ISO 15163:2012

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.