Kunitz method : 

The enzyme assay for the determination of proteolytic activity with casein as substrate was measured according to the method of Kunitz (1965). Reaction mixture contained 1 mL of 1% casein (pH 7.2), 1 mL of culture filtrate and 1 mL of KH2PO4/NaOH buffer (50 mM, pH 7.2). This mixture was incubated for 30 min at 40°C in a water bath. The enzyme reaction was stopped by the addition of 3 ml of 5% trichloroacetic acid (TCA) then placed in the freezer for 10 min and then filtered. A control was run in identical manner except that 3 ml of TCA was added before incubation.

All assays were made in triplicate and average of the three was taken to evaluate the activity units. The absorbance of TCA soluble fraction was read at A 280 nm in 'UV- visible spectrophotometer. One unit of enzyme caseinolytic activity is defined as the amount of enzyme which releases 1 ug of tyrosine under the standard conditions (pH, 7.2, 40 ° C, 30 min)