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PubMed1996

Flocculation of the yeast Candida famata (Debaryomyces hansenii): an essential role for peptone.

Aggregation of Candida famata (Debaryomyces hansenii) is consistent with being a form of lectin-mediated yeast flocculation. Flocculation of C. famata is unusual in that it requires the presence of peptone, either in the growth medium or added later to harvested cells in buffer. Flocculation after peptone addition was rapid, being largely complete within 10 min. Heat-killed cells also flocculated, arguing for direct participation of peptone in the flocculation binding mechanism. Flocculent C. famata cells progressively lost the ability to flocculate when washed with EDTA. Flocculation was fully restored by peptone addition; calcium addition was without effect. C. famata cells were able to agglutinate erythrocytes in the presence or absence of peptone. Pronase E-treated yeast lost both the ability to haemagglutinate and self-flocculate. Haemagglutination was not diminished by progressive EDTA washing, suggesting that surface lectins remained present and active on the yeast cell walls. Non-flocculating C. famata cells mutually flocculated with non-flocculent Schizosaccharomyces pombe cells, shown to have surface-exposed galactose residues. Mutual flocculation was lost following treatment of C. famata with Pronase E. It was concluded that the cell wall of C. famata contains lectins enabling haemagglutination and mutual flocculation but lacks carbohydrate receptors for these lectins. This yeast self-flocculates only via bridging multi-valent carbohydrates; these being present in peptone.

Abrin↗
PubMed2003

Yeast flocculation: what brewers should know.

For many industrial applications in which the yeast Saccharomyces cerevisiae is used, e.g. beer, wine and alcohol production, appropriate flocculation behaviour is certainly one of the most important characteristics of a good production strain. Yeast flocculation is a very complex process that depends on the expression of specific flocculation genes such as FLO1, FLO5, FLO8 and FLO11. The transcriptional activity of the flocculation genes is influenced by the nutritional status of the yeast cells as well as other stress factors. Flocculation is also controlled by factors that affect cell wall composition or morphology. This implies that, during industrial fermentation processes, flocculation is affected by numerous parameters such as nutrient conditions, dissolved oxygen, pH, fermentation temperature, and yeast handling and storage conditions. Theoretically, rational use of these parameters offers the possibility of gaining control over the flocculation process. However, flocculation is a very strain-specific phenomenon, making it difficult to predict specific responses. In addition, certain genes involved in flocculation are extremely variable, causing frequent changes in the flocculation profile of some strains. Therefore, both a profound knowledge of flocculation theory as well as close monitoring and characterisation of the production strain are essential in order to gain maximal control over flocculation. In this review, the various parameters that influence flocculation in real-scale brewing are critically discussed. However, many of the conclusions will also be useful in various other industrial processes where control over yeast flocculation is desirable.

Fermentation↗
PubMed2004

Carbohydrate carbon sources induce loss of flocculation of an ale-brewing yeast strain.

AIMS: To identify the nutrients that can trigger the loss of flocculation under growth conditions in an ale-brewing strain, Saccharomyces cerevisiae NCYC 1195. METHODS AND RESULTS: Flocculation was evaluated using the method of Soares, E.V. and Vroman, A. [Journal of Applied Microbiology (2003) 95, 325]. Yeast growth with metabolizable carbon sources (glucose, fructose, galactose, maltose or sucrose) at 2% (w/v), induced the loss of flocculation in yeast that had previously been allowed to flocculate. The yeast remained flocculent when transferred to a medium containing the required nutrients for yeast growth and a sole nonmetabolizable carbon source (lactose). Transfer of flocculent yeast into a growth medium with ethanol (4% v/v), as the sole carbon source did not induce the loss of flocculation. Even the addition of glucose (2% w/v) or glucose and antimycin A (0.1 mg l(-1)) to this culture did not bring about loss of flocculation. Cycloheximide addition (15 mg l(-1)) to glucose-growing cells stopped flocculation loss. CONCLUSIONS: Carbohydrates were the nutrients responsible for stimulating the loss of flocculation in flocculent yeast cells transferred to growing conditions. The glucose-induced loss of flocculation required de novo protein synthesis. Ethanol prevented glucose-induced loss of flocculation. This protective effect of ethanol was independent of the respiratory function of the yeast. SIGNIFICANCE AND IMPACT OF THE STUDY: This work contributes to the elucidation of the role of nutrients in the control of the flocculation cycle in NewFlo phenotype yeast strains.

Antifungal Agents↗
PubMed2005

Flocculation onset in Saccharomyces cerevisiae: the role of nutrients.

AIMS: To examine the role of the nutrients on the onset of flocculation in an ale-brewing strain, Saccharomyces cerevisiae NCYC 1195. METHODS AND RESULTS: Flocculation was evaluated using the method of Soares, E.V. and Vroman, A. [Journal of Applied Microbiology (2003) 95, 325]. For cells grown in chemically defined medium (yeast nitrogen base with glucose) or in rich medium (containing yeast extract, peptone and fermentable sugars: fructose or maltose), the onset of flocculation occurred after the end of exponential respiro-fermentative phase of growth being coincident with the attainment of the lower level of carbon source in the culture medium. Cells, in exponential respiro-fermentative phase of growth, transferred to a glucose-containing medium without nitrogen source, developed a flocculent phenotype, while these carbon source starved cells, in the presence of all other nutrients that support growth, did not flocculate. In addition, cells in exponential phase of growth, under catabolite repression, when transferred to a medium containing 0.2% (w/v) of fermentable sugar (fructose or maltose) or 2% (v/v) ethanol, showed a rapid triggering of flocculation, while when incubated in 2% (v/v) glycerol did not develop a flocculent phenotype. CONCLUSIONS: The onset of flocculation occurs when a low sugar and/or nitrogen concentration is reached in culture media. The triggering of flocculation is an energetic dependent process influenced by the carbon source metabolism. The presence of external nitrogen source is not necessary for developing a flocculent phenotype. SIGNIFICANCE AND IMPACT OF THE STUDY: This work contributes to the elucidation of the role of nutrients on the onset of flocculation in NewFlo phenotype yeast strains. This information might be useful to the brewing industry, in the control of yeast flocculation, as the time when the onset of flocculation occurs can determine the fermentation performance and the beer quality.

Beer↗
PubMed1992

Yeast flocculation: reconciliation of physiological and genetic viewpoints.

Yeast flocculation results from surface expression of specific proteins (lectins). Two flocculation phenotypes were suggested by physiological and biochemical tests, whereas genetic data suggested a larger number of mechanisms of flocculation. After reviewing the biochemistry, physiology and genetics of flocculation, a new hypothesis combining the data available from these different sources, is proposed. Flocculation results when lectins present on flocculent cell walls bind to sugar residues of neighbouring cell walls. These sugar receptors are intrinsic to the mannan comprising cell walls of Saccharomyces cerevisiae. Two lectin phenotypes were revealed by sugar inhibition studies. The gluco- and mannospecific NewFlo phenotype is not, as yet, found in genetically defined strains. Mannospecific flocculation (Flo1 phenotype) is found in strains containing the genes FLO1, FLO5 and FLO8. This phenotype is also found following mutation of the TUP1 or CYC8 loci, in previously non-flocculent strains. It is therefore proposed that the structural gene for mannospecific flocculation is common or possibly ubiquitous in non-flocculent strains and in consequence, FLO1, FLO5 and FLO8 are probably regulatory genes, exerting positive control over the structural gene. Flocculation expression requires lectin secretion to the cell surface. Many of the observed 'suppressions' of flocculation may be due to mutations of the secretory process, involved in transporting structural proteins to the cell wall. The possible involvement of killer L double-stranded RNA with flocculation is suggested, given the lectin properties of viral coat proteins and an association between L double-stranded RNA and the Flo1 phenotype.

Cell Aggregation↗
PubMed1994

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.

Agglutinins↗
PubMed1996

MIG1 overexpression causes flocculation in Saccharomyces cerevisiae.

MIG1, encoding a C2H2 zinc-finger repressor protein involved in carbon catabolite repression, was found to play a role in non-sexual flocculation of Saccharomyces cerevisiae. Disruption of MIG1 in a flocculent mutant strain of NCYC 227, resulted in a non-flocculent phenotype. Expression of MIG1 on a 2 mu pRS426 vector in a non-flocculent strain, YM 4134, caused flocculation; MIG1 on a high-copy-number LEU2-d plasmid caused intense flocculation in the same strain. Mutations in the SSN6 and TUP1 genes confer a flocculent phenotype in non-flocculent strains of S. cerevisiae, and it has been shown that Mig1 can tether the Ssn6p-Tup1p complex to the regulatory regions of glucose-repressible genes. Mutations in tup1 in a MIG1 background caused flocculation while double mutants of TUP1 and MIG1 did not flocculate. Based on these results, a model for the role of MIG1 in flocculation gene regulation is proposed.

Blotting, Northern↗
PubMed1995

Transcriptional regulation of flocculation genes in Saccharomyces cerevisiae.

Northern analysis showed that DNA from the flocculation gene FLO1 hybridized to mRNA molecules of 4.8 kb. This transcript was specific for the FLO1 gene at the right end of chromosome I since disruption of this gene resulted in the disappearance of the transcript. We further found an absolute correlation between flocculation and the presence of transcripts hybridizing to FLO1 DNA, both in various flocculent and non-flocculent strains and in cells from the non-flocculating and flocculating stages of growth. In all cases transcripts were present in flocculating and absent from non-flocculating cultures. From these results we conclude that the FLO1 gene is transcriptionally regulated. Mutations in TUP1 or SSN6 cause flocculation. Several transcripts hybridizing to FLO1 DNA were present in the mutants but not in the corresponding wild-type strains. Disruption of the FLO1 gene in the tup1 and ssn6 strains showed that one of the transcripts corresponded to the FLO1 gene. Disruption of FLO1 did not abolish flocculation completely but only reduced it, indicating that at least two flocculation genes, including FLO1, are activated or derepressed by mutations in the TUP1/SSN6 regulatory cascade.

Flocculation↗
PubMed1994

Effect of cultural and nutritional conditions on the control of flocculation expression in Saccharomyces cerevisiae.

The effect of cultural (temperature and pH) and nutritional conditions (nitrogen and carbon source) on the flocculation expression of three strains was studied. The strains' flocculation ability was determined by placing the cells in a stationary phase of growth in standard flocculation conditions. The flocculation ability of strain NCYC 1195, recently classified in the literature as the NewFlo phenotype, was more sensitive to growth temperature than Flo1 phenotype strains (NCYC 869 and NRRL Y265). The initial pH of the culture medium did not affect the flocculation ability of Flo1 phenotype strains but in the case of strain NCYC 1195 flocculation was repressed when the initial pH of the culture medium was below 3.5. Flocculation in strain NCYC 1195 was also repressed in defined culture medium; this inhibition was not related to a deficiency in any particular nitrogen source, but rather to the poor buffering capacity of the defined medium. All strains showed strong flocculation when grown in glucose, but were nonflocculent in glycerol. It was clearly demonstrated that the phenotypic expression of flocculation could be induced or repressed by changing cultural and nutritional conditions. Two distinct behaviours were also displayed with regard to the effect of the cultural conditions upon flocculation, namely the effect of pH. These different behaviours can be used to distinguish the two flocculation phenotypes.

Carbon↗
PubMed2006

Flocculation behavior of temperature-sensitive poly(N-isopropylacrylamide) microgels containing polar side chains with -OH groups.

The flocculation behavior of poly(N-isopropylacrylamide) (pNIPAM) microgels containing polar -(OCH(2)CH(2))(3)OH chains, incorporated by the copolymeric components (triethyleneglycol methacrylate, TREGMA), in aqueous NaCl solution was investigated. Determination of the critical flocculation temperatures (CFTs) and the critical flocculation concentrations (CFCs) of the microgels at 45 degrees C shows that polar -(OCH(2)CH(2))(3)OH chains have different influence on the flocculation behavior of the microgels at temperatures below and above their volume phase transition temperatures (VPTTs). The flocculation of the microgels becomes more difficult with the increase of -(OCH(2)CH(2))(3)OH chains below the VPTT. In contrast, the microgels flocculate more easily with more -(OCH(2)CH(2))(3)OH chains above the VPTT. Preliminary investigation on the flocculation kinetics of the microgels further shows that -(OCH(2)CH(2))(3)OH chains have different effects on the flocculation rate at temperatures below and above the VPTT. The flocculating rate of the microgels at 25 degrees C decreases with the increase of -(OCH(2)CH(2))(3)OH chains. While the flocculation rate at 45 degrees C increases with the increase of -(OCH(2)CH(2))(3)OH chains due to their enrichment on the surface of the microgels as a result of the temperature-induced volume-phase transition, which was verified by variable temperature (1)H NMR spectroscopy. The polar -(OCH(2)CH(2))(3)OH chains rich in the surface increase the attractive force between the microgels, promoting the flocculation.

Journal Article↗
PubMed2003

Effect of different starvation conditions on the flocculation of Saccharomyces cerevisiae.

AIMS: To study the effect of different starvation conditions on the flocculation of an ale brewing yeast of Saccharomyces cerevisiae NCYC 1195. METHODS AND RESULTS: Flocculation was assessed by a micro-flocculation technique (Soares and Mota 1997). Carbon-starved cells of a NewFlo phenotype strain did not lose flocculation during a 48 h period. Cells incubated only in the presence of fermentable carbon sources (glucose, galactose and maltose at 2%, w/v), showed a progressive flocculation loss. The incubation of cells in 4% (v/v) ethanol did not induce a flocculation loss. The simultaneous incubation of cells in the presence of 2% (w/v) glucose and 15 microg ml(-1) cycloheximide hindered flocculation loss. The presence of 0.1 mmol l(-1) PMSF or 10 mmol l-1 EDTA prevented partially or completely, respectively, the loss of flocculation in the presence of glucose. CONCLUSIONS: Fermentable sugars induced a flocculation loss, which seems to require de novo protein synthesis and the involvement of different proteases. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings reported here contribute to the elucidation of the role of nutrients on the physiological control of yeast flocculation.

Carbon↗
PubMed1994

Population dynamics of flocculating yeasts.

The problem of understanding the recognition and specific interactions in a population of yeast flocculating cells is discussed. The biochemistry, physiology and genetics of flocculation is briefly reviewed. Yeast flocculation requires the expression of a specific protein (lectin) on flocculent cells, and carbohydrate (receptors) on neighbouring cells. Adhesion experiments performed with cells whose flocculation is repressed by growth conditions, indicating that the inhibition of flocculation is due to inhibition or inactivation of 'lectin-like' component. Additionally, using adhesion experiments, it is demonstrated that cells of non-flocculent strain interact by establishing a true bond with flocculent cells rather than by entrapment inside the floc matrix. As phenotypic expression of flocculation, for several strains, is shown to be repressed, modulated or induced by modifying growth conditions, the constitutiveness and inducibility of flocculation are also discussed.

Cell Wall↗
PubMed1995

Mechanisms of yeast flocculation: comparison of top- 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.

Beer↗