EFFECT OF CONSUMPTION OF LACTOBACILLUS RHAMNOSUS GG AND CALCIUM, IN CARROT-PINEAPPLE JUICE ON DENTAL CARIES RISK IN CHILDREN

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1 International Journal of Probiotics and Prebiotics Vol. 5, No. 4, pp , 2010 ISSN print, Copyright 2010 by New Century Health Publishers, LLC All rights of reproduction in any form reserved EFFECT OF CONSUMPTION OF LACTOBACILLUS RHAMNOSUS GG AND CALCIUM, IN CARROT-PINEAPPLE JUICE ON DENTAL CARIES RISK IN CHILDREN 1 S. Pohjavuori, 1 A.J. Ahola, 2 H. Yli-Knuuttila, 3 L. Piirainen, 4 T. Poussa, 2,5 J.H. Meurman and 1,3 R. Korpela 1 Valio Ltd, R&D, FIN Valio, Helsinki, Finland; 2 Institute of Dentistry, University of Helsinki, FIN University of Helsinki, Helsinki, Finland; 3 Institute of Biomedicine, Pharmacology, University of Helsinki, FIN University of Helsinki, Helsinki, Finland; 4 Stat-Consulting, FIN Tampere, Finland; 5 Department of Oral and Dental Diseases, Helsinki University Central Hospital, FIN HUCH, Helsinki, Finland [Received Month Date, 2010; Accepted May 12, 2010] ABSTRACT: Consumption of milk containing Lactobacillus rhamnosus GG (LGG) has been shown to reduce caries risk in children. We examined whether long-term consumption of juice containing LGG and calcium lactate gluconate (CaLG) affects the caries risk in a double-blind, placebo-controlled setting. The study comprised 530 healthy children, aged 3-6 years. They consumed 5 days a week for 7 months either carrotpineapple juice containing LGG (Valio Ltd, Finland; 5x10 6 cfu/ml) and CaLG or control juice. The children s oral health status was assessed at the baseline and in the end of the study, and salivary Streptococcus mutans counts four times during the study. The caries risk and the need for dental care increased, and high S. mutans counts decreased in both study groups. At the end of the study, the caries risk was not lower (baselineadjusted OR=0.71, 95% CI , p=0.281), but the need of dental care was smaller (baseline-adjusted OR=0.58; 95% CI ; p=0.050), and there were more children who had all the six sextants healthy (baseline-adjusted OR=1.69, 95% CI ; p=0.056) in the LGG-CaLG juice group compared to the control juice group. To conclude, supplementation of juice with LGG and CaLG might be beneficial for dental health in children. KEY WORDS: Calcium, Caries risk, Children, Lactobacillus GG Corresponding Author: Prof. Riitta Korpela, Institute of Biomedicine, Pharmacology, PO Box 63, FIN University of Helsinki, Finland; Fax: ; riitta.korpela@helsinki.fi INTRODUCTION Dental caries is a chronic infectious disease and common in childhood. Although dental health of children in Finland and other Western countries has improved markedly during the past decades (Rytömaa et al., 1980; Seppä et al., 2002), the children with caries are more severely affected than before (Vehkalahti et al., 1997; Mattila et al., 2000). Use of probiotic bacteria to reduce caries has been under increased interest in recent years. Lactobacillus rhamnosus GG (LGG) is one of the most studied probiotic bacteria and shown to have an inhibitory activity against bacterial species including Steptococcus spp. (Silva et al., 1987; Meurman et al., 1995). In laboratory studies LGG has been shown to reduce or not to ferment sucrose and lactose (Saxelin, 1997; Haukioja et al., 2008). In addition, LGG reduces the adhesion of Streptococcus mutans to saliva-coated hydroxyapatite beads (Wei et al., 1992; 2002), possibly by its coaggregation in the dental biofilm (Twetman et al., 2009), which confirms the anticariogenic effect of LGG. We have previously shown that milk containing LGG reduces caries risk in children (Näse et al., 2001). Furthermore, we found that a short-term consumption of cheese containing LGG and Lactobacillus rhamnosus LC 705 showed a trend towards reduction of the risk of the highest level of S. mutans bacteria (Ahola et al., 2002). Also other probiotic bacteria have shown similar positive effects on dental caries (Caglar et al., 2005; 2006; 2008). However, cheese and milk themselves are known to have positive effects on dental caries (Harper et al., 1986; Krobicka et al., 1987; Vacca-Smith and Bowen, 2000; Drummond et al., 2002). In the present study we assessed whether the anticariogenic effects of probiotics could even be seen when using sugar-containing juice, which itself has cariogenic potential, as the vehicle of probiotic administration. Excessive consumption of acid-containing drinks such as soft drinks and juices is associated with an increased risk of dental erosion (Jensdottir et al., 2004; Moazzez et al., 2000). Although water and milk are considered better for dental health, some children occasionally prefer juices. Therefore, formulating sugar-containing acidic drinks with reduced cariogenic and

2 222 Probiotics and caries risk in children erosive potential is a likely approach to support dental health. Enrichment of acidic drinks with minerals such as calcium and phosphorus to reduce their erosive potential has been under increased interest in recent years (Attin et al., 2005; Jensdottir et al., 2005; West et al., 2004). To our knowledge, formulation of an acidic drink with both probiotic bacteria and minerals has not been studied earlier. Therefore, the aim of this doubleblinded, placebo-controlled intervention study was to examine whether long-term consumption of a juice enriched with LGG and calcium lactate gluconate (CaLG) affects the development of caries and caries risk in day care children. MATERIALS AND METHODS Subjects Thirty day care centres, in similar socio-economic areas in the cities of Espoo and Vantaa, Finland, participated in this randomized, double-blind, placebo-controlled multi-centre study. Altogether 530 healthy children aged 3-6 years volunteered to take part. The parents gave their written informed consent prior the study on behalf of their children. The study was ethically conducted in accordance with the Declaration of Helsinki and the Ethics Committee of the Helsinki City Health Department approved the study protocol. Intervention The intervention lasted seven months from September 2002 to May The children were randomised to the LGG- CaLG juice group or to the control juice group according to a computer generated randomly permuted block-method. In randomisation the block size of four was used, and it was stratified according to age (3-4 years or 5-6 years) and the S. mutans count (0-1 or 2-3, where 0: no growth, 1: =10 3 CFU/ ml, 2: 10 4 CFU/ml, 3: =10 5 CFU/ml) from samples taken at inclusion. During the intervention the children drank either carrotpineapple juice (Gefilus, Valio Ltd, Helsinki, Finland) enriched with LGG (5 x 10 6 CFU/ml of strain LGG, ATCC 53103) and calcium (CaLG 120 mg/100g), or control carrot-pineapple juice without LGG and calcium. The juices were delivered to the day care centres in cartons labelled as M and R juices. The study personnel (unless T. P.), the day care personnel, the study children and their families did not know which one was the active product until the end of the study. The day care personnel served the juices with afternoon snacks five days a week and recorded the daily amount of juices consumed by each child. The intended daily dose of study juice was 200 ml. The use of other products containing probiotic bacteria was prohibited for four weeks prior the intervention and throughout the study. Examination of oral health Information about the children s dental health and dietary habits were collected using a structured questionnaire filled in by the parents. The study coordinator checked the completed questionnaires, and supplied missing data with help of the parents. During the study, the parents recorded daily in a symptom diary any illnesses, dental appointments, and medications of the children. The children s oral health was clinically examined at baseline and in the end of the study by an experienced dentist (H. Y.-K.) according to the World Health Organisation criteria from The use of fluoride varnish was forbidden during the intervention, but all necessary dental treatment and fluoride toothpaste were allowed. The S. mutans counts were evaluated four times during the study: at inclusion, at baseline (0 months), in the middle (3.5 months), and in the end of the study (7 months). The samples were taken in the day care centres from all the children and always at the same time, i.e. one hour after breakfast or lunch. The research assistants took samples from the tongue surface by using the Dentocult SM Strip mutans slides (Orion Diagnostica, Espoo, Finland). The S. mutans counts were measured using the following categories: 0: no growth, 1: =10 3 CFU/ml, 2: 10 4 CFU/ml, 3: =10 5 CFU/ml. The caries risk was evaluated at baseline and in the end of the study by using both clinical and microbiological data (S. mutans counts). The sums of decayed, missing and filled teeth in the primary (dmft) and permanent (DMFT) teeth were analysed. The caries risk was considered low if no caries was detected and the S. mutans count was <10 5 CFU/ml; moderate if the child had either a dmft/dmft or initial caries score >0 or the S. mutans count >10 5 CFU/ml; and high if the child had a dmft/dmft or initial caries score >0 and S. mutans count >10 5 CFU/ml. Lactobacillus rhamnosus GG measurements Salivary LGG was analyzed at baseline and in the end of the study in a subgroup of study children (total n=362, LGG-CaLG juice group n=179, control juice group n=183). Saliva samples were cultured on agar and LGG was identified by colony morphology, lactose fermentation test and characteristic cell shapes (Yli-Knuutila et al., 2006). Statistical methods Calculation of the sample size was based on the assumption that the use of LGG-CaLG juice results in 44% reduction in dental caries risk. As a reference data we used our previous study which showed an odds ratio of 0.56 for LGG in preventing caries (Näse et al., 2001). We assumed that after the intervention period the proportion of moderate or high caries risk would be 27% in the LGG-CaLG juice group vs. 40% in the control juice group. It was further estimated that with a two group continuity corrected, Chi-square test with a two-sided significance level would show a 80% power in the detection of the difference between the LGG-CaLG juice group and control juice group (OR=0.56) when the sample size in each is 228. To take the possible dropouts into consideration, 265 subjects per group were randomized. The primary outcome measure was the caries risk measured in the

3 Probiotics and caries risk in children 223 end of the study. In the final analyses the classes moderate and high were combined. Logistic regression analyses were performed to compare the LGG-CaLG juice group to the control juice group with respect to moderate/high caries risk or other binary response variables measured in the end of the study. The corresponding baseline status was included as a categorical covariate. As the baseline-adjusted analysis requires complete data, the main analyses include the subjects who participated in all study phases (baseline, middle and end). The results are given as odds ratios (OR) with 95% confidence intervals (CI). The baseline characteristics were analyzed using t-test for independent samples or Chisquare test, when appropriate. The analyses were performed using SPSS (Version 17.0, the SPSS Inc., Chicago, IL, USA). RESULTS Baseline characteristics and compliance Of the 530 study children, 265 were allocated to the LGG-CaLG juice group and 265 children to the control juice group (Figure 1). Thirty-seven children (14%) in the LGG- CaLG juice group and 43 children (16%) in the control juice group dropped out. The study groups were comparable with respect to the children s oral health habits and dietary habits (Table 1). However, children in the control juice group used slightly more xylitol products (98% in the control juice group vs. 95% in the LGG-CaLG juice group, p=0.061). The average daily consumption of juice in the LGG- FIGURE 1. Flow-chart indicating the progress of participation in the different phases of the 7- month study. TABLE 1. Characteristics of the study children, randomized to consume either juice enriched with Lactobacillus GG and calcium lactate gluconate (LGG-CaLG juice group, n=235) or control juice (control juice group, n=238). Figures are percentages or means (range). LGG-CaLG CONTROL Boys/girls, % 51/49 53/47 Age, years 5.5 ( ) 5.6 ( ) Oral health habits Brushing the teeth, % <1/day 3 4 1/day /day Parental help with brushing, % Age when tooth brushing started, months 11 (0-48) 11 (0-48) Use of fluoride toothpaste, % Use of other fluoride products, % Use of xylitol-products, % Using dummy, % 0 0 Age when dummy eating stopped, months 17 (0-48) 17 (0-48) Using baby bottle, % Age when baby bottle use stopped, months 18 (0-48) 18 (0-72) Dietary habits Drink when thirsty, % Water Milk Juice Sugar snacks, % Number of meals per day 5 (2-7) 5 (2-10) TABLE 2. The number of children with different Streptococcus mutans scores at the baseline, in the middle and in the end of the study in groups consuming either juice enriched with Lactobacillus GG and calcium lactate gluconate (LGG-CaLG juice group, n=211) or control juice (control juice group, n=220). 1 S. mutans scores CFU/ml; 2 S. mutans scores LGG-CaLG CFU/ml; 3 S. mutans =10 5 CFU/ml CONTROL Class Class 2 2 Class 3 3 Class 0-1 Class 2 Class 3 Baseline 190 (90%) 10 (4.7%) 11 (5.2%) 199 (91%) 14 (6.4%) 7 (3.2%) Middle 196 (93%) 10 (4.7%) 5 (2.4%) 202 (92%) 13 (5.9%) 5 (2.3%) End 199 (94%) 8 (3.8%) 4 (1.9%) 207 (94%) 10 (4.5%) 3 (1.4%) CaLG juice and control juice groups were 92 (SD 0.33) and 95 (SD 0.31) ml with no difference in the compliance (46% and 47%). Salivary LGG was analyzed twice in a subgroup of study children (total n=362, LGG-CaLG juice group n=179, control juice group n=183). At baseline, 9.5% of subjects in the LGG-CaLG juice group and 12.0% in the control juice group had detectable amounts of LGG in saliva. At the end of the study the figures were 42.5% versus 17.5% (baseline-adjusted OR=4.09; 95% CI 2.44 to 6.86; p<0.001). Streptococcus mutans counts Only few children showed S. mutans infection (Table 2). During

4 224 Probiotics and caries risk in children the intervention, the number of children with high S. mutans counts (score 3) decreased from 5.2% to 1.9% in the LGG-CaLG juice group and from 3.2% to 1.4% in the control juice group. We combined moderate and high S. mutans count groups (scores 2 to 3) for further analyses. In the middle of the study, the number of children in moderate and high count groups had decreased from 10.0% to 7.1% in the LGG-CaLG juice group and from 9.5% to 8.2% in the control juice group (baseline-adjusted OR 0.75; 95% CI 0.30 to 1.87; p=0.537). In the end of the study the percentage had decreased to 5.7% in the LGG-CaLG juice group and to 5.9% in the control juice group (baselineadjusted OR 0.71; 0.27 to 1.87; p=0.490). The odds ratios indicate a non-significant risk reduction, 25% in the middle of the study and 29% in the end of the study, in favor of the LGG-CaLG group. TABLE 3. Dental caries risk at baseline and in the end of the study in groups consuming either juice enriched with Lactobacillus GG and calcium lactate gluconate (LGG-CaLG juice group, n=197) or control juice (control juice group, n=197). 1 no caries and S. mutans count <10 5 CFU/ml; 2 decayed, missing or filled teeth or initial caries score >0 or S. mutans count >10 5 CFU/ml; 3 decayed, missing or filled teeth or initial caries score >0 and S. mutans count >10 5 CFU/ml. LGG-CaLG CONTROL Low 1 Moderate 2 High 3 Low Moderate High Baseline 144 (73%) 44 (22%) 9 (4.6%) 140 (71%) 51 (26%) 6 (3.0%) End 135 (69%) 58 (29%) 4 (2.0%) 125 (64%) 69 (35%) 3 (1.5%) TABLE 4. The number of healthy sextants at baseline and in the end of the study in the groups consuming either juice enriched with Lactobacillus GG and calcium lactate gluconate (LGG- CaLG juice group, n=197) or control juice (control juice group, n=197). NUMBER BASELINE END LGG-CaLG Control p-value LGG-CaLG Control p-value juice group juice group juice group juice group 0 8 (4.0%) 10 (5.1%) 11 (5.5%) 11 (5.6%) 1 0 (0.0%) 1 (0.5%) 1 (0.5%) 0 (0.0%) 2 7 (3.5%) 5 (2.5%) 4 (2.0%) 9 (4.5%) 3 12 (6.0%) 18 (9.1%) 8 (4.0%) 15 (7.6 %) 4 33 (17%) 35 (28%) 39 (20%) 39 (20%) 5 27 (14%) 30 (15%) 29 (15%) 40 (20%) (56%) 99 (50%) (54%) 84 (42%) FIGURE 2. Number of children at baseline and in the end of the study needing dental care in the groups consuming either juice enriched with Lactobacillus GG and calcium lactate gluconate (LGG- CaLG juice group, n=199) or control juice (control juice group, n=198). Results on clinical dental examination According to the S. mutans counts and clinical examination in the end of the study, the combined high to moderate caries risk increased in both groups during the study, from 27% to 32% in the LGG- CaLG juice group and from 29% to 37% in the control juice group (Table 3). At the end of the study the corresponding percentage was 31% in the LGG-CaLG juice group and 37% in the control juice group, that is, caries risk was non-significantly 29% lower in the LGG-CaLG juice group compared to control juice group (baselineadjusted OR=0.71; 95% CI ; p=0.281). The need of dental care increased in both study groups during the intervention, from 86 (43%) to 91 (46%) in the LGG-CaLG juice group and from 99 (50%) to 114 (58%) in the control juice group (Figure 2). Thus, at the end of the study, the need of dental care was smaller in the LGG-CaLG juice group compared to the controls (baseline-adjusted OR=0.58; 95% CI ; p=0.050). At the end of the intervention there were slightly more children in the LGG-CaLG juice group than in the control juice group who had all the six mouth sextants healthy (baselineadjusted OR=1.69, 95% CI ; p=0.056, Table 4). DISCUSSION In the present study we examined, for the first time, whether long-term consumption of a juice enriched with LGG and CaLG affects the development of caries in children. At the end of the 7-month study, the need for dental care was smaller and there were more children who had all the six sextants healthy in the LGG-CaLG juice group compared with the control juice group. However, no statistically significant differences were found between the study groups in the caries risk and salivary S. mutans counts. Hence a functional mixture of LGG and calcium was able to slightly protect the children s teeth even in a sugar containing juice confirming earlier positive findings on reducing the risk of dental caries with probiotic milk, cheese, yoghurt, lozenge, straws and tablets (Näse et al., 2001; Ahola et al., 2002; Caglar et al., 2005, 2006, 2008). Children commonly consume juices even though their excessive consumption is related to an increased risk of dental erosion and caries (Moazzez et al., 2000; Jensdottir et al., 2004). Therefore, we aimed to formulate juice to reduce its cariogenic and erosive potential.

5 Probiotics and caries risk in children 225 Our positive post-treatment effect confirms earlier observations that LGG may inhibit the colonization of Streptococcus sobrinus (Meurman et al., 1995), a member of the mutans streptococci family. It has also been shown that LGG ferments sucrose very slowly indicating that this bacterium is not cariogenic (Saxelin, 1997; Haukioja et al., 2008). Some probiotic strains seem to inhibit S. mutans in human saliva (Näse et al., 2001; Caglar et al., 2005, 2006, 2008). There are also critical reports (Montalto et al., 2004) and some lactobacilli and bifidobacteria are actually cariogenic (Aas et al., 2008) which emphasizes that the effects are strain specific. In two recent studies LGG is found to ferment glucose (Haukioja et al., 2008; Hedberg et al. 2008). However, amount of glucose in saliva is rather low and LGG did not ferment or fermented only slowly sucrose and lactose in these studies. This characteristic could be one reason why we found stronger positive effects with probiotic milk (Näse et al., 2001) while only a trend with probiotic juice was seen in the present study. Seven-month intervention is a short time for caries lesions to develop. Therefore we assessed the caries risk with both clinical and microbiological data (S. mutans counts) as has been found practical in our earlier study (Näse et al., 2001). The risk of dental caries was classified as low, moderate or high. The high caries risk decreased in both study groups while the moderate caries risk increased during the intervention. At the end of the study the combined moderate to high caries risk was 31% in the LGG- CaLG juice group and 37% in the control juice group which accords with earlier Finnish studies reporting caries prevalence of 28-41% in this age group (Alaluusua et al., 1983; Mattila et al. 1998). Nevertheless, in the end of the present study the need of dental care was smaller and there were more children who had all the six sextants healthy in the LGG-CaLG juice group compared to the control juice group. The S. mutans counts from saliva were assessed from samples taken from the upper surface of the tongue as reported by Meurman and Rantonen (1994) from adult population and now found practical also in the present study with children. The moderate and high S. mutans levels decreased in both study groups, although no statistically significant difference was found between the groups. One explanation might be that there were only few children that had caries and S. mutans infection at all. At baseline, high S. mutans counts were detected in 11 out of 211 children (5.2%) in the LGG-CaLG juice group and only in 7 out of 220 children (3.2%) in the control juice group. Corresponding S. mutans counts in our earlier study were 13% in the LGG milk group and 8.6% in the placebo milk group (Näse et al., 2001). Our study juices contained sucrose because the taste of calcium needed to be covered with flavor and sugar is the natural ingredient of juice. The sugar content might be one reason why no statistically significant decrease in the S. mutans counts could be detected during the study. The high intake of sucrose has been shown to increase the risk of dental caries in children (Ruottinen et al., 2004). In that study the most frequently used food rich in sugar was sweetened dairy products. On the other hand, it has been shown that brushing the teeth twice a day with fluoride toothpaste may have greater impact on caries in young children than restricting sugary foods (Gibson and Williams, 1999). In the present study we found that only 33% of the children brushed their teeth twice a day and 64% brushed once a day. According to a Finnish study only 18% of the 5-year-olds Finns brushes their teeth twice a day (Mattila et al., 1998) which is remarkable less than, for example in the UK, where about 50% of the children brushes their teeth more than once a day (Gibson and Williams, 1999). In addition to probiotics, our study juice also contained calcium in the form of CaLG. This may explain some of the positive effects. The dissolution of mineral from enamel to the surrounding environment is proportionate to the concentration of calcium and phosphorus. Consequently, the addition of calcium and phosphate to a drink in amounts corresponding to the enamel content can prevent enamel from dissolving (Attin et al., 2005; Jensdottir et al., 2005; West et al., 2004). The results of the present study showed that consumption of juice containing LGG and calcium might reduce the need for dental care in children. However, no statistically significant differences were observed between the study groups in the caries risk and salivary S. mutans counts. Nevertheless, in the LGG- CaLG juice group there were more children who had all the six sextants healthy, indicating a post-treatment effect that supplementation of juice with probiotics and calcium might be beneficial for dental health in children. CONFLICT OF INTEREST S. Pohjavuori, A.J. Ahola and R. Korpela were employees of Valio Ltd, sponsoring the study. REFERENCES Aas, J.A., Griffen, A.L., Dardis, S.R., Lee, A.M., Olsen, I., Dewhirst, F.E., Leys, E.J. and Paster, B.J. (2008) Bacteria of dental caries in primary and permanent teeth in children and young adults. Journal of Clinical Microbiology 46, Ahola, A.J., Yli-Knuuttila, H., Suomalainen, T., Poussa, T., Ahlström, A., Meurman, J.H. and Korpela, R. (2002) Short-term consumption of probiotic-containing cheese and its effect on dental caries risk factors. 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6 226 Probiotics and caries risk in children Caglar, E., Sandalli, N., Twetman, S., Kavaloglu, S., Ergeneli, S. and Selvi, S. (2005) Effect of youghurt with Bifidobacterium DN on salivary mutans streptococci and lactobacilli in young adults. Acta Odontologica Scandinavica 63, Drummond, B.K., Chandler, N.P. and Meldrum, A.M. (2002) Comparison of the cariogenicity of some processed cheeses. European Journal of Paediatric Dentristry 3, Gibson, S. and Williams, S. (1999) Dental caries in pre-school children: associations with social class, toothbrushing habit and consumption of sugars and sugar-containing foods. Further analysis of data from the National Diet and Nutrition Survey of children aged years. Caries Research 33, Hara, A.T. and Zero, D.T. (2008) Analysis of the erosive potential of calcium-containing acidic beverages. European Journal of Oral Sciences 116, Harper, D.S., Osborn, J.C., Hefferren, J.J. and Clayton, R. (1986) Cariostatic evaluation of cheeses with diverse physical and compositional characteristics. Caries Research 20, Hedberg, M., Hasslöf, P., Sjöström, I., Twetman, S. and Stecksén- Blicks, C. (2008) Sugar fermentation in probiotic bacteria an in vitro study. Oral Microbiology and Immunology 23, Jensdottir, T., Arnadottir, I.B., Thorsdottir, I., Bardow, A., Gudmundsson, K., Theodors, A. and Holbrook, W.P. (2004) Relationship between dental erosion, soft drink consumption, and gastroesophageal reflux among Icelanders.Clinical Oral Investigations 8, Jensdottir, T., Bardow, A. and Holbrook, P. (2005) Properties and modification of soft drinks in relation to their erosive potential in vitro. Journal of Dentistry 33, Krobicka, A., Bowen, W.H., Pearson, S. and Young, D.A. (1987) The effects of cheese snacks on caries in desalivated rats. Journal of Dental Research 66, Lussi, A., Jäggi, T., Schärer, S. (1993) The influence of different factors on in vitro enamel erosion. Caries Research 27, Mattila, M.L., Rautava, P., Sillanpää, M. and Paunio P (2000) Caries in five-year-old children and associations with family-related factors. Journal of Dental Research 79, Meurman, J.H., Antila, H., Korhonen, A. and alminen, S. (1995) Effect of Lactobacillus rhamnosus strain G (ATCC 53103) on the growth of Streptococcus sobrinus in vitro. European Journal of Oral Sciences 103, Meurman, J.H. and Rantonen, P. (1994) Salivary flow rate, buffering capacity, and yeast counts in 187 consecutive adult patients in Kuopio, Finland. Scandinavian Journal of Dental Research 102, Moazzez, R., Smith, B.G. and Bartlett, D.W. (2000) Oral ph and drinking habit during ingestion of a carbonated drink in a group of adolescents with dental erosion. Journal of Dentistry 28, Montalto, M., Vastola, M., Marigo, L., Covino, M., Graziosetto, R., Curigliano, V., Santoro, L., Cuoco, L., Manna, R. and Gasbarrini, G. (2004) Probiotic treatment increases salivary counts of lactobacilli: a double-blind, randomized, controlled study. Digestion 69, Näse, L., Hatakka, K., Saxelin, M., Pönkä, A., Poussa, T., Korpela, R. and Meurman, J.H. (2001) Effect of long-term consumption of a probiotic bacterium, Lactobacillus rhamnosus GG, in milk on dental caries and caries risk in children. Caries Research 35, Ruottinen, S., Karjalainen, S., Pienihäkkinen, K., Lagström, H., Niinikoski, H., Salminen, M., Rönnemaa, T. and Simell, O. (2004) Sucrose intake since infancy and dental health in 10-year-old children. Caries Research 38, Rytömaa, I., Järvinen, V. and Calonius, P.E. (1980) 44-year dental health survey of Helsinki schoolchildren. Community Dentistry and Oral Epidemiology 8, Saxelin, M. (1997) Lactobacillus GG - a human probiotic strain with thorough clinical documentation. Food Reviews International 13, Seppä, L., Hausen, H., Kärkkäinen, S. and Larmas, M. (2002) Caries occurrence in a fluoridated and a nonfluoridated town in Finland: A retrospective study using longitudinal data from public dental records. Caries Research 36, Silva, M., Jacobus, N.V., Deneke, C. and Gorbach, S.L. (1987) Antimicrobial substance from a human Lactobacillus strain. Antimicrobial Agents and Chemotherapy 31, Twetman, L., Larsen, U., Fiehn, N.E., Stecksen-Blicks, C. and Twetman, S. (2009) Coaggregation between probiotic bacteria and caries-associated strains: An in vitro study. Acta Odontologica Scandinavica 67, Vacca-Smith, A.M., Bowen, W.H. (2000) The effects of milk and kappa-casein on salivary pellicle formed on hydroxyapatite discs in situ. Caries Research 34, Vehkalahti, M., Tarkkonen, L., Varsio, S., Heikkilä, P. (1997) Decrease in and polarization of dental caries occurrence among child and youth populations, Caries Research 31,

7 Probiotics and caries risk in children 227 Wei, H., Loimaranta, V., Tenovuo, J., Rokka, S., Syväoja, E.L., Korhonen, H., Joutsjoki, V. and Marnila, P. (2002) Stability and activity of specific antibodies against Streptococcus mutans and Streptococcus sobrinus in bovine milk fermented with Lactobacillus rhamnosus strain GG or treated at ultra-high temperature. Oral Microbiology and Immunology 17, West, N.X., Hughes, J.A., Parker, D., Weaver, L.J., Moohan, M., De Ath, J. and Addy, M. (2004) Modification of soft drinks with xanthan gum to minimise erosion: A study in situ. British Dental Journal 196, Wongkhantee, S., Patanapiradej, V., Maneenut, C. and Tantbirojn, D. (2006) Effect of acidic food and drinks on surface hardness of enamel, dentine, and tooth-coloured filling materials. Journal of Dentistry 34, Yli-Knuuttila, H., Snäll, J., Kari, K. and Meurman J.H. (2006) Colonization of Lactobacillus rhamnosus GG in the oral cavity. Oral Microbiology and Immunology 21,

8 228 Probiotics and caries risk in children