Brettanomyces susceptibility to antimicrobial agents used in winemaking: in vitro and practical approaches Caur Portugal, Yolanda Senz, Beatriz

Rojo-Bezares, Myriam Zarazaga, Carmen Torres, Juan Cacho & Fernanda Ruiz-Larrea
European Food Research and Technology Zeitschrift fr LebensmittelUntersuchung und -Forschung A ISSN 1438-2377 Eur Food Res Technol DOI 10.1007/s00217-013-2143-2

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Eur Food Res Technol DOI 10.1007/s00217-013-2143-2

OriGiNAL PAper

Brettanomyces susceptibility toantimicrobial agents used inwinemaking: invitro andpractical approaches

CaurPortugal YolandaSenz BeatrizRojo-Bezares MyriamZarazaga CarmenTorres JuanCacho FernandaRuiz-Larrea

Received: 29 September 2013 / Revised: 10 December 2013 / Accepted: 12 December 2013 Springer-Verlag Berlin Heidelberg 2013

Abstract Brettanomyces bruxellensis is a spoiling yeast responsible for developing off-odors in wine described as Brett-character. The objective of this study was to evaluate the antimicrobial activity of four enological compounds against Brettanomyces: potassium metabisulte (PMB), chitosan, enological tannins and dimethyl dicarbonate. Minimal inhibitory concentrations and minimal biocidal concentrations of the antimicrobial agents were determined, and a comparative study between B. bruxellensis and Saccharomyces cerevisiae was performed under in vitro controlled conditions. All tested compounds showed inhibitory effect on the growth of Brettanomyces. Chitosan and the enological tannins showed selectivity

C.Portugal F.RuizLarrea(*) Instituto de Ciencias de la Vid y del Vino (CSICURGR), University ofLa Rioja, Av. Madre de Dios 51, 26006Logroo, Spain e-mail: fernanda.ruiz@unirioja.es Present Address: C.Portugal Laboratory ofTechnology andQuality ofAlcoholic Beverages, College ofAgriculture Luiz de Queiroz, University ofSo Paulo, Av. Pdua Dias 11, Piracicaba13418900, Brazil Y.Senz B.RojoBezares Molecular Microbiology Laboratory, Center forBiomedical Research ofLa Rioja (CIBIR), C/Piqueras 98, 3 Planta, 26006Logroo, Spain M.Zarazaga C.Torres Laboratory ofBiochemistry andMolecular Biology, Department ofFood andAgriculture, University ofLa Rioja, Av. Madre de Dios 51, 26006Logroo, Spain J.Cacho Department ofAnalytical Chemistry, Faculty ofSciences, University ofZaragoza, 50009Zaragoza, Spain

against Brettanomyces, and PMB showed the highest efcacy in concentrations under the currently permitted limits for enological use; consequently, PMB was further evaluated in red wines naturally contaminated by Brettanomyces. Volatile phenol concentrations were determined after long-term storage of the wines treated with PMB. A direct correlation was demonstrated between the concentrations of 4-ethylphenol, 4-ethylguaiacol, 4-propylguaiacol and Brettanomyces populations in the studied wines, and these parameters correlated inversely with the concentrations of PMB employed. This is the rst time that 4-propylguaiacol is shown to correlate with Brettanomyces population in wine. It is of enological signicance that a concentration of 100mg/L of total PMB efciently prevented Brettanomyces growth in the aging red wines of our study and that volatile phenol concentrations were signicantly (p<0.05) higher in those poorly protected wines. Keywords Brettanomyces Potassium metabisulte Volatile phenols Chitosan Enological tannins Dimethyl dicarbonate

Introduction Brettanomyces bruxellensis is a well-known yeast species in wine industry because of the great economic losses it causes. Wine spoilage by yeasts has increased in recent years as a result of the higher microbial susceptibility of wines, partially due to the lower doses of preservatives demanded by consumers and partially due to the increase in wine pH that is emerging in wine producing regions undergoing climate warming trends. Brettanomyces, named Dekkera when growing as its sporulating teleomorph, is responsible for developing off-odors described by most diverse

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descriptors, such as animal, stable, horse sweat, mousy, burnt plastic, medicinal or phenolic taint. Due to the widespread use of the term Brett-character in enology and that the nonsporulating form is the one commonly found in wines, the name Brettanomyces will be used in this article, in spite of recent taxonomy texts that give preference to the designation of the spore forming and teleomorph Dekkera [1]. The principal spoiling compounds associated with the Brett-character are some volatile phenols, mainly 4-ethylphenol, 4-ethylguaiacol, 4-vinylphenol and 4-vinylguaiacol, all of them are secondary metabolites produced by Brettanomyces from the phenolic acids naturally present in wine. This is the reason why monitoring this organoleptic deviation in wineries is routinely performed by analysis of wine volatile phenols by gas chromatography [2]. The production of these volatile phenols by active Brettanomyces cells leads as well to the loss of the fresh and fruity character of wines. The Brett-character in red wines is detected when volatile phenols are above their perception threshold (426g/L for a 1:10 mix of 4-ethylphenol and 4-ethylguaiacol [3]), and this happens once active Brettanomyces in the wine overcome 103cells/mL [4, 5]. Brettanomyces is probably one of the best adapted yeasts to survive in dry red wine, and it is frequently found in wines aging in wooden barrels, vats and equipment that are difcult to sterilize. For winemakers of premium red wines, regular monitoring of Brettanomyces population is mandatory, and when the problem arises, efcient sanitation procedures should be implemented. A number of physicochemical treatments, such as thermal inactivation or high pressures, are currently available to winemakers. Nevertheless, these actions are palliative; their effect is transitory and does not protect wine from further contaminations of Brettanomyces cells. The longest established additive is sulfur dioxide because of its antimicrobial and antioxidant properties, and in the past 10years, alternative inhibitors of the growth of Brettanomyces have been approved or recommended in winemaking. The use of dimethyl dicarbonate (DMDC) is currently allowed exclusively for those wines with a sugar content over 5g/L, and its application should be performed shortly before bottling (EU regulation 643/2006). Chitosan, a deacetylated polysaccharide derived from chitin, has been reported as an efcient antimicrobial agent against Brettanomyces and presents the advantages of being biodegradable and nontoxic [6]. Hydroxycinnamic acids [7], synthetic peptides [8] and some yeast killer toxins isolated from Pichia anomala, Kluyveromyces wickerhamii [9], Pichia membranaefaciens [10] and Ustilago maydis [11] strains have been reported to possess fungicidal effect against Brettanomyces. The objective of this work was to perform a methodical study, carried out under strictly controlled laboratory conditions, to compare the growth inhibitory and biocidal

activities against Brettanomyces of four compounds that are currently available in the market for winemaking: potassium metabisulte (PMB), chitosan, enological tannins and DMDC. The study was performed with 31 enological strains of the species B. bruxellensis and Saccharomyces cerevisiae. We determined minimal inhibitory concentration (MIC) and minimal biocidal concentration (MBC) of the antimicrobial agents both in the presence and in the absence of ethanol. The compound that showed to be the most active in this in vitro screening was further evaluated for its efcacy under winery conditions. Wines naturally contaminated by Brettanomyces were treated with the most effective antimicrobial agent, and microbial populations were monitored over 22months of wine storage. The concentrations of the primary volatile phenols derived from decarboxylation of cinnamic acids were also determined after long-term storage of the contaminated wines.

Materials andmethods Yeast strains andculture conditions A total of 16 B. bruxellensis and 15 S. cerevisiae strains were included in this study; 22 of these strains were recovered from red wines during the period 20052010 and belonged to the collection of enological isolates of the Department of Food and Agriculture of the University of La Rioja; six S. cerevisiae strains were commercial yeast starters for winemaking, and three strains were obtained from the Spanish Culture Type Collection (CECT), as shown in Table1. Yeast species identication was performed by specic PCR analysis: B. bruxellensis, according to the method of Cocolin etal. [12], and S. cerevisiae, according to the method of Fernndez-Espinar etal. [13]. Yeast strains were cultivated from 24h to 48h on YPD agar [10g/L yeast extract (Oxoid Ltd., Basingstoke, UK), 20g/L peptone (Becton, Dickinson Co., LePont de Claix, France), 20g/L glucose (Panreac Qumica S.A., Barcelona, Spain), 20g/L agar (Becton, Dickinson Co.)], and fresh cultures were used for the antimicrobial activity assays described below. Minimal inhibitory concentration (MIC) andminimal biocidal concentration (MBC) Minimal inhibitory concentration was determined by the microtiter dilution method [14], in duplicates and using serial double dilutions of each antimicrobial agent. Yeast cells from a fresh culture were re-suspended in sterile saline solution to reach 1.2 optical density (OD) units at 600nm, and 12.5L was added to a nal volume of 100L in the microtiter assay. Cell growth was determined

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Eur Food Res Technol Table1Yeasts included in the study Species Brettanomyces bruxellensis Dekkera bruxellensis Saccharomyces cerevisiae S. cerevisiae S. cerevisiae S. cerevisiaea S. cerevisiae var. cerevisiaea S. cerevisiae var. bayanus CECT Spanish Culture Type Collection
a a

Source Wine CECT-1009 Wine CECT-1678 CECT-1182 Actiore F33 (Laffort, Bordeaux, France) Lalvin 71B (Lallemand, Blagnac, France) Lalvin BRL97 (Lallemand) Enoferm QA23 (Lallemand) Uvaferm VRB (Lallemand) Uvaferm WAM (Lallemand)

Number of strains 15 1 7 1 1 1 1 1 1 1 1

S. cerevisiae var. bayanusa S. cerevisiae var. cerevisiaea S. cerevisiae var. cerevisiaea

Commercial yeast strain

by OD at 655nm in a Bio-Rad microtiter reader (model 450, Bio-Rad Laboratories, Hercules, California). YPD broth at pH 3.5 was used for standard microtiter assays. MIC was dened as the smallest amount of antimicrobial agent needed to decrease OD more than 50%, when compared with OD of control cells grown without antimicrobial agent, after incubation for 48 to 72h in the microtiter assay. MBC was determined as the minimal concentration of the antimicrobial agent needed to kill more than 99.9% of the initial inoculum after plating 25L of the assayed culture onto YPD agar and incubation for 72h. MIC50 and MIC90 were dened as the MIC that inhibited 50 and 90%, respectively, of the tested microorganisms. Analogous denitions were applied to MBC50 and MBC90. PMB (Dolmar, Gimileo, Spain) was tested in the concentration range from 6.25 to 0.003g/L; enological chitosan (Lallemand, Toulouse, France) was tested from 250 to 0.12mg/L; an enological tannin solution (total polyphenol index=29.2), commercially known as TaniStop (Dolmar), was tested from 4 to 0.002mL/L; and DMDC, commercialized as Velcorin (99.8% purity) (Lanxess, Leverkusen, Germany), was tested from 5 to 0.002g/L. All the antimicrobial agents were assayed in double serial dilutions, they were tested alone and combined with 12.5% ethanol (Panreac Qumica S.A.), and the effect of ethanol alone in the growth medium was also tested. Microtiter assays were carried out in duplicates. All antimicrobial agents were assayed at pH 3.5, and additionally, PMB was also assayed at pH 4.0. Wine production Red wine was produced from Vitis vinifera L. cv. Tempranillo red grapes from local vineyards of the northern Spanish region of La Rioja. Fermentations were carried out following traditional winemaking techniques in 15,000-L wooden tanks. Spontaneous alcoholic fermentation was performed in the presence of grape skins, seeds and stalks, with the indigenous S. cerevisiae yeast strains

and after PMB addition. At the end point of alcoholic fermentation, wine was drawn off from the lees (racking), mixed for homogenization and transferred to wooden barrels for spontaneous malolactic fermentation. Temperature was maintained around 22C. After malolactic fermentation (malic acid content below 0.10g/L), the wine recovered with solid particles from the bottom of one barrel was employed for the experiment as it presented a Brett-like aroma deviation detected by routine sensorial analysis. Turbidity was measured (Turbidimeter 2100N, Hatch Co.), and the main chemical composition of the wine was determined following European Communitys ofcial methods of analysis (1990). After homogenization, the total volume of wine was divided into three equal parts, PMB (Dolmar) was then added to reach the following concentrations: 28, 50 and 100mg/L, and experiments were carried out in 15 repeats. Samples were bottled and stored at 15C under winery conditions for 22months. Microbiological analyses were performed at three stages: just before bottling, after 4months and at the end of storage. Chemical analyses of volatile phenol by-products potentially derived from B. bruxellensis metabolism were also performed at the end of storage. Analysis ofvolatile phenols (SPE andGCIon TrapMS analysis) This analysis was carried out using the method proposed and validated by Lopez etal. [15]. According to the method, 50mL of wine containing 25L of 3-tert-butyl4-hydroxyanisole solution (1:100 in ethanol) and 75L of a surrogate standard solution that included 2-octanol was passed through a cartridge lled with LiChrolut-EN resin (Merck, Darmstadt, Germany) at about 2mL/min. The sorbent was dried by air passing through (0.6bar, 10min), and analytes were recovered by elution with 1.3mL of dichloromethane. An internal standard solution containing 4-hydroxy-4-methyl-2-pentanone and 2-octanol

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(both at 300g/mL of dichloromethane) was added to the eluted sample. The extract was then analyzed by GC with ion trap MS detection under the conditions described. The concentrations of the following volatile phenols were determined: 4-ethylphenol, 4-ethylguaiacol, their respective precursors 4-vinylphenol and 4-vinylguaiacol, as well as 4-propylguaiacol. Microbiological analysis Wine samples were processed for microbiological analysis, and 10mL of wine sample was placed in a sterile tube and centrifuged at 4,000g for 10min to recover microbial cells [16]. The supernatant was discarded and the pellet re-suspended in 1mL of sterile saline solution (0.15M). A volume of 100L of the suspension was spread onto YPD agar plate for total yeasts counting, and the Dekkera/Brettanomyces differential medium (DBDM) [17], modied with 10% (v/v) ethanol, was used for B. bruxellensis counting. Bacteria selective mannitol-agar plates [5g/L yeast extract (Oxoid), 3g/L peptone (Becton, Dickinson Co.), 25g/L d-mannitol (Sigma-Aldrich, Steinheim, Germany)] were employed for acetic acid bacteria (AAB) counting and MRS-agar (Oxoid) under anaerobic conditions for lactic acid bacteria [18]. Isolates obtained from the Brettanomyces-specic medium were species conrmed by PCR analysis [12]. Statistical analysis Statistical methods used for data analysis were as follows: discriminant function analysis of data obtained from PMB-treated wines, including seven variables (three microbial populations and four volatile phenols); and two-sided Pearsons correlation coefcients were used to examine relationships among the studied variables. Analysis of variance (ANOVA) was applied for those data with normal distribution and homogeneous variances, and Kruskal Wallis test was applied to data that did not fulll those requisites. IBM-SPSS Statistics 19.0 software for Windows (IBM-SPSS Inc., Chicago, IL, USA) was used for data processing.

Results andDiscussion Potassium metabisulte antimicrobial activity Figure1 shows MIC values of PMB determined at pH 4.0 (Fig. 1a) and 3.5 (Fig.1b) in the culture broth, and it is clearly shown that the antimicrobial activity of this agent against B. bruxellensis was higher at pH 3.5 than at pH 4.0 (tenfold higher). This increased activity at lower pH is in

accordance with the acid character of metabisulte (pK1 value of 1.81 for sulfurous acid), which in solution yields higher amounts of the molecular and more active SO2 form at lower pH values. Our results at pH 3.5 obtained with 16 B. bruxellensis enological strains in YPD broth (Fig.1b) are consistent with previous studies that reported PMB inhibitory concentrations against B. bruxellensis in the range of 2060mg/L for both synthetic wine [19, 20] and culture broth [21, 22]. Figure2 shows that PMB at pH 3.5 increased its activity in the presence of 12.5% ethanol (MIC50 =24mg/L), when compared with 6% ethanol (MIC50 =96mg/L) or without ethanol (MIC50 =48mg/L) in the growth medium. Biocidal action of PMB was also more powerful in combination with 12.5% ethanol, as shown in Table2 (MBC90 =48mg/L), than without ethanol (MBC90 =96mg/L) or with 6% ethanol (MBC90 =190mg/L, data not shown). Actually, 96mg/L of PMB in the presence of 12.5% ethanol resulted in cell death for all the assayed B. bruxellensis strains under our microtiter test conditions (Table3). The effect of ethanol in potentiating the inhibitory activity of PMB was in accordance with results reported for other PMB sensitive microorganisms, such as lactic acid bacteria [23] that can also spoil wines when their growth is not under strict control. This combined action of PMB and 12.5% ethanol represents an important factor for the winemaker who can reach the biocidal effect of PMB employing low concentrations of this agent (4896mg/L) in a wine with 12.5% ethanol that can be aged and stored with this antiBrettanomyces preservative. When ethanol was assayed in the absence of other antimicrobials, all B. bruxellensis and S. cerevisiae strains of our study showed resistance (data not shown) and no biocidal effect of ethanol could be detected in the assayed range of concentrations (020% ethanol in the culture broth); therefore, ethanol MBC90 and MIC90 were over 20% for both yeast species, as expected from strains recovered from wines. Regarding the effect of PMB on S. cerevisiae, we found that our strains of this species were much more resistant than the B. bruxellensis ones, and they tolerated higher concentrations of PMB in the absence of ethanol (MIC50 and MBC90=780mg/L), as shown in Table2. In spite of that PMB resistance decreased in the presence of 12.5% ethanol (MIC50 =48mg/L, MBC90 =390mg/L). It is important to note that this species itself can produce a certain amount of sulfur dioxide in the range of 1040mg/L as a fermentation by-product [24]. The current rules of the International Organisation of Vine and Wine (OIV) consider 150 and 200mg/L as maximal limits for total SO2 concentrations that can be found in red and white wines, respectively. These concentrations are equivalent to about 260 and 347mg/L of PMB (considering a 55%

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Eur Food Res Technol Fig.1Minimal inhibitory concentrations of potassium metabisulte at different pH values against Saccharomyces and Brettanomyces strains. a pH 4.0, b pH 3.5. Arrows indicate MIC50 for Brettanomyces
15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0

a
Saccharomyces Brettanomyces

Number of strains

50

25

12.50

6.25

3.12

1.56

0.780 0.390 0.190 0.096 0.048 0.024 0.012

Potassium metabisulphite concentration (g/L)

15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0

Number of strains

50

25

12.50

6.25

3.12

1.56

0.780 0.390 0.190 0.096 0.048 0.024 0.012

Potassium metabisulphite concentration (g/L)

yield), and taking into account the results presented here, they can be considered efcient concentrations to inhibit B. bruxellensis growth and biocidal concentrations. It should be also taken into account that PMB efcacy is strongly dependent on wine composition and its acid pH, as discussed above. Antimicrobial activity ofchitosan, enological tannins andDMDC Chitosan Table 2 shows the antimicrobial activity of the three studied compounds that are currently used in winemaking against Brettanomyces: chitosan, enological tannins and DMDC, and Table3 shows the corresponding MIC and MBC of these antimicrobial agents for each strain of this study. Chitosan inhibitory activity against B. bruxellensis was similar, both in the absence and in the presence of 12.5% ethanol in the culture broth, showing the same MIC90 value of 62mg/L (Table2). Its biocidal

concentration MBC90 value could not be determined as it was over the studied range, and its MBC50 value in the presence of 12.5% ethanol was 62mg/L, which means that this concentration was enough to avoid cell growth and kill 50% of the strains. To date, only one study reported the effect of this compound against Brettanomyces and showed that 6g/L of shell crab chitosan was required to inhibit Brettanomyces growth [25]; nevertheless, the currently allowed chitosan for enological practices should be exclusively of fungal origin, which is highly deacetylated, and our results were obtained with the fungal chitosan for enological use, which could explain the difference between our results and the previously reported value. The antifungal activity of chitosan has been associated mainly to its cationic character, which increases at acid pH and with higher degrees of deacetylation, therefore, the higher positive charge density, the stronger antimicrobial activity of chitosan will be observed [6]. Our results showed that ethanol did not modify signicantly chitosan activity against B. bruxellensis, which was expected since ethanol does not modify its electric charge.

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Fig.2Minimal inhibitory concentrations of potassium metabisulte combined with different concentrations of ethanol against Saccharomyces and Brattanomyces strains. a 6% and b 12.5% in the culture broth. Arrows indicate MIC50 for Brettanomyces
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a
Saccharomyces Brettanomyces

Number of strains

6.25

3.12

1.56

0.780 0.390

0.190 0.096 0.048 0.024 0.012 0.006 0.003

Potassium metabisulphite concentration (g/L)

15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0

Number of strains

6.25

3.12

1.56

0.780

0.390 0.190

0.096 0.048 0.024 0.012

0.006 0.003

Potassium metabisulphite concentration (g/L)

Enological tannins Regarding the commercial solution of enological tannins with a 29.2 total polyphenol index (equivalent to 244mg/L of tannic acid, determined according to Mataix and Luque de Castro [26]), Table2 shows that the concentrations required to inhibit 50 and 90% of B. bruxellensis strains drastically decreased in the presence of 12.5% ethanol in the culture broth. In this case, both MIC50 and MIC90 lowered, respectively, from 0.5 and 1, to 0.12mL/L. Tannins presented biocidal activity against 50% of the studied strains at a concentration of 0.25mL/L in the presence of 12.5% ethanol, and as in the case of chitosan, the MBC90 value was above the studied concentration range, which included the legal limit (0.4mL/L) for the use of commercial enological tannins against B. bruxellensis in winemaking. Tannins are known to strongly associate with proteins and glycides and promoting precipitation of large macromolecular aggregates in wine, mainly stabilized by hydrophobic interactions and hydrogen bonds. The antifungal activity of tannins may be associated with this property,

and the effect of ethanol in potentiating their antifungal activity, shown in Table2, supports this hypothesis since ethanol reduces the local dielectric constant and modies hydrogen bonds, thus inducing cell occulation and modifying the membrane properties [27]. DMDC Dimethyl dicarbonate showed inhibitory effect on B. bruxellensis and S. cerevisiae growth in the assayed concentration range (50.002g/L) and under our experimental conditions, as shown in Table2. It has been reported that DMDC way of action is by penetrating into yeast cells and biding to those histidine residues located at the catalytic center of key enzymes for yeast survival [28]. Former studies [29 31] reported inhibitory concentrations of this compound in the range of 100250mg/L against Brettanomyces, assayed either in grape juice or in wines supplemented with glucose, and the MIC values we obtained (78156mg/L) for strains growing in YPD broth are in a similar range as those formerly reported.

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Eur Food Res Technol Table2Values of minimal inhibitory concentrations MIC50 and MIC90 and minimal biocide concentrations MBC50 and MBC90 for the studied antimicrobial agents Antimicrobial agent Range Microorganism MIC MIC50 Ethanol PMB PMB+ethanola Tannins Tannins+ethanol Chitosan Chitosan+ethanola DMDC DMDC+ethanola
a

MBC MIC90 >20% >20% 780mg/L 96mg/L 390mg/L 48mg/L >4mL/L 1mL/L 0.5mL/L 0.125mL/L >250mg/L 62mg/L 125mg/L 62mg/L 312mg/L 156mg/L 30mg/L 60mg/L MBC50 >20% >20% 780mg/L 96mg/L 190mg/L 48mg/L >4mL/L >4mL/L 4mL/L 0.25mL/L >250mg/L 250mg/L 250mg/L 62mg/L 625mg/L 312mg/L 625mg/L 120mg/L MBC90 >20% >20% 780mg/L 96mg/L 390mg/L 48mg/L >4mL/L >4mL/L >4mL/L >4mL/L >250mg/L >250mg/L >250mg/L >250mg/L 1250mg/L 625mg/L 1250mg/L 625mg/L

820% 6.250.003g/L 6.250.003g/L 40.002mL/L 40.002mL/L 2500.12mg/L 2500.12mg/L 50.002g/L 50.002g/L

S. cerevisiae B. bruxellensis S. cerevisiae B. bruxellensis S. cerevisiae B. bruxellensis S. cerevisiae B. bruxellensis S. cerevisiae B. bruxellensis S. cerevisiae B. bruxellensis S. cerevisiae B. bruxellensis S. cerevisiae B. bruxellensis S. cerevisiae B. bruxellensis

>20% >20% 780mg/L 48mg/L 48mg/L 24mg/L >4mL/L 0.5mL/L 0.5mL/L 0.125mL/L >250mg/L 62mg/L 62mg/L 31mg/L 156mg/L 78mg/L 5mg/L 10mg/L

MIC minimum inhibitory concentration, MBC minimum biocide concentration

a

Fixed concentration at 12.5% (v/v)

Both yeast genera of our study, Saccharomyces and Brettanomyces, presented increased sensitivity to the tested antimicrobials (PMB, chitosan, enological tannins and DMD) when combined with 12.5% ethanol in the culture broth. Nevertheless, B. bruxellensis strains were more sensitive to chitosan and enological tannins than S. cerevisiae strains. Both compounds presented specicity against Brettanomyces and did not show biocidal activity against Saccharomyces strains, either in the presence or in the absence of ethanol (Table2) at concentrations equal or under their corresponding legal limits (100mg/L for chitosan and 0.4mL/L for enological tannins). DMDC was able to inhibit the growth of both B. bruxellensis and S. cerevisiae strains (MIC50 values under the legal limit for enological use, i.e., 200mg/L) and showed MBC90 values above the legal limit against both species (Table2). Taking into account the legal limits for the use of these antimicrobials in winemaking, PMB was the agent that showed biocidal activity against all our B. bruxellensis strains (96mg/L of PMB in the presence of 12.5% ethanol) in concentrations permitted in wines. Potassium metabisulte activity againstB. bruxellensis inwines Potassium metabisulte presented the best performance against B. bruxellensis in the previous in vitro microtiter

assays, with the additional advantage of presenting biocidal effect in combination with ethanol at concentrations below the currently permitted limits for winemaking. For these reasons, it was selected for a study in naturally contaminated red dry wines. The initial red wine used for this experiment showed 219 NTU turbidity and the following chemical characteristics: pH 3.8; volatile acidity 0.61g/L; total acidity 3.09g/L; and residual sugars lower than 1.8g/L. Its microbiological analyses before treatments showed 1.6103cfu/mL (counted on YPD agar plates) of total yeast population, no colonies for B. bruxellensis and an initial AAB population of 8.0102cfu/mL (mannitol-agar plates). Figure3 shows the results obtained for total yeast, B. bruxellensis, and AAB populations in the series of bottled wines with total PMB concentrations of 28, 50 and 100mg/L after 4months (Fig.3a) and 22months (Fig.3b) of storage in bottles. No lactic acid bacteria were found in any of the assayed wines. AAB populations remained around 7cfu/mL during the whole period of storage, and no relevant differences were observed among AAB populations of wines treated with different concentrations of PMB. Statistically signicant differences were observed in B. bruxellensis populations among wines treated with 28, 50 and 100mg/L of PMB after 4months of storage (Fig.3a). After 22months of storage, microbiological analyses by colony counting were

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Table3Minimal inhibitory and biocide concentrations of the antimicrobial agents against each of the studied strains MBC Tannins (mL/L) A B A B A B A B A B A B A Chitosan (mg/L) DMDC (g/L) PMB (g/L) Tannins (mL/L) Chitosan (mg/L) DMDC (g/L) B

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B 0.048 0.096 0.390 0.390 0.096 0.012 0.006 n/g 0.048 0.390 0.096 0.012 0.048 0.048 0.048 0.024 0.024 0.012 0.024 0.024 0.048 0.024 0.012 0.048 0.012 0.048 0.048 0.048 0.012 0.096 0.048 0.250 0.125 62.50 31.25 0.078 0.250 1 1 0.5 0.250 0.5 0.125 0.5 2 0.5 0.5 0.031 0.125 0.125 0.125 0.031 0.062 0.016 0.062 0.250 0.008 0.062 31.25 62.50 62.50 62.50 31.25 62.50 31.25 62.50 125 125 62.50 62.50 31.25 31.25 62.50 31.25 62.50 4 62.50 62.50 31.25 31.25 0.078 0.156 0.156 0.156 0.078 0.078 0.156 0.156 0.078 0.078 0.078 0.005 0.019 0.078 0.078 0.010 0.019 0.078 0.039 0.019 0.039 0.005 0.010 0.048 0.097 0.048 0.097 0.780 0.048 0.097 0.097 0.048 0.048 0.190 0.097 0.024 0.048 0.048 0.048 0.048 0.012 0.048 0.048 0.048 0.024 0.097 0.048 1 >4 >4 >4 >4 >4 >4 >4 >4 >4 >4 0.5 0.125 >4 >4 0.250 >4 0.250 n/g 0.250 2 0.125 0.250 0.250 62.50 >250 >250 >250 62.50 250 >250 >250 >250 >250 >250 62.50 62.50 250 >250 >250 62.50 125 31.25 >250 250 >250 62.50 31.25 >4 >4 >4 2 >4 >4 0.5 0.062 >4 >4 >4 2 >4 >4 >4 0.250 0.250 0.250 0.125 0.06 1 0.5 0.5 0.5 0.008 0.016 n/g 0.5 0.5 0.250 0.031 0.5 0.5 0.031 0.125 0.031 0.062 0.125 >250 >250 >250 >250 >250 >250 31.25 31.25 >250 >250 >250 >250 >250 >250 >250 31.25 31.25 31.25 31.25 125 31.25 31.25 31.25 62.50 5 1 >250 62.50 62.50 31.25 62.50 62.50 >250 31.25 31.25 31.25 31.25 31.25 0.312 0.312 0.156 0.312 0.078 0.078 0.156 0.039 0.078 0.312 0.312 0.078 0.156 0.156 0.312 0.039 0.039 0.078 0.019 0.005 0.019 0.039 0.010 0.005 0.002 0.005 0.002 0.005 0.005 0.010 0.005 0.010 0.005 0.010 0.010 0.005 0.002 0.002 0.780 0.780 0.780 0.780 0.390 0.190 0.097 0.097 0.097 0.780 0.780 0.780 0.780 0.780 0.780 0.097 0.097 0.097 0.097 0.190 0.190 0.390 0.390 0.097 0.048 0.024 n/g 0.048 0.190 0.190 0.097 0.190 0.190 0.190 0.097 0.024 0.024 0.024 >4 >4 >4 >4 >4 >4 >4 0.5 >4 >4 >4 2 >4 >4 >4 2 4 0.062 2 1 >4 4 >4 2 >4 >4 n/g >4 >4 0.5 2 1 1 1 2 0.5 0.031 0.250 >250 >250 >250 >250 >250 >250 250 125 >250 >250 >250 >250 >250 >250 >250 62.50 62.50 125 62.50 >250 >250 250 250 125 125 250 125 250 >250 250 >250 >250 >250 250 62.50 62.50 31.25 62.50 1.25 1.25 0.625 1.25 0.312 0.312 1.25 0.156 0.312 1.25 1.25 0.312 0.312 1.25 1.25 0.312 0.312 0.312 0.156 0.312 0.312 0.625 0.625 0.312 0.312 0.625 0.625 0.625 0.625 0.312 0.312 1.25 1.25 0.625 1.25 0.312 0.156 0.625 0.01 0.625 0.625 0.625 0.312 0.625 0.625 1.25 0.078 0.156 0.078 0.078 0.156 0.312 0.312 0.625 0.156 0.312 0.625 0.312 0.312 0.156 0.039 0.156

Strain

MIC

PMB (g/L)

S. cerevisiae

Author's personal copy

B. bruxellensis

F33 WAM QA23 BRL97 C208 C209 C201 C199 71B VRB CNL14a CNL15a CNL16a CNL21a CNL23a BY24 BY48 CP1 CP16

0.780 0.780 0.780 0.780 0.390 0.097 0.048 0.097 0.097 0.780 0.780 0.780 0.780 0.780 0.780 0.097 0.097 0.048 0.048

CP34 NA36f PL126 PL144 PL155 PL2 PL45 PL52 PL80 1009 PL463

0.048 0.048 0.048 0.097 0.780 0.048 0.097 0.097 0.048 0.024 0.097

BY3

0.048

A: Without ethanol, B: With 12.5% ethanol. All assays performed at pH 3.5

Eur Food Res Technol

n/g no growing

Author's personal copy

Eur Food Res Technol Fig.3Microbial populations of wine samples after storage. Means and standard deviations of microbial populations in wine samples after 4months (a) and 22months (b). Bars with different letters have signicant statistical differences (p<0.05)
2

Total yeasts

Brettanomyces

Acetic acid bacteria

Log (cfu/mL)

0 28 50 100

Potassium metabisulphite treatment (mg/L)

2

Log (cfu/mL)

28

50

100

Potassium metabisulphite treatment (mg/L)

negative for B. bruxellensis in those wines treated with 50 and 100mg/L of PMB, and a minimal contaminating B. bruxellensis population was still present in the wines treated with 28mg/L of PMB (Fig.3b). Figure 4 shows volatile phenol concentrations of the wine samples at the end of the storage period (22months). Statistically signicant (p<0.05) lower concentrations of 4-ethylphenol (Fig.4d) were detected in those wines treated with 100mg/L of PMB than in wines with lower PMB concentrations (50 and 28mg/L). The highest concentrations of 4-vinylphenol, which is the precursor molecule for 4-ethylphenol, were found in those wines treated with the lowest PMB concentration (28mg/L), as shown in Fig.4c. Regarding 4-propylguaiacol (Fig.4a) and 4-ethylguaiacol (Fig.4b), statistically signicant differences among mean values were found, and the higher concentration of PMB used to treat the wines, the lower contents of both 4-propylguaiacol and 4-ethylguaiacol were detected in the wines after storage. The existence of 4-propylguaiacol was

previously reported in aged red wines and its concentration correlated with the concentrations of 4-ethylphenol and 4-ethylguaiacol [32, 33], as also evidenced from our results. In those former reports, authors hypothesized that 4-propylguaiacol could have a microbiological origin; however, they could not conrm this assumption. This molecule was also shown to exert a role in the aromatic deviation of wines in an additive effect with 4-ethylphenol and 4-ethylguaiacol [34]. Certain strains of lactic acid bacteria of the species Lactobacillus plantarum, Lactobacillus brevis and Pediococcus pentosaceous [3] have been reported as vinylphenol producers, but since no viable lactic acid bacteria were detected in our wines, their putative contribution to volatile phenol production during wine storage was excluded in our study, and only Brettanomyces microbial population correlated signicantly with the concentrations of the three volatile phenols: 4-propylguaiacol, 4-ethylguaiacol and 4-ethylphenol (Table4). Figure 5 shows the scores for the 45 wine samples on two canonical discriminating functions grouped according

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4-propylguaiacol (g/L) 4-ethylguaiacol (g/L)
5 4 3 2 1 0 28 50 100

Eur Food Res Technol

140 120 100 80 60 40 20 0

b c

b c

28

50

100

Potassium metabisulphite treatment (mg/L) 4-vinylphenol (g/L) 4-ethylphenol (g/L)

120 100 80 60 40 20 0 28 50

Potassium metabisulphite treatment (mg/L)

1,600 1,400 1,200 1,000 800 600 400 200 0

b
100

28

50

100

Potassium metabisulphite treatment (mg/L)

Potassium metabisulphite treatment (mg/L)

Fig.4Results of volatile phenols analysis of the wine samples after storage of 22months. Mean and standard deviation values of concentrations of volatile phenols: a 4-propylguaiacol, b 4-ethylguaiacol,

c 4-vinylphenol, d 4-ethylphenol.Different letters indicate signicant statistical differences (p<0.05)

Table4Pearsons correlation coefcient values among microbial populations at four and 22months storage and concentrations of volatile phenols

4-propylguaiacol 4months Total yeasts Brettanomyces Acetic acid bacteria 22months Total yeasts Brettanomyces Acetic acid bacteria 0.373a 0.566b 0.098 0.122 0.293 0.008

4-ethylguaiacol

4-vinylphenol

4-ethylphenol

0.270 0.436b 0.080 0.099 0.226 0.008

0.274 0.264 0.247 0.019 0.151 0.119

0.250 0.402b 0.042 0.095 0.185 0.033

Signicant correlation at 0.05 Signicant correlation at 0.01

to the three PMB treatments (28, 50 and 100mg/L for groups 1, 2 and 3, respectively) and with seven variables (three microbial populations and four volatile phenols). Functions 1 and 2 explained, respectively, 97.4 and 2.6% of the variance values, and the highest standardized correlation coefcients of function 1 corresponded to the following variables: concentration of 4-ethylguaiacol (coefcient 1.224), concentration of 4-propylguaiacol (coefcient 1.116), concentration of 4-ethylphenol (coefcient 1.919) and B. bruxellensis population after 4months of storage (coefcient 0.999), and these four variables showed as well the highest absolute correlation with function 1 (Table 4). On the contrary, the lowest correlation found was for AAB population (coefcient 0.181). Therefore, our results showed that B. bruxellensis population after storage in glass bottles for four months was dependent upon the PMB treatment and correlated inversely with the PMB

concentration adjusted before storage, being 100mg/L of total PMB enough to prevent B. bruxellensis growth. The levels of 4-ethylguaiacol and 4-ethylphenol, which are the metabolic nal products of the vinylphenol reductase in B. bruxellensis, followed the same trend as B. bruxellensis population, showing the lowest concentrations in those wines that had been treated with 100mg/L of PMB and showed a minimal B. bruxellensis population (0.5cfu/mL) after 4months, and no viable cell counts after 22months of storage. Regarding 4-propylguaiacol, its concentrations followed the same trend as B. bruxellensis population after 4months storage and correlated inversely with the PMB treatment concentration. This result brings new evidences to the previous suggestions that volatile 4-propylguaiacol could be generated by B. bruxellensis, whose enzymatic action upon a precursor molecule extracted from the oak wood, such as ferulic acid, by two successive reduction

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Eur Food Res Technol 3. Chatonnet P, Dubourdie D, Boidron JN, Pons M (1992) The origin of ethylphenols in wines. J Sci Food Agric 60:165178 4. Chatonnet P, Dubordieu D, Boidron JN (1995) The inuence of Brettanomyces/Dekkera sp. yeasts and lactic acid bacteria on the ethylphenol content of red wines. Am J Enol Vitic 46:463468 5. Lonvaud-Funel A, Renouf V (2005) Incidence microbiologicque de lusage de barriques neuves et/ou de barrique usages. Rev Fr dOenol 211:1014 6. Kong M, Chen XG, Xing K, Park HJ (2010) Antimicrobial properties of chitosan and mode of action: a state of the art review. Int J Food Microbiol 144:5163 7. Harris V, Jiranek V, Ford C, Grbin P (2010) Inhibitory effect of hydroxycinnamic acids on Dekkera spp. Appl Microbiol Biotechnol 86:721729 8. Enrique M, Marcos JF, Yuste M, Martnez M, Valls S, Manzanares P (2007) Antimicrobial action of synthetic peptides towards wine spoilage yeasts. Int J Food Microbiol 118:318325 9. Comitini F, Ciani M (2011) Kluyveromyces wickerhamii killer toxin: purication and activity towards Brettanomyces/Dekkera yeasts in grape must. FEMS Microbiol Lett 316:7782 10. Santos A, San Mauro M, Bravo E, Marquina D (2009) PMKT2, a new killer toxin from Pichia membranifaciens, and its promising biotechnological properties for control of the spoilage yeast Brettanomyces bruxellensis. Microbiology 155:624634 11. Santos A, Navascus E, Bravo E, Marquina D (2011) Ustilago maydis killer toxin as a new tool for the biocontrol of the wine spoilage yeast Brettanomyces bruxellensis. Int J Food Microbiol 145:147154 12. Cocolin L, Rantsiou K, Iacumin L, Zironi R, Comi G (2004) Molecular detection and identication of Brettanomyces/Dekkera bruxellensis and Brettanomyces/Dekkera anomalus in spoiled wines. Appl Environ Microbiol 70:13471355 13. Fernndez-Espinar MT, Esteve-Zarzoso B, Querol A, Barrio E (2000) RFLP analysis of the ribosomal internal transcribed spacers and the 5.8S rRNA gene region of the genus Saccharomyces: a fast method for species identication and the differentiation of or yeasts. Antonie Van Leeuwenhoek 78:8797 14. Cavalieri SJ, Harbeck RJ, McCarter YS, Ortez JH, Rankin ID, Sautter RL, Sharp SE, Spiegel CA (2005) Manual of antimicrobial susceptibility testing. In: Coyle MB (ed) American soc microbiol press. http://www.forms.asm.org/ASM/les. Accessed 24 Aug 2012 15. Lpez R, Aznar M, Cacho J, Ferreira V (2002) Determination of minor and trace volatile compounds in wine by solid-phase extraction and gas chromatography with mass spectrometric detection. J Chromatogr 966:167177 16. Lpez I, Tenorio C, Zarazaga M, Dizy M, Torres C, Ruiz-Larrea F (2007) Evidence of mixed wild populations of Oenococcus oeni strains during wine spontaneous malolactic fermentations. Eur Food Res Technol 226:215223 17. Rodrigues N, Gonalves G, Pereira da Silva S, Malfeito-Ferreita M, Loureiro V (2001) Development and use of a new medium to detect yeasts of the genera Dekkera/Brettanomyces. J Appl Microbiol 90:588599 18. Holt JG, Krieg NR, Sneath PHA, Staley JT, Williams ST (1994) Bergeys manual of determinative bacteriology. In: Hensyl WR (ed) Williams and Wilkins, Baltimore 19. Agnolucci M, Rea F, Sbrana C, Cristani C, Fracassetti D, Tirelli A, Nuti M (2010) Sulphur dioxide affects culturability and volatile phenol production by Brettanomyces/Dekkera bruxellensis. Int J Food Microbiol 143:7680 20. Du Toit M, Pretorius IS, Lonvaud-Funel A (2005) The effect of sulphur dioxide and oxygen on the viability and culturability of a strain of Acetobacter pasteurianus and a strain of Brettanomyces bruxellensis isolated from wine. J Appl Microbiol 98: 862871

Fig.5Discriminant function analysis

steps could nally generate 4-propylguaiacol. Nevertheless, further studies are needed to identify and characterize the putative enzymes involved in this process.

Conclusion From the enological point of view, it is important to note that the addition of an adequate PMB concentration was able to efciently control Brettanomyces population, that volatile phenol concentrations correlated with Brettanomyces populations during the rst months of storage and nally, that Brettanomyces viable cell populations were found only in those least protected wines, which in the case of our red wines contained <100mg/L of total PMB.
Acknowledgments This research was nancially supported by Grant CENIT-2008/1002 of the Spanish Ministry of Science and Innovation MICINN-CDTI. Caur B. Portugal was a contractual researcher supported by Grant CENIT-2008/1002. Conflict of interestNone. Compliance with Ethics Requirements This article does not contain any studies with human or animal subjects.

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