Overview
ISO/TS 4985:2023 (IDF/RM 255:2023) specifies a fluorimetric microplate method for determination of alkaline phosphatase (ALP, EC 3.1.3.1) activity in milk and selected milk products. The technique measures enzymatic hydrolysis of the non‑fluorescent substrate 4‑methylumbelliferone phosphate (4‑MUP) to the fluorescent product 4‑methylumbelliferone (4‑MU) at 37 °C over a 15‑minute kinetic read. Results are expressed in milliunits per litre (mU/l) for milk and mU/g for cheese.
Keywords: ISO/TS 4985:2023, alkaline phosphatase, ALP, fluorimetric microplate method, 4‑MUP, milk testing, dairy laboratories.
Key topics and technical requirements
- Scope of application: raw and heat‑treated whole, semi‑skimmed and skimmed milk, cream, flavoured milks, reconstituted milk powder, and soft, semi‑hard and hard cheeses (surface‑mould only). Applicable to cow, sheep and goat milks; extendable to similar species (e.g., buffalo, camelids).
- Analytical principle: continuous fluorimetric direct kinetic assay monitoring formation of 4‑MU.
- Reagents and buffers: examples provided include magnesium chloride, Tergitol 15‑S‑9, AMP buffer and DEA buffer; only analytical‑grade reagents recommended.
- Apparatus and calibration: microplate fluorimeter with defined instrument settings, linearity testing, preparation of working standards and calibration curves (4‑MU standards).
- Sampling and preparation: specific sampling for large hard cheeses (see Clause 7); preparation of ALP‑free controls and test samples for milk and cheese (Clauses 7–9).
- Quality control and controls: negative/positive controls, interfering‑substance tests, heat‑stable microbial ALP control.
- Calculation and reporting: procedures for calibration curve generation, activity calculation and expression of results; precision and interlaboratory data included (Annex A–C).
Applications
- Verification of heat treatment/pasteurization - routine check to confirm effective pasteurization by detecting residual ALP activity.
- Dairy quality control - process monitoring in milk processing, cheese production and reconstitution of milk powder.
- Regulatory and food safety testing - standardized method for compliance testing by food control authorities.
- Research and method development - laboratories conducting enzyme activity studies in dairy matrices.
Who uses this standard
- Dairy plant QC laboratories and production technologists
- National food control and public health laboratories
- Accredited testing laboratories (ISO/IEC 17025)
- Cheesemakers and dairy R&D groups
Related standards
- ISO/TS 4985:2023 is part of ISO/IDF analytical methods for milk. Users should consult other ISO/IDF methods for sampling, microbiological testing and complementary enzyme assays as relevant to their quality program.
This specification delivers a validated, microplate‑based fluorimetric workflow for ALP determination, enabling higher throughput, traceable calibration and robust quality control for dairy testing.