Antimicrobial peptides from Saccharomyces cerevisiae induce physiological changes in Hanseniaspora guilliermondii

Saccharomyces cerevisiae secretes antimicrobial peptides (AMPs) during alcoholic fermentation that are active against other wine-related yeasts (e.g. Hanseniaspora guilliermondii) (Albergaria et al., 2010) and bacteria (e.g. Oenococcus oeni) (Osborne and Edwards, 2007). In the present study we assessed the physiological changes induced by those AMPs on sensitive yeast cells of Hanseniaspora guilliermondii, namely membrane permeability and intracellular pH (pHi) alterations. Membrane permeability was evaluated by staining cells with propidium iodide (PI) and pHi by the fluorescence ratio imaging microscopy (FRIM) technique (Guldfeldt and Arneborg, 1998). Results showed that after 20 min of incubation with inhibitory concentrations of AMPs, the average pHi of cells dropped from 6.5 to 5.4. After 8 h of incubation, 32% of the cells had lost their ĢpH (=pHi-pHext) and after 24 h that percentage rose to 77%. The culturability (plating) and viability (PI staining) of the sensitive yeast cells also decreased in the presence of the AMPs. After 24 h of exposure to AMPs, 61% of the cells were dead (PI-stained) and the number of viable cells fell from 1 ~105 to 1.5 CFU/ml, which means that virtually all cells (99.999%) became unculturable but a sub-population of 39% of cells remained in a viable but non-culturable (VBNC) state. However, those VBNC cells were able to recover their culturability after incubation at optimal growth conditions. Our study revealed that the mode of action of these AMPs seems to be primarily targeted to the cell membranes, reducing their permeability and preventing cells to maintain pH homeostasis

Interactions between Saccharomyces cerevisiae and Hanseniaspora guilliermondii: cell-cell contact mechanism

Several studies have shown that the early death of non-Saccharomyces during wine fermentations are due to yeast-yeast interactions induced by Saccharomyces cerevisiae (Sc) through different mechanisms such as growth arrest mediated by a cell-cell contact mechanism (Nissen et al. 2003) and death mediated by killer-like toxins (Pérez-Nevado et al 2006; Albergaria et al. 2010). Besides, previous work also showed that death of non-Saccharomyces in co-cultivation with Sc is always triggered at the end of exponential growth phase (Pérez-Nevado et al 2006). In order to investigate the role of cell-cell contact in the early death of non-Saccharomyces, we performed assays in which Sc cells pre-grown at enological growth conditions for 12 and 48 h, respectively, were in direct contact with Hanseniaspora guilliermondii (Hg) cells at high cellular density (107-108 cells/ml) in a carbon-free medium. As a negative control we performed similar assays in which Sc and Hg cells were separated by a dialysis tube (pore cut-off of 1000 kDa) and as a positive control a single Hg culture. Results showed that Hg cell density decreased by 2 orders of magnitude (i.e. from 108-106 cells/ml) in contact with 48 h-grown Sc cells, while its viability remained unchanged (108cfu/ml) in the presence of 12 h-grown Sc cells. Moreover, Hg viability was not affected both in the dialysis tube experiments and single culture, which confirmed the death-induced cell-cell contact phenomenon

Isolation and identification of the microbiota of Danish farmhouse and industrially produced surface-ripened cheeses

For studying the microbiota of four Danish surface-ripened cheeses produced at three farmhouses and one industrial dairy, both a culture-dependent and culture-independent approach were used. After dereplication of the initial set of 433 isolates by (GTG)5-PCR fingerprinting, 217 bacterial and 25 yeast isolates were identified by sequencing of the 16S rRNA gene or the D1/D2 domain of the 26S rRNA gene, respectively. At the end of ripening, the cheese core microbiota of the farmhouse cheeses consisted of the mesophilic lactic acid bacteria (LAB) starter cultures Lactococcus lactis subsp. lactis and Leuconostoc mesenteorides as well as non-starter LAB including different Lactobacillus spp. The cheese from the industrial dairy was almost exclusively dominated by Lb. paracasei. The surface bacterial microbiota of all four cheeses were dominated by Corynebacterium spp. and/or Brachybacterium spp. Brevibacterium spp. was found to be subdominant compared to other bacteria on the farmhouse cheeses, and no Brevibacterium spp. was found on the cheese from the industrial dairy, even though B. linens was used as surface-ripening culture. Moreover, Gram-negative bacteria identified as Alcalignes faecalis and Proteus vulgaris were found on one of the farmhouse cheeses. The surface yeast microbiota consisted primarily of one dominating species for each cheese. For the farmhouse cheeses, the dominant yeast species were Yarrowia lipolytica, Geotrichum spp. and Debaryomyces hansenii, respectively, and for the cheese from the industrial dairy, D. hansenii was the dominant yeast species. Additionally, denaturing gradient gel electrophoresis (DGGE) analysis revealed that Streptococcus thermophilus was present in the farmhouse raw milk cheese analysed in this study. Furthermore, DGGE bands corresponding to Vagococcus carniphilus, Psychrobacter spp. and Lb. curvatus on the cheese surfaces indicated that these bacterial species may play a role in cheese ripening

The impact of different ale brewer’s yeast strains on the proteome of immature beer

BACKGROUND: It is well known that brewer’s yeast affects the taste and aroma of beer. However, the influence of brewer’s yeast on the protein composition of beer is currently unknown. In this study, changes of the proteome of immature beer, i.e. beer that has not been matured after fermentation, by ale brewer’s yeast strains with different abilities to degrade fermentable sugars were investigated. RESULTS: Beers were fermented from standard hopped wort (13° Plato) using two ale brewer’s yeast (Saccharomyces cerevisiae) strains with different attenuation degrees. Both immature beers had the same alcohol and protein concentrations. Immature beer and unfermented wort proteins were analysed by 2-DE and compared in order to determine protein changes arising from fermentation. Distinct protein spots in the beer and wort proteomes were identified using Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and MS/MS and revealed common beer proteins, such as lipid transfer proteins (LTP1 and LTP2), protein Z and amylase-protease inhibitors. During fermentation, two protein spots, corresponding to LTP2, disappeared, while three protein spots were exclusively found in beer. These three proteins, all derived from yeast, were identified as cell wall associated proteins, that is Exg1 (an exo-β-1,3-glucanase), Bgl2 (an endo-β-1,2-glucanase), and Uth1 (a cell wall biogenesis protein). CONCLUSION: Yeast strain dependent changes in the immature beer proteome were identified, i.e. Bgl2 was present in beer brewed with KVL011, while lacking in WLP001 beer