Distilled water (dH2O). Centrifuge. Whatman no. 5 paper.
Sterifilter system (Millipore); 0.22-^m membrane filter.
Plate count agar (PCA).
1. de Man-Rogosa-Sharpe (MRS) broth culture medium for lactic acid bacteria: 10 g/L peptone, 10 g/L meat extract, 5 g/L yeast extract, 20 g/L glucose, 5 g/L sodium acetate, 1.08 g/L Tween-80, 2 g/L ammonium citrate, 2 g/L K2HPO4, MgSO4-7H2O (0.2g/L), 0.05 g/L MnSO4-4H2O, pH 6.5. Sterilize in an autoclave at 121°C for 15 min.
2. MC broth culture medium for Staphylococcus sp.: 10 g/L meat extract, 5 g/L yeast extract, 5 g/L NaCL, 1 g/L Na2HPO4, pH 7.0. Sterilize in an autoclave at 121°C for 15 min.
3. Baird-Parker broth culture medium for Kocuria sp.: 10 g/L peptone, 5 g/L meat extract, 1 g/ L yeast extract, 10 g/L sodium pyruvate, 12 g/L glycine, 5 g/L lithium chloride. Sterilize in an autoclave at 121°C for 15 min.
2.3. Ortho-Phthaldialdehyde Method (OPA)
a. Solution A (0.2 M NaH2PO4): Weigh 27.6 g NaH2PO4-H2O and make up to 100 mL with dH2O.
b. Solution B (0.2 MNa2HPO4): Weigh 53.05 g Na2HPO4-7H2O and make up to 100 mL with dH2O.
c. To prepare 1 L of 0.1 M phosphate buffer, pH 7.0, mix 195 mL solution A and 305 mL solution B and bring to 1000 mL with dH2O.
2. Sodium dodecyl sulfate (SDS; 20% w/v): Dissolve 20 g SDS in 100 mL dH2O with gentle stirring. Store at room temperature (see Note 1).
3. 100 mM sodium tetraborate (borax): weigh 3.81 g of Na2B4O7-10H2O and make up to 100 mL with dH2O. This solution is stable for at least 1 mo at 4°C.
4. OPA solution (working reagent): mix 25 mL of 100 mM borax, 2.5 mL 20% SDS, 40 mg OPA (dissolved in 1 mL methanol), and 100 ^L |3-mercaptoethanol. Dilute to a final 50 mL with dH2O. This reagent must be prepared daily.
5. 0.75 NTrichloroacetic acid (TCA): weigh 61.24 g TCA and dilute to 500 mL with dH2O. The solution is stable for at least 2 wk when stored at 4°C.
3.1. Soluble Muscle Extract
1. Weigh 10 g of lean muscle in a stomacher bag (see Note 2).
2. Add 90 mL of 20 mM phosphate buffer, pH 7.
3. Homogenize in a stomacher 400 blender for 3 min.
4. Centrifuge the protein solution (12,000g at 4°C for 20 min).
7. Filter the supernatant containing the proteins through Whatman paper.
8. Filter-sterilize this solution by using a 250-mL capacity filter (Bio-Rad) with a vacuum pump (see Note 4).
9. Add 0.1% Tween-80, previously sterilized (see Note 5).
3.1.1. Sterility Control
1. To quantify the total aerobic organisms, inoculate them in duplicate Petri dishes with 0.5 mL of protein extract.
2. Add 10 mL of the melted PCA and homogenize appropriately.
3. The growth of microorganisms must be negligible, with colony-forming units (CFU) below 1 x 102 CFU/mL.
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