SOP: Determination of Pectinesterase Activity
GIFSQ — Global Institute for Food Safety & Quality
Principle
The opaque cloud and the viscosity of citrus and other fruit juices come largely from their pectin. Hydrolysis of the ester bonds of pectin by the enzyme pectinesterase (PE) causes clarification and gelation of citrus concentrates. The PE activity is measured as the milli-equivalents of ester hydrolysed per minute under standard conditions: the carboxyl groups released by hydrolysis of the ester bond are titrated with alkali while the pH is held at 7.0, and the rate of alkali consumption over the timed period gives the activity. The test is applied to evaporator feed juice, cut-back juice and concentrate, and may be used to measure the efficiency of the pasteurizer. PE units may be expressed per millilitre or per gram of sample soluble solids.
Apparatus required
- pH electrodes with pH meter
- Magnetic stirrer with stirring bar
- Water bath (35 °C)
- Stopwatch
- 250-ml beaker
- Burette
Reagents
a. 2 M sodium chloride stock solution: dissolve 58.45 g of NaCl in 500 ml of water.
b. 1% high-methoxyl pectin–salt solution: dilute 37.5 ml of 2 M sodium chloride solution to 500 ml with water; weigh 5.0 g of high-methoxyl pectin (purified by reprecipitation of aqueous pectin solution with alcohol) into a 1-litre beaker; wet the pectin with a few drops of alcohol; add the 500 ml of salt solution gradually with vigorous stirring; heat nearly to boiling and cool.
c. 0.02 N, 0.1 N and 1.0 N sodium hydroxide solutions.
Procedure
Reconstitute samples of concentrate; note the Brix and apply the correction for temperature and acid; stir the sample vigorously
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Pipette 100 ml of the pectin–salt solution into a 250-ml beaker and bring the temperature to 35 °C in the water bath
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Add 10 ml of the sample juice to the pectin–salt solution; place a magnetic stirring bar in the solution, insert the pH electrodes, and stir slowly at a fixed rate for all samples
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Add 1 N NaOH until the pH is 6.5, then 0.1 N NaOH to pH 7.2; when the pH returns exactly to 7.0, start the stopwatch
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Add 0.02 N NaOH from the burette at a rate that holds the pH as near 7.0 as possible; near 10 minutes, add alkali to bring the pH slightly above 7.0, and stop the watch when the pH returns to 7.0
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Record the millilitres of alkali used and the time
Calculation
PE units × 10° per (g or ml of juice) = (ml of NaOH × N of NaOH × 10°) / (time in minutes × ml of juice)
The “10°” is carried exactly as printed in the book — it is garbled in the available copy (almost certainly 10⁶); check the printed book before critical use. The book also gives the activity expressed per gram of soluble solids, with an expression involving the normality of the alkali, the 10° factor and the °Brix; this second expression is partly garbled in the available copy — it is carried as read. Maximum parameters for different citrus products are: orange concentrate 0.5; pulp wash 0.5; stabilized pulp 1.0 (for testing, use 10 g of stabilized pulp).
Precautions
- Start the timer only when the pH has returned exactly to 7.0 after the preliminary adjustment.
- Keep the stirring rate fixed for all samples — the rate of pH response depends on mixing.
- Check the printed book for the complete per-gram-soluble-solids expression before reporting PE units on that basis.
References
Ranganna, S., Handbook of Analysis and Quality Control for Fruit and Vegetable Products, 2nd edition.
MacDonnell, L. R., Janson, E. F. and Lineweaver, H., Arch. Biochem., 6, 389 (1945) — as cited in the book.
Rouse, A. H. and Atkins, C. D., Univ. Florida Agr. Expt. Sta. Bull., p. 570, 1955 — as cited in the book.