Antifungal hydroxy fatty acids produced during sourdough fermentation: microbial and enzymatic pathways, and antifungal activity in bread

Abstract

Lactobacilli convert linoleic acid to hydroxy fatty acids; however, this conversion has not been demonstrated in food fermentations and it remains unknown whether hydroxy fatty acids produced by lactobacilli have antifungal activity. This study aimed to determine whether lactobacilli convert linoleic acid to metabolites with antifungal activity and to assess whether this conversion can be employed to delay fungal growth on bread. Aqueous and organic extracts from seven strains of lactobacilli grown in modified De Man Rogosa Sharpe medium or sourdough were assayed for antifungal activity. Lactobacillus hammesii exhibited increased antifungal activity upon the addition of linoleic acid as a substrate. Bioassay-guided fractionation attributed the antifungal activity of L. hammesii to a monohydroxy C(18:1) fatty acid. Comparison of its antifungal activity to those of other hydroxy fatty acids revealed that the monohydroxy fraction from L. hammesii and coriolic (13-hydroxy-9,11-octadecadienoic) acid were the most active, with MICs of 0.1 to 0.7 g liter(-1). Ricinoleic (12-hydroxy-9-octadecenoic) acid was active at a MIC of 2.4 g liter(-1). L. hammesii accumulated the monohydroxy C(18:1) fatty acid in sourdough to a concentration of 0.73 ± 0.03 g liter(-1) (mean ± standard deviation). Generation of hydroxy fatty acids in sourdough also occurred through enzymatic oxidation of linoleic acid to coriolic acid. The use of 20% sourdough fermented with L. hammesii or the use of 0.15% coriolic acid in bread making increased the mold-free shelf life by 2 to 3 days or from 2 to more than 6 days, respectively. In conclusion, L. hammesii converts linoleic acid in sourdough and the resulting monohydroxy octadecenoic acid exerts antifungal activity in bread.

Fig 1

LC-APPI-MS extracted-ion chromatogram overlay of organic extract of sourdough fermented with L. hammesii in the presence of 4 g liter⁻¹ linoleic acid (A) and extract of sourdough fermented with L. sanfranciscensis in the presence of 4 g liter⁻¹ linoleic acid (B). Shown are the [M-H]⁻ ions of m/z 279, corresponding to linoleic acid (LA), m/z 293 to 299, corresponding to saturated, mono-, di-, and triunsaturated monohydroxy C₁₈ fatty acids (solid line), m/z 309 to 315, corresponding to saturated, mono-, di-, and triunsaturated dihydroxy C₁₈ fatty acids (dotted line), and m/z 325 to 331, corresponding to saturated, mono-, di-, and triunsaturated trihydroxy C₁₈ fatty acids (dashed line). Separations were performed on a Waters YMC silica column.

Fig 2

Mold-free shelf life in days (d) of bread (control [Cont.]), chemically acidified bread (Chem. acid.), sourdough bread fermented with L. sanfranciscensis (L. sanfran) or L. hammesii (L. hamm.), and bread supplemented with 0.15% coriolic acid (0.15% CA) or 0.4% Ca-propionate (0.4% Prop.). Sourdough was supplemented with 4 g kg⁻¹ linoleic acid and fermented with L. hammesii or L. sanfranciscensis. Bread slices were inoculated with A. niger or P. roqueforti or contaminated by environmental fungal spores during handling and stored until visible mold growth or for 15 days. Data are shown as means ± standard deviations of the results of triplicate independent experiments. Values for bread inoculated with the same mold that do not share a letter are significantly different (P < 0.05).