## 2 Materials and Methods
### Determination of Bio-Physico-Chemical Properties of Unwashed Mince and Surimi  
The pH, Ca\({}^{2+}\)-ATPase activity, SH content, protein surface hydrophobicity, and TCA-soluble peptides were determined using the standard methods as described by [PERSON] et al. [9].  
The pH was obtained using a pH meter (Cyberscan 500, Singapore) after homogenizing the sample with 10 vol of deionized water (\(w/v\)), using an IKA Labortechnik homogenizer (Selangor, Malaysia).  
Ca\({}^{2+}\)-ATPase activity, SH content, and protein surface hydrophobicity were measured employing natural actomyosin (NAM) from unwashed mince and surimi. For the Ca\({}^{2+}\)-ATPase activity, NAM was diluted to 2.5-8 mg/mL with 0.6 M KCl (pH 7.0). A measure of 1 mL NAM solution was combined with 0.6 mL 0.5 M Tris-maleate (pH 7.0) and 1 mL 0.1 M CaCl\({}_{2}\). To make up a total volume of 9.5 mL, deionized water was added. Then, a 0.5 mL solution of 20 mM ATP was added. After an 8-min incubation at 25 \({}^{\circ}\)C, the reaction was stopped by adding 5 mL cold 15% (\(w/v\)) TCA. After that, centrifugation (3500\(\times\)\(g\)/5 min/25 \({}^{\circ}\)C) was performed using a RC-5B plus centrifuge (Sorvall, Norwalk, CT, USA), and the inorganic phosphate liberated in the supernatant was quantified. The activity of the Ca\({}^{2+}\)-ATPase was measured in moles of inorganic phosphate released/mg protein/min. A blank solution was prepared by adding chilled TCA prior to addition of ATP. For the SH content, the NAM sample (0.5 mL, 4 mg/mL) was combined with 4.5 mL of 0.2 M Tris-HCl buffer (pH 6.8) and 0.5 mL of 0.1% DTNB solution in a total volume of 7.5 mL. The absorbance was measured at 412 nm using a Shimadzu UV-2100 spectrophotometer (Shimadzu Scientific Instruments Inc., Columbia, MD, USA) after a 25-min incubation at 40 \({}^{\circ}\)C. By substituting 0.6 M KCl (pH 7.0) for the sample, a blank was created. The absorbance was used to calculate the SH content, which was reported as mol/108 g protein, using the molar extinction of 13,600 M\({}^{-1}\)cm\({}^{-1}\). The hydrophobicity of NAM dispersed in 20 mM phosphate buffer (pH 6) was measured by bromophenol blue sodium salt (BPB) for electrophoresis. To 1 mL of NAM suspension, 200 \(\upmu\)L of 1 mg/mL BPB (in distilled water) was added, and mixed well. A control group was created without NAM. For 10 min, samples and controls were agitated at room temperature (26-28 \({}^{\circ}\)C). The absorbance of the supernatant was measured at 595 nm (A) using a Shimadzu UV-2100 spectrophotometer against a blank of phosphate buffer after centrifugation (2000\(\times\)\(g\)/15 min/room temperature). The following formula was used to calculate the amount of BPB bound:  
\[\text{BPB bound}\ (\upmu\text{g})=200\ \upmu\text{g}\ \times\left[\frac{\text{A}_{ \text{control}}-\ \text{A}_{\text{sample}}}{\text{A}_{\text{control}}}\right] \tag{1}\]  
For the TCA-soluble peptide, the sample was homogenized with 3 vol of 5% (\(w/v\)) TCA before being placed on ice for 1 h. The soluble peptides in the supernatant were measured and recorded as \(\upmu\)mole tyrosine/g sample after centrifugation at 5000\(\times\)\(g\) for 5 min.  
Myoglobin was extracted using a cold 40 mM phosphate buffer, pH 6.8, according to [PERSON] et al. [6]. The spectra in the visible and Soret regions were recorded using a Shimadzu UV-2100 spectrophotometer (Columbia, MD, USA). The following equations were used to calculate the proportions of the three myoglobin variants [12].  
\[\text{[Deoxymyoglobin]}=-0.543\text{P1}+1.594\text{P2}+0.552\text{P3}-1.329 \tag{2}\]  
\[\text{[Oxymyoglobin]}=0.722\text{P1}-1.432\text{P2}-1.659\text{P3}+2.599 \tag{3}\][Metmyoglobin] = \(-0.159\)P1 \(-\) 0.085P2 + 1.262P3 \(-\) 0.520  
where P1 = A582/A525, P2 = A557/A525, and P3 = A503/A525.  
[PERSON]'s method [13] was used to extract lipids. The percentage of lipid reduction was calculated in comparison to the unwashed minec's initial lipid content.