ISO 27105:2009 — Milk and Cheese — Determination of Lysozyme by HPLC

1. Scope and Principle of Lysozyme Determination in Dairy Products

ISO 27105:2016 specifies a high-performance liquid chromatography (HPLC) method for determining hen’s egg white lysozyme content in milk and cheese. Lysozyme, also known as muramidase, is a natural antimicrobial enzyme widely used as a preservative in the dairy industry, particularly in cheese production to prevent late blowing caused by Clostridium tyrobutyricum spores. The method was developed jointly by ISO and the International Dairy Federation (IDF), published as a dual-numbered standard ISO 27105|IDF 216, representing international consensus on analytical methodology for this important dairy additive.

Lysozyme is Generally Recognized as Safe (GRAS) and is preferred over chemical preservatives in many cheese varieties, especially in organic and natural product categories. Its concentration in dairy products typically ranges from 5 to 50 mg/L in milk to 100 to 500 mg/kg in cheese, depending on the dosage rate and cheese type. Accurate determination is critical for both regulatory compliance and quality control — insufficient lysozyme risks late blowing defects while excess levels increase production costs unnecessarily. The HPLC method provides the specificity needed to distinguish intact lysozyme from degraded or denatured forms that may result from processing or aging.

2. Sample Preparation and Chromatographic Procedure

Sample preparation differs between milk and cheese matrices reflecting their different compositions. Milk samples are simply diluted with water and filtered through a 0.45 micrometer membrane filter prior to injection — a straightforward procedure due to milk’s liquid nature and relatively simple matrix. Cheese samples require more extensive preparation: the cheese is grated or homogenized, extracted with a specific buffer solution containing sodium chloride and phosphate at defined pH, followed by centrifugation and filtration to remove fat and insoluble proteins. The extraction efficiency depends critically on buffer composition, pH, temperature, and extraction time, and should be verified using spiked samples.

Chromatographic separation uses reversed-phase HPLC with a C18 column and a gradient of acetonitrile and water containing 0.1 percent trifluoroacetic acid as the ion-pairing agent. Lysozyme is detected by UV absorbance at 220 nm, which provides good sensitivity for the peptide bonds and aromatic amino acids in the protein. Quantification uses either single-point or multi-point calibration depending on the expected concentration range, with purified lysozyme standard of known purity used for calibration. The standard reports precision data from interlaboratory tests with repeatability relative standard deviation of 2 to 6 percent and reproducibility relative standard deviation of 5 to 12 percent depending on concentration and matrix type.

The chromatographic conditions must be optimized to achieve baseline separation of lysozyme from matrix components. Co-eluting peaks from milk proteins or cheese degradation products can interfere with quantification. The standard includes a verification procedure using LC-MS/MS in Annex A for confirmatory analysis when ambiguous results are obtained — particularly valuable for processed cheese or heat-treated dairy products where lysozyme denaturation may have occurred. System suitability criteria include retention time stability within 2 percent, peak asymmetry factor between 0.8 and 1.5, and resolution from nearest peak of at least 1.5.

3. Engineering Insights for Dairy Quality Control Laboratories

Accurate lysozyme quantification is critical for both regulatory compliance and process control. In cheese production, insufficient lysozyme allows Clostridium tyrobutyricum spores to germinate and produce gas, causing late blowing defects characterized by undesirable texture changes, off-flavours, and even can bulging in processed cheese products. Excess lysozyme unnecessarily increases production costs — at typical usage levels of 5 to 25 mg per liter of milk, even small overdosing significantly impacts profitability in large-scale operations. The HPLC method provides the specificity to measure only active lysozyme protein, unlike some microbiological assays that may overestimate due to other antimicrobial compounds present.

Method performance depends heavily on proper column maintenance and mobile phase preparation. The C18 column is subject to fouling from hydrophobic milk proteins and peptides that may not elute under the gradient conditions. Regular column cleaning with strong solvent washes and periodic replacement of the guard column are essential maintenance practices. Laboratories should establish system suitability criteria including retention time stability, peak symmetry, and resolution from adjacent peaks. Regular analysis of a quality control sample — a bulk milk or cheese sample with known lysozyme content stored in aliquots — is recommended to monitor method performance over time and detect drift before it affects routine results.

The standard’s interlaboratory precision data provides a benchmark for laboratory performance assessment. Laboratories should establish their own precision parameters through method validation and ongoing quality control, comparing their performance to the published RSDr and RSDR values. If a laboratory’s precision is significantly worse than the published values, it indicates problems with sample preparation, chromatography, or calibration that require investigation. The method is suitable for milk and cheese within its scope — applicability to other dairy products such as yogurt, cream, or whey products should be verified through method validation including recovery and precision studies before routine use.

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