Stimulated human saliva collection
Clarified stimulated human saliva was used in the present study. The saliva was collected from adult healthy donors, aged 20–60 years, from both genders, who were informed not to eat or drink (except water) for at least 60 min before collection. Donors who smoked or used fixed orthodontic devices were excluded from the collection. The local ethical committee (KEK) considers pooled samples saliva as irreversibly anonymized samples, so prior ethical approval was not required. The saliva collection was performed in the morning, always at the same time. Fifteen donors chewed on paraffin (Paraffin pellets, Ivoclar Vivadent) for 10 min and collect saliva into ice-chilled vials. The stimulated saliva from all donors was immediately pooled, centrifuged (3’000 g, 15 min, 4°C) and the supernatant fraction was collected.To avoid degradation of the salivary proteins, protease inhibitors (phenylmethanesulfonyl fluoride; n-ethylmaleimide; 1,10-phenanthroline monohydrate) were added to the previously prepared human saliva. These inhibitors were initially dissolved in methanol (400 mM) and then compounded till a final concentration of 100 mM per inhibitor. The solution containing all inhibitors was added to the clarified human saliva (1:100). Then, aliquots were prepared and stored at -80°C until the beginning of the experiment.