Antifungal activity of lactic acid bacteria

Enrichment culture techniques produced more than 1200 isolates of lactic acid bacteria (LAB) that were screened for antifungal activity against the indicator mould Aspergillus fumigatus. Approximately 10% of the LAB were active, but only 4% had medium or strong activity in an agar plate assay. The majority of isolates with strong antifungal activity were Lactobacillus coryniformis strains, but Lactobacillus plantarum and Pediococcus pentosaceus were also frequently identified. Some of the isolates lost activity during storage but most maintained their fungal inhibitory effect. Large variations in sensitivity were observed between different moulds and yeasts. Antifungal cyclic dipeptides and phenyllactic acid were detected in culture filtrates from several of the LAB isolates. Lactobacillus coryniformis subsp. coryniformis strain Si3 produced an antifungal compound that lost activity when treated with proteinases. The antifungal peptide(s) was heat stable, with a size of approx. 3kDa and had maximum activity at pH 3.0 to 4.5. Addition of ethanol to the growth medium of strain Si3 prevented a decline in observed antifungal activity during the stationary phase. Glycerol addition to agar plates with L. coryniformis strains, overlaid with soft agar suspensions of yeast cells or fungal spores, strongly enhanced the antifungal effect. This was particularly true with spoilage moulds and yeasts, e.g. Penicillium roqueforti and Pichia anomala, not normally affected by the antifungal metabolites of L. coryniformis. Chemical and genetic data suggested that reuterin (3-hydroxypropionaldehyde) was the cause of this effect. The glycerol/diol dehydratase operon of L. coryniformis was partially elucidated and found to be similar to that Lactobacillus collinoides. Bioassay-guided isolation of new metabolites from LAB revealed that Lactobacillus plantarum MiLAB 14 produces hydroxylated fatty acids with strong antifungal effects. 3-Hydroxydecanoic acid, 3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid and 3-hydroxy-5-cis-dodecenoic acid were characterized from the supernatant of MiLAB 14. The hydroxy fatty acids had total inhibitory effects in the range 10 to >100 µg ml-1 against several moulds and yeasts.