🍎 Alkaline phosphatase (ALP, EC 3.1.3.1) activity in raw and heat-treated milk (whole, semi-skimmed, skimmed, flavoured) and milk-based drinks from cows, sheep and goats, and in reconstituted milk powder — continuous direct kinetic assay: the non-fluorescent substrate 2′-[2-benzothiazolyl]-6′-hydroxybenzothiazole phosphate in diethanolamine buffer pH 10 is hydrolysed by ALP to a highly fluorescent product, read at 38 °C for 3 min (first minute for temperature equilibration), filter fluorimeter with excitation at 440 nm and emission at 520–560 nm, calibration with Fluoroyellow® solutions 0 / 17.24 / 34.48 × 10⁻³ µmol/l; result in mU/l up to 7000 mU/l, above that dilution with ALP-free milk (heated 5 min at 95 °C, < 10 mU/l); PN-EN ISO 11816-1:2024-07

Food chemistry PN-EN ISO 11816-1

Overview

WHAT THE STANDARD COVERS. We read the text of the standard in the iTeh sample of ISO 11816-1:2024 | IDF 155-1:2024 (fourth edition, 2024-01, English text) — PN-EN ISO 11816-1:2024-07 adopts EN ISO 11816-1:2024 and ISO 11816-1:2024 as identical (PKN search): ISO and IDF forewords, clauses 1–8 and clause 9 up to 9.1.1 — the sample ends on page 4. We do not read the rest of instrument verification (9.1.2–9.1.5), the working substrate control (9.2), calibration (9.3), determination (9.4), test-sample-related controls (9.5, including the interfering substance test and the heat-stable microbial phosphatase test), calculation (10), precision (11), the test report (12) or Annex A (interlaboratory study); we know their titles from the table of contents.

ACCORDING TO THE TEXT OF ISO 11816-1:2024. The standard was prepared by ISO/TC 34 "Food products", Subcommittee SC 5 "Milk and milk products", jointly with IDF and in collaboration with CEN/TC 302 (Vienna Agreement); the work was done by IDF/ISO Action Team P19. The fourth edition replaces the third (ISO 11816-1 | IDF 155-1:2013); main changes: the discontinued FLM200 instrument was replaced by the FLM300 version; instructions were revised for the new interface and electronics of the FLM300 — without changing the assay or the test procedure; the heater block is now part of the instrument. Scope (1): fluorimetric determination of alkaline phosphatase (EC 3.1.3.1) activity in raw and heat-treated whole, semi-skimmed, skimmed and flavoured milk; for milk and milk-based drinks from cows, sheep and goats; also for milk powder after reconstitution; the instrument reads activities up to 7000 mU/l, and a sample with higher activity is diluted with ALP-free milk so that the result does not exceed 7000 mU/l. No normative references (2). Definition (3.1): ALP activity — activity of the enzyme in the product determined by the specified procedure, expressed in milliunits per litre of sample (mU/l). Principle (4): continuous fluorimetric direct kinetic assay; a non-fluorescent aromatic monophosphoric ester — 2′-[2-benzothiazolyl]-6′-hydroxybenzothiazole phosphate — in the presence of ALP from the sample undergoes hydrolysis of its phosphate radical, giving a highly fluorescent product; measured at 38 °C over 3 min in a Fluorophos® instrument (pre-incubation of substrate and sample, then multiple kinetic readings of the reaction rate); note: the first minute is equilibration, the actual measurement runs from the beginning of the second to the end of the third minute (2 min). Reagents (5): reagents 5.1–5.5 and apparatus 6.1–6.4 (except 6.3.3) make up the Fluorophos® Test System (trade names given for information; equivalent products acceptable if they give the same results); Fluorophos® substrate — bottles of 144 mg (580 g/mol), 2 years at 2–8 °C in unopened bottles, protected from light; diethanolamine buffer c(DEA) = 2.4 mol/l, pH 10.0, bottles of 240 ml, 2 years at 2–8 °C; working substrate — the content of a buffer bottle (240 ml) added to a substrate bottle (144 mg), mixed by inversion for 3 min (about 1.0 mmol/l, pH 10), amber glass, at least 30 min at room temperature before use; suitability checked by the A/D test (9.2) — the substrate is not used if the reading is above 1200 FLU; stable 60 days at 2–8 °C in the dark or 8 h at 38 °C; 240 ml is enough for about 115 tests; Fluoroyellow® calibrator solutions (2′-(2-benzothiazolyl)-6′-hydroxybenzothiazole) in buffer — A 0, B 17.24 × 10⁻³ and C 34.48 × 10⁻³ µmol/l, 18 months at 2–8 °C; daily instrument control solution 34.48 × 10⁻³ µmol/l. Apparatus (6): filter fluorimeter with a thermostatically controlled cuvette holder (38 ± 1) °C and right-angle optics, excitation 440 nm, emission 520–560 nm; disposable cuvettes of non-fluorescent glass 12 × 75 mm; dispenser 2.0 ml; positive- or air-displacement pipette 0.075 ml — pipetting technique is critical for accuracy; pipettes of 2 and 3 ml; incubator block (38 ± 1) °C; paraffin film; vortex mixer; water bath (63 ± 1) °C and (95 ± 1) °C; volumetric flasks of 100 ml. Sampling (7) is not part of the method — ISO 707 | IDF 50 recommended. ALP-free milk (8.1): a portion of milk of the type tested is dispensed into a tube without touching the rim or sides, heated in the water bath at 95 °C — 5 min from reaching 95 °C at the centre of the portion (thermometer or thermistor) — and cooled rapidly; the ALP activity of the treated milk must be below 10 mU/l. Test sample (8.2): all samples are mixed carefully; prewarming is usually not necessary; pasteurized samples are used as delivered; samples with high activity are diluted with ALP-free milk into the analytical range (< 7000 mU/l). Instrument verification (9.1.1): drift, stray light and stability before analysis; daily A/D test, daily test with the control solution (electronic and optical drift) and recommended external positive, negative and normal controls.

STATUS (as of 03.10.2026). In the PKN search engine (query "PN-EN ISO 11816-1", 03.10.2026) PN-EN ISO 11816-1:2024-07 appears in Polish and English versions, "Introduces: EN ISO 11816-1:2024 [IDT], ISO 11816-1:2024 [IDT]", without a withdrawal note; earlier editions (2014-02, 2007, 2006, 2002) are "Withdrawn and replaced". The record of PN-EN ISO 11816-1:2014-02 (Polish version; read 03.10.2026): publication date 09-12-2015, withdrawal date 01-07-2024, introduces EN ISO 11816-1:2013 [IDT] and ISO 11816-1:2013 [IDT], KT 35 "Milk and Milk Products", replaced by PN-EN ISO 11816-1:2024-07 (English version). What the withdrawal changes for the result: according to the foreword of ISO 11816-1:2024 the changes concern the instrument (FLM300 instead of FLM200, built-in heater block) and the operating instructions, without changing the assay or test procedure.

HOW MANY LABORATORIES AND IN WHAT FORM (copy of the accreditation scope database, load up to 17.09.2026, read 03.10.2026). The number 11816-1 appears in 2 records at 2 laboratories: AB 485 and AB 704. In the current PCA documents (read 03.10.2026; AB 704 issue no. 23 of 20.04.2026, AB 485 issue no. 27 of 19.08.2026) the number appears at the same 2 laboratories; in each document the number of page markers equals the number of PDF pages, without duplicates; searching all PCA documents downloaded from the BIP on 03.10.2026 found no laboratories outside the database copy. Forms: "PN-EN ISO 11816-1:2024-07" (AB 485) and "PN-EN ISO 11816-1:2014-02" (AB 704 — edition withdrawn on 01.07.2024). None of these items is suspended. The "Laboratories" tab (query "PN-EN ISO 11816-1") shows both laboratories — checked by a tab query on the labcoda.pl production server on 03.10.2026.

WHAT THE LABORATORIES TEST (PCA items). AB 485: milk — alkaline phosphatase activity (10–7000) mU/l, fluorimetric method. AB 704: milk and milk-based drinks — alkaline phosphatase activity (10–800) mU/l, fluorimetric method.

WHERE A SEEMINGLY CORRECT RESULT IS EASY TO GET. Range: the instrument reads up to 7000 mU/l — higher activity is diluted with ALP-free milk, not water (1, 8.2.3); AB 704 has a range up to 800 mU/l, so samples above that value require dilution or fall outside its item. Milk for dilutions: of the same type as tested, heated 5 min at 95 °C counted from reaching that temperature at the centre of the portion, without milk touching the rim and sides of the container, and checked (< 10 mU/l) (8.1). Working substrate: at least 30 min before use, A/D test — rejection above 1200 FLU; stable 60 days at 2–8 °C or only 8 h at 38 °C (5.3). Pipetting: 0.075 ml of sample — the technique is critical for accuracy (6.3.2). Instrument: daily A/D test and control solution 34.48 × 10⁻³ µmol/l before analysis (9.1.1). Edition: AB 704 cites the 2014 edition, withdrawn on 01.07.2024 — according to the foreword of the new edition the procedure has not changed, the instrument has (foreword). Other species and products: the method covers milk from cows, sheep and goats and drinks based on it; milk powder only after reconstitution (1).

WHAT WE DO NOT GIVE. We do not reproduce the calibration and determination procedure (9.2–9.5), calculation formulas (10), precision data (11) or the results of the interlaboratory study (Annex A) — they are outside the sample. We do not give criteria for assessing pasteurization efficiency from ALP activity.

Method principle

Alkaline phosphatase from the sample cleaves the phosphate group from a non-fluorescent substrate (2′-[2-benzothiazolyl]-6′-hydroxybenzothiazole phosphate), forming a highly fluorescent product. The sample with substrate in pH 10 buffer is placed in a fluorimeter thermostatted at 38 °C; after one minute of temperature equilibration the instrument measures the increase in fluorescence for two minutes (excitation 440 nm, emission 520–560 nm), and the reaction rate is converted to activity in mU/l on the basis of calibration with solutions of the fluorescent product.

Applications

• Raw and heat-treated milk — whole, semi-skimmed, skimmed and flavoured (1)
• Milk and milk-based drinks from cows, sheep and goats; milk powder after reconstitution (1)
• Milk and milk and milk-based drinks — alkaline phosphatase activity — 2 accreditation scope records of 2 laboratories in PCA

Key parameters

Standard

Standard number

PN-EN ISO 11816-1:2024-07

Title (PL)

Mleko i przetwory mleczne — Oznaczanie aktywności fosfatazy alkalicznej — Część 1: Metoda fluorymetryczna dla mleka i napojów na bazie mleka

Title (EN)

Milk and milk products — Determination of alkaline phosphatase activity — Part 1: Fluorimetric method for milk and milk-based drinks

Step-by-step procedure

1. Preparation of working substrate

   Buffer + substrate, 3 min mixing, ≥ 30 min at room temperature, A/D test ≤ 1200 FLU (5.3). PN-EN ISO 11816-1:2024-07 — no withdrawal note (PKN, 03.10.2026).

2. Instrument verification

   Daily A/D test, control solution, recommended external controls (9.1.1).

3. ALP-free milk

   5 min at 95 °C, rapid cooling, check < 10 mU/l (8.1).

4. Test sample preparation

   Mixing; pasteurized — as delivered; high activity — dilution with ALP-free milk (8.2).

5. Measurement

   38 °C, 3 min, kinetic reading in mU/l (4); details of calibration and determination — outside the sample (9.3, 9.4).

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• Substrate, buffer and calibrators are stored at 2–8 °C and protected from light; working substrate in amber glass (5.1–5.4)

Frequently asked questions