ISO 22160:2007

Overview

ISO 22160:2007 (IDF 209:2007) specifies an Enzymatic Photo-Activated System (EPAS) chemiluminescent method for determining alkaline phosphatase (ALP) activity in milk and milk‑based drinks. The method is validated for pasteurized whole, semi‑skimmed, skimmed milks, cream and flavoured milks from cows, sheep, buffalo and goats, and is suitable for any liquid sample diluted so the ALP activity is below 7 000 mU/L. Results are expressed in milliunits per litre (mU/L).

Key topics and technical requirements

• Principle: ALP hydrolyses a stable aromatic dioxetane‑phosphate substrate at 35 °C ± 1 °C to produce a photo‑activated chemiluminescent product. Signal amplification uses an enhancing macromolecular component and is measured by a luminometer.
• Reagents: non‑chemiluminescent dioxetane ester substrate, stopping solution (0.15 mol/L 2‑amino‑2‑methyl‑1‑propanol with 0.02% benzalkonium chloride, pH 10.7), calibrator tablets and positive control enzyme.
• Calibration & controls: calibration tablets (fluid or cream/flavoured calibrators) create standard curves (typical calibrator activities noted), and a freeze‑dried positive control (e.g., 450 mU/L after rehydration) is used for QA.
• Apparatus: luminometer (operating ~540 nm, with software converting luminescence to enzyme units), mini‑vials, pipettes, incubator/dry block (35 °C ± 1 °C and higher temp baths for controls), water bath, balance, test tubes and printer.
• Sample preparation: mix and test samples at refrigeration temperature (0–7 °C); raw milk requires dilution with ALP‑free milk so diluted activity < 7 000 mU/L. ALP‑free (negative) controls are produced by heating to 95 °C for 1 min.
• Measurement & expression: luminometer readings are converted to ALP units using calibration; unit defined as enzyme catalysing transformation of 1 µmol substrate per minute. Precision and interlaboratory performance data are provided in the standard.

Applications

• Routine quality control and verification of pasteurization efficacy in dairy processing.
• Regulatory compliance testing for pasteurized milk and milk‑based beverages.
• Laboratory methods for dairy research and product development where ALP is a processing indicator.
• Testing of non‑cow milks (sheep, goat, buffalo) and flavored/cream products when validated dilution is applied.

Who uses this standard

• Dairy industry QC laboratories and plant laboratories
• Accredited testing laboratories performing official or contractual analyses
• Regulatory agencies and food safety inspectors
• Dairy R&D teams assessing heat treatment and enzyme residual activity

Related standards

• ISO 707 | IDF 50 - recommended sampling method (sampling not included in ISO 22160 procedure)
• ISO 22160 is published jointly as ISO 22160:2007 / IDF 209:2007 (first edition)

Keywords: ISO 22160:2007, alkaline phosphatase, EPAS, chemiluminescent method, milk testing, luminometer, pasteurization verification, dairy quality control.