Microplate Turbidimetric Assay in Determining Milk-Clotting Activity of Enzyme Preparations

Milk-clotting activity (MCA) is a key quality indicator for enzymes used in cheese production. The traditional method for determining MCA (REMCAT) relies on visual detection of the clotting endpoint. Unfortunately, it is subjective and inefficient for analyzing large numbers of samples. Microplate turbidimetric methods can serve as an alternative, enabling objective and high-throughput MCA measurements. We analyzed the critical parameters governing the reaction (pH, buffer composition, temperature, calcium ion concentration, substrate concentration, and measurement wavelength) to select conditions for effective MCA determination. Our method is a simplified approach for determining the clotting time based on reaching a fixed optical density value. We tested the method using a recombinant chymosin preparation, CHY-MAX Extra. The experiment demonstrated a linear dependence of activity on enzyme concentration (R² > 0.98) in the range of 500–5,000 ng/ml. Calibration with a standard milk-clotting enzyme reference made it possible to express the results in conventional units. The method enables quantitative assessment of total MCA with minimal consumption of enzyme and substrate. The new protocol represents an effective tool for high-throughput screening of novel recombinant and natural milk-clotting enzymes, providing rapid analysis and straightforward data processing.