Overview
EN ISO 11702:2016 - "Animal and vegetable fats and oils - Enzymatic determination of total sterols content" - specifies an enzymatic colourimetric method for quantifying total sterols (free and esterified) in animal and vegetable fats and oils, fatty foods and related products. The method is based on enzymatic oxidation of 3β-hydroxysterols and spectrophotometric measurement of the resulting dye at 405 nm. It applies to test portions containing 1 g to 2 g of fat. Note: the method is not applicable to dark-coloured fats and oils and excludes milk and milk products (and fat from them). The enzyme used oxidizes cholesterol and other 3-hydroxysterols, so results represent total sterols rather than cholesterol only.
Key topics and technical requirements
- Principle: Saponification → extraction of unsaponifiable matter → enzymatic oxidation by cholesterol oxidase → H2O2-mediated formation of a yellow lutidine dye (3,5-diacetyl-1,4-dihydrolutidine) → spectrophotometric measurement at 405 nm.
- Sample size: 1–2 g fat (adjust to give sterol concentration 0.02–0.4 g/L in the test solution).
- Reagents & enzymes: cholesterol oxidase (e.g., from Nocardia erythropolis), catalase, acetylacetone, methanolic KOH, ammonium phosphate buffer; solvents such as isopropanol and acetone.
- Apparatus: spectrophotometer (405 nm), one‑mark volumetric flasks, reflux equipment for saponification, fluted filters.
- Limitations:
- Not suitable for dark-coloured fats and oils.
- Enzyme is not cholesterol-specific - it detects other 3-hydroxysterols (phytosterols included).
- Not validated for products fortified with high levels of sterols.
- Milk fats excluded.
- Quality and reporting: the standard includes requirements for sample preparation (ISO 661 referenced), precision information from interlaboratory testing, and instructions for test reporting.
Applications and users
- Food and oil laboratories performing routine sterol analysis in edible oils, fats, margarines and fatty food products.
- Quality control teams in vegetable oil processors, animal fat processors and food manufacturers.
- Regulatory and compliance agencies assessing sterol content for labelling and compositional standards.
- Research and development groups studying sterol profiles or monitoring phytosterol enrichment (with caution - method not validated for highly fortified products).
Keywords: EN ISO 11702:2016, enzymatic determination, total sterols, cholesterol oxidase, spectrophotometric assay, animal and vegetable fats and oils, fat analysis, food testing.
Related standards
- ISO 661 - Preparation of test sample (fats & oils)
- ISO 3696 - Water for analytical laboratory use
- Method equivalence noted: IUPAC method 2.404 and DGF standard method F‑III 2 (91)
