiochem) or stearic (saturated) (AppliChem, Darmstadt, Germany) fatty acids, at three different concentrations; 0.1, 1 and 10. One group exposed to 10 % sucrose followed by 0.9 % NaCl, but not to fatty acids, and another exposed only to 0.9 % NaCl, in two cycles of 5 min each, served as positive and negative control, respectively. After treatment, biofilms were washed three times with 0.9 % NaCl and returned to the same position in the plate. Culture medium was changed twice per day (9:00 a.m. and 5:00 p.m.). 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). Biofilm acidogenicity As a way to verify acid production by the biofilms formed on enamel, culture medium pH was measured inside each well by a microelectrode (HI 1083B, Hanna instruments, Rumania) coupled to a portable pH meter (HI 9126-02, Hanna instruments, Rumania). Measurements were carried out twice per day in the spent medium, before each medium change and during the entire length of the experiment [21].
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Enamel demineralization
Intra- and extracellular polysaccharides
After the experimental period of 5 days, biofilms were sonicated for 30 s to separate biofilms from the dental substrate and to release adhered bacterial cells. Slab’s SH was measured to estimate demineralization produced throughout the 5 days of the experiment, length reported as sufficient to induce enamel demineralization [22]. SH has been extensively used as a reliable methodology to evaluate demineralization [23] and it has been validated for enamel caries [24]. Briefly, a new enamel SH reading (kg/mm2) was obtained after the experimental phase by a row of three indentations separated by 100 lm from those performed before the experimental phase, using the microindenter. Mean values from the initial and final measurements were used to obtain the percentage of SH loss (%SHL) calculated as: (mean initial SH - mean final SH) 9 100/initial SH.
To assess polysaccharide formation by the S. mutans biofilms, three different fractions were analyzed: soluble (SEPS) and insoluble (IEPS) extracellular and intracellular (IPS) polysaccharides [25]. An aliquot of 200 lL from the biofilm suspension was centrifuged (10,000g for 5 min at 4 °C) to obtain SEPS from the supernatant. For IEPS, the pellet obtained from the previous step was treated with 200 lL of 1 M NaOH, homogenized, centrifuged and stored to extract the polysaccharides from the supernatant [26]. Finally, the pellet from the previous step containing the IPS was incubated with 200 lL 1 M NaOH for 15 min at 100 °C and centrifuged (10,000g for 5 min at 4 °C). Supernatant was used to measure the concentration of IPS. The three supernatants from each extraction step were separately treated with 3 volumes of cold 100 % ethanol and incubated for 30 min at -20 °C. Samples were immediately centrifuged and the resulting pellet was washed with cold 70 % ethanol and centrifuged again (10,000g for 5 min at 4 °C). The resulting pellet of each fraction was resuspended in 1 M NaOH and total carbohydrate concentration was estimated by the sulfuric phenol method [27]. Results were normalized by biofilm dry weight and expressed as percentage of polysaccharides by mg of biomass.
Biofilm analysis After the experimental phase, slabs were washed three times with 0.9 % NaCl and biofilms were detached by shaking the slabs for 30 s with a vortex mixer. A biofilm suspension was formed and aliquoted to evaluate the following biofilm properties: biomass, viable microorganisms [21] and intra and extracellular polysaccharides [25]. A description of each biofilm-dependent variable is briefly provided. Biomass Dry weight of the biofilm eluted from the slab was used to estimate biomass content [19]. Thus, 200 lL of the biofilm suspension was transferred to a pre-weighted tube and incubated with 100 % ethanol at -20 °C for 15 min, centrifuged (10 min at 5,000g and 4 °C) and the resulting pellet was washed with 500 lL of 75 % ethanol. After a second centrifugation, the pellet was dried for 24 h in a desiccator. Biomass was calculated by subtracting the final weight from the initial weight of the empty tube. Biomass content was expressed as mg per mL of biofilm suspension. Number of live bacteria from the biofilms From the biofilm suspension, serial dilutions of the biofilm suspension in 0.9 % NaCl (v/v) were drop-plated on BHI agar plates in duplicate, incubated anaerobically for 24 h at 37 °C and colonies were counted from the dilution that allowed visualization of distinctively isolated colonies. Data of counting was calibrated by the dilution factor and expressed as CFU/mg of biofilm dry weight [25].
Statistical analysis To determine whether the data had parametric distribution, the Kolmogorov–Smirnov test was performed. Means obtained from the different groups in each variable were compared by ANOVA followed by Bonferroni test, using SPSS 15.0 statistical software. Differences were considered significant if p \ 0.05.
Results Biofilms exposed to sucrose alone showed a general trend to significantly decrease medium pH more than biofilms exposed to sucrose first followed by the different fatty acids (Fig. 1a–c). Higher fatty acid concentration led to higher pH values. Higher pH values at an early stage of the experiment were observed for the oleic (Fig. 1a) and linoleic (Fig. 1b) acids in a dose-dependent manner, but not for the saturated fatty acid (Fig. 1c). Regarding demineralization, 1 and 10 mM oleic and linoleic acids exposed to S. mutans biofilms significantly reduced sucrose demineralization as compared to the positive control (Fig. 2). Conversely, stearic acid exposure after the cariogenic challenge failed to show changes in the %SHL with respect to 10 % sucrose only (p [ 0.05).
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Odontology Fig. 1 S. mutans biofilm acidogenicity. Biofilms on the slabs were exposed to sucrose followed by oleic (a), linoleic (b) or stearic acid (c) for 5 min, 39/day. Medium pH was measured 29/day throughout the experiment. Each point in the plot represent mean of 2 independent experiments in triplicate slabs. Medium changes are represented by the arrows. Error bars show SD. Different letters at each time point represent statistical significant differences (p \ 0.05)
Table 1 illustrates the effect of the tested free fatty acids on S. mutans biofilms. Biofilms exposed to 10 mM oleic and linoleic acids showed less biomass than the cariespositive control and the rest of the groups (p \ 0.05). Despite a trend for some antibacterial activity of linoleic acid, only 10 mM oleic acid reduced S. mutans counts when compared with sucrose exposure without fatty acids (p \ 0.05). When compared to the positive control, the highest concentration (10 mM) of oleic and linoleic acid decreased IEPS formation. Saturated stearic acid exposure did not affect any of the S. mutans biofilm properties. None
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of the fatty acids tested appeared to interfere with the effect of sucrose on SEPS and IPS formation by the biofilm.
Discussion It is unquestionable that lipids, triglycerides and fatty acids are non-cariogenic when compared with carbohydrates, particularly sucrose. The question of whether they decrease cariogenicity of sugars if they are presented to a cariogenic biofilm is yet to be clarified. Although several
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Fig. 2 Enamel demineralization in response to fatty acid exposure. Biofilms and slabs were exposed to 10 % sucrose for 5 min followed by either fatty acid for 5 min 39/day. Medium was changed 29/day. Surface microhardness (SH) was measure before and after the experiment to assess percentage of SH loss (%SHL) for slabs exposed
to oleic, linoleic and stearic acid, as indicated each in concentrations of 0.1, 1 and 10. 10 % sucrose was used as (?) control and 0.9 % as NaCl(-) control. Bars represent mean of 2 independent experiments in triplicate (n = 6). Error bars show SD. *p \ 0.05; **p \ 0.001 with respect to the positive control (10 % sucrose ? 0.9 % NaCl)
Table 1 Biofilm properties due to sucrose and free fatty acid exposure Biomass (mg)
Bacteria (CFU/mg biomass)
IEPS (%/mg biomass)
SEPS (%/mg biomass)
IPS (%/mg biomass)
10 % Sucrose ? 0.9 % NaCl
2.50 (0.44)
2.91E?10 (5.24E?09)
5.50 (0.95)
2.50 (0.44)
3.91 (0.72)
10 % Sucrose ? 0.1 mM oleic acid
2.17 (0.29)
2.12E?10 (2.80E?09)
5.18 (1.05)
2.17 (0.29)
3.74 (1.46)
10 % Sucrose ? 1 mM oleic acid
1.92 (0.40)
2.03E?10 (4.16E?09)
5.07 (1.51)
1.92 (0.40)
3.73 (0.58)
10 % Sucrose ? 10 mM oleic acid
1.29 (0.44)**
1.18E110 (6.80E109)*
3.30 (0.34)*
1.29 (0.44)
2.96 (0.91)
10 % Sucrose ? 0.1 mM linoleic acid
2.00 (0.58)
2.79E?10 (9.66E?09)
5.30 (2.62)
2.00 (0.58)
3.46 (1.36)
10 % Sucrose ? 1 mM linoleic acid
1.84 (0.15)
2.05E?10 (6.73E?09)
4.75 (0.99)
1.84 (0.15)
3.51 (0.92)
10 % Sucrose ? 10 mM linoleic acid
1.25 (0.44)**
2.06E?10 (1.06E?10)
2.57 (1.13)*
1.25 (0.44)
2.60 (1.53) 3.85 (1.01)
10 % Sucrose ? 0.1 mM stearic acid
2.17 (0.53)
3.21E?10 (7.93E?09)
5.12 (1.53)
2.17 (0.53)
10 % Sucrose ? 1 mM stearic acid
1.84 (0.72)
3.09E?10 (8.45E?09)
5.40 (0.99)
1.84 (0.72)
4.07 (0.82)
10 % Sucrose ?10 mM stearic acid
1.92 (0.29)
3.36E?10 (7.12E?09)
4.92 (0.83)
1.92 (0.29)
3.86 (0.86)
0.9 % NaCl ? 0.9 % NaCl
0.50 (0.40)**
7.90E109 (4.25E109)*
1.01 (0.53)**
0.93 (0.60)*
0.95 (0.34)*
Mean (SD), n = 6 SEPS soluble extracellular polysaccharides, IEPS insoluble extracellular polysaccharides, IPS intracellular polysaccharides * p \ 0.05; ** p \ 0.001 with respect to the positive control (10 % sucrose ? 0.9 % NaCl)
investigations have suggested an anticaries effect of free fatty acids through an antibacterial mechanism, to the best of our knowledge, this is the first time this fact has been tested using a relevant biofilm–caries model. The chief finding from this study was that S. mutans biofilms’ exposure to sucrose first and then to unsaturated free fatty acids reduces the cariogenicity induced by sucrose. Remarkably, the effect was not only on the acidogenicity (Fig. 1) and demineralization of the enamel (Fig. 2), but also on the biofilm properties (Table 1). Although the drop in pH may seem low, it was enough to lead to significant variations in surface microhardness (Fig. 2). It is important to mention that the reduction in acidogenicity and in
demineralization may not be of clinical relevance and definitive conclusions must be obtained only after clinical studies are conducted. The observed anticaries effect of the free fatty acids might be overcome, once the variability of the oral environment is present. Early investigations from the beginning of the past century on the role of diet on caries stated that although necessary, sucrose is not the sole dietary factor involved in the etiology of caries and dietary lipids may result in an anticariogenic nutrient when combined with carbohydrates (revised in Kabara JJ, 1986 [8]). The demineralization inhibition induced by the presence of fatty acids, as observed here is consistent with the results from classic studies using animal models [28, 29].
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When fatty acids are included in a sugar-containing diet in rats, caries scores significantly decrease when compared with the same diet without the fatty acids [11]. Fatty acids are carboxylic acids with a long aliphatic tail. Most naturally occurring fatty acids have a chain of an even number of carbon atoms, from 4 to 28, which may be either saturated or unsaturated [30]. An antibacterial activity of free fatty acids has been long recognized [31]. A higher antibacterial effect of these substances has been described on Gram-positive than Gram-negative microorganism and when the fatty acid chain is longer [31] and more unsaturated [9]. Likewise, although the monounsaturated (oleic) and the polyunsaturated (linoleic, an omega-6 fatty acid) acids reduced demineralization at two of the concentrations tested, linoleic acid showed lower demineralization at a lower concentration (Fig. 2). Yet, a broad range of fatty acids have shown antibacterial properties against many different Gram-positive and Gram-negative microorganisms [17]. Hence, to compare the fatty acids, we used same carbon chain length fatty acids (C18), but with different saturation. As expected, polyunsaturated and monounsaturated fatty acids resulted in higher antibacterial activity than the saturated stearic acid (Table 1). Despite the reported higher antibacterial effect of unsaturated fatty acids, an in vitro study showed activity of saturated medium chain fatty acids and their esters against the growth and metabolic activities of S. mutans [10]. The study showed that C8 to C15 fatty acids were more effective in inhibiting S. mutans than those with longer carbon chains. The saturated fatty acid used here, stearic acid, is a long chain (C18) fatty acid, which could explain why it showed neither reduction in acidogenicity (Fig. 1), demineralization (Fig. 2), nor an effect on the biofilm properties (Table 1). Importantly, the acidogenicity data were obtained by monitoring pH only twice per day. Thus, there might be several hours in which pH curve from some of the concentrations tested was below the critical pH for enamel demineralization (5.5) and the lower concentrations were above this threshold (Fig. 1a, b). This differential time of exposure to a demineralizing medium might have explained the changes observed in %SHL (Fig. 2). In the case of stearic acid, none of the concentrations tested was capable to maintain pH values above the critical pH (Fig. 1c), which is consistent with the %SHL data (Fig. 2). Our results are also consistent with more recent investigations showing that n-6, n-7 and n-9 fatty acids and their esters have inhibitory activity against several oral pathogenic bacteria [15]. Like in our study (Table 1), oleic and linoleic unsaturated fatty acids had a strong antibacterial effect on S. mutans and stearic saturated free fatty acid failed to show S. mutans inhibitory activity, confirming our findings [15]. In addition to their cardiovascular and systemic benefits when
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consumed in the diet [32], n-3 fatty acids have been also described as having antibacterial activity against oral microorganisms, including S. mutans [14]. We decided to test fatty acids of a similar chain length among them, but further studies should incorporate a broader range of fatty acids with this experimental approach. Oleic and linoleic fatty acid exposure showed less biomass than sucrose alone (Table 1). Since biofilm biomass comprises bacterial cells and polysaccharides [33], we speculate that these unsaturated fatty acids act as both, bacterial killers (Table 1) and metabolic inhibitor substances, resulting in lower EPS production (Table 1). Indeed, growth inhibition or the direct killing of bacteria has been described as the major antibacterial mechanisms of fatty acids [17]. The exact mechanisms of either bactericidal or bacteriostatic activity of fatty acids remain unclear. It has been proposed that fatty acids act on the bacterial cell membrane by detergent properties due to their amphipathic structure. This interaction creates pores that can solubilize the membrane disrupting its structural integrity and interfering with the electron transport chain and disrupting oxidative phosphorylation [34, 35]. Moreover, other processes may take place to contribute to bacterial growth inhibition or death, such as lysis, inhibition of enzyme activity, impairment of nutrient uptake and generation of toxic by-products [17]. In the case of oral microorganisms, it has been suggested that fatty acids would form micelles around the individual bacteria interfering with the adhesion of the cell to the enamel and also with transport across the cell membrane affecting metabolism and, therefore, acid production [11]. Furthermore, oleic and linoleic acids, but not stearic, contained in cocoa bean husk showed two mechanism to act against S. mutans [36]. On the one hand, the fatty acids kill bacteria and secondly, they exhibit an antiglucosyltransferase (GTF) activity, which also decrease caries in infected rats. The enzyme GTF is crucial in the pathogenesis of caries, as it takes part in the synthesis of adhesive glucans [4], one of the most relevant virulence factors of S. mutans [33]. Our data support the hypothesis that free unsaturated fatty acids affect bacterial metabolism, mainly on GTFs activity, with an added antimicrobial activity on S. mutans biofilms, but only at higher doses (Table 1). This design may be considered as a pH-cycling model. By exposing biofilms three times per day to a cariogenic challenge, represented by sucrose, and then keeping them overnight exposed only to a basal glucose concentration in fresh medium, this approach intends to mimic the pH variations to which the dental biofilm is exposed in the oral environment. The fatty acids used here are found in various commonly consumed foods. Oleic acid represents more than 63 and 69 % of the total constituents of olive oil and avocado, respectively. Linoleic acid represents 59 % of corn oil. The amount of fatty acids in foods is highly variable. For example, while a commercial cereal bar contains 0.5 and
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0.2 g of monounsaturated and polyunsaturated fatty acids, respectively, other bar has 2.1 and 0.1 g of monounsaturated and polyunsaturated fatty acids, respectively. The most efficient fatty acids to antagonize demineralization induced by sucrose and to have antagonist activity against the biofilm were oleic and linoleic at 10 mM concentration, which is equivalent to 2.8 g of mono and 2.7 g of polyunsaturated fatty acids. The concentrations of fatty acids used here, therefore, appear reasonable for a potential use of the fatty acids as anticaries agents. It has been reported that the structure of the fatty acids determines their antibacterial activity. Thus, free fatty acids are apparently more active than the fatty acids bound to their esters [17]. This assertion has been challenged in recent studies showing an equally effective activity of fatty acids and their esters against S. mutans in vitro [15]. In this study, we used free fatty acids, knowing that dietary lipids are not mainly found as free fatty acids. Oral bacteria, specifically S. mutans, are endowed with enzymes that have been reported to have lipolytic activity [37], nonetheless. Hence, potential nutritional and clinical applications may take both approaches; as the use of purified unsaturated free fatty acids or as dietary lipids. The state of the research on this field precludes the immediate use of this evidence in the clinic, albeit it certainly supports further investigation. Additional in vivo studies would be required to show a clinically relevant anticaries effect of unsaturated free fatty acids.
Conclusion Linoleic and oleic free fatty acids seem to interfere with the cariogenicity of S. mutans biofilms, when they are presented after highly cariogenic challenges with sucrose. Saturated stearic fatty acid shows no effect in inhibiting sucrose cariogenicity. The effect of the unsaturated free fatty acids appears to be metabolic, rather than antibacterial, albeit more studies are necessary to elucidate the mechanism. Acknowledgments Part of this work was presented at the 59th ORCA Congress, Cabo Frio, Brazil, 2012. Authors thank Ms. Gloria Correa-Beltra´n for statistical assistance. This manuscript was submitted in partial fulfillment of the requirements for the DDS degree by PJ. Conflict of interest These investigations were funded by a Chilean government grant Fondecyt #11100005 to Rodrigo A: Giacaman. The authors declare that they have no conflict of interest.
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