## 3 Results and Discussion
### Effect of HPP on Protcolytic Indexes
Given the lower ripening extension index by 15 months of storage, the HPP P1 cheeses were in fact firmer and harder compared to Ch\({}_{\text{C}}\). This was confirmed by both instrumental (TPA profile) and sensorial analysis by the trained panel (see further details in Sections 3.3 and 3.4, respectively). Figure 1: _Cont._  
A significant progressive increase in the value of TCA/TN, understood as a ripening depth index (\(p<0.001\)), was observed for all cheeses during the 15 months of refrigerated storage (Figure 1A); an intermediate plateau similar to that previously observed for WSN/TN was also registered. This proteolysis index expresses the presence of medium- and small-sized peptides (with a chain length between 2 and 20 amino acids residues) and free amino acids, which may result from the strong proteolytic action of _C. cardunculus_ extract [6; 23] but is mainly derived from peptidase activity from viable or lysed lactic acid bacteria [4; 5; 7] and/or from psychotrophic bacteria [19]. In this study, the viable cell numbers of psychotropic bacteria were higher in the Ch\({}_{\text{C}}\) and P2 and P3 cheeses compared to the P1 cheeses [24]. The obtained TCA/TN values are in agreement with other reported results; [PERSON] and [PERSON] (1997) [19] reported a TCA/TN value of 5.5-6.2% at 35 days of ripening for _Serra da Estrela_ cheeses, [PERSON] et al. (2003) [6] reported values of 7-16% at 35-180 days of ripening, while [PERSON] and [PERSON] (2011) [7] reported values of 3.7% at 60 days of ripening. In general, there were no significant differences in the TCA/TN indexes between the Ch\({}_{\text{C}}\) cheeses and the HPP P2 and P3 cheeses (450 MPa/6 and 9 min, respectively). On the other hand, significantly lower TCA/TN indexes were obtained for the HPP P1 cheeses treated at 600 MPa/6 min than for the Ch\({}_{\text{C}}\) cheeses (\(p<0.001\)) over the whole storage period. This observation may be consequence of a reduced microbial peptidase activity that could be linked with the reduction in microbial viable cell numbers caused by HPP, as observed in a previous work [24]. This effect was also observed in HPP-treated Casar chees (600 MPa/20 min) at 60 days of ripening and after being stored for 240 days, with [PERSON] et al. (2015) [9] suggested that HPP at 600 MPa led to a reduction in the production of medium to small-sized peptides. On the other hand, [PERSON], [PERSON], [PERSON] and [PERSON] (2012) [25] observed an increase in TCA/TN in l  
Figure 1: (**A**) Ripening extension index (WSN/TN), (**B**) ripening depth index (TCA/TN) and (**C**) free amino acid index (PTA/TN) of Serra da Estrela cheese at 45, 135, 225 and 500 days of manufacture for non-processed chees (\(\blacklozenge\), Ch\({}_{\text{C}}\)) and HPP-treated cheeses (\(\blacksquare\) P1, \(\blacklozenge\) P2 and \(\blacklozenge\) Y3). -milk) HPP-treated (400 and 600 MPa/7 min) at 50 days of ripening compared to control cheeses, possibly due to the intracellular release of proteinases/peptidases [25]. These results seem to demonstrate that high-pressure treatments may accelerate or decelerate cheese proteolysis based on the cheese type and the pressure conditions used [26]. The PTA/TN ratio, used as a free amino acid (FAA) index, is related to the final product of proteolysis, the FAA and very small peptides (containing less than six amino acid residues). Once again, the PTA/TN ratio significantly (\(p<0.05\)) increased progressively during refrigerated storage for all the non-treated and HPP-treated cheeses (Figure 1C). In the case of the Ch\({}_{\rm C}\) cheeses, the values of PTA/TN increased almost 4-fold, going from 1.4 \(\pm\) 0.2 at 0 months storage to 5.4 \(\pm\) 0.1 at 15 months of storage. These values are within the range previously reported by [PERSON] et al. (2003) [6], i.e., 3-12% at 60-180 days of ripening, and they are also in line with the increase from 0.56 to 2.6% during the first 60 days of ripening reported by [PERSON] and [PERSON] (2011) [7] for the same type of cheese.