SOP: Determination of Thermal Inactivation Time of Peroxidase

GIFSQ — Global Institute for Food Safety & Quality

Principle

Peroxidase is the most heat-resistant of the enzyme systems in fruits and is used as the index organism-equivalent for thermal process schedules based on enzyme inactivation. The thermal inactivation time (TIT) — the minimum heating time at a given temperature at which peroxidase activity disappears — is determined by heating tubes of the prepared material at different temperatures, cooling them rapidly, and testing for residual peroxidase activity.

Apparatus required

• TIT tubes of Corning glass, 13.5 cm length, 0.9 cm outer diameter, 0.7 cm inner diameter
• Wide-mouthed pipette
• Temperature-controlled water bath
• Needle-type thermocouple, 10.5 cm long, fitted with spacers to position it in the centre of the tube; potentiometer for noting the temperature (the author used a Leeds and Northrup potentiometer calibrated in °F)
• Chilled water bath (for rapid cooling)
• Colorimeter (420 nm for clear solutions; reflectance attachment for pulps and syrup homogenates)

Reagents

a. 0.3% hydrogen peroxide (H₂O₂) — 1 ml per test.

b. 1% solution of guaiacol in ethanol — 1 ml per test (for the standard test).

c. Alcoholic 1% pyrocatechol — 1 ml per test (for cherry syrup homogenates, as used by Dastur et al.).

Sample preparation

Prepare the material as described for thermal inactivation time work (pulp, syrup homogenate, juice or puree adjusted to the canned product’s pH and °Brix).

Procedure — Determination of TIT

Using the wide-mouthed pipette, transfer 2 ml of the sample into each TIT tube

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Place the requisite number of tubes at a specified temperature in the temperature-controlled water bath; take out 3 tubes at different intervals of heating time and immediately plunge them into chilled water to cool; perform the experiment 3 times

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Determine the come-up time using the 10.5 cm needle-type thermocouple fitted with spacers; note the temperature at different intervals of time (seconds) using the potentiometer; carry out the experiment in triplicate for each temperature

Procedure — Test for peroxidase activity (clear solutions)

To 2 ml of the sample add 1 ml of 0.3% H₂O₂ and 1 ml of the 1% solution of guaiacol in ethanol; allow the colour to develop for 15 min at room temperature; measure the colour of clear solutions at 420 nm

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For cherry syrup homogenates: to each tube add in sequence 1 ml of 0.3% H₂O₂ and 1 ml of alcoholic 1% pyrocatechol; stir, hold the tubes for 15 min to permit colour development; filter the solution and measure the colour at 585 nm

Procedure — Extent of inactivation and TIT

Pipette 2 ml of the unheated preparation into 6 tubes; develop the colour in 3 tubes directly; inactivate the enzymes in the remaining 3 tubes by heating in a boiling water bath for 5 min; after cooling, add the reagents — the two sets of tubes provide comparison of maximum activity and no activity

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Take the minimum heating time at a temperature at which the reading or the colour is similar to the sample having no activity as the inactivation time (TIT)

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For syrup homogenates of fruit or fruit puree, in which the colour cannot be measured directly by transmittance: measure the colour by reflectance, using the sample having no activity to set the instrument to 100% reflectance; note the reflectance at different wavelengths and find the wavelength of reflectance minimum; after colour development, using the sample having no activity to set the instrument to 100% at the wavelength of minimum reflectance, measure the reflectance of the samples heated for different time intervals at a temperature; the inactivation time is the minimum heating time at a temperature at which the reading is 100%

Procedure — Determination of TIT and thermal resistance of pectinesterase

The procedures are similar to those for peroxidase, except for the following: to determine the activity, at least 10 ml of the sample is required — take 10 ml of the sample in Pyrex glass tubes of 13.5 cm length, 1.6 cm outer diameter and 1.4 cm inner diameter; the come-up time may range from 5 to 6 min

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For the determination of activity and the quantitative estimation of pectinesterase, use 10 ml of the sample and make the determinations as described in Chapter 28 of the book

Precautions

• Plunge the heated tubes into chilled water immediately on removal — cooling must be fast so that no further inactivation occurs after the timed heating.

References

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

Dastur, K., K.G. Weckel & J.H. von Elbe, Food Technol. (Chicago), 22, 177 (1968) — cited in the book.

Nath, N. & S. Ranganna, J. Food Sci. & Technol., 14, 113 (1977) — cited in the book.