Overview
ISO 15163:2012 - "Milk and milk products - Calf rennet and adult bovine rennet - Determination by chromatography of chymosin and bovine pepsin contents" - is a reference analytical method published jointly by ISO and IDF. It specifies a chromatography-based procedure to quantify the amounts of chymosin and bovine pepsin in calf and adult bovine rennets, and can be applied to mixtures containing fermentation-produced bovine chymosin (FPC). Results are reported as percentage activities or as mg/L of active enzyme, supporting interpretation of milk‑clotting characteristics relevant to cheese production.
Key topics and technical requirements
- Analytical principle: Desalting of rennet followed by separation of enzymes using anion-exchange chromatography; individual fractions are assayed for milk‑clotting activity using ISO 11815/IDF 157.
- Manual and automated options: Procedures are provided for a manual Fractogel DEAE column setup and an automated set-up (e.g., Mono Q, FPLC/ÄKTA).
- Reagents and buffers: Specified reagents include piperazine buffers, graded NaCl elution buffers, urea and ethanol options for column treatment; buffer preparation, degassing and preservation guidance are included.
- Apparatus: Typical laboratory glassware, pH meter, peristaltic pump (manual), chromatography column or prepacked Mono Q (automated), and equipment for clotting-time determination.
- Procedure elements: Sampling guidance (reference to ISO 707), desalting (dialysis or desalting columns), column preparation/regeneration, fraction collection, and clotting time determination.
- Calculations and reporting: Conversion of clotting activity into International Milk‑Clotting Units (IMCU), expression of chymosin/pepsin as % of total activity or mg/L active enzyme; requirements for test reports.
- Quality and performance: Method precision data, including repeatability and reproducibility from interlaboratory tests, are provided. The standard is a reference method - changes require validation and documentation in the test report.
- Annexes: Informative annexes cover qualitative checks (double immunodiffusion) and interlaboratory test data.
Practical applications and users
- Dairy laboratories performing quality control of liquid and powdered rennets.
- Cheese manufacturers evaluating enzyme composition to predict curd formation, proteolysis and product suitability.
- Rennet and enzyme suppliers verifying composition of calf/bovine rennet and FPC-containing blends.
- Regulatory and standards bodies requiring traceable, reproducible quantification of milk-clotting enzymes.
- Research groups studying the effects of enzyme composition on cheesemaking performance.
Related standards and references
- ISO 11815 / IDF 157 - Determination of total milk‑clotting activity (used to assay separated fractions).
- ISO 707 / IDF 50 - Recommended sampling methods for rennet products.
- The standard also notes reference rennet powders with total activity fixed at 1 000 IMCU/g for calibration consistency.
Keywords: ISO 15163:2012, chymosin, bovine pepsin, calf rennet, chromatography, milk-clotting activity, FPC, anion exchange, dairy quality control.
Open preview (PDF)Milk and milk products — Calf rennet and adult bovine rennet — Determination by chromatography of chymosin and bovine pepsin contents
