A rapid and selective assay for measuring cell surface hydrophobicity of brewer's yeast cells.

A rapid and selective assay was developed to measure cell surface hydrophobicity of brewer's yeast cells. During this so-called magnobead assay, bottom-fermenting yeast cells adhere to paramagnetic, polystyrene-coated latex beads which can easily be removed from the cell suspension by using a (samarium-cobalt) magnet. At pH 4 center dot 5, electrostatic repulsion between yeast cells and latex beads was found to be minimal and yeast cell adhesion was predominantly based on hydrophobic interactions. The percentage of cells adhering to the beads could be calculated and provided a measure for cell surface hydrophobicity. Cell surface hydrophobicity measured by the magnobead assay was found to yield similar results, as did determination of contact angles of water droplets on a layer of yeast cells, a standard method for measuring surface hydrophobicity. However, the magnobead assay has the following advantages: (i) it is a quick and simple method, and, more significantly, (ii) hydrophobicity can be measured under physiological conditions. Use of the magnobead assay confirmed that a higher level of cell surface hydrophobicity is correlated with stronger flocculence of brewer's lager yeast cells.

Isolation and partial purification of mannose-specific agglutinin from brewer's yeast involved in flocculation.

Yeast cell-agglutinating activity, designated agglutinin (possible lectin), was isolated from cell walls of both non-flocculent and flocculent brewer's yeast cells. Agglutinin-mediated aggregation of yeast cells in a manner similar to flocculation with respect to specific mannose-sensitivity, pH-dependence and calcium-dependence. Agglutinating activity was found to be heat-stable and protease-insensitive. Furthermore, addition of agglutinin to flocculent cells strongly stimulated the flocculation ability of the cells, whereas addition to non-flocculent cells rendered these cells weakly flocculent. Agglutinin was found to be released from flocculent cells during the course of a flocculation assay, but not from non-flocculent cells. Presence of mannose during the assay inhibited release of agglutinin. Our results suggest that (i) mannose-specific agglutinin is continuously synthesized during growth of brewer's yeast cells, (ii) agglutinin is present in cell walls of non-flocculent cells but is unable to bind its ligand on other cells, and (iii) the ability of yeast cells to flocculate in a flocculation assay depends, among other factors, on release of agglutinin from the cells. A 10-kDa polypeptide might represent one form of agglutinin.

Purification and partial characterization of a flocculin from brewer's yeast.

Analysis of a shear supernatant from flocculent, "fimbriated" Saccharomyces cerevisiae brewer's yeast cells revealed the presence of a protein involved in flocculation of the yeast cells and therefore designated a flocculin. The molecular mass of the flocculin was estimated to be over 300 kDa, as judged from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel permeation chromatography of the flocculin yielded an aggregate with an apparent molecular weight of > 2,000. The flocculin was found to be protease sensitive, and the sequence of its 16 N-terminal amino acids revealed at least 69% identity with the predicted N terminus of the putative protein encoded by the flocculation gene FLO1. The flocculin was isolated from flocculent S. cerevisiae cells, whereas only a low amount of flocculin, if any, could be isolated from nonflocculent cells. The flocculin was found to stimulate the flocculation ability of flocculent yeast cells without displaying lectinlike activity (that is, the ability to agglutinate yeast cells).

Cause and control of flocculation in yeast.

Flocculation is an important characteristic of microorganisms which can be problematical, but may also be exploited in fermentation processes. The molecular mechanism of flocculation is still poorly understood although, recently, cell-surface hydrophobicity of brewer's yeast has been shown to play a key role. Regulation of this factor could enable control of flocculation during fermentation.

Determinants of flocculence of brewer's yeast during fermentation in wort.

Ability of Saccharomyces cerevisiae MPY3 cells to flocculate during fermentation in wort was found to be triggered after growth limitation by oxygen shortage and to coincide with a sharp increase in cell surface hydrophobicity of the cells. Presence of oxygen in the pitching wort influenced final cell number, flocculence of the cells and cell surface hydrophobicity. Flocculation ability of cells grown in air-depleted pitching wort was hampered, concomitant with a decrease in final cell number and in final cell surface hydrophobicity. Addition of ergosterol and Tween 80 to air-depleted wort restored normal growth of the cells as well as flocculation ability and the increase in cell surface hydrophobicity. The same parameters increased in value after addition of ergosterol and Tween 80 to a fermentation with air-saturated pitching wort. Hydrophobicity of a non-flocculent mutant of S. cerevisiae strain MPY3, fermenting in air-saturated pitching wort, did not increase at cell division arrest. These results support the hypothesis that cell surface hydrophobicity is a major determinant for yeast cells to become flocculent, and suggest that shortage of sterols and unsaturated fatty acids precedes flocculence under brewing conditions.

Flocculence of Saccharomyces cerevisiae cells is induced by nutrient limitation, with cell surface hydrophobicity as a major determinant.

Initiation of flocculation ability of Saccharomyces cerevisiae MPY1 cells was observed at the moment the cells stop dividing because of nitrogen limitation. A shift in concentration of the limiting nutrient resulted in a corresponding shift in cell division and initiation of flocculence. Other limitations also led to initiation of flocculence, with magnesium limitation as the exception. Magnesium-limited S. cerevisiae cells did not flocculate at any stage of growth. Cell surface hydrophobicity was found to be strongly correlated with the ability of the yeast cells to flocculate. Hydrophobicity sharply increased at the end of the logarithmic growth phase, shortly before initiation of flocculation ability. Treatments of cells which resulted in a decrease in hydrophobicity also yielded a decrease in flocculation ability. Similarly, the presence of polycations increased both hydrophobicity and the ability to flocculate. Magnesium-limited cells were found to be strongly affected in cell surface hydrophobicity. A proteinaceous cell surface factor(s) was identified as a flocculin. This heat-stable component had a strong emulsifying activity, and appears to be involved in both cell surface hydrophobicity and in flocculation ability of the yeast cells.