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Acid production from sorbitol in human dental plaque
ACID
D.
PRODUCTION FROM SORBITOL DENTAL PLAQUE
RIRKHED.
Departments
S.
EDWARDSSON,B.
SVENSSON,
IN HUMAN
F. M~SKOVITZ and G. FROSTELI.*
of Oral Microbiology and Cariology. University of Lund. School of Dentistry. S-214 21 MalmB. Sweden
production from glucose and sorbitol in dental plaque suspensions and the pH changes in dental plaque in oiuo after mouth rinses with 10 per cent solutions of glucose and sorbitol were studied before and after 4-6 weeks of frequent daily mouth rinses with sorbitol in 18 subjects. The mean acid production from sorbitol, in per cent of that from glucose, increased about 21 per cent (JJ < 0.001) and mean initial (resting) plaque pH values were approximately 0.2 units higher (p < 0.01) after the sorbitol adaptation period. The pHdecreases from sorbitol were significantly more pronounced after the 6-week adaptation period (p < 0.01). Acid production activity from sorbitol and the pH-decreases after mouth rinses with sorbitol were considerably smaller than the corresponding values found with glucose before as well as after the adaptation period.
Summary---Acid
INTRODUCTION Sorbitol is utilized as a low cariogenic sugar substitute in many products such as chewing-gums and candies. Some people use these products frequently; the risk of adaptation of the oral flora to metabolize sorbitol in a more cariogenic way has been discussed by Frostell (1965), Giilzow (1971) and Mgkinen (1972). After frequent consumption of sorbitol, such an adaptation may take place depending on whether oral microorganisms utilize the substrate by alternative metabolic routes or on an increased number of sorbitol-fermenting microorganisms in the mouth. Sorbitol is fermented by some oral microorganisms, e.g. Streprococm mutms, enterococci and lactobacilli (Shockley, Randles and Dodd, 1956; Carlsson, 1968: Edwardsson, Birkhed and MejBre, 1977). Acid production from sorbitol by lactobacilli (Shockley rt al., 1956) and the sorbitol-6-phosphate dehydrogenase activity of Strep. nnltans have been shown to be adaptive; the presence of glucose. which is metabolized by constitutive enzymes, acts as repressor to the degradation of sorbitol (Brown and Wittenberger. 1973). Human diets contain various concentrations of glucose and other carbohydrates. which thus may have an effect on the oral microorganisms in situ. Hence, we have studied acid production from sorbitol before and after a period of adaptation with frequent exposure of the mouth to this sugar alcohol.
MATERl.&LAND METHODS Eighteen subjects, 25~.40 years of age, were studied. Prior to the study, ihey all used sorbitol-containing dentifrices daily but consumed only sporadically sorbitol-containing products, such as chewing-gums and candies. * Present address: Department of Cariology, Karolinska Institute. Odontological Clinic. Fack, S-141 04 Huddinge, Sweden.
The subjects were instructed not to clean their teeth for 2 days before the experiment and not to eat or drink anything on the morning of the examination. No professional cleaning was performed before this 2-day period. The examinations were always carried out in the forenoon of the third day. Each subject rinsed his mouth with tap-water for 10s to remove loose debris. Plaque material was scraped off as completely as possible from the buccal, lingual and approximal surfaces of the teeth with a blunt instrument and was immediately transferred to a plastic spoon kept in a moist chamber until the wet weight of plaque was determined to the nearest 0.1 mg. The pooled material was homogenized at 4-6 C in 0.01 M sodium potassium phosphate buffer, pH 6.X (plaque concentration approx 10mg wet wtjml), in a glass mortar hy pushing the Teflon pestle IO times to the bottom of the mortar with one rotation each time. I.0 ml aliquots of the suspension were immediately distributed into each of three l-ml micro-titration glass vessels by aid of an I-ml pipette with a wide opening and kept at 4 6 C until tested. The storage time of the suspensions never exceeded 7 h. The equipment for the titration experiment was manufactured by Radiometer A/S. Copenhagen. Denmark. The micro-titration vessel with I.Oml of the plaque suspension was placed in a thermostat jacket of a micro-titration assembly maintained at 37 C. An automatic burette delivering approximately 0.01 ml 0.002 M NaOH at each impulse was fitted to the titration vessel. The auto-burette was titted with a titrator and a recorder. The titrator. used for pH-stat work at pH 6.8. was regularly checked with a standard buffer at pH 6.50. After the plaque suspension had reached the desired temperature. about 3 min, the titrator was set on up scale and the apparatus kept the pH constant at about 6.8; the amounts of alkali required being considered to give an estimate of the endogenous acid production of the plaque suspension. After endogenous acid production had been followed
972
D. Birkhed, S. Edwardsson, B. Svensson, F. Moskovitz and G. Frostell
for about 6 min, 1.0 ml of an aqueous solution, 37 C, of 0.2 M glucose was added and acid production was allowed to go on for another 6min. Acid production activity is expressed as E x 10e9 (E = equivalent wt) of acid per mg plaque per min. Endogenous activity was deducted from the value. The acid production experiment was also performed with 1.0 ml of 0.2 M sorbitol as a substrate. To check the acid production stability of the plaque suspension, another experiment with glucose, the glucose control, was performed. The automatic titration method we used has been described in detail (Birkhed, 1978). Changes
in pH qfdental
plmque with in-vivo
incubation
One week after the in-oitro acid production experiment, the subjects were again instructed not to clean their teeth for 2 days. On the forenoon of the third day, the subjects rinsed their mouths with tap-water to remove loose debris, before rinsing for 30s with 10 ml of 10 per cent glucose. Samples of dental plaque for pH-determinations were taken immediately before (initial plaque pH-value) and 2, 5, 10, 20 and 30min after the rinse (Frostell, 1970). Analysis was carried out using the pH-values or the differences between the pH-values (Frostell, 1973). After one week, the experiment was repeated using a rinse of 1Oml of 10 per cent sorbitol. Ten per cent test solutions of glucose, pH 7.3, and sorbitol, pH 7.2, were chosen because this concentration can be expected initially in the saliva after consumption of candies or chewing-gum containing these carbohydrates. Adaptation
period
The subjects were instructed to rinse the mouth 6 times per day for 6 weeks, each time for approximately 2min with 10ml of a 10 per cent sorbitol solution without swallowing it. 3 times between meals and 3 times immediately after mealtimes. No other dietary changes were prescribed. After 4 weeks, the subjects were instructed not to clean their teeth for 2 days. On the morning of the Table
1. Dental
plaque
third day no sorbitol rinsing, eating or drinking were allowed and dental plaque was scraped off for determination of in-citro acid production from glucose and sorbitol as described. After 5 and 6 weeks of adaptation, the pH-changes in uioo in dental plaque were measured after mouth rinses with glucose and sorbito1. Statistical
methods
For practical reasons, two groups with 9 subjects per group were run separately with approximately an g-month interval. As both groups were treated in the same way, the data were pooled; the results before and after the adaptation period were compared using a paired t-test. RESULTS
The error, coefficient of variation, for the titration method was calculated to 11.2 per cent using 36 double experiments with glucose. The error of the single observation of the pH of dental plaque, judged from 144 measurements at pH 5.0 and 432 at pH 6.0, was kO.06 and _tO.OS, respectively, when the pHmeter was calibrated against the standard buffers. Table 1 shows the plaque weights and acid production activities with glucose and sorbitol. The wet weight of the plaque material increased approximately 5 per cent and the acid production from glucose was about 1 per cent higher after the period allowed for sorbitol adaptation. These differences were not statistically significant. The acid production activity from sorbitol expressed either as E x 10m9/ min per mg plaque (wet wt) or as per cent of that of glucose increased significantly (p < 0.001) after the adaptation period. Seventeen of the 18 persons studied showed higher acid production activity in the plaque suspensions from sorbitol after the adaptation period than before. The effect of mouth rinses with a 10 per cent solution of glucose and sorbitol on the mean pH-values of dental plaque is presented in Tables 2 and 3 and Fig. 1. All pH-values in the experiment with glucose
wet weight and titrable acid production activity (*SD) from glucose in 18 subjects before and after the sorbitol adaptation period Acid production Dental plaque (mg)
Before
63.1
( _t 11.2) After
66.3
(+ 19.6) Difference
3.2 (* 17.5)
t-value Level of significance
0.800
Glucose A 12.4 (k5.6) 12.5 ( IL4.9) 0.1 (k5.3) 0.022
activity
Sorbitol A
Per cent
1.4 (& 1.3) 3.8
11.3 (k8.2) 30.4 (* 15.0)
( f 2.0) 2.4 (& 1.8) 5.068 < 0.001
19.1
(& 15.4) 5.676
and sorbitol
Glucose
(control)
A
Per cent
12.7 (i6.1) 12.4 (k5.0) - 0.3 (k6.2) 0.200
102.4 (f 14.5) 99.2 ( f 19.6)
-3.2
( + 25.5) 0.527
-
(p)
A = acid production or glucose (control)/A
activity calculated as E x IO-‘/min from glucose; x 100.
per mg plaque (wet wt); per cent = A from sorbitol
Acid production
from sorbitol
in human
dental
plaque
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973
974
D. Birkhed.
S. Edwardsson,
B. Svensson,
I
F. Moskovitz
and G. Frostell
I
I
7.0
zG,o;;y 50-
I 0
I IO
I 20
Time,
I 30
min
in the pH of dental plaques after a mouth rinse for 30s with 10 per cent solutions and sorbitol (O-O) before and with glucose (U-m) and sorbitol (04) after the sorbitol adaptation period. Mean values from experiments with 18 subjects.
Fig. 1. Changes
of glucose (O---Cl)
were higher after than before the adaptation period, the greatest difference in pH, but not [HI’, being at the initial pH (0 mm). No statistically-significant difference was found at the recorded pH minimum, i.e. 1Omin. The mouth rinse with glucose caused almost identical pH-decreases at 2, 5 and lOmin, 0.14 pH-units more pronounced at 20min and 0.16 pHunits more pronounced at 30 min after the adaptation period, The differences were not statistically significant. Sorbitol caused lower pH-values in dental plaque after the adaptation period, especially at 5, 10 and 20min, than before. The initial pH-value in the experiments with sorbitol was 0.16 units higher after the adaptation period. DISCUSSION
The increase in in-&~ acid production from sorbitol, following the period of increased intake of sorbito1 from approximately 1.4 to 3.8 E x 10-9/mg plaque material wet wt per min or 171.4 per cent, shows that adaptation occurred. This difference is more than 15 times larger than the calculated coefficient of variation of the titration method used. Corresponding figures for the acid production from glucose were within the error of the method used. Sorbitol rinsing did not affect acid production from glucose, indicating no inhibitory effects and making valid the expression of results in terms of per cent acid production from glucose, i.e. treating the glucose values as controls. The initial plaque pH-values in the in-oivo experiments were a little higher after the adaptation period than before, both in the experiments with glucose (Table 2, Fig. 1) and with sorbitol (Table 3, Fig. 1). These differences may have influenced the statistical analyses of the pH-differences. However, the average glucose-pH curves (Fig. 1) ran almost parallel to each other, whereas the sorbitol_pH curves were divergent. Moreover, both the calculated pH-differences and the raw pH-values in the experiments
with sorbitol, especially at 10 and 20min, showed statistically significant differences in contrast to the results with glucose. Thus, both the in-vitro and the in-uiuo experiments suggest that the fermentability of sorbitol was more pronounced after the adaptation period than before. The acid production activity from sorbitol and the pH-decreases after mouth rinses with sorbitol did not in any case reach the level of that with glucose. This is in agreement with results both with crude plaque material (Graf and Miihlemann, 1966; Giilzow, 1971; Frostell, 1973; Birkhed, 1978) and with oral microorganisms (Shockley et al., 1956). The pH-values obtained after a mouth rinse with 10 per cent solution of sorbitol never reached values below pH 6.0, even after adaptation. The experimental method we used permits measurement mainly in superficial plaque. Previous observations show that rinsings with sucrose or glucose solutions give rise to larger pH-decreases in deeper parts of plaque (StrBlfors, 1950; Graf and Miihlemann, 1966) and in carious dentine (Dirksen, Little and Bibby, 1963) than in superficial plaque (Frostell, 1973). Hence, it may be suspected that larger pH-decreases also may take place in deep plaque and in carious lesions after rinses with sorbitol solutions. Such a concept is supported by the presence in carious dentine of large numbers of Strep. mutans and Lactobacillus casei (Loesche, Hocket and Syed, 1973; Edwardsson, 1974), species known to be sorbitol-fermenting (Edwardsson et al., 1977). An adaptation to an increased acid production from sorbitol in the mouth may theoretically depend on one of the following mechanisms or by a combination of them: (1) Ecologic changes; increase in numbers of sorbitol-fermenting microorganisms, e.g. Strep. mutans, lactobacilli, enterococci, or colonization of sorbitol-fermenting microorganisms normally not found in the mouth. (2) Induction; increased production of bacterial enzymes involved in sorbitol
Acid production
from sorbitol
such as sorbitol-6-phosphate dehydrogenase. (3) Mutation; mutants of non-fermenting oral microorganisms synthesize sorbitol-degrading enzymes. The adaptation of the oral flora in situ to sorbitol has been investigated by Frostell (1965) Giilzow (1968) and Cornick and Bowen (1972). From those studies and the present one, it is not possible to evaluate which of the above-mentioned mechanisms apply. It&fro studies show that sorbitol metabolism in sorbitol-fermenting bacteria is inducible (Shockley et ul., 1956: Brown and Wittenberg, 1973). Cornick and Bowen (1972) found no selection of specific sorbitol-fermenting bacteria in plaque material after frequent consumption of sorbitol in macaque monkeys. This may indicate that an induction is a probable explanation to the increased acid production We found higher initial plaque pH-values after the sorbitol mouth rinse-period; the pH-curve caused by glucose did not reach as low a pH-value after the adaptation period as it did before. However, there was no statistically-significant difference at the minimum record value, i.e. pH at 10min. and the corresponding acid production activity from glucose in the irwitro experiments was not altered. On the other hand, Soderling ef ~1. (1975) observed increased buffering capacity and elevation of pH in whole saliva after stimulation by sorbitol chewing-gum. Sorbitol-containing products are usually not consumed with such frequency as our experimental mouth rinses. However, some individuals do consume sorbitol-containing products regularly. e.g. persons suffering of dryness in the mouth depending on insufficiencies of the salivary glands, Studies of such individuals could help to show whether sorbitol is noncariogenic in man. For the time being, sorbitol can be regarded as a satisfactory low-cariogenic substitute for the readily-fermentable sugars, such as sucrose. fructose and glucose.
metabolism,
Ac,knoa’/rdyrmr,~t.s~~ We acknowledge the excellent technical assistance of Mrs. Elisabeth Thornqvist. The investigation was supported by Patentmedelsfonden for odontologisk profylaxforskning.
REFERENCES
Birkhed D. 1978. Automatic titration method for determination of the acid production from sugars and sugar
in human
dental
975
plaque
alcohols in small samples of dental plaque material. Curies Rrs. 12, 128-136. Brown A. T. and Wittenberger C. L. 1973. Mannitol and sorbitol catabolism in Srreptococcus mtcttrns. 4rch.j ortrl Biol. 18, 117-126. Carlsson J. 1968. A numerical taxonomic study of human oral streptococci. Odonr. Rrcy 19, 137 160. Cornick D. E. and Bowen W. H. 1972. The effect of sorhi tol on the microbiology of dental plaque in monkeys (Macucu
irus) Archs
orul Bid.
17, 1637. 1648.
Dirksen T. R.. Little M. F. and Bibby B. G. lY63. The pH of carious cavittesII..The pH at different depths in isolated cavittes. ,4r~~h.\ ortrl B&l. 8, 9 I 07. Edwardsson S. 1974. Bacteriological studies on deep areas of carious dentine. Odont. Rrry 25, suppl. 32. Edwardsson S., Birkhed D. and Mejare B. 1977. Acid production from LycasinE. maltitol, sorbitol and xylitol by some oral streptococci and lactobacilli. .4~,1(( odour. scrrnd. 35, 257-263. Frostell G. 1965. Substitution of fermentable sugars in sweets. In: Srrnposiu of’ fltr Swedish Nutrition Foundution 111. :Vurrition trnrl CariwPrcwwrion (Edited by Bl~x G.) pp. 6&66. Almqvist and Wiksell, Uppsala. Sweden. Frostell G. 1970. A method for evaluation of acid potentialities of foods. ilcrtr o
D.
PRODUCTION FROM SORBITOL DENTAL PLAQUE
RIRKHED.
Departments
S.
EDWARDSSON,B.
SVENSSON,
IN HUMAN
F. M~SKOVITZ and G. FROSTELI.*
of Oral Microbiology and Cariology. University of Lund. School of Dentistry. S-214 21 MalmB. Sweden
production from glucose and sorbitol in dental plaque suspensions and the pH changes in dental plaque in oiuo after mouth rinses with 10 per cent solutions of glucose and sorbitol were studied before and after 4-6 weeks of frequent daily mouth rinses with sorbitol in 18 subjects. The mean acid production from sorbitol, in per cent of that from glucose, increased about 21 per cent (JJ < 0.001) and mean initial (resting) plaque pH values were approximately 0.2 units higher (p < 0.01) after the sorbitol adaptation period. The pHdecreases from sorbitol were significantly more pronounced after the 6-week adaptation period (p < 0.01). Acid production activity from sorbitol and the pH-decreases after mouth rinses with sorbitol were considerably smaller than the corresponding values found with glucose before as well as after the adaptation period.
Summary---Acid
INTRODUCTION Sorbitol is utilized as a low cariogenic sugar substitute in many products such as chewing-gums and candies. Some people use these products frequently; the risk of adaptation of the oral flora to metabolize sorbitol in a more cariogenic way has been discussed by Frostell (1965), Giilzow (1971) and Mgkinen (1972). After frequent consumption of sorbitol, such an adaptation may take place depending on whether oral microorganisms utilize the substrate by alternative metabolic routes or on an increased number of sorbitol-fermenting microorganisms in the mouth. Sorbitol is fermented by some oral microorganisms, e.g. Streprococm mutms, enterococci and lactobacilli (Shockley, Randles and Dodd, 1956; Carlsson, 1968: Edwardsson, Birkhed and MejBre, 1977). Acid production from sorbitol by lactobacilli (Shockley rt al., 1956) and the sorbitol-6-phosphate dehydrogenase activity of Strep. nnltans have been shown to be adaptive; the presence of glucose. which is metabolized by constitutive enzymes, acts as repressor to the degradation of sorbitol (Brown and Wittenberger. 1973). Human diets contain various concentrations of glucose and other carbohydrates. which thus may have an effect on the oral microorganisms in situ. Hence, we have studied acid production from sorbitol before and after a period of adaptation with frequent exposure of the mouth to this sugar alcohol.
MATERl.&LAND METHODS Eighteen subjects, 25~.40 years of age, were studied. Prior to the study, ihey all used sorbitol-containing dentifrices daily but consumed only sporadically sorbitol-containing products, such as chewing-gums and candies. * Present address: Department of Cariology, Karolinska Institute. Odontological Clinic. Fack, S-141 04 Huddinge, Sweden.
The subjects were instructed not to clean their teeth for 2 days before the experiment and not to eat or drink anything on the morning of the examination. No professional cleaning was performed before this 2-day period. The examinations were always carried out in the forenoon of the third day. Each subject rinsed his mouth with tap-water for 10s to remove loose debris. Plaque material was scraped off as completely as possible from the buccal, lingual and approximal surfaces of the teeth with a blunt instrument and was immediately transferred to a plastic spoon kept in a moist chamber until the wet weight of plaque was determined to the nearest 0.1 mg. The pooled material was homogenized at 4-6 C in 0.01 M sodium potassium phosphate buffer, pH 6.X (plaque concentration approx 10mg wet wtjml), in a glass mortar hy pushing the Teflon pestle IO times to the bottom of the mortar with one rotation each time. I.0 ml aliquots of the suspension were immediately distributed into each of three l-ml micro-titration glass vessels by aid of an I-ml pipette with a wide opening and kept at 4 6 C until tested. The storage time of the suspensions never exceeded 7 h. The equipment for the titration experiment was manufactured by Radiometer A/S. Copenhagen. Denmark. The micro-titration vessel with I.Oml of the plaque suspension was placed in a thermostat jacket of a micro-titration assembly maintained at 37 C. An automatic burette delivering approximately 0.01 ml 0.002 M NaOH at each impulse was fitted to the titration vessel. The auto-burette was titted with a titrator and a recorder. The titrator. used for pH-stat work at pH 6.8. was regularly checked with a standard buffer at pH 6.50. After the plaque suspension had reached the desired temperature. about 3 min, the titrator was set on up scale and the apparatus kept the pH constant at about 6.8; the amounts of alkali required being considered to give an estimate of the endogenous acid production of the plaque suspension. After endogenous acid production had been followed
972
D. Birkhed, S. Edwardsson, B. Svensson, F. Moskovitz and G. Frostell
for about 6 min, 1.0 ml of an aqueous solution, 37 C, of 0.2 M glucose was added and acid production was allowed to go on for another 6min. Acid production activity is expressed as E x 10e9 (E = equivalent wt) of acid per mg plaque per min. Endogenous activity was deducted from the value. The acid production experiment was also performed with 1.0 ml of 0.2 M sorbitol as a substrate. To check the acid production stability of the plaque suspension, another experiment with glucose, the glucose control, was performed. The automatic titration method we used has been described in detail (Birkhed, 1978). Changes
in pH qfdental
plmque with in-vivo
incubation
One week after the in-oitro acid production experiment, the subjects were again instructed not to clean their teeth for 2 days. On the forenoon of the third day, the subjects rinsed their mouths with tap-water to remove loose debris, before rinsing for 30s with 10 ml of 10 per cent glucose. Samples of dental plaque for pH-determinations were taken immediately before (initial plaque pH-value) and 2, 5, 10, 20 and 30min after the rinse (Frostell, 1970). Analysis was carried out using the pH-values or the differences between the pH-values (Frostell, 1973). After one week, the experiment was repeated using a rinse of 1Oml of 10 per cent sorbitol. Ten per cent test solutions of glucose, pH 7.3, and sorbitol, pH 7.2, were chosen because this concentration can be expected initially in the saliva after consumption of candies or chewing-gum containing these carbohydrates. Adaptation
period
The subjects were instructed to rinse the mouth 6 times per day for 6 weeks, each time for approximately 2min with 10ml of a 10 per cent sorbitol solution without swallowing it. 3 times between meals and 3 times immediately after mealtimes. No other dietary changes were prescribed. After 4 weeks, the subjects were instructed not to clean their teeth for 2 days. On the morning of the Table
1. Dental
plaque
third day no sorbitol rinsing, eating or drinking were allowed and dental plaque was scraped off for determination of in-citro acid production from glucose and sorbitol as described. After 5 and 6 weeks of adaptation, the pH-changes in uioo in dental plaque were measured after mouth rinses with glucose and sorbito1. Statistical
methods
For practical reasons, two groups with 9 subjects per group were run separately with approximately an g-month interval. As both groups were treated in the same way, the data were pooled; the results before and after the adaptation period were compared using a paired t-test. RESULTS
The error, coefficient of variation, for the titration method was calculated to 11.2 per cent using 36 double experiments with glucose. The error of the single observation of the pH of dental plaque, judged from 144 measurements at pH 5.0 and 432 at pH 6.0, was kO.06 and _tO.OS, respectively, when the pHmeter was calibrated against the standard buffers. Table 1 shows the plaque weights and acid production activities with glucose and sorbitol. The wet weight of the plaque material increased approximately 5 per cent and the acid production from glucose was about 1 per cent higher after the period allowed for sorbitol adaptation. These differences were not statistically significant. The acid production activity from sorbitol expressed either as E x 10m9/ min per mg plaque (wet wt) or as per cent of that of glucose increased significantly (p < 0.001) after the adaptation period. Seventeen of the 18 persons studied showed higher acid production activity in the plaque suspensions from sorbitol after the adaptation period than before. The effect of mouth rinses with a 10 per cent solution of glucose and sorbitol on the mean pH-values of dental plaque is presented in Tables 2 and 3 and Fig. 1. All pH-values in the experiment with glucose
wet weight and titrable acid production activity (*SD) from glucose in 18 subjects before and after the sorbitol adaptation period Acid production Dental plaque (mg)
Before
63.1
( _t 11.2) After
66.3
(+ 19.6) Difference
3.2 (* 17.5)
t-value Level of significance
0.800
Glucose A 12.4 (k5.6) 12.5 ( IL4.9) 0.1 (k5.3) 0.022
activity
Sorbitol A
Per cent
1.4 (& 1.3) 3.8
11.3 (k8.2) 30.4 (* 15.0)
( f 2.0) 2.4 (& 1.8) 5.068 < 0.001
19.1
(& 15.4) 5.676
and sorbitol
Glucose
(control)
A
Per cent
12.7 (i6.1) 12.4 (k5.0) - 0.3 (k6.2) 0.200
102.4 (f 14.5) 99.2 ( f 19.6)
-3.2
( + 25.5) 0.527
-
(p)
A = acid production or glucose (control)/A
activity calculated as E x IO-‘/min from glucose; x 100.
per mg plaque (wet wt); per cent = A from sorbitol
Acid production
from sorbitol
in human
dental
plaque
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973
974
D. Birkhed.
S. Edwardsson,
B. Svensson,
I
F. Moskovitz
and G. Frostell
I
I
7.0
zG,o;;y 50-
I 0
I IO
I 20
Time,
I 30
min
in the pH of dental plaques after a mouth rinse for 30s with 10 per cent solutions and sorbitol (O-O) before and with glucose (U-m) and sorbitol (04) after the sorbitol adaptation period. Mean values from experiments with 18 subjects.
Fig. 1. Changes
of glucose (O---Cl)
were higher after than before the adaptation period, the greatest difference in pH, but not [HI’, being at the initial pH (0 mm). No statistically-significant difference was found at the recorded pH minimum, i.e. 1Omin. The mouth rinse with glucose caused almost identical pH-decreases at 2, 5 and lOmin, 0.14 pH-units more pronounced at 20min and 0.16 pHunits more pronounced at 30 min after the adaptation period, The differences were not statistically significant. Sorbitol caused lower pH-values in dental plaque after the adaptation period, especially at 5, 10 and 20min, than before. The initial pH-value in the experiments with sorbitol was 0.16 units higher after the adaptation period. DISCUSSION
The increase in in-&~ acid production from sorbitol, following the period of increased intake of sorbito1 from approximately 1.4 to 3.8 E x 10-9/mg plaque material wet wt per min or 171.4 per cent, shows that adaptation occurred. This difference is more than 15 times larger than the calculated coefficient of variation of the titration method used. Corresponding figures for the acid production from glucose were within the error of the method used. Sorbitol rinsing did not affect acid production from glucose, indicating no inhibitory effects and making valid the expression of results in terms of per cent acid production from glucose, i.e. treating the glucose values as controls. The initial plaque pH-values in the in-oivo experiments were a little higher after the adaptation period than before, both in the experiments with glucose (Table 2, Fig. 1) and with sorbitol (Table 3, Fig. 1). These differences may have influenced the statistical analyses of the pH-differences. However, the average glucose-pH curves (Fig. 1) ran almost parallel to each other, whereas the sorbitol_pH curves were divergent. Moreover, both the calculated pH-differences and the raw pH-values in the experiments
with sorbitol, especially at 10 and 20min, showed statistically significant differences in contrast to the results with glucose. Thus, both the in-vitro and the in-uiuo experiments suggest that the fermentability of sorbitol was more pronounced after the adaptation period than before. The acid production activity from sorbitol and the pH-decreases after mouth rinses with sorbitol did not in any case reach the level of that with glucose. This is in agreement with results both with crude plaque material (Graf and Miihlemann, 1966; Giilzow, 1971; Frostell, 1973; Birkhed, 1978) and with oral microorganisms (Shockley et al., 1956). The pH-values obtained after a mouth rinse with 10 per cent solution of sorbitol never reached values below pH 6.0, even after adaptation. The experimental method we used permits measurement mainly in superficial plaque. Previous observations show that rinsings with sucrose or glucose solutions give rise to larger pH-decreases in deeper parts of plaque (StrBlfors, 1950; Graf and Miihlemann, 1966) and in carious dentine (Dirksen, Little and Bibby, 1963) than in superficial plaque (Frostell, 1973). Hence, it may be suspected that larger pH-decreases also may take place in deep plaque and in carious lesions after rinses with sorbitol solutions. Such a concept is supported by the presence in carious dentine of large numbers of Strep. mutans and Lactobacillus casei (Loesche, Hocket and Syed, 1973; Edwardsson, 1974), species known to be sorbitol-fermenting (Edwardsson et al., 1977). An adaptation to an increased acid production from sorbitol in the mouth may theoretically depend on one of the following mechanisms or by a combination of them: (1) Ecologic changes; increase in numbers of sorbitol-fermenting microorganisms, e.g. Strep. mutans, lactobacilli, enterococci, or colonization of sorbitol-fermenting microorganisms normally not found in the mouth. (2) Induction; increased production of bacterial enzymes involved in sorbitol
Acid production
from sorbitol
such as sorbitol-6-phosphate dehydrogenase. (3) Mutation; mutants of non-fermenting oral microorganisms synthesize sorbitol-degrading enzymes. The adaptation of the oral flora in situ to sorbitol has been investigated by Frostell (1965) Giilzow (1968) and Cornick and Bowen (1972). From those studies and the present one, it is not possible to evaluate which of the above-mentioned mechanisms apply. It&fro studies show that sorbitol metabolism in sorbitol-fermenting bacteria is inducible (Shockley et ul., 1956: Brown and Wittenberg, 1973). Cornick and Bowen (1972) found no selection of specific sorbitol-fermenting bacteria in plaque material after frequent consumption of sorbitol in macaque monkeys. This may indicate that an induction is a probable explanation to the increased acid production We found higher initial plaque pH-values after the sorbitol mouth rinse-period; the pH-curve caused by glucose did not reach as low a pH-value after the adaptation period as it did before. However, there was no statistically-significant difference at the minimum record value, i.e. pH at 10min. and the corresponding acid production activity from glucose in the irwitro experiments was not altered. On the other hand, Soderling ef ~1. (1975) observed increased buffering capacity and elevation of pH in whole saliva after stimulation by sorbitol chewing-gum. Sorbitol-containing products are usually not consumed with such frequency as our experimental mouth rinses. However, some individuals do consume sorbitol-containing products regularly. e.g. persons suffering of dryness in the mouth depending on insufficiencies of the salivary glands, Studies of such individuals could help to show whether sorbitol is noncariogenic in man. For the time being, sorbitol can be regarded as a satisfactory low-cariogenic substitute for the readily-fermentable sugars, such as sucrose. fructose and glucose.
metabolism,
Ac,knoa’/rdyrmr,~t.s~~ We acknowledge the excellent technical assistance of Mrs. Elisabeth Thornqvist. The investigation was supported by Patentmedelsfonden for odontologisk profylaxforskning.
REFERENCES
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in human
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alcohols in small samples of dental plaque material. Curies Rrs. 12, 128-136. Brown A. T. and Wittenberger C. L. 1973. Mannitol and sorbitol catabolism in Srreptococcus mtcttrns. 4rch.j ortrl Biol. 18, 117-126. Carlsson J. 1968. A numerical taxonomic study of human oral streptococci. Odonr. Rrcy 19, 137 160. Cornick D. E. and Bowen W. H. 1972. The effect of sorhi tol on the microbiology of dental plaque in monkeys (Macucu
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Dirksen T. R.. Little M. F. and Bibby B. G. lY63. The pH of carious cavittesII..The pH at different depths in isolated cavittes. ,4r~~h.\ ortrl B&l. 8, 9 I 07. Edwardsson S. 1974. Bacteriological studies on deep areas of carious dentine. Odont. Rrry 25, suppl. 32. Edwardsson S., Birkhed D. and Mejare B. 1977. Acid production from LycasinE. maltitol, sorbitol and xylitol by some oral streptococci and lactobacilli. .4~,1(( odour. scrrnd. 35, 257-263. Frostell G. 1965. Substitution of fermentable sugars in sweets. In: Srrnposiu of’ fltr Swedish Nutrition Foundution 111. :Vurrition trnrl CariwPrcwwrion (Edited by Bl~x G.) pp. 6&66. Almqvist and Wiksell, Uppsala. Sweden. Frostell G. 1970. A method for evaluation of acid potentialities of foods. ilcrtr o