Anders Broberg

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

ABSTRACT We have isolated a Lactobacillus plantarum strain (MiLAB 393) from grass silage that produces broad-spectrum antifungal compounds, active against food- and feed-borne filamentous fungi and yeasts in a dual-culture agar plate assay. Fusarium sporotrichioides and Aspergillus fumigatus were the most sensitive among the molds, and Kluyveromyces marxianus was the most sensitive yeast species. No inhibitory activity could be detected against the mold Penicillium roqueforti or the yeast Zygosaccharomyces bailii. An isolation procedure, employing a microtiter well spore germination bioassay, was devised to isolate active compounds from culture filtrate. Cell-free supernatant was fractionated on a C18 SPE column, and the 95% aqueous acetonitrile fraction was further separated on a preparative HPLC C18 column. Fractions active in the bioassay were then fractionated on a porous graphitic carbon column. The structures of the antifungal compounds cyclo(l-Phe-l-Pro), cyclo(l-Phe-trans-4-OH-l-Pro) and 3-phenyllactic acid (l/d isomer ratio, 9:1), were determined by nuclear magnetic resonance spectroscopy, mass spectrometry, and gas chromatography. MIC values against A. fumigatus and P. roqueforti were 20 mg ml−1 for cyclo(l-Phe-l-Pro) and 7.5 mg ml−1 for phenyllactic acid. Combinations of the antifungal compounds revealed weak synergistic effects. The production of the antifungal cyclic dipeptides cyclo(l-Phe-l-Pro) and cyclo(l-Phe-trans-4-OH-l-Pro) by lactic acid bacteria is reported here for the first time.

Antifungal 3-hydroxy fatty acids from Lactobacillus plantarum MiLAB 14

ABSTRACT We report the identification and chemical characterization of four antifungal substances, 3-(R)-hydroxydecanoic acid, 3-hydroxy-5-cis-dodecenoic acid, 3-(R)-hydroxydodecanoic acid and 3-(R)-hydroxytetradecanoic acid, from Lactobacillus plantarum MiLAB 14. The concentrations of the 3-hydroxy fatty acids in the supernatant followed the bacterial growth. Racemic mixtures of the saturated 3-hydroxy fatty acids showed antifungal activity against different molds and yeasts with MICs between 10 and 100 μg ml−1.

Insight into trade???off between wood decay and parasitism from the genome of a fungal forest pathogen

Parasitism and saprotrophic wood decay are two fungal strategies fundamental for succession and nutrient cycling in forest ecosystems. An opportunity to assess the trade-off between these strategies is provided by the forest pathogen and wood decayer Heterobasidion annosum sensu lato. We report the annotated genome sequence and transcript profiling, as well as the quantitative trait loci mapping, of one member of the species complex: H. irregulare. Quantitative trait loci critical for pathogenicity, and rich in transposable elements, orphan and secreted genes, were identified. A wide range of cellulose-degrading enzymes are expressed during wood decay. By contrast, pathogenic interaction between H. irregulare and pine engages fewer carbohydrate-active enzymes, but involves an increase in pectinolytic enzymes, transcription modules for oxidative stress and secondary metabolite production. Our results show a trade-off in terms of constrained carbohydrate decomposition and membrane transport capacity during interaction with living hosts. Our findings establish that saprotrophic wood decay and necrotrophic parasitism involve two distinct, yet overlapping, processes.

Metabolite Profiles of Lactic Acid Bacteria in Grass Silage

ABSTRACT The metabolite production of lactic acid bacteria (LAB) on silage was investigated. The aim was to compare the production of antifungal metabolites in silage with the production in liquid cultures previously studied in our laboratory. The following metabolites were found to be present at elevated concentrations in silos inoculated with LAB strains: 3-hydroxydecanoic acid, 2-hydroxy-4-methylpentanoic acid, benzoic acid, catechol, hydrocinnamic acid, salicylic acid, 3-phenyllactic acid, 4-hydroxybenzoic acid, (trans, trans)-3,4-dihydroxycyclohexane-1-carboxylic acid, p-hydrocoumaric acid, vanillic acid, azelaic acid, hydroferulic acid, p-coumaric acid, hydrocaffeic acid, ferulic acid, and caffeic acid. Among these metabolites, the antifungal compounds 3-phenyllactic acid and 3-hydroxydecanoic acid were previously isolated in our laboratory from liquid cultures of the same LAB strains by bioassay-guided fractionation. It was concluded that other metabolites, e.g., p-hydrocoumaric acid, hydroferulic acid, and p-coumaric acid, were released from the grass by the added LAB strains. The antifungal activities of the identified metabolites in 100 mM lactic acid were investigated. The MICs against Pichia anomala, Penicillium roqueforti, and Aspergillus fumigatus were determined, and 3-hydroxydecanoic acid showed the lowest MIC (0.1 mg ml−1 for two of the three test organisms).

Application of nano-probe NMR for structure determination of low nanomole amounts of arabinoxylan oligosaccharides fractionated by analytical HPAEC-PAD.

A methodology for NMR analysis of low nanomole amounts of oligosaccharides fractionated by analytical HPAEC is presented. Arabinoxylan derived oligosaccharides purified by HPAEC-PAD on an analytical column, by single injections, were analyzed with nano-probe NMR and MALDI-TOF MS to provide full structural assignment. The NMR data were obtained with a 500 MHz NMR spectrometer equipped with a 1H-observe nano-probe. Both one- and two-dimensional experiments on arabinoxylan samples in the low nanomole range were performed, including 1H-1H DQF-COSY, 1H-1H TOCSY and 1H-1H ROESY. These experiments allowed, in combination with MALDI-TOF MS and literature NMR data, a complete structural determination of several tetra-, penta-, hexa- and heptasaccharides. Two new structures: alpha-L-Araf-(1 --> 2)-beta-D-Xylp-(1 --> 4)-beta-D-Xylp-(1 --> 4)-beta-D-Xylp-(1 --> 4)-D-Xylp and alpha-L-Araf-(1 --> 2)[alpha-L-Araf-(1 --> 3)]-beta-D-Xylp-(1 --> 4)-beta-D-Xylp-(1 --> 4)-beta-D-Xylp-(1 --> 4)-D-Xylp) were characterized, as well as some previously published structures.

Metabolite profiles of the biocontrol yeast Pichia anomala J121 grown under oxygen limitation.

Abstract The biocontrol yeast Pichia anomala J121 prevents mould growth during the storage of moist grain under low oxygen/high carbon dioxide conditions. Growth and metabolite formation of P. anomala was analyzed under two conditions of oxygen limitation: (a) initial aerobic conditions with restricted oxygen access during the growth period and (b) initial microaerobic conditions followed by anaerobiosis. Major intra- and extracellular metabolites were analyzed by high-resolution magic-angle spinning (HR-MAS) NMR and HPLC, respectively. HR-MAS NMR allows the analysis of major soluble compounds inside intact cells, without the need for an extraction step. Biomass production was higher in treatment (a), whereas the specific ethanol production rate during growth on glucose was similar in both treatments. This implies that oxygen availability affected the respiration and not the fermentation of the yeast. Following glucose depletion, ethanol was oxidized to acetate in treatment (a), but continued to be produced in (b). Arabitol accumulated in the culture substrate of both treatments, whereas glycerol only accumulated in treatment (b). Trehalose, arabitol, and glycerol accumulated inside the cells in both treatments. The levels of these metabolites were generally significantly higher in treatment (b) than in (a), indicating their importance for P. anomala during severe oxygen limitation/anaerobic conditions.

In-situ identification of major metabolites in the red alga Gracilariopsis lemaneiformis using high-resolution magic angle spinning nuclear magnetic resonance spectroscopy

Abstract. The content of low-molecular-weight compounds in the red alga Gracilariopsis lemaneiformis [(Bory) Dawson, Acleto, et Foldvik] has been analysed in-situ using high-resolution magic angle spinning (HR-MAS) nuclear magnetic resonance (NMR) spectroscopy. The major heteroside was shown to be floridoside, but digeneaside and isofloridoside were also detected in the alga. Other major components were isethionic acid and the amino acids taurine and citrulline. The results from the HR-MAS NMR analysis were confirmed with high-resolution NMR spectroscopy, high-resolution fast atom bombardment mass spectrometry (FABMS) and GC-MS, on material isolated from the studied alga, but also on authentic samples.

Presence of microthecin in the red alga Gracilariopsis lemaneiformis and its formation from 1,5-anhydro-D-fructose

Microthecin, a pyrone isolated from Gracilariopsis lemaneiformis, is formed from 1,5-anhydro-D-fructose through two sequential elimination reactions; presumably by the action of a single enzyme.

Biosynthesis of fomannoxin in the root rotting pathogen Heterobasidion occidentale

Fomannoxin is a biologically active benzohydrofuran, which has been suggested to be involved in the pathogenicity of the root rotting fungus Heterobasidion annosum sensu lato. The biosynthesis of fomannoxin was investigated through an isotopic enrichment study utilizing [1-¹³C]glucose as metabolic tracer. ¹³C NMR spectroscopic analysis revealed the labeling pattern and showed that the isoprene building block originates from the mevalonic acid pathway, whereas the aromatic motif is formed via the shikimic acid route by elimination of pyruvate from chorismic acid. A natural product, 4-hydroxy-3-(3-methylbut-2-enyl)benzaldehyde, was isolated and characterized, and was suggested to be a key intermediate in the biosynthesis of fomannoxin and related secondary metabolites previously identified from the H. annosum fungal species complex.

Viridin-like steroids from Hymenoscyphus pseudoalbidus

Three furanosteroids were isolated from the ash dieback causing fungus Hymenoscyphus pseudoalbidus along with the known compounds viridiol and demethoxyviridiol. The compounds were characterized by 1D and 2D NMR spectroscopy, LC-HRMS and polarimetry.