Proteolytic Activity of Lactic Acid Bacteria in the Soluble Muscle Extract

3.2.1. Culture Conditions of the Strains

1. Propagate Lactobacillus and Staphylococcus/Kocuria sp. strains in the specific media and incubate at 30°C.

2. Harvest the cells at logarithmic phase (overnight culture) and wash twice with 20 mM phosphate buffer (pH 7.0).

3.2.2. Growth in the Soluble Muscle Extract

1. Inoculate 30 mL of the soluble muscle extract medium with an overnight culture to yield an initial number of 105 CFU/mL corresponding to an OD680 of 0.15 (see Note 6).

3. Take samples every 24 h for pH, bacterial growth, and proteolytic activity analysis.

4. Measure the pH values with a pH meter using 2 mL of the sample.

5. Determine bacterial cell counts using 0.1% peptone water as the diluent.

6. Plate 0.5 mL of each dilution in duplicate in Petri dishes adding the medium corresponding to each organism.

3.2.3. OPA Spectrophotometric Assay

1. Add 500 ^L of 0.75 N TCA to 250 ^L of homogenized sample.

2. Vortex vigorously and allow to stand for 15 min at 4°C.

3. Centrifuge the solution at 13,000g for 10 min and transfer the supernatant to a clean Eppendorf tube with a micropipet (see Note 7).

4. Add 1 mL of OPA reagent to 50 ^L supernatant in a 1-mL quartz cuvet.

5. Mix briefly by inversion and incubate for 5 min at room temperature.

6. Measure the absorbance at 340 nm against the blank (sterile soluble muscle extract).

7. Calculate the millimoles per liter of the a-amino acid released (mM) from the following relationship: mM = eAA340F, where AA340 is the experimentally observed change of absorbance at 340 nm using a 1-cm light path, F is the dilution factor corresponding to the assay procedure, and e is the molar absorption coefficient (6000 M/cm).

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