The Influence of Sucralose on Bacterial Metabolism D.A. YOUNG and W.H. BOWEN University of Rochester, Rochester Cariology Center, Department of Dental Research, 601 Elmwood Avenue, Rochester, New York 14642
Sucralose (1',4',6' trideoxy-trichloro-galactosucrose) is a nontoxic, intensely sweet sucrose derivative that has been shown to be non-cariogenic in experimental animals. The purpose of this study was to determine whether certain oral bacteria could utilize sucralose. Sucralose, as a sole carbon source, was unable to support growth of ten strains of oral bacteria and dental plaque. When sucralose was incorporated into a liquid medium containing glucose or sucrose, all organisms tested displayed similar pH falls, compared with controls. The incorporation of 126 mmol/L sucralose into glucose agar medium caused total inhibition of growth of Streptococcus sobrinus 6715-17, Streptococcus sanguis 10904, Streptococcus sanguis Challis, Streptococcus salivarius, and Actinomyces viscosus WVU627. Sucralose had no effect on IPS production. Sucralose was not bound to, nor taken up by, cells. Sucralose inhibited the formation of glucan and fructan polymers in proportion to the sucralose-to-enzyme ratio, but independent of the sucrose concentration in the assay mixture. No radioactive polymer was formed from '4C-U-sucralose added to mixtures containing glucosyltransferase (GTF) or fructosyltransferase (FTF). Inhibition of GTF and FTF by sucralose was removed following dialysis of the enzyme/sucralose mixture. These results show that sucralose was not utilized by the oral bacteria tested and that the inhibitory effect of sucralose on GTF and FTF was non-competitive and reversible. The results further support the concept that sucralose is non-cariogenic. J Dent Res 69(8):1480-1484, August, 1990
Introduction. The search for suitable non-cariogenic derivatives of sucrose has resulted in limited success. In the last decade, however, it has been found that specific halogenation of sucrose can yield intensely sweet compounds (Hough and Khan, 1978). One such sucrose derivative, 1',4',6' trideoxy-trichloro-galactosucrose (sucralose), is 600 times sweeter than sucrose and appears to be metabolically inert in many strains of oral bacteria, thus making it an attractive substitute for the sweetness of sucrose. The frequent ingestion of sucrose may promote caries in several ways. For example, sucrose may promote growth and acid production by plaque bacteria. It can also be a substrate for the synthesis of intra- and extracellular polysaccharide. Clearly, any assessment of a sucrose substitute should include a study of its effects on these phenomena in bacteria. Plaque harbors a wide range of bacteria, but the microbial etiology of dental caries is associated primarily with the metabolic activities of Streptococcus mutans. Clearly, though, other microorganisms in dental plaque may contribute to the pathogenesis of dental caries. We therefore chose to study the ability of a range of Gram-positive micro-organisms -commonly associated with the onset and progress of dental caries in humans Received for publication November 9, 1989 Accepted for publication March 2, 1990
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and experimental animals (Menaker and McGhee, 1982)-to utilize sucralose. Because of the importance of glucosyltransferase (GTF) and fructosyltransferase (FTF) in the formation and growth of plaque (Gibbons, 1983; R0lla et al., 1983; Hamada, 1983) and, in addition, because sucralose has some structural features in common with sucrose, it was appropriate to be determined whether sucralose can be utilized as a substrate for these enzymes and be synthesized to glucan or fructan polymer or, alternatively, whether it can act as an inhibitor (reversible or irreversible) for these enzymes.
Materials and methods. Micro-organisms. -The following organisms were included in our study: Streptococcus mutans GS-5; Streptococcus sobrinus 6715-17; Streptococcus rattus FA-1; Streptococcus sanguis 10904; Streptococcus sanguis Challis; Streptococcus salivarius; Staphylococcus aureus H; Actinomyces viscosus OMZ1O5E; Actinomyces viscosus WVU627; Lactobacillus casei 393; Lactobacillus casei 4646; and plaque from fasted sub-
jects.
Sucralose.-Sucralose was obtained from McNeil Specialty Products Company (Skillman, NJ) and was determined by gas chromatography to be 99.3% pure. Media.-Inocula were routinely prepared in tryptone-yeast extract (2.5% tryptone, 1.5% yeast extract, 0.5% K2HPO4, 0.1% MgSO4 plus indicated carbohydrate) (TYE) broth. For preparation of growth curves, a modified FMC defined medium (Terleckyj et al., 1975) was utilized that contained 21.0 g Casamino acids (Difco Laboratories, Detroit, MI), 1.85 g (NH4)2SO4, 0.56 g MgSO4-7H2O, 9.52 g KH2PO4, 6.10 g K2HPO4, and 0.5 mg para amino benzoic acid per liter. The pH was adjusted to 7.0 and 10.0 mL of BME vitamin mixture (Gibco Laboratories, Grand Island, NY) was added, and the medium was filter-sterilized with a 0.2->Lm filter. Organisms displayed normal morphology during growth in this medium. Carbohydrate utilization. -Growth curves were derived for determination of whether the micro-organisms were able to utilize sucralose as a sole carbohydrate source and whether sucralose could influence growth via inhibition of glucose uptake. So that no unintended carbohydrate was added to the test culture by way of inoculum, starter cultures were grown overnight in TYE, and the cells were harvested by centrifugation and washed twice with PKMN buffer (Gerhardt, 1981), which contained 60 mmol/L KC1, 60 mmol/L NaCl, 5 mmol/L MgSO4, and 50 mmol/L potassium phosphate buffer (pH 7.0). The cells were re-suspended in 0.85% NaCl and used as the inoculum. Culture tubes containing defined medium plus or minus the indicated concentrations of glucose and/or sucralose were used, as shown in Table 1. Uninoculated medium was incubated overnight at 37°C in an atmosphere of 10% CO2. Tubes then received a 3.3% v/v inoculum and were kept at 37°C in a heater block for the duration of the experiment. Growth was monitored for 24 h by measurement of absorbance at 700 nm (A700) (SequoiaTurner Model 340 spectrophotometer), and growth rates were determined by logarithmic regression analysis.
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Acid production. -The influence of sucralose on acid production by the test bacteria from sucrose or glucose was examined as follows. Cells were grown overnight in TYE medium, centrifuged at 10,000 g, re-suspended in PKMN buffer, and then incubated at 37TC for two h so that intracellular polysac-
charides would be catabolized (Gibbons and Socransky, 1962). Following incubation, cells were again washed and re-suspended in PKMN buffer. Sucralose, PKMN buffer, and sucrose were added to 4 mL
of standardized cell suspension to yield the following final mixtures: control, no carbohydrate; 16.7 mmol/L sucrose; 16.7 mmol/L sucralose; and 16.7 mmol/L sucrose plus 16.7 mmol/ L sucralose. Tubes were incubated in a heater block at 370C and the pH monitored with Beckman Model pH140 pH meter. After one h, glucose (final concentration, 16.4 mmol/L) was added to the control and sucralose-only tubes, and the pH was monitored over time for determination of whether pre-incubation with sucralose affected acid production.
Growth
on
solid medium and intracellular polysaccharide
production (IPS). -The test organisms and a sample of plaque were inoculated onto TYE (without glucose) plus 1.6% agar (Difco) containing appropriate amounts and combinations of sucralose and glucose. Presence of growth was observed over 48 h. Because some organisms appeared sensitive to the presence of sucralose, we decided to determine whether the effect could be attributed to increased osmolality of the medium. Comparable concentrations of sorbitol and mannitol were included, and their effect on growth was observed. The amounts of glucose and sucralose added to the control medium, free of added carbohydrates, were as follows: 2.5% glucose (139 mmol/ L); 5.0% glucose (278 mmol/L); 2.5% sucralose (63 mmol/ L); 5.0% sucralose (126 mmol/L); 2.5% glucose (139 mmol/ L) + 2.5% sucralose (63 mmol/L); 5.0% glucose (278 mmol/ L) + 5.0% sucralose (126 mmol/L); 2.5% glucose (139 mmol/ L) + 5.0% sucralose (126 mmol/L); and 5.0% glucose (278 mmol/L) + 2.5% sucralose (63 mmol/L). The concentrations of sorbitol and mannitol followed the same pattern. Intracellular polysaccharide production (IPS) was assayed by a modified method of Gibbons and Socransky (1962). Following incubation, plates upon which growth had occurred were flooded with Gram's iodine. The iodophilic IPS-containing colonies were identified by a darkening in color and scored, from I-C , indicating no change or a yellow color, to " + + + + ", indicating dark purple or black-staining colonies.
GTF and FTFpreparation.-S. sobrinus 6715-17 (for GTF) and S. salivarius (for FTF) were grown in dialysis tubing (Spectrum Medical Industries, Inc., Los Angeles, CA) (15,000 molecular weight cut-off) containing approximately 250 mL of TYE medium suspended in a liter of the same medium to concentrate the extracellular GTF and FTF enzymes. The contents of the dialysis bags were centrifuged for removal of cells, and the supernatant was used as the GTF and FTF preparations. Polymer assay. -Synthesis of glucan and fructan was carried out with use of the methods described in Robrish et al. (1972). Incorporation of sucralose into polymer was studied with use of a 14C universally-labeled preparation of the sweetener in the test system. All assays were incubated in 1.5-mL polypropylene microcentrifuge tubes at 37°C. Aliquots were taken at the beginning and at the 1.5-, 3.0-, and 4.5-hour time points. Radio-labeled polymer was precipitated over glass fiber filters and washed three times with cold methanol in a vacuum manifold (Millipore). Filters were placed in vials of liquid scintillation cocktail and radioactivity counted for quantitation of incorporation. Inhibition assay.-The effects of sucralose on the rates of glucan and fructan polymer formation were determined by the
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concentration of 14C-labeled sucrose being kept constant and by variation of the concentrations of sucralose in the assay system. The assays were incubated and sampled, and polymer was quantitated as previously described. Additional experiments were performed for determination of whether the observed inhibition by sucralose could be relieved by an increase in the sucrose concentration in the assay and whether the inhibition was reversible. Sucrose concentrations of 50-400 mmol/ L were added to enzyme (FTF or GTF) sucralose (50-150 mmol/L) mixtures. Increasing amounts of sucrose were added to each separate concentration of sucralose. For determination of whether sucralose-associated inhibition of glucan polymer formation was reversible, GTF preparation (previously described) was incubated at 370C for four h in the presence of 100 mmol/L sucralose. After four h, the GTF + sucralose preparation was dialyzed in 15,000 mw cut-off dialysis tubing against 1000 times its volume of 50 mmol/L potassium phosphate buffer pH 7.0 at 40C with stirring. GTF preparation without sucralose, exposed to the same conditions, was used as a control. Following dialysis, the two GTF preparations (exposed and unexposed to sucralose) were assayed by two different approaches. Sucralose was included in the first series of assays, and it was omitted in the second series. The results were compared for determination of whether dialysis of the sucralose + GTF preparation would give similar glucan yields as did the dialyzed control (thus, no remaining sucralose and no inhibition), and whether the addition of sucralose to the GTF assay with dialyzed GTF preparation would give similar inhibition levels as with the undialyzed control assay.
Binding or uptake of sucralose by micro-organisms. Several experiments were conducted for determination of whether sucralose was bound or taken up by bacterial cells. To this end, S. mutans (sobrinus) 6715-17 was grown overnight in TYE medium and treated as before. 14C-labeled sucralose, PKMN buffer, glucose, and sucrose were added to 200 pL of standardized cell preparation to yield the following solutions: (A) 100 mmol/L sucralose, or (B) 100 mmol/L sucralose plus 100 mmol/L glucose, or (C) 100 mmol/L sucralose plus 100 -
TABLE 1 EFFECT OF SUCRALOSE ON GROWTH RATE OF ORAL MICROORGANISMS Percent Control Growth Rate (S.D.)*t 13..88 mmol/L 27.75 mmol/L 55.5 mmol/L Bacterial Strain sucralose sucralose sucralose S. mutans GS-5 1211.3 ( 7.3) 111.6 ( 4.9) 96.9 ( 0.4)t S. sobrinus 6715-17 13C).5 (13.6) 84.5 ( 7.6) 62.4 ( 0.7) S. sanguis 10904 .4 94.1 (24.2) 68.7 (23.8) (18.2) 124 S. sanguis Challis ).3 (14.2) 107.8 (16.3) 91.0 (12.7) S. salivarius .1 ( 7.4) 108.5 ( 8.9) 89.7 ( 1.1)4 S. aureus H 10( ).9 ( 0.6) 86.4 ( 1.0)t 71.2 ( 3.8)4 A. viscosus OMZ105E ioc3.4( 5.3) 102.3 ( 4.4) 90.5 ( 2.1) 103 A. viscosus WVU627 98.2 ( 4.9) 1.6 ( 7.9) 83.3 ( 4.5) L. case 393 1065.1 ( 4.6) 96.3 (1.3) 87.2 ( 0.1)4 L. case 4646 104U2 ( 5.9) 99.8 (1.6) 97.2 ( 6.3) 13.7 (13.6) 80.8 Plaque 55.5 (10.0) 7.2) *Bacteria were grown overnight in TYE medium, washed twice in PKMN buffer, re-suspended in sterile saline, and added to modified FMC medium containing 27.75 mmol/L glucose (control) or to the same medium plus the indicated concentration of sucralose. Growth was monitored by measurement of absorbance at 700 nm, and growth rate was determined by logarithmic regression analysis. t% control growth rate is expressed relative to control without added sucralose, whose value is equal to 100%. tSignificantly different from control via Student t test at the 99% confidence limit. -
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YOUNG & BOWEN GUOSE ADDED a control
7.0 9
A A A
A i 5
2 6.0 -
x
sucralose
*
sucrose
0
sucrose
A
9
plus sucralose
a
x 0
5.0 a
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I 3.0 1 0.0
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I
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TABLE 2 EFFECT OF SUCRALOSE ON INTRACELLULAR POLYSACCHARIDE (IPS) PRODUCTION IPS Production* Bacterial Strain 0.0% sucralose 2.5% sucralose 5.0% sucralose ++ S. sobrinus 6715-17 NA +/_ +++ S. mutans GS-5 ++ + ++++ S. sanguis 10904 ++ ++ NA +++ S. rattus FA-1 +++ +++ ++ S. sanguis Challis ++ NA + ++ S. aureus H + ++++ ++++ S. salivarius NA A. viscosus OMZ1O5E ++++ + +±+ ++ A. viscosus WVU627 ++ ++ NA L. casei 393 ++ + ++ L. casei 4646 ++ ++ ++ * Bacteria were grown for 24 h on TYE plates containing 5.0% glucose plus the indicated concentration of sucralose. Plates were flooded with iodine, and colonies were scored for degree of IPS formation, from -, indicating yellow color or no change, to + + + +, indicating deep purple or black staining. NA-not applicable, since no growth occurred in the presence of 5.0% sucralose.
HOURS Fig. -Influence of sucralose on acid production by S. sobrinus 6715.
mmol/L sucrose. Control assays contained cells plus 100 mmol/ L 14C glucose or 14C sucrose. All assay mixtures were incubated, sampled, and cell-associated radiolabel quantitated, as described in the polymer assay section.
Results. Carbohydrate utilization.-The ability of micro-organisms to grow in the defined medium plus glucose, as well as signs of morphological changes, were continuously monitored by phase-contrast microscopy. None of the cultures displayed any apparent cellular abnormalities; growth achieved optical densities ranging from A700 0.3 to 2.0, with 90% of strains reaching an A700 of 1.0 within 8 to 10 h. Addition of sucralose to the defined medium containing glucose appeared to cause a slight increase in cell size and a decrease in chain length in all
organisms examined.
The micro-organisms examined apparently could not utilize sucralose as a carbohydrate source. Although S. aureus and S. mutans GS-5 as well as plaque displayed slight growth equivalent to one or two doublings, this apparent growth was not always reproducible or to the same extent each time (data not shown). When sucralose was used in combination with glucose, few effects were observed. However, five organisms, S. mutans GS-5, S. sobrinus 6715-17, S. salivarius, S. aureus (H), and L. casei 393 all displayed a slightly decreased growth rate in glucose medium plus 55.5 mmol/L sucralose (Table 1). Acid production. -Cell preparations in PKMN buffer and those in sucralose plus buffer displayed no decline in pH values in one hour. When glucose was added to the buffer containing sucralose, the pH values decreased and were not significantly different from a buffered control containing the added glucose. Suspensions of cells in buffered sucrose and in sucrose plus sucralose also resulted in acid production (Fig.). Solid medium growth and IPS production. -Five organisms-S. sobrinus 6715-17, S. sanguis 10904, S. sanguis Challis, S. salivarius, and A. viscosus WVU627-failed to grow on solid medium containing 5.0% sucralose, 5.0% sucralose plus 5.0% glucose, and 5.0% sucralose plus 2.5% glucose. These organisms grew on medium containing 2.5%
sucralose and lower concentrations of sucralose and glucose. Growth of the other five organisms and plaque was not inhibited on any of the solid media examined. The five organisms inhibited when sucralose was in the medium all grew on medium containing comparable concentrations of sorbitol and mannitol (Table 2). The IPS assay performed on all the micro-organisms revealed that sucralose did not interfere with IPS production on media containing glucose. The organisms grown on the medium containing only sucralose did not synthesize iodophilic IPS, whereas those grown on medium containing glucose or any combination of glucose and sucralose displayed the presence of IPS. Glucosyltransferase and fructosylItransferase studies. -The S. salivarius medium supernate was assayed for GTF and FTF activity with 14C-glucose-labeled sucrose and 3H-fructose-labeled sucrose, respectively, as substrates in the assay mixture. It displayed activities for both enzymes. When 14C-labeled sucralose was added to the assay mixtures as the sole labeled carbohydrate, no incorporation was found either in the presence or absence of unlabeled sucrose (Table 4). This assay procedure was repeated with an S. sobrinus 6715-15 preparation used as a source of GTF. When sucrose concentration was varied from 0 to 300 mmol/L, no incorporation of radiolabeled sucralose into the resultant polymer was observed. Sucralose inhibited the formation of glucan polymer by the S. sobrinus 6715-15 GTF preparation (Table 3). When extracellular FTF preparations from S. salivarius were assayed in the same manner, a similar inhibition was observed (Table 3). The degree of inhibition was related to the concentration of sucralose in the test systems, and addition of excess sucrose did not overcome the inhibition. Reversibility of inhibition of GTF by sucralose.-When the GTF preparation was dialyzed against buffer, the glucan-forming activity decreased by approximately 10%, as compared with that in undialyzed controls. However, when sucralose was added to the dialyzed GTF preparation, the percent inhibition of glucan production was similar to that induced by sucralose added to undialyzed controls. When the GTF preparation, incubated four h with sucralose, was dialyzed and then incubated with substrate, the resultant rate of glucan production was very similar to that of its dialyzed control (Table 4), showing that the inhibitory effect of sucralose could be removed by dialysis.
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SUCRALOSE AND BACTERIAL METABOLISM
TABLE 3 EFFECT OF SUCRALOSE ON GLUCOSYL- AND FRUCTOSYLTRANSFERASE ACTIVITIES % Inhibition % Inhibition Dextransucrase Sucralose of GTF Activity of FTF Activity Added to Assays (S.D.)* (S.D.)t mmol/L 22.4 ( 5.7) 8.4 (4.4) 50 150 43.8 (1.3) 25.0 (2.1) 64.4 (11.2) 44.8 (1.3) 300 * Glucosyltransferase (GTF) prepared from S. sobrinus 6715 was incubated with 150.0 mmol/L 14C-glucosyl sucrose plus the indicated concentration of sucralose at 37'C and was sampled at 1.5, 3.0, and 4.5 h. Aliquots were assayed for incorporation of '4C-glucose into methanolinsoluble polymer, and reaction rates were determined by regression analysis. Data are expressed as % inhibition of the reaction rate of control samples containing 150.0 mmol/L 14C-glucosyl sucrose, without sucralose. tInhibition experiments with fructosyltransferase (FTF) prepared from S. salivarius were conducted in the same manner, except that 3H-fructosyl sucrose was used as substrate for FTF, and samples were assayed for the incorporation of 3H-fructose into methanol-insoluble polymer. No polymer was formed from 14C sucralose alone.
The study was repeated on three occasions; a typical result is shown. Binding. -Significant levels of '4C glucose and 14C sucrose were found associated with cells. However, in all cases, 14C sucralose was neither bound to nor taken up by cells, as demonstrated by the absence of radiolabel in the filters.
Discussion. The metabolism of sucralose in animals and man and its potential for toxicity have been studied extensively. Sucralose is poorly absorbed, and most of it passes through the alimentary canal unchanged. In man, less than 5% of the oral dose is biotransformed to a glucuronide conjugate and is eliminated in the urine. All human biotransformed products are produced in animal species, and over 100 studies in these species demonstrate that sucralose is safe (Grice, 1990). Sucralose appears to have no potential for supporting growth of the ten strains of oral bacteria and plaque examined in this study. None of the organisms tested appeared to have the ability to produce acid from sucralose. Our results differ from other work (Drucker and Verran, 1980; Drucker, 1983) that showed sucralose to be anti-acidogenic when tested in combination with sucrose with use of a cell system in 0.85% saline and 50 mmol/L sucralose. Physiological saline is osmotically hypotonic to bacterial cells and does not contain the magnesium necessary for preservation of membrane integrity. This could yield cells sensitive to assay conditions. We suspended our cells in a sodium, magnesium, potassium-phosphate-buffered system with 16.7 mmol/L sucralose. It is also possible that differences in the results arose from the use of different strains. Five strains of bacteria tested failed to grow on solid medium containing 5.0% sucralose with or without glucose; however, these organisms grew well on medium containing higher molar quantities of mannitol and sorbitol, thus suggesting that the inhibition was probably not due to a difference in osmotic pressure. The reason for this inhibition is unclear. It is conceivable that the failure to grow results from differences in osmolality of media, even though we attempted to control for this variable. However, it should be realized that because of the intense sweetness of sucralose, clinical use of such high concentrations is unlikely. Sucralose did not interfere with IPS production, an obser-
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TABLE 4 REVERSIBILITY OF SUCRALOSE INHIBITION OF GLUCOSYLTRANSFERASE (GTF) GTF Assay Conditions* Enzyme pLmol '4C-glucose Treatment Dialysis Substrate Incorporated per Hourt None Sucrose 0.099 - Sucrose + Sucralose None 0.055 + None Sucrose 0.080 + Sucralose Sucrose 0.079 + Sucrose + Sucralose None 0.041 + Sucrose + Sucralose Sucralose 0.038 *GTF prepared from S. sobrinus 6715-17 was incubated for 4.0 h at 37'C with buffer or 100.0 mmol/L sucralose, followed by dialysis against 1000 volumes of buffer in 15,000 molecular weight cut-off dialysis tubing (Spectrum). The enzyme preparations were then incubated with 150.0 mmol/L '4C-glucosyl-sucrose plus or minus 150.0 mmol/L sucralose and sampled at hourly intervals. tAliquots taken at hourly intervals were measured for incorporation of 14C-glucose into methanol-insoluble polymer, and the rate of incorporation was determined by regression analysis.
vation which was not unexpected because sucralose does not interfere with bacterial metabolism, as indicated by its lack of effect on acid production and on growth rates of the test organisms. The inability of cells to take up, or bind, sucralose further supports the concept that sucralose is metabolically inert. Glucosyltransferase and fructosyltransferase preparations were found to be inhibited proportional to the concentration of sucralose added. Other investigators (Thaniyavarn et al., 1983; Binder and Robyt, 1985) have reported that many substituted sucrose derivatives act as competitive inhibitors of GTF. Halogenated sucrose derivatives (Bhattacharjee and Mayer, 1985) have been, in some cases, shown to be reversible inhibitors of GTF. Apparently, sucralose binds weakly to the enzymes because the inhibition can be readily and effectively removed by dialysis. The mechanism by which sucralose inhibits GTF and FTF is unclear. It is unlikely, however, that sucralose covalently modifies the GTF and FTF enzymes because the inhibition was reversible by dialysis. Because sucralose inhibition was not of a competitive nature, it appears that sucralose does not occupy the active site of the enzyme. However, this does not preclude the possibility of sucralose modifying the active site allosterically and reversibly. REFERENCES BHATIACHARJEE, M.K. and MAYER, R.M. (1985): Interaction in Deoxyhalosucrose Derivatives with Dextransucrase, Carbohydr Res 142:277-284. BINDER, T.P. and ROBYT, J.F. (1985): Inhibition of Streptococcus mutans 6715 Glucosyltransferases by Sucrose Analogs Modified at Positions 6 and 6', Carbohydr Res 140:9-20. DRUCKER, D.B. (1983): Comparative Effects of Five Chlorosucrose Analogues on Acidogenicity and Adherence of the Oral Bacterium Streptococcus mutans in vitro, Arch Oral Biol 28:833-837. DRUCKER, D.B. and VERRAN, J. (1980): Comparative Effects of the Substance-Sweeteners Glucose, Sorbitol, Sucrose, Xylitol and Trichlorosucrose in Lowering the pH by Two Oral Streptococcus mutans Strains in vitro, Arch Oral Biol 24:965-970. GERHARDT, P.D. (1981): Diluents and Biomass Measurement. In: Manual of Methods for General Bacteriology, P. Gerhardt, R.G.E. Murray, R.N. Costilow, E.W. Nester, W.A. Wood, N.R. Krieg, and G.B. Phillips, Eds., Washington, DC: American Society for Microbiology, p. 504. GIBBONS, R.J. (1983): Importance of Glucosyltransferase in the
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Colonization of Oral Bacteria. In: Proceedings, Glucosyltransferases, Glucans, Sucrose and Dental Caries, R.J. Doyle and J.E. Ciardi, Eds., Sp. Supp. Chemical Senses, Washington, DC: IRL Press, pp. 11-19. GIBBONS, R.J. and SOCRANSKY, S.S. (1962): Intracellular Polysaccharide Storage by Organisms in Dental Plaques, Arch Oral Biol 7:73-80. GRICE, H.A. (1990): Sucralose- -An Overview of the Toxicity Data, Food and Chemical Toxicology (in press). HAMADA, S. (1983): Role of Glucosyltransferase and Glucan in Bacterial Aggregation and Adherence to Smooth Surfaces. In: Proceedings, Glucosyltransferases, Glucans, Sucrose and Dental Caries, R.J. Doyle and J.E. Ciardi, Eds., Sp. Supp. Chemical Senses, Washington, DC: IRL Press, pp. 37-38. HOUGH, L. and KHAN, R. (1978): Intensification of Sweetness, Trends Biochem Sci 3:61-63. MENAKER, L. and McGHEE, J.R. (1982): Dental Caries: In: Dental Microbiology, J.R. McGhee, S.M. Michalek, and G.H. Cassell, Eds., Philadelphia, PA: Harper and Row Publishers, pp. 691713.
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ROBRISH, S.A.; REID, W.; and KRICHEVSKY, M.I. (1972): Distribution of Enzymes Forming Polysaccharide from Sucrose and the Composition of Extracellular Polysaccharide Synthesized by Streptococcus mutans, Appl Microbiol 24:184-190. R0LLA, G.; CIARDI, J.E.; EGGEN, K.H.; BOWEN, W.H.; and AFSETH, J. (1983): Free Glucosyl- and Fructosyltransferase in Human Saliva and Adsorption of these Enzymes to Teeth in vivo. In: Proceedings, Glucosyltransferases, Glucans, Sucrose and Dental Caries, R.J. Doyle and J.E. Ciardi, Eds., Sp. Supp. Chemical Senses, Washington, DC: IRL Press, pp. 21-29. TERLECKYJ, B.; WILLE7T, N.P.; and SHOCKMAN, G.D. (1975): Growth of Several Cariogenic Strains of Oral Streptococci in a Chemically Defined Medium, Infect Immun 11:649-655. THANIYAVARN, S.; SINGH, S.; TAYLOR, K.G.; and DOYLE, R.J. (1983): Kinetic Analysis for the Inhibition of Dextransucrase by Amino Sugars. In: Proceedings, Glucosyltransferases, Glucans, Sucrose and Dental Caries, R.J. Doyle and J.E. Ciardi, Eds., Sp. Supp. Chemical Senses, Washington, DC: IRL Press, pp. 161-170.
Sucralose (1',4',6' trideoxy-trichloro-galactosucrose) is a nontoxic, intensely sweet sucrose derivative that has been shown to be non-cariogenic in experimental animals. The purpose of this study was to determine whether certain oral bacteria could utilize sucralose. Sucralose, as a sole carbon source, was unable to support growth of ten strains of oral bacteria and dental plaque. When sucralose was incorporated into a liquid medium containing glucose or sucrose, all organisms tested displayed similar pH falls, compared with controls. The incorporation of 126 mmol/L sucralose into glucose agar medium caused total inhibition of growth of Streptococcus sobrinus 6715-17, Streptococcus sanguis 10904, Streptococcus sanguis Challis, Streptococcus salivarius, and Actinomyces viscosus WVU627. Sucralose had no effect on IPS production. Sucralose was not bound to, nor taken up by, cells. Sucralose inhibited the formation of glucan and fructan polymers in proportion to the sucralose-to-enzyme ratio, but independent of the sucrose concentration in the assay mixture. No radioactive polymer was formed from '4C-U-sucralose added to mixtures containing glucosyltransferase (GTF) or fructosyltransferase (FTF). Inhibition of GTF and FTF by sucralose was removed following dialysis of the enzyme/sucralose mixture. These results show that sucralose was not utilized by the oral bacteria tested and that the inhibitory effect of sucralose on GTF and FTF was non-competitive and reversible. The results further support the concept that sucralose is non-cariogenic. J Dent Res 69(8):1480-1484, August, 1990
Introduction. The search for suitable non-cariogenic derivatives of sucrose has resulted in limited success. In the last decade, however, it has been found that specific halogenation of sucrose can yield intensely sweet compounds (Hough and Khan, 1978). One such sucrose derivative, 1',4',6' trideoxy-trichloro-galactosucrose (sucralose), is 600 times sweeter than sucrose and appears to be metabolically inert in many strains of oral bacteria, thus making it an attractive substitute for the sweetness of sucrose. The frequent ingestion of sucrose may promote caries in several ways. For example, sucrose may promote growth and acid production by plaque bacteria. It can also be a substrate for the synthesis of intra- and extracellular polysaccharide. Clearly, any assessment of a sucrose substitute should include a study of its effects on these phenomena in bacteria. Plaque harbors a wide range of bacteria, but the microbial etiology of dental caries is associated primarily with the metabolic activities of Streptococcus mutans. Clearly, though, other microorganisms in dental plaque may contribute to the pathogenesis of dental caries. We therefore chose to study the ability of a range of Gram-positive micro-organisms -commonly associated with the onset and progress of dental caries in humans Received for publication November 9, 1989 Accepted for publication March 2, 1990
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and experimental animals (Menaker and McGhee, 1982)-to utilize sucralose. Because of the importance of glucosyltransferase (GTF) and fructosyltransferase (FTF) in the formation and growth of plaque (Gibbons, 1983; R0lla et al., 1983; Hamada, 1983) and, in addition, because sucralose has some structural features in common with sucrose, it was appropriate to be determined whether sucralose can be utilized as a substrate for these enzymes and be synthesized to glucan or fructan polymer or, alternatively, whether it can act as an inhibitor (reversible or irreversible) for these enzymes.
Materials and methods. Micro-organisms. -The following organisms were included in our study: Streptococcus mutans GS-5; Streptococcus sobrinus 6715-17; Streptococcus rattus FA-1; Streptococcus sanguis 10904; Streptococcus sanguis Challis; Streptococcus salivarius; Staphylococcus aureus H; Actinomyces viscosus OMZ1O5E; Actinomyces viscosus WVU627; Lactobacillus casei 393; Lactobacillus casei 4646; and plaque from fasted sub-
jects.
Sucralose.-Sucralose was obtained from McNeil Specialty Products Company (Skillman, NJ) and was determined by gas chromatography to be 99.3% pure. Media.-Inocula were routinely prepared in tryptone-yeast extract (2.5% tryptone, 1.5% yeast extract, 0.5% K2HPO4, 0.1% MgSO4 plus indicated carbohydrate) (TYE) broth. For preparation of growth curves, a modified FMC defined medium (Terleckyj et al., 1975) was utilized that contained 21.0 g Casamino acids (Difco Laboratories, Detroit, MI), 1.85 g (NH4)2SO4, 0.56 g MgSO4-7H2O, 9.52 g KH2PO4, 6.10 g K2HPO4, and 0.5 mg para amino benzoic acid per liter. The pH was adjusted to 7.0 and 10.0 mL of BME vitamin mixture (Gibco Laboratories, Grand Island, NY) was added, and the medium was filter-sterilized with a 0.2->Lm filter. Organisms displayed normal morphology during growth in this medium. Carbohydrate utilization. -Growth curves were derived for determination of whether the micro-organisms were able to utilize sucralose as a sole carbohydrate source and whether sucralose could influence growth via inhibition of glucose uptake. So that no unintended carbohydrate was added to the test culture by way of inoculum, starter cultures were grown overnight in TYE, and the cells were harvested by centrifugation and washed twice with PKMN buffer (Gerhardt, 1981), which contained 60 mmol/L KC1, 60 mmol/L NaCl, 5 mmol/L MgSO4, and 50 mmol/L potassium phosphate buffer (pH 7.0). The cells were re-suspended in 0.85% NaCl and used as the inoculum. Culture tubes containing defined medium plus or minus the indicated concentrations of glucose and/or sucralose were used, as shown in Table 1. Uninoculated medium was incubated overnight at 37°C in an atmosphere of 10% CO2. Tubes then received a 3.3% v/v inoculum and were kept at 37°C in a heater block for the duration of the experiment. Growth was monitored for 24 h by measurement of absorbance at 700 nm (A700) (SequoiaTurner Model 340 spectrophotometer), and growth rates were determined by logarithmic regression analysis.
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SUCRALOSE AND BACTERIAL METABOLISM
Acid production. -The influence of sucralose on acid production by the test bacteria from sucrose or glucose was examined as follows. Cells were grown overnight in TYE medium, centrifuged at 10,000 g, re-suspended in PKMN buffer, and then incubated at 37TC for two h so that intracellular polysac-
charides would be catabolized (Gibbons and Socransky, 1962). Following incubation, cells were again washed and re-suspended in PKMN buffer. Sucralose, PKMN buffer, and sucrose were added to 4 mL
of standardized cell suspension to yield the following final mixtures: control, no carbohydrate; 16.7 mmol/L sucrose; 16.7 mmol/L sucralose; and 16.7 mmol/L sucrose plus 16.7 mmol/ L sucralose. Tubes were incubated in a heater block at 370C and the pH monitored with Beckman Model pH140 pH meter. After one h, glucose (final concentration, 16.4 mmol/L) was added to the control and sucralose-only tubes, and the pH was monitored over time for determination of whether pre-incubation with sucralose affected acid production.
Growth
on
solid medium and intracellular polysaccharide
production (IPS). -The test organisms and a sample of plaque were inoculated onto TYE (without glucose) plus 1.6% agar (Difco) containing appropriate amounts and combinations of sucralose and glucose. Presence of growth was observed over 48 h. Because some organisms appeared sensitive to the presence of sucralose, we decided to determine whether the effect could be attributed to increased osmolality of the medium. Comparable concentrations of sorbitol and mannitol were included, and their effect on growth was observed. The amounts of glucose and sucralose added to the control medium, free of added carbohydrates, were as follows: 2.5% glucose (139 mmol/ L); 5.0% glucose (278 mmol/L); 2.5% sucralose (63 mmol/ L); 5.0% sucralose (126 mmol/L); 2.5% glucose (139 mmol/ L) + 2.5% sucralose (63 mmol/L); 5.0% glucose (278 mmol/ L) + 5.0% sucralose (126 mmol/L); 2.5% glucose (139 mmol/ L) + 5.0% sucralose (126 mmol/L); and 5.0% glucose (278 mmol/L) + 2.5% sucralose (63 mmol/L). The concentrations of sorbitol and mannitol followed the same pattern. Intracellular polysaccharide production (IPS) was assayed by a modified method of Gibbons and Socransky (1962). Following incubation, plates upon which growth had occurred were flooded with Gram's iodine. The iodophilic IPS-containing colonies were identified by a darkening in color and scored, from I-C , indicating no change or a yellow color, to " + + + + ", indicating dark purple or black-staining colonies.
GTF and FTFpreparation.-S. sobrinus 6715-17 (for GTF) and S. salivarius (for FTF) were grown in dialysis tubing (Spectrum Medical Industries, Inc., Los Angeles, CA) (15,000 molecular weight cut-off) containing approximately 250 mL of TYE medium suspended in a liter of the same medium to concentrate the extracellular GTF and FTF enzymes. The contents of the dialysis bags were centrifuged for removal of cells, and the supernatant was used as the GTF and FTF preparations. Polymer assay. -Synthesis of glucan and fructan was carried out with use of the methods described in Robrish et al. (1972). Incorporation of sucralose into polymer was studied with use of a 14C universally-labeled preparation of the sweetener in the test system. All assays were incubated in 1.5-mL polypropylene microcentrifuge tubes at 37°C. Aliquots were taken at the beginning and at the 1.5-, 3.0-, and 4.5-hour time points. Radio-labeled polymer was precipitated over glass fiber filters and washed three times with cold methanol in a vacuum manifold (Millipore). Filters were placed in vials of liquid scintillation cocktail and radioactivity counted for quantitation of incorporation. Inhibition assay.-The effects of sucralose on the rates of glucan and fructan polymer formation were determined by the
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concentration of 14C-labeled sucrose being kept constant and by variation of the concentrations of sucralose in the assay system. The assays were incubated and sampled, and polymer was quantitated as previously described. Additional experiments were performed for determination of whether the observed inhibition by sucralose could be relieved by an increase in the sucrose concentration in the assay and whether the inhibition was reversible. Sucrose concentrations of 50-400 mmol/ L were added to enzyme (FTF or GTF) sucralose (50-150 mmol/L) mixtures. Increasing amounts of sucrose were added to each separate concentration of sucralose. For determination of whether sucralose-associated inhibition of glucan polymer formation was reversible, GTF preparation (previously described) was incubated at 370C for four h in the presence of 100 mmol/L sucralose. After four h, the GTF + sucralose preparation was dialyzed in 15,000 mw cut-off dialysis tubing against 1000 times its volume of 50 mmol/L potassium phosphate buffer pH 7.0 at 40C with stirring. GTF preparation without sucralose, exposed to the same conditions, was used as a control. Following dialysis, the two GTF preparations (exposed and unexposed to sucralose) were assayed by two different approaches. Sucralose was included in the first series of assays, and it was omitted in the second series. The results were compared for determination of whether dialysis of the sucralose + GTF preparation would give similar glucan yields as did the dialyzed control (thus, no remaining sucralose and no inhibition), and whether the addition of sucralose to the GTF assay with dialyzed GTF preparation would give similar inhibition levels as with the undialyzed control assay.
Binding or uptake of sucralose by micro-organisms. Several experiments were conducted for determination of whether sucralose was bound or taken up by bacterial cells. To this end, S. mutans (sobrinus) 6715-17 was grown overnight in TYE medium and treated as before. 14C-labeled sucralose, PKMN buffer, glucose, and sucrose were added to 200 pL of standardized cell preparation to yield the following solutions: (A) 100 mmol/L sucralose, or (B) 100 mmol/L sucralose plus 100 mmol/L glucose, or (C) 100 mmol/L sucralose plus 100 -
TABLE 1 EFFECT OF SUCRALOSE ON GROWTH RATE OF ORAL MICROORGANISMS Percent Control Growth Rate (S.D.)*t 13..88 mmol/L 27.75 mmol/L 55.5 mmol/L Bacterial Strain sucralose sucralose sucralose S. mutans GS-5 1211.3 ( 7.3) 111.6 ( 4.9) 96.9 ( 0.4)t S. sobrinus 6715-17 13C).5 (13.6) 84.5 ( 7.6) 62.4 ( 0.7) S. sanguis 10904 .4 94.1 (24.2) 68.7 (23.8) (18.2) 124 S. sanguis Challis ).3 (14.2) 107.8 (16.3) 91.0 (12.7) S. salivarius .1 ( 7.4) 108.5 ( 8.9) 89.7 ( 1.1)4 S. aureus H 10( ).9 ( 0.6) 86.4 ( 1.0)t 71.2 ( 3.8)4 A. viscosus OMZ105E ioc3.4( 5.3) 102.3 ( 4.4) 90.5 ( 2.1) 103 A. viscosus WVU627 98.2 ( 4.9) 1.6 ( 7.9) 83.3 ( 4.5) L. case 393 1065.1 ( 4.6) 96.3 (1.3) 87.2 ( 0.1)4 L. case 4646 104U2 ( 5.9) 99.8 (1.6) 97.2 ( 6.3) 13.7 (13.6) 80.8 Plaque 55.5 (10.0) 7.2) *Bacteria were grown overnight in TYE medium, washed twice in PKMN buffer, re-suspended in sterile saline, and added to modified FMC medium containing 27.75 mmol/L glucose (control) or to the same medium plus the indicated concentration of sucralose. Growth was monitored by measurement of absorbance at 700 nm, and growth rate was determined by logarithmic regression analysis. t% control growth rate is expressed relative to control without added sucralose, whose value is equal to 100%. tSignificantly different from control via Student t test at the 99% confidence limit. -
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YOUNG & BOWEN GUOSE ADDED a control
7.0 9
A A A
A i 5
2 6.0 -
x
sucralose
*
sucrose
0
sucrose
A
9
plus sucralose
a
x 0
5.0 a
pH
K
0 0
4.0-
0
a
0
I 3.0 1 0.0
9
I
I
0.5
1.0
1.5
TABLE 2 EFFECT OF SUCRALOSE ON INTRACELLULAR POLYSACCHARIDE (IPS) PRODUCTION IPS Production* Bacterial Strain 0.0% sucralose 2.5% sucralose 5.0% sucralose ++ S. sobrinus 6715-17 NA +/_ +++ S. mutans GS-5 ++ + ++++ S. sanguis 10904 ++ ++ NA +++ S. rattus FA-1 +++ +++ ++ S. sanguis Challis ++ NA + ++ S. aureus H + ++++ ++++ S. salivarius NA A. viscosus OMZ1O5E ++++ + +±+ ++ A. viscosus WVU627 ++ ++ NA L. casei 393 ++ + ++ L. casei 4646 ++ ++ ++ * Bacteria were grown for 24 h on TYE plates containing 5.0% glucose plus the indicated concentration of sucralose. Plates were flooded with iodine, and colonies were scored for degree of IPS formation, from -, indicating yellow color or no change, to + + + +, indicating deep purple or black staining. NA-not applicable, since no growth occurred in the presence of 5.0% sucralose.
HOURS Fig. -Influence of sucralose on acid production by S. sobrinus 6715.
mmol/L sucrose. Control assays contained cells plus 100 mmol/ L 14C glucose or 14C sucrose. All assay mixtures were incubated, sampled, and cell-associated radiolabel quantitated, as described in the polymer assay section.
Results. Carbohydrate utilization.-The ability of micro-organisms to grow in the defined medium plus glucose, as well as signs of morphological changes, were continuously monitored by phase-contrast microscopy. None of the cultures displayed any apparent cellular abnormalities; growth achieved optical densities ranging from A700 0.3 to 2.0, with 90% of strains reaching an A700 of 1.0 within 8 to 10 h. Addition of sucralose to the defined medium containing glucose appeared to cause a slight increase in cell size and a decrease in chain length in all
organisms examined.
The micro-organisms examined apparently could not utilize sucralose as a carbohydrate source. Although S. aureus and S. mutans GS-5 as well as plaque displayed slight growth equivalent to one or two doublings, this apparent growth was not always reproducible or to the same extent each time (data not shown). When sucralose was used in combination with glucose, few effects were observed. However, five organisms, S. mutans GS-5, S. sobrinus 6715-17, S. salivarius, S. aureus (H), and L. casei 393 all displayed a slightly decreased growth rate in glucose medium plus 55.5 mmol/L sucralose (Table 1). Acid production. -Cell preparations in PKMN buffer and those in sucralose plus buffer displayed no decline in pH values in one hour. When glucose was added to the buffer containing sucralose, the pH values decreased and were not significantly different from a buffered control containing the added glucose. Suspensions of cells in buffered sucrose and in sucrose plus sucralose also resulted in acid production (Fig.). Solid medium growth and IPS production. -Five organisms-S. sobrinus 6715-17, S. sanguis 10904, S. sanguis Challis, S. salivarius, and A. viscosus WVU627-failed to grow on solid medium containing 5.0% sucralose, 5.0% sucralose plus 5.0% glucose, and 5.0% sucralose plus 2.5% glucose. These organisms grew on medium containing 2.5%
sucralose and lower concentrations of sucralose and glucose. Growth of the other five organisms and plaque was not inhibited on any of the solid media examined. The five organisms inhibited when sucralose was in the medium all grew on medium containing comparable concentrations of sorbitol and mannitol (Table 2). The IPS assay performed on all the micro-organisms revealed that sucralose did not interfere with IPS production on media containing glucose. The organisms grown on the medium containing only sucralose did not synthesize iodophilic IPS, whereas those grown on medium containing glucose or any combination of glucose and sucralose displayed the presence of IPS. Glucosyltransferase and fructosylItransferase studies. -The S. salivarius medium supernate was assayed for GTF and FTF activity with 14C-glucose-labeled sucrose and 3H-fructose-labeled sucrose, respectively, as substrates in the assay mixture. It displayed activities for both enzymes. When 14C-labeled sucralose was added to the assay mixtures as the sole labeled carbohydrate, no incorporation was found either in the presence or absence of unlabeled sucrose (Table 4). This assay procedure was repeated with an S. sobrinus 6715-15 preparation used as a source of GTF. When sucrose concentration was varied from 0 to 300 mmol/L, no incorporation of radiolabeled sucralose into the resultant polymer was observed. Sucralose inhibited the formation of glucan polymer by the S. sobrinus 6715-15 GTF preparation (Table 3). When extracellular FTF preparations from S. salivarius were assayed in the same manner, a similar inhibition was observed (Table 3). The degree of inhibition was related to the concentration of sucralose in the test systems, and addition of excess sucrose did not overcome the inhibition. Reversibility of inhibition of GTF by sucralose.-When the GTF preparation was dialyzed against buffer, the glucan-forming activity decreased by approximately 10%, as compared with that in undialyzed controls. However, when sucralose was added to the dialyzed GTF preparation, the percent inhibition of glucan production was similar to that induced by sucralose added to undialyzed controls. When the GTF preparation, incubated four h with sucralose, was dialyzed and then incubated with substrate, the resultant rate of glucan production was very similar to that of its dialyzed control (Table 4), showing that the inhibitory effect of sucralose could be removed by dialysis.
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SUCRALOSE AND BACTERIAL METABOLISM
TABLE 3 EFFECT OF SUCRALOSE ON GLUCOSYL- AND FRUCTOSYLTRANSFERASE ACTIVITIES % Inhibition % Inhibition Dextransucrase Sucralose of GTF Activity of FTF Activity Added to Assays (S.D.)* (S.D.)t mmol/L 22.4 ( 5.7) 8.4 (4.4) 50 150 43.8 (1.3) 25.0 (2.1) 64.4 (11.2) 44.8 (1.3) 300 * Glucosyltransferase (GTF) prepared from S. sobrinus 6715 was incubated with 150.0 mmol/L 14C-glucosyl sucrose plus the indicated concentration of sucralose at 37'C and was sampled at 1.5, 3.0, and 4.5 h. Aliquots were assayed for incorporation of '4C-glucose into methanolinsoluble polymer, and reaction rates were determined by regression analysis. Data are expressed as % inhibition of the reaction rate of control samples containing 150.0 mmol/L 14C-glucosyl sucrose, without sucralose. tInhibition experiments with fructosyltransferase (FTF) prepared from S. salivarius were conducted in the same manner, except that 3H-fructosyl sucrose was used as substrate for FTF, and samples were assayed for the incorporation of 3H-fructose into methanol-insoluble polymer. No polymer was formed from 14C sucralose alone.
The study was repeated on three occasions; a typical result is shown. Binding. -Significant levels of '4C glucose and 14C sucrose were found associated with cells. However, in all cases, 14C sucralose was neither bound to nor taken up by cells, as demonstrated by the absence of radiolabel in the filters.
Discussion. The metabolism of sucralose in animals and man and its potential for toxicity have been studied extensively. Sucralose is poorly absorbed, and most of it passes through the alimentary canal unchanged. In man, less than 5% of the oral dose is biotransformed to a glucuronide conjugate and is eliminated in the urine. All human biotransformed products are produced in animal species, and over 100 studies in these species demonstrate that sucralose is safe (Grice, 1990). Sucralose appears to have no potential for supporting growth of the ten strains of oral bacteria and plaque examined in this study. None of the organisms tested appeared to have the ability to produce acid from sucralose. Our results differ from other work (Drucker and Verran, 1980; Drucker, 1983) that showed sucralose to be anti-acidogenic when tested in combination with sucrose with use of a cell system in 0.85% saline and 50 mmol/L sucralose. Physiological saline is osmotically hypotonic to bacterial cells and does not contain the magnesium necessary for preservation of membrane integrity. This could yield cells sensitive to assay conditions. We suspended our cells in a sodium, magnesium, potassium-phosphate-buffered system with 16.7 mmol/L sucralose. It is also possible that differences in the results arose from the use of different strains. Five strains of bacteria tested failed to grow on solid medium containing 5.0% sucralose with or without glucose; however, these organisms grew well on medium containing higher molar quantities of mannitol and sorbitol, thus suggesting that the inhibition was probably not due to a difference in osmotic pressure. The reason for this inhibition is unclear. It is conceivable that the failure to grow results from differences in osmolality of media, even though we attempted to control for this variable. However, it should be realized that because of the intense sweetness of sucralose, clinical use of such high concentrations is unlikely. Sucralose did not interfere with IPS production, an obser-
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TABLE 4 REVERSIBILITY OF SUCRALOSE INHIBITION OF GLUCOSYLTRANSFERASE (GTF) GTF Assay Conditions* Enzyme pLmol '4C-glucose Treatment Dialysis Substrate Incorporated per Hourt None Sucrose 0.099 - Sucrose + Sucralose None 0.055 + None Sucrose 0.080 + Sucralose Sucrose 0.079 + Sucrose + Sucralose None 0.041 + Sucrose + Sucralose Sucralose 0.038 *GTF prepared from S. sobrinus 6715-17 was incubated for 4.0 h at 37'C with buffer or 100.0 mmol/L sucralose, followed by dialysis against 1000 volumes of buffer in 15,000 molecular weight cut-off dialysis tubing (Spectrum). The enzyme preparations were then incubated with 150.0 mmol/L '4C-glucosyl-sucrose plus or minus 150.0 mmol/L sucralose and sampled at hourly intervals. tAliquots taken at hourly intervals were measured for incorporation of 14C-glucose into methanol-insoluble polymer, and the rate of incorporation was determined by regression analysis.
vation which was not unexpected because sucralose does not interfere with bacterial metabolism, as indicated by its lack of effect on acid production and on growth rates of the test organisms. The inability of cells to take up, or bind, sucralose further supports the concept that sucralose is metabolically inert. Glucosyltransferase and fructosyltransferase preparations were found to be inhibited proportional to the concentration of sucralose added. Other investigators (Thaniyavarn et al., 1983; Binder and Robyt, 1985) have reported that many substituted sucrose derivatives act as competitive inhibitors of GTF. Halogenated sucrose derivatives (Bhattacharjee and Mayer, 1985) have been, in some cases, shown to be reversible inhibitors of GTF. Apparently, sucralose binds weakly to the enzymes because the inhibition can be readily and effectively removed by dialysis. The mechanism by which sucralose inhibits GTF and FTF is unclear. It is unlikely, however, that sucralose covalently modifies the GTF and FTF enzymes because the inhibition was reversible by dialysis. Because sucralose inhibition was not of a competitive nature, it appears that sucralose does not occupy the active site of the enzyme. However, this does not preclude the possibility of sucralose modifying the active site allosterically and reversibly. REFERENCES BHATIACHARJEE, M.K. and MAYER, R.M. (1985): Interaction in Deoxyhalosucrose Derivatives with Dextransucrase, Carbohydr Res 142:277-284. BINDER, T.P. and ROBYT, J.F. (1985): Inhibition of Streptococcus mutans 6715 Glucosyltransferases by Sucrose Analogs Modified at Positions 6 and 6', Carbohydr Res 140:9-20. DRUCKER, D.B. (1983): Comparative Effects of Five Chlorosucrose Analogues on Acidogenicity and Adherence of the Oral Bacterium Streptococcus mutans in vitro, Arch Oral Biol 28:833-837. DRUCKER, D.B. and VERRAN, J. (1980): Comparative Effects of the Substance-Sweeteners Glucose, Sorbitol, Sucrose, Xylitol and Trichlorosucrose in Lowering the pH by Two Oral Streptococcus mutans Strains in vitro, Arch Oral Biol 24:965-970. GERHARDT, P.D. (1981): Diluents and Biomass Measurement. In: Manual of Methods for General Bacteriology, P. Gerhardt, R.G.E. Murray, R.N. Costilow, E.W. Nester, W.A. Wood, N.R. Krieg, and G.B. Phillips, Eds., Washington, DC: American Society for Microbiology, p. 504. GIBBONS, R.J. (1983): Importance of Glucosyltransferase in the
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Colonization of Oral Bacteria. In: Proceedings, Glucosyltransferases, Glucans, Sucrose and Dental Caries, R.J. Doyle and J.E. Ciardi, Eds., Sp. Supp. Chemical Senses, Washington, DC: IRL Press, pp. 11-19. GIBBONS, R.J. and SOCRANSKY, S.S. (1962): Intracellular Polysaccharide Storage by Organisms in Dental Plaques, Arch Oral Biol 7:73-80. GRICE, H.A. (1990): Sucralose- -An Overview of the Toxicity Data, Food and Chemical Toxicology (in press). HAMADA, S. (1983): Role of Glucosyltransferase and Glucan in Bacterial Aggregation and Adherence to Smooth Surfaces. In: Proceedings, Glucosyltransferases, Glucans, Sucrose and Dental Caries, R.J. Doyle and J.E. Ciardi, Eds., Sp. Supp. Chemical Senses, Washington, DC: IRL Press, pp. 37-38. HOUGH, L. and KHAN, R. (1978): Intensification of Sweetness, Trends Biochem Sci 3:61-63. MENAKER, L. and McGHEE, J.R. (1982): Dental Caries: In: Dental Microbiology, J.R. McGhee, S.M. Michalek, and G.H. Cassell, Eds., Philadelphia, PA: Harper and Row Publishers, pp. 691713.
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ROBRISH, S.A.; REID, W.; and KRICHEVSKY, M.I. (1972): Distribution of Enzymes Forming Polysaccharide from Sucrose and the Composition of Extracellular Polysaccharide Synthesized by Streptococcus mutans, Appl Microbiol 24:184-190. R0LLA, G.; CIARDI, J.E.; EGGEN, K.H.; BOWEN, W.H.; and AFSETH, J. (1983): Free Glucosyl- and Fructosyltransferase in Human Saliva and Adsorption of these Enzymes to Teeth in vivo. In: Proceedings, Glucosyltransferases, Glucans, Sucrose and Dental Caries, R.J. Doyle and J.E. Ciardi, Eds., Sp. Supp. Chemical Senses, Washington, DC: IRL Press, pp. 21-29. TERLECKYJ, B.; WILLE7T, N.P.; and SHOCKMAN, G.D. (1975): Growth of Several Cariogenic Strains of Oral Streptococci in a Chemically Defined Medium, Infect Immun 11:649-655. THANIYAVARN, S.; SINGH, S.; TAYLOR, K.G.; and DOYLE, R.J. (1983): Kinetic Analysis for the Inhibition of Dextransucrase by Amino Sugars. In: Proceedings, Glucosyltransferases, Glucans, Sucrose and Dental Caries, R.J. Doyle and J.E. Ciardi, Eds., Sp. Supp. Chemical Senses, Washington, DC: IRL Press, pp. 161-170.
