Purification and characterization of novel antifungal compounds from the sourdough Lactobacillus plantarum strain 21B

Abstract

Sourdough lactic acid bacteria were selected for antifungal activity by a conidial germination assay. The 10-fold-concentrated culture filtrate of Lactobacillus plantarum 21B grown in wheat flour hydrolysate almost completely inhibited Eurotium repens IBT18000, Eurotium rubrum FTDC3228, Penicillium corylophilum IBT6978, Penicillium roqueforti IBT18687, Penicillium expansum IDM/FS2, Endomyces fibuliger IBT605 and IDM3812, Aspergillus niger FTDC3227 and IDM1, Aspergillus flavus FTDC3226, Monilia sitophila IDM/FS5, and Fusarium graminearum IDM623. The nonconcentrated culture filtrate of L. plantarum 21B grown in whole wheat flour hydrolysate had similar inhibitory activity. The activity was fungicidal. Calcium propionate at 3 mg ml(-1) was not effective under the same assay conditions, while sodium benzoate caused inhibition similar to L. plantarum 21B. After extraction with ethyl acetate, preparative silica gel thin-layer chromatography, and chromatographic and spectroscopic analyses, novel antifungal compounds such as phenyllactic and 4-hydroxy-phenyllactic acids were identified in the culture filtrate of L. plantarum 21B. Phenyllactic acid was contained at the highest concentration in the bacterial culture filtrate and had the highest activity. It inhibited all the fungi tested at a concentration of 50 mg ml(-1) except for P. roqueforti IBT18687 and P. corylophilum IBT6978 (inhibitory concentration, 166 mg ml(-1)). L. plantarum 20B, which showed high antimold activity, was also selected. Preliminary studies showed that phenyllactic and 4-hydroxy-phenyllactic acids were also contained in the bacterial culture filtrate of strain 20B. Growth of A. niger FTDC3227 occurred after 2 days in breads started with Saccharomyces cerevisiae 141 alone or with S. cerevisiae and Lactobacillus brevis 1D, an unselected but acidifying lactic acid bacterium, while the onset of fungal growth was delayed for 7 days in bread started with S. cerevisiae and selected L. plantarum 21B.

FIG. 1

Kinetics of growth, fermentation end products (lactic and acetic acids and ethanol), and antifungal activity of Lactobacillus plantarum 21B. Antifungal activity is expressed as percent inhibition of spore germination of Endomyces fibuliger IBT605. Each value is the mean from three replicates ± standard deviation.

FIG. 2

GC profiles of ethyl acetate extract (a) of culture filtrate from Lactobacillus plantarum 21B and (b) of active fraction C obtained from its purification. Metabolites were identified by GC/MS analysis and compared with standard samples.

FIG. 3

Growth of Aspergillus niger FTDC3227 in bread started with S. cerevisiae 141 and Lactobacillus plantarum 21B after 7 days of storage (A) and in bread started with Saccharomyces cerevisiae 141 alone after 2 days of storage (B).