*4.4. Lysozyme-Like Activity*

Normally, a spectrophotometric method is used to demonstrate the occurrence of lysozyme activity [17]; however, the standard assay on inoculated Petri dishes can be used as an alternative method to demonstrate the occurrence of lysozyme activity [18]. Here, the presence of lysozyme activity was detected by the standard assay on Petri dishes, which resulted as a quick, sensitive, low-cost, and therefore very versatile method [16,18,20,77–79]. Briefly, 700 μL of 5 mg/mL of dried *Micrococcus luteus* cell walls (Sigma, Saint Louis, MO, USA) were suspended in 7 mL of 0.05 M PB-agarose (1.2%, pH 5.0) then spread on a Petri dish. Four wells of 6.3 mm diameters were sunk in agarose gel and each filled with 30 μL of sample (oocyte lysate). After overnight incubation at 37 ◦C, the diameter of the cleared zone of at least four replicates was measured. Diameters of lysis were compared with those of reference obtained with known amounts of standard hen egg-white lysozyme (Merck, Darmstadt, Germany). The effects of pH, ionic strength (I), and temperature were assessed for each sample. The pH effect was tested by dialyzing (7000-MW cut-off), the samples in PB 0.05 M, ionic strength, I = 0.175, adjusted at pH 4, 5, 6, 7, 8, and by dissolving agarose in PB at the same I and pH values. The ionic strength effect was tested in PB 0.05 M (pH 6.0), adjusted at I = 0.0175, 0.175, 1.75. Agarose was dissolved in PB at the same I values. The temperature effect was evaluated by performing the Petri dish assays (in PB, at pH 6.0, and I = 0.175) and incubating the plates at 5, 15, 22, and 37 ◦C. The dose-response curve of lysozyme-like activity was constructed by using Petri dish assays (in PB, at pH 6.0, and I = 0.175) with different amounts of sample (10, 20, 30, 40, 50, 60, or 80 μL of sample in each well in triplicate).
