Overview
EN ISO 22160:2007 (ISO 22160:2007 / IDF 209:2007) specifies a standardized laboratory method for measuring alkaline phosphatase (ALP) activity in pasteurized milk and milk-based drinks using an Enzymatic Photo-Activated System (EPAS) - a chemiluminescent assay. The method applies to pasteurized whole, semi-skimmed and skimmed milk, cream and flavoured milks from cows, sheep, buffalo and goats, and to other liquid samples when diluted so ALP activity is below 7 000 mU/L.
Key topics and technical requirements
- Principle: A stable dioxetane-phosphate substrate is hydrolysed by ALP at 35 °C ± 1 °C to produce a chemiluminescent product. Photo-activation (chemiluminescence) is amplified and measured by a luminometer.
- Incubation: Enzymatic hydrolysis stopped after a specified incubation time (3 minutes).
- Calibration: Luminometer calibration uses commercially supplied calibration tablets (dried milk with known ALP activity) rehydrated in ALP‑free milk to produce standard points (examples given for milk and cream/flavoured milk). Calibration checks and control tests are specified.
- Reagents & handling:
- Non‑chemiluminescent dioxetane ester substrate (store 0–7 °C; stable ~6 months at 4 °C; short stability at elevated temperature).
- Stopping solution: 0.15 mol/L 2‑amino‑2‑methyl‑1‑propanol with 0.02 % benzalkonium chloride, pH 10.7 (room temperature use; storage guidance provided).
- Sample scope: Validated for cow, sheep, buffalo and goat milks (including skim and cream), and suitable for diluted liquid matrices.
- Quality & performance: The standard includes procedures for calibration adjustment, control tests, and reports precision data from interlaboratory trials (repeatability and reproducibility considerations).
Applications and users
This EN ISO 22160:2007 method is used for:
- Dairy quality control: Verifying adequacy of pasteurization (ALP as heat‑treatment indicator).
- Regulatory testing: Compliance with food safety and pasteurization standards.
- Analytical laboratories: Routine testing in commercial and third‑party labs using luminometers and EPAS reagents.
- Dairy processors and manufacturers: In-house monitoring of pasteurized milk, cream and flavoured products.
- Test-kit and instrument vendors: Developing and validating chemiluminescent ALP assay kits and luminometer calibration systems.
Related standards
- Supersedes EN ISO 11816‑1:2000 (see standard document for references and bibliographic notes).
- Pertinent for laboratories following ISO/IDF microbiological and chemical testing frameworks.
Keywords: EN ISO 22160, ISO 22160:2007, alkaline phosphatase, EPAS, chemiluminescent assay, milk testing, pasteurized milk, dairy quality control, luminometer, calibration tablets.