BS EN ISO 11816-2:2024
Milk and milk products. Determination of alkaline phosphatase activity - Fluorimetric method for cheese

Standard No.

BS EN ISO 11816-2:2024

Release Date

2024

Published By

British Standards Institution (BSI)  GB  /  BSI

Latest

BS EN ISO 11816-2:2024

Introduction

Analysis of the core content of the standard

This part of the standard specifies the method principle for determining the activity of alkaline phosphatase (ALP) in cheese using continuous fluorescence kinetic method: using a non-fluorescent aromatic monophosphate substrate 2′-[2-benzothiazolyl]-6′-hydroxybenzothiazole phosphate, it is hydrolyzed under the action of ALP to produce a highly fluorescent product, and the fluorescence is detected at 38°C for 3 minutes using a Fluorophos® instrument.

---

Technology Evolution and Major Changes

Version	BS EN ISO 11816-2:2016	BS EN ISO 11816-2:2024
Instrument Requirements	FLM200 Model	Upgrade to FLM300 (Integrated Heating Block)
Calibration Procedure	Step-by-Step	Optimization of User Interface and Electronics
Detection Limit	7,000 mU/kg	Maintain the same sensitivity

---

Key operating procedures

Sample preparation specifications

Special processing requirements for different types of cheese:

• Large hard cheese: Take a 1cm thick sample 0.5cm below the rind of the wheel edge (to avoid uneven ALP activity in the core area)
• Soft mold cheese: Remove the rind but keep the subcutaneous fat layer
• Semi-hard cheese: Conventional grinding and mixing is sufficient

Instrument calibration points

Use three calibration solutions (A:0μmol/L, B:17.24×10⁻³μmol/L,

---

Quality Control Requirements

Control Type	Qualified Standard	Frequency
A/D Test	302 FLU±4 FLU	Daily Startup
Instrument Control Solution	602 FLU±12 FLU	Daily Testing
Negative control	<10 mU/kg	Each batch

---

Result calculation and expression

ALP activity calculation formula:
Ap = (Ap1 × f2 × 25)/m
Wherein:
Ap1=supernatant ALP activity (mU/L), f2=dilution factor, m=sample mass (g)

Precision requirements

• Repeatability limit: ≤12% average value (same laboratory)
• Reproducibility limit: ≤29% average value (between laboratories)

---

Implementation suggestions

Handling of special situations

Verification steps when the test value is abnormal:

1. Interfering substance test: Use calibration solution A for test, and re-sample when the reading is >20 mU/kg
2. Thermotolerant microorganism ALP test: Detect residual activity after heating at 63°C for 30 minutes
3. Highly active samples: Over 7,000 mU/kg, dilute with extraction buffer

Note on the scope of standard application

This method is not suitable for cheeses containing mold inside (such as blue cheese), and special attention should be paid to the sampling location for large hard cheeses that have been blanched at more than 50°C.

BS EN ISO 11816-2:2024 history

• 2024 BS EN ISO 11816-2:2024 Milk and milk products. Determination of alkaline phosphatase activity - Fluorimetric method for cheese
• 2016 BS EN ISO 11816-2:2016 Milk and milk products. Determination of alkaline phosphatase activity. Fluorimetric method for cheese
• 2003 BS EN ISO 11816-2:2003 Milk and milk products. Determination of alkaline phosphatase activity using a fluorimetric method. Fluorometric method for cheese

Topics on standards and specifications

Standard and Specification