Abstract
In this study the efficacy of two different methods for extracting lipopeptides produced by five Bacillus strains-ethyl acetate extraction, and acid precipitation followed by methanol extraction—was investigated using mass spectrometry. High performance thin layer chromatography (HPTLC) was also used for the simultaneous separation of complex mixtures of lipopeptide extracts and for the determination of antimicrobial activity of their components. The mass spectra clearly showed well-resolved groups of peaks corresponding to different lipopeptide families (kurstakins, iturins, surfactins, and fengycins). The ethyl acetate extracts produced the most favorable results. The extracts of SS-12.6, SS-13.1, and SS-38.4 showed the highest inhibition zones. An iturin analog is responsible for the inhibition of Xanthomonas arboricola and Pseudomonas syringae phytopathogenic strains. HPTLC bioautography effectively identified the active compounds from a mixture of lipopeptide extracts, proving in situ its potential for use in direct detection and determination of antimicrobials. In the test of potential synergism among individual extracts used in different mixtures, stronger antimicrobial effects were not observed. Biochemical and phylogenetic analysis clustered isolates SS-12.6, SS-13.1, SS-27.2, and SS-38.4 together with Bacillus amyloliquefaciens, while SS-10.7 was more closely related to Bacillus pumilus.
Highlights
Environmental contamination caused by the widespread use of pesticides and the appearance of multiple resistant strains has led to public awareness, and a demand for a reduction in the use of artificial chemical compounds
Five lipopeptide-producing Bacillus strains (SS-10.7, SS-12.6, SS13.1, SS-27.2, and SS-38.4) were grown under pre-established conditions in LB medium for 24 h and their extracts obtained either by ethyl acetate extraction or by a combination of acid precipitation followed by methanol extraction
Analysis of the mass spectra of all the extracts tested, extracts of isolates SS-10.7, SS-27.2, and SS-38.4, clearly showed distinct groups of peaks at m/z = 850–950 which correspond to the kurstakin family, m/z = 1000– 1150 which includes representatives of the surfactin and iturin families, and m/z = 1,450–1,550 which represents the fengycin family
Summary
Environmental contamination caused by the widespread use of pesticides and the appearance of multiple resistant strains has led to public awareness, and a demand for a reduction in the use of artificial chemical compounds. The ability to produce various antimicrobial substances, and their sporulation capacity, give Bacillus strains a significant advantage in terms of their survival in different habitats. Bacillus species produce a wide array of biologically active compounds, including polyketides, lipopeptides, siderophores, and peptides (Stein, 2005; Ongena and Jacques, 2008; Hamdache et al, 2011). Many studies have shown that lipopeptides from the fengycin, surfactin, and iturin families have significant potential for fighting plant pathogens (Stein, 2005; Romero et al, 2007; Ongena and Jacques, 2008; Raaijmakers et al, 2010; Roongsawang et al, 2010; Romano et al, 2011; Dimkicet al., 2013, 2015).
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