## 3 Results and Discussion  
### Effect of HPP on Protcolytic Indexes  
The assessment of the extent of proteolysis in cheese is of interest as an index of cheese maturity and quality. For each type of cheese, the ideal ripening time is established based on the achievement of the desired texture, aroma and flavor properties. The evolution of the three proteolytic indexes of the _Serra da Estrela_ cheeses, immediately upon HPP and throughout 15 months of refrigerated storage, is shown in Figure 1. The ripening extension index (WSN/TN ratio) is typically used to follow the aging of cheese, with it being proportional to the proteolytic enzyme activity. It reflects the direct action of rennet, which is retained in the curd after manufacturing, on casein, and it consists of large to medium-sized peptides [18]. At the beginning of storage (0 months), the Ch\({}_{\text{C}}\) cheeses were characterized by a WSN/TN ratio of \(29\pm 2.0\%\). This index varies throughout ripening and all through the cheese-making season, with [PERSON] and [PERSON] (1997) [4; 19] reporting WSN/TN values of between 27 and 36% for _Serra da Estrela_ cheese at 35 days of ripening During the 15 months of refrigerated storage, this index increased steadily up to 1.5 months of storage,stabilized between 1.5 and 6 months of storage and then increased steadily again up to a final WSN/TN value of 45 \(\pm\) 1.1% at 15 months of storage. Similar values, between 23-59%, were reported for this type of cheese within 35-180 days of ripening. In terms of the HPP-treated _Serra da Estrela_ cheeses, different behaviors were observed compared to the control Ch\({}_{\text{C}}\) cheese. At the beginning of storage (0 months), immediately after the HPP treatments with 6 min of holding time, independently of the pressure intensity (P1 and P2), there were no significant changes in the WSN/TN index (\(p>\) 0.05) compared to the Ch\({}_{\text{C}}\) cheese. On the other hand, HPP treatment for a longer period of time (P3--450 MPa/9 min) caused a significant 17% increase in the corresponding WSN/TN index (\(p<\) 0.05) immediately after HPP, yet no further changes were observed over the 15 months storage of these P3 cheeses (\(p>\) 0.05). This observation may be related to the fact that a longer period of exposure (9 min) may have promoted further conformational changes in the casein matrix, making it more susceptible to proteolytic enzymes as compared to the 6 min exposure [20; 21]. In general, a deceleration of the ripening extension index was observed for all the HPP-treated cheeses during the 15 months of storage, especially for those treated under higher pressure (P1, 600 MPa) (\(p>\) 0.05). This behavior was also observed by [PERSON] et al. (2015) [9] for HPP-treated Casar cheeses (600 MPa/20 min) at 60 days of ripening and after being stored for 240 days. Application of HPP at the end of ripening (42-60 days) also led to lower proteolysis levels for Ibores and La Serena cheeses [12; 22]. Notably, the P1 cheeses kept their WSN/TN index stable during the whole storage period, with values ranging between 27-30%, i.e., values similar to those of the Ch\({}_{\text{C}}\) cheese at 0 months (29% obtained at 45 days of ripening was considered to be the best condition to generate ideal sensorial properties). Such results indicate that this HPP treatment (600 MPa/6 min), when applied to 45-days-ripened _Serra da Estrela_ cheese (0 months storage), may halt proteolysis during 15 months of storage, keeping cheese properties at ideal levels that are comparable to those of 45-day-ripened Ch\({}_{\text{C}}\) cheeses. This observation is of considerable importance, since texture, aroma and flavor are influenced by proteolytic activity. The cheese matrix is in itself a network of casein particles, which disintegrates as proteolytic enzymes take action. A lower level of proteolysis leads to a more consistent cheese, whereas a higher level of proteolysis increases the softening and meltability of cheese [9].