Overview - ISO 13875:2005 (IDF 178:2005)
ISO 13875:2005 specifies a reverse‑phase HPLC method for the quantitative determination of acid‑soluble β‑lactoglobulin (β‑LG) in liquid milk. The method measures the fraction of β‑LG that remains soluble after isoelectric precipitation at pH 4.6 and is validated over a range of 0 to 3 500 mg β‑LG per litre. It is designed to distinguish different categories of heat‑treated milks (raw, pasteurized, UHT, sterilized) and provides procedures for sample preparation, HPLC analysis, calibration and result expression.
Key topics and technical requirements
- Principle: caseins and denatured whey proteins are precipitated at pH 4.6; the acid whey supernatant is analyzed by reverse‑phase HPLC to quantify soluble β‑LG.
- Sample preparation: pH adjustment to 4.6, centrifugation (2 000 g), filtration and dilution into phosphate buffer; different dilution factors depending on milk heat treatment.
- HPLC conditions: reverse‑phase column (PLRP‑S 150 × 4.6 mm, 5 or 8 µm or equivalent), gradient elution with HPLC‑grade water, acetonitrile and trifluoroacetic acid; UV detection at 205 nm or 280 nm; injection typically 20 µl; column temperature 40 °C.
- Calibration: single‑point or multi‑point calibration against a standardized reference sample (freeze‑dried milk reference) and a pure β‑LG standard (A+B genetic variants). The standard details preparation and periodic standardization of reference material.
- Reagents & safety: HPLC‑grade solvents and high‑purity trifluoroacetic acid; safety precautions for potentially hazardous elution solvents are recommended.
- Apparatus: pH‑meter, centrifuge, microfiltration devices (0.22 µm), analytical balance and HPLC system with integrator/software for peak area measurement.
- Quality and precision: the standard includes precision data and results from interlaboratory trials (see Annex A). It also defines reporting and expression of results.
Applications and who uses it
- Dairy quality control labs monitoring protein denaturation or heat damage in liquid milk.
- Milk processors comparing thermal processing regimes (pasteurization, UHT, sterilization).
- Regulatory and food safety laboratories requiring standardized, traceable β‑LG measurements.
- Research laboratories studying whey protein behavior, heat treatment effects and milk protein composition.
Benefits include reliable differentiation of heat‑treated milk categories, traceable quantification of soluble β‑LG and compatibility with routine HPLC instrumentation.
Related standards
- ISO 8968‑1 / IDF 20‑1 and ISO 8968‑2 / IDF 20‑2 (nitrogen/protein determination - Kjeldahl / block digestion) - normative references in ISO 13875.
- ISO 707 (recommended sampling methods for milk) - sampling guidance referenced by the method.
Keywords: ISO 13875:2005, β‑lactoglobulin, beta‑lactoglobulin, reverse‑phase HPLC, acid‑soluble, liquid milk, dairy testing, heat‑treated milk, HPLC method.
Open preview (PDF)Liquid milk — Determination of acid-soluble beta-lactoglobulin content — Reverse-phase HPLC method
