## 3 Results and Discussion
### Effect of HPP on the Free Amino Acids Profile of Beef During Storage
[46], the valine content in beef increased 6-fold during storage. According to [PERSON] et al. [44] the greatest increases during beef storage were observed for the amino acids serine, threonine, leucine, isoleucine, methionine, valine, and tryptophan. The content of isoleucine in beef on day 1 was 34.21-37.40 mg/kg, and on day 56 it ranged from 129.57 to 270.23 mg/kg. The content of isoleucine on day 1 was not differentiated (\(p>0.05\)) by the pressure used (Table 2). A significant decrease in the isoleucine content compared to the control meat was already observed from the 7 th day of storage in the case of the HPP500 meat, from 21 st day in the case of the HPP400 meat, and from 28 th day in the case of the HPP300 meat. From the 21 st day of storage, the content of isoleucine in the HPP300 meat was significantly higher compared to the meat treated by 400 and 500 MPa. On the last day, the isoleucine content in the HPP500 beef was more than 2-fold lower than in the control meat (129.57 vs. 270.23 mg/kg, respectively). Similarly, [PERSON] et al. [1] observed a significant reduction in the content of essential amino acids, i.e., leucine and isoleucine, in lamb meat subjected to HPP treatment at a pressure of 600 MPa. According to [PERSON] et al. [7], muscle proteins are easily subjected to oxidation processes that cause the losses of EAAs. Protein oxidation usually occurred at pressure values similar to those at which lipid oxidation occurred (>300 MPa). This is in agreement with our studies, as stronger lipid oxidation occurred at higher HPP levels (400 and 500 MPa, Table 1). An effect (\(p\leq 0.05\)) of storage time on the isoleucine content of beef was observed. The growth dynamics, similar to the dynamics described for other amino acids, varied and were slower when higher pressure was applied. For the control meat, an approximately 8-fold increase in the isoleucine content was observed, from 36.49 (day 1) to 270.23 mg/kg (day 56), while in the HPP500 meat, an approximately 4-fold increase was observed, from 34.21 (day 1) to 129.57 mg/kg (day 56). [PERSON] et al. [4] and [PERSON] et al. [42] also observed an increase in the isoleucine content in beef. In the beef treated with HPP 500 MPa (20 \({}^{\circ}\)C, 5 min), the increase in the isoleucine content after 14 days of refrigerated storage was smaller than that for the control meat [4]. [PERSON] et al. [44] found that phenylalanine, methionine, lysine, leucine, and isoleucine were the amino acids for which the content increased the most with the extension of the beef conditioning time. The leucine content on day 1 was 55.34-68.41 mg/kg, and at the end of the storage time of skin-packed beef, it ranged from 256.46 to 551.66 mg/kg. From day 7 to day 21, the HPP-treated meat was characterized by a significantly (\(p\leq 0.05\)) lower content of this amino acid compared to the control meat. Starting from day 21, the HPP500 meat had the lowest (\(p\leq 0.05\)) leucine content (Table 2). Moreover, it was observed that from day 28 of storage up to day 56, the leucine content in the HPP300 meat was higher or significantly higher than in the control, HPP400, and HPP500 meat. Considering that HPP reduces aminopeptidase activity, a reduced leucine concentration was expected in all pressurized samples [20; 45]. However, taking into account that HPP can inactivate microorganisms [47; 48], a lower concentration of leucine in the control than in the HPP300 samples could be a result of some degradation reactions catalyzed by endogenous bacteria proteases, which occurred in the unpressurized (control) meat. The ability of leucine catabolism is frequent among bacteria, and leucine is a widely known precursor of flavor compounds [48; 49]. Additionally, it can be used by microorganisms as an alternative source of carbon or nitrogen in the case of a deficiency of these elements in the habitat [49]. The storage time affected the leucine content in beef (\(p\leq 0.05\)). The highest leucine content was found on the 56 th day of beef storage (Table 2).