Scand. J. Dent. Res. 1976: 84: 372-376 (Key words: dental plaque; sucrose)
Relative effects of suerolytic enzymes in human dental plaque A. AKSNES Institute of Physiology, University of Bergen, Bergen, Norway ABSTRACT - The relative effects in human dental plaque material from the three main extracellular suerolytic enzymes from bacterial origin, invertase, dextransucrase and levansucrase, have been investigated by means of quantitative determination of products with sucrose as the substrate. Twenty young men having carious lesions and harboring plaque material on the tooth surfaces, were selected. One gram (wet weight) of plaque material was obtained and divided in five samples. 0.2 g each, for different investigations and controls. Twice as much fructan as glucan was found in plaque. Invertase activity was found to dominate sucrolysis within plaque with 99.67 % of the total activity.
Prolonged intake of sucrose gives rise to voluminous plaque formation in humans and experimental animals and promotes the development of carious lesions (GusTAFSSON, OUENZEL, SwENANDER, L A N K E ,
LuNDQuisT, CRAHNEN, BONOW & KRASSE 1954, KEYES 1962, CARLSSON 1965). The formation of polysaccharide and its role in the caries process have been emphasized by the isolation of cariogenic organisms that require a high proportion of sucrose in the diet of the hosts (FITZGERALD & KEYES 1960,
ZINNER, JABLON,
& SusLow 1965). In the hamster, organisms establish themselves more easily on a diet rich in sucrose than on one rich in glucose. This suggests that the metabolism of glucose in plaque may be different from that of sucrose (KRASSE 1965). The first evidence of the occurrence of extracellular polysaccharide in dental plaque was provided by MCDOUGALL
ARAN
(1964). He concluded that this polysaccharide is a levan and amounts to 0.3— 2.9 % of the total dry weight of plaque material. The polymerization of dietary sugar by dental plaque has been investigated by CRITGHLEY, WOOD, SAXTON & LEAGH (1967) with a method including the incubation of plaque material in a sucrose solution. The conclusion of CRITCHLEY et al. (1967) was that 1-2 % of the total dry weight of plaque is a levan, but that the raajor polysaccharide polymerized by enzymes from plaque bacteria is a dextran. However, these authors did not study other products formed by the action of plaque on sucrose solutions. Hydrolysis by invertase is the dominant reaction in the effect of plaque on sucrose and must be included in any quantitative study of oral suerolytic processes. The aim of this work was to study quantitatively the products formed upon
SUCROLYTIC ENZYMES IN PLAQUE incubation of plaque material with sucrose solutions. The products include: (a) the monosaccharides formed by invertase action and by levan- and dextransucrase; and (b) the polysaccharides formed by the polymerizing action of the two sucrases. For this purpose methods have been developed and adapted for the separate analysis of bacterial levans and dextrans. Since individual plaque-forming bacteria may possess characteristic patterns of extracellular enzyme content, plaque action on sucrose solutions may give information about the dominant organism active in the plaque. iVIaterial and methods All chemicals were obtained from E. Merck AG, Darmstadt. The glucose-oxidase reagent was Glox® from Kabi AB, Stockholm. Dextran T 10 was supplied by Pharmacia AB, Stockholm. Measurements were made with a Hitachi 101, spectrophotometer and glass cuvettes.
CARBOHYDRATE GHEMISTRY Ho WELL & JORDAN (1967) have proposed that levan be hydrolyzed using 0.1 N sulfuric acid for 1 h at 100°C. In this study, 0.1 N hydrochloric acid at 100°C was used. By continuing this hydrolysis for more than 1 h, more fructose from dental plaque was obtained until hydrolysis had lasted for about 4 h, when 10 % of the glucan was also hydrolyzed. Full hydrolysis of glucan was done using 1 N HCl for 4 h at 100°C (D.^HLQUIST, KRASSE,
OLSSON & GARDELL 1967). In my experience
the fructose content was reduced to 1/6 by this hydrolysis. In this study hydrolysis with 0.1 N HCl for 4/2 h is called Step 1, and hydrolysis with 1 N HCl for 4 h Step 2. For qualitative determinations tlie resorcinol method of ROE (1957) and the Glox® method were used. For quantitative analyses the Glox® method and the Somogyi-Nelson method (NELSON 1957) were combined. All samples were neutralized with NaOH before determination. To find out if extraction of polysaccharide with 0.5 N sodium hydroxide should be done.
373
bacterial polysaccharide from S. mutans and dextran T 10 were divided into two equal parts. One of these parts was treated overnight with NaOH 0.5 N, and after neutralization with HCl was precipitated from solution by ethanol and centrifuged (CRITGHLEY et al. 1967). The remaining part was precipitated directly in 70 % ethanol and centrifuged in the same way, 15,000 rev/min. Both parts were hydrolyzed as in Step 2. No difference was observed in the yield of glucose between the samples being treated with NaOH and those being precipitated directly.
METHOD OF ASSAY Dental plaque was collected from 20 young men in military service. Those having bad oral hygiene and several untreated cavities were chosen, and plaque was collected with a dental probe from decalcified and cariesprone dental surfaces. The wet weight of all this plaque amounted to 1.0 g, and after weighing it was divided into five parts by weight. Precautious pooling was done to ensure that all parts were as equal as possible. The first of these samples, 0.2 g wet weight, was incubated with 100 ml of sucrose solution 0.4 M at 37°C for 72 h. The sucrose solution for incubation was not autoclaved because that would have given a brown color. Instead, it was boiled for 20 min and cooled to 37 °C just before use to avoid risk of reinfection. No indication of bacterial growth changes in population and enzyme equilibrium was seen during incubation. The enzyme effect, being determined by measurement of glucose and fructose production, remained constant in relation and rate throughout the incubation period. The second sample was incubated in sterilized 0.1 M phosphate buffer at the same temperature and for the same length of time to see whether changes in polysaccharide content would occur due to dextranase or levanase effect. The third sample was not incubated but was hydrolyzed at once. After Step 1, the monohexose and the glucose recovered from polysaccharide were determined. After hydrolysis Step 2 the glucose recovered was determined. At the end of the incubation period the water insoluble matter in the first and second samples was precipitated by centrifugation at 5,000 rev/min. The content of monohexose in
AKSNES
374
supernatant was determined and the polysaccharide was precipitated with ethanol as previously described. In sample two no soluble polysaccharide could be precipitated. The polysaccharide from supernatant in the first sample (incubated with sucrose) was resuspended in water and centrifuged four times to remove the sugars. The water-insoluble plaque, after being incubated in sucrose, was treated in the same way for the same reason. The plaque material from the first and second sample and the precipitated polysaccharide was hydrolyzed as described before, like the untreated plaque, and the recovery of monohexose and its glucose part was determined. Each determination to be made was performed in triplicate, three assays and three zeros. The mean values from those three determinations are given under Results. To see how much dissimilation of the glucose and the fructose produced by the suerolytic enzymes would occur during incubation, the fourth and the fifth sample of 0.2 g plaque were incubated with glucose and fructose solutions 0.4 M under similar conditions. Results
The sucrose content of 100 ml of 0.4 M sucrose solution is 14.1 g. 0.2 g wet weight of plaque produced 6.5 g of monohexose from this sucrose within 72 h of incubation at 37°C. Glucose content was 3.2 g. The recovery of fructose on hydrolysis Step 1 of ethanol-precipitated polysaccharide from supernatant was 1.4 mg. No glucose was detected by this step. The recovery of glucose after hydrolysis Step 2 was 0.7 mg. The recovery of fructose from plaque material incubated in sucrose was 8.4 mg axi^ the recovery of glucose 3.5 mg. For the plaque material directly hydrolyzed without incubation in sucrose the fructose content was 2.0 mg and the glucose content 0.5 mg. The increase in levan during incubation within plaque was thus 8.4 mg - 2.0 mg = 6.4 mg. In dextran, the corresponding increase was 3.5 mg — 0.5 mg = 3 mg. If the poly-
saccharide from supernatant is added, the quantity of levan was 6.4 mg -f 1.4 mg = 7.8 mg. The dextran formed within and outside plaque was 3.0 mg -f 0.7 mg = 3.7 mg. Loss of dextran due to plaque dextranase was not detected but a loss of levan due to levanase was found to be 0.3 mg at the end of the incubation period with 0.1 M phosphate buffer. Taking this into account, the levan produced was calculated to be 8.1 mg. The approximate results may be written as 12 mg polysaccharide, 4 mg dextran and 8 mg levan. If, due to the effect of glucolytic and fructolytic enzymes within plaque bacteria, the loss of monohexose during incubation is taken into account, the total monohexose formed was about 7.24 g and the total glucose formed, 3.63 g. Recovery of polysaccharide-bound monohexose was 12 mg and sucrose split by sucrase must therefore be 24 mg. Percentages calculated are: 99.67 % for invertase, 0.11 % for dextransucrase, and 0.22 % for levansucrase. One U is the enzyme power sufficient to split 1 jxM substrate per minute. 7,240 mg of sucrose was split in 72 h by 0.2 g plaque, mol. wt. 342, which makes 4.9 U. Per g of plaque it is 24.5 U : 23.69 for invertase, 0.27 for dextransucrase, and 0.54 U for levansucrase. Discussion
According to a report by AKSNES (1977) based on the average of 356 young men, 1 ml of unstimulated saliva contains 14 mU. In the present investigation 1 g (wet weight) of plaque, gathered from the tooth surface by means of a probe, was found to contain 24 U (about 1,500 times more). Specific weight of plaque is a little
SUCROLYTIC ENZYMES IN PLAQUE more than 1, but here it is assumed that 1 ml of saliva and 1 g of plaque (wet weight) may be compared. RAUGH (1961) has shown that normal unstimulated saliva secretion from the combined salivary gland system is about 0.3 ml per min. If estimated plaque content on a person's tooth surface is 20 [il ci:; 20 mg, the combined net enzyme power regarding all suerolytic enzymes is 0.49 U according to results in this work. According to the author's observation based on a group of 356 young men, the unstimulated saliva secreted for 1 h contains 0.28 U. This level of activity can be accounted for if all the suerolytic power of 20 yd of plaque is transferred to the saliva secreted during a couple of hours under unstimulated conditions. In addition to the accumulated enzyme content within plaque, the production of extracellular suerolytic enzyme must be assumed to continue in living bacteria at any time where the medium permits metabolism. Determined by their effect on sucrose the combined effect of sucrases was 0.33 % of total suerolytic activity. In 20 ^1 of dental plaque this would be 0.33 % of 0.49 U = 1.45 mU, and the quantity of polysaccharide in 1 h would be 36.5 Hg with sucrose available. If we assume that the interrelationship between invertase and the sucrases is the same in extracellular fluid of plaque and in saliva and that a person has 2 ml of unstimulated saliva in contact with plaque containing 14 mU per ml, the produced polysaccharide in this saliva would be 1 ug in 1 h of continuous sucrose access. This plaque and saliva system produces 37.5 ug polysaccharide per hour including 25 j.ig levan. The dry weight of 20 iil c^ 20 mg is about 1 mg. The percentage given by MGDOUGALL (1964) is 0.5-2.9% of dry
375
weight. The levan of this plaque may thus be estimated to be 10-60 |j,g. This quantity can be formed within an hour or more. In the present work 8 mg fructose was recovered from 0.2 g plaque (wet weight) (20 |il c^ 0.2 g/10). Thus the content of 20 mg is 800 |ig, or about 4 %. This indicates that levan can accumulate where plenty of substrate is available for a long period of time. A quantity of 20 \.d c^ 20 mg of sucrose incubated plaque material was shown to contain 350 ^.ig of glucose on hydrolysis or 1.75 % of plaque (wet weight). The addition of 12.5 \,ig from the plaque saliva system per hour indicates that dextran can also accumulate for a long period of time if access to substrate is maintained. References
A.: Suerolytic enzymes from human dental plaque in saliva. Scand. J. Dent. Res. 1977: in press. CARLSSON, J.: Zooglea-forming streptococci resembling Streptococcus sanguis isolated from dental plaque in man. Odontol. Revy 1965: 16: 349-358. AKSNES,
CRITCHLEY, P., WOOD, J. M., SAXTON, C . A.
& LEAGH, S. A.: The polymerization of die-
tary sugars by dental plaque. Caries Res. 1967: 1: 112-129. D.AHLQuisT, A., KRASSE, B., OLSSON, E . & G.A.RDELL, S.: Extracellular polysaccharides formed by caries-inducing streptococci. Helv. Odontol. Acta 1967: 11: 15-21. FITZGERALD, R . J. & KEYES, P. H.: Demon-
stration of the etiologic role of streptococci in experimental caries in hamster. / . Am. Dent. Assoc. 1960: 61: 9-19. GUSTAFSSON, B. E., QUENZEL, C. E., SwEN.ANDER LANKE, L . , LUNDQUIST, C , GRAHNEN,
H., BONOW, B. & KRASSE, B.: The Vipeholm
dental caries study. Acta Odontol. Scand. 1954: 11: 232-388. Ho WELL, A., JR. & JORDAN, H . V.: Production of an extracellular levan by Odontomyces viscosus. Arch. Oral Biol. 1967: 12: 571-573. KEYES, P. H.: Bacteriological findings and biological implications. Int. Dent. J. 1962: 12: 443-464. KRASSE, B.: The effect of caries-inducing strep-
376
AKSNES
tococci in hamsters fed diets with sucrose or glucose. Arch. Oral Biol. 1965: 10: 223-226. MCDOUGALL, W . A.: Studies on the dental plaque. IV. Levans and the dental plaque. Aust. Dent. J. 1964: 9: 1-5. NELSON, N . : Arsenomolybdate method of Nelson. In: CoLowicK, S. P. & KAPLAN, N . O .
(ed.): Methods in enzymology III. Academic Press, New York 1957, p. 85. RAUCH, S.: Physiologie und Pathologie der Regulationen der Speicheldriisentatigkeit. Desch. Zahnaerztl. Z. 1961: 16: 153-165. ROE, Resorcinol method of Roe. In: COLO WICK.
S. P. & KAPLAN, N . O . (ed.): Methods in enzymology III. Academic Press, New York 1957, p. 75. ZINNER, D . D . , JABLON, J. M., ARAN, A. P. &
SusLow, M. S.: Experimental caries induced in animals by Streptococci of human origin. Proc. Soc. Exp. Biol. 1965: 118: 766-774. Address: Department of Physiology Arstadveien 19 5000 Bergen Norway
Relative effects of suerolytic enzymes in human dental plaque A. AKSNES Institute of Physiology, University of Bergen, Bergen, Norway ABSTRACT - The relative effects in human dental plaque material from the three main extracellular suerolytic enzymes from bacterial origin, invertase, dextransucrase and levansucrase, have been investigated by means of quantitative determination of products with sucrose as the substrate. Twenty young men having carious lesions and harboring plaque material on the tooth surfaces, were selected. One gram (wet weight) of plaque material was obtained and divided in five samples. 0.2 g each, for different investigations and controls. Twice as much fructan as glucan was found in plaque. Invertase activity was found to dominate sucrolysis within plaque with 99.67 % of the total activity.
Prolonged intake of sucrose gives rise to voluminous plaque formation in humans and experimental animals and promotes the development of carious lesions (GusTAFSSON, OUENZEL, SwENANDER, L A N K E ,
LuNDQuisT, CRAHNEN, BONOW & KRASSE 1954, KEYES 1962, CARLSSON 1965). The formation of polysaccharide and its role in the caries process have been emphasized by the isolation of cariogenic organisms that require a high proportion of sucrose in the diet of the hosts (FITZGERALD & KEYES 1960,
ZINNER, JABLON,
& SusLow 1965). In the hamster, organisms establish themselves more easily on a diet rich in sucrose than on one rich in glucose. This suggests that the metabolism of glucose in plaque may be different from that of sucrose (KRASSE 1965). The first evidence of the occurrence of extracellular polysaccharide in dental plaque was provided by MCDOUGALL
ARAN
(1964). He concluded that this polysaccharide is a levan and amounts to 0.3— 2.9 % of the total dry weight of plaque material. The polymerization of dietary sugar by dental plaque has been investigated by CRITGHLEY, WOOD, SAXTON & LEAGH (1967) with a method including the incubation of plaque material in a sucrose solution. The conclusion of CRITCHLEY et al. (1967) was that 1-2 % of the total dry weight of plaque is a levan, but that the raajor polysaccharide polymerized by enzymes from plaque bacteria is a dextran. However, these authors did not study other products formed by the action of plaque on sucrose solutions. Hydrolysis by invertase is the dominant reaction in the effect of plaque on sucrose and must be included in any quantitative study of oral suerolytic processes. The aim of this work was to study quantitatively the products formed upon
SUCROLYTIC ENZYMES IN PLAQUE incubation of plaque material with sucrose solutions. The products include: (a) the monosaccharides formed by invertase action and by levan- and dextransucrase; and (b) the polysaccharides formed by the polymerizing action of the two sucrases. For this purpose methods have been developed and adapted for the separate analysis of bacterial levans and dextrans. Since individual plaque-forming bacteria may possess characteristic patterns of extracellular enzyme content, plaque action on sucrose solutions may give information about the dominant organism active in the plaque. iVIaterial and methods All chemicals were obtained from E. Merck AG, Darmstadt. The glucose-oxidase reagent was Glox® from Kabi AB, Stockholm. Dextran T 10 was supplied by Pharmacia AB, Stockholm. Measurements were made with a Hitachi 101, spectrophotometer and glass cuvettes.
CARBOHYDRATE GHEMISTRY Ho WELL & JORDAN (1967) have proposed that levan be hydrolyzed using 0.1 N sulfuric acid for 1 h at 100°C. In this study, 0.1 N hydrochloric acid at 100°C was used. By continuing this hydrolysis for more than 1 h, more fructose from dental plaque was obtained until hydrolysis had lasted for about 4 h, when 10 % of the glucan was also hydrolyzed. Full hydrolysis of glucan was done using 1 N HCl for 4 h at 100°C (D.^HLQUIST, KRASSE,
OLSSON & GARDELL 1967). In my experience
the fructose content was reduced to 1/6 by this hydrolysis. In this study hydrolysis with 0.1 N HCl for 4/2 h is called Step 1, and hydrolysis with 1 N HCl for 4 h Step 2. For qualitative determinations tlie resorcinol method of ROE (1957) and the Glox® method were used. For quantitative analyses the Glox® method and the Somogyi-Nelson method (NELSON 1957) were combined. All samples were neutralized with NaOH before determination. To find out if extraction of polysaccharide with 0.5 N sodium hydroxide should be done.
373
bacterial polysaccharide from S. mutans and dextran T 10 were divided into two equal parts. One of these parts was treated overnight with NaOH 0.5 N, and after neutralization with HCl was precipitated from solution by ethanol and centrifuged (CRITGHLEY et al. 1967). The remaining part was precipitated directly in 70 % ethanol and centrifuged in the same way, 15,000 rev/min. Both parts were hydrolyzed as in Step 2. No difference was observed in the yield of glucose between the samples being treated with NaOH and those being precipitated directly.
METHOD OF ASSAY Dental plaque was collected from 20 young men in military service. Those having bad oral hygiene and several untreated cavities were chosen, and plaque was collected with a dental probe from decalcified and cariesprone dental surfaces. The wet weight of all this plaque amounted to 1.0 g, and after weighing it was divided into five parts by weight. Precautious pooling was done to ensure that all parts were as equal as possible. The first of these samples, 0.2 g wet weight, was incubated with 100 ml of sucrose solution 0.4 M at 37°C for 72 h. The sucrose solution for incubation was not autoclaved because that would have given a brown color. Instead, it was boiled for 20 min and cooled to 37 °C just before use to avoid risk of reinfection. No indication of bacterial growth changes in population and enzyme equilibrium was seen during incubation. The enzyme effect, being determined by measurement of glucose and fructose production, remained constant in relation and rate throughout the incubation period. The second sample was incubated in sterilized 0.1 M phosphate buffer at the same temperature and for the same length of time to see whether changes in polysaccharide content would occur due to dextranase or levanase effect. The third sample was not incubated but was hydrolyzed at once. After Step 1, the monohexose and the glucose recovered from polysaccharide were determined. After hydrolysis Step 2 the glucose recovered was determined. At the end of the incubation period the water insoluble matter in the first and second samples was precipitated by centrifugation at 5,000 rev/min. The content of monohexose in
AKSNES
374
supernatant was determined and the polysaccharide was precipitated with ethanol as previously described. In sample two no soluble polysaccharide could be precipitated. The polysaccharide from supernatant in the first sample (incubated with sucrose) was resuspended in water and centrifuged four times to remove the sugars. The water-insoluble plaque, after being incubated in sucrose, was treated in the same way for the same reason. The plaque material from the first and second sample and the precipitated polysaccharide was hydrolyzed as described before, like the untreated plaque, and the recovery of monohexose and its glucose part was determined. Each determination to be made was performed in triplicate, three assays and three zeros. The mean values from those three determinations are given under Results. To see how much dissimilation of the glucose and the fructose produced by the suerolytic enzymes would occur during incubation, the fourth and the fifth sample of 0.2 g plaque were incubated with glucose and fructose solutions 0.4 M under similar conditions. Results
The sucrose content of 100 ml of 0.4 M sucrose solution is 14.1 g. 0.2 g wet weight of plaque produced 6.5 g of monohexose from this sucrose within 72 h of incubation at 37°C. Glucose content was 3.2 g. The recovery of fructose on hydrolysis Step 1 of ethanol-precipitated polysaccharide from supernatant was 1.4 mg. No glucose was detected by this step. The recovery of glucose after hydrolysis Step 2 was 0.7 mg. The recovery of fructose from plaque material incubated in sucrose was 8.4 mg axi^ the recovery of glucose 3.5 mg. For the plaque material directly hydrolyzed without incubation in sucrose the fructose content was 2.0 mg and the glucose content 0.5 mg. The increase in levan during incubation within plaque was thus 8.4 mg - 2.0 mg = 6.4 mg. In dextran, the corresponding increase was 3.5 mg — 0.5 mg = 3 mg. If the poly-
saccharide from supernatant is added, the quantity of levan was 6.4 mg -f 1.4 mg = 7.8 mg. The dextran formed within and outside plaque was 3.0 mg -f 0.7 mg = 3.7 mg. Loss of dextran due to plaque dextranase was not detected but a loss of levan due to levanase was found to be 0.3 mg at the end of the incubation period with 0.1 M phosphate buffer. Taking this into account, the levan produced was calculated to be 8.1 mg. The approximate results may be written as 12 mg polysaccharide, 4 mg dextran and 8 mg levan. If, due to the effect of glucolytic and fructolytic enzymes within plaque bacteria, the loss of monohexose during incubation is taken into account, the total monohexose formed was about 7.24 g and the total glucose formed, 3.63 g. Recovery of polysaccharide-bound monohexose was 12 mg and sucrose split by sucrase must therefore be 24 mg. Percentages calculated are: 99.67 % for invertase, 0.11 % for dextransucrase, and 0.22 % for levansucrase. One U is the enzyme power sufficient to split 1 jxM substrate per minute. 7,240 mg of sucrose was split in 72 h by 0.2 g plaque, mol. wt. 342, which makes 4.9 U. Per g of plaque it is 24.5 U : 23.69 for invertase, 0.27 for dextransucrase, and 0.54 U for levansucrase. Discussion
According to a report by AKSNES (1977) based on the average of 356 young men, 1 ml of unstimulated saliva contains 14 mU. In the present investigation 1 g (wet weight) of plaque, gathered from the tooth surface by means of a probe, was found to contain 24 U (about 1,500 times more). Specific weight of plaque is a little
SUCROLYTIC ENZYMES IN PLAQUE more than 1, but here it is assumed that 1 ml of saliva and 1 g of plaque (wet weight) may be compared. RAUGH (1961) has shown that normal unstimulated saliva secretion from the combined salivary gland system is about 0.3 ml per min. If estimated plaque content on a person's tooth surface is 20 [il ci:; 20 mg, the combined net enzyme power regarding all suerolytic enzymes is 0.49 U according to results in this work. According to the author's observation based on a group of 356 young men, the unstimulated saliva secreted for 1 h contains 0.28 U. This level of activity can be accounted for if all the suerolytic power of 20 yd of plaque is transferred to the saliva secreted during a couple of hours under unstimulated conditions. In addition to the accumulated enzyme content within plaque, the production of extracellular suerolytic enzyme must be assumed to continue in living bacteria at any time where the medium permits metabolism. Determined by their effect on sucrose the combined effect of sucrases was 0.33 % of total suerolytic activity. In 20 ^1 of dental plaque this would be 0.33 % of 0.49 U = 1.45 mU, and the quantity of polysaccharide in 1 h would be 36.5 Hg with sucrose available. If we assume that the interrelationship between invertase and the sucrases is the same in extracellular fluid of plaque and in saliva and that a person has 2 ml of unstimulated saliva in contact with plaque containing 14 mU per ml, the produced polysaccharide in this saliva would be 1 ug in 1 h of continuous sucrose access. This plaque and saliva system produces 37.5 ug polysaccharide per hour including 25 j.ig levan. The dry weight of 20 iil c^ 20 mg is about 1 mg. The percentage given by MGDOUGALL (1964) is 0.5-2.9% of dry
375
weight. The levan of this plaque may thus be estimated to be 10-60 |j,g. This quantity can be formed within an hour or more. In the present work 8 mg fructose was recovered from 0.2 g plaque (wet weight) (20 |il c^ 0.2 g/10). Thus the content of 20 mg is 800 |ig, or about 4 %. This indicates that levan can accumulate where plenty of substrate is available for a long period of time. A quantity of 20 \.d c^ 20 mg of sucrose incubated plaque material was shown to contain 350 ^.ig of glucose on hydrolysis or 1.75 % of plaque (wet weight). The addition of 12.5 \,ig from the plaque saliva system per hour indicates that dextran can also accumulate for a long period of time if access to substrate is maintained. References
A.: Suerolytic enzymes from human dental plaque in saliva. Scand. J. Dent. Res. 1977: in press. CARLSSON, J.: Zooglea-forming streptococci resembling Streptococcus sanguis isolated from dental plaque in man. Odontol. Revy 1965: 16: 349-358. AKSNES,
CRITCHLEY, P., WOOD, J. M., SAXTON, C . A.
& LEAGH, S. A.: The polymerization of die-
tary sugars by dental plaque. Caries Res. 1967: 1: 112-129. D.AHLQuisT, A., KRASSE, B., OLSSON, E . & G.A.RDELL, S.: Extracellular polysaccharides formed by caries-inducing streptococci. Helv. Odontol. Acta 1967: 11: 15-21. FITZGERALD, R . J. & KEYES, P. H.: Demon-
stration of the etiologic role of streptococci in experimental caries in hamster. / . Am. Dent. Assoc. 1960: 61: 9-19. GUSTAFSSON, B. E., QUENZEL, C. E., SwEN.ANDER LANKE, L . , LUNDQUIST, C , GRAHNEN,
H., BONOW, B. & KRASSE, B.: The Vipeholm
dental caries study. Acta Odontol. Scand. 1954: 11: 232-388. Ho WELL, A., JR. & JORDAN, H . V.: Production of an extracellular levan by Odontomyces viscosus. Arch. Oral Biol. 1967: 12: 571-573. KEYES, P. H.: Bacteriological findings and biological implications. Int. Dent. J. 1962: 12: 443-464. KRASSE, B.: The effect of caries-inducing strep-
376
AKSNES
tococci in hamsters fed diets with sucrose or glucose. Arch. Oral Biol. 1965: 10: 223-226. MCDOUGALL, W . A.: Studies on the dental plaque. IV. Levans and the dental plaque. Aust. Dent. J. 1964: 9: 1-5. NELSON, N . : Arsenomolybdate method of Nelson. In: CoLowicK, S. P. & KAPLAN, N . O .
(ed.): Methods in enzymology III. Academic Press, New York 1957, p. 85. RAUCH, S.: Physiologie und Pathologie der Regulationen der Speicheldriisentatigkeit. Desch. Zahnaerztl. Z. 1961: 16: 153-165. ROE, Resorcinol method of Roe. In: COLO WICK.
S. P. & KAPLAN, N . O . (ed.): Methods in enzymology III. Academic Press, New York 1957, p. 75. ZINNER, D . D . , JABLON, J. M., ARAN, A. P. &
SusLow, M. S.: Experimental caries induced in animals by Streptococci of human origin. Proc. Soc. Exp. Biol. 1965: 118: 766-774. Address: Department of Physiology Arstadveien 19 5000 Bergen Norway
