Portugal Et Al_2013

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European Food Research andTechnologyZeitschrift fr Lebensmittel-Untersuchung und -Forschung A ISSN 1438-2377 Eur Food Res TechnolDOI 10.1007/s00217-013-2143-2

Brettanomyces susceptibility toantimicrobial agents used in winemaking:in vitro and practical approaches

Caur Portugal, Yolanda Senz, BeatrizRojo-Bezares, Myriam Zarazaga,Carmen Torres, Juan Cacho & FernandaRuiz-Larrea

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Eur Food Res TechnolDOI 10.1007/s00217-013-2143-2

ORIgInal PaPER

Brettanomyces susceptibility to antimicrobial agents used in winemaking: in vitro and practical approaches

Caur Portugal Yolanda Senz Beatriz RojoBezares Myriam Zarazaga Carmen Torres Juan Cacho Fernanda RuizLarrea

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

against Brettanomyces, and PMB showed the highest effi-cacy in concentrations under the currently permitted limits for enological use; consequently, PMB was further evalu-ated in red wines naturally contaminated by Brettanomy-ces. 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 first time that 4-propylguai-acol is shown to correlate with Brettanomyces population in wine. It is of enological significance that a concentration of 100 mg/l of total PMB efficiently prevented Brettano-myces growth in the aging red wines of our study and that volatile phenol concentrations were significantly (p < 0.05) higher in those poorly protected wines.

Keywords Brettanomyces Potassium metabisulfite Volatile phenols Chitosan Enological tannins Dimethyl dicarbonate

Introduction

Brettanomyces bruxellensis is a well-known yeast spe-cies 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 under-going climate warming trends. Brettanomyces, named Dek-kera when growing as its sporulating teleomorph, is respon-sible for developing off-odors described by most diverse

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 com-pounds against Brettanomyces: potassium metabisulfite (PMB), chitosan, enological tannins and dimethyl dicar-bonate. Minimal inhibitory concentrations and minimal biocidal concentrations of the antimicrobial agents were determined, and a comparative study between B. brux-ellensis 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. Ruiz-larrea (*) Instituto de Ciencias de la Vid y del Vino (CSIC-UR-gR), University of la Rioja, av. Madre de Dios 51, 26006 logroo, Spaine-mail: [email protected]

Present Address: C. Portugal laboratory of Technology and Quality of alcoholic Beverages, College of agriculture luiz de Queiroz, University of So Paulo, av. Pdua Dias 11, Piracicaba 13418-900, Brazil

Y. Senz B. Rojo-Bezares Molecular Microbiology laboratory, Center for Biomedical Research of la Rioja (CIBIR), C/Piqueras 98, 3 Planta, 26006 logroo, Spain

M. Zarazaga C. Torres laboratory of Biochemistry and Molecular Biology, Department of Food and agriculture, University of la Rioja, av. Madre de Dios 51, 26006 logroo, Spain

J. Cacho Department of analytical Chemistry, Faculty of Sciences, University of Zaragoza, 50009 Zaragoza, Spain

Author's personal copy

Eur Food Res Technol

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descriptors, such as animal, stable, horse sweat, mousy, burnt plastic, medicinal or phenolic taint. Due to the wide-spread 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-ethyl-phenol, 4-ethylguaiacol, 4-vinylphenol and 4-vinylguaiacol, all of them are secondary metabolites produced by Brettan-omyces from the phenolic acids naturally present in wine. This is the reason why monitoring this organoleptic devia-tion 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 (426 g/l for a 1:10 mix of 4-ethylphenol and 4-ethylguaiacol [3]), and this happens once active Brettanomyces in the wine over-come 103 cells/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 difficult to sterilize. For winemakers of premium red wines, regular monitoring of Brettanomyces population is mandatory, and when the problem arises, efficient sanita-tion procedures should be implemented. a number of phys-icochemical treatments, such as thermal inactivation or high pressures, are currently available to winemakers. nev-ertheless, these actions are palliative; their effect is transi-tory 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 10 years, alternative inhibitors of the growth of Brettanomyces have been approved or recommended in winemaking. The use of dimethyl dicar-bonate (DMDC) is currently allowed exclusively for those wines with a sugar content over 5 g/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 efficient antimicrobial agent against Brettanomyces and presents the advantages of being biodegradable and nontoxic [6]. Hydroxycinnamic acids [7], synthetic peptides [8] and some yeast killer tox-ins isolated from Pichia anomala, Kluyveromyces wick-erhamii [9], Pichia membranaefaciens [10] and Ustilago maydis [11] strains have been reported to possess fungi-cidal effect against Brettanomyces.

The objective of this work was to perform a methodi-cal 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 metabisulfite (PMB), chitosan, enological tan-nins and DMDC. The study was performed with 31 eno-logical strains of the species B. bruxellensis and Saccha-romyces 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 evalu-ated for its efficacy under winery conditions. Wines natu-rally contaminated by Brettanomyces were treated with the most effective antimicrobial agent, and microbial popula-tions were monitored over 22 months 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 and methods

Yeast strains and culture conditions

a total of 16 B. bruxellensis and 15 S. cerevisiae strains were included in this study; 22 of these strains were recov-ered 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 Table 1. Yeast species identification was per-formed by specific PCR analysis: B. bruxellensis, accord-ing to the method of Cocolin et al. [12], and S. cerevisiae, according to the method of Fernndez-Espinar et al. [13]. Yeast strains were cultivated from 24 h to 48 h on YPD agar [10 g/l yeast extract (Oxoid ltd., Basingstoke, UK), 20 g/l peptone (Becton, Dickinson Co., lePont de Claix, France), 20 g/l glucose (Panreac Qumica S.a., Barcelona, Spain), 20 g/l agar (Becton, Dickinson Co.)], and fresh cultures were used for the antimicrobial activity assays described below.

Minimal inhibitory concentration (MIC) and minimal 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 ster-ile saline solution to reach 1.2 optical density (OD) units at 600 nm, and 12.5 l was added to a final volume of 100 l in the microtiter assay. Cell growth was determined



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by OD at 655 nm 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 defined as the smallest amount of antimicrobial agent needed to decrease OD more than 50 %, when com-pared with OD of control cells grown without antimicro-bial agent, after incubation for 48 to 72 h 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 25 l of the assayed culture onto YPD agar and incubation for 72 h. MIC50 and MIC90 were defined as the MIC that inhibited 50 and 90 %, respectively, of the tested microorganisms. analogous defi-nitions were applied to MBC50 and MBC90. PMB (Dolmar, gimileo, Spain) was tested in the concentration range from 6.25 to 0.003 g/l; enological chitosan (lallemand, Tou-louse, France) was tested from 250 to 0.12 mg/l; an eno-logical tannin solution (total polyphenol index = 29.2), commercially known as TaniStop (Dolmar), was tested from 4 to 0.002 ml/l; and DMDC, commercialized as Vel-corin (99.8 % purity) (lanxess, leverkusen, germany), was tested from 5 to 0.002 g/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 dupli-cates. 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. Tem-pranillo red grapes from local vineyards of the northern Spanish region of la Rioja. Fermentations were car-ried out following traditional winemaking techniques in 15,000-l wooden tanks. Spontaneous alcoholic fermenta-tion 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 fer-mentation, wine was drawn off from the lees (racking), mixed for homogenization and transferred to wooden bar-rels for spontaneous malolactic fermentation. Temperature was maintained around 22 C. after malolactic fermenta-tion (malic acid content below 0.10 g/l), the wine recov-ered 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 deter-mined following European Communitys official 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 100 mg/l, and experiments were carried out in 15 repeats. Samples were bottled and stored at 15 C under winery conditions for 22 months. Microbiological analy-ses were performed at three stages: just before bottling, after 4 months and at the end of storage. Chemical analy-ses of volatile phenol by-products potentially derived from B. bruxellensis metabolism were also performed at the end of storage.

analysis of volatile phenols (SPE and gC-Ion Trap-MS analysis)

This analysis was carried out using the method proposed and validated by lopez et al. [15]. according to the method, 50 ml of wine containing 25 l of 3-tert-butyl-4-hydroxyanisole solution (1:100 in ethanol) and 75 l of a surrogate standard solution that included 2-octanol was passed through a cartridge filled with liChrolut-En resin (Merck, Darmstadt, germany) at about 2 ml/min. The sorbent was dried by air passing through (0.6 bar, 10 min), and analytes were recovered by elution with 1.3 ml of dichloromethane. an internal standard solution containing 4-hydroxy-4-methyl-2-pentanone and 2-octanol

Table 1 Yeasts included in the study

CECT Spanish Culture Type Collectiona Commercial yeast strain

Species Source number of strains

Brettanomyces bruxellensis Wine 15Dekkera bruxellensis CECT-1009 1Saccharomyces cerevisiae Wine 7S. cerevisiae CECT-1678 1S. cerevisiae CECT-1182 1S. cerevisiaea actiflore F33 (laffort, Bordeaux, France) 1S. cerevisiae var. cerevisiaea lalvin 71B (lallemand, Blagnac, France) 1S. cerevisiae var. bayanusa lalvin BRl97 (lallemand) 1S. cerevisiae var. bayanusa Enoferm Qa23 (lallemand) 1S. cerevisiae var. cerevisiaea Uvaferm VRB (lallemand) 1S. cerevisiae var. cerevisiaea Uvaferm WaM (lallemand) 1



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(both at 300 g/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 deter-mined: 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 anal-ysis, and 10 ml of wine sample was placed in a ster-ile tube and centrifuged at 4,000 g for 10 min to recover microbial cells [16]. The supernatant was discarded and the pellet re-suspended in 1 ml of sterile saline solution (0.15 M). a volume of 100 l of the suspension was spread onto YPD agar plate for total yeasts counting, and the Dekkera/Brettanomyces differential medium (DBDM) [17], modified with 10 % (v/v) ethanol, was used for B. bruxellensis counting. Bacteria selective mannitol-agar plates [5 g/l yeast extract (Oxoid), 3 g/l peptone (Becton, Dickinson Co.), 25 g/l d-mannitol (Sigma-aldrich, Stein-heim, 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-specific medium were species con-firmed by PCR analysis [12].

Statistical analysis

Statistical methods used for data analysis were as fol-lows: discriminant function analysis of data obtained from PMB-treated wines, including seven variables (three micro-bial populations and four volatile phenols); and two-sided Pearsons correlation coefficients 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 KruskalWallis test was applied to data that did not fulfill those req-uisites. IBM-SPSS Statistics 19.0 software for Windows (IBM-SPSS Inc., Chicago, Il, USa) was used for data processing.

Results and Discussion

Potassium metabisulfite antimicrobial activity

Figure 1 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 metabisulfite (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 2060 mg/l for both synthetic wine [19, 20] and culture broth [21, 22].

Figure 2 shows that PMB at pH 3.5 increased its activ-ity in the presence of 12.5 % ethanol (MIC50 = 24 mg/l), when compared with 6 % ethanol (MIC50 = 96 mg/l) or without ethanol (MIC50 = 48 mg/l) in the growth medium. Biocidal action of PMB was also more powerful in combination with 12.5 % ethanol, as shown in Table 2 (MBC90 = 48 mg/l), than with-out ethanol (MBC90 = 96 mg/l) or with 6 % ethanol (MBC90 = 190 mg/l, data not shown). actually, 96 mg/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 (Table 3). The effect of etha-nol in potentiating the inhibitory activity of PMB was in accordance with results reported for other PMB sensi-tive 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 (4896 mg/l) in a wine with 12.5 % ethanol that can be aged and stored with this anti-Brettanomyces 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 = 780 mg/l), as shown in Table 2. In spite of that PMB resistance decreased in the presence of 12.5 % ethanol (MIC50 = 48 mg/l, MBC90 = 390 mg/l). It is important to note that this species itself can produce a cer-tain amount of sulfur dioxide in the range of 1040 mg/l as a fermentation by-product [24]. The current rules of the International Organisation of Vine and Wine (OIV) consider 150 and 200 mg/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 347 mg/l of PMB (considering a 55 %



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yield), and taking into account the results presented here, they can be considered efficient concentrations to inhibit B. bruxellensis growth and biocidal concentrations. It should be also taken into account that PMB efficacy is strongly dependent on wine composition and its acid pH, as discussed above.

antimicrobial activity of chitosan, enological tannins and DMDC

Chitosan

Table 2 shows the antimicrobial activity of the three studied compounds that are currently used in winemak-ing against Brettanomyces: chitosan, enological tannins and DMDC, and Table 3 shows the corresponding MIC and MBC of these antimicrobial agents for each strain of this study. Chitosan inhibitory activity against B. bruxel-lensis was similar, both in the absence and in the pres-ence of 12.5 % ethanol in the culture broth, showing the same MIC90 value of 62 mg/l (Table 2). 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 62 mg/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 6 g/l of shell crab chitosan was required to inhibit Brettanomyces growth [25]; nevertheless, the cur-rently 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 activ-ity of chitosan will be observed [6]. Our results showed that ethanol did not modify significantly chitosan activity against B. bruxellensis, which was expected since ethanol does not modify its electric charge.

Fig. 1 Minimal inhibitory concentrations of potassium metabisulfite at different pH values against Saccharomyces and Brettanomyces strains. a pH 4.0, b pH 3.5. Arrows indicate MIC50 for Brettanomyces

Saccharomyces Brettanomyces

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50 25 12.50 6.25 3.12 1.56 0.780 0.390 0.190 0.096 0.048 0.024 0.012

Num

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Potassium metabisulphite concentration (g/L)

a

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50 25 12.50 6.25 3.12 1.56 0.780 0.390 0.190 0.096 0.048 0.024 0.012

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b



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Enological tannins

Regarding the commercial solution of enological tannins with a 29.2 total polyphenol index (equivalent to 244 mg/l of tannic acid, determined according to Mataix and luque de Castro [26]), Table 2 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 0.12 ml/l. Tan-nins presented biocidal activity against 50 % of the studied strains at a concentration of 0.25 ml/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 (0.4 ml/l) for the use of commer-cial enological tannins against B. bruxellensis in winemak-ing. Tannins are known to strongly associate with proteins and glycides and promoting precipitation of large macro-molecular aggregates in wine, mainly stabilized by hydro-phobic 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 Table 2, supports this hypothesis since ethanol reduces the local dielectric constant and modifies hydrogen bonds, thus inducing cell flocculation and modi-fying the membrane properties [27].

DMDC

Dimethyl dicarbonate showed inhibitory effect on B. brux-ellensis and S. cerevisiae growth in the assayed concentra-tion range (50.002 g/l) and under our experimental condi-tions, as shown in Table 2. 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 [2931] reported inhibitory concentrations of this compound in the range of 100250 mg/l against Brettanomyces, assayed either in grape juice or in wines supplemented with glu-cose, and the MIC values we obtained (78156 mg/l) for strains growing in YPD broth are in a similar range as those formerly reported.

Fig. 2 Minimal inhibitory concentrations of potassium metabisulfite 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

Saccharomyces Brettanomyces

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6.25 3.12 1.56 0.780 0.390 0.190 0.096 0.048 0.024 0.012 0.006 0.003

6.25 3.12 1.56 0.780 0.390 0.190 0.096 0.048 0.024 0.012 0.006 0.003

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Both yeast genera of our study, Saccharomyces and Bret-tanomyces, 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 sen-sitive to chitosan and enological tannins than S. cerevisiae strains. Both compounds presented specificity against Bret-tanomyces and did not show biocidal activity against Sac-charomyces strains, either in the presence or in the absence of ethanol (Table 2) at concentrations equal or under their corresponding legal limits (100 mg/l for chitosan and 0.4 ml/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., 200 mg/l) and showed MBC90 values above the legal limit against both species (Table 2). 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 (96 mg/l of PMB in the presence of 12.5 % ethanol) in concentrations permitted in wines.

Potassium metabisulfite activity against B. bruxellensis in wines

Potassium metabisulfite 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 contami-nated red dry wines.

The initial red wine used for this experiment showed 219 nTU turbidity and the following chemical characteris-tics: pH 3.8; volatile acidity 0.61 g/l; total acidity 3.09 g/l; and residual sugars lower than 1.8 g/l. Its microbiologi-cal analyses before treatments showed 1.6 103 cfu/ml (counted on YPD agar plates) of total yeast population, no colonies for B. bruxellensis and an initial aaB population of 8.0 102 cfu/ml (mannitol-agar plates). Figure 3 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 100 mg/l after 4 months (Fig. 3a) and 22 months (Fig. 3b) of storage in bottles. no lactic acid bacteria were found in any of the assayed wines. aaB populations remained around 7 cfu/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 significant differences were observed in B. bruxellensis populations among wines treated with 28, 50 and 100 mg/l of PMB after 4 months of storage (Fig. 3a). after 22 months of stor-age, microbiological analyses by colony counting were

Table 2 Values of minimal inhibitory concentrations MIC50 and MIC90 and minimal biocide concentrations MBC50 and MBC90 for the studied antimicrobial agents

MIC minimum inhibitory concentration, MBC minimum biocide concentrationa Fixed concentration at 12.5 % (v/v)

antimicrobial agent Range Microorganism MIC MBC

MIC50 MIC90 MBC50 MBC90

Ethanol 820 % S. cerevisiae >20 % >20 % >20 % >20 %B. bruxellensis >20 % >20 % >20 % >20 %

PMB 6.250.003 g/l S. cerevisiae 780 mg/l 780 mg/l 780 mg/l 780 mg/lB. bruxellensis 48 mg/l 96 mg/l 96 mg/l 96 mg/l

PMB + ethanola 6.250.003 g/l S. cerevisiae 48 mg/l 390 mg/l 190 mg/l 390 mg/lB. bruxellensis 24 mg/l 48 mg/l 48 mg/l 48 mg/l

Tannins 40.002 ml/l S. cerevisiae >4 ml/l >4 ml/l >4 ml/l >4 ml/lB. bruxellensis 0.5 ml/l 1 ml/l >4 ml/l >4 ml/l

Tannins + ethanola 40.002 ml/l S. cerevisiae 0.5 ml/l 0.5 ml/l 4 ml/l >4 ml/lB. bruxellensis 0.125 ml/l 0.125 ml/l 0.25 ml/l >4 ml/l

Chitosan 2500.12 mg/l S. cerevisiae >250 mg/l >250 mg/l >250 mg/l >250 mg/lB. bruxellensis 62 mg/l 62 mg/l 250 mg/l >250 mg/l

Chitosan + ethanola 2500.12 mg/l S. cerevisiae 62 mg/l 125 mg/l 250 mg/l >250 mg/lB. bruxellensis 31 mg/l 62 mg/l 62 mg/l >250 mg/l

DMDC 50.002 g/l S. cerevisiae 156 mg/l 312 mg/l 625 mg/l 1250 mg/lB. bruxellensis 78 mg/l 156 mg/l 312 mg/l 625 mg/l

DMDC + ethanola 50.002 g/l S. cerevisiae 5 mg/l 30 mg/l 625 mg/l 1250 mg/lB. bruxellensis 10 mg/l 60 mg/l 120 mg/l 625 mg/l



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Tabl

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M

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and

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con

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ratio

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inst

each

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per

form

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t pH

3.5

n/g

no g

row

ing

Stra

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ICM

BC

PMB

(g/l

)Ta

nnin

s (ml

/l)

Chito

san

(mg/l

)D

MD

C (g/

l)PM

B (g

/l)

Tannin

s (ml

/l)

Chito

san

(mg/l

)D

MD

C (g/

l)a

Ba

Ba

Ba

Ba

Ba

Ba

Ba

B

S. c

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isia

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30.

780

0.04

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40.

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250

125

0.31

20.

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0.78

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190

>4

1>

250

>25

01.

251.

25W

aM

0.78

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096

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250

31.2

50.

312

0.01

90.

780

0.19

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4>

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250

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01.

251.

25Qa

230.

780

0.39

0>

40.

5>

250

31.2

50.

156

0.03

90.

780

0.39

0>

44

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025

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625

0.62

5B

Rl9

70.

780

0.39

02

0.5

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0.31

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1 3

negative for B. bruxellensis in those wines treated with 50 and 100 mg/l of PMB, and a minimal contaminating B. bruxellensis population was still present in the wines treated with 28 mg/l of PMB (Fig. 3b).

Figure 4 shows volatile phenol concentrations of the wine samples at the end of the storage period (22 months). Statistically significant (p < 0.05) lower concentrations of 4-ethylphenol (Fig. 4d) were detected in those wines treated with 100 mg/l of PMB than in wines with lower PMB concentrations (50 and 28 mg/l). The highest con-centrations of 4-vinylphenol, which is the precursor mol-ecule for 4-ethylphenol, were found in those wines treated with the lowest PMB concentration (28 mg/l), as shown in Fig. 4c.

Regarding 4-propylguaiacol (Fig. 4a) and 4-ethylguai-acol (Fig. 4b), statistically significant 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 concentra-tion 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; how-ever, they could not confirm 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-eth-ylguaiacol [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 bac-teria were detected in our wines, their putative contribu-tion to volatile phenol production during wine storage was excluded in our study, and only Brettanomyces microbial population correlated significantly with the concentrations of the three volatile phenols: 4-propylguaiacol, 4-ethylguai-acol and 4-ethylphenol (Table 4).

Figure 5 shows the scores for the 45 wine samples on two canonical discriminating functions grouped according

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

Total yeasts Brettanomyces Acetic acid bacteria

0

1

2

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(cfu/m

L)

Potassium metabisulphite treatment (mg/L)

a

0

1

2

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b



1 3

to the three PMB treatments (28, 50 and 100 mg/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 cor-relation coefficients of function 1 corresponded to the fol-lowing variables: concentration of 4-ethylguaiacol (coeffi-cient 1.224), concentration of 4-propylguaiacol (coefficient 1.116), concentration of 4-ethylphenol (coefficient 1.919) and B. bruxellensis population after 4 months of stor-age (coefficient 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 (coefficient 0.181). Therefore, our results showed that B. bruxellensis population after stor-age in glass bottles for four months was dependent upon the PMB treatment and correlated inversely with the PMB

concentration adjusted before storage, being 100 mg/l of total PMB enough to prevent B. bruxellensis growth. The levels of 4-ethylguaiacol and 4-ethylphenol, which are the metabolic final products of the vinylphenol reductase in B. bruxellensis, followed the same trend as B. bruxellen-sis population, showing the lowest concentrations in those wines that had been treated with 100 mg/l of PMB and showed a minimal B. bruxellensis population (0.5 cfu/ml) after 4 months, and no viable cell counts after 22 months of storage. Regarding 4-propylguaiacol, its concentrations followed the same trend as B. bruxellensis population after 4 months 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

020406080

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4-et

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b b

c a

0200400600800

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4-et

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heno

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aad b

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

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

Table 4 Pearsons correlation coefficient values among microbial populations at four and 22 months storage and concentrations of volatile phenols

a Significant correlation at 0.05

b Significant correlation at 0.01

4-propylguaiacol 4-ethylguaiacol 4-vinylphenol 4-ethylphenol

4 months Total yeasts 0.373a 0.270 0.274 0.250 Brettanomyces 0.566b 0.436b 0.264 0.402b

acetic acid bacteria 0.098 0.080 0.247 0.04222 months Total yeasts 0.122 0.099 0.019 0.095 Brettanomyces 0.293 0.226 0.151 0.185 acetic acid bacteria 0.008 0.008 0.119 0.033



1 3

steps could finally 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 efficiently control Brettanomyces population, that volatile phenol concentrations correlated with Brettano-myces populations during the first months of storage and finally, that Brettanomyces viable cell populations were found only in those least protected wines, which in the case of our red wines contained


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21. Barata a, Caldeira J, Botelheiro R, Pagliara a, Malfeito-Ferreira M, loureiro V (2008) Survival patterns of Dekkera bruxellen-sis in wines and inhibitory effect of sulphur dioxide. Int J Food Microbiol 121:201207

22. Conterno l, lucy Joseph CM, arvik TJ, Henick-Kling T, Bisson lF (2006) genetic and physiological characterization of Brettan-omyces bruxellensis strains isolated from wines. am J Enol Vitic 57:139147

23. Rojo-Bezares B, Senz Y, Zarazaga M, Torres C, Ruiz-larrea F (2007) antimicrobial activity of nisin against Oenococcus oeni and other wine bacteria. Int J Food Microbiol 116:3236

24. Romano P, Suzzi g (1993) Sulphur dioxide and wine microor-ganisms. In: Fleet g (ed) Wine microbiology and biotechnology. Harwood academic Publishers, Chur, pp 373393

25. gmez-Rivas l, Escudero-abarca BI, aguilar-Uscanda Mg, Hayward-Jones PM, Mendoza P, Ramrez M (2004) Selective antimicrobial action of chitosan against spoilage yeasts in mixed culture fermentations. J Ind Microbiol Biot 31:1622

26. Mataix E, luque de Castro MD (2001) Simultaneous (or sequen-tial) determination of the total polyphenol index (or I280) and density in wines by flow injection. analyst 126:251255

27. Soares EV (2011) Flocculation in Saccharomyces cerevisiae: a review. J appl Microbiol 110:118

28. Hornsey IS (2007) Chemistry and biology of winemaking. The Royal Society of Chemistry, Cambridge

29. Costa a, Barata a, Malfeito-Ferreira M, loureiro V (2008) Eval-uation of the inhibitory effect of dimethyl dicarbonate (DMDC) against wine microorganisms. Food Microbiol 25:422427

30. Delfini C, gaia P, Schellino R, Strano M, Pagliara a, ambr S (2002) Fermentability of grape must after inhibition with dime-thyl dicarbonate (DMDC). J agric Food Chem 50:56055611

31. Renouf V, Strehaiano P, lonvaud-Funel a (2008) Effectiveness of dimethyldicarbonate to prevent Brettanomyces bruxellensis growth in wine. Food Control 19:208216

32. aznar M, lpez R, Cacho J, Ferreira V (2003) Prediction of aged red wine aroma properties from aroma chemical composi-tion: partial least squares regression models. J agric Food Chem 51:27002707

33. Ferreira V, Jarauta I, Cacho J (2006) Physicochemical model to interpret the kinetics of aroma extraction during wine aging in wood: Model limitations suggest the necessary existence of bio-chemical processes. J agric Food Chem 54:30473054

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Brettanomyces susceptibility toantimicrobial agents used inwinemaking: invitro andpractical approachesAbstract IntroductionMaterials andmethodsYeast strains andculture conditionsMinimal inhibitory concentration (MIC) andminimal biocidal concentration (MBC)

Wine productionAnalysis ofvolatile phenols (SPE andGC-Ion Trap-MS analysis)Microbiological analysisStatistical analysis

Results andDiscussionPotassium metabisulfite antimicrobial activityAntimicrobial activity ofchitosan, enological tannins andDMDCChitosanEnological tanninsDMDC

Potassium metabisulfite activity againstB. bruxellensis inwines

ConclusionAcknowledgments References

Portugal Et Al_2013

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