## 4 Discussion  
This pilot study explored how the mastication of carrots in different shapes, sliced (CS) and grated (CG), affects the salivary secretion and proteome (proportions of salivary proteins) compared to a non-food control (parafilm). The results showed almost no changes in salivation after parafilm mastication and an increase in salivation after carrot mastication, although this was statistically significant only in the case of CG. These results indicated that chewing food (carrots) tends to induce higher increases in salivation compared to non-food (parafilm), with the grated carrot tending to induce higher salivation after mastication. Previous studies have observed similar results with a lower secretion of saliva after the mastication of non-food products (parafilm) compared to foods (e.g., celery, bread, toast, cake), with no statistical differences in salivation among food types [9; 27]; see the review in [PERSON], 2022 [1]. When looking at the changes in salivary protein profiles, it was interesting to observe that chewing the non-flavoured material (parafilm) produced almost no effects at the salivary protein profile level, with only one spot (Ig \(\alpha\)-1 chain C region protein) presenting statistically significant differences between the period before and after chewing parafilm. These results suggested that the chewing itself has minor effects on the salivary proteome. Although different studies showing the relevance of chewing force to healthy salivary gland function [28] and despite it being known that chewing results in increased salivary secretion due to activation of the masticatory-salivary reflex [29], as parafilm is an inert material, which has neither nutrients nor flavours, it appears to be less effective in stimulating the active secretion of salivary proteins that usually occurs in food consumption. The chewing of carrots, both in slices (CS) and grated (CG), induced variations in the levels of serum albumin (decrease) and amylase and Ig kappa chain C regions (increase) in saliva. Thus, chewing real foods such as carrots produced more changes in the salivary proteome than chewing parafilm. This could be explained by a higher activation of the salivary glands by the mechanical stimulation of mastication combined with sensory and nutritional stimulation from real foods, which has previously been observed to result in a higher secretion of saliva and proteins [30]. In addition to that, when comparing the differences in salivary protein levels depending on the shape of carrots (CS vs. CG), higher differences were observed for CS than for CG, as 9 protein spots changed in CG compared to 37 spots in CS due to mastication. These results indicated that chewing CS, which presented a harder texture and involved more chewing movements, affected the salivary proteome more than the consumption of CG, which could explain why more protein changes were observed in CS than in CG. A higher mastication need is well accepted to induce a higher saliva volume, with researchers such as [PERSON] (1916) and [PERSON] (1961) (both cited by [PERSON], 1999 [30]) demonstrating that receptors within the periodontal ligament were responsible for afferent information inducing salivary secretion (for more detail, please see [PERSON], 1999, and [PERSON] et al. (1999) [30; 31]). Since CS and CG are associated with similar sensory stimulation, higher mastication results in a higher amount of saliva produced, possibly also resulting in more changes at the salivary protein profile level. Among salivary proteins, increased levels of some proteoforms (molecular forms of the same protein) of amylase, Ig chains, and prolactin-inducible protein (PIP) were observed due to CS mastication. Regarding amylase, although some proteoforms of this protein increased due to the mastication of both types of carrot, a higher number of spots increased only after chewing CS. These results were not surprising, as amylase is a key enzyme in saliva involved in oral digestion, as it begins to break down carbohydrates in the mouth.