## 4 Discussion
The relatively increased levels of amylase in CS than in CG suggest that the food-shape characteristics have a significant influence on its secretion, and not only the composition of the chewing product (food vs. non-food). Foods that are in harder and larger portions (sliced vs. grated) and that required a higher chewing effort exhibited greater increases in the levels of amylase in the saliva. In a previous study evaluating the impact of non-food (parafilm) and food chewing (celery or bread) on salivary amylase, the authors also found a higher amylase activity in the saliva after the consumption of foods than of non-foods [27]. Among foods, they found more amylase secretion after bread than celery consumption, which was higher after mastication of larger pieces of bread than smaller ones [27], which agrees with the findings from this study. Thus, the increase in amylase levels in saliva could be explained not only by the food composition itself (e.g., the starch content), but also due to the physical characteristics of the food (shape or hardness) and the mastication force. In spite of this, and according to a previous systematic review, other authors have found a different impact of chewing on salivary amylase [15]. For instance, some authors have observed a decrease, other authors an increase, and most authors have found no effect of mastication on amylase activity [15]. Thus, no consensus has been reached about the impact of mastication on amylase activity, although these studies used different chewing materials (such as chewing gum, plastic pieces, and dental prints) that were not always food. In addition, most of these studies evaluated the total amylase activity present in saliva using diverse kits, but they did not evaluate the variations in the amylase proteoforms present in saliva, not all of which necessarily present the same enzymatic activity [32]. Indeed, these proteoforms may present slightly different structures due to modifications (e.g., glycosylation, phosphorylation) which might lead to variations in substrate affinity or catalytic activity, thus affecting their enzymatic activity efficiency [32]. Thus, it is not to be discarded that the diverse results about the impact of mastication on salivary amylase could be due to different effects that chewing may have on amylase proteoforms, influencing the total salivary amylase activity in a different way, depending on the chewing stimuli (e.g., food properties). In addition, and interestingly, the spots that increased with CS are spots with a lower molecular mass and higher isoelectric point than the native form of amylase. A previous study from [PERSON] et al. (2005) [26] used mass spectrometry to study the different protein spots of amylase separated by 2-DE. The authors observed that the spots in the gel region corresponding to the spots of amylase which increased with CS mastication are truncated forms of amylase, resulting from protease activity. The unresolved question is whether proteolysis occurs before or after secretion in the mouth. In addition to the effect that amylase modifications may have on its enzymatic activity [26], it was previously observed that different stimuli result in increases in different amylase proteoforms [32], reinforcing the different stimulation that the different types of chewing produce. The complexity of salivary-alpha amylase is increased by the high variation among individualsin the number of copies of the gene that codifies this protein (AMY1), which has been linked to human dietary evolution for starch intake [33]. On the other hand, and concerning regions of Ig kappa, they are associated with immune functions in the oral cavity. Their increase due to CS mastication could be solely due to the active chewing movements, which could reflect the activation of the immune response associated with the protection of the oral mucosa against food mastication, food particles, or potential pathogens.