## 3 Results and Discussion
### Antibacterial and Antifungal Activity of Bacillus amyloliquefaciens \(\mathrm{M}\)
amyloliquefaciens_ M is a promising biocontrol agent against phytopathogenic fungi. The antibiotic substances produced by _B. amyloliquefaciens_ M are best secreted into the medium at pH = 5 or are associated with the cell surface. Determination of the R\({}_{\rm f}\) Value of the Antibiotic Substances Produced by B. amyloliquefaciens M  
B. amyloliquefaciens M synthesizes antibiotic substances with antibacterial and antifungal activity. The antibiotic substances were separated in the solvent system: n-butanol/acetic acid/water (3:1:1, \(v/v/v\)) by chromatographic analysis. Spots with the following R\({}_{\rm f}\) values were found on the thin-layer chromatogram: 4 spots with the following R\({}_{\rm f}\) values--0.47 (inhibyrin-positive), 0.55 (inhibyrin-positive), 0.67 (inhibyrin-negative), and 0.75 (inhibyrin-negative). Two spots with R\({}_{\rm f}\) values of 0.47 (inhibyrin-positive) and 0.55 (inhibyrin-positive) were determined on the biochromatogram of _B. amyloliquefaciens_ M with _Aspergillus flavus_ ATCC 9643. The antibiotic substances formed were peptides, as detected with ninhydrin to establish a proteinaceous component by the characteristic pink-violet color. The spots with R\({}_{\rm f}\) values R\({}_{\rm f}\) = 0.67 and R\({}_{\rm f}\) = 0.75 were ninhydrin-negative, but according to [PERSON] and [PERSON], 1998 [38], the antibiotics belong to the iturnis group. Both substances with R\({}_{\rm f}\) values of 0.47 and 0.55 were close to those obtained by [PERSON] et al., 1986, [PERSON], 1991 [39; 40], in the biochromatogram demonstrated to be active against _Aspergillus flavus_ ATCC 9643, ninhydrin-positive and, according to the authors, were cyclic peptides and were very similar in properties to iturin A produced by _Bacillus subtilis_. As a result of these experimental studies, it has been proven that the antibiotic substances produced by _B. amyloliquefaciens_ M were proteinaceous substances. Sephadex G200 gel (Hangzhou J&H Chemical Co., Ltd., Hangzhou, China) chromatography of the acellular supernatant (ASN) obtained as described in Section 2.9 was performed, and 4 peaks were detected (Figure 1). SDS polyacrylamide gel electrophoresis (SDS-PAGE) was performed on the resulting four peaks. The fractional profiles of the four peaks and their R\({}_{\rm f}\) values as well as the share of individual protein fractions in the total protein profile were determined (Figures 2-4). Figure 1: Elution profile in Sephadex G200 gel chromatography. Figure 3: Fractional profiles of the obtained 4 peaks (1—peak II; 2—peak III; 3—peak IV; 4—peak I; 5—markers). (1—250 kDa; 2—150 kDa; 3—100 kDa; 4—75 kDa; 5—50 kDa; 6—37 kDa; 7—25 kDa; 8—20 kDa; 9—15 kDa; 10—10 kDa). Figure 2: SDS-PAGE of the obtained 4 peaks: 1—peak II; 2—peak III; 3—peak IV; 4—peak I; 5—markers, MW, kDa. Significant differences were observed in the profiles of the four peaks, with the majority of fractions eluting in the first two peaks (Figure 2). Peak 1 and peak 2 included 6 fractions each, the highest molecular weight being registered in peak 1 and having a molecular weight (Mw) of 96 kDa. In contrast, in peak 2, the highest molecular weight fraction was in the low molecular weight range with Mw = 49 kDa. A serious impression makes the high share of the lowest molecular weight fraction (Mw = 10 kDa)--68.25% compared to the other five fractions (Figure 4). The fractions with molecular weights of 16 kDa and 21 kDa, which occupied 10.44% and 11.77%, respectively, were in a higher quantity at this peak. As the molecular weight of this peak increased, the amounts of the fractions (Mw = 26, Mw = 40, and Mw = 49 kDa) decreased. The comparison in the percentage ratio of individual fractions at peak I and peak II from Figure 4a,d shows that, unlike peak II, peak I was dominated by the highest molecular weight fraction (Mw = 96 kDa)--25.57%, followed by the next one with higher molecular weight (Mw = 29 kDa)--19.22%. The lowest molecular weight fractions at peak I (Mw = 15 and Mw = 17 kDa) occupied approximately the same share--16.98% and 16.16%.