Enology

What kind of sanitation should be applied to remove Brettanomyces bruxellensis biofilms? Original language of the article: English.

The capacity for five Brettanomyces bruxellensis strains to form biofilm on stainless steel was confirmed, and the sanitation of these biofilms was tested using a solution of lactic acid and a reference method (a solution of foaming caustic soda and peroxide at 5 %). Different responses were observed depending on the strain: lactic acid solution induced a slight reduction in cell population, while the reference method resulted in the elimination of the adhered cells for three strains, but generated VBNC states for two others. The effects of sanitation on the biofilm formed is strain-dependent.

Introduction

Winery sanitation is at the heart of guidelines for controlling microbial contaminations and, in particular, contamination by Brettanomyces bruxellensis. Indeed, this spoilage yeast is able to survive in stressful conditions; for example, in the presence of sulphites1 2 3. The ability to form biofilm is a potential resistance strategy developed by some yeast4, although little attention has been given to the case of B. bruxellensis so far. Biofilms are defined as a community of one or more types of microorganisms, which adhere to biotic and abiotic surfaces that can grow in all three dimensions, and which are embedded in a self-produced matrix called EPS (Extra Polymeric Substances)5. A recent study carried out at the IUVV (Dijon, France) described the biofilm of B. bruxellensis as a thin structure consisting of different cell morphologies, including yeast cells and pseudo-filamentous cells embedded in an extracellular matrix (Figure 1)6. Moreover, the persistence of biofilm cells on surfaces over time was demonstrated.

Currently, the most commonly used process for cleaning tanks is to apply a mixture of diluted chemical solutions containing alkaline detergents and acid solutions7. Eco-friendly alternatives that are effective in combatting all survival forms of B. bruxellensis are nevertheless sought to reduce the use of chemicals in the wine industry. In this context, we aimed to study the impact of two different methods of sanitation (a solution of foaming caustic soda and peroxide at 5 % and a solution of lactic acid at 5 %) on strains of Brettanomyces developed in biofilm.

Figure 1. SEM observations (x 500) of one-month old biofilm of Brettanomyces bruxellensis developed on stainless steel chip on a YPD medium. The surface is covered in microcolonies containing yeast and filamentous cells (indicated by white arrow).

Seeking removal of Brettanomyces bruxellensis biofilms

Five strains of B. bruxellensis from different origins were used (Table 1) and for each strain fourteen-day-old biofilms were formed on stainless steel chips in a synthetic medium (Yeast Extract- Peptone-Dextrose (YPD) medium), commonly used for growing yeasts in research laboratories, to evaluate two different sanitation methods.

Before biofilm formation, starter cultures ensured that cells were in the same physiological state, allowing the accurate comparison of the ability of different strains to form biofilms. Indeed, growth kinetics is strain-dependent with strain BR17 being slower compared to the other strains (Table 1). On the other hand, strain GS04 was the fastest to grow. However, the ability of all investigated strains to adhere to and form a biofilm on stainless steel chips was confirmed with a starting population in the biofilm on the chip of around 10cells/cm² for all the strains after 14 days of incubation (Figure 2).

Table 1. Origins and growth parameters of B. bruxellensis strains.

Figure 2. Concentration of viable or culturable cell populations (cells/cm²) of five B. bruxellensis strains over winery method (A) and lactic acid 5 % (B) sanitation process. The term “adhered cells” corresponds to the cells bound to the surface of the chip after treatment. Different letters represent significant difference (p-values ≤ 0.05) between cell population adhered to chips. Detection limit of the technics: 706 cells/cm² (cytometer: viable; dotted line), 12 cells/cm² (plate: culturable; full line).

. The effect of the winery method

The winery method, or reference method used by the Inter Rhône cellar, consists of applying a solution of foaming caustic soda and peroxide at 5 %. Biofilms were therefore exposed to this solution for 15 min, and populations on the chip were determined on agar media (culturable population) and by flow cytometry (viable population) at the end of the cleaning process. No remaining cells on the chips were detected after the treatment of three strains S11, GS04 and GS12, thereby demonstrating the effectiveness of this method (Figure 2A). However, for BR17 and S14 strains, some viable cells were detected by flow cytometry, thereby revealing cells in a viable but non culturable (VBNC) state8.

. The effect of lactic acid

To investigate an alternative cleaning procedure for winemaking equipment, an eco-friendly approach was tested. Biofilms were treated with a solution of 5 % lactic acid for 15 min, and populations on stainless steel chips were determined as previously described. The results show a significant reduction in the population of culturable cells on the chips after the lactic acid treatment for all strains, except for strain GS12 (Figure 2B). However, this treatment did not eliminate all the cells.

These assays demonstrate different strain- and treatment-dependent behaviour. Strain GS12 appears to be slightly more tolerant than the other strains to the lactic acid method. On the other hand, strains BR17 and S14 seem to be more tolerant to the winery method with the induction of the VBNC state, while populations of the other three strains were totally eliminated. This VBNC state could explain the resistance of these two strains to different chemical products and could be the cause of the year-on-year recontamination of tanks, as cells may resuscitate from the VBNC state when the stress conditions disappear.

Conclusion

All the tested strains, regardless of origin, were able to form biofilms on stainless steel (around 10cells/cm²) after 14 days of incubation in a YPD medium. Currently, the lactic acid solution is not a harsh enough treatment to eradicate B. bruxellensis, but it is a promising approach, which could be improved by increasing contact time or the concentration of the solution. The winery method demonstrated its effectiveness, but two strains exhibited VBNC cells after treatment. These results show that as for SO29 10 11, sanitation represents a stress for yeast with a strain-dependent response. The development of cheap and reliable detection methods which are reproducible at strain level could allow B. bruxellensis contamination to be predicted and controlled.

The translation of this article into English was offered to you by Moët Hennessy.

NOTES

  • Avramova, M., Vallet-Courbin, A., Maupeu, J., Masneuf-Pomarède, I., Albertin, W., (2018). Molecular diagnosis of Brettanomyces bruxellensis’ sulfur dioxide sensitivity through genotype specific method. Frontiers in Microbiology, vol. 9:1260.
  • Longin, C., Degueurce, C., Julliat, F., Guilloux-Benatier, M., Rousseaux, S., and Alexandre, H. (2016). Efficiency of population-dependent sulfite against Brettanomyces bruxellensis in red wine. Food Res. Int. 89(Pt 1), 620–630.
  • Serpaggi, V., Remize, F., Recorbet, G., Gaudot-Dumas, E., Sequeira-Le Grand, A., Alexandre, H., (2012). Characterization of the ‘Viable but Nonculturable’ (VBNC) state in the wine spoilage yeast Brettanomyces. Food Microbiology, vol. 30, no. 2, 2012, pp. 438–447.
  • Tek, E.L., Sundstrom, J.F., Gardner, J.M., Oliver, S.G., Jiranek, V., 2018. Evaluation of the ability of commercial wine yeasts to form biofilms (mats) and adhere to plastic: implications for the microbiota of the winery environment. FEMS Microbiol. Ecol. 94.
  • Flemming, H.-C., Wingender, J., (2010). The biofilm matrix. Nat. Rev. Microbiol. 8, 623–633.
  • Lebleux, Manon, Abdo, H., Coelho, C., Basmaciyan, L., Albertin, W., Maupeu, J., Laurent, J., Roullier-Gall, C., Alexandre, H., Guilloux-Benatier, M., Weidmann, S., Rousseaux, S., (2020). New Advances on the Brettanomyces bruxellensis biofilm mode of life. International Journal of Food Microbiology, vol. 318, p. 108464.
  • Institut Français du Vin, (2016). Guide de bonnes pratiques d’hygiène filière vins. R36.5, p.66-73.
  • Serpaggi, V., Remize, F., Recorbet, G., Gaudot-Dumas, E., Sequeira-Le Grand, A., Alexandre, H., (2012). Characterization of the ‘Viable but Nonculturable’ (VBNC) state in the wine spoilage yeast Brettanomyces. Food Microbiology, vol. 30, no. 2, 2012, pp. 438–447.
  • Longin, C., Degueurce, C., Julliat, F., Guilloux-Benatier, M., Rousseaux, S., and Alexandre, H. (2016). Efficiency of population-dependent sulfite against Brettanomyces bruxellensis in red wine. Food Res. Int. 89(Pt 1), 620–630.
  • Serpaggi, V., Remize, F., Recorbet, G., Gaudot-Dumas, E., Sequeira-Le Grand, A., Alexandre, H., (2012). Characterization of the ‘Viable but Nonculturable’ (VBNC) state in the wine spoilage yeast Brettanomyces. Food Microbiology, vol. 30, no. 2, 2012, pp. 438–447.
  • Tek, E.L., Sundstrom, J.F., Gardner, J.M., Oliver, S.G., Jiranek, V., 2018. Evaluation of the ability of commercial wine yeasts to form biofilms (mats) and adhere to plastic: implications for the microbiota of the winery environment. FEMS Microbiol. Ecol. 94.

Authors


Manon Deluchat

Affiliation : InterRhône, Service technique, 2260 route de grès, 84100 Orange, France
Country : France


Claire Lhomme

Affiliation : InterRhône, Service technique, 2260 route de grès, 84100 Orange, France
Country : France


Claudine Degueurce

Affiliation : InterRhône, Service technique, 2260 route de grès, 84100 Orange, France
Country : France


Virginie Serpaggi

Affiliation : InterRhône, Service technique, 2260 route de grès, 84100 Orange, France
Country : France


Romain Lacroix

Affiliation : Syndicat des vignerons des Côtes du Rhône, Service technique, 2260 Route de grès, 84100 Orange, France
Country : France


Manon Lebleux

Affiliation : UMR Procédés Alimentaires et Microbiologiques, Equipe VAlMiS (Vin, Aliments, Microbiologie, Stress), AgroSup Dijon - Université Bourgogne Franche-Comté, IUVV, Dijon, France
Country : France


Stéphanie Weidmann

Affiliation : UMR Procédés Alimentaires et Microbiologiques, Equipe VAlMiS (Vin, Aliments, Microbiologie, Stress), AgroSup Dijon - Université Bourgogne Franche-Comté, IUVV, Dijon, France
Country : France


Sandrine Rousseaux

Affiliation : UMR Procédés Alimentaires et Microbiologiques, Equipe VAlMiS (Vin, Aliments, Microbiologie, Stress), AgroSup Dijon - Université Bourgogne Franche-Comté, IUVV, Dijon, France
Country : France

sandrine.rousseaux@u-bourgogne.fr

References

  • Avramova, M., Vallet-Courbin, A., Maupeu, J., Masneuf-Pomarède, I., Albertin, W., (2018). Molecular diagnosis of Brettanomyces bruxellensis’ sulfur dioxide sensitivity through genotype specific method. Frontiers in Microbiology, vol. 9:1260.
  • Longin, C., Degueurce, C., Julliat, F., Guilloux-Benatier, M., Rousseaux, S., and Alexandre, H. (2016). Efficiency of population-dependent sulfite against Brettanomyces bruxellensis in red wine. Food Res. Int. 89(Pt 1), 620–630.
  • Serpaggi, V., Remize, F., Recorbet, G., Gaudot-Dumas, E., Sequeira-Le Grand, A., Alexandre, H., (2012). Characterization of the ‘Viable but Nonculturable’ (VBNC) state in the wine spoilage yeast Brettanomyces. Food Microbiology, vol. 30, no. 2, 2012, pp. 438–447.
  • Tek, E.L., Sundstrom, J.F., Gardner, J.M., Oliver, S.G., Jiranek, V., 2018. Evaluation of the ability of commercial wine yeasts to form biofilms (mats) and adhere to plastic: implications for the microbiota of the winery environment. FEMS Microbiol. Ecol. 94.
  • Flemming, H.-C., Wingender, J., (2010). The biofilm matrix. Nat. Rev. Microbiol. 8, 623–633.
  • Lebleux, Manon, Abdo, H., Coelho, C., Basmaciyan, L., Albertin, W., Maupeu, J., Laurent, J., Roullier-Gall, C., Alexandre, H., Guilloux-Benatier, M., Weidmann, S., Rousseaux, S., (2020). New Advances on the Brettanomyces bruxellensis biofilm mode of life. International Journal of Food Microbiology, vol. 318, p. 108464.
  • Institut Français du Vin, (2016). Guide de bonnes pratiques d’hygiène filière vins. R36.5, p.66-73.

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