Evaluating the Impact of Various Dietary Nitrate Supplements on Oral Health

Prior to and following the intervention, participants will provide an unstimulated saliva sample for pH measurement. The pH of each saliva sample will be measured using a pre-calibrated pH meter (±0.01 pH accuracy). Measurements will be performed in three independent replicates for each sample, with the three values recorded and then averaged. This average is used for the final pH value of the sample, helping to reduce the influence of random errors and improve the accuracy and reliability of the results. The device is recalibrated prior to measurements using standard calibration solutions to ensure accurate readings.

Prior to and following the intervention, participants will provide unstimulated saliva and tongue swab samples. DNA will be extracted from both the saliva and tongue swab samples, and 16S rRNA sequencing will be performed to identify and quantify changes in the oral microbiome.

Prior to and following the intervention, participants will provide an unstimulated saliva sample to determine saliva nitrate and nitrite levels using ozone-based chemiluminescence. Home-collected saliva samples will also be analysed for three consecutive days to ensure compliance.

Participants will provide an unstimulated saliva sample prior to and following the intervention. The saliva volume collected (in mL) will be divided by the time taken (in min) to obtain the flow rate in ml/min.

Participants will provide an unstimulated saliva sample prior to and following the intervention. If sufficient sample volume is available, exploratory analysis of salivary biomarkers related to inflammation may be considered.

The impact of the study interventions on dry mouth symptom severity pre- and post-intervention will be assessed using the Summated Xerostomia Inventory-Dutch Version (SXI-D) questionnaire (Thomson et al., 2011), which has been validated in clinical and epidemiological research. Participants will provide information about their oral symptoms over the previous four weeks (e.g., feeling dry mouth or difficulty eating dry foods). Participants will be asked to select one of three options, each of which is associated with a score (Never (1); Occasionally (2) and Often, (3)). The SXI-D score range is 5-15, with higher scores indicating more severe dry mouth.

Participants will provide information on their oral hygiene behaviours before and after the intervention using the Oral Hygiene Behaviour Index questionnaire (Buunk-Werkhoven et al., 2011). The score ranges from 0 to 16, with a high total score indicating a high level of oral hygiene practices.

Before and after the intervention, participants will provide information about their intake of nitrate containing foods through a validated digital food frequency questionnaire(McMahon et al., 2023).

The acceptability of the interventions will be assessed after the study is completed through a questionnaire designed to measure acceptability dimensions according to the Theoretical Framework of Acceptability (Sekhon et al., 2017), using a 5-point Likert scale to assess different aspects such as overall acceptability, palatability, and ease of use.

Compliance will be assessed using a participant-completed compliance log over a 4-week period. Participants will be asked to record:

• The date and time of supplement intake.
• How it was consumed. Additionally, any unused supplement sachets/bottles will be collected at the final visit to help estimate adherence.