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Bioassay-guided isolation and characterisation of antifungal metabolites

studies of lactic acid bacteria and propionic acid bacteria

Sjögren, Jörgen (2005). Bioassay-guided isolation and characterisation of antifungal metabolites. Diss. (sammanfattning/summary) Uppsala : Sveriges lantbruksuniv., Acta Universitatis Agriculturae Sueciae, 1652-6880 ; 2005:17
ISBN 91-576-7016-1
[Doctoral thesis]

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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.

Authors/Creators:Sjögren, Jörgen
Title:Bioassay-guided isolation and characterisation of antifungal metabolites
Subtitle:studies of lactic acid bacteria and propionic acid bacteria
Series Name/Journal:Acta Universitatis Agriculturae Sueciae
Year of publishing :2005
Number of Pages:65
ALLI. Ström, K., J. Sjögren, A. Broberg & J. Schnürer. 2002. Lactobacillus plantarum MiLAB 393 produces the antifungal cyclic dipeptides cyclo(L-Phe-L-Pro) and cyclo(L-Phe-trans-4-OH-L-Pro) and 3-phenyllactic acid. Applied and Environmental Microbiology 68, 4322-4327. II. Magnusson, J., K. Ström, S. Roos, J. Sjögren & J. Schnürer. 2003. Broad and complex antifungal activity among environmental isolates of lactic acid bacteria. FEMS Microbiology Letters 219, 129-135. III. Sjögren, J., J. Magnusson, A. Broberg, J. Schnürer & L. Kenne. 2003 Antifungal 3-hydroxy fatty acids from Lactobacillus plantarum MiLAB 14. Applied and Environmental Microbiology 69, 7554-7557. IV. Sjögren, J., H. Lind, A. Broberg, J. Schnürer & L. Kenne. Antifungal metabolites from the type strains of five propionibacteria species.Submitted to Applied and Environmental Microbiology. V. Lind, H., J. Sjögren, A. Broberg, S. Gohil, L. Kenne & J. Schnürer. Antifungal peptides in the sodium lactate medium for dairy propionibacteria. Submitted to Journal of Agricultural and Food Chemistry.
Place of Publication:Uppsala
ISBN for printed version:91-576-7016-1
Publication Type:Doctoral thesis
Full Text Status:Public
Agris subject categories.:X Agricola extesions > X30 Life sciences
Subjects:Not in use, please see Agris categories
Agrovoc terms:lactobacillus, propionibacterium, lactic acid bacteria, propionic acid bacteria, metabolites, antifungal properties, bacteriocins, moulds, bioassays, hplc, nmr spectroscopy, mass spectrometry
Keywords:Diketopiperazine, cyclic dipeptides, lactic acid bacteria, propionibacteria, hydroxy fatty acids, HPLC-MS, NMR, metabolites, antifungal, mould, phenyllactic acid
Permanent URL:
ID Code:798
Department:(NL, NJ) > Dept. of Chemistry (until 131231)
Deposited By: Jörgen Sjögren
Deposited On:24 Mar 2005 00:00
Metadata Last Modified:02 Dec 2014 10:07

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