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Strive-The Student View The Effect of Splenda and Xylitol on the Growth of Streptococcus mutans
By Amanda Hogan, BS Introduction Dental caries is one of the most common diseases of the mouth. Cavities became widespread in the 17th century due to the development of sucrose.[1] Sucrose is broken down by the oral bacteria Streptococcus mutans. S. mutans uses sucrose as a food source by the formation of glucose and fructose through the enzyme invertase. Fructose is broken down into lactic acid by fructosyltransferase (FTF); lactic acid causes dental caries. Glucose is broken down by glucosyltransferase (GTF) into dextran, which becomes dental plaque.[1] Scientists have been examining ways other than eliminating the intake of sucrose to prevent dental caries. In clinical research, some sweeteners, also known as sugar alcohols, have been shown to prevent the formation of cavities. Those sweeteners include xylitol, sorbitol, mannitol, lactitol, erythritol and sucralose.[2] Extensive studies have been conducted to determine which sugar alcohol is most effective in preventing dental caries. In 1988, Makinen and Isokangas suggested that xylitol was the most effective in reducing caries. However, these scientists stated that further testing was required to determine the ability of different mixtures of sweeteners to prevent caries.[3] In 2004, Van Loveren tried to determine if xylitol was superior to other sweeteners in preventing dental decay. The studies mainly focused on xylitol gum versus sorbitol gum. These results were inconclusive, though, because only two out of the four studies showed xylitol as the better inhibitor against oral bacterial growth.[2] Another study determined that different doses of xylitol in chewing gum resulted in the same levels of growth inhibition. Therefore, xylitol’s ability to inhibit bacteria is not dose dependent at the concentrations tested.[4] A 1995 article by Trahan showed that xylitol prevented most oral bacteria from producing cariogenic acids.[5] Further experiments illustrated xylitol’s ability to inhibit S. mutans’ growth, which reduced the amount of potentially cariogenic bacteria present in saliva and dental plaque.[5] A study conducted by Tapiainen and others in 2001 described that a 5 percent xylitol solution was able to inhibit the growth of Streptococcus pneumoniae.[6] It had been previously observed that xylitol’s ability to inhibit the growth of S. mutans was negatively affected by the presence of fructose.[7] In the Tapiainen study, xylitol could not inhibit the growth of S. pneumoniae in the presence of fructose.[6] Both xylitol and fructose are brought into the cell by a fructose phosphotransferase system. When the cell takes in xylitol through the fructose phosphotransferase system, it is metabolized into xylitol-5-phosphate. The cell is not able to use xylitol-5-phosphate, which may be toxic to the bacteria. The fructose phosphotransferase system, however, has greater affinity for fructose over xylitol, which eliminates xylitol’s effectiveness in inhibiting the growth of S. pneumoniae. The scientists were able to conclude that fructose prevented xylitol from inhibiting the growth of S. pneumoniae.[6] Sucralose, or trichlorogalactosucrose (TGS), is a 12 carbon non-cariogenic compound that is not able to be utilized by oral bacteria. According to Young and Bowen, sucralose did not bind to the bacterial cell and was not taken up by several different oral bacteria.[8] Sucralose inhibits the formation of glucan and fructan polymers. These polymers are used by oral bacteria to produce lactic acid, which is the major cause of dental caries. Young and Bowen were able to conclude that sucralose is noncompetitive and reversible in inhibiting the effects of glucosyltransferase (GTF) and fructosyltransferase (FTF), preventing the formation of the polymers.[8] Wunder and Bowen later found that S. mutans GTF was inhibited by sucralose by reducing enzyme activity in an in vitro assay. This study suggested that sucralose binds to the active site in GTF.[9] According to this study, sucralose is a competitive inhibitor of GTF, whereas Young and Bowen previously stated the opposite. This is an obvious conflict in the literature that needs to be resolved.[9] The present investigation was conducted to analyze whether xylitol and the sucralose-based low-calorie sweetener, Splenda (McNeil Nutritionals, LLC), are able to act together, either additively or synergistically, to inhibit S. mutans.
Materials and Methods Tryptic soy broth (TSB) and tryptic soy agar (TSA) were used to culture the bacteria. An autoclave at 121° C for 15 minutes was used to mix and sterilize the TSB and TSA. S. mutans (Ward’s, Rochester, N.Y.) was rehydrated with TSB. In order to determine the number of bacteria, a standard growth curve for S. mutans was established. A standard calibration curve consists of optical density (OD) readings from a SFM 35 spectrophotometer at a wavelength of 550 nm compared to the number of colonies produced after 24 hours from each of a series of different dilutions of S. mutans. TSA with sheep’s blood (Carolina Biological Supplies, Burlington, NC) was used to provide an ideal growth environment for colony counting. S. mutans grows more proficiently in low-oxygen environments, which the hemoglobin in sheep’s blood provided by binding to the oxygen present.[10] Thus, sheep’s blood created the ideal conditions for S. mutans to grow. The numbers of colonies found in each Petri dish were then converted to colony-forming units (CFUs) per milliliter. A scatter plot was made of cell numbers (CFU/mL) versus the absorbance at 550 nm (Figure 1). The equation from a best-fit line based on a proportional, linear relationship of bacterial dilutions was used to determine the number of bacteria present at a certain OD reading. Artificial sweeteners were added to TSB using sterile technique as described in Table I. The amount of TSB present was adjusted so that each tube would have a total volume of 3 mL. The bacteria were added last. The volume of the bacteria required was determined by first finding the OD of the stock S. mutans and using the standard growth curve equation to determine the volume needed for 10,000 CFU per tube. The tubes were incubated for 36 hours at 37°C. The OD of each tube was measured every 12 hours. The equation found from the standard growth curve was then used to determine the number of CFU in each tube. This experiment was performed in duplicate and repeated five times to determine statistical significance. Gram stains were done after several experiments to ensure S. mutans was still present. Significance was determined using analysis of variance with Tukey’s Honestly Significant difference test in SPSS. The 10 tests were averaged together for each tube, and the standard deviation was determined. The line graphs presented were made of the averages with the standard deviation. The first graph compared ingredients in Splenda for the ability to affect the standard growth of S. mutans. The second graph illustrates the growth of S. mutans in the presence and absence of the different sweeteners.
Results Sheep’s blood resulted in the most countable plates, which gave the most accurate equation for the standard calibration curve. The standard calibration curve results in a best-fit line with the equation y = 1*107x - 19654 (Figure 1). This equation was used to determine the volume of stock bacteria needed for each experiment. The gram stain was positive after several tests, indicating S. mutans was present in all tubes. There was little difference in the growth of S. mutans in all the tubes comparing Splenda ingredients after 12 hours (Figure 2). When comparing the ingredients in Splenda, maltodextrin had higher CFU per mL (mean: 9721346 CFU/mL) values than Splenda (mean: 8401346 CFU/mL) at 24 hours. However, it had lower values than TSB with only S. mutans. There was no difference between the growth of S. mutans in all the tubes comparing different sweeteners after 12 hours (Figure 3). The tubes with artificial sweeteners had lower CFU per mL than TSB with only S. mutans at 24 and 36 hours. After 24 hours, the combination of xylitol and Splenda had lower CFU per mL (mean: 4786346 CFU/mL) values than xylitol (mean: 6177346 CFU/mL) and Splenda (mean: 8401346 CFU/mL). Each tube continued along the same trend after 36 hours, but Splenda had lower CFU per mL (mean: 7963346 CFU/mL), while xylitol (mean: 7066346 CFU/mL) and both xylitol and Splenda (mean: 5874346 CFU/mL) increased the number of bacteria present slightly. At 12 hours, there was no significant difference for the treatments (F = 0.560; p = 0.691; df 4,45). After 24 hours, there was significant difference in all the tubes with bacteria, except the two controls that had no bacteria (Table II). There also was not a significant difference between TSB with S. mutans and TSB with S. mutans and maltodextrin at 24 hours (p = 0.133). After 36 hours, there was significant difference in all the tubes with bacteria. However, there was no significant difference between the two controls (p = 1.000).
Discussion At 36 hours, the tubes with sweeteners had lower CFU values than TSB with S. mutans, which signified inhibition of S. mutans growth. Maltodextrin treatment of S. mutans inhibited the growth only slightly since it developed significant differences compared to other treatments only after 36 hours. The modest inhibition caused by maltodextrin indicated the other ingredient in Splenda, which was sucralose, did the majority of the inhibition because the tube with Splenda had a greater inhibition than maltodextrin. Xylitol showed greater inhibition than Splenda, which adds to Van Loveren’s inconclusive results.[2] Van Loveren tested the claim that xylitol was superior to other sweeteners in the prevention of dental caries. Splenda can be added to the list of sweeteners that xylitol is superior to in preventing dental decay.[2] Both xylitol and Splenda (54.96 percent) inhibited the growth of S. mutans better than either sweetener alone, which signifies an additive effect. Xylitol by itself had a 41.87 percent inhibition effect on the growth of S. mutans. Splenda had 20.94 percent inhibition ability. For an additive growth inhibition, both xylitol and Splenda had to equal 62.87 percent inhibition rate. The probability of error between the Splenda and xylitol added together and the combination of both sweeteners is low (12.58 percent error). Since the actual amount of inhibition created by the combination of xylitol and Splenda had a low percent error when compared to the expected inhibition, xylitol and Splenda had an additive effect on the growth of S. mutans. This additive effect of the sweeteners could indicate different mechanisms of inhibition. Since xylitol’s effectiveness was not dose sensitive at higher concentrations, the addition of a sweetener that inhibited through the same mechanism should not result in increased inhibition.[4] Xylitol inhibits S. mutans through the fructose phosphotransferase system.11 Splenda, therefore, likely did not inhibit through fructose phosphotransferase because it resulted in a greater inhibition of S. mutans growth when added to xylitol.
References
Additional Resources Loesche W. Role of streptococcus mutans in human dental decay. Microbiol Rev. 1986; 353-80. Amanda Hogan, BS, is in her final year at Midlands Technical College in Columbia, South Carolina for an associate degree in dental hygiene. She looks forward to practicing dental hygiene for a few years and getting her master’s. She hopes to continue to do more research.
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