Screening of Hydrogen Peroxide Generating Lactic Acid Bacteria

3.2.1. Qualitative Method on Plate

1. Solution A was prepared with 12.5 mg of TMB (see Note 3) and 3 mL of methanol (see Note 4; 17.34 mM TMB), with shaking to complete dissolution.

2. Solution B was prepared with 0.5 mg of peroxidase and 1 mL sterilized distilled water (0.5 mg/mL or 100 U/mL peroxidase; see Note 5).

3. Preparation of TMB-agar plates: MRS medium was melted at 45°C. Then 10 mL of MRS agar were mixed with a 0.6-mL aliquot of solution A and a 0.2-mL aliquot of solution B. This mixture was added to plates and allowed to solidify (see Note 6). The final concentrations were: 1 mM TMB and 10 ^g/mL (2 U/mL) peroxidase.

4. Assay: The lactobacilli, grown as described in Subheading 3.1.2., step 1, were spread over the surface of TMB agar plates. The plates were then incubated microaerophically at 37°C, for 48 h. After 48 h, plates were exposed to air. Colonies able to produce hydrogen peroxide turn blue or brown (see Notes 7-9).

3.2.2. Quantitative Method: Spectrophotometric Method Modified by Using o-Dianisidine Horseradish Peroxidase

3.2.2.1. Preparation of Reagent Solution

1. Solution C was prepared with 1 mg of o-dianisidine (see Note 10) per mL of 0.4% Triton X 100 (3.2 mM o-dianisidine).

2. Solution D was prepared with 120 ^g of peroxidase per mL of 10 mM phosphate buffer (120 ^g/mL or 24 U/mL peroxidase; see Note 11).

3. The reagent solution (20 mL) was prepared by using: a 1-mL aliquot of solution C, a 0.3 mL aliquot of solution D, 0.2 mL of 0.4% Triton X-100, and 18.5 mL of 10 mM phosphate buffer (pH 7.4). The final concentrations were: 0.16 mM o-dianisidine and 1.8 ^g/mL (0.36 U/mL) peroxidase (see Notes 12 and 13).

3.2.2.2. Standard Curve for the Quantification of H2O2

1. An H2O2 solution (8.8 mM) was prepared with 5 ^L of 30% H2O2 solution filled up to 5 mL with distilled water (see Note 14). The solutions with different H2O2 concentrations (from 0.00176 to 2.64 mM) were prepared from 8.8 mM H2O2 solution (see Note 15).

2. An 0.8-mL aliquot of reagent solution was mixed with a 0.2-mL aliquot of each H2O2 concentration, or with 0.2 mL of distilled water (reference). These mixtures were incubated at 25°C for 15 min.

3. The absorbance was measured at 430 nm, subtracting the optical density (OD) of the reference.

4. The OD values were plotted against the H2O2 concentrations (in mM). The slope of the linear part of the standard curve is the transformation factor of OD values to millimolar H2O2 concentrations.

3.2.2.3. Detection of the Levels of H2O2 Produced During the Growth of Microorganisms

1. The microorganisms were grown twice in LAPTg broth without agitation at 37°C and a third time (15 h, 37°C) with agitation (at 50 opm).

2. The supernatant fluids of the third cultures were separated by centrifugation (2000g, 10 min) (see Note 16).

3. A 0.2-mL aliquot of the supernatant fluids of lactobacilli was mixed with a 0.8-mL aliquot of reagent solution, and the assay was performed as described above (see Subheading 3.2.2.1.) (see Note 17).

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