Flocculation of Saccharomyces cerevisiae: inhibition by sugars
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C. L. MASY,A. HENQUINET, AND M. M. MESTDAGH Unite' de chimie des interfaces, Universite' catholique de Louvain, Place Croix du Sud 2, boite 18, B-1348 Louvain-la-Neuve, Belgium
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Received May 1, 1992 Revision received July 22, 1992 Accepted July 3 1, 1992 A., and MESTDAGH, M. M. 1992. Flocculation of Saccharomyces cerevisiae: inhibition by MASY,C. L., HENQUINET, sugars. Can. J . Microbiol. 38: 1298- 1306. Flocculation is governed by the competition between electrostatic repulsion (nonspecific interactions) and polysaccharide-protein bonds (specific interactions). In our study, the inhibition of flocculation by sugars for 12 strains of Saccharomyces cerevisiae leads us to extend the classification described in the literature and to define three groups of yeasts: flocculation mannose sensitive (MS), flocculation glucose-mannose sensitive (GMS), and flocculation mannose insensitive (MI). Only the first two groups showed specific interactions between proteins and mannans. In the MI group, the sugars tested did not inhibit flocculation. To characterize the particularities of the stereochemistry of the cell-wall proteic receptors oaf strains belonging to the MS and GMS groups, 31 sugars were used as inhibitor probes on two representative strains. The results show that the lectin specificity of strains belonging to the GMS group is less restricted regarding C-1 and C-2 hydroxyl groups than the lectin from strains belonging to the MS group, which interacts with all of the hydroxyl groups of mannopyranose. The two groups also differ with respect to inhibition by sugars: strains belonging to the MS group are partially inhibited whereas strains of the GMS group are completely inhibited. We observed that the presence of ethanol increases sugar fixation by strains from the MS group, but not from the GMS group. Moreover, both receptors interact with disaccharides, provided the two monomers are linked by an a(144), a(143), or a(14 2) bond. Key words: yeast flocculation, proteic receptors, sugars, lectins. MASY,C. L., HENQUINET, A., et MESTDAGH,M. M. 1992. Flocculation of Saccharomyces cerevisiae: inhibition by sugars. Can. J. Microbiol. 38 : 1298-1306. La floculation des levures est regie par la competition entre la repulsion electrostatique (interactions non-specifiques) et les liens polysaccharides-proteines (interactions specifiques). Dans cette etude, les auteurs ont examine l'inhibition de la floculation par les sucres chez 12 souches de Saccharomyces cerevisiae, ce qui leur a permis d'etendre la classification decrite dans la litterature et de definir trois groupes de levures : floculation sensible au mannose (MS), floculation sensible au glucose (GMS) et floculation insensible au mannose (MI). Seuls les deux premiers groupes montrent des interactions specifiques entre les proteines et les mannanes. Chez le troisieme groupe (MI), il n'y a pas d'inhibition de la floculation provoquee par les sucres analyses. Trente et un sucres ont ete choisis comme sondes inhibitrices envers des souches representatives, afin de caracteriser les particularites stereochimiques des recepteurs de la paroi cellulaire des souches appartenant aux groupes MS et GMS. Les resultats montrent que la specificite de la lectine des souches appartenant au groupe GMS est moins limitee, relativement aux groupements hydroxyles C-1 et C-2, que la lectine des souches appartenant au groupe MS, laquelle interagit avec tous les groupements hydroxyles du mannopyranose. Les deux groupes different quant a leur inhibition par les sucres, les souches du groupe MS subissant une inhibition partielle, tandis que celles du groupe GMS sont inhibees completement. Les auteurs ont observe que la fixation du sucre est augmentee en presence d'ethanol chez les souches du groupe MS, contrairement aux souches du groupe GMS. De plus, les deux recepteurs interagissent avec des disaccharides, pourvu que les deux monomeres soient rattaches par une liaison a(l --4), a(14 3), ou a(14 2). Mots cle's : floculation des levures, recepteurs proteiques, sucres, lectines.
Introduction Flocculation of yeasts is the intercellular process by which yeasts come together and stick into clumps that easily separate from the medium at the end of the fermentation process (Calleja 1984; Stewart and Russell 1987). This phenomenon is of great interest for brewers because the time of occurrence and intensity of flocculation affect the degree of fermentation and, therefore, the alcohol level, the carbohydrate content, and the flavor of the beer (Johnston and Reader 1983; Stewart and Russell 1987; Thornton 1985; Bautista et al. 1986). Flocculation also allows immobilization and accumulation of the biocatalyst in the reactor (Chen and Chen-Shung 1986; Barre 1988). The inhibition of flocculation by sugars (Eddy 1955a; Kihn et al. 1988a) and the specific influence of calcium ions ' ~ u t h o rto whom all correspondence should be addressed. Prlntcd ~n Canada / lrnpr~rneau Canada
(Mill 1964; Kihn et al. 1988a; Stratford and Keenan 1988; Strat ford et al. 1988; Masy et al. 1991; Masy 1991) have led many authors to describe flocculation by means of a model that involves specific lectin interactions between cells (Miki et al. 1982; Taylor and Orton 1978). Stratford (1989) distinguished two groups of yeasts according to their inhibitory pattern with sugars. One group, called "Flol phenotype," was partially inhibited but only by mannopyranose. It included strains containing FLOI, FL04, F L 0 5 , FL08, and TUPl genes. The strains of the other group, called "NewFlo phenotype," were completely inhibited by mannose, glucose, maltose, or sucrose. From these observations, the author concluded that there were two different mechanisms. The first mechanism, characterizing the Flol phenotype group, involved a lectin specific to mannose whereas the
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second one, the NewFlo phenotype group, involved a broadspecificity lectin. This paper describes the differences between the flocculation mannose sensitive (MS) and the flocculation glucosemannose sensitive (GMS) groups in terms of the stereochemical specificity of sugars and the effect of ethanol. A third group, called flocculation mannose insensitive (MI), which is not inhibited by sugars, is also described. Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by UNIVERSITY OF MICHIGAN on 11/10/14 For personal use only.
Materials and methods Microorganisms and cultures The strains used, listed in Table 1, were kept at - 20°C in liquid medium (25 g/L glucose, 20 g/L yeast extract, and glycerol 25% v/v). Cells were plated on YD medium (30 g/L glucose, 20 g/L yeast extract, and 25 g/L agar) in Petri dishes. A sample of a single colony was streaked for single cells on YD agar, which after incubation was kept at 4°C for no more than 1 week. The culture media contained 20 g/L yeast extract and 50 g/L glucose. The cells were cultivated in two steps as follows: a preculture (10 mL in 50-mL Erlenmeyer flasks, 18 h) was used to inoculate the culture (200 mL in 1-L Erlenmeyer flasks with two vertical baffles) to a final concentration of 2 x lo6 cells/mL. The cell concentration was determinated by direct microscopic counting. The following culture conditions were chosen to obtain flocculation in the culture media. Top fermentation strains (cf. Table 2, Saccharomyces cerevisiae var. cerevisiae) were grown at 30°C and bottom fermentation strains (cf. Table 1, Saccharomyces cerevisiae var. uvarum) at 11"C. Cells were harvested at the stationary phase: 24 h for all the strains, except NCYC 1195 and MUCL 28323 (48 h), and MUCL 29759 and MUCL 28285 (10 days). The cells were harvested by centrifugation (2500 x g, 4"C, 5 min) and were washed three times with distilled water, once with EDTA (2 mM, pH 8), and finally once with distilled water. The cells were suspended in distilled water and stored at 4°C for a maximum of 12 h. Flocculation test Flocculation tests were performed using a modified procedure of Kihn et al. (1988b), in which acetate buffer (10 mM acetic acid with 1 M NaOH; pH 4) was added to avoid pH variations, as this can modify drastically the flocculation behavior of some strains (Masy et al. 1991). The samples were prepared in test tubes (16 x 100 mm) and unless otherwise specified had the following composition: 10 mM acetate buffer (10 mM acetic acid with NaOH; pH 4), 0.5 mM calcium, possibly another substance (such as sugar, ethanol) as specified in the text, yeast cells (lo8 cells/mL), and distilled water to a final volume of 5 mL. The cells were added last of all under vigorous stirring (15 s). The tubes were sealed and agitated on a rotary shaker turning at 60 rpm. The duration of the agitation corresponded to the steady state between flocs and free cell concentration (FCC) defined in Kihn et al. (1988b). This was found to be 30 min for strains MUCL 29759, MUCL 28285, MUCL 28733, or MUCL 28323, and 5 min for the other strains. After agitation, the samples were allowed to stand undisturbed for 5 min (NCYC 869) or 15 min (all the other strains) after which the absorbance at 660 nm was measured (Bausch & Lomb, Spectronic 700). Results are expressed as residual absorbance or as FCC. Cell wall preparation Cell walls were prepared using the method described by Kihn et al. (1987). For the flocculation test, a suspension of 2.69 g/L of isolated cell walls was used; the procedures were otherwise identical. For strain NCYC 869, a steady state was reached after 5 min of agitation and 5 min of sedimentation. Chemicals All chemicals used were analytical grade except agar and yeast extract. They included CaC1, (Merck); EDTA (tetrasodium salt,
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FIG. 1. Effect of sugars of FCC expressed in cell concentration of the strains (A) NCYC 869 (MS group), (B) NCYC 1195 (GMS group), and (C) 27c (MI group), at 500 mM sugar concentrations and pH 4. Analysed sugars: man, mannopyranose; me-man, methyl-a-D-mannopyranose; malt, maltose; glu, glucose; gal, galactose; suc, sucrose; gly, glycerol; sorb, sorbitol. The experimental curves reported are for mannopyranose (m) and galactose (0).
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FIG. 2. Inhibition by sugars of yeast MS group containing different flocculation genes: (A) NCYC 870 (FLOI), (B) STX347-1D (FL05), and (C) YIY 261 (FL08), at 5 mM calcium concentration and pH 4. Analysed sugars: mannose and methyl-a-D-mannopyranose (m); glucose, galactose, maltose, glycerol, and sorbitol (0). The experimental curves reported are for mannopyranose (m) and glucose (0). Aldrich); yeast extract (Difco); agar (Pastagar A, Pasteur); acetic acid (Janssen); sodium hydroxide (Merck); hydrochloric acid (Merck); N-acetyl-a-D-glucosamine (Janssen); N-acetyl-P-Dmannosamine (Janssen); D-altrose (Janssen); p-aminophenyl-a-Dmannopyranose (Sigma); D-cellobiose (Fluka); D-fructose (Merck); D-galactose (Janssen); D-glucose (Janssen); a-D-glucose-1-phosphate (disodium salt; Sigma); D-glucose-6-phosphate (sodium salt; Sigma); glycerol (UCB, Union Chimique Belge); D-lactose (UCB); D-lyxose (Janssen); maltose (Janssen); maltotriose (Sigma); D-mannitol (Janssen); D-mannopyranose (Janssen); L-mannopyranose (Janssen); 3-0-a-D-mannopyranosyl-a-D-mannopyranose (Sigma); D-mannosamine (Sigma); a-D-mannose-1-phosphate (sodium salt, Sigma); D-mannose-6-phosphate (disodium salt; Sigma); methyl-a-D-glucopyranose (Sigma); methyl-P-D-glucopyranose (Sigma); methyl-a-D-mannopyranose (Janssen); methyl-2-O-c~-~-mannopyranosyl-c~-~-rnannopyranose (Sigma); methyl-4-O-c~-~-mannopyranosyl-c~-~-rnannopyranose (Sigma); p-nitrophenyl-a-D-mannopyranose (Sigma); p-nitrophenyl-P-D-
mannopyranose (Sigma); D-sorbitol (Merck); ribose (Sigma); sucrose (Janssen); and D-talose (Janssen).
Results Although calcium is known to play an important role in flocculation, at the concentration used in our experiments (0.5 mM), it failed to cause flocculation in the nonflocculent strains (strains D273- lob and KL 14-4A). To ascertain whether the calcium concentration we used was too low, calcium concentrations of 0.5-500 mM were tested. The results show, however, that even at the maximum concentration, no flocculation was observed when absorbance was measured.
Inhibition groups of yeasts When flocculation was measured in the presence of various sugars (monosaccharides or disaccharides) differ-
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ing in their O H configuration of substituents, only some of the sugars inhibited flocculation. The inhibitory effects of various sugars on the flocculation of the yeasts from the MS, GMS, and MI groups are illustrated in Figs. l A , lB, and l C , respectively. For yeasts from the MS and GMS groups, either noninhibitory or inhibitory curves were observed (Figs. 1A and 1B). Flocculation of the MS group was inhibited by only D-mannopyranose and methyl-a-Dmannopyranose, while that of GMS was inhibited by D-glucopyranose, D-mannopyranose, maltose, sucrose, and methyl-a-D-mannopyranose. The group of strains for which no sugar inhibited flocculation was called flocculation mannose insensitive (MI) (Fig. 1C). The strains in Table 1 all displayed the behaviors characteristic of their particular group. Results are presented as FCC. They show that inhibition of flocculation was complete for the strains of the GMS group (10' cells recovered) and incomplete for those of the MS group. Sugar inhibition of the flocculation of isolated cells walls instead of whole cells (MS group) was examined for strain NCYC 869. The results indicate that flocculation of isolated cells walls was inhibited by the same sugars that inhibited the corresponding entire cells. Flocculation, therefore, could be considered to be a process that involves only the cell wall (Eddy 1958; Nishihara et al. 1982), suggesting that sugar inhibition is not due to a metabolic effect. Figure 2 shows the inhibition of flocculation in the presence of increasing sugar concentration, for yeast containing different flocculation genes (MS group). The inhibitory capacity of mannopyranose and methyl-a-D-mannopyranose varies greatly between strains. The following table presents the order of inhibition, with the highest FCC obtained by using sugar concentrations up to 700 mM and the minimal sugar concentration needed to reach this FCC. Gene Strains FCC (ceIIs/mL) Sugar concentration (mM)
FL08 YIY2616 3 x 10 700
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FL05 > FL08. The FL08 strain was hardly inhibited by sugar at all. Thus, one can wonder if specific interactions are important for flocculation of this strain. Moreover, it was noted that the degree of inhibition by sugars corresponds to the order of pH sensitivity (Masy 1991). The mechanism by which ethanol promotes flocculation is unknown but a hypothesis has been proposed (Eddy 1955b). Amory et al. (1988) suggested that ethanol either acts as a surfactant by lowering water surface tension or modifies the conformation and properties of surface polymers. However, one should keep in mind that in these studies, the influence of pH may not have been well controlled. The reason why ethanol had an effect on sugar inhibition of strains from the MS group only but not on flocculation in our study remains to be explained.
Stereochemistry of the proteic receptors Inhibition by sugars indicates some crucial points concerning the interaction of sugar with lectinlike receptors of strains belonging to the MS or GMS group. In Fig. 5, the carbohydrate is presented in the C-1 chair conformation, which is the most probable conformation for a - and P-D-glucopyranosides and mannopyranosides in solution. Both groups of strains needed the pyranose ring configuration and an equatorial position of the hydroxyl on C-3, C-4, and C-6 (Stratford and Assinder 1991). The main differences are on C-1 and C-2, where no special configuration of the hydroxyl groups is required for strains belonging to the GMS group. An axial configuration is required for strains belonging to the MS group (Fig. 5). However, even in the GMS group, C-2 hydroxyl function represents a destabilizing factor (Stratford and Assinder 1991). Thus, strains from the GMS group show a broad specificity but only for C-2 and C-1. Some residues can influence the fixation: for example, a phenyl group increases the inhibitory power of .the compound for strains of the MS (Kihn et al. 1988a) and GMS groups. This suggests that a hydrophobic region exists near the mannose-binding site. For strains belonging to the MS group one can conclude that the receptor binds two or more residues of mannose in a(1- 2), a(1 -- 3), or a(1 -- 4) configurations. For strains from the GMS group, the receptor binds two residues with a(1 -- 2) or a(1 -- 4) bonds. Both receptors seem to be able to bind lateral chains of mannans that contain a(1 -- 2) and a ( l -- 3) bonds (Ballou 1976). Proteic receptors of strains from the GMS group present an inhibitory pattern similar to concanavalin A lectin; the proteic receptor of strain NCYC 869 has an inhibitory pattern like that of Escherichia coli type I lectin (Lis and Sharon 1986) that is specific for D-mannose but differs in the inhibitory pattern of oligomers. Of course, it is not possible to generalize on the basis of the two strains, but our results suggest that a more systematic study is worthwhile. Some preliminary results obtained on strain MUCL 29759, another strain from the GMS group (Kihn et al. 1988b), confirmed the results presented here (complementary results not shown). In conclusion, yeast strains can be grouped according to the inhibition of their flocculation by sugars. The MI group, which is not inhibited by mannose or glucose, is added to the MS and GMS groups already defined as Flol phenotype and NewFlo phenotype, respectively. (Stratford 1989; Stratford and Assinder 1991). The main differences between
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the receptors of strains belonging to the MS and GMS groups are the hydroxyl groups on C-1 and C-2. In the GMS group, these two positions are less important for fixation of the sugar. In the MS group, all hydroxyl groups are necessary for fixation to occur. The inhibition by sugar is not complete for the receptors of strains belonging to the MS group and this may be due to the higher specificity of these receptors for sugars. For these strains, ethanol enhances inhibition but the initial cell concentration is not entirely recovered.
Acknowledgements The authors gratefully acknowledge gifts of yeast strains (YIY 261) and (27c) from I. Yamashita of Hiroshima University (Japan) and from C. Ghommidth of the Centre de genie et technologie alimentaire (microbiologie industrielle), University of Montpellier (France), respectively. The authors thank the Service de la programmation de la politique scientifique (Concerted Action Physical Chemistry of Interfaces and Biotechnology) for financial support. Amory, D.E., Dufour, J.-P., and Rouxhet, P.G. 1988. Flocculence of brewery yeasts and their surface properties: chemical composition, electrostatic charge, and hydrophobicity. J. Inst. Brew. 94: 79-84. Ballou, C. 1976. Structure and biosynthesis of the mannan component of the yeast cell envelope. Adv. Microb. Physiol. 14: 93-158. Barre, P. 1988. Control of alcoholic fermentation in wine making. Int. Biotechnol. Symp. 8th 1988, 3: 899-909. Bautista, J., Chico, E., and Machado, A. 1986. Cell removal from fermentation broth by flocculation-sedimentation. Biotechnol. Lett. 8: 315-318. Bell, G.I., Dembo, M., and Bongrand, P. 1984. Cell adhesion: competition between nonspecific repulsion and specific bonding. Biophys. J. 52: 1051-1064. Bell, G.I. 1988. Models of cell adhesion involving specific binding. In Physical basis of cell-cell adhesion. Edited by P. Bongrand. CRC Press, Inc. Boca Raton, Fla. pp. 227-256. Calleja, G.B. 1984. Microbial aggregation. CRC Press, Inc., Boca Raton, Fla. p. 266. Chen, L., and Chen Shung, G. 1986. Continuous ethanol production using induced yeast aggregates. Appl. Microbiol . Biotechnol. 25: 208-212. Eddy, A.A. 1955a. Flocculation characteristics of yeasts. 11. Sugars as dispersing agents. J . Inst. Brew. 61: 313-317. Eddy, A.A. 1955b. Flocculation characteristics of yeasts. 111. General role of flocculating agents and special characteristics of two yeasts flocculated by alcohol. J . Inst. Brew. 61: 318-320. Eddy, A.A. 1958. Composite nature of the flocculation process of top and bottom strains of Saccharomyces. J. Inst. Brew. 64: 143-151. Esser, K., Kues, V., and Hinrichs, J. 1987. Genetic control of flocculation of yeast with respect to application in biotechnology. In Flocculation in biotechnology and separations systems. Edited by Y.A. Attia. Elsevier Science Publishers B.V., Amsterdam. pp. 393-398. Ghommidth, C., Fourtot, C., and Navarro, J.M. 1987. Fermentation in Saccharomyces cerevisiae and mitochondria1 DNA structure. Appl. Microbiol. Biotechnol. 29: 48-54. Johnston, J.R., and Reader, H.P. 1983. Genetic control of flocculation. In Yeast genetics: fundamental and applied aspects. Edited by J.F.T. Spencer, D.H. Spencer, and A. R. W. Smith. Springer-Verlag, New York. pp. 205-224.
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Kamada, K., and Murata, M. 1984. On the mechanism of brewer's yeast flocculation. Agric. Biol. Chem. 48: 2423-2433. Kihn, J.-C., Mestdagh, M.M., and Rouxhet, P.G. 1987. ESR study of copper(I1) retention by entire cell, cell walls and protoplasts of Saccharomyces cerevisiae. Can. J. Microbiol. 33: 777-782. Kihn, J.-C., Masy, C.L., and Mestdagh, M.M. 1988a. Yeast flocculation: competition between nonspecific and specific bonding in cell adhesion. Can. J. Microbiol. 34: 773-778. Kihn, J.-C., Masy, C.L., Mestdagh, M.M., and Rouxhet, P.G. 1988b. Yeast flocculation: factors affecting the measurement of flocculence. Can. J . Microbiol. 34: 779-781. Lewis, D.W., and Johnston, J.R. 1983. Flocculation genes in Saccharomyces cerevisiae. Microb. Genet. Bull. No. 35. p. 11. Lis, H., and Sharon, N. 1986. Lectins as molecules and as tools. Annu. Rev. Biochem. 55: 35-67. Masy, C.L., Kockerols, M., and Mestdagh, M.M. 1991. Calcium activity versus "calcium threshold" as the key factor in the induction of yeast flocculation in simulated industrial fermentations. Can. J . Microbiol. 37: 295-303. Masy, C.L. 1991. Incidence de facteurs physico-chimiques et genetiques sur les interactions specifiques intervenant dans la floculation de levures Saccharomyces cerevisiae. Ph.D thesis, Universite catholique de Louvain, Belgium. Miki, B.L.A., Poon, N.H., James, A.P., and Seligy, V.L. 1982. Possible mechanism for flocculation interactions governed by gene FLO 1 in Saccharomyces cerevisiae. J . Bacteriol. 150: 878-889. Mill, P.J. 1964. The nature of the interaction between flocculent cells in the flocculation of Saccharomyces cerevisiae. J. Gen. Microbiol. 35: 3 1-68. Nishihara, H., Toraya, T., and Fukui, S. 1982. Flocculation of cell walls of brewer's yeast and effects of metals ions, protein denaturants, and enzyme treatments. Arch. Microbiol. 131: 112-115. Poretz, D., and Goldstein, I.J. 1970. An examination of the topography of the saccharide binding sites of Concanavalin A and the forces involved in the complexation. Biochemistry, 9: 2890-2896. So, L.L., and Goldstein, I. 1968. Protein-carbohydrate interaction. XX. On the number of combining sites on Concanavalin A, the phytohemagglutinin of the jack bean. Biochim. Biophys. Acta, 165: 398-404. Stewart, G.G., and Russell, I. 1977. The identification, characterization, and mapping of a gene for flocculation in Saccharomyces sp. Can. J. Microbiol. 23: 441-447. Stewart, G.G., and Russell, I. 1987. The relevance of the flocculation properties of yeast on today's brewing industry. Monogr. Eur. Brew. Conv. 12: 53-70. Stratford, M. 1989. Evidence for two mechanisms of flocculation in Saccharomyces cerevisiae. Yeast, 5: S441-445. Stratford, M., and Assinder, S. 1991. Yeast flocculation: F101 and NewFlo phenotypes and receptor structure. Yeast, 7: 559-574. Stratford, M., and Keenan, M.H.J. 1988. Yeast flocculation: quantification. Yeast, 4: 107-1 15. Stratford, M., Coleman, H.P., and Keenan, M.H.J. 1988. Yeast flocculation: a dynamic equilibrium. Yeast, 4: 199-208. Taylor, N. W., and Orton, W.L. 1978. Aromatic compounds and sugars in flocculation of Saccharomyces cerevisiae. J . Inst. Brew. 84: 113-114. Thornton, R. J . 1985. The introduction of flocculation into homothallic wine yeast. A practical example of the modification of wine making properties by the use of genetic techniques. Am. J . Enol. Vitic. 36: 47-49. Yamashita, I., and Fukui, S. 1983. Mating signals control expression of both starch fermentation genes and a novel flocculation gene FL08 in the yeast Saccharomyces. Agric. Biol. Chem. 47: 2889-2896.
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C. L. MASY,A. HENQUINET, AND M. M. MESTDAGH Unite' de chimie des interfaces, Universite' catholique de Louvain, Place Croix du Sud 2, boite 18, B-1348 Louvain-la-Neuve, Belgium
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Received May 1, 1992 Revision received July 22, 1992 Accepted July 3 1, 1992 A., and MESTDAGH, M. M. 1992. Flocculation of Saccharomyces cerevisiae: inhibition by MASY,C. L., HENQUINET, sugars. Can. J . Microbiol. 38: 1298- 1306. Flocculation is governed by the competition between electrostatic repulsion (nonspecific interactions) and polysaccharide-protein bonds (specific interactions). In our study, the inhibition of flocculation by sugars for 12 strains of Saccharomyces cerevisiae leads us to extend the classification described in the literature and to define three groups of yeasts: flocculation mannose sensitive (MS), flocculation glucose-mannose sensitive (GMS), and flocculation mannose insensitive (MI). Only the first two groups showed specific interactions between proteins and mannans. In the MI group, the sugars tested did not inhibit flocculation. To characterize the particularities of the stereochemistry of the cell-wall proteic receptors oaf strains belonging to the MS and GMS groups, 31 sugars were used as inhibitor probes on two representative strains. The results show that the lectin specificity of strains belonging to the GMS group is less restricted regarding C-1 and C-2 hydroxyl groups than the lectin from strains belonging to the MS group, which interacts with all of the hydroxyl groups of mannopyranose. The two groups also differ with respect to inhibition by sugars: strains belonging to the MS group are partially inhibited whereas strains of the GMS group are completely inhibited. We observed that the presence of ethanol increases sugar fixation by strains from the MS group, but not from the GMS group. Moreover, both receptors interact with disaccharides, provided the two monomers are linked by an a(144), a(143), or a(14 2) bond. Key words: yeast flocculation, proteic receptors, sugars, lectins. MASY,C. L., HENQUINET, A., et MESTDAGH,M. M. 1992. Flocculation of Saccharomyces cerevisiae: inhibition by sugars. Can. J. Microbiol. 38 : 1298-1306. La floculation des levures est regie par la competition entre la repulsion electrostatique (interactions non-specifiques) et les liens polysaccharides-proteines (interactions specifiques). Dans cette etude, les auteurs ont examine l'inhibition de la floculation par les sucres chez 12 souches de Saccharomyces cerevisiae, ce qui leur a permis d'etendre la classification decrite dans la litterature et de definir trois groupes de levures : floculation sensible au mannose (MS), floculation sensible au glucose (GMS) et floculation insensible au mannose (MI). Seuls les deux premiers groupes montrent des interactions specifiques entre les proteines et les mannanes. Chez le troisieme groupe (MI), il n'y a pas d'inhibition de la floculation provoquee par les sucres analyses. Trente et un sucres ont ete choisis comme sondes inhibitrices envers des souches representatives, afin de caracteriser les particularites stereochimiques des recepteurs de la paroi cellulaire des souches appartenant aux groupes MS et GMS. Les resultats montrent que la specificite de la lectine des souches appartenant au groupe GMS est moins limitee, relativement aux groupements hydroxyles C-1 et C-2, que la lectine des souches appartenant au groupe MS, laquelle interagit avec tous les groupements hydroxyles du mannopyranose. Les deux groupes different quant a leur inhibition par les sucres, les souches du groupe MS subissant une inhibition partielle, tandis que celles du groupe GMS sont inhibees completement. Les auteurs ont observe que la fixation du sucre est augmentee en presence d'ethanol chez les souches du groupe MS, contrairement aux souches du groupe GMS. De plus, les deux recepteurs interagissent avec des disaccharides, pourvu que les deux monomeres soient rattaches par une liaison a(l --4), a(14 3), ou a(14 2). Mots cle's : floculation des levures, recepteurs proteiques, sucres, lectines.
Introduction Flocculation of yeasts is the intercellular process by which yeasts come together and stick into clumps that easily separate from the medium at the end of the fermentation process (Calleja 1984; Stewart and Russell 1987). This phenomenon is of great interest for brewers because the time of occurrence and intensity of flocculation affect the degree of fermentation and, therefore, the alcohol level, the carbohydrate content, and the flavor of the beer (Johnston and Reader 1983; Stewart and Russell 1987; Thornton 1985; Bautista et al. 1986). Flocculation also allows immobilization and accumulation of the biocatalyst in the reactor (Chen and Chen-Shung 1986; Barre 1988). The inhibition of flocculation by sugars (Eddy 1955a; Kihn et al. 1988a) and the specific influence of calcium ions ' ~ u t h o rto whom all correspondence should be addressed. Prlntcd ~n Canada / lrnpr~rneau Canada
(Mill 1964; Kihn et al. 1988a; Stratford and Keenan 1988; Strat ford et al. 1988; Masy et al. 1991; Masy 1991) have led many authors to describe flocculation by means of a model that involves specific lectin interactions between cells (Miki et al. 1982; Taylor and Orton 1978). Stratford (1989) distinguished two groups of yeasts according to their inhibitory pattern with sugars. One group, called "Flol phenotype," was partially inhibited but only by mannopyranose. It included strains containing FLOI, FL04, F L 0 5 , FL08, and TUPl genes. The strains of the other group, called "NewFlo phenotype," were completely inhibited by mannose, glucose, maltose, or sucrose. From these observations, the author concluded that there were two different mechanisms. The first mechanism, characterizing the Flol phenotype group, involved a lectin specific to mannose whereas the
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second one, the NewFlo phenotype group, involved a broadspecificity lectin. This paper describes the differences between the flocculation mannose sensitive (MS) and the flocculation glucosemannose sensitive (GMS) groups in terms of the stereochemical specificity of sugars and the effect of ethanol. A third group, called flocculation mannose insensitive (MI), which is not inhibited by sugars, is also described. Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by UNIVERSITY OF MICHIGAN on 11/10/14 For personal use only.
Materials and methods Microorganisms and cultures The strains used, listed in Table 1, were kept at - 20°C in liquid medium (25 g/L glucose, 20 g/L yeast extract, and glycerol 25% v/v). Cells were plated on YD medium (30 g/L glucose, 20 g/L yeast extract, and 25 g/L agar) in Petri dishes. A sample of a single colony was streaked for single cells on YD agar, which after incubation was kept at 4°C for no more than 1 week. The culture media contained 20 g/L yeast extract and 50 g/L glucose. The cells were cultivated in two steps as follows: a preculture (10 mL in 50-mL Erlenmeyer flasks, 18 h) was used to inoculate the culture (200 mL in 1-L Erlenmeyer flasks with two vertical baffles) to a final concentration of 2 x lo6 cells/mL. The cell concentration was determinated by direct microscopic counting. The following culture conditions were chosen to obtain flocculation in the culture media. Top fermentation strains (cf. Table 2, Saccharomyces cerevisiae var. cerevisiae) were grown at 30°C and bottom fermentation strains (cf. Table 1, Saccharomyces cerevisiae var. uvarum) at 11"C. Cells were harvested at the stationary phase: 24 h for all the strains, except NCYC 1195 and MUCL 28323 (48 h), and MUCL 29759 and MUCL 28285 (10 days). The cells were harvested by centrifugation (2500 x g, 4"C, 5 min) and were washed three times with distilled water, once with EDTA (2 mM, pH 8), and finally once with distilled water. The cells were suspended in distilled water and stored at 4°C for a maximum of 12 h. Flocculation test Flocculation tests were performed using a modified procedure of Kihn et al. (1988b), in which acetate buffer (10 mM acetic acid with 1 M NaOH; pH 4) was added to avoid pH variations, as this can modify drastically the flocculation behavior of some strains (Masy et al. 1991). The samples were prepared in test tubes (16 x 100 mm) and unless otherwise specified had the following composition: 10 mM acetate buffer (10 mM acetic acid with NaOH; pH 4), 0.5 mM calcium, possibly another substance (such as sugar, ethanol) as specified in the text, yeast cells (lo8 cells/mL), and distilled water to a final volume of 5 mL. The cells were added last of all under vigorous stirring (15 s). The tubes were sealed and agitated on a rotary shaker turning at 60 rpm. The duration of the agitation corresponded to the steady state between flocs and free cell concentration (FCC) defined in Kihn et al. (1988b). This was found to be 30 min for strains MUCL 29759, MUCL 28285, MUCL 28733, or MUCL 28323, and 5 min for the other strains. After agitation, the samples were allowed to stand undisturbed for 5 min (NCYC 869) or 15 min (all the other strains) after which the absorbance at 660 nm was measured (Bausch & Lomb, Spectronic 700). Results are expressed as residual absorbance or as FCC. Cell wall preparation Cell walls were prepared using the method described by Kihn et al. (1987). For the flocculation test, a suspension of 2.69 g/L of isolated cell walls was used; the procedures were otherwise identical. For strain NCYC 869, a steady state was reached after 5 min of agitation and 5 min of sedimentation. Chemicals All chemicals used were analytical grade except agar and yeast extract. They included CaC1, (Merck); EDTA (tetrasodium salt,
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FIG. 1. Effect of sugars of FCC expressed in cell concentration of the strains (A) NCYC 869 (MS group), (B) NCYC 1195 (GMS group), and (C) 27c (MI group), at 500 mM sugar concentrations and pH 4. Analysed sugars: man, mannopyranose; me-man, methyl-a-D-mannopyranose; malt, maltose; glu, glucose; gal, galactose; suc, sucrose; gly, glycerol; sorb, sorbitol. The experimental curves reported are for mannopyranose (m) and galactose (0).
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FIG. 2. Inhibition by sugars of yeast MS group containing different flocculation genes: (A) NCYC 870 (FLOI), (B) STX347-1D (FL05), and (C) YIY 261 (FL08), at 5 mM calcium concentration and pH 4. Analysed sugars: mannose and methyl-a-D-mannopyranose (m); glucose, galactose, maltose, glycerol, and sorbitol (0). The experimental curves reported are for mannopyranose (m) and glucose (0). Aldrich); yeast extract (Difco); agar (Pastagar A, Pasteur); acetic acid (Janssen); sodium hydroxide (Merck); hydrochloric acid (Merck); N-acetyl-a-D-glucosamine (Janssen); N-acetyl-P-Dmannosamine (Janssen); D-altrose (Janssen); p-aminophenyl-a-Dmannopyranose (Sigma); D-cellobiose (Fluka); D-fructose (Merck); D-galactose (Janssen); D-glucose (Janssen); a-D-glucose-1-phosphate (disodium salt; Sigma); D-glucose-6-phosphate (sodium salt; Sigma); glycerol (UCB, Union Chimique Belge); D-lactose (UCB); D-lyxose (Janssen); maltose (Janssen); maltotriose (Sigma); D-mannitol (Janssen); D-mannopyranose (Janssen); L-mannopyranose (Janssen); 3-0-a-D-mannopyranosyl-a-D-mannopyranose (Sigma); D-mannosamine (Sigma); a-D-mannose-1-phosphate (sodium salt, Sigma); D-mannose-6-phosphate (disodium salt; Sigma); methyl-a-D-glucopyranose (Sigma); methyl-P-D-glucopyranose (Sigma); methyl-a-D-mannopyranose (Janssen); methyl-2-O-c~-~-mannopyranosyl-c~-~-rnannopyranose (Sigma); methyl-4-O-c~-~-mannopyranosyl-c~-~-rnannopyranose (Sigma); p-nitrophenyl-a-D-mannopyranose (Sigma); p-nitrophenyl-P-D-
mannopyranose (Sigma); D-sorbitol (Merck); ribose (Sigma); sucrose (Janssen); and D-talose (Janssen).
Results Although calcium is known to play an important role in flocculation, at the concentration used in our experiments (0.5 mM), it failed to cause flocculation in the nonflocculent strains (strains D273- lob and KL 14-4A). To ascertain whether the calcium concentration we used was too low, calcium concentrations of 0.5-500 mM were tested. The results show, however, that even at the maximum concentration, no flocculation was observed when absorbance was measured.
Inhibition groups of yeasts When flocculation was measured in the presence of various sugars (monosaccharides or disaccharides) differ-
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ing in their O H configuration of substituents, only some of the sugars inhibited flocculation. The inhibitory effects of various sugars on the flocculation of the yeasts from the MS, GMS, and MI groups are illustrated in Figs. l A , lB, and l C , respectively. For yeasts from the MS and GMS groups, either noninhibitory or inhibitory curves were observed (Figs. 1A and 1B). Flocculation of the MS group was inhibited by only D-mannopyranose and methyl-a-Dmannopyranose, while that of GMS was inhibited by D-glucopyranose, D-mannopyranose, maltose, sucrose, and methyl-a-D-mannopyranose. The group of strains for which no sugar inhibited flocculation was called flocculation mannose insensitive (MI) (Fig. 1C). The strains in Table 1 all displayed the behaviors characteristic of their particular group. Results are presented as FCC. They show that inhibition of flocculation was complete for the strains of the GMS group (10' cells recovered) and incomplete for those of the MS group. Sugar inhibition of the flocculation of isolated cells walls instead of whole cells (MS group) was examined for strain NCYC 869. The results indicate that flocculation of isolated cells walls was inhibited by the same sugars that inhibited the corresponding entire cells. Flocculation, therefore, could be considered to be a process that involves only the cell wall (Eddy 1958; Nishihara et al. 1982), suggesting that sugar inhibition is not due to a metabolic effect. Figure 2 shows the inhibition of flocculation in the presence of increasing sugar concentration, for yeast containing different flocculation genes (MS group). The inhibitory capacity of mannopyranose and methyl-a-D-mannopyranose varies greatly between strains. The following table presents the order of inhibition, with the highest FCC obtained by using sugar concentrations up to 700 mM and the minimal sugar concentration needed to reach this FCC. Gene Strains FCC (ceIIs/mL) Sugar concentration (mM)
FL08 YIY2616 3 x 10 700
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FL05 > FL08. The FL08 strain was hardly inhibited by sugar at all. Thus, one can wonder if specific interactions are important for flocculation of this strain. Moreover, it was noted that the degree of inhibition by sugars corresponds to the order of pH sensitivity (Masy 1991). The mechanism by which ethanol promotes flocculation is unknown but a hypothesis has been proposed (Eddy 1955b). Amory et al. (1988) suggested that ethanol either acts as a surfactant by lowering water surface tension or modifies the conformation and properties of surface polymers. However, one should keep in mind that in these studies, the influence of pH may not have been well controlled. The reason why ethanol had an effect on sugar inhibition of strains from the MS group only but not on flocculation in our study remains to be explained.
Stereochemistry of the proteic receptors Inhibition by sugars indicates some crucial points concerning the interaction of sugar with lectinlike receptors of strains belonging to the MS or GMS group. In Fig. 5, the carbohydrate is presented in the C-1 chair conformation, which is the most probable conformation for a - and P-D-glucopyranosides and mannopyranosides in solution. Both groups of strains needed the pyranose ring configuration and an equatorial position of the hydroxyl on C-3, C-4, and C-6 (Stratford and Assinder 1991). The main differences are on C-1 and C-2, where no special configuration of the hydroxyl groups is required for strains belonging to the GMS group. An axial configuration is required for strains belonging to the MS group (Fig. 5). However, even in the GMS group, C-2 hydroxyl function represents a destabilizing factor (Stratford and Assinder 1991). Thus, strains from the GMS group show a broad specificity but only for C-2 and C-1. Some residues can influence the fixation: for example, a phenyl group increases the inhibitory power of .the compound for strains of the MS (Kihn et al. 1988a) and GMS groups. This suggests that a hydrophobic region exists near the mannose-binding site. For strains belonging to the MS group one can conclude that the receptor binds two or more residues of mannose in a(1- 2), a(1 -- 3), or a(1 -- 4) configurations. For strains from the GMS group, the receptor binds two residues with a(1 -- 2) or a(1 -- 4) bonds. Both receptors seem to be able to bind lateral chains of mannans that contain a(1 -- 2) and a ( l -- 3) bonds (Ballou 1976). Proteic receptors of strains from the GMS group present an inhibitory pattern similar to concanavalin A lectin; the proteic receptor of strain NCYC 869 has an inhibitory pattern like that of Escherichia coli type I lectin (Lis and Sharon 1986) that is specific for D-mannose but differs in the inhibitory pattern of oligomers. Of course, it is not possible to generalize on the basis of the two strains, but our results suggest that a more systematic study is worthwhile. Some preliminary results obtained on strain MUCL 29759, another strain from the GMS group (Kihn et al. 1988b), confirmed the results presented here (complementary results not shown). In conclusion, yeast strains can be grouped according to the inhibition of their flocculation by sugars. The MI group, which is not inhibited by mannose or glucose, is added to the MS and GMS groups already defined as Flol phenotype and NewFlo phenotype, respectively. (Stratford 1989; Stratford and Assinder 1991). The main differences between
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the receptors of strains belonging to the MS and GMS groups are the hydroxyl groups on C-1 and C-2. In the GMS group, these two positions are less important for fixation of the sugar. In the MS group, all hydroxyl groups are necessary for fixation to occur. The inhibition by sugar is not complete for the receptors of strains belonging to the MS group and this may be due to the higher specificity of these receptors for sugars. For these strains, ethanol enhances inhibition but the initial cell concentration is not entirely recovered.
Acknowledgements The authors gratefully acknowledge gifts of yeast strains (YIY 261) and (27c) from I. Yamashita of Hiroshima University (Japan) and from C. Ghommidth of the Centre de genie et technologie alimentaire (microbiologie industrielle), University of Montpellier (France), respectively. The authors thank the Service de la programmation de la politique scientifique (Concerted Action Physical Chemistry of Interfaces and Biotechnology) for financial support. Amory, D.E., Dufour, J.-P., and Rouxhet, P.G. 1988. Flocculence of brewery yeasts and their surface properties: chemical composition, electrostatic charge, and hydrophobicity. J. Inst. Brew. 94: 79-84. Ballou, C. 1976. Structure and biosynthesis of the mannan component of the yeast cell envelope. Adv. Microb. Physiol. 14: 93-158. Barre, P. 1988. Control of alcoholic fermentation in wine making. Int. Biotechnol. Symp. 8th 1988, 3: 899-909. Bautista, J., Chico, E., and Machado, A. 1986. Cell removal from fermentation broth by flocculation-sedimentation. Biotechnol. Lett. 8: 315-318. Bell, G.I., Dembo, M., and Bongrand, P. 1984. Cell adhesion: competition between nonspecific repulsion and specific bonding. Biophys. J. 52: 1051-1064. Bell, G.I. 1988. Models of cell adhesion involving specific binding. In Physical basis of cell-cell adhesion. Edited by P. Bongrand. CRC Press, Inc. Boca Raton, Fla. pp. 227-256. Calleja, G.B. 1984. Microbial aggregation. CRC Press, Inc., Boca Raton, Fla. p. 266. Chen, L., and Chen Shung, G. 1986. Continuous ethanol production using induced yeast aggregates. Appl. Microbiol . Biotechnol. 25: 208-212. Eddy, A.A. 1955a. Flocculation characteristics of yeasts. 11. Sugars as dispersing agents. J . Inst. Brew. 61: 313-317. Eddy, A.A. 1955b. Flocculation characteristics of yeasts. 111. General role of flocculating agents and special characteristics of two yeasts flocculated by alcohol. J . Inst. Brew. 61: 318-320. Eddy, A.A. 1958. Composite nature of the flocculation process of top and bottom strains of Saccharomyces. J. Inst. Brew. 64: 143-151. Esser, K., Kues, V., and Hinrichs, J. 1987. Genetic control of flocculation of yeast with respect to application in biotechnology. In Flocculation in biotechnology and separations systems. Edited by Y.A. Attia. Elsevier Science Publishers B.V., Amsterdam. pp. 393-398. Ghommidth, C., Fourtot, C., and Navarro, J.M. 1987. Fermentation in Saccharomyces cerevisiae and mitochondria1 DNA structure. Appl. Microbiol. Biotechnol. 29: 48-54. Johnston, J.R., and Reader, H.P. 1983. Genetic control of flocculation. In Yeast genetics: fundamental and applied aspects. Edited by J.F.T. Spencer, D.H. Spencer, and A. R. W. Smith. Springer-Verlag, New York. pp. 205-224.
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Kamada, K., and Murata, M. 1984. On the mechanism of brewer's yeast flocculation. Agric. Biol. Chem. 48: 2423-2433. Kihn, J.-C., Mestdagh, M.M., and Rouxhet, P.G. 1987. ESR study of copper(I1) retention by entire cell, cell walls and protoplasts of Saccharomyces cerevisiae. Can. J. Microbiol. 33: 777-782. Kihn, J.-C., Masy, C.L., and Mestdagh, M.M. 1988a. Yeast flocculation: competition between nonspecific and specific bonding in cell adhesion. Can. J. Microbiol. 34: 773-778. Kihn, J.-C., Masy, C.L., Mestdagh, M.M., and Rouxhet, P.G. 1988b. Yeast flocculation: factors affecting the measurement of flocculence. Can. J . Microbiol. 34: 779-781. Lewis, D.W., and Johnston, J.R. 1983. Flocculation genes in Saccharomyces cerevisiae. Microb. Genet. Bull. No. 35. p. 11. Lis, H., and Sharon, N. 1986. Lectins as molecules and as tools. Annu. Rev. Biochem. 55: 35-67. Masy, C.L., Kockerols, M., and Mestdagh, M.M. 1991. Calcium activity versus "calcium threshold" as the key factor in the induction of yeast flocculation in simulated industrial fermentations. Can. J . Microbiol. 37: 295-303. Masy, C.L. 1991. Incidence de facteurs physico-chimiques et genetiques sur les interactions specifiques intervenant dans la floculation de levures Saccharomyces cerevisiae. Ph.D thesis, Universite catholique de Louvain, Belgium. Miki, B.L.A., Poon, N.H., James, A.P., and Seligy, V.L. 1982. Possible mechanism for flocculation interactions governed by gene FLO 1 in Saccharomyces cerevisiae. J . Bacteriol. 150: 878-889. Mill, P.J. 1964. The nature of the interaction between flocculent cells in the flocculation of Saccharomyces cerevisiae. J. Gen. Microbiol. 35: 3 1-68. Nishihara, H., Toraya, T., and Fukui, S. 1982. Flocculation of cell walls of brewer's yeast and effects of metals ions, protein denaturants, and enzyme treatments. Arch. Microbiol. 131: 112-115. Poretz, D., and Goldstein, I.J. 1970. An examination of the topography of the saccharide binding sites of Concanavalin A and the forces involved in the complexation. Biochemistry, 9: 2890-2896. So, L.L., and Goldstein, I. 1968. Protein-carbohydrate interaction. XX. On the number of combining sites on Concanavalin A, the phytohemagglutinin of the jack bean. Biochim. Biophys. Acta, 165: 398-404. Stewart, G.G., and Russell, I. 1977. The identification, characterization, and mapping of a gene for flocculation in Saccharomyces sp. Can. J. Microbiol. 23: 441-447. Stewart, G.G., and Russell, I. 1987. The relevance of the flocculation properties of yeast on today's brewing industry. Monogr. Eur. Brew. Conv. 12: 53-70. Stratford, M. 1989. Evidence for two mechanisms of flocculation in Saccharomyces cerevisiae. Yeast, 5: S441-445. Stratford, M., and Assinder, S. 1991. Yeast flocculation: F101 and NewFlo phenotypes and receptor structure. Yeast, 7: 559-574. Stratford, M., and Keenan, M.H.J. 1988. Yeast flocculation: quantification. Yeast, 4: 107-1 15. Stratford, M., Coleman, H.P., and Keenan, M.H.J. 1988. Yeast flocculation: a dynamic equilibrium. Yeast, 4: 199-208. Taylor, N. W., and Orton, W.L. 1978. Aromatic compounds and sugars in flocculation of Saccharomyces cerevisiae. J . Inst. Brew. 84: 113-114. Thornton, R. J . 1985. The introduction of flocculation into homothallic wine yeast. A practical example of the modification of wine making properties by the use of genetic techniques. Am. J . Enol. Vitic. 36: 47-49. Yamashita, I., and Fukui, S. 1983. Mating signals control expression of both starch fermentation genes and a novel flocculation gene FL08 in the yeast Saccharomyces. Agric. Biol. Chem. 47: 2889-2896.
