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.The major problem for long-term shelf life of baked goods is contamination with fungi. Although they are used as the main chemical preservatives, salts of propionic, sorbic, and benzoic acids have shown several drawbacks. Reports on the appearance of cancer-like tumors in rats fed with propionic acid at concentrations of up to 4% led to prohibition of the use of calcium propionate in some European countries (31). Prolonged preservation of breads with high concentrations of calcium propionate stimulated the growth of resistant strains of Penicillium roqueforti (42). Since a few years ago, the European directive on preservatives (18) decreased the permitted concentrations of sorbate and propionate to 0.2 and 0.3% (wt/wt), respectively. Ethanol is also permitted as an antifungal preservative (2% [wt/wt]). Its inhibitory effect varies depending on the fungal species, and in some cases, it is not sufficient to prevent contamination (14).Several studies have focused on the antifungal activity of compounds from natural sources and on biopreservation as effective alternatives to chemical preservatives. Several antimicrobial compounds have been identified, especially proteins or peptides from plants, as well as metabolites derived from fermentation by lactic acid bacteria (12,23,25,42,43). Besides acidification during sourdough fermentation, lactic acid bacteria also synthesize a wide range of low-molecular-weight compounds (27), peptides (28), and proteins (10, 37) with antifungal activity. Unfortunately, only a few studies have assessed their inhibitory activity under bread-making conditions. Recent studie...

Antifungal Hydroxy Fatty Acids Produced during Sourdough Fermentation: Microbial and Enzymatic Pathways, and Antifungal Activity in Bread

“…The antifungal activity of metabolites from lactic acid bacteria in bread has, to date, been attributed not to a single compound but, rather, to their synergistic activity with substrate-or yeastderived compounds (4,5,8,10). This study demonstrated that enzymatic and microbial activities generate antifungal hydroxy fatty acids from linoleic acid.…”

“…ourdough bread has an extended mold-free storage life compared to that of conventionally leavened products (1, 2), and metabolites from specific strains of lactobacilli contribute to the prolonged storage life of sourdough bread (3,4,5). While the fermentation microbiota of traditional sourdough is controlled by the fermentation conditions and the choice of raw materials, the industrial production of sourdough often relies on single strains of lactobacilli with defined metabolic properties (6, 7).…”

Strategies to Extend Bread and GF Bread Shelf-Life: From Sourdough to Antimicrobial Active Packaging and Nanotechnology

“…Wickerhamomyces anomalus LCF1695 is, for instance, used as a mixed starter in combination with Lb. plantarum 1A7 [43]; Meyerozyma guilliermondii LCF1353 harbour marked antifungal activity toward P. roqueforti DPPMAF1; sourdough fermented with a combined starter culture-M. guilliermondii LCF1353, W. anomalus LCF1695 and Lb. plantarum 1A7 strain-allows to obtain excellent results in terms of extended shelf-life [44].…”

Long-Term Fungal Inhibition by Pisum sativum Flour Hydrolysate during Storage of Wheat Flour Bread

“…Several recent studies have addressed the use of metabolites from sourdough lactic acid bacteria and yeasts alone and in combination with natural preservatives, which were extracted from plant materials or were contained in pseudocereals used in the formula for making baked goods (1,2,6,7,8,12,13). Despite promising results, the problem of fungal contamination, of great concern to both consumers and bakeries, still remains unsolved.…”

“…Sourdough was also considered for extending the shelf life of baked goods. In particular, sourdough lactic acid bacteria synthesize a broad range of antifungal compounds, such as phenyllactic acid, peptides, cyclic dipeptides, and short or medium-chain fatty acids (2,6,7,8,9,10,11).Several recent studies have addressed the use of metabolites from sourdough lactic acid bacteria and yeasts alone and in combination with natural preservatives, which were extracted from plant materials or were contained in pseudocereals used in the formula for making baked goods (1,2,6,7,8,12,13). Despite promising results, the problem of fungal contamination, of great concern to both consumers and bakeries, still remains unsolved.…”