Epsilon Open Archive

Abstract

A bioassay-guided isolation method was developed with Aspergillus fumigatus and Rhodotorula mucilaginosa as target organisms. The bioassay was a dilution bioassay and was performed after each fractionation step. The isolation procedure was based on fractionation of cell-free supernatants using solid phase extraction (SPE) and reversed-phase high performance liquid chromatography (RP-HPLC). Cell-free supernatant was fractionated on a C18 SPE column, and the 95% aqueous acetonitrile fraction was further fractionated on a preparative HPLC C18 column. Fractions active in the bioassay were further separated using preparative HPLC with a C18 column or a porous graphitized carbon column. In the second HPLC step the conditions were optimised for each active fraction. The method was used for analysis of cell-free supernatants from strains of lactic acid bacteria (LAB), propionic acid bacteria (PAB), and uninoculated sodium lactate (SL) medium. The structures of the isolated antifungal metabolites were characterised using nuclear magnetic resonance (NMR), mass spectrometry (MS) and gas chromatography-mass spectrometry (GC-MS). The studies of LAB strains resulted in the isolation and identification of several antifungal diketopiperazines (DKPs); cyclo(L-Leu-cis-4-OH-D-Pro), cyclo(L-Leu-trans-4-OH-L-Pro), cyclo(L-Phe-cis-4-OH-D-Pro), cyclo(L-Phe-trans-4-OH-L-Pro) and cyclo(L-Phe-L-Pro), and 3-hydroxy fatty acids (3-OH-FAs); (R)-3-hydroxydecanoic acid, (R)-3-hydroxydodecanoic acid, (R)-3-hydroxytetradecanoic acid and 3-hydroxy-5-cis-dodecenoic acid, and D-3-phenyllactic acid (PLA) and L-3-PLA. The antifungal metabolites isolated or identified from dairy PAB were; cyclo(L-Phe-L-Pro) and cyclo(L-Ile-L-Pro) and D-3-PLA and L-3-PLA. From SL medium, used for cultivation of dairy PAB, seven antifungal peptides, His-Pro-Leu-Pro-Leu, Phe-Leu-Pro-Tyr-Pro, Gly-Pro-Phe-Pro-Ile, Gly-Pro-Phe-Pro-Leu, Gly-Pro-Phe-Pro-Leu-Val, Val-Tyr-Pro-Phe-Pro-Gly-Pro-Ile and Val-Ala-Pro-Phe-Gly-Val-Ala-Val-Phe-Gly were isolated and characterised by FAB-MS/MS. The minimal inhibitory concentration (MIC) against A. fumigatus was 20 mg/ml for cyclo(L-Phe-L-Pro) and cyclo(L-Ile-L-Pro), 25 µg/ml for 3-OH-dodecanoic acid, 100 µg/ml for 3-OH-decanoic acid and 7.5 mg/ml for PLA. A high performance liquid chromatography electrospray mass spectrometry (HPLC-ES-MS) method was developed for identification of DKPs in a complex cultivation medium. For determination of the absolute configuration of 3-OH-FAs a GC-MS method was developed. The method was based on the analysis of a 3-O-trimethylsilyl N-(S)-phenylethylamide derivatives of the isolated 3-OH-FAs.