*2.7. Determination of FREE SH Content*

The free SH content of OVM samples was determined according to the method of Ellman [20] with slight modifications. Ellman's reagent was prepared according to the following procedure: first, 0.369 g of DTNB was dissolved in 50 mmol/L Na2HPO4 (pH 7.0); then, the volume was adjusted to 100 mL; the reagent was stored at 4 ◦C in the dark. The protein samples were diluted to 2 mg/mL with 0.1 mol/L phosphate buffer (pH 8.0) containing 0.086 mol/L Tris-HCl, 0.09 mol/L Gly, 4 mmol/L EDTA and 8 mol/L urea. The diluted protein solution (1 mL) was mixed with buffer solution (8 mL), and centrifuged at 8000 r/min for 20 min. Following this, 4.5 mL of supernatant was added to 0.5 mL of Ellman's reagent. The mixed solution was reacted at room temperature for 15 min after evenly oscillated. The absorption at 412 nm was measured. The free SH content was calculated according to the following formula:

$$\text{SH} \, (\text{\textquotedblleft} \text{\textquotedblright} \text{\textquotedblright} \text{\textquotedblleft} \text{\textquotedblright}) = \text{\textquotedblleft} 3.53 \times \text{A}\_{412} / \text{\textquotedblleft} \text{\textquotedblright} \text{\textquotedblright} \tag{2}$$

where 73.53 = 106/(1.36 × <sup>10</sup>4); 1.36 × <sup>10</sup><sup>4</sup> is the molar absorption coefficient of DTNB; C is the protein concentration (mg/mL).
