4.1. Study Participant and Saliva Sample Collection

This pilot study included five participants, aged 24–38 year (32.4 ± 2.7) from Oral Health Center, School of Dentistry, The University of Queensland, Australia. Periodontal charting was performed for each participant to determine their periodontal pocket depth (PPD) and bleeding on probing (BOP). This study was approved by the University of Queensland Human Ethics Research Committee (HREC No. 2018001225) and informed consent was obtained from all participants. All participants were non-smokers, with no underlying systemic diseases (details shown in Table 1).

Participants refrained from eating and drinking for at least one hour prior to saliva sample collection. Prior to sample collection, the participants were asked to rinse their mouth with ~10 mL of water to remove the food debris. Whole unstimulated saliva samples were collected from each participant using two different methods: spitting and drooling [22,23], with a 30-min interval on the same day between the two collections. Spitting samples were collected by asking participants to spit saliva directly into a 50 mL sterile falcon tube (Sarstedt, Mawson Lakes, Australia); drooling samples were collected 30 min later by asking participants to pool saliva in the mouth (2–5 min) before drooling into a sterile 50 mL falcon tube. Saliva samples were collected within a 2–3 min timeframe for a volume of 1–3 mL. Fresh saliva samples were processed for downstream analysis. The saliva flow rate was calculated as flow rate = saliva volume/collection time. The salivary density was calculated as total protein (μg/mL) × flow rate (mL/min) as a protein secretion rate (total protein output per unit of time).

Participants were asked to respond to the question ‘The saliva sample collection procedure was comfortable’ immediately after each saliva collection procedure, by marking a visual analogue scale (VAS) from 0 (disagree) to 9 (agree).