## 3 Results and Discussion
### Effect of HPP on Aminopeptidase Activity  
HPP is capable of inactivating microorganisms, but it can also affect enzyme activity due to their (in)activation or to changes in their substrates' conformation [21]. It is well established that aminopeptidase activity contributes significantly to proteolysis in cheese [27], and so its activity was quantified on both Lys-p-NA and Leu-p-NA substrates, and the results are listed in Table 1. The activity decreased during the refrigerated storage for both substrates and for all cheeses, with the exception of the P1 cheeses, which showed a significant increment in the activity on Leu-p-NA from 0 to 6 months of storage (\(p<0.001\)). Aminopeptidase activity on Lys-p-NA was significantly higher (\(p<0.001\)) in the P1 (17% higher) and P2 (+59%) chees at 0 months than in the Ch\({}_{\rm C}\) cheeses. A similar behavior was observed throughout the 15 months of storage, although no significant activity variation was observed during the last 9 months of storage, for all the cheeses. On the other hand, all the cheeses registered a similar activity for Leu-p-NA at 0 months (\(p>0.05\)); however, during the 15 months of refrigerated storage, the associated aminopeptidase activity decreased significantly (\(p<0.05\)) in the Ch\({}_{\rm C}\), P2 and P3 cheeses. It is important to note that aminopeptides contribute toward the ripening depth index, taking into account the amount of nitrogen in the TCA fraction per TN. The TCA fraction allows for the quantification of small peptides containing between 2 and 20 amino acid residues and free amino acids resulting from secondary proteolysis, which is brought about by the enzymes produced by the starter cultures and released thereby upon lysis [28]. [PERSON] et al. (2000) [29] suggested that HPP treatment could release intracellular enzymes due to incremented cell membrane permeability and microbial cells lysis, favoring the release of intracellular material, including peptidases. In general, an inverse relationship was observed between the proteolytic indexes and aminopeptidase activity during storage; as the values of the proteolytic indexes increased, the aminopeptidase activity decreased. Thus, aminopeptidase activity is not an adequate indicator of proteolysis in _Serra da Estrela_ cheese. Indeed, other proteolytic enzymes will have their activities increased during storage and/or HPP, and storage may increase the proteolytic susceptibility of proteins. For instance, changes in cheese casein conformation or casein aggregation were reported to occur during HPP [12]. A higher aminopeptidase activity in HPP-treated cheeses (400 MPa/10 min) was also observed for La Serena cheese treated at 50 days of ripening and analyzed 10 days later, even though a similar level of proteolysis was quantified [12]. Furthermore, [PERSON] et al. (2007) [30] also observed this tendency in HPP-treated ewe's cheese during ripening; the authors tested two HPP treatments, i.e., 400 MPa and 500 MPa for 10 min each, and although some aminopeptidase inactivation was observed at the higher HPP treatment (500 MPa/10 min), both HPP-treated cheeses revealed similar WSN contents compared to a control.