Antifungal activity of Wickerhamomyces anomalus and Lactobacillus plantarum during sourdough fermentation: identification of novel compounds and long-term effect during storage of wheat bread

Abstract

This study aimed at investigating the antifungal activity of Wickerhamomyces anomalus and sourdough lactic acid bacteria to extend the shelf life of wheat flour bread. The antifungal activity was assayed by agar diffusion, growth rate inhibition, and conidial germination assays, using Penicillium roqueforti DPPMAF1 as the indicator fungus. Sourdough fermented by Lactobacillus plantarum 1A7 (S1A7) and dough fermented by W. anomalus LCF1695 (D1695) were selected and characterized. The water/salt-soluble extract of S1A7 was partially purified, and several novel antifungal peptides, encrypted into sequences of Oryza sativa proteins, were identified. The water/salt-soluble extract of D1695 contained ethanol and, especially, ethyl acetate as inhibitory compounds. As shown by growth inhibition assays, both water/salt-soluble extracts had a large inhibitory spectrum, with some differences, toward the most common fungi isolated from bakeries. Bread making at a pilot plant was carried out with S1A7, D1695, or a sourdough started with a combination of both strains (S1A7-1695). Slices of the bread manufactured with S1A7-1695 did not show contamination by fungi until 28 days of storage in polyethylene bags at room temperature, a level of protection comparable to that afforded by 0.3% (wt/wt) calcium propionate. The effect of sourdough fermentation with W. anomalus LCF1695 was also assessed based on rheology and sensory properties.

Fig. 1.

Conidial germination of Penicillium roqueforti DPPMAF1 incubated in WFH for 12 and 24 h at 25°C under different conditions. Treatments were as follows: Ct, WFH alone (control); CP, WFH supplemented with 0.3% (wt/vol) calcium propionate; S1A7, WFH supplemented with 30% water/salt-soluble extract of sourdough fermented with Lactobacillus plantarum 1A7; D1695, WFH supplemented with 30% water/salt-soluble extract of dough fermented with Wickerhamomyces anomalus LCF1695; and S1A7-1695, WFH supplemented with 30% water/salt-soluble extract of sourdough fermented with L. plantarum 1A7 and W. anomalus LCF1695. Data are the means for three independent experiments, and the values within series marked with different letters are statistically significantly different (P < 0.05).

Fig. 2.

Antifungal activity of water/salt-soluble extract of dough fermented with Wickerhamomyces anomalus LCF1695 (D1695), as determined by hyphal radial growth inhibition (44) after 8 days of incubation at 25°C. (A) PDA alone (control); (B) PDA containing water/salt-soluble extract of D1695 (30% [vol/vol]).