Annals of Microbiology

, Volume 60, Issue 3, pp 549–556 | Cite as

Susceptibility of wine spoilage yeasts and bacteria in the planktonic state and in biofilms to disinfectants

  • Mariana Tristezza
  • António Lourenço
  • André Barata
  • Luísa Brito
  • Manuel Malfeito-FerreiraEmail author
  • Virgílio Loureiro
Original Article


The aim of this work was to determine the ability of six yeast and two bacterial species associated with wine spoilage to form biofilms in mono- or co-culture using the Calgary Biofilm Device (CBD). Moreover, the efficacy of several disinfectants was evaluated against these spoilage microorganisms, both in the planktonic and the biofilm states. Results showed that Dekkera bruxellensis, Saccharomyces cerevisiae, Saccharomycodes ludwigii, Schizosaccharomyces pombe and Acetobacter aceti formed biofilms both in wine and in synthetic medium. Zygosaccharomyces bailii formed biofilm only in wine and Pichia guilliermondii and Lactobacillus hilgardii formed biofilms only in synthetic medium. In wine, D. bruxellensis presented the same biofilm population when grown in pure culture or in mixed culture with acetic acid bacteria. There was a 3-log increase in biofilm formed by A. aceti in mixed culture with L. hilgardii. Alkaline chlorine-based disinfectant was the most effective in decontaminating spoilage yeast and bacteria both in planktonic and biofilm tests. Sodium hydroxide-based detergents and peracetic-based disinfectant were also efficient against suspended cells, but at least 10-fold more concentrated solutions were needed to remove the biofilms. Furthermore, the results showed that, except for the neutral detergent VK10, the tested agents were actually effective when used under the conditions recommended by manufacturers. In any case, biofilms showed greater tolerance to biocides when compared to the same microorganisms in the planktonic state. To our knowledge, this is the first study in which the CBD is used to assess the ability of wine spoilage microorganisms to form biofilms and their susceptibilities to disinfectant agents.


Biofilms Disinfectants Spoilage yeasts Acetic acid bacteria Lactic acid bacteria 


  1. Bloomfield SF (1988) Cosmetics and pharmaceuticals: biodeterioration and disinfectants. In: Houghton DR, Smith RN, Eggins HOW (eds) Biodeterioration 7. Elsevier, Essex, pp 135–145Google Scholar
  2. Bloomfield SF, Arthur M, Klingeren B, van Pullen W, Holah JT, Elton R (1994) An evaluation of the repeatability and reproducibility of a surface test for the activity of disinfectants. J Appl Bacteriol 76:86–94PubMedGoogle Scholar
  3. Brackett RE (1992) Shelf stability and safety of fresh produce as influenced by sanitation and disinfection. J Food Prot 55:808–814Google Scholar
  4. Carpentier B, Cerf O (1993) Biofilms and their consequences, with particular reference to hygiene in the food industry. J Appl Bacteriol 75:499–511PubMedGoogle Scholar
  5. Cerf O, Carpentier B, Sanders P (2010) Test for determining in-use concentration of antibiotics and disinfectants are based on entirely different concepts: “Resistance” has different meanings. Int J Food Microbiol 136:247–254CrossRefPubMedGoogle Scholar
  6. Ceri H, Olson ME, Stremick C, Read RR, Morck DW, Buret AG (1999) The Calgary Biofilm Device: new technology for rapid determination of antibiotic susceptibilities in bacterial biofilms. J Clin Microbiol 37:1771–1776PubMedGoogle Scholar
  7. Ceri H, Olson ME, Morck DW, Storey D, Read RR, Buret AG, Olson B (2001) The MBEC assay system: multiple equivalent biofilms for antibiotic and biocide susceptibility testing. Methods Enzymol 337:377–384CrossRefPubMedGoogle Scholar
  8. Czechowski MH, Banner M (1992) Control of biofilms in breweries through cleaning and sanitizing. Tech Q - Master Brew Assoc Am 29(3):86–88Google Scholar
  9. Donlan RM, Costerton JW (2002) Biofilms: survival mechanisms of clinically relevant microorganisms. Clin Microbiol Rev 15:167–193CrossRefPubMedGoogle Scholar
  10. du Toit M, Pretorius IS (2000) Microbial spoilage and preservation of wine: using weapons from nature's own arsenal—a review. S Afr J Enol Vitic 21:74–96Google Scholar
  11. Enrique M, Marcos JF, Yuste M, Martínez M, Vallés S, Manzanares P (2007) Antimicrobial action of synthetic peptides towards wine spoilage yeasts. Int J Food Microbiol 118(3):318–325CrossRefPubMedGoogle Scholar
  12. Frank JF, Koffi RA (1990) Surface-adherent growth of Listeria monocytogenes is associated with increased resistance to surfactant sanitizers and heat. J Food Prot 53:550–554Google Scholar
  13. Han Y, Guentert AM, Smith RS, Linton RH, Nelson PE (1999) Efficacy of chlorine dioxide gas as a sanitizer for tanks used for aseptic juice storage. Food Microbiol 16(1):53–61CrossRefGoogle Scholar
  14. Joseph LCM, Kumar G, Su E, Bisson LF (2007) Adhesion and biofilm production by wine isolates of Brettanomyces bruxellensis. Am J Enol Vitic 58(3):373–378Google Scholar
  15. Kawarai T, Furukawa S, Ogihara H, Yamasaki M (2007) Mixed-species biofilm formation by lactic acid bacteria and rice wine yeasts. Appl Environ Microbiol 73(14):4673–4676CrossRefPubMedGoogle Scholar
  16. Kumar CG, Anand SK (1998) Significance of microbial biofilms in food industry: a review. Int J Food Microbiol 42:9–27CrossRefPubMedGoogle Scholar
  17. Loureiro V, Malfeito-Ferreira M (2003) Spoilage yeasts in the wine industry. Int J Food Microbiol 86:23–50CrossRefPubMedGoogle Scholar
  18. Mattila-Sandholm T, Wirtanen G (1992) Biofilm formation in the industry: a review. Food Rev Int 8:573–603CrossRefGoogle Scholar
  19. McGrath K, Odell DE, Davenport RR (1991) The sensitivity of vegetative cells and ascospore of some food spoilage yeasts to sanitisers. Int Biodeterior 27:313–326CrossRefGoogle Scholar
  20. Mosteller TM, Bishop JR (1993) Sanitizer efficacy against attached bacteria in a milk biofilm. J Food Prot 56:34–41Google Scholar
  21. Parahitiyawa NB, Samaranayake YH, Samaranayake LP, Ye J, Tsang PW, Cheung BP, Yau JY, Yeung SK (2006) Interspecies variation in Candida biofilm formation studied using the Calgary biofilm device. Acta Pathol Microbiol Immunol Scand 114(4):298–306Google Scholar
  22. Parsek MR, Greenberg EP (2005) Sociomicrobiology: the connections between quorum sensing and biofilms. Trends Microbiol 13:27–33CrossRefPubMedGoogle Scholar
  23. Peeters E, Nelis HJ, Coenye T (2008) Comparison of multiple methods for quantification of microbial biofilms grown in microtiter plates. J Microbiol Meth 72(2):157–165CrossRefGoogle Scholar
  24. Salo S, Wirtanen G (2005) Disinfectant efficacy on foodborne spoilage yeast strains. Food Bioprod Process 83(4):288–296CrossRefGoogle Scholar
  25. Storgårds E, Pihlajamäki O, Haikara A (1997) Biofilms in the brewing process—a new approach to hygiene management, in Proc 26th Congr Eur Brew Conv, Maastricht, The Netherlands, 717–724Google Scholar
  26. Winniczuk PP, Parrish ME (1997) Minimum inhibitory concentrations of antimicrobials against micro-organisms related to citrus juice. Food Microbiol 14:373–381CrossRefGoogle Scholar
  27. Wirtanen G (1995) Biofilm formation and its elimination from food processing equipment, VTT Publications 251. VTT Offsetpaino, EspooGoogle Scholar
  28. Wirtanen G, Salo S (2003) Disinfection in food processing – efficacy testing of disinfectants. Rev Environ Sci Biotechnol 2:293–306CrossRefGoogle Scholar

Copyright information

© Springer-Verlag and the University of Milan 2010

Authors and Affiliations

  • Mariana Tristezza
    • 2
  • António Lourenço
    • 1
  • André Barata
    • 1
  • Luísa Brito
    • 1
  • Manuel Malfeito-Ferreira
    • 1
    Email author
  • Virgílio Loureiro
    • 1
  1. 1.Laboratório de Microbiologia, CBAA/ Departamento de Botânica e Engenharia BiológicaInstituto Superior de AgronomiaLisboaPortugal
  2. 2.C.N.R., Institute of Sciences of Food Production, Unit of LecceLecceItaly

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