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 analogue 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.

New information has come to light about the biological activity of propolis and the quality of natural products which requires a rapid and reliable assessment method such as High Performance Thin-Layer Chromatography (HPTLC) fingerprinting. This study investigates chromatographic and chemometric approaches for determining the antimicrobial activity of propolis of Serbian origin against various bacterial species. A linear multivariate calibration technique, using Partial Least Squares, was used to extract the relevant information from the chromatographic fingerprints, i.e. to indicate peaks which represent phenolic compounds that are potentially responsible for the antimicrobial capacity of the samples. In addition, direct bioautography was performed to localize the antibacterial activity on chromatograms. The biological activity of the propolis samples against various bacterial species was determined by a minimum inhibitory concentration assay, confirming their affiliation with the European poplar type of propolis and revealing the existence of two types (blue and orange) according to botanical origin. The strongest antibacterial activity was exhibited by sample 26 against Staphylococcus aureus, with a MIC value of 0.5 mg/mL, and Listeria monocytogenes, with a MIC as low as 0.1 mg/mL, which was also the lowest effective concentration observed in our study. Generally, the orange type of propolis shows higher antimicrobial activity compared to the blue type. PLS modelling was performed on the HPTLC data set and the resulting models might qualitatively indicate compounds that play an important role in the activity exhibited by the propolis samples. The most relevant peaks influencing the antimicrobial activity of propolis against all bacterial strains were phenolic compounds at R_F values of 0.37, 0.40, 0.45, 0.51, 0.60 and 0.70. The knowledge gained through this study could be important for attributing the antimicrobial activity of propolis to specific chemical compounds, as well as the verification of HPTLC fingerprinting as a reliable method for the identification of compounds that are potentially responsible for antimicrobial activity. This is the first report on the activity of Serbian propolis as determined by several combined methods, including the modelling of antimicrobial activity by HPTLC fingerprinting.