## 3 Results and Discussion
### Protein Profile Analysis Using SDS-PAGE  
Figure 3 shows the SDS-PAGE profiles of sarcoplasmic (a) and myofibrillar (b) proteins extracted from vacuum-packed wild red deer meat treated with HHP (150-600 MPa) and a control (0 MPa) on Day 1 and Day 14 of storage at 4 \(\pm\) 1 \({}^{\circ}\)C. The molecular weight standards in Lane 1 provided a reference for identifying key protein bands from 250 kDa to 10 kDa.  
Proteins in meat, including sarcoplasmic and myofibrillar proteins, undergo significant changes during high-pressure treatment, primarily due to aggregation and denaturation [16; 35]. Sarcoplasmic proteins, which are water-soluble, include enzymes from the glycolytic pathway and myoglobin, a key pigment responsible for meat color [16]. Myoglobin is one of the most critical sarcoplasmic proteins, making its analysis essential for understanding color changes in meat. On Day 1, sarcoplasmic protein profiles remained relatively consistent across treatments up to 500 MPa, with no major visual differences in band intensity observed between the control, 150 MPa, 450 MPa, and 500 MPa sam  
Figure 3: SDS-PAGE patterns of (**a**) sarcoplasmic and (**b**) myofibrillar proteins of vacuum-packed wild red deer meat subjected to HHP treatments (150–600 MPa) and control (0 MPa) on Day 1 and Day 14 of storage at 4 \(\pm\) 1 \({}^{\circ}\)C. (Lane 1: molecular weight standard; Lane 2: control (0 MPa); Lane 3: 150 MPa; Lane 4: 200 MPa; Lane 5: 250 MPa; Lane 6: 300 MPa; Lane 7: 350 MPa; Lane 8: 400 MPa; Lane 9: 450 MPa; Lane 10: 500 MPa; Lane 11: 550 MPa; Lane 12: 600 MPa; Ald = Aldolase; GaPPh = Glyceraldehyde 3-phosphate dehydrogenase; Mk = Myokinase; Mb = Myoglobin; Tm = Tropomyosin; MHC = Myosin heavy chain; MLC = Myosin light chain).  
ples. Noticeable reductions in band intensity were more apparent at 550 and 600 MPa. High-pressure treatment induces conformational changes in myoglobin, such as denaturation of the globin component or oxidation of ferrous ions, which can result in meat discoloration [23; 35; 36]. As [PERSON] et al. [37] reported, this discoloration is closely linked to spontaneous structural changes in myoglobin.  
Interestingly, the sample treated at 250 MPa (Lane 5) on Day 1 and Day 14 exhibited an unexpectedly strong reduction in band intensity similar to the 600 MPa treatment (Lane 12). This non-linear behavior suggests that 250 MPa may represent a transitional pressure level where both pressure-induced tenderization and early stages of protein denaturation coexist. Although considered a moderate pressure, 250 MPa may still be sufficient to disrupt the solubility of heat- or pressure-sensitive sarcoplasmic proteins. This non-linear behavior highlights the complexity of protein responses under varying pressure conditions, where unfolding and solubility loss may not correlate directly with pressure intensity.  
Day 14, the degradation of sarcoplasmic proteins was more pronounced across all treatments. The control sample (Lane 2) still retained identifiable bands, but higher-pressure treatments (500-600 MPa; Lanes 10-12) exhibited marked reductions in band intensity, with the near disappearance of lower-molecular-weight proteins.  
The SDS-PAGE analysis of myofibrillar proteins in Figure 3b also demonstrated significant changes due to HHP and storage duration. The majority of myofibrillar proteins consist of myosin, which is composed of two heavy chains and three light chains. As shown in Figure 3b, the intensity of the MLC-1 band decreased at higher pressures, suggesting potential denaturation or reduced solubility as a result of high-pressure treatment. In contrast, MLC-2 remained detectable even after exposure to 600 MPa. At lower pressures (150 and 200 MPa), the intensity of myofibrillar proteins, as observed in the SDS-PAGE analysis, was comparable to that of the control. However, at pressures of 250 MPa or higher, a noticeable reduction in protein intensity was observed. This decline in intensity is attributed to protein aggregation caused by the high-pressure treatment.