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Cariogenicity of soluble starch in oral in vitro biofilm and experimental rat caries studies: a comparison

2008; Oxford University Press; Volume: 105; Issue: 3 Linguagem: Inglês

10.1111/j.1365-2672.2008.03810.x

1365-2672

Thomas Thurnheer, Elin Giertsen, Rudolf Gmür, B. Guggenheim,

Dental Erosion and Treatment

Journal of Applied MicrobiologyVolume 105, Issue 3 p. 829-836 Free Access Cariogenicity of soluble starch in oral in vitro biofilm and experimental rat caries studies: a comparison T. Thurnheer, T. Thurnheer Institute for Oral Biology, Section for Oral Microbiology and General Immunology, University of Zürich, Zürich, SwitzerlandSearch for more papers by this authorE. Giertsen, E. Giertsen Institute of Clinical Dentistry, Department of Cariology and Gerodontology, Faculty of Dentistry, University of Oslo, Oslo, NorwaySearch for more papers by this authorR. Gmür, R. Gmür Institute for Oral Biology, Section for Oral Microbiology and General Immunology, University of Zürich, Zürich, SwitzerlandSearch for more papers by this authorB. Guggenheim, B. Guggenheim Institute for Oral Biology, Section for Oral Microbiology and General Immunology, University of Zürich, Zürich, SwitzerlandSearch for more papers by this author T. Thurnheer, T. Thurnheer Institute for Oral Biology, Section for Oral Microbiology and General Immunology, University of Zürich, Zürich, SwitzerlandSearch for more papers by this authorE. Giertsen, E. Giertsen Institute of Clinical Dentistry, Department of Cariology and Gerodontology, Faculty of Dentistry, University of Oslo, Oslo, NorwaySearch for more papers by this authorR. Gmür, R. Gmür Institute for Oral Biology, Section for Oral Microbiology and General Immunology, University of Zürich, Zürich, SwitzerlandSearch for more papers by this authorB. Guggenheim, B. Guggenheim Institute for Oral Biology, Section for Oral Microbiology and General Immunology, University of Zürich, Zürich, SwitzerlandSearch for more papers by this author First published: 19 August 2008 https://doi.org/10.1111/j.1365-2672.2008.03810.xCitations: 16 Thomas Thurnheer, Institute for Oral Biology, Section for Oral Microbiology and General Immunology, University of Zürich, Plattenstrasse 11, CH-8032 Zürich, Switzerland. E-mail: thomas.thurnheer@zzmk.uzh.ch AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract Aims: Common belief suggests that starch is less cariogenic than sugar; however, the related literature is quite controversial. We aimed to compare cariogenic and microbiological effects of soluble starch in both a standard animal model and an oral biofilm system, and to assess the possible substitution of the animal model. Methods and Results: Six-species biofilms were grown anaerobically on enamel discs in saliva and medium with glucose/sucrose, starch (average molecular weight of 5000, average polymerization grade of 31), or mixtures thereof. After 64·5 h of biofilm formation, the microbiota were quantitated by cultivation and demineralization was measured by quantitative light-induced fluorescence. To assess caries incidence in rats, the same microbiota as in the biofilm experiments were applied. The animals were fed diets containing either glucose, glucose/sucrose, glucose/sucrose/starch or starch alone. Results with both models show that demineralization was significantly smaller with starch than sucrose. Conclusions: The data demonstrate that soluble starch is substantially less cariogenic than glucose/sucrose. Significance and Impact of the Study: By leading to the same scientific evidence as its in vivo counterpart, the described in vitro biofilm system provides an interesting and valuable tool in the quest to reduce experimentation with animals. Introduction In 1890, Miller reported that acid produced after incubating a bread–saliva mixture with oral micro-organisms was capable of demineralizing tooth enamel. Based on these in vitro studies, Miller concluded that starch was more cariogenic than sugar (Miller 1973; reprinted from the original work from 1890). Ever since, cariogenicity of starch is a matter of debate. Epidemiological studies indicated, for example, lower caries prevalence in a South African rural population consuming starch as the main carbohydrate source in comparison with other groups living in a more urban environment and eating a mixture of starch and sucrose (Staz 1938; Retief et al. 1975; Cleaton-Jones et al. 1984). These findings suggested that caries incidence might be lower in populations consuming traditional starch-based foodstuffs than in urban populations with a sucrose-rich refined diet characteristic of many urban environments (Schmid et al. 1987), clearly contradicting Miller's original hypothesis. In the past decades, a large number of studies addressed the cariogenicity of starch (reviewed by Rugg-Gunn 1993; Grenby 1997; Moynihan 1998; Lingström et al. 2000; Moynihan 2005). The inconsistent results obtained were in part explained by the fact that different textures or degrees of hydrolysation of starch were investigated and compared (Rugg-Gunn 1993; Moynihan 1998 for review). In animal experiments, starch was found to lead to lower caries incidence compared with sucrose, glucose or fructose (Guggenheim et al. 1966; Moynihan 1998). On the other hand, animal and in situ studies with mixtures of starch and sucrose revealed enhanced cariogenicity compared with starch alone (Shaw 1980; Firestone et al. 1982; Ribeiro et al. 2005). In vitro studies by Brudevold et al. (1988) indicated that the cariogenicity of starch is related to both its digestion by salivary amylase and the presence of an acidogenic plaque. The effect of low molecular weight degradation products of starch on both glucan synthesis produced from sucrose by Streptococcus mutans and microbial adherence has been discussed controversially on the basis of results from in vitro test systems (Gibbons and Nygaard 1968; Newbrun et al. 1977; Balekjian et al. 1980; Vacca-Smith et al. 1996). The objective of this study was to address the hypothesis that soluble starch is less cariogenic than sugars in biofilms growing in vitro or on rat teeth. We investigated the demineralization potential of starch, sucrose and glucose, in vitro and in vivo. In addition, in the in vitro biofilm tests carried out with the 'Zürich biofilm model' (Guggenheim et al. 2001a,b; Shapiro et al. 2002; Gmür et al. 2006), the effect of these carbohydrates on the microbial composition of the biofilms were determined. In an animal experiment, the effect of these carbohydrates on fissure caries, smooth surface caries and dental plaque formation was examined. The application of an in vitro and in vivo assay allowed a comparison between the results of these two test systems. Materials and methods In vitro biofilm experiments The procedures to produce six-species biofilms have been described in detail (Shapiro et al. 2002; Thurnheer et al. 2003). In brief, Actinomyces naeslundii OMZ 745, Candida albicans OMZ 110, Fusobacterium nucleatum KP-F2 (OMZ 596), Streptococcus oralis SK 248 (OMZ 607), Streptococcus sobrinus OMZ 176, and Veillonella dispar ATCC 17748T (OMZ 493) were used for biofilm formation. Biofilms were grown in 24-well polystyrene cell-culture plates on bovine enamel discs of 6·8 mm Ø (Gmür et al. 2006) that had been preconditioned for pellicle formation in whole unstimulated pooled saliva (in the following termed saliva). The processing of batches of saliva has been described in detail by Guggenheim et al. (2001a). To initiate a biofilm experiment, the discs were covered for the first 16·5 h with 1·6 ml of growth medium containing 70% saliva, 30% modified fluid universal medium (mFUM) (Guggenheim et al. 2001a) and 200 μl of a cell suspension prepared from equal volumes and densities of each strain. During the initial culture period, the carbohydrate component of the medium was 0·3% glucose. Thereafter, the growth medium contained besides 70% saliva either 30% strongly buffered mFUM for assays A–C, or weakly buffered FUM (Gmür and Guggenheim 1983) for assays D–F (see footnotes of Table 1). The growth medium was supplemented with glucose, sucrose and/or Zulkowsky's soluble starch in various combinations amounting to 0·3% or 1% (Table 1). Soluble starch with an average molecular weight of 5000 and an average polymerization grade of 31 (Zulkowsky 1880) was purchased from Merck (Darmstadt, Germany). Biofilm-covered discs were washed after 16·5, 20·5, 24·5, 40·5, 44·5 and 48·5 h by three consecutive dips in 2 ml of water (1 min per dip). They were incubated anaerobically at 37°C. At the end of the experiment (64·5 h), the pH of the culture supernatant was measured and biofilms were harvested at room atmosphere in 1 ml 0·9% NaCl by vigorous vortexing. The total CFU, streptococci and all taxa were assessed by anaerobic culture using selective and nonselective media (Guggenheim et al. 2001a; van der Ploeg and Guggenheim 2004). Table 1. Biofilm composition (mean CFU ± SD, ×106) and enamel mineralization (mean ΔF ± SD) after growth in medium containing glucose and sucrose (assays A and D), glucose, sucrose and starch (B and E), or starch alone (C and F) Assays*,† Investigated microbiota Demineralization All bacteria Streptococci A. naeslundii F. nucleatum S. oralis S. sobrinus V. dispar C. albicans QLF‡ A 598·9 ± 137·8§ 176·0 ± 53·6 192·2 ± 56·7 94·6 ± 83·3 144·9 ± 41·7 32·4 ± 19·1 22·9 ± 25·4 0·01 ± 0·01 0·8 ± 6·0 B 554·4 ± 206·8 180·0 ± 55·0 195·3 ± 97·9 47·4 ± 48·7 129·3 ± 38·4 49·9 ± 28·6 24·2 ± 30·8 0·02 ± 0·01 −0·8 ± 4·5 C 174·3 ± 92·5 67·8 ± 34·9 51·6 ± 33·9 10·1 ± 11·7 43·0 ± 29·6 26·0 ± 10·6 14·0 ± 15·8 0·01 ± 0·01 −0·4 ± 4·3 D 45·3 ± 44·7 21·4 ± 34·6 0·3 ± 0·3 <0·01 0·65 ± 0·44 22·3 ± 34·6 1·2 ± 1·6 0·01 ± 0·01 −26·5 ± 5·5 E 87·6 ± 74·4 24·7 ± 20·5 3·8 ± 5·0 <0·01 1·00 ± 0·55 23·6 ± 20·1 17·8 ± 24·2 <0·01 −19·4 ± 2·6 F 104·6 ± 59·5 12·7 ± 14·5 2·1 ± 1·6 <0·01 0·29 ± 0·27 12·4 ± 14·4 18·8 ± 24·5 <0·01 −13·0 ± 3·3 A., Actinomyces; F., Fusobacterium; S., Streptococcus; V., Veillonella; C., Candida. *Carbohydrate concentrations used in assays: A, 0·15% glucose (g) + 0·15% sucrose (s); B, 0·075% g + 0·075% s + 0·15% starch (st); C, 0·3% st; D, 0·5% g + 0·5% s; E, 0·25% g + 0·25% s + 0·5% st; F, 1·0% st. †Assays A–C were done with strongly buffered saliva/medium, assays D–F with weakly buffered saliva/medium as growth medium. ‡QLF, quantitative light-induced fluorescence. §Data originated from three independent experiments run in triplicate (n = 9). Determination of amylase activity α-Amylase activity present in saliva and biofilm culture supernatants was determined with the Phadebas® test kit (Pharmacia, Dübendorf, Switzerland) according to the instructions of the manufacturer. Besides saliva, fractions of unprocessed saliva and freshly pasteurized saliva were tested for amylase activity. The amount of enzyme catalysing the hydrolysis of 1 μmol glucosidic linkage per minute at 37°C is defined as 1 unit (U) of amylase activity. Bacterial strains were screened for amylase activity as follows (Kilian and Nyvad 1990): single strains were grown anaerobically for 72 h at 37°C on mFUM agar with 1% soluble starch and stained by covering the surface with Lugol's iodine. Strains that showed a cleared zone around colonies were scored amylase positive. Demineralization of bovine enamel discs Enamel discs were harvested after 64·5 h and freed of any biofilm remnants by maximum vortexing. Demineralization was measured by in vitro quantitative light-induced fluorescence (QLF) as described (Gmür et al. 2006) and expressed by ΔF, which is defined as the per cent change in fluorescence radiance in each image point averaged over the entire analysis area of the disc. In vivo rat study The animal study was approved by the 'Veterinäramt des Kantons Zürich' and conformed to the Swiss laws on animal protection. The experiment was conducted on 10 litters of Osborne–Mendel rats distributed at random among five treatments (Table 2). To avoid fissure impaction with food and bedding particles, the pups and their dams were transferred on day 13 after birth to stainless-steel screen-bottom cages without bedding and fed a finely ground stock diet (diet No. 890; Nafag, Gossau, Switzerland). Tap water was given ad libitum. On day 20 after birth, 50 pups were weaned and offered 3 days' drinking water with 2% glucose and 2% sucrose and the modified cariogenic diet 2000a (Guggenheim et al. 1966) containing 40% sucrose, 28% skim milk, 24% wheat flour, 5% brewer's yeast, 2% Gevral protein (Whitehall-Robins, Zug, Switzerland) and 1% NaCl. On days 21 and 22, all rats were associated orally, twice daily, with the mixture of strains described earlier by inoculating 200 μl of a freshly prepared bacterial suspension using a 1 ml syringe without a needle. On day 23, the pups were distributed at random among five treatments (Table 2), one animal per cage. Cages were connected to a programmed feeding machine (König et al. 1968) (König-Hofer FAG 72 KT; Aathal-Seegräben, Switzerland) delivering 36 meals per day. Each meal consisted of 400 mg of the basic diet (control group) or supplemented in the four test treatments with glucose, sucrose and/or soluble starch in various combinations (Table 2). Drinking water, without glucose and sucrose, was available ad libitum. Table 2. Mean of plaque extent and caries incidence in 10 rats receiving as sole source of nourishment 36 daily meals containing glucose, sucrose and/or soluble starch in various combinations over a 40-day experimental period Plaque extent (Δ)* Initial dentinal fissure lesions (ΔΔ) Advanced dentinal fissure lesions (ΔΔ) Smooth surface caries (ΔΔΔ) Treatments 1 Diet† 2·1 ± 0·74 5·6 ± 2·99 2·0 ± 2·11 1·0 ± 1·83 2 15% sucrose + diet‡ 2·0 ± 0·67 9·4 ± 0·52 7·5 ± 1·18 2·5 ± 2·68 3 7·5% sucrose + 7·5% glucose + diet‡ 2·1 ± 0·74 8·7 ± 0·95 6·7 ± 1·77 1·8 ± 1·48 4 3·75% sucrose + 3·75% glucose +7·5% starch + diet‡ 2·1 ± 0·57 9·1 ± 1·45 6·7 ± 1·89 1·2 ± 0·79 5 15% starch + diet‡ 2·4 ± 0·52 6·3 ± 2·31 3·1 ± 2·51 0·4 ± 0·97 Statistical analyses Significance level of variance ratio, F: pF ns§ <0·001 <0·001 ns Least significant differences at P < 0·05 – 1·69 1·75 – P < 0·001 – 2·95 3·06 – *Symbols: Δ, 4 units at risk; ΔΔ, 12 fissures at risk; ΔΔΔ, 20 units at risk. †Basic diet with 64% wheat flour, 28% skim milk, 5% brewer's yeast, 2% Gevral protein, 1% NaCl. ‡Adjusted basic diet with 49% wheat flour, 28% skim milk, 5% brewer's yeast, 2% Gevral protein, 1% NaCl. §ns, not significant. To allow most of the care and feeding of the animals to take place during normal working hours, the circadian rhythm was reversed between days 16 and 19 by prolonging the active phase of the rats by 3 h each day. By day 19, the active phase for the animals was from 10·00 to 22·00 h. During this period, the animals received their 36 meals, one every 20 min. This feeding regime continued for 40 days until the animals were 62 days old. On day 62, the animals were anaesthetized with CO2 and decapitated. The upper and lower jaws were dissected and immersed in fixative (10% phosphate-buffered formalin) for a minimum of 72 h. Erythrosin-stained maxillary molars were evaluated for plaque extent (Regolati and Hotz 1972) and smooth surface caries (Keyes 1958). Mandibular molars were sectioned and scored for fissure caries (König et al. 1958). Statistical analyses The null hypothesis that there was no microbiological difference between biofilms grown with different carbohydrate supplements was tested by paired t-tests. Data on enamel demineralization measured by QLF were tested analogously. Data from the rat study were compared by two-way analysis of variance and calculation of least significant differences. All statistical analyses were done with Statview 5·01 (SAS Institute, Cary, NC, USA). Results Biofilm experiments Only A. naeslundii and F. nucleatum produced amylase activity when grown planktonically. Salivary amylase activity decreased slightly during saliva processing from 164 267 ± 924, 143 200 ± 8119, to 119 467 ± 11 602 U for unprocessed, pasteurized, and processed saliva, respectively. In biofilm culture supernatants, the amylase activity reached 184 267 ± 128 430, 231 200 ± 127 245 and 204 267 ± 109 601 U after 16·5, 40·5 and 64·5 h of growth (mean values and standard deviations from triplicates). The effects of the different concentrations of glucose, sucrose and starch (see footnotes of Table 1) on the microbial composition of the biofilms were studied with a strongly and a weakly buffered medium (assays A–C and D–F, respectively). Among strongly buffered biofilms exposed to a total carbohydrate concentration of 0·3%, no major microbial shifts occurred (Fig. 1), although the differences between assays A and C in total CFU, CFU of total streptococci, CFU of A. naeslundii, F. nucleatum and S. oralis, but notably not S. sobrinus, were significant (Tables 1 and 3). With biofilms grown in weakly buffered saliva/FUM and a total carbohydrate supplement of 1% (composed of glucose/sucrose, glucose/sucrose/starch or starch; assays D–F), microbial biofilm populations differed between treatments (Fig. 1, Table 1). Differences in all bacteria, A. naeslundii and V. dispar were significant owing to increased CFU numbers (assays D vs F), whereas S. oralis and C. albicans CFU were decreased (P = 0·06 and 0·08, respectively; Table 3). Apart from this carbohydrate effect, all biofilms formed in weakly buffered medium revealed a smaller total count of bacteria than those propagated in strongly buffered saliva/medium (Fig. 1). Actinomyces naeslundii and S. oralis were affected by several log steps, and F. nucleatum dropped to or below the detection limit under these weakly buffered conditions. Exceptional and remarkable was the large variation of S. sobrinus CFU among individual biofilms exposed to 1% starch under weakly buffered conditions. Figure 1Open in figure viewerPowerPoint Box plot diagram showing the effect of starch on the microbial composition of biofilms. Concentrations of glucose (G), sucrose (S) and starch (ST): () 0·15% S G + 0·15% S; () 0·075% G + 0·075% S + 0·15% ST; () 0·3% ST; () 0·5% G + 0·5% S; () 0·25% G + 0·25% S + 0·5% ST; () 1% ST. The last three assays contained no additional buffer in the medium (see footnote to Table 1). Horizontal bars within boxes are median values; whiskers indicate 25% and 75% percentiles. Data originate from three independent experiments run in triplicate. Table 3. Statistical comparison of biofilm composition and demineralization after growth in medium containing glucose and sucrose (assays A and D), glucose, sucrose and starch (B and E), or starch alone (C and F) Compared assays* P values from paired t-tests for investigated microbiota (CFU) and enamel mineralization (ΔF)† All bacteria Streptococci An‡ Fn So Ss Vd Ca QLF, A (g/s) vs B (g/s/st) 0·5989 0·8777 0·9353 0·1616 0·4224 0·1467 0·9248 0·0025 0·5244 B (g/s/st) vs C (st) 0·0001§ <0·0001 0·0007 0·0400 <0·0001 0·0319 0·3594 0·0002 0·8287 A (g/s) vs C (st) <0·0001 <0·0001 0·0001 0·0082 <0·0001 0·3891 0·3838 0·1083 0·6406 D (g/s) vs E (g/s/st) 0·1633 0·8102 0·0498 0·3516 0·1826 0·9245 0·0574 0·3196 0·0028 E (g/s/st) vs F (st) 0·5988 0·1899 0·3357 0·4534 0·0060 0·2106 0·9317 0·0726 0·0003 D (g/s) vs F (st) 0·0295 0·5193 0·0050 0·4327 0·0622 0·4622 0·0471 0·0812 <0·0001 *See footnotes to Table 1. †Data for the calculation of p-values originated from three independent experiments run in triplicate (n = 9). ‡An, Act. naeslundii; Fn, Fus. nucleatum; So, Strep. oralis; Ss, Strep. sobrinus; Vd, V. dispar; Ca, C. albicans; QLF, quantitative light-induced fluorescence. §Bold font indicates statistical significance at P < 0·05. Independent of the carbohydrate supplement, no demineralization was observed when biofilms were grown for 64·5 h in strongly buffered medium (Fig. 2). In contrast, differential demineralization was noted underneath biofilms grown in weakly buffered medium. The least demineralization was found with biofilms grown in the presence of starch alone (Fig. 2, Table 1). Greatest mineral loss occurred when biofilms were propagated in the presence of 0·5% glucose and 0·5% sucrose (assay D), whereas significantly less mineral loss was observed in assay E (Tables 1 and 3). Medium harvested after 64·5 h from assays A–F showed respective average pH values of 6·47, 6·56, 6·65, 4·56, 4·67 and 4·84 with narrow scattering, the latter three values being well below the 'critical pH' of enamel demineralization. Figure 2Open in figure viewerPowerPoint Box plot diagram showing the effect of different carbohydrate composition and concentration on the extent of demineralization of bovine enamel discs underneath biofilms. Measurements were made using quantitative light-induced fluorescence (QLF) and are expressed by ΔF. Horizontal bars within boxes are median values; whiskers indicate 25% and 75% percentiles. Data originate from three independent experiments run in triplicate. Assays A–F: see footnotes to Table 1. Animal experiment At the end of the experimental period, all animals were in good health. With few, randomly distributed exceptions, the animals consumed all meals offered. With respect to smooth surface plaque formation, there were no significant differences among the treatments. The incidence of initial dentinal fissure lesions was significantly less in treatment group 5 (15% starch) in comparison with groups 2 (sucrose; P < 0·001), 3 (sucrose + glucose; P < 0·01), and 4 (sucrose, glucose and starch; P < 0·01). Treatments 1 (flour diet) and 5 did not differ (Table 2). The data for initial and advanced dentinal fissure lesion incidence were similar. However, in treatment groups 1 and 5, the advanced dentinal fissure lesion incidence was significantly lower (P < 0·001) in comparison with the three treatments receiving sucrose (Table 2). Smooth surface caries incidence followed a similar pattern but inter-treatment differences remained insignificant. Discussion Variations in the types of starch or starchy food are reasons for the controversial results regarding starch cariogenicity (reviewed by Moynihan 1998). Cariogenicity of raw starch is very low (Rugg-Gunn 1993; Grenby 1997). Therefore, and because raw starch is not consumed by men, soluble starch was tested in the present study, and as starch-mediated effects will likely be modulated by amylase present in the test system, amylase activity was investigated. In plaque, microbial amylase activity was shown to be low compared with that of salivary origin (Birkhed and Skude 1978). Plaque and in vitro biofilms are not directly comparable, but our tests proved that amylase activity was present in biofilms and in the applied processed saliva. The portion of processed saliva present in the medium contained around 70% of the amylase activity of fresh saliva. Considering the amylase activity in biofilm supernatants after 64·5 h, it follows that at least 40% of the activity was attributable to biofilm-generated amylase. These findings imply that the applied starch was hydrolysed by the biofilm. Some studies suggested that initial adherence of S. mutans and S. sobrinus is in fact inhibited by hydrolysation products of starch, such as maltose (Gibbons and Nygaard 1968; Newbrun et al. 1977; Balekjian et al. 1980), whereas, using a resting cell in vitro test system, it was shown that glucosyltransferases and amylase increase initial adherence (Vacca-Smith et al. 1996). However, results of modulation of initial adherence by such compounds in static adherence test systems cannot be compared with growing biofilms, as in in vitro biofilms, the growth conditions determine the microbial composition in these consortia. The effect of partial adherence inhibition is annulated rapidly during the following growth phase. In the present in vitro biofilm experiments, S. sobrinus colonized equally well with all three carbohydrate medium supplements (glucose + sucrose, glucose/sucrose + starch, or starch alone) concentrated at 0·3%. These findings clearly fail to provide evidence for an inhibition of mutans streptococci colonization by starch breakdown products. On the other hand, A. naeslundii, F. nucleatum and S. oralis CFU were significantly reduced with starch as the lone carbohydrate supplement. Such differences are possibly explained by reduced co-adherence in the absence of extracellular polysaccharides produced from sucrose or may reflect less favourable growth conditions in the absence of these sugars. Effects on enamel demineralization were not observed under these strongly buffered experimental conditions. In biofilms grown in unbuffered medium with 1% carbohydrate of any sort, A. naeslundii, F. nucleatum, S. oralis (and to a lesser extent C. albicans and S. sobrinus), but not V. dispar, were reduced substantially compared with biofilms grown in buffered medium. This drop is explained by lower acid tolerance. In addition, the presence of starch affected the microbial ecology in a subtle species-specific manner and resulted in highly significantly less enamel demineralization. Under unbuffered conditions, A. naeslundii and V. dispar reached significantly higher cell numbers in the presence of starch instead of glucose + sucrose, whereas S. oralis and C. albicans CFU dropped. The production of comparatively less acid in the presence of starch may be an important factor (pH 4·84 with starch opposed to 4·56 with glucose + sucrose), but can explain hardly the entire phenomena observed. Their causes are most likely multifactorial and must be clarified in future experiments. To validate the in vitro biofilm analysis and to verify our observation that soluble starch is less cariogenic than sugars, we studied the effects of glucose, sucrose and soluble starch also in an animal experiment. The basic experimental design was similar to the one used by Firestone et al. (1982), who reported that raw starch had little cariogenic potential, cooked starch was more cariogenic than raw starch and a 1 : 1 mixture of sucrose and starch was equally or more cariogenic than sucrose alone. Our findings show that the mixture of dietary starch and sugars resulted in more dentinal fissure caries than starch alone, confirming the previous results (Firestone et al. 1982). However, they do not corroborate studies showing that foods containing 1% or more hydrolysable starch in combination with sucrose or other sugars resulted in enhanced cariogenicity (Mundorff et al. 1990), and that a small amount of added starch increases sucrose cariogenicity (Ribeiro et al. 2005). Importantly, the results from the present in vivo study with rats correspond well with those gained with unbuffered in vitro biofilms where starch alone was barely cariogenic, whereas mixtures of glucose and sucrose with or without the presence of starch caused significantly more enamel demineralization. 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