Debaryomyces hansenii Strains Isolated From Danish Cheese Brines Act as Biocontrol Agents to Inhibit Germination and Growth of Contaminating Molds.

The antagonistic activities of native Debaryomyces hansenii strains isolated from Danish cheese brines were evaluated against contaminating molds in the dairy industry. Determination of chromosome polymorphism by use of pulsed-field gel electrophoresis (PFGE) revealed a huge genetic heterogeneity among the D. hansenii strains, which was reflected in intra-species variation at the phenotypic level. 11 D. hansenii strains were tested for their ability to inhibit germination and growth of contaminating molds, frequently occurring at Danish dairies, i.e., Cladosporium inversicolor, Cladosporium sinuosum, Fusarium avenaceum, Mucor racemosus, and Penicillium roqueforti. Especially the germination of C. inversicolor and P. roqueforti was significantly inhibited by cell-free supernatants of all D. hansenii strains. The underlying factors behind the inhibitory effects of the D. hansenii cell-free supernatants were investigated. Based on dynamic headspace sampling followed by gas chromatography-mass spectrometry (DHS-GC-MS), 71 volatile compounds (VOCs) produced by the D. hansenii strains were identified, including 6 acids, 22 alcohols, 15 aldehydes, 3 benzene derivatives, 8 esters, 3 heterocyclic compounds, 12 ketones, and 2 phenols. Among the 71 identified VOCs, inhibition of germination of C. inversicolor correlated strongly with three VOCs, i.e., 3-methylbutanoic acid, 2-pentanone as well as acetic acid. For P. roqueforti, two VOCs correlated with inhibition of germination, i.e., acetone and 2-phenylethanol, of which the latter also correlated strongly with inhibition of mycelium growth. Low half-maximal inhibitory concentrations (IC50) were especially observed for 3-methylbutanoic acid, i.e., 6.32-9.53 × 10-5 and 2.00-2.67 × 10-4 mol/L for C. inversicolor and P. roqueforti, respectively. For 2-phenylethanol, a well-known quorum sensing molecule, the IC50 was 1.99-7.49 × 10-3 and 1.73-3.45 × 10-3 mol/L for C. inversicolor and P. roqueforti, respectively. For acetic acid, the IC50 was 1.35-2.47 × 10-3 and 1.19-2.80 × 10-3 mol/L for C. inversicolor and P. roqueforti, respectively. Finally, relative weak inhibition was observed for 2-pentanone and acetone. The current study shows that native strains of D. hansenii isolated from Danish brines have antagonistic effects against specific contaminating molds and points to the development of D. hansenii strains as bioprotective cultures, targeting cheese brines and cheese surfaces.

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.

The Effects of NaCl and Temperature on Growth and Survival of Yeast Strains Isolated from Danish Cheese Brines.

Yeasts play an important role in cheese making, by contributing to microbial community establishment and improving flavor. This study aimed at investigating the impact of NaCl and temperature on growth and survival of 20 strains belonging to the yeast species Candida intermedia (2 strains), Debaryomyces hansenii (11), Kluyveromyces lactis (1), Papiliotrema flavescens (1), Rhodotorula glutinis (1), Sterigmatomyces halophilus (2) and Yamadazyma triangularis (2) isolated from Danish cheese brines. All yeasts could grow in Malt Yeast Glucose Peptone (MYGP) medium with low NaCl (≤ 4%, w/v) concentrations at 25 °C and 16 °C. Further, none of the strains, except for one strain of D. hansenii (KU-9), were able to grow under a condition mimicking cheese brine (MYGP with 23% (w/v) NaCl and 6.3 g/L lactate) at 25 °C, while all yeasts could grow at 16 °C, except for the two strains of C. intermedia. In the survival experiment, D. hansenii, S. halophilus and Y. triangularis survived in MYGP with 23% (w/v) NaCl throughout 13.5 days at 25 °C, with Y. triangularis and S. halophilus being the most NaCl tolerant, while the remaining yeasts survived for less than 7 days. These results enable the selection of relevant yeasts from cheese brines for potential use in the cheese industry.

Probiotic potential of Saccharomyces cerevisiae and Kluyveromyces marxianus isolated from West African spontaneously fermented cereal and milk products.

The yeast species Saccharomyces cerevisiae and Kluyveromyces marxianus are associated with fermentation of West African indigenous foods. The aim of this study was to characterize potential probiotic properties of S. cerevisiae and K. marxianus isolates from the West African milk products lait caillé and nunu and a cereal-based product mawè. The strains (14 in total) were identified by 26S rRNA gene sequencing and characterized for survival at gastrointestinal stress (bile salts and low pH) and adhesion to Caco-2 intestinal epithelial cells. Selected yeast isolates were tested for their effect on the transepithelial electrical resistance (TEER), using the intestinal epithelial cell line Caco-2 and for maintenance of intracellular pH (pHi ) during perfusion with gastrointestinal pH (3.5 and 6.5). All tested yeasts were able to grow in bile salts in a strain-dependent manner, exhibiting a maximum specific growth rate (µmax ) of 0.58-1.50 h-1 . At pH 2.5, slow growth was observed for the isolates from mawè (µmax of 0.06-0.80 h-1 ), whereas growth of yeasts from other sources was mostly inhibited. Yeast adhesion to Caco-2 cells was strain specific and varied between 8.0% and 36.2%. Selected strains of S. cerevisiae and K. marxianus were able to maintain the pHi homeostasis at gastrointestinal pH and to increase TEER across the Caco-2 monolayers, indicating their potential to improve intestinal barrier functions. Based on overall results, strains of K. marxianus and S. cerevisiae from mawè exhibited the highest probiotic potential and might be recommended for further development as starter cultures in West African fermented products.

Technological properties of indigenous Lactococcus lactis strains isolated from Lait caillé, a spontaneous fermented milk from Burkina Faso.

The experiments reported in this research paper aimed to determine the technological properties of indigenous Lactococcus lactis strains isolated from Lait caillé, a spontaneous fermented milk, from the perspective of starter culture development. Fermentations were conducted to determine the acidification patterns. The ropy character, growth in 0.04 g/ml NaCl and citrate metabolism were additionally tested. Furthermore, the rheological properties of samples from selected strains and the impact of cold storage were evaluated. Based on the rate of acidification, the indigenous strains were divided into 2 groups depending on their fermentation time, i.e. 10-13 h (fast acidifier), and up to 72 h (slow acidifier), respectively. The physiological tests suggested that most of these strains produced exopolysaccharides but none could ferment citrate. The flow properties of the samples inoculated by the fast acidifier strains showed a time-dependent shear thinning behaviour, while their viscoelastic properties corresponded structurally to those of weak gels. Cold storage decreased the viscosity and CFU counts for most of the indigenous strains tested. This study is a step towards the definition of starter cultures for African spontaneous fermented milks such as Lait caillé.

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.