Journal of Industrial Microbiology & Biotechnology

, Volume 39, Issue 12, pp 1821–1832 | Cite as

TRFLP analysis reveals that fungi rather than bacteria are associated with premature yeast flocculation in brewing

  • Mandeep KaurEmail author
  • John P. Bowman
  • Doug C. Stewart
  • Megan Sheehy
  • Agnieszka Janusz
  • R. Alex Speers
  • Anthony Koutoulis
  • David E. Evans
Environmental Microbiology


Premature yeast flocculation (PYF) is a sporadic fermentation problem in the brewing industry that results in incomplete yeast utilization of fermentable sugars in wort. Culture-independent, PCR-based fingerprinting techniques were applied in this study to identify the associations between the occurrence of the PYF problem during brewery fermentation with barley malt-associated microbial communities (both bacteria and fungi). Striking differences in the microbial DNA fingerprint patterns for fungi between PYF positive (PYF +ve) and negative (PYF −ve) barley malts were observed using the terminal restriction fragment length polymorphism (TRFLP) technique. The presence of terminal restriction fragments (TRFs) of 360–460 bp size range, for fungal HaeIII restriction enzyme-derived TRFLP profiles appeared to vary substantially between PYF +ve and PYF −ve samples. The source of the barley malt did not influence the fungal taxa implicated in PYF. TRFLP analysis indicates bacterial taxa are unlikely to be important in causing PYF. Virtual digestion of fungal sequences tentatively linked HaeIII TRFs in the 360–460 bp size range to a diverse range of yeast/yeast-like species. Findings from this study suggest that direct monitoring of barley malt samples using molecular methods could potentially be an efficient and viable alternative for monitoring PYF during brewery fermentations.


Barley malt Brewery fermentation Community fingerprinting Microbial communities Yeast 



We greatly acknowledge Adam Smolenski (Central Science Laboratory—Research, University of Tasmania, Tasmania, Australia) for his assistance in TRFLP analysis. Joseph Lake (Food Science and Technology, Dalhousie University, Nova Scotia, Canada) for performing the small-scale fermentation test. Ms. Kaur was the recipient of an Australian Research Council Industry Linkage scholarship (LP0560329) that was supported by Viterra Ltd.

Supplementary material

10295_2012_1188_MOESM1_ESM.docx (830 kb)
Supplementary material 1 (DOCX 829 kb)


  1. 1.
    Anderson MJ, Gorley RN, Clarke KR (2008) PERMANOVA+ for PRIMER: guide to software and statistical methods. PRIMER-E, PlymouthGoogle Scholar
  2. 2.
    Armstrong K, Bendiak D (2007) PYF malt: practical brewery observations of fermentability. Tech Q Master Brew Assoc Am 44:40–46Google Scholar
  3. 3.
    Axcell BC, Tulej R, Mulder CJ (1986) The influence of the malting process on malt fermentability performance. In: Proceedings of the convention of the institute guild of brewing, Australia and New Zealand section, Hobart, Australia, pp 63–69Google Scholar
  4. 4.
    Axcell BC, van Nierop SNE, Vundla W (2000) Malt-induced premature yeast flocculation. Tech Q Master Brew Assoc Am 37:501–504Google Scholar
  5. 5.
    Blechová P, Havlová P, Havel J (2005) The study of premature yeast flocculation and its relationship with gushing of beer. Monatsschrift Brauwiss 59:64–78Google Scholar
  6. 6.
    Bony M, Thines-Sempoux D, Barre P, Blondin B (1997) Localization and cell surface anchoring of the Saccharomyces cerevisiae flocculation protein Flo1p. J Bacteriol 179:4929–4936PubMedGoogle Scholar
  7. 7.
    Bray JR, Curtis JT (1957) An ordination of the upland forest communities of southern Wisconsin. Ecol Monogr 27:325–349CrossRefGoogle Scholar
  8. 8.
    Clarke KR (1993) Non-parametric multivariate analyses of changes in community structure. Aust J Ecol 18:117–143CrossRefGoogle Scholar
  9. 9.
    Demain A, Phaff H, Kurtzman C (1998) The industrial and agricultural significance of yeasts. In: Kurtzman C, Fell J (eds) The yeasts, a taxonomic study. Elsevier Science BV, Amsterdam, pp 13–30CrossRefGoogle Scholar
  10. 10.
    Egert M, Friedrich MW (2003) Formation of pseudo-terminal restriction fragments, a PCR-related bias affecting terminal restriction fragment length polymorphism analysis of microbial community structure. Appl Environ Microbiol 69:2555–2562PubMedCrossRefGoogle Scholar
  11. 11.
    Esteban A, Abarca ML, Bragulat MR, Cabaňes FJ (2006) Effect of water activity on ochratoxin A production by Aspergillus niger aggregate species. Int J Food Microbiol 108:188–195PubMedCrossRefGoogle Scholar
  12. 12.
    Flannigan B (1996) The microflora of barley and malt. In: Priest FG, Campbell I (eds) Brewing microbiology. Chapman and Hall, London, pp 83–126Google Scholar
  13. 13.
    Fujino S, Yoshida T (1976) Premature flocculation of yeast induced by some wort constituents. Rep Res Lab Kirin Brew Co 19:45–53Google Scholar
  14. 14.
    Gorjanović S, Sužnjević D, Beljanski M, Ostojić S, Gorjanović R, Vrvić M, Hranisavljević J (2004) Effects of lipid-transfer protein from malting barley grain on brewer’s yeast fermentation. J Inst Brew 110:297–302CrossRefGoogle Scholar
  15. 15.
    Griggs D, Fisher G, Walker S (2008) Factors that promote premature yeast flocculation condition in malt. In: Proceedings of the world brewing congress, Honolulu, USA, Presentation # 49, CD–ROMGoogle Scholar
  16. 16.
    Gurtler V, Stanisich VA (1996) New approaches to typing and identification of bacteria using the 16S–23S rDNA spacer region. Microbiol 142:3–16CrossRefGoogle Scholar
  17. 17.
    Hall TA (1999) BioEdit: a user—friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41:95–98Google Scholar
  18. 18.
    Hammond J (2000) Yeast growth and nutrition. In: Smart K (ed) Brewing yeast fermentation performance, 1st edn. Blackwell Scientific, Oxford, pp 77–85Google Scholar
  19. 19.
    Helin TRM, Slaughter JC (1977) Minimum requirements for zinc and manganese in brewers’ wort. J Inst Brew 83:17–19Google Scholar
  20. 20.
    Herrera VE, Axcell BC (1991) Induction of premature yeast flocculation by polysaccharide fraction isolated from malt husk. J Inst Brew 97:359–366Google Scholar
  21. 21.
    Inagaki H, Yamazumi K, Uehara H, Mochzuki K (1994) Determination of fermentation behaviour-malt evaluation system based on the original small scale fermentation test. In: European brewery convention monograph 23, Malting Technology Andernach Fachverlag Hans Carl, Nürnberg, Germany, pp 111–136Google Scholar
  22. 22.
    Jibiki M, Sasaki K, Kaganami N, Kawatsura K (2006) Application of a newly developed method for estimating the premature yeast flocculation potential of malt samples. J Am Soc Brew Chem 64:79–85Google Scholar
  23. 23.
    Kennedy N, Edwards S, Clipson N (2005) Soil bacterial and fungal community structure across a range of unimproved and semi-improved upland grasslands. Microb Ecol 50:463–473PubMedCrossRefGoogle Scholar
  24. 24.
    Koizumi H, Ogawa T (2005) Rapid and sensitive method to measure premature yeast flocculation activity in malt. J Am Soc Brew Chem 63:147–150Google Scholar
  25. 25.
    Koizumi H, Kato Y, Ogawa T (2008) Barley malt polysaccharides inducing premature yeast flocculation and their possible mechanism. J Am Soc Brew Chem 66:137–142Google Scholar
  26. 26.
    Lake JC, Speers RA (2008) A discussion of malt-induced premature yeast flocculation. Tech Q Master Brew Assoc Am 45:253–262Google Scholar
  27. 27.
    Lake JC, Speers RA, Porter AV, Gill TA (2008) Miniaturizing the fermentation assay: effects of fermentor size and fermentation kinetics on detection of premature yeast flocculation. J Am Soc Brew Chem 66:94–102Google Scholar
  28. 28.
    Laitila A, Wilhelmson A, Kotaviita E, Olkku J, Home S, Juvonen R (2006) Yeasts in an industrial malting ecosystem. J Ind Microbiol Biotechnol 33:953–966PubMedCrossRefGoogle Scholar
  29. 29.
    Lemos JLS, de Fontes MCA, Pereira N (2001) Xylanase production by Aspergillus awamori in solid-state fermentation and influence of different nitrogen sources. Appl Biochem Biotechnol 91(93):681–689PubMedCrossRefGoogle Scholar
  30. 30.
    Liliya G, Bourque NT, Bärlocher F (2005) Fungal diversity during initial stages of leaf decomposition in a stream. Mycol Res 109:246–253CrossRefGoogle Scholar
  31. 31.
    Lindahl BD, Ihrmark K, Boberg J, Trumbore SE, Högberg P, Stenlid J, Finlay RD (2007) Spatial separation of litter decomposition and mycorrhizal nitrogen uptake in a boreal forest. New Phytol 173:611–620PubMedCrossRefGoogle Scholar
  32. 32.
    Lozupone C, Hamady M, Knight R (2006) UniFrac—an online tool for comparing microbial community diversity in a phylogenetic context. BMC Bioinf 7:371CrossRefGoogle Scholar
  33. 33.
    McCabe JT (1999) The practical brewer. Master Brewers Association of the Americas, Wauwatosa, WIGoogle Scholar
  34. 34.
    Nakamura T, Chiba K, Asahara Y, Tada S (1997) Prediction of barley which causes premature yeast flocculation. In: Proceedings of the European brewery convention congress, Maastricht, The Netherlands, pp 53–60Google Scholar
  35. 35.
    Noll M, Matthies D, Frenzel P, Derakshani M, Liesack W (2005) Succession of bacterial community structure and diversity in a paddy soil oxygen gradient. Environ Microbiol 7:382–395PubMedCrossRefGoogle Scholar
  36. 36.
    Noots I, Delcour JA, Michiels CW (1999) From field barley to malt: detection and specification of microbial activity for quality aspects. Crit Rev Microbiol 25:121–153PubMedCrossRefGoogle Scholar
  37. 37.
    O’Donnell K (1993) Fusarium and its near relatives. In: Reynolds DR, Taylor JW (eds) The fungal holomorph: mitotic, meiotic and pleomorphic speciation in fungal systematic. CAB International, Wallingford, pp 225–233Google Scholar
  38. 38.
    Rees GN, Baldwin DS, Watson GO, Perryman S, Nielsen DL (2004) Ordination and significance testing of microbial community composition derived from terminal restriction fragment length polymorphisms: application of multivariate statistics. Antonie Van Leeuwenhoek 86:339–347PubMedCrossRefGoogle Scholar
  39. 39.
    Sasaki K, Yamashita H, Kono K, Kitagawa Y (2008) Investigation of the causes of PYF malt using a modified analytical method for the PYF potential. In: Proceedings of the world brewing congress, Honolulu, USA, Poster # 183, CD–ROMGoogle Scholar
  40. 40.
    Singh BK, Nunan N, Ridgway KP, McNicol J, Young JPW, Daniell TJ, Prosser JI, Millard P (2007) Relationship between assemblages of mycorrhizal fungi and bacteria on grass roots. Environ Microbiol 10:534–541PubMedCrossRefGoogle Scholar
  41. 41.
    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
  42. 42.
    Smith CJ, Danilowicz BS, Clear AK, Costello FJ, Wilson B, Meijer WG (2005) T-Align, a Web-based tool for comparison of multiple terminal restriction fragment length polymorphism profiles. FEMS Microbiol Ecol 54:375–380PubMedCrossRefGoogle Scholar
  43. 43.
    Speers RA, Tung MA, Durance TD, Stewart GG (1992) Biochemical aspects of yeast flocculation and its measurement: a review. J Inst Brew 98:293–300Google Scholar
  44. 44.
    Stewart GG, Russell I (1986) Centenary review: one hundred years of yeast research and development in the brewing industry. J Inst Brew 92:537–558Google Scholar
  45. 45.
    Stratford M (1989) Yeast flocculation: calcium specificity. Yeast 5:487–496CrossRefGoogle Scholar
  46. 46.
    Stratford M (1996) Yeast flocculation: restructuring the theories in line with recent research. Cerevisia 21:38–45Google Scholar
  47. 47.
    Stratford M, Carter AT (1993) Yeast flocculation: lectin synthesis and activation. Yeast 9:371–378PubMedCrossRefGoogle Scholar
  48. 48.
    Thies FL, König W, König B (2007) Rapid characterization of the normal and disturbed vaginal microbiota by application of 16S rRNA gene terminal RFLP fingerprint. J Med Microbiol 56:755–761PubMedCrossRefGoogle Scholar
  49. 49.
    van Nierop SNE, Cameron-Clarke A, Axcell BC (2004) Enzymatic generation of factors from malt responsible for premature yeast flocculation. J Am Soc Brew Chem 62:108–116Google Scholar
  50. 50.
    van Nierop SNE, Axcell BC, Cantrell IC, Rautenbach M (2008) Optimised quantification of the antiyeast activity of different barley malts towards a lager brewing yeast strain. Food Microbiol 25:895–901PubMedCrossRefGoogle Scholar
  51. 51.
    Verstrepen KJ, Derdelinckx G, Verachtert H, Delvaux FR (2003) Yeast flocculation: what brewers should know. Appl Microbiol Biotechnol 61:197–205PubMedGoogle Scholar
  52. 52.
    Widmer F, Hartmann M, Frey B, Kölliker R (2006) A novel strategy to extract specific phylogenetic sequence information from community T-RFLP. J Microbiol Methods 66:512–529PubMedCrossRefGoogle Scholar
  53. 53.
    Wood DA, Gill TA, Speers RA, Jenkins C (2005) Impact of malted barley quality and wort composition on the occurrence of premature yeast flocculation. In: Proceedings of the European brewery convention congress, Prague, Czech Republic, Poster # 64, CD-ROMGoogle Scholar
  54. 54.
    Yang C, Qi Li, Wang J, Zhao Y (2007) Study on premature yeast flocculation induced by the contamination of filamentous fungi of malt (in Chinese). Liquor-Making Sci Technol 151:50–55Google Scholar
  55. 55.
    Yoshida T, Yamada K, Fujino S, Koumegawa J (1979) Effect of pressure on physiological aspects of germinating barleys and quality of malts. J Am Soc Brew Chem 37:77–84Google Scholar
  56. 56.
    Zarattini RA, Williams JW, Ernandes JR, Stewart GG (1993) Bacterial-induced flocculation in selected brewing strains of Saccharomyces. Cerevisia Biotechnol 18:65–70Google Scholar

Copyright information

© Society for Industrial Microbiology and Biotechnology 2012

Authors and Affiliations

  • Mandeep Kaur
    • 1
    • 2
    Email author
  • John P. Bowman
    • 1
  • Doug C. Stewart
    • 3
  • Megan Sheehy
    • 3
  • Agnieszka Janusz
    • 3
  • R. Alex Speers
    • 4
  • Anthony Koutoulis
    • 2
  • David E. Evans
    • 2
  1. 1.Food Safety Centre, Tasmanian Institute of AgricultureUniversity of TasmaniaHobartAustralia
  2. 2.School of Plant ScienceUniversity of TasmaniaHobartAustralia
  3. 3.Viterra LtdAdelaideAustralia
  4. 4.Food Science and TechnologyDalhousie UniversityHalifaxCanada

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