SOP: Test for Inactivation of Pectinesterase
GIFSQ — Global Institute for Food Safety & Quality
Principle
The test is based on the increase in viscosity of a juice containing medium-methoxyl pectin and calcium on incubation — an increase caused by the activity of residual pectinesterase in the juice. Residual enzyme de-esterifies the pectin, and the resulting low-methoxyl pectin forms a calcium gel that raises the viscosity. A juice sample heated in boiling water (no enzyme activity) serves as the control. Gelation may also be observed in juices that have not been adequately pasteurized.
Apparatus required
- 100-ml stoppered conical flasks
- Viscometer
- Water bath or incubator (34 °C)
Reagents
a. Medium-methoxyl (6.0%) pectin: to a 2% solution of high-methoxyl (9–10%) pectin in water, add concentrated ammonia until the pH is 10.5; let stand at room temperature for 60 minutes for de-esterification; at the end of 60 minutes acidify to pH 0.5 with concentrated HCl and precipitate the pectin with alcohol; separate the precipitate by straining through nylon cloth; wash successively with 50, 60 and 70% ethanol and finally with absolute ethanol; dry the residue overnight at 60 °C, powder, and pass through a 52-mesh sieve. Determine the methoxyl content as given in Chapter 2 — it should range between 6 and 6.5%. Prepare a 0.5% solution of the pectin in water.
b. 1.0 N calcium chloride solution.
c. 0.1 N sodium hydroxide solution.
d. Toluene.
Procedure
To 10 ml of the 0.5% pectin solution add 10 ml of the pasteurized juice
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Adjust the pH to 7.0 with 0.1 N NaOH and make up the volume to 60 ml with water
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Add 1 ml of calcium chloride solution and mix
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Add a few drops of toluene, transfer to a 100-ml stoppered conical flask, and incubate at 34 °C for 2 days
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Prepare a control sample having no enzyme activity in the same way, using juice heated for 10 minutes in boiling water
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Measure the viscosity with a suitable viscometer — any increase in the viscosity of the sample after incubation, as compared with the value before incubation or with the control sample incubated similarly, is indicative of the presence of active enzyme
Precautions
- The control must be treated identically in every respect except for the boiling-water heating.
- Add the toluene to prevent microbial growth during the 2-day incubation.
References
Ranganna, S., Handbook of Analysis and Quality Control for Fruit and Vegetable Products, 2nd edition.
Nath, N. and Ranganna, S., J. Food Technol., 12, 411 (1977) — as cited in the book.