SOP: Quantitative Estimation of Peroxidase in Vegetables

GIFSQ — Global Institute for Food Safety & Quality

Principle

The AOAC procedure for the quantitative estimation of peroxidase is based on the oxidation of ascorbic acid in a coupled reaction: ascorbic acid reduces indophenol, peroxidase combines with hydrogen peroxide, and the peroxide–enzyme complex re-oxidises the reduced indophenol. The decrease in ascorbic acid concentration, measured by titration with indophenol, is used as a measure of peroxidase activity. The overall reactions are: (i) Ascorbic acid + indophenol → H₂ indophenol + Dehydroascorbic acid; (ii) Peroxidase + H₂O₂ → Peroxidase·H₂O₂; (iii) Peroxidase·H₂O₂ + H₂ indophenol → indophenol + H₂O + Peroxidase.

Apparatus required

• High speed blender
• Glass wool and cotton gauze
• 400-ml beaker
• Magnetic stirrer
• Quick-delivery pipettes
• 125-ml conical flasks

Reagents — relatively stable

a. Phosphate-oxalate buffer, 0.1 M, pH 6.0: dissolve 14.2 g of sodium phosphate, dibasic (Na₂HPO₄, or 26.81 g of Na₂HPO₄·7H₂O) and 12.6 g of oxalic acid (H₂C₂O₄·2H₂O) in water with heat, cool, adjust the pH to 6.0 with 1.0 N NaOH, and dilute to 1 litre.

b. Oxalic acid, 1.0 M: dissolve 142 g of oxalic acid (H₂C₂O₄·2H₂O) in water and dilute to 1 litre.

c. Sodium chloride, 2.0% solution: dissolve 20 g of NaCl in water, dilute to 1 litre, and store at 0°C.

Reagents — relatively unstable

d. Hydrogen peroxide, 0.1 N (H₂O₂): dilute 0.58 ml of 30% H₂O₂ to 100 ml with cold water. Standardise as follows: to 25 ml of 0.1 N H₂O₂, add 10 ml of 4 N H₂SO₄, 6 ml of 1 N KI and 3 drops of 1 N ammonium molybdate ((NH₄)₂Mo₇O₂₄); titrate with 0.1 N sodium thiosulphate using starch as indicator. Store the H₂O₂ solution in a refrigerator; prepare weekly.

e. Indophenol solution, 0.001 N: dissolve 200 mg of 2,6-dichlorophenol-indophenol, or 175 mg of its sodium salt, or equivalent, in water and dilute to 1 litre. Standardise against 0.01 N Na₂S₂O₃ by titrating the iodine liberated by 50 ml of dye after addition of 10 ml of 1 N potassium iodide solution and 10 ml of 4 N H₂SO₄. Store in a refrigerator; prepare fresh weekly.

f. Ascorbic acid, 0.05 M: dissolve 880 mg of l-ascorbic acid in 0.1 M buffer and dilute to 100 ml; prepare fresh daily.

Sample preparation

To 50 g of fresh or frozen material, add 200 ml of cold 2% NaCl solution and blend in a high speed blender. Strain through glass wool initially and then through cotton gauze to remove coarse particles. Assay within 30 min.

Procedure

Take 75 ml of buffer, 50 ml of dye and 5 ml of ascorbic acid in a 400-ml beaker

↓

Add enough water to give a total volume of 250 ml after the addition of the tissue extract and H₂O₂

↓

Adjust the temperature to 25 ± 5°C (the print reads “25 + 5° C”; carried as ±, flagged)

↓

Place the beaker on a magnetic stirrer and stir the mixture for 30 sec at a speed that does not cause excessive aeration

↓

Pipette 1–3 ml of extract (corresponding to 0.2–0.4 g of the sample), add 5 ml of H₂O₂ solution from a quick-delivery pipette, and start the stopwatch immediately on addition of H₂O₂; continue mixing during the addition

↓

Remove a 25 ml aliquot of the reaction mixture with a quick-delivery pipette and discharge it into a 125-ml conical flask containing 5 ml of oxalic acid

↓

Take 4–5 samples similarly at 1 min intervals and discharge into flasks containing 5 ml of acid each; record the reaction time as the time when the pipette is emptied out

↓

Titrate the residual ascorbic acid with dye solution and calculate the first-order reaction rate constant

Calculation

If V₀ = ml of indophenol solution required to oxidise the ascorbic acid present in 25 ml of complete reaction mixture at time 0, and Vₜ = ml of indophenol solution required to oxidise the ascorbic acid remaining in 25 ml of reaction mixture after t min, the first-order rate constant k₁ is calculated using:

k₁ = (2.303 / t) × log₁₀(V₀ / Vₜ)

• Worked example from the book: if V₀ is 45 ml and V₂ is 40 ml after 2 min,
  k₁ = 2.303 × (1/2) × log₁₀(1.125) = 0.0589, and K₁ = 5.89 × 10⁻².

Note: the printed general expression for k₁ is garbled in the available copy; the expression above is read from the book’s own worked example, flagged for checking against the printed book.

K₁ per gram of sample is calculated from K₁, the volume of the reaction mixture, the volume of the aliquot taken, the total volume of the blended sample, the volume of extract used, and the weight of sample taken. Worked example from the book: to 50 g of sample, 200 ml of water was added for blending; if 1 ml of this extract was made to 250 ml and 25 ml aliquots were used for titration, K₁/g = (0.0589 × 250 × 250 × 1) / (25 × 1 × 50) = 2.94.

Note: the printed general expression for K₁/g is garbled in the available copy; the book’s column labels and the worked example above are carried as printed, flagged for checking against the printed book.

Precautions

• Carry out all titrations beyond the fading end point to a clearly visible pink end point permanent for 1 min.
• Adjust all titrations to the same end point by comparison with the first flask of each run.
• Add the dye rapidly at first and more slowly later.
• Adjust the enzyme activity so that the constant is of the order of magnitude of 15–20, as printed (carried as printed, flagged).

References

Ranganna, S., Handbook of Analysis and Quality Control for Fruit and Vegetable Products, 2nd edition. Masure, M.P. and H. Campbell, Fruit Prod. J., 23, 369 (1944).