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Oral Microbiol Immunol 2001: 16: 94–99 Copyright C Munksgaard 2001

Printed in Denmark . All rights reserved

ISSN 0902-0055

S. Takahashi-Abbe1, K. Abbe2,
Inhibitory effect of sorbitol on N. Takahashi1, Y. Tamazawa3,
T. Yamada1

sugar metabolism of
1
Department of Oral Biochemistry,
3
Department of Geriatric Dentistry, Tohoku
University School of Dentistry, 2Division of
Life Science, Kansei Fukushi Research

Streptococcus mutans in vitro Center, Tohoku Fukushi University, Sendai,


Japan

and on acid production in dental


plaque in vivo
Takahashi-Abbe S, Abbe K, Takahashi N, Tamazawa Y, Yamada T. Inhibitory
effect of sorbitol on sugar metabolism of Streptococcus mutans in vitro and on acid
production in dental plaque in vivo.
Oral Microbiol Immunol 2001: 16: 94–99. C Munksgaard, 2001.

This study was conducted to find out whether sorbitol inhibits the sugar
metabolism of Streptococcus mutans in vitro and the acid production in dental
plaque in vivo. S. mutans NCIB 11723 was anaerobically grown in sorbitol-
containing medium. The rate of acid production from sugars was estimated
with a pH stat. The rate of acid production from glucose or sucrose was not
changed at various concentrations of oxygen. By the addition of sorbitol to sugar, Key words: dental plaque-pH; acid
however, the acid production was decreased with increasing levels of oxygen. production; Streptococcus mutans; sorbitol;
Intracellular NADH/NADπ ratio and (dihydroxyacetone- inhibition; sugar metabolism; sugar-alcohol;
pyruvate formate-lyase; NADH/NADπ ratio;
phosphateπglyceraldehyde-phosphate)/3-phosphoglycerate ratio were high in-dwelling telemetry
whenever the acid production was inhibited by sorbitol. Sorbitol also inhibited
the acid production in dental plaque in vivo. These results suggest that the Shoko Takahashi-Abbe, Department of Oral
increased NADH/NADπ ratio during sorbitol metabolism through the Biochemistry, Tohoku University School of
Dentistry, 4-1 Seiryo-machi, Sendai,
inactivation of pyruvate formate-lyase by oxygen inhibited glyceraldehyde- 980-8575 Japan
phosphate dehydrogenase and then the acid production of S. mutans and the one
in dental plaque. Accepted for publication August 24, 2000

A variety of sugar-alcohols has been tol is sometimes used as a negative con- also inhibits the acid production in den-
used as sugar substitutes in foods. The trol for in vivo acidogenicity-test of tal plaque in vivo.
replacement of fermentable sugars with foods (3, 9, 10, 19, 24, 25).
non- or low-fermentable sweeteners in In addition to the low acidogenicity,
Material and methods
confectioneries is considered to be a sorbitol inhibits the sugar metabolism
Bacterial strain and growth conditions
practical way to prevent dental caries. of Actinomyces, one of the dominant
Sorbitol is one of the frequently used acidogenic bacteria in dental plaque, S. mutans NCIB 11723 (7) was grown
sugar substitutes to be applied in con- when carbon dioxide is hardly available at 35æC in a modified D87 medium (39)
fectioneries because of its low ferment- (32). Sorbitol is also reported to inhibit containing 1% sorbitol (wt/vol) as a car-
ability and physicochemical properties. the growth on glucose of the sorbitol- bon source under strictly anaerobic
Although mutans streptococci are metabolizing mutant of Streptococcus conditions in an anaerobic glove box
known to produce organic acids from sanguis (11). Therefore, this study was (atmosphere; N2, 80%; H2, 10%; CO2,
sorbitol (6), the acidogenicity of sorbi- conducted to find out whether sorbitol 10%: type NHC) as described pre-
tol is found negligibly small by measur- inhibits the sugar metabolism of Strep- viously (2, 29, 33). The cells were har-
ing plaque pH in vivo (in-dwelling tel- tococcus mutans, one of the most cario- vested in the logarithmic growth phase
emetry methods) (15, 17–19, 23). Sorbi- genic bacteria in dental plaque, and and washed three times with 4 mM pot-
Sorbitol-inhibition on sugar metabolism 95

assium phosphate buffer (pH 7.0) con- bated at 55æC for 1 min in air. The alka- All six subjects were asked not to al-
taining 150 mM KCl and 5 mM MgCl2 line solution was neutralized and centri- ter their dietary habits and not to brush
(washing buffer) under strictly anaer- fuged as reported previously (31). the areas where the electrodes were
obic conditions. Glycolytic intermediates except 3- placed. The subjects were also asked to
phosphoglycerate were assayed accord- suck one sorbitol-candy (2.5 g; 97% sor-
ing to the methods of Minakami et al. bitol, the rest consists of polydextrose
Fermentation of sugar
(22). The amount of 3-phosphoglycer- and flavors) three times daily for 1 week
The reaction mixture (6 ml) contained ate was assayed by the method of Czok before the pH-telemetry experiment.
washed cells (1.5–2.7 mg dry weight per (8). NADπ and NADH were assayed by Dental plaque was allowed to accumu-
ml), 100 mM KCl, 3.3 mM MgCl2 and the methods of Klingenberg (20) and late over the tip of the electrode in vivo
0.93 mM potassium phosphate buffer Takahashi et al. (31), respectively. for 3 to 6 days. The subject did not eat
(pH 7.0). After preincubation at 35æC or drink except water for 2 h prior to
for 5 min, the reaction was started by the telemetry experiment. A reference
Plaque-pH telemetry in vivo
the addition of sugar and sugar alcohol electrode (Type PH-8005, Nihon Kohd-
at a final concentration of 20 mM each An appliance for plaque-pH telemetry en Kogyo Co., Tokyo, Japan) was at-
under strictly anaerobic conditions in contained a miniaturized ion-sensitive tached to the arm of the subject.
an anaerobic glove box (atmosphere; field-effect transistor (ISFET) pH-elec- Changes in pH of dental plaque were
N2, 90%; H2, 10%: type NH). The rate trode (Type PH-6010, Nihon Kohden studied before and after the 1-week sor-
of acid production was estimated at pH Kogyo Co., Tokyo, Japan) on human bitol-adaptation period. The subject
7.0 by titrating with 50 mM KOH with enamel as described previously with rinsed with water and chewed neutral
a pH stat (AUTO pH STAT; AUT-211S, some modifications (13, 14, 37, 38). paraffin wax (ParafilmA; American Na-
TOA Electronics Ltd., Tokyo, Japan) One subject (S.A.: female, age 41) tional CanTM, Milwaukee, Wisconsin)
placed in an NH-type box. had an appliance attached on the buc- before each measurement. After the pH
Various volumes of air-saturated cal surface of a permanent upper first became neutral and stable, the subject
water (oxygen concentration at 35æC: molar using orthodontic ST-LOCK rinsed with 15 ml of sugar solution for
220 mM) (12) were added to the reaction (Sankin Industry Co., Tochigi, Japan). 2 min and the plaque pH was continu-
mixtures containing washed cells as re- Each of the other subjects (M.Y.: fe- ously monitored for 20 or 30 min.
ported previously (39). male, age 36; F.A.: female, age 66; K.T.:
female, age 62; T.S.: female, age 66; and
Results
H.S.: female, age 47) had a mandibular
Assay for end products Effect of sorbitol and oxygen on sugar
prosthesis incorporating ISFET pH-
metabolism of S. mutans
After 15 min of incubation, a part of electrode at the interproximal area that
the reaction mixture (1.5 ml) was con- was created with human enamel in the The rate of acid production from glu-
secutively filtered through a MILLEXA missing teeth area of the prosthesis. cose or sucrose by the washed cells was
AP20 Prefilter (Millipore, Tokyo, Ja-
pan) and a DISMIC Cellulose Acetate
Filter of pore size 0.2 mm (Advantic
Toyo, Tokyo, Japan) to stop the reac-
tion. A portion of the filtrate was di-
luted with 0.2 M HCl and assayed for
organic acids with a carboxylic acid
analyzer (model S-14; Tokyo Rikakikai
Ltd., Tokyo, Japan) as reported pre-
viously (31). The rest of the filtrate was
enzymatically assayed for ethanol (4).

Assay for glycolytic intermediates, NADπ


and NADH

After 15 min of the incubation, 3.0 ml


of the reaction mixture was sampled
and thoroughly mixed with 0.30 ml of
6.6 N perchloric acid to stop the reac-
tion. The acid solution was neutralized
and centrifuged as reported previously
(39). The supernatants were used for Fig. 1. Effect of sorbitol and oxygen on acid production by S. mutans under various concen-
the assay of glycolytic intermediates trations of oxygen. The mean value (∫standard deviation) of Hπ ion production in three
different experiments is shown. A. Washed cells were incubated with the reaction mixtures
and NADπ.
containing 20 mM glucose and various concentrations of oxygen (0, 30, 60 mM). B. Washed
For the assay of NADH, another 1.0 cells were incubated with the reaction mixtures containing glucose-sorbitol mixture (20 mM
ml of the reaction mixture was sampled each) and various concentrations of oxygen (0, 30, 60 mM). C. Washed cells were incubated
and was thoroughly mixed with 0.40 ml with the reaction mixtures containing 20 mM sucrose and various concentrations of oxygen
of 1.67 M alcoholic KOH (83% etha- (0, 30, 60 mM). D. Washed cells were incubated with the reaction mixtures containing sucrose-
nol) and then the solution was incu- sorbitol mixture (20 mM each) and various concentrations of oxygen (0, 30, 60 mM).
96 Takahashi-Abbe et al.

not affected by oxygen (Fig. 1A, C).


Total amount of lactate, formate and
acetate produced from glucose (Fig. 2
left) or sucrose (data not shown) did
not change, irrespective of the presence
of oxygen. However, the cells produced
more lactate and less formate, acetate
and ethanol with increasing levels of
oxygen (Fig. 2 left).
Sorbitol had no effect on the rate of
acid production from glucose in the ab-
sence of oxygen (Fig. 1A, B, O2: 0 mM).
However, the rate of acid production
from glucose-sorbitol mixture de-
creased with increasing concentrations
of oxygen (Fig. 1B, O2: 0–60 mM). Not
only volatiles (formate, acetate and
ethanol) but also lactate production de-
creased (Fig. 2 right). Sorbitol also in-
hibited the acid production from su-
crose by sorbitol-grown cells (Fig. 1D). Fig. 2. Effect of sorbitol and oxygen on end products from glucose by S. mutans under various
Sorbitol-inhibition was not observed on concentrations of oxygen. The mean value (∫standard deviation) of end products in three
the acid production from glucose by different experiments is shown.
glucose-grown cells (data not shown).
This means that sorbitol did not inhibit
the glucose metabolism of S. mutans if level of NADH was extremely low, ir- creased greatly and were 9.3–10 times
not adapted to sorbitol. respective of the presence of oxygen, re- higher than those in the absence of oxy-
Other sugar alcohols, xylitol, erythri- sulting in low NADH/NADπ ratios gen during the fermentation of glucose-
tol or maltitol, had no effect on the acid (Table 1, glucose). In addition, sorbitol mixture. The (DHAPπGalP)/
production from glucose or sucrose by [dihydroxyacetone-phosphate (DHAP) 3PG ratios also increased 3.7–4.3 times
sorbitol-grown S. mutans (data not π glyceraldehyde-phosphate (GalP)]/3- (Table 1, gluπsor). During the fermen-
shown). phosphoglycerate (3PG) ratios were tation of sucrose-sorbitol mixture in-
low. When the cells were incubated with stead of glucose-sorbitol mixture, simi-
glucose-sorbitol mixture in the absence lar results were obtained (Table 1,
of oxygen, both NADH/NADπ and sucπsor). That is, both NADH/NADπ
Effect of sorbitol and oxygen on glycolytic
(DHAPπGalP)/3PG ratios were also and (DHAPπGalP)/3PG ratios were
intermediates, NADπ and NADH
low (Table 1, gluπsor, O2: 0 mM). In the high whenever the acid production was
When glucose was metabolized as an presence of oxygen (O2: 30 and 60 mM), inhibited by sorbitol (Fig. 1C, D, Table
only available sugar, the intracellular however, NADH/NADπ ratios in- 1, sucrose, sucπsor).

Table 1. Effect of sorbitol and oxygen on glycolytic intermediates, NADπ and NADH in S. mutans
Oxygen concentration
Incubated with (mM) DHAPπGalP 3PG NADπ NADH (DHAPπGalP)/3PG NADH/NADπ
glucoseb 0 14∫3.5a 25∫8.5 8.0∫0.45 0.69∫0.22 0.56∫0.41 0.09∫0.03
30 14∫1.5 24∫9.0 7.1∫1.6 1.0∫0.13 0.58∫0.54 0.14∫0.01
60 18∫2.0 19∫3.5 6.4∫0.15 1.1∫0.06 0.95∫0.35 0.17∫0.02
gluπsorc 0 16∫2.5 18∫7.0 6.8∫0.70 0.95∫0.35 0.89∫0.54 0.14∫0.06
30 13∫0.58 4.0∫1.3 2.9∫0.23 3.7∫0.45 3.3∫0.95 1.3∫0.25
60 11∫3.3 2.9∫1.4 2.7∫0.25 3.9∫0.45 3.8∫0.87 1.4∫0.31
sucrosed 0 16∫2.3 25∫9.6 5.2∫1.9 0.61∫0.22 0.64∫0.30 0.12∫0.01
30 16∫1.5 24∫9.0 5.4∫1.6 0.69∫0.13 0.67∫0.34 0.13∫0.02
60 15∫0.58 22∫6.7 5.0∫1.7 0.79∫0.36 0.68∫0.23 0.16∫0.02
sucπsore 0 16∫3.8 15∫3.8 3.3∫0.49 2.0∫0.58 1.1∫0.50 0.61∫0.24
30 13∫2.0 5.8∫1.2 2.7∫0.40 3.7∫0.85 2.2∫0.15 1.4∫0.55
60 13∫3.9 4.4∫0.35 1.8∫0.31 3.7∫0.93 3.0∫0.65 2.1∫0.26
a
Mean value (nmol/mg dry wt of cells)∫ standard deviation of three different experiments with duplicate determinations on each occasion.
b
Cells were incubated with 20 mM glucose in the reaction mixture.
c
Incubated with glucose-sorbitol mixture (20 mM each).
b
Incubated with 20 mM sucrose.
c
Incubated with sucrose-sorbitol mixture (20 mM each).
DHAP, dihydroxyacetone-phosphate; GalP, glyceraldehyde-phosphate; 3PG, 3-phosphoglycerate.
Sorbitol-inhibition on sugar metabolism 97

increasing levels of oxygen (Fig. 2 left)


clearly indicates the inactivation of PFL
by oxygen (Fig. 4, white arrow). Instead
of the inactivated PFL, LDH seemed to
function efficiently during the glucose
fermentation. The low NADH/NADπ
ratio during the glucose or sucrose fer-
mentation (Table 1) suggests that the
LDH pathway could oxidize the
NADH generated by GalPDH, even if
the PFL pathway was inhibited (Fig. 4).
In the presence of sorbitol, however,
oxygen inhibited not only the volatiles
Fig. 3. Effect of sorbitol on acid production from sucrose in dental plaque in vivo. After the production but also the lactate produc-
1-week adaptation period, the subject (S.A.) rinsed with 15 ml of solution for 2 min. A. Plaque tion (Fig. 1B, D, Fig. 2, right). These
pH. B. Hπ ion concentration (mmol/l) calculated from A. Sorbitol, 10% sorbitol solution; results indicate that both PFL and
sucπsor, sucrose-sorbitol mixture (10% each); sucrose, 10% sucrose solution. LDH did not function efficiently.
Therefore, in addition to inactivation of
PFL by oxygen, further consideration
also have lactate dehydrogenase (LDH) should be required to explain the de-
Effect of sorbitol on acid production
which catalyzes lactate production (1, crease of acid production from the glu-
in vivo
2, 5, 28–30, 33, 35, 36, 39) (Fig. 4). The cose-sorbitol mixture or sucrose-sorbi-
The in vivo pH-fall after rinsing with su- NADH produced through the EMP tol mixture.
crose-sorbitol mixture was smaller than pathway should be re-oxidized to Sorbitol has two more hydrogen
that after rinsing with sucrose only NADπ by the PFL- and the LDH-path- atoms in the molecule than those of
(Fig. 3A). Inhibitory effect of sorbitol way. The former pathway includes two glucose or sucrose, and these hydrogen
was more clearly expressed in Hπ ion dehydrogenases, acetaldehyde dehydro- atoms generate additional NADH by
concentration (mmol/l) (Fig. 3B). The genase and alcohol dehydrogenase (Fig. the reaction of sorbitol 6-phosphate de-
integral of Hπ ion concentration for 30 4). hydrogenase (Fig. 4). Therefore, when
min after rinsing with sucrose-sorbitol The observation that S. mutans pro- S. mutans cells were incubated with glu-
mixture and sucrose solution were 191 duced more lactate and less formate, cose-sorbitol mixture or sucrose-sorbi-
and 747 mmol ¡ min ¡ lª1, respectively. acetate and ethanol from glucose with tol mixture, the LDH pathway is not
This means that the addition of sorbitol
to the rinse solution decreased the acid
production to 26%. In another subject Table 2. Inhibitory effecta of sorbitol in dental plaque in vivo before and after adaptation to
(MY), a similar result was obtained sorbitol
(Table 2, experiment B). Smaller inhibi- Experiment A (before adaptation) Experiment B (after adaptation)
tory effects were observed in the other
sucroseπ sucroseπ
three subjects (FA, KT, and TS) (Table
Subject sorbitolb sorbitolc sucrosed sorbitol sorbitol sucrose
2, experiment B). Only one subject (HS)
had no inhibition. SA NTe NT NT 9f 150 507
Inhibitory effect of sorbitol was ob- (2%)g (30%) (100%)
served to some extent even before the MY 17 481 665 8 49 174
experimental adaptation to sorbitol (3%) (72%) (100%) (5%) (28%) (100%)
(Table 2, experiment A). Some subjects FA 21 531 684 41 361 717
might take sorbitol from their daily (3%) (78%) (100%) (6%) (50%) (100%)
foods (fruits, candy etc.) in dietary KT 6 215 205 42 598 996
habits. In this study, their dietary habits (3%) (105%) (100%) (4%) (60%) (100%)
were not controlled. 20 1,230 1,330
(2%) (92%) (100%)
Discussion TS 32 448 510 54 722 875
(6%) (88%) (100%) (6%) (83%) (100%)
Mutans streptococci metabolize glu-
HS NT NT NT 73 958 933
cose or sucrose to pyruvate through
(8%) (103%) (100%)
the Embden-Meyerhof-Parnas (EMP)
pathway along with the production of
a
Inhibitory effect of sorbitol expressed in the integral of Hπ ion concentration measured
NADH by a glycolytic enzyme, glycer- during 22 min.
b
Rinse with 10% sorbitol solution.
aldehyde-phosphate dehydrogenase c
Rinse with sucrose-sorbitol mixture (10% each).
(GalPDH) (Fig. 4). These bacteria have d
Rinse with 10% sucrose solution.
an oxygen-sensitive enzyme, pyruvate e
Not tested.
formate-lyase (PFL), which catalyzes f
mmol Hπ ¡ min ¡ lª1.
the first step of conversion of pyruvate g
The integral of Hπ ion concentration for 22 min on rinsing with sucrose solution was re-
into formate, acetate and ethanol, and garded as 100%.
98 Takahashi-Abbe et al.

Acknowledgments
This study was supported by Grant-in-
Aid for Scientific Research (B) (2) No.
10557168, (B) (2) No. 10470387 and (C)
No. 10671764 from the Ministry of
Education, Science, Sports and Culture,
Japan.

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