We have isolated a Lactobacillus plantarum strain (MiLAB 393) from grass silage that produces broadspectrum antifungal compounds, active against food-and feed-borne filamentous fungi and yeasts in a dualculture 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 C 18 SPE column, and the 95% aqueous acetonitrile fraction was further separated on a preparative HPLC C 18 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.
More than 1200 isolates of lactic acid bacteria isolated from different environments were screened for antifungal activity in a dualculture agar plate assay. Approximately 10% of the isolates showed inhibitory activity and 4% showed strong activity against the indicator mould Aspergillus fumigatus. The antifungal spectra for 37 isolates with strong activity and five isolates with low or no activity were determined. Several of the strains showed strong inhibitory activity against the moulds A. fumigatus, Aspergillus nidulans, Penicillium commune and Fusarium sporotrichioides, and also against the yeast Rhodotorula mucilaginosa. Penicillium roqueforti and the yeasts Pichia anomala and Kluyveromyces marxianus were not inhibited. Several isolates showed reduced antifungal activity after storage and handling. The majority of the fungal inhibitory isolates were identified by 16S rDNA sequencing as Lactobacillus coryniformis. Lactobacillus plantarum and Pediococcus pentosaceus were also frequently identified among the active isolates. The degree of fungal inhibition was not only related to production of lactic or acetic acid. In addition, antifungal cyclic dipeptides were identified after HPLC separation and several other active fractions were found suggesting a highly complex nature of the antifungal activity.
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).Postharvest spoilage of food and animal feed by molds and yeasts is a problem worldwide. Apart from giving the food and feed an unpleasant smell, taste, or appearance, these fungi may also produce a wide array of mycotoxins, making the food and animal feed unsuitable for consumption. One traditional way of controlling pathogenic fungi in food and animal feed is to use lactic acid bacteria (LAB) to produce a variety of fermented foodstuffs, including sauerkraut and a multitude of dairy products, as well as silage for use as animal feed (27). This biological approach to control spoilage fungi is particularly interesting because of the general desire to reduce the use of chemical preservatives and fungicides in the food and feed industries, as well as in agriculture. Moreover, many LAB are generally regarded as safe, which makes these organisms particularly suitable for use in food and animal feed applications. In silage making, often the LAB naturally present in the crop are enough to initiate fermentation. However, the use of a biological silage additive will ensure that a high enough number of LAB always is present. With an additive, it is also possible to use strains with desired properties, such an antifungal activity, to further improve the final product.The antimicrobial activity of LAB is commonly explained by the synthesis of small organic acids such as lactic, acetic, and formic acids, which may exert the biological effect either directly or by acidification of the growth medium (reviewed in reference 23). Other substances produced by LAB have been reported to have antimicrobial effects, e.g., 2,3-butadione, reuterin (3-hydroxypropionaldehyde...
The existence of a second IgG-binding protein, protein Sbi, in Staphy/ococcus aureus has been reported previously. Later data indicated that protein Sbi also bound another serum component. This component has now been affinitypurified on immobilized protein Sbi and identified as P,-glycoprotein I (/?,-GPI), also known as apolipoprotein H. The minimal P,-GPI-binding domain was identified by shotgun phage display and the binding was shown to be mediated by a region of 57 amino acids, clearly separated from the IgGbinding domain. It is also shown that protein Sbi, and thus the P,-GPI-binding activity, is expressed on the staphylococcal cell surface at levels varying between strains.
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