*4.5. Enzymatic Activity of Bacillus strains*

The enzymatic activity of antagonistic bacterial strains was carried out using generally accepted methods [64]. Chitinase, lipase, and protease activity was determined. To determine the chitinolytic activity, a synthetic medium of the following composition was used, g/l: sucrose—20.0; NaNO3—3.0; KH2PO4—1.0; MgSO4—0.3; chalk—10.0; and agar—20.0. The medium was sterilized by autoclaving, poured into Petri dishes, and cooled. Inoculating of bacterial strains was performed by streaking. Petri dishes were incubated for 7–10 days at a temperature of +28.0 ◦C. Chitinase activity was judged by the formation of clearing zones around the colonies.

Lipolytic activity was determined on yolk agar of the following composition, g/l: peptone—40.0; glucose—2.0; Na2HPO4—5.0; NaCl—2.0; MgSO4 0.5% solution—2.0 mL; and agar—25.0. 4. The medium was sterilized by autoclaving and cooled to +60.0 ◦C. The egg shell was disinfected with alcohol and allowed to dry. The egg was broken and the yolk was separated from the egg white. The yolk, in compliance with the rules of asepsis, was transferred into molten agar and stirred until a homogeneous suspension was obtained, which was poured into Petri dishes and left to solidify. Inoculating of bacterial strains was performed by streaking. Petri dishes were incubated for 14 days at a temperature of +28.0 ◦C. Then the lid of the Petri dish was removed and the surface was carefully examined under oblique illumination. Lipolytic activity was judged by the formation of an oily, glistening, or nacreous layer above and around the bacterial colony on the agar surface.

To determine protease activity, sterile (autoclaved) skim milk was mixed at +50.0 ◦C with an equal volume of 4% molten aqueous agar. Inoculating of bacterial strains was performed by streaking. Petri dishes were incubated for 14 days. Protease activity was judged by the formation of clearing zones around the colonies.

Gelatinase activity was tested on meat-peptone gelatin, g/l: meat-peptone broth—39.0; and gelatin—150.0. The medium was poured into test tubes, sterilized by autoclaving, and cooled at room temperature. Inoculating of bacterial strains was carried out by injection. The tubes were incubated for 7–10 days at room temperature. The liquefaction of the gelatin was observed visually. The intensity and form of liquefaction were indicated.
