Saliva Collection and Processing Protocol

The NSS and SS from the 10 volunteers (five women and five men) between 25 and 67 years of age were collected in the morning between 9 and 11 a.m. and for 5 min (Figure 1). The NSS was naturally collected in 15 ml centrifuge tubes (VWR, PA, USA), while for the SS, the volunteers chewed a piece of Parafilm™ during the collection (30 (link)). After this, the saliva samples were centrifuged at 15,000 g and 4°C during 15 min (31 (link)). The supernatants from the NSS and SS were pooled into two different tubes. The pH was determined in both the saliva mixtures SS (pH = 7.7) and NSS (pH = 7.6). Then, aliquots of 1.5 ml of saliva were prepared in Eppendorf tubes (VWR, PA, USA) and were frozen (−80°C) until the experiments. This storage technique has been previously proven not to affect the saliva EA (32 (link)). Figure 1 shows a schematic summary of the experimental procedure.
The volunteers did not report oral diseases. The donors were not allowed to eat or drink anything 1 h before the saliva collection. The participants were informed about the purpose of the study, and they gave their written consent to participate. These experiments were authorized by the Bioethical Committee of the Spanish National Council of Research (CSIC), Spain.