## 3 Results and Discussion
### Bio-Physico-Chemical Properties
#### 3.2.3 Myoglobin Content, Derivatives, and Absorption Spectra in the Soret Region  
Myoglobin is a heme protein that is responsible for the quality of fish muscle, particularly for the development of color and rancidity. Surimi quality factors (e.g., whiteness, lipid oxidation, microbial growth, and shelf life) are all affected by the occurrence of residual heme proteins in surimi, especially dark-fleshed fish [21; 22]. Table 1 shows the myoglobin content and myoglobin derivatives of unwashed mackerel mince and surimi produced using various washing cycles. Myoglobin content in unwashed mince was 6.77 mg/g sample, whereas it was 2.56, 1.63, and 1.61 mg/g sample in surimi after washing with W1, W2, and W3. The W2 and W3 samples had the same residual myoglobin levels (\(p>0.05\)). Sarooplasmic proteins, such as myoglobin, can be dissolved in water, and be washed out of the muscle during water washing [9]. By washing minced fish in cold water, most sarcoplasmic proteins, including myoglobin, hemoglobin, and other undesired compounds, may be eliminated, and the whiteness of surimi can be improved [23]. Parts of myoglobin, on the other hand, have the ability to interact with myofibrillar proteins [7]. The interaction of myoglobin with muscle constituents, such as myofibrillar proteins and/or membranes, can result in impaired extractability, according to [PERSON] and [PERSON] [22]. As a result, increasing the number of washing cycles from two to three did not result in a greater capacity for myoglobin removal.  
Deoxymyoglobin (purple red), oxymyoglobin (bright red), and metmyoglobin (tan or brown) are the three major forms of myoglobin, which differ in their exposure to oxygen and the chemical state of iron. W3 sample had the highest amount of deoxymyoglobin, followed by W2 and W1, while unwashed mince had the lowest (\(p<0.05\)). In all of the samples, there was no significant variation in the proportion of oxymyoglobin (\(p>0.05\)). All of the samples contained a high percentage of oxidized metmyoglobin (\(\sim\)73-76%), which was quite likely to happen during sample handling, processing, and storage. However, increasing the number of washing cycle up to two cycles can lower metmyoglobin levels. Increasing the number of washing cycles can prevent the deoxymyoglobin form of surimi from converting to metmyoglobin, resulting in a delay in lipid oxidation and a reduction in TBARS value (see Section 3.4.4). Deoxymyoglobin and oxymyoglobin can both be oxidized to metmyoglobin. The conversion of reduced myoglobin to oxidized forms resulted in a greyish-brown color in fish muscle, which was related to lipid oxidation [24]. Both a superoxide anion and a hydrogen peroxide are formed during the oxidation of oxymyoglobin, which then react with iron to produce hydroxyl radicals. These hydroxyl radicals have the capacity to permeate the muscle's hydrophobic lipid area, facilitating lipid oxidation [22; 25].  
The absorption spectra in the Soret region (380-450 nm) of myoglobin extracted from mackerel unwashed mince and surimi are depicted in Figure 1. The content and oxidation state of the myoglobin can be observed by absorption bands in the ultraviolet and visible wavelengths. [PERSON] and [PERSON] [26] stated that the heme is responsible for the intense peak at about 410 nm. The peak in the Soret band of all samples was identified at 407 nm, according to the results. Metmyoglobin predominance was revealed by the blue shift from 410 nm to 407 nm. This result confirmed the metmyoglobin content shown in Table 1. During iced storage, [PERSON] et al. [25] observed a shift from 410 nm to 408 nm in oxeye scad (Selar boops) muscle, which was associated with a substantial rise in metmyoglobin concentration. The sample that had not been washed exhibited the highest Soret peak. Surimi made by increasing the number of washing cycles, especially W2 and W3, showed a decrease in Soret peak, demonstrating the removal effectiveness of myoglobin from minced fish.