Inulin and S. Salivarius Reduce Halitosis
Condition: Halitosis; Tongue Diseases
MeSH terms: Tongue Diseases; Halitosis
Study type: Interventional
Phase: Phase 2

Summary: 
      Halitosis associated with tongue coating results from the production of volatile sulfur
      compounds by bacterial action. The use of prebiotics and probiotics might be useful in
      treating such condition. The aim of this study was to evaluate the effect of the prebiotic
      inulin combined with the probiotic Streptococcus salivarius (SS) on halitosis by coating.
    

Detailed description: 
      Participants In this randomized, controlled, double-blind, parallel, phase II clinical trial,
      adult patients (≥18 years old) who complained of halitosis in the presence of tongue coating
      were invited to participate. They were identified with invitations in the form of posters
      distributed at the Dental School clinics, University of Passo Fundo (UPF), from August to
      November, 2015. The Dental School is located on the main campus of UPF,Passo Fundo, a city
      with >200.000 habitants. Oral halitosis was initially diagnosed by oral examination for
      tongue coating identification, followed by organoleptic test to confirm the halitosis. Total
      sample size was estimated in 45 participants, considering a difference of 80% in Halimeter
      reduction between treatments, for tests with α = 5% and power = 80%. Halitosis was defined by
      Halimeter values ≥ 75 ppb. All participants gave written consent before entering the study.
      The research protocol was approved by the local ethics committee (CAAE:
      40334914.0.0000.5342).

      Clinical examination Oral examination was performed by a trained dentist (CRM), identifying
      dental cavities, plaque, gingivitis, periodontal pockets and any dental prosthesis. Coating
      was described after visual inspection of the tongue and coating index was classified into 4
      levels: 0 - no coating; 1 - thin coating on 1/3; 2 - thin coating on more than 1/3 or thick
      coating on only 1/3; 3 - thick coating on more than 1/3.5

      Evaluation of halitosis After clinical examination, halitosis was quantified by organoleptic
      and Halimeter‡ tests. These were performed by a trained and calibrated examiner (CRM), with
      levels of agreement and reproducibility greater than 80% for organoleptic test and coating
      index, using test and retest comparisons with an expert judge (CKR). All patients were
      examined between 8 and 11 am. Patients were asked to avoid eating garlic, onion and other
      food condiments for two days before testing and avoid overeating margarine, milk, fry,
      sardines, salami, bologna, sausage, red meat, cheeses, foods with sulfur in the composition
      (cabbage, broccoli, cauliflower, egg), drinking alcohol and smoking, following protocols
      reported elsewhere.5, 13, 15 In the examination morning, patients should avoid using candies
      and gums, but were allowed to have breakfast and tooth brushing with water only. Once
      included in the study, patients were asked to avoid gums other than those provided in the
      trial.

      Organoleptic test Patients at rest were instructed to hold air in the mouth while breathing
      through the nose for 1 min. They blow through mouth with the examiner (CRM) positioned 10 cm
      from the patient mouth. Patient breath was rated from 0 to 5, as follows: 0 - no smell, 1 -
      poor barely perceptible odour, 2 - slight stench, 3 - moderate smelly, 4 - strong stench, and
      5 - severe stench.21 With the purpose of selecting patients, halitosis was considered present
      when this score was equal or higher than 1 in the presence of tongue coating.

      Halimeter Patients had their breath objectively measured using a Halimeter, capable of
      measuring the VSC level. Halitosis is identified when the device registers ≥75 ppb.1, 22 The
      measurements were made by inserting a disposable straw into the patient mouth above the
      tongue. According to the manufacturer, the final result was obtained as the average of three
      measurements.

      Prebiotic, probiotic and placebo Lactobacillus salivarius G60, inulin and placebo§ (composed
      of gomagron) were prepared in the form of gum‖, identical in physical appearance, odour,
      taste and consistency making the following experimental groups: LS+inulin: Lactobacillus
      salivarius G60 1 billion colony forming units (CFU) + inulin 1 g; LS: Lactobacillus
      salivarius G60 1 billion CFU; and placebo. Patients were instructed to use 1 gum every 12 h
      (after breakfast and after dinner) for 10 days.

      Quality of life assessment For oral related quality of life (QOL), we applied the
      questionnaire OHIP-14, translated and validated to Brazilian Portuguese.23 The final score
      range from 0 (best) to 56 (worst). Participants responded the questionnaires on days 0 and
      14.

      Study protocol Patients were evaluated in a dental clinic, following the steps illustrated in
      Figure 2. After signing the informed consent, clinical and quality of life data were
      recorded, followed by oral examination to assess tongue coating, and organoleptic test to
      confirm halitosis. Afterwards, the Halimeter was used to objectively measure halitosis.

      Enrolled patients were randomized by the examiner (CRM) using an envelope code that was
      computer generated, organized by the gums manufacturer in blocks for 3 patients 1:1:1, each
      containing one of the 3 treatments (LS+inulin, LS and placebo). Therefore, examiner and
      participants were blinded to treatments. Patients were instructed to use 1 gum every 12 hours
      starting on the fourth day after the interview, and interrupting 2 weeks after the first
      interview (10 days treatment). This delay of 4 days before starting the treatment allowed an
      interval of 14 days for attenuation of recall bias with OHIP-14. Each gum was placed in the
      mouth to complete dissolution, allowing gentle mastication. The final examination was
      performed in the last day of treatment, 12 hours after using the last gum, repeating coating
      index, organoleptic and Halimeter tests, and quality of life assessment (OHIP-14).
      Instructions about oral hygiene were provided by the examiner (CRM): patients were asked to
      avoid any new approach that could alter halitosis, such as tongue cleaning or changes in
      routine tooth brushing. Treatment adherence was assessed as the number of gums not used in
      relation to the total number of gums received. Safety was characterized according to side
      effects described by the patients, including headache, oral complaints and abdominal symptoms
      (yes/not). Primary outcomes were organoleptic test, Halimeter and coating index, whereas
      secondary outcomes were quality of life and treatment adherence/safety. Allocation into the
      three treatment groups was revealed only after termination of data analysis.

      Metabolism of inulin by Lactobacillus salivarius G60 The capacity of Lactobacillus salivarius
      G60 to metabolize inulin as a carbon source was tested by inoculating 50 ml of bacterial
      suspension LS (1x10⁸-⁹ cells/ml) in buffered physiological saline solution in 5 ml of
      environment half-defined by the limit of detection of the method (LDM) without glucose and
      sucrose but supplemented with inulin. The following culture mediums were prepared: PYG
      (peptone yeast + 1% glucose), PYI (peptone yeast + 1% inulin) and PY (peptone yeast). The
      latter served as negative control. The LS G60 previously cultivated was included in these
      three solutions respectively, and incubated at 30°C for 24 hours.

      Spectrophotometry (Genesys 10S VIS, made Madison, USA) analyses using 600 mm for optical
      density (OD) were performed. To measure absorbance, the solution with LS G60 was adjusted to
      OD=1, according to the formula C1.V1=C2.V2 (C1: initial concentration, V1: initial volume,
      C2: final concentration and V2: final volume). The measurements were performed at the initial
      time (zero), and after 2, 4, 6, 8, 10 and 12 hours. All tests were repeated for confirmation.

      Inhibition of pathogenic bacteria by Lactobacillus salivarius G60 Well diffusion test was
      performed. Three drops of 10 ml of bacterial suspension (1x10⁸-⁹ cells/ml of LS G60) in
      buffered saline were placed in soy triptic agar and incubated at 30°C for 3 hours under
      optimal anaerobic conditions. The plates were covered with 10 ml of soft soy triptic agar
      (0.7% agar) and inoculated with 1 ml of bacterial suspension (1x10⁸-⁹ cells/ml) in buffered
      saline of halitosis-causing pathogens (Porphyromonas gingivalis and Prevotella intermedia)
      under anaerobic conditions. They were incubated at 30°C and zones of inhibition halo of
      microbial growth around the droplets were examined at 24, 48 and 72 hours.

      Statistical analysis Data was statistically analyzed by one of the authors (SMCJ) blinded to
      the interventions. Initial comparisons among groups of patients were done using ANOVA (age)
      and chi-square (gender) tests. Treatment values on days 0 and 14 were first compared in each
      group by Wilcoxon or t tests for paired data. We employed generalized linear models (GZLM) to
      compare treatment means adjusting for pre-intervention values. The inclusion of covariable
      gender or age was decided based on Akaike´s index. Gender entered as covariable in the
      analysis of Halimeter, age was included in the models for OHIP-14, whereas no covariable was
      used when analyzing organoleptic score. We used Gama distribution and log link for the
      analyses of Halimeter and OHIP-14, as well as Poisson distribution and log link for
      organoleptic scores and coating index. The constant 1 was added to OHIP-14 values to make
      them amenable to the use of logarithms. Pairwise multiple comparisons among groups were made
      using Least Significant Difference. Results from metabolization of inulin by Lactobacillus
      salivarius G60 were analyzed using ANOVA and Tukey post hoc test. The statistical analyses
      were performed using Graph Prism 4.0 and SPSS v.18 softwares. The limit considered for
      statistical significance was 5%.
    

Intervention name(s): Inulin+SS; S salivarius; Placebo
Intervention type(s): Drug; Drug; Drug
Intervention description(s): Experimental treatment: A gum of inulin 1g plus Streptococcus salivarius 1 billion CFU per oral each 12 hours for 10 days; Active comparator: A gum of Streptococcus salivarius 1 billion CFU per oral each 12 hours for 10 days; Placebo 1 gum each 12 hours for 10 days
Other intervention name(s): Inulin plus Streptococcus salivarius; Streptococcus salivarius
Allocation: Randomized
Intervention model: Parallel Assignment
Masking: Double (Participant, Investigator)
Primary purpose: Treatment