Chromogenic Method

3.3.1. Substrate: a- or fi-Naphthyl Derivatives of Fatty Acids

In this method, the substrate naphthyl derivatives are hydrolyzed, releasing a- or p-naphthol, which is coupled with Fast Garnet GBC salt (diazotized salts) to give a colored product.

The method is as follows:

1. To 100 ^L of intracellular or extracellular fraction, add 160 ^L of 100 mM sodium phosphate buffer, pH 7.0, and 20 ^L of a- and |3-naphthyl substrate (see Note 4).

2. Mix gently and incubate at 30°C (mesophilic bacteria) or 37°C (thermophilic bacteria) for 1 h.

3. Add 0.6 mL of Fast Garnet GBC solution for color development of (red).

4. Incubate at room temperature for 15 min (see Note 5).

5. Measure the absorbance at 560 nm.

6. The concentration of color complex formed can be calculated using a standard a- or |3-naphthol curve.

One unit of enzyme activity is defined as the amount of enzyme that released 1 ^mol of a- and p-naphthol per min under the conditions of the assay.

3.3.2. Substrate: p-Nitrophenyl Derivatives of Fatty Acids

In this method, the amount of pNP released from the pNP derivatives of fatty acids by the action of LAB esterases or lipases is measured at 410 nm. The method is as follows:

1. To 200 ^L of the intracellular or extracellular fraction, add 400 ^L of 150 mM sodium phosphate buffer, pH 7.0, 775 ^L of solvent mixture, and 125 ^L of the pNP derivative substrate (see Notes 4 and 6).

2. Mix gently and incubate at 30°C (mesophilic bacteria) or 37°C (thermophilic bacteria) for 1 h.

3. Stop the reaction by addition of 175 ^L of 80% (v/v) acetic acid.

4. Centrifuge for 5 min at 13,000g.

5. Measure the release of pNP at 410 nm.

6. The concentration of pNP released can be calculated from the derived value of molar adsorption of pNP at 410 nm:

where AA410 is the experimentally observed change of absorbance at 410 nm using a 1-cm light path, and F is the dilution factor corresponding to the assay procedure. One unit of enzyme activity is defined as the amount of enzyme that released 1 ^mol of pNP per min under conditions of the assay.

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