Elsevier

Food Microbiology

Volume 30, Issue 2, June 2012, Pages 438-447
Food Microbiology

Characterization of the “viable but nonculturable” (VBNC) state in the wine spoilage yeast Brettanomyces

https://doi.org/10.1016/j.fm.2011.12.020Get rights and content

Abstract

Although the viable but not culturable (VBNC) state has been studied in detail in bacteria, it has been suggested that maintenance of viability with loss of culturability also exists in eukaryotic cells, such as in the wine spoilage yeast Brettanomyces. To provide conclusive evidence for the existence of a VBNC state in this yeast, we investigated its capacity to become viable and nonculturable after sulfite stress, and its ability to recover culturability after stressor removal. Sulfite addition induced loss of culturability but maintenance of viability. Increasing the medium pH to decrease the concentration of toxic SO2 allowed yeast cells to become culturable again, thus demonstrating the occurrence of a VBNC state in Brettanomyces upon SO2 exposure. Relative to culturable Brettanomyces, VBNC yeast cells were found to display a 22% decrease in size on the basis of laser granulometry. Assays for 4-ethylguaiacol and 4-ethylphenol, volatile phenols produced by Brettanomyces, indicated that spoilage compound production could persist in VBNC cells. These morphological and physiological changes in VBNC Brettanomyces were coupled to extensive protein pattern modifications, as inferred by comparative two-dimensional electrophoresis and mass spectrometric analyses. Upon identification of 53 proteins out of the 168 spots whose abundance was significantly modified in treated cells relative to control, we propose that the SO2-induced VBNC state in Brettanomyces is characterized by a reduced glycolytic flux coupled to changes in redox homeostatis/protein turnover-related processes. This study points out the existence of common mechanisms between yeast and bacteria upon entry to the VBNC state.

Highlights

► The wine yeast Brettanomyces enters a VBNC state after a sulfite stress. ► Sulfite removal allows yeast cell to become culturable again. ► In VBNC state cell size is reduced by 20%. ► VBNC state cells are able to produce 4-ethylphenol. ► VBNC cells still synthesize proteins.

Introduction

The wine spoilage yeast Brettanomyces is responsible for producing volatile phenols that are associated with unpleasant aromas, and which may thus compromise beverage quality and productivity (Du Toit et al., 2005). Although a large number of culture-based techniques are currently available for assessing the presence of this undesirable spoilage yeast during winemaking processes, in most cases, Brettanomyces is not detected. However, some wines nonetheless present volatile phenol compound-associated aromas a few months or years later, a dilemma which could potentially be explained by the ability of Brettanomyces to enter a viable but nonculturable (VBNC) state (Agnolucci et al., 2010).

The VBNC phenotype, which is characterized by an inability of cells to divide on bacteriological media, even though they are still alive and maintain metabolic or cellular activity, has been widely observed and described in bacteria. Evidence for the presence of cells that are viable but not culturable is provided by viability assays (Kell et al., 1998), which involve the direct investigation of various cell functions, such as respiration, membrane potential or integrity and enzymatic activity (Lleò et al., 2000; Rudi et al., 2005; Divol and Lonvaud-Funel, 2005). Fluorescent probes also can be used to count viable cells by microscopy or flow cytometry (FCM). VBNC bacteria frequently differ from normal culturable cells in terms of their size and the chemical composition of the cell wall (Byrd et al., 1992; Oliver, 2005). Additionally, macromolecular synthesis and respiration rates decrease as cells enter a VBNC state (Lai et al., 2009). By contrast, ATP levels and membrane potential remain generally high (Lindback et al., 2010).

The basis for a VBNC hypothesis therefore involves demonstration of the recovery of culturability in VBNC cells. This “resuscitation” has been the subject of heated debate (Kell et al., 1998), but recovery of cell division in the VBNC population has been unambiguously described for numerous bacterial species (Zhong et al., 2009; Dhiaf et al., 2008). According to Barcina and Arana (2009), the VBNC phenotype may be considered as an integral part of the life cycle of non-differentiating bacteria.

Most studies on VBNC cells have focused on pathogenic bacteria, including Vibrio, Campylobacter, Shigella and Enterococcus (Bogosian et al., 2000; Tholozan et al., 1999; Rahman et al., 1996; Heim et al., 2002), which, despite some controversy (Jacob, 2010), are considered to generate a potential public health risk (Oliver, 2005). Chemical and environmental factors have been reported to induce a VNBC state, including nutrient starvation, extreme temperatures and osmotic concentrations, oxygen, and food preservatives (Oliver, 2010).

In contrast to bacteria, the VBNC state in other microorganisms, including particularly eukaryotes, has received much less attention. However, it has been suggested that in wine, yeast cells may attain a state in which they are viable but not culturable (Millet and Lonvaud-Funel, 2000). In particular, recent studies have indicated that sulfur dioxide (SO2), an antimicrobial agent used in food preservation, induces a VBNC state in the wine spoilage yeast Brettanomyces (Du Toit et al., 2005; Agnolucci et al., 2010). These latter authors reported a capacity of several strains of Brettanomyces to enter a VBNC state after a sulfite treatment ranging from 0.2 to 1.0 mg l−1 and they mentioned a potential regain of culturability after stressor removal. Unfortunately, neither the protocol nor the data relative to resuscitation have been provided to strengthen this conclusion. Consequently, the VBNC state existence in Brettanomyces has never been conclusively demonstrated to exist, nor fully characterized.

A variety of microorganisms are exposed to the toxin sulfide (representing the sum of H2S, HS and S2), but most studies dedicated to the effect of SO2 on living organisms have focused on animals. Sulfur dioxide has been shown to form adducts with aldehydes, ketones, thiamine, and cysteine residues in proteins; and sulfite forms adducts with NAD, nucleosides and nucleotides. Sulfite also lyse disulfide bonds in native proteins, a process called sulfitolysis (WHO Food Additives Series 21). With regard to yeasts, transcript profiling changes in Saccharomyces cerevisiae cells upon sulfite exposure in their exponential growth phase have been shown to include up-regulation of genes involved in carbohydrate metabolism and down-regulation of genes having function in protein biosynthesis and transcription (Park and Hwang, 2008). By contrast, irreversible inhibition of colony formation in S. cerevisiae by sulfite has been ascribed to the SO2-driven activation of ATP hydrolysis (Schimz and Holzert, 1979; Schimz, 1980), and to inhibition of the enzyme glyceraldehyde-3-phosphate dehydrogenase (Hinze and Holzer, 1986).

Taking into account the fact that sulfite has been hypothesized to induce a VBNC state in Brettanomyces cells (Du Toit et al., 2005; Agnolucci et al., 2010), the aim of this study was to check this hypothesis. In this regard, we have tested the ability of cells (i) to enter VBNC state, (ii) to exit this state, (iii) to conserve their capacity to produce volatile phenols and (iv) to maintain their size. Furthermore, in order to determine if “communal metabolic pathways” of bacteria exist in the VBNC state, we performed a comparative proteomic analysis of control and VNBC cells, which allowed us to suggesting putative mechanisms for the loss of cultivability upon sulfite exposure.

Section snippets

Yeast strain and culture conditions

Brettanomyces bruxellensis strain LO2E2, isolated from Burgundy red wine by the Institut Technique de la Vigne et du Vin (Beaune, France) was used in the current study. It was grown on YPD agar (10 g l−1 yeast extract, 10 g l−1 Bacto-peptone, 20 g l−1 glucose, 20 g l−1 agar) at 28 °C for 5 day as starter inocula. VBNC studies were performed in a synthetic wine medium (10% v/v ethanol, 3 g l−1 d-l malic acid, 0.01% acetic acid, 0.1 g l−1 potassium sulfate, 0.025 g l−1 magnesium sulfate, 1 g l−1

Evidence for a VBNC state (induction and exit)

Sulfite has been suggested to induce the VBNC state in Brettanomyces (Du Toit et al., 2005; Agnolucci et al., 2010). We therefore used this compound as a stress factor for studying VBNC in this yeast. To monitor the conversion of Brettanomyces cells to the VBNC state, flow cytometry counts of viable cells and CFU (colony forming unit) counts on YPD agar were compared.

In the absence of sulfite, all cells remained viable and culturable in medium simulating wine over a period of 11 days at 28 °C (

Discussion

Sulfur dioxide (SO2) is the preservative most commonly added to wine as a way to control the growth of spoilage microbes during winemaking, but its effects on B. bruxellensis populations are poorly understood. Although the existence of VBNC-like phenomena in B. bruxellensis upon SO2 addition has been suggested by Du Toit et al. (2005), Millet et al. (1995) and Agnolucci et al. (2010), to date neither the ability to regain culturability, nor morphological and large-scale metabolic changes so far

Acknowledgments

We thank Christelle Guillier for technical assistance in proteomic experiments, the Clinical and Innovation Proteomic Platform for MALDI experiments and Maud Darsonval for microbiological technical assistance. We also thank Jeannine Lherminier and Aline Bonnotte for SEM experiments. This work was supported by BIVB, Inter-Rhône, FranceAgriMer and Regional Council of Burgundy. We thank Dr learmonth from University of Southern Queensland for his proofreading assistance.

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      Considering sugar utilization, the differences highlighted in the results between the two strains at the different time points indicate that the usage of sugars undergoes a strain-specific consumption dynamic. Regarding the release of VPs they were not produced after the SO2 pulse, in disagreement to what was observed by Serpaggi et al. (2012) who reported the cells can produce 4-ethyl-phenol, although in a lower amount than control cells, entering in a SO2-induced VBNC state. The last observation suggests that a VBNC state is not triggered by the SO2 treatment under the investigated experimental conditions.

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    Present address: CTCPA, ZA aéroport, site Agroparc, BP 21203, F-84009 Avignon cedex, France.

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