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The aim of this study was to evaluate the effect of drops containing probiotic bacteria on the recurrence of dental caries in preschool children. The study employed a randomized, placebo-controlled, double-blinded design with two parallel arms. 38 preschool children were enrolled after comprehensive restorative treatment under general anesthesia or conscious sedation (baseline), and they were followed up after 6 and 12 months. Parents of children in the test group were instructed to give 5 daily drops containing two strains of Limosilactobacillus reuteri (DSM 17938 and ATCC PTA 5289) at bedtime. The placebo drops were identically composed but lacked bacteria. The duration of the intervention was 12 months. The primary endpoint was recurrence of new caries lesions on subject level (yes/no), and secondary endpoints were presence of dental plaque and gingivitis. We found high rate of recurrent moderate and extensive lesions after 12 months (67%) but there were no significant differences between the groups. We observed no beneficial effects on dental plaque or gingival inflammation. The findings were however uncertain and inconclusive due to lack of power, a consequence of the COVID-19 pandemic. ClinTrials.gov Identifier: (NCT04929340), June 18, 2021; retrospectively registered.

The World Health Organization has defined probiotics as “Live microorganisms which, when administered in adequate amounts, confer a health benefit to the host.” Traditionally, probiotic microorganisms (mainly Lactobacillus ssp. and Bifidobacterium ssp.) have been used to prevent or treat diseases in the gastrointestinal tract. In the past 20 years, there has been an increased interest in possible oral health effects of probiotics. In vitro studies have shown promising results with growth inhibition of mutans streptococci (MS) and Candida albicans. There are only a few clinical studies with caries development as the primary outcome while more studies have been focusing on control of caries risk factors or so-called surrogate outcomes. Several studies have evaluated the effects of probiotic bacteria on MS in saliva and/or plaque, and a number of probiotic strains show ability to reduce the number of MS. Probiotic bacteria have not been shown to permanently colonize the oral cavity; in early-in-life interventions or in subjects with a mature microbiota. To date investigated strains are transiently present in saliva during and shortly after an intervention. There are eight randomized controlled clinical trials with dental caries as outcome and probiotic strains, administration, duration of the intervention, and target group varied. In a majority of the studies (75%), the interventions resulted in caries reduction in the treatment groups. Although a majority of these studies suggest a caries-preventive effect of probiotic bacteria, more long-term clinical studies are needed in this field before probiotics could be recommended for preventing or treating dental caries.

Inflammation and infection postpartum threaten the mother and her infant. Human milk provides a defense for the infant, but inflammatory complications like mastitis may lead to the cessation of breastfeeding. Antisecretory factor (AF) has a role in the regulation of secretory processes and inflammation. The objective of the study was to describe AF-levels in plasma and breast milk, and in relation to breast complications. Breastfeeding mothers (n = 95) were consecutively recruited at a Well Baby Clinic in Umeå, Sweden. At inclusion four weeks postpartum, samples of venous blood (10 mL) and breast milk (10 mL) were collected. Active AF was analyzed with ELISA using a monoclonal antibody mAb43, and was detected in all samples of plasma and breast milk with a positive correlation (Spearman coefficient = 0.40, p < 0.001; Pearson correlation = 0.34, p < 0.01). High AF-levels in plasma correlated with high AF-levels in breast milk. The results suggest a co-regulation between active AF in plasma and breastmilk, and/or a local regulation of AF in the breast. Further studies are needed to determine the pathways for the activation of AF-levels in breast milk and plasma.

Pregnancy and post-partum represent a period of susceptibility for vitamin D insufficiency. This study investigated S-25 [OH] D levels in women in northern Sweden 4 weeks post-partum and its association with selected background factors. Blood from 100 healthy women were analyzed for iron status and serum levels of S-25[OH] D using ionization-mass spectrometry (HPLC-APCI-MS). <50 nmol/L was categorized as insufficiency and <25 nmol/L as deficiency. Maternal BMI, dietary habits, fungal infections during pregnancy, and infant birth characteristics were collected using questionnaires and medical charts. 58% were vitamin D insufficient whereas 10% had deficiency. Insufficiency was most common during winter (OR = 2.77; 95% CI = 1.1-6.96) and women with deficiency reported lower milk consumption; 11.3 ± 22.8 intakes per months vs. 34.0 ± 28.9 for those above 25 nmol/L (p < 0.05). Vitamin D-insufficient women had lower serum ferritin levels (p < 0.01) and higher serum transferrin levels (p < 0.05). A history of vaginal fungal infection during pregnancy was associated with insufficiency (OR = 5.10; 95% CI = 1.01-25.73), however, the confidence interval of the estimate was wide, resulting in uncertainty. It is concluded that vitamin D insufficiency 4 weeks post-partum was common in women living at 63°49'N. The odds of being insufficient were increased during winter whereas milk consumption was negatively associated with deficiency. The low vitamin D-levels particularly during winter is a public health concern. From a public health perspective it has to be considered whether dietary advices alone should be modified or if supplementation with vitamin D during pregnancy and the post-partum period also is needed.

Objective: To assess the urinary fluoride excretion in preschool children after drinking fluoridated milk with 0.185 mg F and 0.375 mg F and to study the impact of use of fluoride toothpaste. Basic research design: Double-blind cross-over study. Participants: Nine healthy children, 2.5-4.5 years of age. Intervention: In a randomized order, participants drank 1.5 dl milk once daily for 7 days with no fluoride added (control), 0.185 mg fluoride added and 0.375 mg fluoride added. The experiment was performed twice with (Part I) and without (Part II) parental tooth brushing with 1,000 ppm fluoride toothpaste. The fluoride content in the piped drinking water was 0.5 mg F/L. Main outcome measure: Urinary fluoride excretion. Results: The 24-hour urinary fl uoride excretion/kg body weight varied from 0.014 mg F for the placebo intervention and non-fluoride toothpaste to 0.027 mg F for the 0.375 mg intervention with use of 1,000 ppm fluoride toothpaste. The difference compared with the placebo intervention was not statistically significant for any of the interventions when fluoride toothpaste was used (p⟩0.05) while it was statistically significantly different when non-fluoride toothpaste was used (p⟨0.05). Conclusions: All sources of fluoride must be considered when designing community programs. With 0.5 mg F/L in the drinking water and daily use of fluoride toothpaste, most children had a fluoride intake optimal for dental health. In this setting, additional intake of fluoride milk was within safe limits up to 0.185 mg/day while conclusions about the safety of 0.375 mg/day were uncertain.

BACKGROUND: Children with severe congenital heart disease (CHD) need considerable nutritional support to reach normal growth. The actual intake of macro- and micronutrients in outpatient CHD infants over a 6-month period in infancy is not described in the literature. The present study aimed to prospectively investigate the distribution between macro- and micronutrient intake, meal frequency and growth in children with CHD. METHODS: At 6, 9 and 12 months of age, a 3-day food diary and anthropometric data were collected in 11 infants with severe CHD and 22 healthy age- and feeding-matched controls. Macro- and micronutrient intake, meal frequency and growth were calculated. RESULTS: Compared to the healthy controls, CHD infants had a statistically significantly higher intake of fat at 9 months of age (4.8 versus 3.6 g kg(-1) day(-1) ), a higher percentage energy (E%) from fat, (40.6% versus 34.5%) and a lower E% from carbohydrates (46.1% versus 39.6%) at 12 months of age, and a lower intake of iron (7.22 versus 9.28 mg day(-1) ) at 6 months of age. Meal frequency was significantly higher at 6 and 9 months of age (P < 0.01). Mean Z-score weight for height, weight for age and body mass index for age were significant lower (P < 0.01) at all time points. CONCLUSIONS: Despite a higher intake of energy from fat and a higher meal frequency, the intake does not meet the needs for growth, and the results may indicate a low intake of micronutrients in CHD infants.

Concepts of caries prevention Erupting teeth are healthy, and the first carious lesion and its restoration marks the initiation of a series of treatments that during the tooth’s lifetime will end up in more and more complicated restorations. Today there is sufficient scientific knowledge about the etiology of caries and about factors that interfere in this process in order for us to develop effective preventive strategies. There is no excuse not to use this knowledge in attempts to control caries. Therefore, caries prevention should be given a high priority by the dental profession as well as by governmental authorities. In this context, the pediatric dentist has a very specific responsibility in meeting the challenge of keeping children and adolescents free from caries. For example, very young children with early signs of caries should be given special attention, as they tend to develop more caries compared to children without early signs of caries.

OBJECTIVES: To identify, appraise and summarize existing knowledge and knowledge gaps in practice-relevant questions in pediatric dentistry. METHODS: A systematic mapping of systematic reviews was undertaken for domains considered important in daily clinical practice. The literature search covered questions in the following domains: behavior management problems/dental anxiety; caries risk assessment and caries detection including radiographic technologies; prevention and non-operative treatment of caries in primary and young permanent teeth; operative treatment of caries in primary and young permanent teeth; prevention and treatment of periodontal disease; management of tooth developmental and mineralization disturbances; prevention and treatment of oral conditions in children with chronic diseases/developmental disturbances/obesity; diagnosis, prevention and treatment of dental erosion and tooth wear; treatment of traumatic injuries in primary and young permanent teeth and cost-effectiveness of these interventions. Abstracts and full text reviews were assessed independently by two reviewers and any differences were solved by consensus. AMSTAR was used to assess the risk of bias of each included systematic review. Reviews judged as having a low or moderate risk of bias were used to formulate existing knowledge and knowledge gaps. RESULTS: Out of 81 systematic reviews meeting the inclusion criteria, 38 were judged to have a low or moderate risk of bias. Half of them concerned caries prevention. The quality of evidence was high for a caries-preventive effect of daily use of fluoride toothpaste and moderate for fissure sealing with resin-based materials. For the rest the quality of evidence for the effects of interventions was low or very low. CONCLUSION: There is an urgent need for primary clinical research of good quality in most clinically-relevant domains in pediatric dentistry

Background: Mutations in the EDAR-gene cause hypohidrotic ectodermal dysplasia with defects in ectodermal appendage development including teeth, skin, exocrine glands and hair. Hair defects are sparsely described in genetically defined samples. The aim of this study was to investigate hair structures in three families with a heterozygous c.1072C > T mutation in the EDAR gene using scanning electron microscopy. Methods: Three Swedish families, where some members had a known c.1072C > T mutation in the EDAR gene with an autosomal dominant inheritance (AD) were included (n = 37) of which 17 carried the mutation and 20 did not. Thirty-two age and gender matched not related individuals served as a reference group. Confirmation of the c.1072C > T mutation in the EDAR gene was performed by genomic sequencing. Hairs were subjected to blinded scanning electron microscopy examination and hair defects were categorized and scored. Results: The minimum and maximum diameters of hairs were lower in the mutation group compared to the reference group. Subjects in the mutation group had to greater extent deep deformations in hair shafts compared to the non-mutation group and the reference group (p < 0.001). Conclusions: Individuals with a c.1072C > T mutation in the EDAR-gene displayed more hair shaft deformations confirming the role of EDAR for human hair follicle development and postnatal hair follicle cycling.

Colonisation of the gastrointestinal tract is influenced by primary microbial exposure and bioactive factors in breastmilk. The aim was to explore the prevalence of oral Candida in the first year of life in relation to selected exposures. Oral Candida was studied in 100 healthy infants at 4 and 8 weeks, 3, 6 and 12 months of age and related to delivery mode, birth weight, infant health and feeding, antibiotics, antimycotics, steroids and probiotics in mother and infant, living conditions, maternal smoking and infections The association between lactoferrin and antisecretory factor in breastmilk and maternal serum haemoglobin, transferrin, and ferritin levels in relation to oral Candida was also explored. About 11% to 15% of the infants had oral Candida at the respective age. Colonisation was fairly stable until 6 months of age. There was no conclusive impact of the investigated exposures at entry. Infants with a furry pet at home had a lower frequency of Candida at 3 months, (P < 0.05) whereas all but one colonised infant had older siblings at 12 months (P < 0.01). Lactoferrin in breastmilk was negatively associated with colonisation at 6 months of age. It is concluded that 11 to 15% had oral Candida. Exposure to furry pets and siblings impacted oral Candida.

OBJECTIVES: To identify, appraise and summarize existing knowledge and knowledge gaps in practice-relevant questions in pediatric dentistry.METHODS: A systematic mapping of systematic reviews was undertaken for domains considered important in daily clinical practice. The literature search covered questions in the following domains: behavior management problems/dental anxiety; caries risk assessment and caries detection including radiographic technologies; prevention and non-operative treatment of caries in primary and young permanent teeth; operative treatment of caries in primary and young permanent teeth; prevention and treatment of periodontal disease; management of tooth developmental and mineralization disturbances; prevention and treatment of oral conditions in children with chronic diseases/developmental disturbances/obesity; diagnosis, prevention and treatment of dental erosion and tooth wear; treatment of traumatic injuries in primary and young permanent teeth and cost-effectiveness of these interventions. Abstracts and full text reviews were assessed independently by two reviewers and any differences were solved by consensus. AMSTAR was used to assess the risk of bias of each included systematic review. Reviews judged as having a low or moderate risk of bias were used to formulate existing knowledge and knowledge gaps.RESULTS: Out of 81 systematic reviews meeting the inclusion criteria, 38 were judged to have a low or moderate risk of bias. Half of them concerned caries prevention. The quality of evidence was high for a caries-preventive effect of daily use of fluoride toothpaste and moderate for fissure sealing with resin-based materials. For the rest the quality of evidence for the effects of interventions was low or very low.CONCLUSION: There is an urgent need for primary clinical research of good quality in most clinically-relevant domains in pediatric dentistry.

BACKGROUND: Mutations in the EDAR-gene cause hypohidrotic ectodermal dysplasia, however, the oral phenotype has been described in a limited number of cases. The aim of the present study was to clinically describe individuals with the c.1072C > T mutation (p. Arg358X) in the EDAR gene with respect to dental signs and saliva secretion, symptoms from other ectodermal structures and to assess orofacial function. METHODS: Individuals in three families living in Sweden, where some members had a known c.1072C > T mutation in the EDAR gene with an autosomal dominant inheritance (AD), were included in a clinical investigation on oral signs and symptoms and self-reported symptoms from other ectodermal structures (n = 37). Confirmation of the c.1072C > T mutation in the EDAR gene were performed by genomic sequencing. Orofacial function was evaluated with NOT-S. RESULTS: The mutation was identified in 17 of 37 family members. The mean number of missing teeth due to agenesis was 10.3 ± 4.1, (range 4-17) in the mutation group and 0.1 ± 0.3, (range 0-1) in the non-mutation group (p < 0.01). All individuals with the mutation were missing the maxillary lateral incisors and one or more of the mandibular incisors; and 81.3% were missing all four. Stimulated saliva secretion was 0.9 ± 0.5 ml/min in the mutation group vs 1.7 ± 0.6 ml/min in the non-mutation group (p < 0.01). Reduced ability to sweat was reported by 82% in the mutation group and by 20% in the non-mutation group (p < 0.01). The mean NOT-S score was 3.0 ± 1.9 (range 0-6) in the mutation group and 1.5 ± 1.1 (range 0-5) in the non-mutation group (p < 0.01). Lisping was present in 56% of individuals in the mutation group. CONCLUSIONS: Individuals with a c.1072C > T mutation in the EDAR-gene displayed a typical pattern of congenitally missing teeth in the frontal area with functional consequences. They therefore have a need for special attention in dental care, both with reference to tooth agenesis and

Why should primary teeth be restored? Caries in the primary dentition is a public health concern and a challenge for the dental care system. An assumption has traditionally been that primary teeth with carious lesions should be restored by removing infected tissue and replacing it with an appropriate dental material. This practice, however, has been questioned. This paper reviews consequences of untreated caries and the benefits of restoring primary teeth. Treatment of caries in primary teeth has positive implications for the child, both in the short and long term. The rapid caries progression in primary teeth suggests a more operative approach compared to permanent teeth since in this case caries progresses more slowly. Deep restorations in primary teeth increase the risk of pulpal involvement. There is no conclusive scientific support for treatment of caries in the primary dentition, but there is some support that stepwise or partial caries removal reduces the incidence of pulp exposure in symptomless, vital, carious primary teeth.

Abstract saknas, Oral health in children with congenital heart disease

The aim of this project was to study possible influences of Turner syndrome (TS) karyotype and the number of X chromosomes with intact short arm (p-arm) on dental crown width. Primary and permanent mesio-distal crown width was measured on plaster casts from 112 TS females. The influence on crown width of four karyotypes: 1. monosomy (45,X), 2. mosaic (45,X/46,XX), 3. isochromosome, and 4. other, and the number of intact X chromosomal p-arms were investigated. In comparisons between karyotypes, statistically significant differences were found for isochromosome karyotype maxillary second premolars, canines, laterals, mandibular first premolars, and canines, indicating that this karyotype was the most divergent as shown by the most reduced crown width. When each karyotype group were compared versus controls, all teeth in the isochromosome group were significantly smaller than controls (P < 0.01-0.001). The 45,X/46,XX karyotype expressed fewer and smaller differences from controls, while 45,X individuals seemed to display an intermediate tooth width compared with 45,X/46,XX and isochromosomes. No significant difference in crown width was found comparing the groups with one or two intact X chromosomal p-arms. Both primary and permanent teeth proved to have a significantly smaller crown width in the entire group of TS females compared to healthy females. We conclude that the isochromosome group deviates most from other karyotypes and controls, exhibiting the smallest dental crown width, while individuals with 45,X/46,XX mosaicism seemed to have a less affected crown width. An influence of the number of intact p-arms on crown width could not be demonstrated in this study.

Karies är ett folkhälsoproblem som kan kopplas både till biologi och till frågor som rör sociala förhållanden och livsstil. Trots att det finns starkt vetenskapligt stöd för hur karies kan förebyggas – kvarstår problematiken och många drabbas. Karies är fortfarande ett stort problem hos barn med en tydlig och ökande polarisering mot socioekonomiskt svaga grupper.

Objective: Co-aggregation and growth inhibition abilities of probiotic bacteria may play a key role in their interference with the oral biofilm. The aim was to investigate the in vitro ability of selected commercial probiotic lactobacilli to co-aggregate and inhibit growth of oral mutans steptococci isolated from adults with contrasting levels of caries. Materials and methods: Mutans streptococci (MS) strains were isolated from caries-free (n = 3) and caries-susceptible (n = 5) young adults and processed with eight commercial probiotic lactobacilli strains. One laboratory reference strain (S. mutans Ingbritt) was selected as control. Co-aggregation was determined spectrophotometrically and growth inhibition was assessed with the agar overlay technique. Results: All probiotic lactobacilli showed an ability to co-aggregate with the isolated MS strains. Statistically significant differences (p < 0.05) were found between strains from different individuals when compared with the reference strain. The selected lactobacilli inhibited MS growth, but the ability varied between the strains and was clearly related to pH. No differences were observed between the different MS strains from caries-free and caries-susceptible individuals. Conclusions: The selected lactobacilli displayed co-aggregation activity and inhibited growth of clinical mutans streptococci. The growth inhibition was strain-specific and dependent on pH and cell concentration. The findings indicate that the outcome of lactobacilli-derived probiotic therapy might vary between individuals and depend on the specific strain used.

Objective. Co-aggregation and growth inhibition abilities of probiotic bacteria may play a key role in their interference with the oral biofilm. The aim was to investigate the in vitro ability of selected commercial probiotic lactobacilli to co-aggregate and inhibit growth of oral mutans steptococci isolated from adults with contrasting levels of caries. Materials and methods. Mutans streptococci (MS) strains were isolated from caries-free (n = 3) and caries-susceptible (n = 5) young adults and processed with eight commercial probiotic lactobacilli strains. One laboratory reference strain (S. mutans Ingbritt) was selected as control. Co-aggregation was determined spectrophotometrically and growth inhibition was assessed with the agar overlay technique. Results. All probiotic lactobacilli showed an ability to co-aggregate with the isolated MS strains. Statistically significant differences (p <0.05) were found between strains from different individuals when compared with the reference strain. The selected lactobacilli inhibited MS growth, but the ability varied between the strains and was clearly related to pH. No differences were observed between the different MS strains from caries-free and caries-susceptible individuals. Conclusions. The selected lactobacilli displayed co-aggregation activity and inhibited growth of clinical mutans streptococci. The growth inhibition was strain-specific and dependent on pH and cell concentration. The findings indicate that the outcome of lactobacilli-derived probiotic therapy might vary between individuals and depend on the specific strain used.

AIM: To study the saliva profiles in children with severe heart disease taking heart failure medication compared with the saliva from healthy age and gender matched controls. STUDY DESIGN: Cross sectional case-control design. METHODS: Twenty-four age and gender matched pairs of children, mean age 12.0 years participated. Stimulated saliva was collected in a standardized way before lunchtime and the subjects were asked to refrain from all eating, drinking and tooth brushing 90 mins before sampling. Stimulated salivary secretion rate, buffering capacity, total salivary viable count of bacteria, mutans streptococci and lactobacilli, calcium, chloride, magnesium, potassium, sodium and salivary IgA were determined. RESULTS: There were 7 of the 24 children in the cardiac group who had secretions below 0.5 ml/min compared with no child in the control group (p<0.01). Lower [corrected] total viable counts of bacteria (TVC) were detected in the cardiac group 1.4x10⁶ ± 1.2x10⁷ vs. 2.7x10⁶ ± 2.9x10⁷ in the control group (p<0.05). Mutans streptococci (MS) in the cardiac group were 5.2x10⁴ ± 1.5x10⁵ vs. 8.1 x10³ ± 1.3x10⁴ in the control group, (p>0.05) and MS ratio of TVC constituted 0.11±0.35 per cent compared to 0.01±0.02 per cent for the control group (p>0.05). STATISTICS: Continuous data were analysed by an analysis of variance (ANOVA) and categorical data by chi-square test. CONCLUSION: Reduced salivary secretion could be a caries risk factor in children taking heart failure medication.

The selected probiotic strains showed a significant but somewhat varying ability to inhibit growth of oral mutans streptococci and Candida albicans in vitro.

The primary aims were to investigate the total number of radiographs and the reason for dental radiography in children and adolescents in the Public Dental Health Service. Secondary aims were to study the influence of caries experience and orthodontic treatment on the number of radiographs. For this retrospective study, 544 adolescents regularly attending three Public Dental Health clinics in the county of Västerbotten, northern Sweden were selected. The number of radiographs exposed each year from 3 to 19 years of age was registered. Information on reason for the radiographic examination was extracted from dental records as well as the caries experience at 19 years of age. The attrition rate was 7% due to incomplete data. The total mean number of radiographs exposed was 23+/-6 of which 1+/-2 were extra-oral radiographs. Bitewing radiographs for caries registration constituted 87% of the intra-oral radiographs with a mean number of 19+/-4 exposures. There was a statistically significant difference (p<0.01) between subjects with caries experience (20+/-4) and those with no caries (16+/-4). No gender-related differences were displayed. During preschool ages, bitewing radiographs were taken in less than 10% of the children. Children treated at specialist clinics in orthodontics displayed higher number of radiographic examinations than non-referred children (p<0.01). CONCLUSION: A mean of 23 radiographs were exposed and bitewings for caries detection were the most common radiographic examination. Fewer bitewing radiographs were exposed in caries-free subjects over time but its use during the preschool ages was low. Orthodontics treatment increased the frequency of radiography significantly.

INTRODUCTION: Food supplemented with probiotic bacteria is a rapidly growing sector of the market. The aim of the present study was to evaluate and compare the acid production of selected probiotic strains available in commercial products. METHODS: Six Lactobacillus strains (Lactobacillus plantarum 299v and 931; Lactobacillus rhamnosus GG and LB21; Lactobacillus paracasei subsp. paracasei F19, and Lactobacillus reuteri PTA 5289) were cultivated at 37 degrees C in an anaerobic atmosphere on Man, Rogosa, Shape (MRS) agar for 48 h or MRS broth for 16 h. After centrifugation, the cells were washed and resuspended in sterile phosphate-buffered saline and immediately subjected to a fermentation assay with 12 different carbohydrates (nine sugars and three sugar alcohols) in microtiter plates with a pH indicator. The plates were examined for color changes after 24, 48, and 72 h of incubation under aerobic and anaerobic conditions. Three scores were used: negative (pH > 6.8); weak (pH 5.2-6.8), and positive (pH < 5.2). The strains were characterized with the API 50 CH system to confirm their identity. RESULTS: L. plantarum fermented all the sugars except for melibiose, raffinose, and xylitol. Both L. rhamnosus strains were generally less active although L. rhamnosus GG was slightly more active than strain LB21 in the 5% CO(2) setting. The latter strain exhibited negative reactions for sucrose, maltose, arabinose, and sorbitol under anaerobic conditions. The assays with L. paracasei and L. reuteri had negative or weak reactions for all tested sugars under both aerobic and anaerobic conditions. CONCLUSION: The metabolic capacity to form acid from dietary sugars differed significantly between the various probiotic strains.