Applied Microbiology and Biotechnology

, Volume 61, Issue 3, pp 197–205 | Cite as

Yeast flocculation: what brewers should know

  • K. J. VerstrepenEmail author
  • G. Derdelinckx
  • H. Verachtert
  • F. R. Delvaux


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.


Flocculation Pitching Rate Lager Yeast Lager Strain Flocculation Rate 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



The authors wish to thank Dr. F. Bauer, Prof. I.S. Pretorius, Prof. J.M. Thevelein and Prof. J. Winderickx for the many fruitful discussions on yeast flocculation. K.J. Verstrepen wishes to thank the Fund for Scientific Research Flanders (FWO-Vlaanderen) for the financial support of his work.


  1. Axcell BC, Van Nierop S, Vundla W (2000) Malt induced premature flocculation of yeast. In: Abstracts, World Brewing Congress, Orlando, Fla., 29 July–2 August 2000. ASBC St. Paul, Minn., pp 69Google Scholar
  2. Barker MG, Smart KA (1996) Morphological changes associated with the cellular ageing of a brewing yeast strain. J Am Soc Brew Chem 54:121–126Google Scholar
  3. Barney MC, Jansen GP, Helber GR (1980) Use of genetic transformation for the introduction of flocculence into yeast. J Am Soc Brew Chem 38:71–74Google Scholar
  4. Barton AB, Bussey H, Storms RK, Kaback DB (1997) Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: characterization of the 54 kb right terminal CDC15-FLO1-PHO11 region. Yeast 13:1251–1263CrossRefGoogle Scholar
  5. Bendiak D, Van der Aar P, Barbero F, Benzing P, Berndt R, Carrick K, Dull C, Dunn S, Eto M, Gonzalez M, Hayashi N, Lawrence D, Miller J, Phare K, Pugh T, Rashel L, Rossmore K, Smart KA, Sobczak J, Speers A, Casey G (1996) Yeast flocculation by absorbance. J Am Soc Brew Chem 54:245–248Google Scholar
  6. Bidard F, Bony M, Blondin B, Dequin S, Barre P (1995) The Saccharomyces cerevisiae FLO1 flocculation gene encodes a cell surface protein. Yeast 11:809–822PubMedGoogle Scholar
  7. Bony M, Thines-Sempoux D, Barre P, Blondin B (1997) Localisation and cell surface anchoring of the Saccharomyces cerevisiae flocculation protein Flo1p. J Bacteriol 179:4929–4936PubMedGoogle Scholar
  8. Bony M, Thines-Sempoux D, Barre P, Blondin B (1998) Distribution of the flocculation protein, Flop, at the cell surface during yeast growth: the availability of Flop determines the flocculation level. Yeast 14:25–35CrossRefPubMedGoogle Scholar
  9. Bossier P, Goethals P, Rodrigues-Pousada C (1997) Constitutive flocculation in Saccharomyces cerevisiae through overexpression of the GTS1 gene, coding for a 'Glo'-type Zn-finger-containing protein. Yeast 13:717–725CrossRefPubMedGoogle Scholar
  10. Costa MJ, Moradas-Ferreira P (2001) S. cerevisiae flocculation: identification of specific cell wall proteins. Proc Congr Eur Brew Conv 24:283–290Google Scholar
  11. Deans K, Pinder A, Catley BJ, Hodgson JA (1997) Effects of cone cropping and serial re-pitch on the distribution of cell ages in brewery yeast. Proc Congr Eur Brew Conv 26:469–476Google Scholar
  12. De Clerck J (1984) Cours de brasserie, 2nd edn. Academic Press, LeuvenGoogle Scholar
  13. Dengis PB, Rouxhet PG (1997) Flocculation mechanism of top and bottom fermenting brewing yeast. J Inst Brew 103:257–261Google Scholar
  14. D'Hautcourt O, Smart KA (1999) Measurement of brewing yeast flocculation. J Am Soc Brew 57:123–128Google Scholar
  15. Eddy AA (1955) Flocculation characteristics of yeast II: sugars as dispersing agents. J Inst Brew 61:313–317Google Scholar
  16. Fleming AB, Pennings S (2001) Antagonistic remodelling by Swi-Snf and Tup1-Ssn6 of an extensive chromatin region forms the background for FLO1 gene regulation. EMBO J 20:5219–5231CrossRefPubMedGoogle Scholar
  17. Gagiano M, van Dyk D, Bauer FF, Lambrechts MG, Pretorius IS (1999a) Divergent regulation of the evolutionary closely related promoters of the Saccharomyces cerevisiae STA2 and MUC1 genes. J Bacteriol 181:6497–6508PubMedGoogle Scholar
  18. Gagiano M, van Dyk D, Bauer FF, Lambrechts MG, Pretorius IS (1999b) Msn1p/Mss10p, Mss11p and Muc1p/Flo11p are part of a signal transduction pathway downstream of Mep2p regulating invasive growth and pseudohyphal differentiation in Saccharomyces cerevisiae. Mol Microbiol 31:103–116PubMedGoogle Scholar
  19. Garsoux G, Haubursin S, Bilbault S, Dufour J-P (1993) Yeast flocculation: biochemical characterization of yeast cell wall components. Proc Congr Eur Brew Conv 24:275–282Google Scholar
  20. Gilliand R (1951) The flocculation characteristics of brewing yeast during fermentation. Proc Congr Eur Brew Conv 3:3557Google Scholar
  21. Gilliand R (1978) Deterioration and improvement of brewing yeast. Eur Brew Conv Monogr 5:51–65Google Scholar
  22. Gonzales MG, Fernandez S, Sierra JA (1996) Effect of temperature in the evaluation of yeast flocculation ability by the Helm's method. J Am Soc Brew Chem 54:29–31Google Scholar
  23. Hammond JRM (1991) The development of brewing processes: the impact of European biotechnology regulations. Proc Congr Eur Brew Conv 23:393–400Google Scholar
  24. Hammond JRM (1995) Genetically-modified brewing yeasts for the 21st century. Yeast 11:1613–1627PubMedGoogle Scholar
  25. Heggart HM, Margaritis A, Pilkington H, Stewart RJ, Dowhanick TM, Russel I (1999) Factors affecting yeast viability and vitality characteristics: a review. Tech Q Master Brew Assoc Am 36:383–406Google Scholar
  26. Helm E, Nohr B, Thorne RSW (1953) The measurement of yeast flocculence and its significance in brewing. Wallerstein Lab Commun 16:315–325Google Scholar
  27. Herrera VE, Axcell BC (1989) The influence of barley lectins on yeast flocculation. J Am Soc Brew Chem. 47:29–34Google Scholar
  28. Herrera VE, Axcell BC (1991a) Induction of premature yeast flocculation caused by a polysaccharide fraction isolated from malt husk. J Inst Brew 97:359–366Google Scholar
  29. Herrera VE, Axcell BC (1991b) Studies on the binding between yeast and a malt polysaccharide that induces heavy yeast flocculation. J Inst Brew 97:367–373Google Scholar
  30. Ishida-Fujii K, Goto S, Sugiyama H, Takagi Y, Saiki T, Takagi M (1998) Breeding of flocculent industrial alcohol yeast strains by self-cloning of the flocculation gene FLO1 and repeated-batch fermentation by transformants. J Gen Appl Microbiol 44:347–353PubMedGoogle Scholar
  31. Javadekar VS, Silvaraman H, Sainkar SR, Khan MI (2000) A mannose binding protein from the cell surface of flocculent Saccharomyces cerevisiae (NCIM 3528): its role in flocculation. Yeast 16:99–110CrossRefPubMedGoogle Scholar
  32. Jibiki M, Ishibiki T, Yuuki T, Kagami N (2001) Application of polymerase chain reaction to determine the flocculation properties of brewer's lager yeast. J Am Soc Brew 59:107–110Google Scholar
  33. Jin Y-L, Speers A (2000) Effect of environmental conditions on the flocculation of Saccharomyces cerevisiae. J Am Soc Brew Chem 58:108–116Google Scholar
  34. Jin Y-L, Ritcey LL, Speers RAR, Dolphin PJ (2001) Effect of cell-surface hydrophobicity, charge and zymolectin density on the flocculation of Saccharomyces cerevisiae. J Am Soc Brew Chem 59:1–9Google Scholar
  35. Johnston JR, Reader HP (1983) Genetic control of flocculation. In: Spencer JFT, Spencer DM, Smith ARW (eds) Yeast genetics, fundamental and applied aspects. Springer, Berlin Heidelberg New York, pp 205–222Google Scholar
  36. Kamada K, Murata M (1984) On the mechanism of brewer's yeast flocculation. Agric Biol Chem 48:2423–2433Google Scholar
  37. Kempers J, Van der Aar P, Krotjé J (1991) Flocculation of brewer's yeast during fermentation. Proc Congr Eur Brew Conv 23:249–256Google Scholar
  38. Kobayashi O, Hayashi N, Sone H (1995) The FLO1 genes determine two flocculation phenotypes distinguished by sugar inhibition. Proc Congr Eur Brew Conv 16:361–367Google Scholar
  39. Kobayashi O, Suda H, Ohtani T, Sone H (1996) Molecular cloning and analysis of the dominant flocculation gene FLO8 from Saccharomyces cerevisiae. Mol Gen Genet 251:707–715CrossRefPubMedGoogle Scholar
  40. Kobayashi O, Hayashi N, Kuroki R, Sone H (1998) Region of Flo1 proteins responsible for sugar recognition. J Bacteriol 180:6503–6510PubMedGoogle Scholar
  41. Kobayashi O, Hiroyuki Y, Sone H (1999) Analysis of the genes activated by the FLO8 gene in Saccharomyces cerevisiae. Curr Genet 36:256–261PubMedGoogle Scholar
  42. Lievens K, Devogel D, Iserentant D, Verachtert H (1994) Evidence for a factor produced by Saccharomyces cerevisiae which causes flocculation of Pediococcus damnosus 12A7 cells. Colloid Surface B Biointerfaces 2:189–198CrossRefGoogle Scholar
  43. Lipke PN, Hull-Pillsbury C (1984) Flocculation of Saccharomyces cerevisiae tup1 mutants. J Bacteriol 159:797–799PubMedGoogle Scholar
  44. Masy CL, Henquinet A, Mestdagh MM (1992) Flocculation of Saccharomyces cerevisiae: inhibition by sugars. Can J Microbiol 38:1298–1306PubMedGoogle Scholar
  45. Miki BLA, Poon NH, Seligy VL (1982) Repression and induction of flocculation interactions in Saccharomyces cerevisiae. J Bacteriol 150:890–899PubMedGoogle Scholar
  46. Mochaba F, Cantrell I, Vundla W (2001) The use of concanavalin A to investigate the mechanism and onset of flocculation by a brewing yeast strain. Proc Congr Eur Brew Conv 28:397–406Google Scholar
  47. Nakamura T, Chiba K, Ashara Y, Tada S (1997) Prediction of barley which produces premature yeast flocculation. Proc Congr Eur Brew Conv 26:53–60Google Scholar
  48. Nishihara H, Kio K, Imamura M (2000) Possible mechanism of co-flocculation between non-flocculent yeasts. J Inst Brew 106:7–10Google Scholar
  49. Nishihara H, Miyake K, Kageyama Y (2002) Distinctly different characteristics of flocculation in yeast. J Inst Brew 108:187–192Google Scholar
  50. Pan XW, Heitman J (1999) Cyclic-AMP-dependent protein kinase regulates pseudohyphal differentiation in Saccharomyces cerevisiae. Mol Cell Biol 19:4874–4887PubMedGoogle Scholar
  51. Patelakis SJJ, Ritcey L, Speers RA (1998) Density of lectin-like receptors in the FLO1 phenotype of Saccharomyces cerevisiae. Lett Appl Microbiol 26:279–282CrossRefPubMedGoogle Scholar
  52. Peng X, Sun J, Iserentant D, Michiels C, Verachtert H (2001a) Flocculation and coflocculation of bacteria by yeasts. Appl Microbiol Biotechnol 55:777–781CrossRefPubMedGoogle Scholar
  53. Peng X, Sun J, Michiels C, Iserentant D, Verachtert H (2001b) Coflocculation of Escherichia coli and Schizosaccharomyces pombe. Appl Microbiol Biotechnol 57:175–181CrossRefPubMedGoogle Scholar
  54. Powell CD, Van Zandycke SM, Quain DE, Smart KA (2000) Replicative ageing and senescence in Saccharomyces cerevisiae and the impact on brewing fermentations. Microbiology 146:1023–1034PubMedGoogle Scholar
  55. Pretorius IS (2000) Tailoring wine yeast for the new millennium: novel approaches to the ancient art of winemaking. Yeast 16:675–729PubMedGoogle Scholar
  56. Quain DE, Powell CD, Hamilton A, Ruddlesden D, Box W (2001) Why warm cropping is best. Proc Congr Eur Brew Conv 28:388–395Google Scholar
  57. Reboredo NM, Siero C, Blanco P, Villa TG (1996) Isolation and characterization of a mutant of Saccharomyces cerevisiae affected in the FLO1 locus. FEMS Microbiol Lett 137:57–61CrossRefPubMedGoogle Scholar
  58. Rhymes MR, Smart KA (2001) Effect of storage conditions on the flocculation and cell wall characteristics of an ale brewing yeast strain. J Am Soc Brew 59:32–38Google Scholar
  59. Robertson RS, Fink G (1998) The three yeast A kinases have specific signalling functions in pseudohyphal growth. Proc Natl Acad Sci USA 95:13783–13787PubMedGoogle Scholar
  60. Rupp S, Summers E, Lo HJ, Madhani, H, Fink G (1999) MAP kinase and cAMP filamentation signaling pathways converge on the unusually large promoter of the yeast FLO1 gene. EMBO J 18:1257–1269PubMedGoogle Scholar
  61. Russel I, Stewart GG, Reader HP, Johnston JR, Martin PA (1980) Revised nomenclature of genes that control yeast flocculation. J Inst Brew 80:116–121Google Scholar
  62. Sato M, Watari J, Shinotsuka K (2001) Genetic instability in flocculation of bottom-fermenting yeast. J Am Soc Brew Chem 59:130–134Google Scholar
  63. Sato M, Maeba H, Watari J, Takashio M (2002) Analysis of an inactivated Lg-FLO1 gene present in bottom-fermenting yeast. J Biosci Bioeng 93:395–398CrossRefGoogle Scholar
  64. Sieiro C, Reboredo NM, Villa TG (1995) Flocculation of industrial and laboratory strains of Saccharomyces cerevisiae. J Ind Microbiol 14:461–466PubMedGoogle Scholar
  65. Sieiro C, Reboredo NM, Blanco P, Villa TG (1997) Cloning of a new FLO gene from the flocculating Saccharomyces cerevisiae IM1–8b strain. FEMS Microbiol Lett 146:109–115CrossRefPubMedGoogle Scholar
  66. Smart KA (1999) Ageing in brewing yeast. Brew Guardian 128:19–24Google Scholar
  67. Smart KA, Whisker S (1996) Effect of serial repitching on the fermentation properties and condition of brewing yeast. J Am Soc Brew 54:41–44Google Scholar
  68. Smit G, Straver MH, Lugtenberg JJ, Kijne JW (1992) Flocculence of Saccharomyces cerevisiae cells is induced by nutrient limitation, with cell surface hydrophobicity as a major determinant. Appl Environ Microbiol 58:3709–3714PubMedGoogle Scholar
  69. Smith RL, Johnson AD (2000) Turning genes off by Tup1-Ssn6: a conserved system of transcriptional repression in eukaryotes. Trends Biochem Sci 25:325–330CrossRefPubMedGoogle Scholar
  70. Soares EV, Mota M (1996) Flocculation onset, growth phase and genealogical age in Saccharomyces cerevisiae. Can J Microbiol 42:539–547PubMedGoogle Scholar
  71. Soares EV, Texeira JA, Mota M (1994) Effect of cultural and nutritional conditions on the control of flocculation expression in Saccharomyces cerevisiae. Can J Microbiol 40:851–857PubMedGoogle Scholar
  72. Stewart GG, Russel I (1986) The relevance of flocculation properties of yeast in today's brewing industry. Eur Brew Conv Monogr 7:53–68Google Scholar
  73. Stratford M (1989) Yeast flocculation: calcium specificity. Yeast 5:487–496Google Scholar
  74. Stratford M (1992) yeast flocculation: a new perspective. Adv Microbiol Physiol 33:2–71Google Scholar
  75. Stratford M, Assinder S (1991) Yeast flocculation: Flo1 and newFlo phenotypes and receptor structure. Yeast 7:559–574PubMedGoogle Scholar
  76. Stratford M, Keenan MHJ (1988) Yeast flocculation: quantification. Yeast 4:107–115PubMedGoogle Scholar
  77. Straver MH, Kijne JW (1996) A rapid and selective assay for measuring cell surface hydrophobicity of brewer's yeast cells. Yeast 12:207–213CrossRefPubMedGoogle Scholar
  78. Straver MH, Aar PC, van der Smit G, Kijne JW (1993) Determinants of flocculence of brewer's yeast during fermentation in wort. Yeast 9:527–532PubMedGoogle Scholar
  79. Straver MH, Smit G, Kijne JW (1994a) Purification and partial characterization of a flocculin from brewer's yeast. Appl Environ Microbiol 60:2754–2758PubMedGoogle Scholar
  80. Straver MH, Traas VM, Smit G, Kijne JW (1994b) Isolation and partial purification of mannose-specific agglutinin from brewer's yeast involved in flocculation. Yeast 10:1183–1193PubMedGoogle Scholar
  81. Tamaki H, Miwa T, Shinozaki M (2000) GPR1 regulates filamentous growth through FLO1 in the yeast Saccharomyces cerevisiae. Biochem Biophys Res Commun 267:164–168CrossRefPubMedGoogle Scholar
  82. Taylor NW, Orton WI (1978) Aromatic compounds and sugars in flocculation of Saccharomyces cerevisiae. J Inst Brew 84:113–114Google Scholar
  83. Teunissen A, Holub E, Van Der Hucht J, Van Den Bergh JA, Steensma HY (1993a) Sequence of the open reading frame of the FLO1 gene from Saccharomyces cerevisiae. Yeast 9:423–427PubMedGoogle Scholar
  84. Teunissen A, Van Den Bergh JA, Steensma HY (1993b) Physical localization of the flocculation gene FLO1 on chromosome I of Saccharomyces cerevisiae. Yeast 9:1–10Google Scholar
  85. Teunissen A, Van Den Bergh JA, Steensma HY (1995) Transcriptional regulation of the flocculation genes in Saccharomyces cerevisiae. Yeast 11:435–446PubMedGoogle Scholar
  86. Texeira JM, Texeira JA, Mota M, Manuela M, Guerra B, Machado Cruz JM, S'Almeida AM (1991) The influence of cell wall composition of a brewer's flocculant lager yeast on sedimentation during successive industrial fermentations. Proc Congr Eur Brew Conv 23:241–248Google Scholar
  87. Van den Bremt K, Iserentant D, Verachtert H (1997a) Induction of flocculation in Pediococcus damnosus by different yeast strains. Biotechnol Tech 11:879–884Google Scholar
  88. Van den Bremt K, Nuyens F, Iserentant D, Verachtert H (1997b) Identification of a Saccharomyces cerevisiae factor inducing flocculation of Pediococcus damnosus. Med Fac Landbouww Univ Gent 62:1185–1192Google Scholar
  89. Van der Aar P, Straver MH, Teunissen A (1993) Flocculation of brewer's lager yeast. Proc Congr Eur Brew Conv 14:259–266Google Scholar
  90. Verhasselt P, Volckaert G (1997) Sequence analysis of a 37.6 kbp cosmid clone from the right arm of Saccharomyces cerevisiae chromosome XII, carrying YAP3, HOG1, SNR6, tRNA-Arg3 and 23 new open reading frames, among which several homologies to proteins involved in cell division control and to mammalian growth factors and other animal proteins are found. Yeast 13:241–250CrossRefPubMedGoogle Scholar
  91. Verstrepen KJ, Bauer FF, Michiels C, Derdelinckx G, Delvaux FR, Pretorius IS (1999) Controlled expression of FLO1 in Saccharomyces cerevisiae. Eur Brew Conv Monogr 28:30–42Google Scholar
  92. Verstrepen KJ, Bauer FF, Winderickx J, Derdelinckx G, Dufour J-P, Thevelein JM, Pretorius IS, Delvaux FR (2001a) Genetic modification of Saccharomyces cerevisiae: fitting the modern brewer's needs. Cerevisia 26:89–97Google Scholar
  93. Verstrepen KJ, Michiels C, Derdelinckx G, Delvaux FR, Winderickx J, Thevelein JM, Bauer FF, Pretorius IS (2001b) Late fermentation expression of FLO1 in Saccharomyces cerevisiae. J Am Soc Brew Chem 59:69–76Google Scholar
  94. Watari J, Takata Y, Ogawa M, Nishikawa N, Kamimura M (1989) Molecular cloning of a flocculation gene in Saccharomyces cerevisiae. Agric Biol Chem 53:901–903Google Scholar
  95. Watari J, Kudo M, Nishikawa N, Kamimura M (1990) Construction of flocculent yeast cells (Saccharomyces cerevisiae) by mating or protoplast fusion using a yeast cell containing the flocculation gene FLO5. Agric Biol Chem 54:1677–1681Google Scholar
  96. Watari J, Nomura M, Sahara H, Koshino S, Keranen S (1994a) Construction of flocculent brewer's yeast by chromosomal integration of the yeast flocculation gene FLO1. J Inst Brew 100:73–77Google Scholar
  97. Watari J, Takata Y, Ogawa M, Sahara H, Koshino S, Onnela ML, Airaksinen U, Jaatinen R, Pentillä M, Keränen S (1994b) Molecular cloning and analysis of the yeast flocculation gene FLO1. Yeast 10:211–225PubMedGoogle Scholar
  98. Watari J, Sato M, Ogawa M, Shinotsuka K (1999) Genetic and physiological instability of brewing yeast. Eur Brew Conv Monogr 28:148–160Google Scholar
  99. Wilcocks KL, Smart KA (1995) The effect of surface charge and hydrophobicity for the flocculation of chain-forming brewing yeast strains and resistance of these parameters to acid washing. FEMS Microbiol Lett 134:293–297CrossRefPubMedGoogle Scholar
  100. Yamashita I, Fukui S (1983) Mating signals control expression of both starch fermentation genes and a novel flocculation gene FLO8 in the yeast Saccharomyces. Agric Biol Chem 47:2889–2896Google Scholar

Copyright information

© Springer-Verlag 2003

Authors and Affiliations

  • K. J. Verstrepen
    • 1
    Email author
  • G. Derdelinckx
    • 1
  • H. Verachtert
    • 1
  • F. R. Delvaux
    • 1
  1. 1.Centre for Malting and Brewing Science, Department of Food and Microbial TechnologyK.U. LeuvenLeuven (Heverlee)Belgium

Personalised recommendations