Jo urnal of Radiation Research

and Applied Sciences

J. Rad. Res. Appl. Sci., Vol. 2, No. 5, pp. 1003-1026 (2009)

Antimicrobial Effects of Sodium Fluoride, Xylitol and

Metals Salts on in Vitro Growth Inhibition, Acid
Production and Ultrastructure of Streptococcus mutans.
T. M. El-Mongy and A. B. Abd EI-Aziz
Microbiology Department, National Centre for Radiation Research and Technology,
Atomic Energy Authority, , P.O. Box 29 Nasr City, Cairo, Egypt.
Email: abdelazizamany@gmail.com
Received: 26/10/2009. Accepted: 21/12/2009.

ABSTRACT
This study aimed to evaluate the effects of sodium fluoride (NaF), dietary
sugars, sugar alcohols (xylitol and sorbitol) and different metals salts either
separately or in combination, by different concentrations at different pH, on the
growth inhibition, acid production and ultra structure of Streptococcus mutans.
NaF was more effective at low pH, when NaF was added to actively growing
Streptococcus mutans broth culture, the growth rate was unaffected by 75 ppm
F-, slowed by 150 ppm F-, and immediately arrested by 300 or 600 ppm F-. On
the other hand, the best effect of xylitol was at high pH. The effect of xylitol
was more marked in the presence of NaF as the acid production was inhibited
and the pH did not fall to 5.0. The response of Streptococcus mutans to metals
salts was typical of this organism's response to a number of trace metals above
optimum concentrations of which may be inhibitory. Synergistic effect
observed by addition of metals salts by concentration ranged from 0.2 to
5.0mML-1, 300 ppm NaF and 5% xylitol. This formula can work at any pH
value and causes no drop of the broth culture pH to below 5.0 which is the
optimal pH for growth and multiplication of Streptococcus mutans, so this
formula worked as pH buffer regulation and growth inhibition for S. mutans.
Low concentration of this combined formula after 5 min only at 5.0 and 7.0 pH
values caused effective complete destruction of the bacterial viable cells and
this effect was observed clearly by Electron Microscope photo graph.
Key words: Sodium Fluoride, Xylitol, Metals Salts, Streptococcus mutans.

INTRODUCTION
When the equilibrium of the oral environment changes, the proliferation of
various bacterial species may hazardously increase and act together to initiate

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T. M. EL-MONGY& A. B. ABD EI-AZIZ / J. Rad. Res. Appl. Sci., Vol. 2, No. 5 (2009)

and further aggravate certain oral diseases, as periodontal diseases, dental caries
(1)
, abnormal test acuity or halitosis with the growth of the dental plaque(2).
Many investigative studies have documented the inhibition of plaque growth
and the reduction of bacterial acid formation by the use of antibacterial agents
added to mouthrinses or toothpaste preparations (2). According to their chemical
characteristics, mouthrinses commercially available contain cationic, anionic
and nonionic active ingredients which may, alter the bacterial membrane
function. Certain metal ions may alter the function of the cells membrane and
the enzymatic activity within the cell, impairing the production of acids during
the glycolysis process (3). Among the cationic agents some divalent metal ions
like Cu+2, Zn+2 and Sn+2, are most widely used. They are electro statically
attracted by oral surfaces known to carry negatively charged groups, thus
increasing the residence time of the active ingredient in the oral cavity (3). In the
presence of xylitol and NaF, Streptococcus mutans (S. mutans), the oral
organism nearly always used in such assays, is unable to acquire the nutrients
necessary for its survival and reproduction (4). In order to be effective, the active
ingredient (s) in a mouthrinse preparation must show a high substantivity
(ability to maintain an effective concentration for prolonged periods of time)
and to be able to interfere with the metabolism of targeted microorganisms.
Additionally, its bactericidal effect would last as long as the agents active form
is present at effective levels and should be harmless towards the oral mucosa
and of low toxicity to humans, since certain volume of the substance may be
swallowed during rinsing (1).
The aim of this study was to evaluate in vitro the antimicrobial activity of
sodium fluoride, xylitol combined with different metals salts at differing
concentrations and initial pH on the growth, ultra structure and acid production
by Streptococcus mutans and investigate whether this combination could result
in an additive synergetic effects.
MATERIALS AND METHODS
Organism
Streptococcus mutans an oral pathogen was obtained from the medical lab
of the Department of Microbiology at Ain Shams University.

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Growth Medium
The medium used was brain heart infusion (BHI) broth (Difco
Laboratories). Streptococcus mutans was cultured in (BHI) broth containing
0.2% glucose, buffered with 0.36 % KH2PO4 and adjusted to pH 7.0 at 37C up
to the exponential phase of growth (optical density [OD], 0.2 to 0.3). Three
hundred micro liters of this suspension was transferred into 3 ml of test medium
containing various sugar alcohols, dietary sugar or metal salts at different
concentrations. The test tubes were incubated at 37C for 48 hrs. Each test was
carried out in triplicate. All the medium components were autoclaved
separately. All compounds included in the media as potential inhibitors were
filter sterilized.
Maintenance Medium
The organism was maintained by weekly subculture on brain heart
infusion agar plates.
Analytical Determinations
Growth measurement
Absorbance at 650 nm against the standard medium was used as a measure
of growth on a LW-V-200-RS spectrophotometer, with the measurements being
performed every 1 to 2 hrs during the logarithmic phase of growth. The OD
results were calculated as the means of three measurements.
Measurement of acid production
All pH measurements were performed by means of a glass electrode pH
meter type with a Fisher pencil probe.
Test materials comprised:
1. Commercial sugars (glucose, sucrose, maltose and fructose) and sugar
alcohols (xylitol and sorbitol).
2. Aqueous sodium fluoride.
3. Pure metal salts of copper, magnesium, calcium, zinc and nitrite.
Sugars or sugar alcohols
Xylitol, sorbitol (Sigma Chemical Co., St. Louis, Mo.), fructose, glucose,
maltose or sucrose (Merck, Darmstadt, Germany), was sterilized by filtration

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and separately added (5% wt/vol) or in mixture (5% tested sugar and 5% xylitol
wt/vol) to the basic medium from a 50 % (wt/vol) stock solution. The inhibitory
effect of sugars and sugars combined with xylitol was investigated by
measurement of the acid production rate. Antimicrobial effectiveness was
assessed by measuring reduction in optical density at 650 nm using a visible
spectrophotometer.
Aqueous sodium fluoride
Freshly prepared aqueous solutions of NaF were sterilized by filtration and
serially diluted in tubes with (BHI) broth to yield final fluoride concentrations
of 0, 75, 150, 300, 600, 1200 or 1500 ppm and the broth pH was adjusted to
values 7.0 and above with 1 N NaOH or to pH values below 7.0 with 1 N NaCl.
Initial and terminal pH values after 24 and 48 hrs of incubation of agent-broth
solutions were determined. In experiments to determine the effect of pH on
fluoride inhibition, the buffer used was 1% KH2PO4 and the pH was adjusted as
indicated in each experiment. OD of cell suspensions was determined during the
time-course of the incubation.
Metals Salts
The antimicrobial efficacy of different metal salts against Streptococcus
mutans was tested in (BHI) liquid medium. Zinc chloride, calcium phosphate,
calcium carbonate, magnesium sulfate, cupper sulfate and sodium nitrite was
added to the medium to final concentrations of 0, 0.2, 0.5, 1, and 5 mML-1 and
incubated for 48 hours. The optical densities (OD) of cell suspensions were
determined and the cultures pH was measured after 24 and 48hrs during the
time-course of the incubations.
Ultra structure
Streptococcus mutans tested strain was grown in 5% xylitol, 5% glucose,
5% fructose, 5% sorbitol, control medium (BHI) and in the effective inhibition
concentrations of NaF or tested metal salts (calcium, zinc and nitrite salts) for
24 hrs and the ultra structure of bacteria examined by electron microscope. S.
mutans broth cultures were exposed to the combined formula of xylitol, NaF
and effective metals salts at pH 5.0 and pH 7.0 for only 5 min and the bacterial
ultra structure examined by electron microscope.

T. M. EL-MONGY& A. B. ABD EI-AZIZ / J. Rad. Res. Appl. Sci., Vol. 2, No. 5 (2009)
1007

Statistical Analysis
Experimental data were subjected to analysis of variance (ANOVA) using
SAS program 6.1., SAS institute, Cary, NC (5). One-way analysis of variance
(ANOVA) was used to test the differences between the groups at each point in
time. A significance level of =0.0001 was set for comparison of the groups.
The data are the means of 3 independent experiments. Vertical bars indicate
standard deviations.
RESULTS
In the present study, all pH and OD measurements were monitored to
ensure that comparisons were made between cultures of comparable growth
phase. The preliminary experiments indicated that the OD measurements gave
good agreement with the direct count during the period of growth, so OD alone
was used to estimate total cell numbers.
The mean OD for the Streptococcus mutans after 10 hrs of cultivation in
different broth media varies significantly (P< 0.0001) from one culture to
another. It was 0.85 and 0.62 with 5% glucose and maltose respectively (Fig.1),
whereas it was 0.86 in the control medium (BHI) and 0.75 in 5% sucrose
(Fig.2).
The mean OD values for the Streptococcus mutans remained less in
xylitol-containing medium than in the control medium throughout the
observation period for all media tested (P < 0.0001).Tested oral Streptococcus
strain was inhibited by xylitol which exhibited a dose-related inhibition of
Streptococcus mutans in the (BHI) medium. The difference was greater after 10
hrs of incubation, where the mean OD measurements for the S. mutans were 0.
46, 0.28 and 0.20 in 2.5, 5 and 10 % xylitol, i.e., it was 46.51%, 67.45% and
76.74% less in the xylitol-containing medium than the control culture (BHI),
respectively (Fig. 2).
The addition of glucose, maltose, or sucrose at concentration 5% did not
alter the effect of xylitol, and growth inhibition was detected (Fig.1). The
difference was greater after 8-15 hrs of the beginning of the cultivation. The
mean OD measurements after 10 hrs for the Streptococcus mutans were 0.45 ,
0.54 and 0.49 in 5% of glucose, maltose and sucrose combined with 5%
xylitol respectively; i.e., it was 47.67, 37.20 and 43.02 % less in the xylitol-

T. M. EL-MONGY& A. B. ABD EI-AZIZ / J. Rad. Res. Appl. Sci., Vol. 2, No. 5 (2009)

1008

containing medium than the control medium (R2 = 0.99, [ P < 0.0001]),
(Figs.1and 2). Meanwhile the OD was 0.67 by using 5 % fructose. Xylitolinduced growth inhibition was prevented by the addition of 5 % fructose (OD,
0.62) (Fig.1). Sorbitol at concentration of 5% had no inhibition effect on the
growth of Streptococcus mutans (OD, 0.51). Xylitol at a concentration of 5%
was as effective alone as it was in combination with 5% sorbitol (Fig.2). The
mean OD for the Streptococcus mutans after 10 hrs of cultivation was 0.30 in
the medium containing both 5% sorbitol and xylitol.
0.9
0.8
glucose+xy
fructose+xy
sucrose+xy
maltose+xy
glucose 5%
fructose 5%
sucrose 5%
Maltose 5%

0.7
0.6

OD

0.5
0.4
0.3
0.2
0.1
0

10

15

20

24

48

Time (hrs)

Fig. 1. Growth of Streptococcus mutans in media containing different sugars and

xylitol measured in terms of OD counts over 48 hrs. *Error bars represent
standard deviation. *Means of data are significantly different (P <
0.0001)].

The OD values for the cultures grown on the experimental media

containing different metal salts differed significantly (P < 0.0001) from those
for cultures grown on the control medium. Growth of Streptococcus mutans,
measured in terms of OD, in media containing (per Liter) 1.0 mM of ZnCl2 , 5.0
mM of CaPO4, or 0.5 mM of NaNO2 over 48 hrs was compared with in control
medium (BHI) at pH 7.0. Inhibition of 41.05 %, 77.32 % and 58.02 %
(P<0.0001), were observed with ZnCl2, CaPO4, and NaNO2, respectively
(Fig.3). MgSO4 at concentration 0.5 mM inhibited the growth of Streptococcus
mutans by 37.79 %. Meanwhile, CaCO3 and CuSO4 at concentration 5.0 mM

T. M. EL-MONGY& A. B. ABD EI-AZIZ / J. Rad. Res. Appl. Sci., Vol. 2, No. 5 (2009)
1009

and 0.2 mM were having little effect on S. mutans activity (28.95% and 33.48
%, respectively) (Fig.3).
1
0.9
0.8
BHI

0.7

xylitol 2.5%
xylitol 5%

0.6

xylitol10%

OD

sorbitol 5%

0.5

sorb+xy

0.4
0.3
0.2
0.1
0
0

10

15

20

24

48

Time (hrs)

Fig. 2. Growth of Streptococcus mutans in media containing different sugars and

xylitol measured in terms of OD counts over 48 hrs. *Error bars represent
standard deviation. *Means of data are significantly different (P <
0.0001)].

Table 1 reveals the effect of different treatments on the final pH of growth

media. Xylitol was reported to inhibit the growth of Streptococcus mutans and
the rate of acid production from glucose. These results were notable at pH 5.57.0. After 48 hrs of incubation demonstrated that maltose, glucose and fructose
gave final pH values of 4.24, 4.15 and 4.92 compared with sucrose (final pH
3.95). Addition of 5% xylitol to the sorbitol cultures gave a final pH 6.27;
whereas the final pH values with different tested xylitol concentrations ranged
between 6.40 and 6.47); sorbitol-xylitol challenges gave less acid production
than sorbitol (final pH 6.12); challenges alone (Table 1). It was concluded that
the media which have high pH values were those containing xylitol alone or
mixtures of xylitol and sorbitol. Xylitol was not able to inhibit the acid
production from the easily fermented glucose (final pH of glucose/xylitol
mixture 5.43); and fructose (final pH fructose/xylitol mixture 4.96).

T. M. EL-MONGY& A. B. ABD EI-AZIZ / J. Rad. Res. Appl. Sci., Vol. 2, No. 5 (2009)

1010

0.7
CaPO4

0.6

CaCO3
NaNO2
MgSO4

0.5

CuSO4
ZnCl2

OD

0.4

0.3

0.2

0.1

0
0

10

15

20

24

48

Time (hrs)

Fig. 3. Effect of different metal salts on Streptococcus mutans growth. *Error bars
represent standard deviation. *Means of data are significantly different
(P < 0.0001)].

This study compared the effects of different concentrations of sodium

fluoride on S. mutans growth (Fig.4).The addition of 300 ppm F- to actively
broth cultures almost totally arrested the growth of the S. mutans (OD, 0.08) at
pH 5.5. The addition of 1200 ppm F immediately terminated S. mutans growth
(OD, 0.04). (R2 = 0.99, [P< 0.0001]).
The inhibitory concentration of fluoride ions progressively diminished as
the acidity increased, the inhibitory effect of fluoride was maximal at pH 5.5
and gradually decreased at pH values (6.0-7.5). The streptococci were inhibited
by low fluoride concentrations (75 ppm) at pH 5.5 than (1500 ppm) at pH 7.5,
where the OD was 0.13 and 0.31, respectively. The inhibitory effects of fluoride
on acid production significantly increased (P< 0.0001) when the pH dropped
from 7.0 to 5.5 (Fig. 4).

T. M. EL-MONGY& A. B. ABD EI-AZIZ / J. Rad. Res. Appl. Sci., Vol. 2, No. 5 (2009)
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Table 1. Effect of different treatments on the final pH of Streptococcus mutans

growth media.
Treatment
BHI (control)
Glucose 5%
Glucose+xylitol (5%)
Fructose 5%
Fructose+xylitol (5%)
Sucrose 5%
Sucrose + xylitol (5%)
Maltose 5%
Maltose+xylitol (5%)
Sorbitol 5%
Sorbitol +xylitol (5%)
Xylitol 2.5%
Xylitol 5%
Xylitol 10%
ZnCl2
NaNO2
MgSO 4
CuSO4
CaPO4
CaCO3

OD
10hrs
0.860
0.853
0.450
0.669
0.619
0.674
0.485
0.615
0.540
0.51
0.30
0.46
0.275
0.206
0.507
0.361
0.535
0.572
0.195
0.611

SD
0.028
0.015
0.007
0.009
0.050
0.009
0.007
0.006
0.014
0.021
0.032
0.042
0.021
0.005
0.003
0.021
0.007
0.006
0.007
0.002

48hrs
0.483
0.645
0.305
0.403
0.383
0.481
0.225
0.325
0.117
0.213
0.176
0.119
0.103
0.092
0.471
0.300
0.361
0.455
0.099
0.523

pH
SD
0.014
0.007
0.006
0.013
0.046
0.003
0.007
0.005
0.010
0.028
0.002
0.014
0.001
0.003
0.001
0.014
0.016
0.002
0.001
0.005

10hrs
4.81
5.67
5.74
5.65
5.69
5.72
5.61
5.55
5.33
5.95
6.08
6.10
6.17
6.22
4.97
6.31
5.26
5.82
6.17
5.00

24hrs
4.72
4.75
5.51
5.09
5.08
4.16
5.19
4.60
5.67
6.05
6.20
6.38
6.40
6.43
4.62
6.44
4.78
6.31
6.57
4.54

48hrs
4.70
4.15
5.43
4.92
4.96
3.95
5.12
4.24
5.72
6.12
6.27
6.40
6.45
6.47
4.52
7.08
4.57
6.44
6.69
4.39

Initial pH 5.5. * Data are recorded as mean of 3 observations SD. * Significance change
from control group at P< 0.0001.

At pH 7.0 there was significant decrease (P< 0.0001) in final pH with the
increase of F- concentration. The final pH was 5.89 with 1500 ppm F-, where it
was 5.66 with 1200 ppm F- and it decreased to reach 4.84 with 75 ppm F after
48hrs (Fig.5). The greatest effect of NaF was at pH 5.5 for all concentrations
tested. As the pH decreased the inhibition effect of NaF increased and the
smallest concentration used (75 ppm F-) gave OD 0.125, where it gave OD
0.705 at pH 7.5. By increasing the concentration of NaF from 75 ppm to 300
ppm at pH 5.5 the OD was 0.08 and it was 0.046 with 1500 ppm F- after 48 hrs
(Fig.5).
Three hundred ppm NaF was taken as the lowest effective concentration of
F and its inhibitory effect in combination with others antimicrobial agents was
tested. From Table 2. Fluoride (300 ppm) and xylitol 5% together have
-

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T. M. EL-MONGY& A. B. ABD EI-AZIZ / J. Rad. Res. Appl. Sci., Vol. 2, No. 5 (2009)

synergistic inhibitory effects on the mutans streptococci growth as the inhibition

percentage increased by 87.14 more than NaF alone (300 ppm) as control
medium at pH 7.0.
0.9
0.8
0.7
0.6
control
75ppm
150ppm
300ppm
600ppm
1200ppm
1500ppm

OD

0.5
0.4
0.3
0.2
0.1
0

5.5

6.5

7.5

pH
Fig. 4. Effect of different NaF concentrations on Streptococcus mutans growth at
various initial pH after 48 hrs. *Error bars represent standard
deviation. *Means of data are significantly different (P < 0.0001)].
8
7

pH

4
BHI
F75ppm

F150ppm
F300ppm

F600ppm
F1200ppm

F1500ppm

0
0

10

14

20

24

48

Times (hrs)

Fig. 5. Effect of different NaF concentrations on pH values of Streptococcus mutans

growth media.

T. M. EL-MONGY& A. B. ABD EI-AZIZ / J. Rad. Res. Appl. Sci., Vol. 2, No. 5 (2009)
1013

Table 2. Comparison of metal salts and xylitol inhibition effect after 48 hrs at pH
7.0 with (300 ppm NaF) as control medium.
Metals salts

Inhibition %

1.0 mM ZnCl2

68.59

5.0 mM CaPO4

84.53

0.5 mM NaNO3

53.13

0.5 mM MgSO4

27.81

5.0 mM CaCO3

2.65

0.2 mM CuSO4

21.09

5% xylitol
87.14
*Significance change from control group at P< 0.0001.

Table 3. ANOVA Statistics

Treatment

ANOVA
SS

Mean

Mean
Square

BHI (control)
Glucose (5%)
Glucose + xylitol (5%)
Fructose 5%
Fructose + xylitol (5%)
Sucrose (5%)
Sucrose + xylitol (5%)
Maltose 5%
Maltose+ xylitol (5%)
Sorbitol (5%)
Sorbitol + xylitol (5%)
Xylitol (2.5%)
Xylitol (5%)
Xylitol (10%)
ZnCl2
NaNO2
MgSO 4
CuSO4
CaPO4
CaCO3

0.931
1.263
0.323
0.573
0.449
0.758
0.486
0.845
0.693
0.231
0.077
0.281
0.092
0.049
0.209
0.095
0.186
0.220
0.019
0.368

0.519
0.610
0.306
0.413
0.389
0.449
0.386
0.318
0.297
0.280
0.193
0.236
0.158
0.130
0.428
0.317
0.371
0.432
0.137
0.498

0.133
0.105
0.027
0.048
0.037
0.063
0.040
0.070
0.058
0.019
0.011
0.040
0.013
0.007
0.030
0.013
0.027
0.031
0.003
0.053

Root
MSE
0.018
0.018
0.012
0.012
0.021
0.011
0.023
0.009
0.020
0.012
0.016
0.020
0.011
0.006
0.004
0.013
0.009
0.006
0.006
0.012

F-ratio R-Square

CV

404.84
309.69
194.60
332.33
84.59
527.71
78.46
869.10
148.80
141.94
41.54
95.19
111.74
177.91
201.51
83.91
326.48
743.13
84.11
346.79

3.49
3.02
3.83
2.90
5.40
2.43
5.88
2.83
6.63
4.15
8.46
8.70
6.89
4.85
0.89
4.00
2.43
1.50
4.14
2.47

0.997
0.996
0.994
0.996
0.987
0.997
0.986
0.998
0.992
0.992
0.976
0.989
0.991
0.994
0.999
0.988
0.996
0.998
0.988
0.997

Metal ions were evaluated in combination with NaF as potential

antimicrobial agents for growth inhibition of S. mutans. The inhibition
percentage increased by 68.59, 84.53 and 53.13 with 1.0 mM of ZnCl2, 5.0 mM
of CaPO4 and 5.0 mM of NaNO2, respectively. Meanwhile, MgSO4 and CuSO4
at concentration 0.5 mM and 0.2 mM with NaF having little inhibitory effect by

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27.81% and 21.09%, respectively and the synergetic effect of 5.0 mM CaCO3
was 2.65% and can be neglected (Table 2).
The final pH of tested bacteria broth culture in the presence of nitrite
resulted in a higher pH (6.31) than was found in controls (pH 4.81) after 10 hrs.
Presence of nitrite resulted in considerable reduction of acid production with the
tested strain. With ZnCl2, CaPO4, and NaNO2, the pHs were pH 4.52, 6.69 and
7.08, respectively after 48 hrs of cultivation. Meanwhile, with MgSO4 and
CuSO4 the final pH values were 4.57 and 6.44, respectively (Table 1).
Ultra structure
Examination of S. mutans exposed to xylitol, glucose, fructose and
sorbitol at concentration 5%, NaF (300 ppm) and different metals salts which
gave high synergetic effect with NaF (calcium, zinc and nitrite) or control
medium (BHI) for 24 hrs by electron microscopy (EM photos,1-12) indicated
that a xylitol-containing reaction mixture caused distinct alterations in bacterial
ultra structure without notable effect on the total viability of the strain and the
cell wall of S. mutans became more diffuse, the proportion of damaged S.
mutans increased. Degrading cells, autolysis, and intracellular vacuoles were
frequently seen in the reaction mixtures after exposure to xylitol for 24 hrs, but
not after exposure to other sugars or control medium.
The Streptococcus mutans in the medium containing xylitol and sorbitol
remained viable until the end of the test. The autolysis of Streptococcus mutans
was seen in BHI and in the medium containing 5% sorbitol, which is a typical
phenomenon in streptococci cultures after rapid logarithmic growth and which
is thought to be mediated by autolytic enzymes. The bacteria in the media
containing an extra carbon source for growth, i.e., fructose, glucose, or sucrose
at concentration of 5%, had high OD values at the end of the experiment
without autolysis.
Fluoride ion (F-) was more effective in growth inhibition than the non
xylitol(X), non (X)Non F-. XF- was more effective than F- alone, and
combination of metals salts with F- X was the most effective in the inhibition of
Streptococcus mutans growth and the rate of acid production. Examination of S.
mutans exposed to F- X- metal salts mixture for 5min, at pH 5.0 and 7.0, by
electron microscopy indicated complete damaged of the microbial cells.

T. M. EL-MONGY& A. B. ABD EI-AZIZ / J. Rad. Res. Appl. Sci., Vol. 2, No. 5 (2009)
1015

1- Streptococcus mutans on BHI medium 2-Effect of glucose on Streptococcus

mutans
(control)

3-Effect of CaPO4 on Streptococcus mutans 4-Effect of ZnCl2 on Streptococcus mutans

5-Effect of Sorbitol on Streptococcus

mutans

6-Effect of Fructose on Streptococcus

mutans

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T. M. EL-MONGY& A. B. ABD EI-AZIZ / J. Rad. Res. Appl. Sci., Vol. 2, No. 5 (2009)

7-Effect of NaNO3 on Streptococcus mutans 8-Effect of xylitol on Streptococcus mutans

9-Effect of NaF (300 ppm F) at pH 7.0 on

Streptococcus mutans

11-Effect of combined formula at pH 5.0

on Streptococcus mutans

10-Effect of NaF (300 ppm F) at pH 5.0 on

Streptococcus mutans

12-Effect of combined formula at pH 7.0

on Streptococcus mutans

Effect of different treatments on Streptococcus mutans Ultra structure. (Electron

microscope photos, 1-12).

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DISCUSSION
Sugars and other fermentable carbohydrates, after being hydrolyzed by
salivary amylase, provide substrate for the action of oral bacteria, which in turn
lower the plaque and salivary pH. The resultant action is the beginning of tooth
demineralization (6).
Our study indicates that xylitol causes marked inhibition of S. mutants
growth while, glucose, sucrose, and maltose had no effect on this inhibition
effect of xylitol which was eliminated by fructose. Sorbitol as sugar alcohol has
no effect on the bacterial growth in this study (Fig.1). Acid production by S.
mutants was increased by tested dietary sugars, and reduced by tested sugars
alcohols (Table 1). Also, the present data revealed that glucose and maltose give
similar falls in pH, while fructose had only slightly smaller effects compared
with sucrose.
In other study pure culture of S. mutants was found to produce a moderate
to large decrease in pH with glucose meanwhile, galactose produces a
significantly smaller decrease in pH than does glucose (7).
In another study the sugar alcohols, sorbitol and mannitol reported to
slowly fermented to acid by oral bacterial, and xylitol is virtually nonfermentable (8).
From Table 1, it is clear that sorbitol gave a small pH drop whereas xylitol
caused a negligible decrease in pH and addition of xylitol to the bacterial
suspension caused inhibition of acid production from sorbitol by S. mutans. Our
present data are in agreement with previous study, where Streptococcus mutans
cells grown on a medium containing xylitol and the mixture of sorbitol and
xylitol formed less acid from glucose(9).
In addition, from the present data in Table 1, it was noticed that the only
mixtures which increased the pH values of the suspensions were those
containing xylitol alone or mixtures of xylitol and sorbitol. Xylitol was not able
to inhibit the acid production from the easily fermented glucose and fructose.
Addition of small amounts of xylitol to the sorbitol cultures gave a growth
below that of cultures with no extra carbon (10).
The first step of xylitol metabolism in mutans group streptococci is entry
of xylitol into the bacterial cell via the fructose phosphotransferase system and
xylitol does not cause growth inhibition in the presence of fructose(8). Xylitol

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retarded the growth of mutans streptococci in the presence of glucose,

galactose, maltose, lactose (L) or sucrose as an energy source. The addition of
sorbitol at concentrations of 1, 2.5 or 5% to the growth medium did not affect
the growth of Streptococcus pneumoniae and neither inhibited nor enhanced the
xylitol induced growth impairment (9). Xylitol inhibited acid production by
washed cells of Streptococcus mutans from glucose, galactose, maltose, lactose
or sucrose (12-83% inhibition). However, in the presence of Fr, no inhibition of
acid production was observed (10).
Xylitol was reported to inhibit the glycolysis and growth of S. mutans
(direct inhibition) and inhibit the rate of acid production from glucose, with a
decrease in the intracellular level of fructose 1, 6-bisphosphate and an
intracellular accumulation of xylitol 5-phosphate (X5P) (indirect inhibition) (11).
Many elements have been examined for their inhibitory effects upon
caries, but relatively few have shown significant results. More than any other
element, fluorine has been the subject of attention and investigation.
This study compared the effect of different concentrations of NaF at
different initial pH and the present data reveled that, as the acidity of the growth
culture increased the effect of low concentrations of F- increased (Figs. 4, 5).
In comparison, the inhibitory effect of NaF, SnF2, and SnC12 on the
growth of Streptococcus mutans was studied in vitro and the results showed
that, sodium fluoride arrested the growth at concentrations 300 ppm and 600
ppm F- and it showed some bactericidal activity at 150 and 300 ppm F-, and at
600 ppm F- was totally bactericidal (12).
Previous report has shown that the cell membrane is impermeable to the
fluoride anion and that intracellular accumulation of fluoride depends on
translocation of hydrogen fluoride across the membrane (13).
Also, it was investigated that the inhibitory effects of fluoride on acid
production increased when the pH dropped from 5.5 to 4.0. A concentration of
5 ppm fluoride inhibited the acid production activity of Streptococcus mutants
at pH 5 and below. These results suggest that fluoride in dental plaque may
affect acid production below pH 5.5 (14). In another study 1 mML-1 fluoride,
used with the glucose or provided continuously, reduced both the rate of change
and the degree of fall in pH, and in doing so prevented the enrichment of S.
mutans in the culture (15).

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This study indicates that fluoride and xylitol together have synergistic
inhibitory effects on the acid production of S. mutans and xylitol has the
potential to enhance the inhibitory effects of low concentrations of fluoride
(Table 2).
It is clear that xylitol effect was most marked in the presence of fluoride.
Under these conditions, the rate of lactate production was reduced at least 3fold, the pH did not fall to 5.0 and only about 50% of the added glucose was
consumed. This suggests that xylitol can augment the metabolic effects on S.
mutans of low levels of fluoride (16).
In a previous study, demineralization is inhibited and remineralization is
accelerated when 5% xylitol is used with (500 ppmF -) fluoride, compared to
toothpastes containing 500 ppm F- only in vitro. Toothpaste containing 500 ppm
F- and 5% xylitol might be beneficial, both with respect to its caries inhibiting
effect and decrease in the risk of dental fluorosis (17).
In another study, the combination of fluoride and xylitol (fluoride (0-6.4
mM) and/or xylitol (60 mM)) inhibited acid production more effectively than
fluoride or xylitol alone. Analyses of intracellular glycolytic intermediates
revealed that xylitol inhibited the upper part of the glycolytic pathway, while
fluoride inhibited the lower part. This study indicates that fluoride and xylitol
together have synergistic inhibitory effects on the acid production of mutans
streptococci and suggests that xylitol has the potential to enhance inhibitory
effects of low concentrations of fluoride (18).
Inhibition of the formation and metabolism of dental plaque by zinc salts
has been well documented (3, 19). The mechanisms remain unclear, but studies
suggest that the inhibition of acid production is correlated with adsorption of
zinc on the bacterial cell wall (19). Factors other than concentration may affect on
zinc action such as the difference in zinc salts applied and methods of
application. Zinc may also reduce plaque bulk by inhibiting extra cellular
polysaccharide-producing enzymes of plaque organisms (20).
The effect of various metallic salts upon growth and acid production has
been investigated. In the present study, inhibition was high with CaPO4, and
NaNO2, respectively, moderate with ZnCl2 and MgSO4 and low with CaCO3
and CuSO4 (Fig.3). Other study reported that copper, nickel; gold, silver, and
mercury salts exerted the greatest inhibitory action on acid production.

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Magnesium, cobalt, manganese, aluminum, iron, and chromic salts showed little
or no activity. Copper at a concentration of 0.25 mg. per 100 ml. of saliva
containing sucrose definitely inhibited acid formation while a concentration of 3
to 4 mg per 100 ml of saliva caused complete inhibition of acid production (21).
In the present study, the final pH of tested bacteria broth culture in the
presence of nitrite resulted in a higher pH (6.31) than was found in controls (pH
4.81) after 10 hrs (Table 1). It has been reported that nitrite at acidic pH has
been shown to be antibacterial particularly against the highly cariogenic species
Streptococcus mutans (22). At an acid pH, nitrite is converted to nitrous acid and
hence to nitric oxide (NO) and nitrogen dioxide (NO2) radicals which are
harmful to bacteria S. mutans (23).
The NO radical can react with various other substances, e.g. super oxide
(O2), iron and thiols. All of these interactions prove damaging to various
cellular targets. NO2 is also able to interact with DNA causing deamination or
cross-linking (24). At acidity levels below pH 5.0, low concentrations of nitrite
(0.2 mM) caused effective complete killing of the periodontal bacteria (25).
Calcium has been implicated in cariostasis by the formation of labile
reaction products with fluoride enhancing its uptake (26). The acidic calcium
phosphate (containing 0. 7 M Ca, 1.9 M PO4) treatment markedly enhanced the
ability of the enamel to acquire fluoride and their protection effect could be
conferred by direct and indirect effects and can exert antimicrobial effects,
including glycolysis inhibition (27).
Mg2+ and phosphate also were important for inhibition and they
formulated a modulus for predicting inhibition based on the concentrations of
three key agents, F-, Mg2+, and phosphate. In a previous study, inhibition has
been found to occur at 5 mM inorganic phosphate and 2 mM Mg2+ in the
presence of 16-54 mM fluoride (28).
It has been investigated that xylitol, NaF and ZnCl2 in combination
inhibited the growth of S. sobrinus OMZ 176 when added to Brain Heart
Infusion broth. Fluoride was bactericidal only at pH 4.0 (29). However, the
combination of zinc plus fluoride was strongly bactericidal at all pH values that
were tested. Glucose uptake was reduced; the glycolysis inhibited at the glucose
6-phosphate and fructose-1.6-diphosphate level and the accumulation of xylitol
metabolites was increased. These effects in combination probably accounted for

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1021

the inhibition of growth and it suggested that xylitol can augment the metabolic
effects on S. mutans of low levels of fluoride (30).
In the present study the ultra structure of S. mutans bacteria revealed that,
xylitol has a harmful effect on the bacterial cell wall and the proportion of
damage increased after exposure to xylitol for 2 hrs, as compared with other
sugars or control medium
S. mutans exposed to 5% xylitol for 2 hrs failed to form the longer chains
comprising 4 to 6 cocci at a time that were seen in the control media. The ultra
structure of the bacteria was not damaged after exposure to 5% glucose or 5%
fructose, but after sorbitol exposure the cell wall structure became slightly more
diffuse as compared with xylitol and control media (BHI).
Previous reports have shown that the cell wall of five strains of
pneumococci exposed to 0.5%-5% xylitol and glucose, fructose and sorbitol at
concentration 5% or control medium (BHI) for 0.5-2 hrs became more diffuse
and less well defined; the main diameter of the polysaccharide capsule became
ragged and small as compared with the control media after exposure to xylitol
for 2 h, but not after exposure to other sugars or control medium. The
phenotype of all pneumococcal strains was opaque before xylitol exposure and
became almost transparent both in xylitol and in control medium during the
experiment (31).
In another study, a xylitol-containing reaction mixture caused distinct
alterations in bacterial ultra structure without notable effect on the total viability
of the strain. Incubations in media containing 50 mg/ml of glucose, fructose,
sucrose, lactose, sorbitol or mannitol as the primary carbon source did not affect
bacterial ultra structure. Xylitol degrading cells and autolysis, intracellular
vacuoles and lamellate formations in the cytoplasmic membrane were
frequently seen independent of the concentration of xylitol in the reaction
mixtures. Despite the alterations in ultra structure of the xylitol-incubated
bacteria, there was no difference in their viability when compared to the
controls (32).
CONCLUSION
The experimental results in our study support the combined effectiveness
of the ingredients that the literature suggests. Calcium phosphate reduces
demineralization of teeth and plaque formation through its bactericidal effect on

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anaerobic microorganisms and work as remineralizing agent. Zinc chloride

reduces plaque and decalcification via its extra- and intra-cellular attachment,
disrupting the metabolic activity of the microbiota responsible for plaque.
Xylitol inhibits bacteriological growth. Zinc chloride enhances the activity of
the sodium fluoride and adds its own antimicrobial properties, and sodium
nitrite serves as a desensitizer that also complexes the metals, enhancing their
activity on the enzymes of the microbiota. The five ingredients work together to
reduce etiological factors, microbiota and their by-products, plaque and
decalcification. In this way, they combine to aid in the maintenance of a normal
host/parasite balance.
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