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The application of flow cytometry in microbiological monitoring during winemaking: two case studies

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Abstract

In this work, we exploit a general flow cytometry technique involved in the differentiation of live and dead yeast cells for two applications in winemaking. The discrimination of yeast populations is achieved using two fluorescent dyes that measure the metabolic activity and membrane integrity of the yeast. This analytical approach is first applied for quality control of active dry yeast. Results are discussed in comparison with the Codex Oenologique International (International Oenological Codex) of the International Organisation of Vine and Wine (OIV), demonstrating that analysis using flow cytometry is a valuable alternative, given the ease of execution and the high quality of results obtained in terms of reproducibility, repeatability, and confidence interval. In the second case, we apply flow cytometry as a technique for monitoring the production of sparkling wines using the “Champenoise” method, and describe the evolution of yeast through the production process. In this case, results are directly compared with those obtained with the two methods (plate counts and direct microscopic count) listed in the OIV standards, in order to ensure a thorough understanding of the improvements related to the use of flow cytometry.

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References

  • Attfield PV, Kletsas S, Veal DA, van Rooijen R, Bell PJL (2000) Use of flow cytometry to monitor cell damage and predict fermentation activity of dried yeasts. J Appl Microbiol 89(2):207–214

    Article  CAS  PubMed  Google Scholar 

  • Bouix M, Ghorbal S (2013) Rapid enumeration of Oenococcus oeni during malolactic fermentation by flow cytometry. J Appl Microbiol 114(4):1075–1081

    Article  CAS  PubMed  Google Scholar 

  • Boyd AR, Gunasekera TS, Attfield PV, Simic K, Vincent SF, Veal DA (2003) A flow-cytometric method for determination of yeast viability and cell number in a brewery. FEMS Yeast Res 3:11–16

    Article  CAS  PubMed  Google Scholar 

  • Branco P, Monteiro M, Moura P, Albergaria H (2012) Survival rate of wine-related yeasts during alcoholic fermentation assessed by direct live/dead staining combined with fluorescence in situ hybridization. Int J Food Microbiol 158(1):49–57

    Article  CAS  PubMed  Google Scholar 

  • Buratti S, Ballabio D, Giovanelli G, Dominguez CMZ, Moles A, Benedetti S, Sinelli N (2011) Monitoring of alcoholic fermentation using near infrared and mid infrared spectroscopies combined with electronic nose and electronic tongue. Anal Chim Acta 697(1–2):67–74

    Article  CAS  PubMed  Google Scholar 

  • Cahill JT, Carroad PA, Kunkee RE (1980) Cultivation of yeast under carbon-dioxide pressure for use in continuous sparkling wine production. Am J Enol Vitic 31(1):46–52

    CAS  Google Scholar 

  • Carrascosa AV, Martinez-Rodriguez A, Cebollero E, Gonzalez R (2011) Carrascosa AV, Munoz R, Gonzalez R eds. Yeast. Saccharomyces II. Second fermentation yeasts. Molecular Wine Microbiology. Elsevier Academic Press Inc. (CA) pp. 33–49

  • Casey GP, Ingledew WMM (1986) Ethanol tolerance in yeasts. Crit Rev Microbiol 13(3):219–280

    Article  CAS  PubMed  Google Scholar 

  • Di Egidio V, Sinelli N, Giovanelli G, Moles A, Casiraghi E (2010) NIR and MIR spectroscopy as rapid methods to monitor red wine fermentation. Eur Food Res Technol 230(6):947–955

    Article  CAS  Google Scholar 

  • Diaz M, Herrero M, Garcia LA, Quiros C (2010) Application of flow cytometry to industrial microbial bioprocesses. Biochem Eng J 48(3):385–407

    Article  CAS  Google Scholar 

  • Fiala J, Lloyd DR, Rychtera M, Kent CA, Al-Rubeai M (1999) Evaluation of cell numbers and viability of Saccharomyces cerevisiae by different counting methods. Biotechnol Tech 13(11):787–795

    Article  CAS  Google Scholar 

  • Fleet GH (2007) Yeasts in foods and beverages: impact on product quality and safety. Curr Opin Biotechnol 18(2):170–175

    Article  CAS  PubMed  Google Scholar 

  • França MB, Panek AD, Eleutherio ECA (2007) Oxidative stress and its effects during dehydration. Comp Biochem Physiol A Mol Integr Physiol 146(4):621–631

  • Frohman CA, de Orduna RM (2012) Advantages of modern process control and fed-batch fermentations in oenology. Am J Enol Vitic 63(3):450–455

  • Gerbaux V, Berger JL (2009) Practical use of flow cytometry for monitoring yeasts in oenology. Bull de l’OIV 82:357–366

    CAS  Google Scholar 

  • Gonzalez R, Vian A, Carrascosa AV (2008) Morphological changes in Saccharomyces cerevisiae during the second fermentation of sparkling wine. Food Sci Technol Int 14(4):393–398

  • Ibarra-Junquera V, Escalante-Minakata P, Mancilla-Margalli NA, Murguia JS, de la Rosa LA, Rosu HC (2010) Krause J, Fleischer O. eds. Strategies to monitor alcoholic fermentation processes. Industrial fermentation: food processes, nutrient sources and production strategies. Nova Science Publishers, Inc. (NY) pp. 151–185

  • International Fruit Juice Producers U (1996). IFU-MB06 - Mesophilic & Thermoduric - Thermophilic Bacteria: Spores Count

  • ISO/IEC 17025. (1999). General requirements for the competence of testing and calibration laboratories.

  • ISO 5725–1. (1994). Accuracy (trueness and precision) of measurement methods and results. Part 1: General principles and definitions

  • Jepras RI, Carter J, Pearson SC, Paul FE, Wilkinson MJ (1995) Development of a robust flow cytometric assay for determining numbers of viable bacteria. Appl Environ Microbiol 61(7):2696–2701

    PubMed Central  CAS  PubMed  Google Scholar 

  • Kunkee RE, Ough CS (1966) Multiplication and fermentation of Saccharomyces cerevisiae under carbon dioxide pressure in wine. Appl Microbiol 14(4):643–648

    PubMed Central  CAS  PubMed  Google Scholar 

  • Malacrino P, Zapparoli G, Torriani S, Dellaglio F (2001) Rapid detection of viable yeasts and bacteria in wine by flow cytometry. J Microbiol Methods 45(2):127–134

    Article  CAS  PubMed  Google Scholar 

  • Mazzei P, Spaccini R, Francesca N, Moschetti G, Piccolo A (2013) Metabolomic by H-1 NMR spectroscopy differentiates “Fiano di Avellino” white wines obtained with different yeast strains. J Agric Food Chem 61(45):10816–10822

    Article  CAS  PubMed  Google Scholar 

  • OIV Codex Oenologique International (2014a) Techniques analytiques et de controle microbiologiques, analyses communes a toutes les monographies (Oeno 17/2003, OIV-Oeno 329–2009)

  • OIV - Recueil international des méthodes d’analyses (2014b) Volume 2. Recueil international des méthodes d’analyses. Analyse microbiologique des vins et des moûts (MA-AS4-01)

  • Oliver JD (2005) The viable but nonculturable state in bacteria. J Microbiol 43:93–100

    PubMed  Google Scholar 

  • Penacho V, Valero E, Gonzalez R (2012) Transcription profiling of sparkling wine second fermentation. Int J Food Microbiol 153(1–2):176–182

    Article  CAS  PubMed  Google Scholar 

  • Portell X, Ginovart M, Carbo R, Vives-Rego J (2011) Differences in stationary-phase cells of a commercial Saccharomyces cerevisiae wine yeast grown in aerobic and microaerophilic batch cultures assessed by electric particle analysis, light diffraction and flow cytometry. J Ind Microbiol Biotechnol 38(1):141–151

    Article  CAS  PubMed  Google Scholar 

  • Pozo-Bayon MA, Martinez-Rodriguez A, Pueyo E, Moreno-Arribas MV (2009) Chemical and biochemical features involved in sparkling wine production: from a traditional to an improved winemaking technology. Trends Food Sci Technol 20(6–7):289–299

    Article  CAS  Google Scholar 

  • Quiros C, Herrero M, Garcia LA, Diaz M (2012) Effects of SO2 on lactic acid bacteria physiology when used as a preservative compound in malolactic fermentation. J Inst Brew 118(1):89–96

    Article  CAS  Google Scholar 

  • Reed G, Chen SL (1978) Evaluating commercial active dry wine yeasts by fermentation activity. Am J Enol Vitic 29(3):165–168

    CAS  Google Scholar 

  • Ribéreau-Gayon P, Glories Y, Maujean A, Dubourdieu D (2006) Handbook of Enology, Volume 1. The Microbiology of Wine and Vinifications. John Wiley & Sons, Hoboken (NJ)

    Google Scholar 

  • Romera-Fernandez M, Berrueta LA, Garmon-Lobato S, Gallo B, Vicente F, Moreda JM (2012) Feasibility study of FT-MIR spectroscopy and PLS-R for the fast determination of anthocyanins in wine. Talanta 88:303–310

    Article  CAS  PubMed  Google Scholar 

  • Ross DD, Joneckis CC, Ordonez JA, Sisk AM, Wu RK, Hamburger AW, Nora RE (1989) Estimation of cell survival by flow cytometric quantification of fluorescein diacetate/propidium iodide viable cell number. Cancer Res 49:376–3782

    Google Scholar 

  • Salma M, Rousseaux S, Sequeira-Le Grand A, Divol B, Alexandre H (2013) Characterization of the viable but nonculturable (VBNC) state in Saccharomyces cerevisiae. PLoS ONE 8(10):e77600

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Torresi S, Frangipane MT, Anelli G (2011) Biotechnologies in sparkling wine production. Interesting approaches for quality improvement: a review. Food Chem 129(3):1232–1241

    Article  CAS  PubMed  Google Scholar 

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Correspondence to Raffaele Guzzon.

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Guzzon, R., Larcher, R. The application of flow cytometry in microbiological monitoring during winemaking: two case studies. Ann Microbiol 65, 1865–1878 (2015). https://doi.org/10.1007/s13213-014-1025-6

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  • DOI: https://doi.org/10.1007/s13213-014-1025-6

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