SOP: Peroxidase Test for Enzyme Activity in Dehydrated Vegetables

GIFSQ — Global Institute for Food Safety & Quality

Principle

If peroxidase has survived dehydration, it oxidises guaiacol in the presence of hydrogen peroxide, and the reconstituted tissue develops a reddish-brown colour. The colour observed after 5 and 10 min shows whether the enzyme is active. For dehydrated beetroot the same reaction is shown with benzidine, which gives a bluish-black colour when positive.

Apparatus required

• Test tube (1 inch in diameter)

Reagents

a. Guaiacol solution, 1% in 95% alcohol — dissolve 1 g of guaiacol in 95% alcohol and make up to 100 ml with 95% alcohol.

b. Hydrogen peroxide solution, 0.5%, freshly prepared — add 10 ml of “10 volume” (3%) hydrogen peroxide to 50 ml of water. Prepare fresh for each run.

c. Benzidine solution, 0.5% in 95% alcohol (for dehydrated beetroot only) — dissolve 0.5 g of benzidine in 95% alcohol and make up to 100 ml with 95% alcohol.

Sample preparation

Take a small portion of the dehydrated vegetable for reconstitution. Do not use the ground sample-preparation procedure for this test.

Procedure

Reconstitute a small portion of the sample in cold water for 10–15 min; pour off the excess water

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Add small pieces of the reconstituted sample to a test tube 1 inch in diameter, to a depth of about 1 inch

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Add 10 ml of water, 1 ml of the 1% guaiacol solution in 95% alcohol, and 1 ml of the fresh 0.5% hydrogen peroxide solution

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Shake the test tube to mix; note the reaction after 5 min and again after 10 min

Interpretation

• Negative — no discolouration; or reddish-brown specks, or a few larger spots, especially on the veins.
• Light positive — light brown markings scattered throughout the tissue, or heavy browning of a few pieces not restricted to the veins.
• Positive — reddish-brown colouration more pronounced than in the light positive.

For dehydrated beetroot, use the 0.5% benzidine solution in 95% alcohol in place of the guaiacol solution; a bluish-black colouration indicates a positive test.

Reagent check

Check the effectiveness of the reagents by carrying out the test on a fresh vegetable and on the same vegetable after boiling for 5 min and cooling. The fresh vegetable should give a strong positive reaction and the boiled vegetable a negative reaction.

Precautions

• Prepare the 0.5% hydrogen peroxide solution fresh for every run.
• Read and record the reaction at both 5 min and 10 min.
• Check the effectiveness of the reagents by carrying out the test on fresh vegetables, and on the same vegetables after boiling for 5 min and cooling: the fresh vegetable should give a strong positive reaction and the boiled one a negative reaction.

References

Ranganna, S., Handbook of Analysis and Quality Control for Fruit and Vegetable Products, 2nd edition.