Effect of Prebiotics-Enhanced Probiotics on the Growth of Streptococcus mutans

Streptococcus mutans predominantly creates an acidic environment in an oral cavity. This results in dental demineralization and carious lesions. The probiotics are beneficial microorganisms that modulate the bacterial balance in the digestive system. Prebiotics are defined as nondigestible oligosaccharides that are utilized for the selective stimulation of the beneficial microorganisms. The objective of this study was to evaluate the efficacy of the prebiotics, galactooligosaccharides (GOS) and fructooligosaccharides (FOS), for enhancing the probiotic Lactobacillus acidophilus ATCC 4356, for inhibiting Streptococcus mutans (A32-2) for the prevention of dental caries. The growth rate of the S. mutans significantly decreased when cocultured with L. acidophilus in the GOS-supplemented medium at 3%, 4%, and 5%. In the FOS-supplemented medium, the growth rate of S. mutans significantly decreased in all concentrations when cocultured with L. acidophilus. There was no significant difference in the growth rate of L. acidophilus in all concentrations of either GOS or FOS. It can be concluded that the growth rate of S. mutans was significantly retarded when cocultured with L. acidophilus and the proper concentration of prebiotics. These prebiotics have potential for a clinical application to activate the function of the naturally intraoral L. acidophilus to inhibit S. mutans.


Introduction
Humans are host to oral microbiomes in a positive relationship which is a critical determinant for the regulation of oral symbiosis [1]. e alteration of the oral environment, caused by the frequent consumption of carbohydrates, has been beneficial to acidogenic microorganisms with the consequential creation of a low pH environment in the oral cavity.
is altered condition enhances the activities and growth of acidogenic and aciduric microbes, predominantly S. mutans and Lactobacillus spp. In the homeostasis condition, only 2% of S. mutans is contained in the dental biofilm. When the balanced condition is transformed to an acidic environment, the percentage of S. mutans and Lactobacillus spp. substantially increases [2]. Some strains of Lactobacillus spp. are forms of probiotics that are beneficial for balancing the microorganism environment in the gastrointestinal tract [3][4][5]. However, to be beneficial in the oral cavity, probiotics must firstly aggregate and attach to the oral tissue, which then creates a protective barrier to prevent the colonization of the pathogenic microorganism [6]. e growth and activity of probiotics are enhanced by nondigestible oligosaccharides, namely, prebiotics [6,7] which are unable to be digested by the host, but do enhance the beneficial effects of probiotics by selectively stimulating the growth and activities of the probiotics [8].
e aims of this study are to investigate the effect of probiotics on S. mutans after the probiotics received the prebiotics. e prebiotics in this study, galactooligosaccharides (GOS) and fructooligosaccharides (FOS), have been approved by the FDA. Douglas, University of Sheffield. e probiotic was L. acidophilus type strain ATCC 4356 T (DSMZ 20079 T ).

Effect of the Prebiotics on S. mutans and L. acidophilus.
An equal number of S. mutans and L. acidophilus (10 6 cells) were cocultured in the MRS medium supplemented with 1%, 2%, 3%, 4%, and 5% of GOS (v/v) and FOS (w/v). A control group was formed with the culturing media not supplemented by prebiotics. e effect of the prebiotics on the S. mutans and L. acidophilus both separately, and as a coculture, was determined by measuring the growth rate at 3, 6, and 12 h. e growth rate was calculated from the formula: where N t : number of bacteria at time point "t," N 0 : number of bacteria at the initial time point (time point "0"), t: duration of observing time, and t 0 : initial time point (time point "0").

Effect of Prebiotics on the Cellular Fatty Acids.
After coculturing between S. mutans and L. acidophilus 10 6 cells each for 6 h, the cellular fatty acid of the S. mutans and L. acidophilus, separately and cocultured, was analyzed by gas chromatography using the MIDI Sherlock ™ microbial identification system. Bacterial pallets 0.06 g or supernatant 0.5 ml from the samples were independently vigorously mixed with 1 ml of Reagent 1 (3.75 N NaOH in deionized water and methanol) for 10 s, and the solutions were then heated in 100°C water for 25 min and then plunged into an ice bath for chilling. For the methylation, 2 ml of Reagent 2 (6.0 N HCl in methanol) was vigorously mixed with the cold sample for 10 s. e samples were then incubated in 80°C water for 10 min and cooled in ice. For the fatty acid extraction, 1.25 ml of Reagent 3 (hexane: methyl tert-butyl ether (1 : 1 v/v)) was added and consecutively mixed for 10 min. e upper part of the solution was collected and mixed with Reagent 4 (0.3 N NaOH) for 5 min, and the supernatant collected for the fatty acid analysis.

Suitable
Culturing Medium for the Coculture of S. mutans and L. acidophilus. S. mutans could survive for 14 h in the BHI and 10 h in the TYE media, and the S. mutans grew in the MRS culturing medium throughout the 24-hour growth phase. L. acidophilus grew in the TYE for only three hours and only one hour in the BHI medium but grew in the MRS medium with similar patterns as S. mutans (see Figures 1(a) and 1(b)). Both S. mutans and L. acidophilus grew well in the MRS medium. erefore, the MRS medium was selected for the coculture between S. mutans and L. acidophilus.
During the first 8-hour period in the MRS medium, the number of viable bacteria in each was similar, but for the subsequent 16 h, the number of L. acidophilus consistently exceeded that of S. mutans (see Figure 1(c)).

Determination of the Influence of Prebiotics Supplementation on pH.
Organic acids are one of the common metabolic products from both S. mutans and L. acidophilus. In the individual culture, the pH of the FOS-supplemented medium was insignificantly higher than that of GOS-supplemented medium for both S. mutans and L. acidophilus (P > 0.05). In the coculture, there was also no statistical difference in the pH between GOS-and FOS-supplemented media (P > 0.05). e pH of the individual S. mutans culture (pH � 5.9-6.2) was higher than that of either the individual L. acidophilus (pH � 5-5.4) culture or the coculture (pH � 5.1-5.4) (see Table 1).

Effect of the Prebiotics on the Growth of S. mutans and L. acidophilus.
e individual cultures of S. mutans, grown in the prebiotics-free medium, showed a growth rate of 0.9921 ± 0.14 h −1 . When supplemented with the different concentrations of GOS, the growth rates of the individual cultures of S. mutans were similar to those of the control group (P > 0.05). e most significant decrease in the growth rate of S. mutans was observed in 3% FOS as compared to the control (P � 0.032) (see Table 2).
When the FOS was added into the coculture, the growth rate of S. mutans significantly decreased in all concentrations (P � 0.034) as compared to the control. e minimum growth rate was found in the 3% FOS (0.2281 ± 0.12 h −1 ) (see Table 2).
For L. acidophilus, the growth rate of both the individual and cocultures in all concentrations of the GOS-and FOSsupplemented media was insignificantly different from the control (P > 0.05) and among the concentrations (see Table 3).

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International Journal of Microbiology

Effect of Prebiotics on the Cellular Fatty Acids.
In this study, the cellular fatty acids and the secreted fatty acids were observed in the coculture after being cultured for 6 h. When S. mutans and L. acidophilus were grown in the prebiotics-supplemented culturing medium, the percentage of cellular fatty acid was similar to that of the control group. For the cocultures, the percentages of cellular saturated fatty acids and unsaturated fatty acid from the mixed pellets were also not different from those of the control group (see Table 4).
When S. mutans and L. acidophilus were individually grown or cocultured in the prebiotics-supplemented culturing media, the percentage of secreted fatty acid was similar to the control group (see Table 5).

Discussion
Dental caries is a major oral problem in all ages. S. mutans are the vital cariogenic bacteria, metabolizing sugar with      Note. e statistics compared within each type of prebiotics. e uppercase letters indicate the significant difference within each column. e lowercase letters indicate the significant difference within each row. their acidogenic and aciduric properties. Several studies have been reported that attempted to modify the S. mutans pathogenicity by adding probiotics such as L. acidophilus, suggesting that probiotics could generate a protective barrier, inhibit pathogenic microorganisms, and stimulate the host defensive mechanisms [11]. L. acidophilus is also naturally found in small amounts in the oral cavity [12]. e oral probiotics must be able to adhere and to colonize the oral tissue and to compete with pathogenic microorganisms at the binding site [13]. Lactobacillus spp. are well-known probiotics which have the ability to adhere to epithelial cells [14] and produce several bacteriocin effects on other microorganisms [15]. L. acidophilus (ATCC4356 T � DSMZ 20079 T ) was the probiotic applied in this study, as it is the type strain most found in the human body and is normally about 3%-9% of the bacteria found in oral cavities. It has also been previously reported as the preferred probiotics in many studies [16].
For this experiment, the two distinct strains S. mutans and L. acidophilus were used and cultured in the same media. First, however, it was important that the appropriate culturing conditions for both strains be determined. Based on previous studies, the appropriate culture media for L. acidophilus are MRS media and BHI [17,18] and tryptic soy are suitable for S. mutans cultivation [19]. L. acidophilus are anaerobic bacteria and require an oxygen-free condition for propagation. However, L. acidophilus can be cultured in 5% CO 2 at 37°C [20]. According to many studies, the interaction between S. mutans and L. acidophilus occurs in the agar diffusion process under anaerobic conditions [18,21]. For this study, S. mutans and L. acidophilus were cocultured in culturing broth under conditions of 5% CO 2 . e results showed that S. mutans seemed to be able to grow in almost all types of culturing media. However, the S. mutans receded from the TYE or BHI cultures. L. acidophilus, which are fastidious microorganisms, need more complex nutrients such as amino acids and peptides [22,23], trace elements [24], vitamins, oleic acids [25], buffering agents, and polysorbates, for growing and cell activity. All these substances are in the MRS, but not in the BHI and TYE.
According to Singh et al., when the subjects consumed probiotic ice cream containing freeze-dried L. acidophilus La5, the number of salivary mutans streptococci significantly decreased [26]. Moreover, L. acidophilus DSM 20079 T suppressed the ability of S. mutans ATCC 35668 to adhere to the microtiter plates, indicating that when L. acidophilus were introduced into the system before the S. mutans were added, the suppression of salivary mutans streptococci was more efficient [27]. e L. acidophilus DSM 20079 T also has the ability to decrease the viable count of S. mutans [28,29].
is is the first study to determine the growth rate of S. mutans when cocultured with L. acidophilus in the prebiotics-supplemented media. Other previous studies only determined the viable count of S. mutans, not the growth rate [28,29]. e bacterial growth rate reflects the ability of bacteria to multiply. In order to simulate the oral situation by applying prebiotics before bed, a six-hour incubation period was selected for determining the growth rates.
After GOS was added into the individual culture, the growth rate of S. mutans was not statistically different from the control. However, when the S. mutans was cocultured with L. acidophilus, the growth rate of the S. mutans significantly decreased in the 3%, 4%, and 5% concentrations of GOS as compared to the control group (no prebiotics). It is indicating that the 3% GOS was the optimum concentration that could decrease the growth rate of S. mutans when cocultured with L. acidophilus. e growth of S. mutans decreased after receiving the FOS, in both the individual culture and the coculture. e minimum growth rate was seen in the 3% FOS in both the individual culture and the coculture. In the coculture, the growth rate in all concentrations decreased, while in the single culture, only the 3% FOS resulted in a decrease. ese results indicate that the efficacy of FOS to decrease the growth rate of S. mutans was potentiated when L. acidophilus was present in the culture.
Even though L. acidophilus can utilize both GOS [30] and FOS [31], there is the limitation in GOS digestion, resulting in the metabolized and acidic products being limited [30]. According to this study, the growth rates of L. acidophilus were not affected by GOS and FOS in both the individual culture and the coculture. When L. acidophilus is cocultured with S. mutans in the prebiotic-supplemented media, the growth rates of S. mutans decreased. is finding indicates that GOS and FOS might enhance the cell activity of L. acidophilus to reduce the growth of S. mutans. However, the mechanism of GOS and FOS dominated by L. acidophilus has not been identified and needs further studies.
GOS and FOS are resistant to gastric digestion in humans. e energy from GOS is 1.7 kcal/g and from FOS is 1.5 kcal/g, indicating that they can be used as sweeteners in food and beverages. However, excessive intake probably results in abdominal discomfort, flatulence, and diarrhea [32]. e recommended dose ranges between 8 and 20 g/d.
A lipid bilayer is the main composition of the cell membrane. Its components are affected by the composition of the fatty acids. e saturated fatty acid chains, which are less than 12 carbon atoms, have been detected in small amounts in bacteria. e bigger saturated fatty acids, more than 16 carbon atoms, have been found in larger proportions than the small fatty acids, and so the unsaturated fatty acids are a major portion of all the fatty acids of the bacteria. e culturing conditions have an influence on the rearrangement of the membrane fatty acids [33].
S. mutans altered their membrane fatty acids during growth in low pH conditions. At pH 6.5, the cellular fatty acids of S. mutans UA159 were composed of 65.4% saturated fatty acids and 34.6% unsaturated fatty acids. When the pH drops to 5.5, the saturated fatty acids reduced to 53.3% and the unsaturated fatty acids increased to 46.7% [34]. Bender et al. mentioned that the unsaturated fatty acids of S. mutans increased in acidic conditions especially for C18 : 1 and C20 : 1 [35].
In 1991, Sato et al. found that the lipids composition of the cell membrane of S. mutans markedly changed when fatty acids were added into the growth medium [36]. Even International Journal of Microbiology though the fatty acids are the cell composition, they have an antimetabolite effect on several microorganisms [37]. e half-time for pH equilibration of S. mutans significantly increased when lauric acid and palmitic acid were added, but it slightly increased when arachidic acid was added [38]. Carson and Daneo-Moore found that the oleic acid was able to induce the lysis of bacterial membrane due to the susceptibility of the double bond of unsaturated fatty acids to the oxidizing agent [39]. In this study, the growth rate of S. mutans in 3% FOS significantly decreased compared with the other groups, but there was no significant difference on each kind of fatty acids in that group. e prebiotics had no effect on the fatty acid composition of the cell membrane.
e results from this study showed that GOS and FOS, which are polysaccharide prebiotics, do not support the growth rate of S. mutans and L. acidophilus. On the contrary, the 3% FOS decreased the growth rate of S. mutans. When considering the clinical applications, the 3% FOS might be useful as a treatment for high caries-risk patients to reduce the growth rate of S. mutans in the oral cavity. In addition, the 3%, 4%, and 5% of GOS and 1%, 2%, 3%, 4%, and 5% of FOS have potential as a clinical application to activate the function of the intraoral L. acidophilus, which is the naturally occurring probiotic, resulting in a decrease in the growth rate of S. mutans, but with no increase in the number of cariogenic bacteria. However, the mechanism of action is obscure. To identify the specific mechanism, further studies are required.

Conclusions
e prebiotics, GOS and FOS, had no effect on the growth rates of L. acidophilus ATCC 4356, and the 3% FOS significantly decreased the growth rate of S. mutans A32-2. e 3% concentration of GOS and the 1% of FOS seem to have the potential to decrease the growth rate of S. mutans when applied together with the probiotics L. acidophilus.

Data Availability
e data used to support the findings of this study are included within the article.

Conflicts of Interest
e authors declare that they have no conflicts of interest.