Multifunctional properties and safety evaluation of lactic acid bacteria and yeasts associated with fermented cereal doughs.

Spontaneous cereal fermentations involve diverse lactic acid bacteria (LAB) and yeasts which may include multifunctional and safe or unsafe strains. This study assessed acidification ability, safety, antifungal activity and free amino acids release ability of LAB and yeasts previously isolated from spontaneously fermented cereal doughs in Benin. Fourteen LAB and thirteen yeast strains were studied in liquid media and/or in a model cereal dough prepared in laboratory conditions. Antifungal activity was assessed against Candida glabrata in liquid medium. Amino acids were determined by pre-column derivatization and separation with reversed-phase HPLC. Antimicrobial susceptibility was analysed by minimum inhibitory concentration determination. The acidification ability was higher for LAB compared to yeast strains. All LAB strains retarded the growth of C. glabrata Cg1 with the highest inhibition recorded for Weissella confusa Wc1 and Wc2. The highest free amino acid content was found in the doughs fermented with Pichia kudriavzevii Pk2 and Pk3. All the LAB strains were susceptible to ampicillin, chloramphenicol, erythromycin, but displayed phenotypic resistance to kanamycin, streptomycin and tetracycline. Positive PCR amplicon of resistance genes were detected in the following cases: 2 LAB strains were positive for kanamycin (aph(3)III), 5 strains were positive for streptomycin (aadA and/or strA and/or strB) and 3 strains were positive for tetracycline (tet (L) and/or tet (M)). For yeasts, most of the P. kudriavzevii strains were resistant to amphotericin B, fluconazole and itraconazole opposite to K. marxianus and Saccharomyces cerevisiae strains which were susceptible. The results obtained are valuable for selecting safe and multifunctional strains for cereal fermentation in West Africa.

Occurrence and Importance of Yeasts in Indigenous Fermented Food and Beverages Produced in Sub-Saharan Africa.

Indigenous fermented food and beverages represent a valuable cultural heritage in sub-Saharan Africa, having one of the richest selections of fermented food products in the world. In many of these indigenous spontaneously fermented food and beverages, yeasts are of significant importance. Several factors including raw materials, processing methods, hygienic conditions as well as the interactions between yeasts and other commensal microorganisms have been shown to influence yeast species diversity and successions. Both at species and strain levels, successions take place due to the continuous change in intrinsic and extrinsic growth factors. The selection pressure from the microbial stress factors leads to niche adaptation and both yeast species and strains with traits deviating from those generally acknowledged in current taxonomic keys, have been isolated from indigenous sub-Saharan African fermented food products. Yeasts are important for flavor development, impact shelf life, and nutritional value and do, in some cases, even provide host-beneficial effects. In order to sustain and upgrade these traditional fermented products, it is quite important to obtain detailed knowledge on the microorganisms involved in the fermentations, their growth requirements and interactions. While other publications have reported on the occurrence of prokaryotes in spontaneously fermented sub-Saharan food and beverages, the present review focuses on yeasts considering their current taxonomic position, relative occurrence and successions, interactions with other commensal microorganisms as well as beneficial effects and importance in human diet. Additionally, the risk of opportunistic yeasts is discussed.

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

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 (EtOHpH3.4), (iii) 285 mM lactic acid (LApH3.4) and (iv) 150 mM acetic acid (AApH3.4) 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 (pHi), 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 (EtOHpH3.4). 285 mM lactic acid (LApH3.4) reduced the specific growth rate (µmax) 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 (AApH3.4) as well as the combinations (LA + AA)pH 3.4 and (LA + AA+EtOH)pH 3.4 reduced µmax 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 pHi to near physiological range (pHi = 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.