## 3 Results and Discussion
### Gelling Characteristics
#### 3.4.6 SDS-PAGE Protein Patterns  
Figure 4 shows the SDS-PAGE protein patterns of unwashed mince, surimi, and their gels. The predominant bands were determined to be myosin heavy chain (MHC) at 205 kDa and actin at 45 kDa. Myosin is the major protein responsible for the development of the gel network among them [61; 62; 63]. In the absence of \(\beta\)ME in all samples, some polymerized proteins populated the stacking gel in the non-gelled samples (Figure 4a), suggesting the sensitivity of mackerel muscle proteins to aggregation during handling and surimi production. Most polymerized proteins can be dissociated in the presence of \(\beta\)ME. However, a part of the protein aggregates remained in the stacking gel, suggesting the presence of other bonds, such as hydrophobic interactions. When compared to surimi, the unwashed mince revealed some protein bands below 25 kDa, indicating the existence of sarcoplasmic proteins in this sample. Surimi, on the other hand, showed no noticeable changes in MHC band intensity. Myosin is a salt-soluble protein family. As a result, it can be retained after being washed with water. When thorough washing (e.g., 4-cycle water washing) was used, however, some myosin was lost [17].  
MHC bands intensity decreased after gelation (Figure 4b) in the presence and absence of \(\beta\)ME, relative to that seen in the original unwashed mince and surimi, presumably to polymerization during heating. The reduction in MHC bands was thought to be caused by protein cross-linking during setting, which resulted in reduced band intensity [61]. The result was accompanied by an increase in gel strength (Table 2). In all of the samples, there were no discernible changes in actin bands. Actin (45 kDa) was shown to be the main protein in the gel in the current investigation, while [PERSON] and [PERSON] [63] made a similar observation, reporting that actin could not be polymerized during gelation. In general, actin may not be a TGase substrate, and the tropomyosin and troponin fractions did not contribute to the gel formation, hence their levels did not alter after the gel was formed [64]. In the absence of \(\beta\)ME, the polymerized proteins appeared and hung on the stacking gel for all gels (Figure 4b), but their intensities were reduced in the presence of \(\beta\)ME. According to the findings, all of the gels were stabilized by the disulfide bond as well as other bonds such as hydrophobic interaction.  
Figure 4: SDS-PAGE patterns before (**a**) and after gelation (**b**) of proteins from unwashed mince (U) and surimi from Indian mackerel prepared by one washing cycle (W1), two washing cycles (W2), and three washing cycles (W3). MHC, myosin heavy chain; AC, actin; M, protein markers; \(-\beta\)ME, without \(\beta\)-mercaptoethanolhanol; \(+\beta\)ME, with \(\beta\)-mercaptoethanolhanol. Samples (15 \(\mu\)g protein) were loaded onto the polyacrylamide gels comprising a 10% running gel and a 4% stacking gel, and subjected to electrophoresis at a constant current of 15 mA/gel.  
Figure 3: FTIR spectra of gels from unwashed mince (U) and surimi from Indian mackerel prepared by one washing cycle (W1), two washing cycles (W2), and three washing cycles (W3).