Flocculation mechanisms of top and bottom fermenting brewing yeast

The flocculation behaviour of a large set of top and bottom fermenting brewing yeasts was investigated. Bottom and top fermenting strains flocculated according to different mechanisms. Bottom strains flocculated in the stationary growth phase in the presence of sufficiently high Ca2+ and sufficiently low sugar concentrations; these strains possessed a lectin-mediated flocculation mechanism. Top strains flocculated in the stationary growth phase without addition of Ca2+, only in the presence of sufficiently high concentrations of ethanol. Some of the top strains were inhibited with mannose, but not with sucrose or galactose, while others were not inhibited by any of these sugars. The different sensitivity of flocculation of top and bottom strains with respect to ethanol may be related to the hydrophobicity of the cell surface. Flocculation models for bottom and top fermenting yeasts were proposed. It is suggested that, besides the sugar inhibition pattern, the sensitivity of flocculation with respect to ethanol should be included as an additional parameter for classification of brewing yeasts

Preparation of yeast cells for surface analysis by XPS

Analysis of the chemical composition of the surface of microbial cells by X-ray photoelectron spectroscopy (XPS) requires drying. The influence of the sample preparation procedure (washing, freezing, freeze drying, storage) has been examined with brewing yeasts. The recommended procedure involves washing at least once and pouring a concentrated suspension into a vial, precooled at liquid nitrogen temperature. The freeze-drying conditions are not critical provided that no melting occurs; this would cause cell lysis and release of intracellular compounds which alter the XPS results. Prolonged storage of freeze-dried samples prior to surface analysis leads to oxidation of surface groups. The cooling rate is high enough to prevent migration of intracellular constituents to the surface; however removal of surface polymers or appendages during washing or rearrangements of the polymer network at the surface, resulting from freezing, recrystallization or water removal, cannot be excluded

Performance of XPS analysis of model biochemical compounds

The performance of the XPS analysis of the chemical functions of biosurfaces has been assessed by examining model biochemical compounds (homopeptides, glucose related compounds,...). The components of the C1s, O1s and N1s peaks may be attributed to classes of chemical functions. The FWHM of the components is controlled by sample charging heterogeneity and the same FWHM can usually be used for the different components of a given peak. The reproducibility of the binding energy (same function, different compounds) is better than 0.15 eV for most of chemical functions. The repeatability of the concentration ratios (same compound) is within a few percent. Internal consistency is demonstrated by the agreement between atom concentration ratios deduced from total intensities, on the one hand, and relative intensities of the C1s components, on the other hand. A chemical composition in the range C15H26-30O4-6 is representative of common contamination overlayers. Modelling the effect of surface contamination overlayers indicate that the underestimation of O/C and N/C due to contamination is practically compensated by neglecting the kinetic energy dependence of the transmission function of the SSX-100 spectrometer. The accuracy of the O/C and N/C atom concentration ratiO1s (underestimation of about 10 %) is within the range of the precision obtained when the whole set of model compounds is taken into consideration

X-ray Photoelectron-spectroscopy Analysis of Biosurfaces - Examination of Performances With Yeast-cells and Related Model Compounds

Two brewer's yeast strains were analyzed by X-ray photoelectron spectroscopy (XPS). The C-1s peak was split into components representing carbon related to different chemical functions. Sample manipulation, spectrometer use and data treatment are responsible for variation coefficients of less than 3% for the atom fractions of oxygen, total carbon and major types of carbon. The possible influence of surface degradation and contamination on the XPS results was investigated using model compounds (quartz, porous silica, sorbitol, starch). The degradation of yeast during XPS analysis is marked by a decrease in the amount of oxygen and of carbon singly bound to oxygen or nitrogen, and by an increase in the amount of carbon only bound to carbon and hydrogen. This is due to X-rays or photoelectrons and not to flood gun electrons. The degradation increases proportionally with X-ray intensity and varies according to the strain. For both of the strains studied, the surface concentration of carbon only bound to carbon and hydrogen is much higher than expected from the composition of whole cell walls. Correcting the XPS data for contamination and degradation, and accounting for proteins still leaves a high concentration of hydrocarbon-like compounds at the surface; this may either reflect the real surface composition or be due to the migration of lipids through the cell wall during freeze-drying

Mechanisms of Yeast Flocculation - Comparison of Top-fermenting and Bottom-fermenting Strains

The flocculation of two brewing yeast strains, top-fermenting strain Saccharomyces cerevisiae MUCL 38485 and bottom-fermenting strain Saccharomyces carlsbergensis MUCL 28285, has been investigated by means of a turbidimetric test. The two strains showed different electrical properties, a different hydrophobicity, and a different surface chemical composition. They flocculated according to completely different mechanisms; however, no correlation between the cell physicochemical properties and the onset of flocculation was found for either strain. Flocculation of the bottom strain was governed by a lectin-mediated mechanism. It was inhibited by mannose and some other sugars, required calcium specifically, occurred in a narrow pH range different from the isoelectric point, and was not influenced by ethanol. The onset of flocculation at the end of the exponential phase was controlled both by the appearance of ''active'' lectins at the cell surface and by the decrease in sugar concentration in the solution. Flocculation of the top strain was not inhibited by mannose, did not require the addition of calcium, and took place at the cell isoelectric point. Low concentrations of ethanol broadened the pH range in which the cells flocculated, and flocculation was favored by an increase of ionic strength. Adsorbed ethanol may induce flocculation by reducing the electrostatic repulsion between cells, by decreasing steric stabilization, and/or by allowing the protrusion of polymer chains into the liquid phase. The onset of flocculation was controlled by both a change of the cell surface and an increase in ethanol concentration. The only evidence for an adhesin-mediated mechanism was the specific requirement for a small amount of calcium

Performance of Xps Analysis of Model Biochemical-compounds

The performance of the XPS analysis of the chemical functions of biosurfaces has been assessed by examining model biochemical compounds (homopeptides, glucose related compounds,...). The components of the C-1s, O-1s and N-1s peaks may be attributed to classes of chemical functions. The FWHM of the components is controlled by sample charging heterogeneity and the same FWHM can usually be used for the different components of a given peak. The reproducibility of the binding energy (same function, different compounds) is better than 0.15 eV for most of chemical functions. The repeatability of the concentration ratios (same compound) is within a few percent. Internal consistency is demonstrated by the agreement between atom concentration ratios deduced from total intensities, on the one hand, and relative intensities of the C-1s components, on the other hand. A chemical composition in the range C15H26-30O4-6 is representative of common contamination overlayers. Modelling the effect of surface contamination overlayers indicate that the underestimation of O/C and N/C due to contamination is practically compensated by neglecting the kinetic energy dependence of the transmission function of the SSX-100 spectrometer. The accuracy of the O/C and N/C atom concentration ratios (underestimation of about 10 %) is within the range of the precision obtained when the whole set of model compounds is taken into consideration

Flocculation in ale brewing strains of Saccharomyces cerevisiae : re-evaluation of the role of cell surface charge and hydrophobicity

Flocculation is an eco-friendly process of cell separation, which has been traditionally exploited by the brewing industry. Cell surface charge (CSC), cell surface hydrophobicity (CSH) and the presence of active flocculins, during the growth of two (NCYC 1195 and NCYC 1214) ale brewing flocculent strains, belonging to the NewFlo phenotype, were examined. Ale strains, in exponential phase of growth, were not flocculent and did not present active flocculent lectins on the cell surface; in contrast, the same strains, in stationary phase of growth, were highly flocculent (>98%) and presented a hydrophobicity of approximately three to seven times higher than in exponential phase. No relationship between growth phase, flocculation and CSC was observed. For comparative purposes, a constitutively flocculent strain (S646-1B) and its isogenic non-flocculent strain (S646-8D) were also used. The treatment of ale brewing and S646-1B strains with pronase E originated a loss of flocculation and a strong reduction of CSH; S646-1B pronase E-treated cells displayed a similar CSH as the non-treated S646-8D cells. The treatment of the S646-8D strain with protease did not reduce CSH. In conclusion, the increase of CSH observed at the onset of flocculation of ale strains is a consequence of the presence of flocculins on the yeast cell surface and not the cause of yeast flocculation. CSH and CSC play a minor role in the auto-aggregation of the ale strains since the degree of flocculation is defined, primarily, by the presence of active flocculins on the yeast cell wall.Manuela D. Machado gratefully acknowledges the post-doctoral grant from Fundacao para a Ciencia e a Tecnologia (FCT) from Portuguese Government (SFRH/BPD/72816/2010)