## 3 Results and Discussion
### Effect of HPP on the Free Amino Acids Profile of Beef During Storage  
Beef provides significant amounts of nine essential amino acids (EAAs) such as threonine, methionine, valine, isoleucine, leucine, phenylalanine, lysine, histidine, and tryptophan [20; 38]. Therefore, this discussion is focused on changes in the profile of these amino acids, presented in Table 2.  
In our study, for all nine analyzed EAAs, an effect (\(p\leq 0.05\)) of HPP treatment and storage time was observed (Table 2). Compared to the control meat, the application of HPP reduced the content of most of the analyzed free amino acids (except leucine, phenylalanine, and lysine), and exposure to higher pressure caused greater losses of given amino acids. However, the dynamics of changes in the content of individual amino acids varied. In most cases, the increase in content of free amino acids during beef storage was higher for the control and HPP300 meat, and lower for the HPP400 and HPP500 meat. Depending on the HPP treatment and storage time, changes in the amino acid content can be pronounced by residual microbiological activity. For example, in the study by [PERSON] et al. [31], the higher the applied pressure, the slower the growth of bacteria, mainly Enterobacteriaceae and lactic acid bacteria, in skin-packed beef. In untreated meat, the rate of microbial growth was significantly higher than in HPP meat, which may influence changes in the amino acid content. Moreover, [PERSON] et al. [39] and [PERSON] et al. [40] reported that the consequences of protein oxidation caused by HPP could lead to the depletion of essential nutrients like amino acids, reducing their bioavailability and reduced protein digestibility. The threonine content in beef at the beginning of storage (day 1) was 45.76-55.22 mg/kg, while at the end of storage time it ranged from 75.26 to 262.31 mg/kg. The HPP had an effect (\(p\leq 0.05\)) on the threonine content. Differences (\(p\leq 0.05\)) between the control and HPP-treated samples were already visible on day 1 of storage. Significant losses of this amino acid were already observed at the lowest pressure of 300 MPa, compared to the control meat. From day 14 of storage, it was observed that exposure to higher pressures resulted in greater losses in this amino acid content compared to the control meat. In each of the analyzed storage days, the HPP500 meat had the lowest (\(p\leq 0.05\)) threonine content, and it was even 2-3-fold lower than that observed in the control (75.26 mg/kg vs. 262.31 mg/kg on day 56, respectively). [PERSON] et al. [4] proved that the HPP treatment changes the amounts of free amino acids, including threonine, and the content of free amino acids was reduced by applying a higher pressure, such as 500 MPa. This may be due to the reduced protein extraction after the pressure treatment at 500 MPa caused by the changes in its structure, as stated by [PERSON] et al. [41]. A significant (\(p\leq 0.05\)) effect of the storage time on the threonine content in beef was found (Table 2). An increase (\(p\leq 0.05\)) in the threonine content was observed from the 7 th day of storage compared to 1 st day, regardless of the treatment. During the meat storage, this gradual increase continued, but with different dynamics. On day 56 of storage, the threonine content for the control meat was almost 5-fold higher than on day 1 (262.31 vs. 55.22 mg/kg), for the HPP300 meat it was over 4-fold higher, and for the HPP400 meat it was approximately 2.5-fold higher. After the application of the 500 MPa pressure, the observed increase in the threonine content during storage was the slowest. Between days 14 and 56, the threonine content in HPP500 meat ranged from 70.32 to 75.26 mg/kg (Table 2). Also, [PERSON] et al. [42] found an increase in the threonine content in beef during storage. The gradual release of amino acids during storage is typical for meat proteolysis during aging, as beef in skin packages ages. The increase in amino acids is proportional to the aging time and starts after 14 days of storage [43; 44].