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

Antimicrobial Effects of Sodium Fluoride, Xylitol andMetals Salts on in Vitro Growth Inhibition, Acid

Production and Ultrastructure ofStreptococcus mutans.

T. M. El-Mongy and A. B. Abd EI-Aziz.

Microbiology Department, National Center for Radiation Research and Technology,

Atomic Energy Authority, Nasr City, Cairo, Egypt.

Email:[email protected]

Received: 26/10/2009. Accepted: 21/12/2009.

ABSTRACT

This study aimed to evaluate the effects of sodium fluoride (NaF), dietarysugars, sugar alcohols (xylitol and sorbitol) and different metals salts eitherseparately or in combination, by different concentrations at different pH, on thegrowth 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 ppmF

-, 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 xylitolwas more marked in the presence of NaF as the acid production was inhibitedand the pH did not fall to 5.0. The response ofStreptococcus mutans to metalssalts was typical of this organism's response to a number of trace metals aboveoptimum concentrations of which may be inhibitory. Synergistic effect

observed by addition of metals salts by concentration ranged from 0.2 to5.0mML

-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 theoptimal pH for growth and multiplication of Streptococcus mutans, so thisformula 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 pHvalues caused effective complete destruction of the bacterial viable cells andthis 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)1004

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 differingconcentrations 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). Threehundred 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 sugaralcohols (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 filtrationand separately added (5% wt/vol) or in mixture (5% tested sugar and 5% xylitol


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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.


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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 indifferent 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-containing medium than the control medium (R

2= 0.99, [ P < 0.0001]),


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(Figs.1and 2). Meanwhile the OD was 0.67 by using 5 % fructose. Xylitol-

induced 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.

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


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0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 2 6 10 15 20 24 48

OD

BHI

xylitol 2.5%

xylitol 5%

xylitol10%

sorbitol 5%

sorb+xy

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)].

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 2 6 10 15 20 24 48

OD

CaPO4

CaCO3

NaNO2

MgSO4

CuSO4

ZnCl2

Time (hrs)

Fig. 3. Effect of different metal salts on Streptococcus mutans growth. [(-)Errorbars represent standard deviation. (-)Means of data are significantly

different (P < 0.0001)].


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

media. Xylitol was reported to inhibit the growth ofStreptococcus mutans and

the rate of acid production from glucose. These results were notable at pH 5.5-

7.0. 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).

Table 1. Effect of different treatments on the final pH of Streptococcus mutans

growth media.

TreatmentOD pH

10hrs SD 48hrs SD 10hrs 24hrs 48hrs

BHI (control) 0.860 0.028 0.483 0.014 4.81 4.72 4.70

Glucose 5% 0.853 0.015 0.645 0.007 5.67 4.75 4.15

Glucose+xylitol (5%) 0.450 0.007 0.305 0.006 5.74 5.51 5.43

Fructose 5% 0.669 0.009 0.403 0.013 5.65 5.09 4.92

Fructose+xylitol (5%) 0.619 0.050 0.383 0.046 5.69 5.08 4.96

Sucrose 5% 0.674 0.009 0.481 0.003 5.72 4.16 3.95

Sucrose + xylitol (5%) 0.485 0.007 0.225 0.007 5.61 5.19 5.12

Maltose 5% 0.615 0.006 0.325 0.005 5.55 4.60 4.24

Maltose+xylitol (5%) 0.540 0.014 0.117 0.010 5.33 5.67 5.72

Sorbitol 5% 0.51 0.021 0.213 0.028 5.95 6.05 6.12

Sorbitol +xylitol (5%) 0.30 0.032 0.176 0.002 6.08 6.20 6.27

Xylitol 2.5% 0.46 0.042 0.119 0.014 6.10 6.38 6.40

Xylitol 5% 0.275 0.021 0.103 0.001 6.17 6.40 6.45

Xylitol 10% 0.206 0.005 0.092 0.003 6.22 6.43 6.47

ZnCl2 0.507 0.003 0.471 0.001 4.97 4.62 4.52

NaNO2 0.361 0.021 0.300 0.014 6.31 6.44 7.08

MgSO4 0.535 0.007 0.361 0.016 5.26 4.78 4.57

CuSO4 0.572 0.006 0.455 0.002 5.82 6.31 6.44

CaPO4 0.195 0.007 0.099 0.001 6.17 6.57 6.69

CaCO3

0.611 0.002 0.523 0.005 5.00 4.54 4.39

Initial pH 5.5. * Data are recorded as mean of 3 observations SD. * Significance change from

control group at P< 0.0001.


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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).

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

5.5 6 6.5 7 7.5

OD

control

75ppm

150ppm

300ppm

600ppm

1200ppm

1500ppm

pH

Fig. 4. Effect of different NaF concentrations on Streptococcus mutans growth at

various initial pH after 48 hrs.

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 thesmallest concentration used (75 ppm F

-) gave OD 0.125, where it gave OD


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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).

0

1

2

3

4

5

6

7

8

0 2 4 6 8 10 14 20 24 48

pH

BHI

F75ppm

F150ppm

F300ppm

F600ppm

F1200ppm

F1500ppm

Times (hrs)

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

growth media.

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

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.

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

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


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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 (Table1).

Table 2. Comparison of metal salts and xylitol inhibition effect after 48 hrs at pH

7.0 with (300 ppm NaF) as control medium.

*Significance change from control group at P< 0.0001.

Table 3. ANOVA Statistics

Metals salts Inhibition %

1.0 mM ZnCl2 68.59

5.0 mM CaPO4 84.53

0.5 mMNaNO3 53.13

0.5 mMMgSO4 27.81

5.0 mM CaCO3 2.65

0.2 mM CuSO4 21.09

5% xylitol 87.14

Treatment

ANOVA

SS

Mean

Mean

Square

Root

MSE

F-ratio

R-Square

CV

BHI (control) 0.931 0.519 0.133 0.018 404.84 0.997 3.49

Glucose (5%) 1.263 0.610 0.105 0.018 309.69 0.996 3.02

Glucose + xylitol (5%) 0.323 0.306 0.027 0.012 194.60 0.994 3.83

Fructose 5% 0.573 0.413 0.048 0.012 332.33 0.996 2.90

Fructose + xylitol (5%) 0.449 0.389 0.037 0.021 84.59 0.987 5.40

Sucrose (5%) 0.758 0.449 0.063 0.011 527.71 0.997 2.43

Sucrose + xylitol (5%) 0.486 0.386 0.040 0.023 78.46 0.986 5.88

Maltose 5% 0.845 0.318 0.070 0.009 869.10 0.998 2.83

Maltose+ xylitol (5%) 0.693 0.297 0.058 0.020 148.80 0.992 6.63

Sorbitol (5%) 0.231 0.280 0.019 0.012 141.94 0.992 4.15

Sorbitol + xylitol (5%) 0.077 0.193 0.011 0.016 41.54 0.976 8.46

Xylitol (2.5%) 0.281 0.236 0.040 0.020 95.19 0.989 8.70

Xylitol (5%) 0.092 0.158 0.013 0.011 111.74 0.991 6.89

Xylitol (10%) 0.049 0.130 0.007 0.006 177.91 0.994 4.85

ZnCl2 0.209 0.428 0.030 0.004 201.51 0.999 0.89

NaNO2 0.095 0.317 0.013 0.013 83.91 0.988 4.00

MgSO4 0.186 0.371 0.027 0.009 326.48 0.996 2.43

CuSO4 0.220 0.432 0.031 0.006 743.13 0.998 1.50

CaPO4 0.019 0.137 0.003 0.006 84.11 0.988 4.14

CaCO3 0.368 0.498 0.053 0.012 346.79 0.997 2.47


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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,1-8) indicatedthat 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 streptococcicultures 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 ofS.

mutans exposed to F

-

X- metal salts mixture for 5min, at pH 5.0 and 7.0, byelectron microscopy indicated complete damaged of the microbial cells.


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

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

5-Effect of Sorbitol on Streptococcusmutans

6-Effect of Fructose on Streptococcusmutans


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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 onStreptococcus mutans

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

Streptococcus mutans

11-Effect of combined formula 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. (Electronmicroscope photos, 1-12).


15/24


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 ofS. mutants was found to produce a moderateto 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 non-

fermentable(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 ofStreptococcus pneumoniae and neither inhibited nor enhanced the

xylitol induced growth impairment(9)

. Xylitol inhibited acid production by

washed cells ofStreptococcus 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 300ppm 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 therate 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 3-

fold, 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 CaPO 4, and

NaNO2, respectively, moderate with ZnCl2 and MgSO4 and low with CaCO3

and CuSO4 (Fig.3). Other study reported that copper, nickel; gold, silver, andmercury salts exerted the greatest inhibitory action on acid production.


18/24


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 thepresence 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 (NO 2) 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-, Mg

2+, 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 xylitolmetabolites was increased. These effects in combination probably accounted for


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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 ofS. 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 othersugars 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 diffuseand 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 reducesdemineralization of teeth and plaque formation through its bactericidal effect on


20/24


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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