Effects of intrinsic microbial stress factors on viability and physiological condition of yeasts isolated from spontaneously fermented cereal doughs

Abstract

Fermented cereal doughs constitute a predominant part of West African diets. The environment of fermented doughs can be hostile for microbial survival due to high levels of microbial metabolites such as weak carboxylic organic acids and ethanol. In order to get a better understanding of the intrinsic factors affecting the microbial successions of yeasts during dough fermentation, survival and physiological responses of the yeasts associated with West African fermented cereal doughs were investigated at exposure to relevant concentrations of microbial inhibitory compounds. Three strains each of the predominant species, i.e. Saccharomyces cerevisiae, Kluyveromyces marxianus, Pichia kudriavzevii as well as the opportunistic pathogen Candida glabrata were studied. The strains were exposed to individual stress factors of cereal doughs, i.e. (i) pH 3.4, (ii) 3% (v/v) ethanol (EtOH_pH3.4), (iii) 285 mM lactic acid (LA_pH3.4) and (iv) 150 mM acetic acid (AA_pH3.4) as well as to combinations of these stress factors, i.e. (v) (LA + AA)_{pH 3.4} and (vi) (LA + AA+EtOH)_{pH 3.4}. Growth and single cell viability were studied by flow cytometry using combined SYTO 13 and propidium iodide (PI) staining. Intracellular pH (pH_i), plasma membrane integrity and micro-colony development of stressed cells were studied by fluorescence microscopy using PI and carboxyfluorescein diacetate succinimidyl ester (CFDA-se). Viability of the yeast strains was not affected by pH 3.4 and 3% (v/v) ethanol (EtOH_pH3.4). 285 mM lactic acid (LA_pH3.4) reduced the specific growth rate (μ_max) from 0.27-0.41 h^-1 to 0.11-0.26 h^-1 and the viability from 100% to 2.6-41.7% at 72 h of exposure in most yeast strains, except for two strains of C. glabrata. 150 mM acetic acid (AA_pH3.4) as well as the combinations (LA + AA)_{pH 3.4} and (LA + AA+EtOH)_{pH 3.4} reduced μ_max to 0.0 h^-1 and induced significant cell death for all the yeast strains. Exposed to (LA + AA+EtOH)_{pH 3.4}, the most resistant yeast strains belonged to S. cerevisiae followed by P. kudriavzevii, whereas C. glabrata and K. marxianus were more sensitive. Strain variations were observed within all four species. When transferred to non-stress conditions, i.e. MYGP, pH 5.6, after exposure to (LA + AA+EtOH)_{pH 3.4} for 6 h, 45% of the single cells of the most resistant S. cerevisiae strain kept their plasma membrane integrity, recovered their pH_i to near physiological range (pH_i = 6.1-7.4) and resumed proliferation after 3-24 h of lag phase. The results obtained are valuable in order to change processing conditions of the dough to favor the survival of preferable yeast species, i.e. S. cerevisiae and K. marxianus and inhibit opportunistic pathogen yeast species as C. glabrata.

Keywords: Flow cytometry; Fluorescence microscopy; Intracellular pH; Mawè doughs; Plasma membrane integrity; Yeast survival.