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Effectiveness of dimethlydicarbonate to prevent Brettanomyces bruxellensis growth in wine
Food Control 19 (2008) 208–216 www.elsevier.com/locate/foodcont
Effectiveness of dimethlydicarbonate to prevent Brettanomyces bruxellensis growth in wine Vincent Renouf a
a,*
, Pierre Strehaiano b, Aline Lonvaud-Funel
a
UMR Œnologie 1219 Universite´ Bordeaux 2 Victor Se´galen, INRA, ISVV 351, cours de la libe´ration, 33405 Talence Cedex, France b Laboratoire de Ge´nie Chimique, UMR INP, CNRS, 5503, 5, rue Paulin Talabot, BP 1301, 31106 Toulouse Cedex, France Received 1 January 2007; received in revised form 17 March 2007; accepted 23 March 2007
Abstract The aim of this study was to investigate the anti-microbial properties of the dimethyldicarbonate (DMDC) towards the wine spoilage yeast Brettanomyces bruxellensis at different winemaking stages. DMDC anti-microbial activity was estimated in red must for diverse wine microorganisms including different strains of B. bruxellensis. DMDC effect before alcoholic fermentation, before malolactic fermentation, and in finished wine were investigated. DMDC was also tested on lees. Microbial monitoring was done by epifluorescence observation and plate numeration. The identification of yeast species and the specific detection of B. bruxellensis were performed with molecular tools. DMDC stopped B. bruxellensis growth at different winemaking stages. But it could also act on fermenting species like Saccharomyces cerevisiae and Oenococcus oeni. Therefore, its use before the end of fermentations should be avoided. On the other hand, the DMDC action was shown to be transitory. Therefore, single addition during ageing could be insufficient. Finally, DMDC could be used just before bottling as an ultimate anti-microbial tool. 2007 Elsevier Ltd. All rights reserved. Keywords: Brettanomyces; Wine; Stabilization; Dimethyldicarbonate; Volatiles phenols
1. Introduction Brettanomyces bruxellensis (B. bruxellensis) is considered a wine spoilage yeast species due to its ability to produce volatile phenols conferring off-odours and losses of fruity sensorial qualities in wines (Suarez, Surez-Lepe, Morata, & Claderon, 2007). Studies were focused on the development of efficient and sensitive tools of B. bruxellensis species (Ibeas, Lozano, Perdigones, & Jimenez, 1996)
Abbreviations: AAB, acetic acid bacteria; AF, alcoholic fermentation; DMDC, dimethyldicarbonate; LAB, lactic acid bacteria; MIC, Minimal Inhibitory concentration; MLF, malolactic fermentation; NS, non-Saccharomyces (yeast); TPI, total phenolic acids; TY, total yeast * Corresponding author. Present address: UMR EGV 1287 ENITAB, ISVV, 1 cours du ge´ne´ral de Gaulle, CS 40201, 33175 Gradignan Cedex, France. Tel.: +33 666214709. E-mail address: [email protected] (V. Renouf). 0956-7135/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2007.03.012
and strains identification (Miot-Sertier & Lonvaud-Funel, 2007) with also quantitative data by real time molecular analysis (Delaherche, Claisse, & Lonvaud-Funel, 2004). Other considered the possible routes for B. bruxellensis contamination (Renouf & Lonvaud-Funel, 2007). B. bruxellensis is a constant wine resident but it develops mainly at the end of fermentation, when other microbial species decline (Renouf, Gindreau, Claisse, & Lonvaud-Funel, 2005; Renouf, Falcou et al., 2006). It is a strong resistant species standing nutrients deprivation (Uscanga, Delia, & Strehaiano, 2000) and high ethanol degrees (Medawar, Strehaiano, & De´lia, 2003). Traditional practices such as racking (Renouf & Lonvaud-Funel, 2004) and fining (Murat & Dumeau, 2003) are partially on a transitorily efficient to limit B. bruxellensis growth. SO2 is generally used to control microbial spoilage. Its effectiveness depends on the pH and on the phenolic compounds level (Barbe, de Revel, Joyeux, Lonvaud-Funel, &
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Bertrand, 2000). Only molecular SO2 is active against microbial growth (Sudraut & Chauvet, 1985). Licker, Acree, and Henick-Kling (1999) required a minimum molecular SO2 concentration of 0.625 mg/L to have significant impact on B. bruxellensis. Hence, for high pH wines, a free SO2 concentration over than 50 mg/L is required (Renouf, Walling, Coulon, & Lonvaud-Funel, 2006). But high SO2 concentration confers off-odours and also causes allergic responses in some consumer (Stevenson & Simon, 1981). Therefore, the wine industry is seeking ways to minimize SO2 addition. Among the molecules tested, sorbic acid has a significant impact on cell viability (Terrell, Morris, Johnson, Gbur, & Makus, 1993) by disrupting homeostasis pH (Plumridge et al., 2004). But its degradation by certain lactic acid bacteria leads to the formation of 2-ethoxycarbonyl-3,5-hexadiene, which cause disagreeable ‘‘geranium’’ tastes (Crowell & Guymon, 1975). Chitosan (Roller & Covill, 1999) interacts with anionic groups on the yeast cell surface and limits the diffusion of essential solutes, such as sugars (Raltson, Tracey, & Wrench, 1964) and heavy metal cations (copper, cobalt, cadmium, . . .) (Brady, Stoll, Starke, & Duncan, 1994). However its effectiveness is strongly variable according to the pH. Vanillin is used against spoilage yeast in fruit juices and dairy products (Fitzgeral, Stratford, & Narbad, 2003). It inhibits enzymes implicated in cell energy production (Conner, Beuchat, Worthington, & Hitchcock, 1984). It also disrupts membrane functions (Rico-Munoz, Bargiota, & Davidson, 1987). However, some species are less sensitive to vanillin due to their ability to convert it to its alcohol and acid derivatives (De Wulf et al., 1986). It is notably the case for Brettanomyces sp. (Edlin, Narbad, Dickinson, & Lloyd, 1995). Moreover to insure an effective anti-microbial action, the level of vanillin required (30–100 mg/L) is one thousand higher (Cerrutti & Alzamora, 1996) than usual wine concentrations (De Revel, Bloem, Augustin, Lonvaud-Funel, & Bertrand, 2005). Its excessive use would lead to a modification of wine aroma. Nisin is a natural product of the bacteria Lactococcus lactis exhibiting anti-microbial activities towards a wide range of Gram positive bacteria (Ogden & Waites, 1986) by forming pores into the cytoplasmic membrane and allowing the efflux of essential cellular. But some strains present high tolerance due to nisinase activity (Daeschel, Jung, & Watson, 1991). A similar resistance phenomenon may occur for yeast for which the inhibitory effect of nisin is limited (Radler, 1990). Esters of pyrocarbonic acid are used as anti-microbial agent for sterilizing fermented beverages. The diethylpyrocarbonate (DEPC) is very efficient but it is banished due its ability to form ethyl carbamate, which may affect consumer’s safety (Larsen, 1974; Pound & Lawson, 1976). The dimethyldicarbonate (DMDC, Velcorin) is an alternative because its by-products in alcoholic beverages, methanol and methyl carbamate, are less formed (Peterson & Ough, 1979; Stafford & Ough, 1976). DMDC acts by inhibiting some glycolytic enzymes (Temple & Ough,
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1978) notably the alcohol-dehydrogenase and the glyceraldehyde-3-phosphate-dehydrogenase by methoxycarbonylation of the nucleophilic residues (imidazoles, amines, thiols) (Ough, 1983). The other hydroxyl-amino and thiols group of wine constituents may also be affected by the methoxylcarbonylation through reaction with DMDC, but the concentration levels of the resulting derivatives are however very low and DMDC yields no residual odours or flavours (Ough, 1983). In addition, its effects is not directly pH dependent (Threlfall & Morris, 2001). The aim of this work was to evaluate the effectiveness of DMDC towards B. bruxellensis in red wines. First, we evaluated the Minimal Inhibitory Concentration (MIC) of DMDC in red grape juice for several species and different strains of yeast and bacteria commonly found in wine. Then, DMDC was added (i) at several levels in grape juice before alcoholic fermentation (AF) containing mixtures of Saccharomyces cerevisiae, B. bruxellensis and other nonSaccharomyces species, (ii) in wine after AF and before malolactic fermentation (MLF) with mixture of Oenococcus oeni and B. bruxellensis and in post-fermented wines with only B. bruxellensis species (iii). Stabilization of lees containing high B. bruxellensis populations was also tested (iv). 2. Materials and methods 2.1. Strains and culture media Strains with their collection number are listed in Table 1. Yeast were cultivated on YPG plates (yeast extract 10 g/L, bactotryptone 10 g/L, glucose 20 g/L, agar 20 g/L, pH adjusted to 4.8 using orthophosphoric acid). For total yeast (TY) cultures the medium was supplemented with biphenyl (Fluka) (0.015% w/v) and chloramphenicol (Sigma– Aldrich) (0.01% w/v) to prevent mould and bacteria growth respectively. The addition of 0.01% (w/v) cycloheximide (Sigma–Aldrich) eliminated the Saccharomyces sp. and allowed the enumeration of non-Saccharomyces (NS) yeast population. Incubation was carried out at 25 C during 5 days for TY and 10 days for NS. Lactic acid bacteria (LAB) were cultivated on grape juice medium plates (red grape juice 250 mL/L, yeast extract 5 g/L, Tween 80 mL/L, pH adjusted to 4.8 with NaOH 5 N). During co-culture with B. bruxellensis, yeast growth was inhibited by adding 100 mg/L of pimaricine (Delvocid, DSM Food Specialities). The plates were incubated at 25 C for 7 days in anaerobic conditions, using an anaerobic system envelope with palladium catalyst (BBL). To count Acetic Acid Bacteria (AAB) the same medium was used but penicillin (15 mg/L) was added to prevent the Gram positive bacteria growth. AAB plates incubation lasted 5 days at 25 C in aerobic condition. Comparison between cultivable populations and cell viability was made by epifluorescence measurement according to Millet and Lonvaud-Funel (2000).
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Table 1 Minimum inhibitory concentration of DMDC for several oenological yeast and bacteria strains in red grape juice Microorganism
Species
Designationa
Minimum inhibitory concentration (mg/L)
Yeast
Aureobasidium pullulans Brettanomyces bruxellensis Brettanomyces bruxellensis Brettanomyces bruxellensis Brettanomyces bruxellensis Brettanomyces bruxellensis Brettanomyces bruxellensis Brettanomyces bruxellensis Brettanomyces bruxellensis Brettanomyces bruxellensis Brettanomyces bruxellensis Candida cantarelli Candida ethanolica Cryptococcus albus Hanseniaspora uvarum Lipomyces spencermartinsiae Metschnikowia fructicola Pichia anomala Pichia anomala Rhodotorula mucilaginosa Saccharomyces cerevisiae Saccharomyces cerevisiae Saccharomyces cerevisiaee Zygosaccharomyces bailii
IOEBL0448 IOEBL0308 IOEBL0407 IOEBL0411 IOEBL0447 IOEBL0453 IOEBL0463 IOEBL0465 IOEBL0512 IOEBL0542 CLIB300 IOEBL0404 IOEBL0504 CLIB373 IOEBL0401 IOEBL0450 IOEBL0530 IOEBL0426 CLIB284 CLIB370 IOEBL0434 IOEBL0437 IOEBL0440 IOEBL0310
50 150 150 150 150 150 150 150 150 150 150 50 100 250 50 50 250 400 400 400 200 150 100 250
Bacteria
Lactobacillus brevis Lactobacillus collinoides Lactobacillus hilgardii Lactobacillus plantarum Leuconostoc mesenteroides Oenococcus oeni Oenococcus oeni Oenococcus oeni Pediococcus damnosus Pediococcus dextrinicus Pediococcus parvulus Pediococcus pentosaceus
IOEBL0019 IOEBL0203 IOEBL0021 IOEBL0401 ATCC8293 ATCC23277 ATCC23279 IOEBL0025 ATCC25248 ATCC33087 ATCC19371 ATCC33316
600 600 250 250 100 150 150 250 250 250 400 400
a
IOEBL: Levures de l’Institut d’Oenologie de Bordeaux, Talence, France; IOEBB: Bacte´ries de l’Institut d’Oenologie de Bordeaux, Talence, France; CLIB: Collection de Levures d’Inte´reˆt Biotechnologique INRA, Thiverval Grignon, France, ATCC: American Type Culture Collection, Manassas, USA.
2.2. Yeast species identification The yeast species identification was performed by molecular tools supporting RFLP analysis of the 5.8S rRNA gene and the two ribosomal internal transcribed spacers (ITS1 and ITS2) using CfoI, HaeIII and HinfI endonucleases (Esteve-Zarzoso, Belloch, Uruburu, & Querol, 1999). Twenty colonies on plates carrying between 30 and 300 colonies were tested. This method allowed for the estimation of the percentage of each species (Clemente-Jimenez, Mingorance-Cazorla, Martinez-Rodriguez, Las-Heras Vazquez, & Rodriguez-Vico, 2004). To detect specifically B. bruxellensis, the species-specific PCR developed by Ibeas et al. (1996) was used. 2.3. Chemical analyses Conventional analyses: pH, alcohol content, free and total SO2 and total polyphenol index (TPI) were carried
out by the usual methods recommended by the international organization of the Vine and Wine (OIV). L-malic acid, glucose and fructose concentrations were measured by enzymatic methods (Biopharm). Volatile phenols were measured after dichloromethane extraction (Rodriguez, Gonc¸alves, Pereira-da-silva, Malfeito-Ferreira, & Loureiro, 2001) followed by gas chromatography (Chatonnet & Boidron, 1988). 2.4. Drug sensitivity assays The DMDC sensitivity was assayed by measuring the minimal inhibitory concentration (MIC) in a red grape juice liquid medium (pH = 4.0, glucose + fructose 180 g/ L, L-malic acid 2 g/L, TPI = 37) containing different drug concentrations comprised between 10 g/L and 10 mg/L (10, 5, 2.5, 1.25, 0.6, 0.4, 0.25, 0.2, 0.15, 0.100, 0.050, 0.025, 0.010 g/L). Viable yeast and bacteria were inoculated at the initial concentration of 103 cells/mL. The incu-
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bation was made in spectrophotometer tank previously sterilized by UV irradiation. In all cases, DMDC resistance was scored by measuring the optical density at 600 nm after 6 days of growth at 25 C and by comparison with a control without DMDC for each strain. For each concentration, assays were made on triplicate. 2.5. DMDC experiments All assays were carried out in triplicate. The wine storage temperature was 25 C. The flasks containing grape juice or wine sample were closed with healthy and sterilized caps. Each sampling was carried out in sterile conditions. Controls without microorganisms were also performed. 2.5.1. Influence of DMDC on grape must fermentation Grape juice was sterilized by filtration (0.22 lm). The sugar concentration (glucose + fructose) was standardized to 190 g/L. The pH was adjusted to 3.7. 200 mL of grape juice were inoculated with different single species and yeast mixtures S. cerevisiae (IOEBL0434), B. bruxellensis, (IOEBL0447), Candida cantarelli (IOEBL0404) and Hanseniaspora uvarum (IOEBL0401) at the initial concentration of 103 CFU/mL. Assays were conducted with different concentrations of DMDC (0, 75, 150, 250, 500 and 1000 mg/ L). Cell viability was estimated by epifluorescence measurement just after yeast inoculation, homogenisation before DMDC addition and 24 hours after DMDC addition. Volumes analysed by epilfluorescence were comprised between 0.1 mL and 10 mL and are chosen according to the viable cells population. The threshold of this technique was 102 ¢/mL. The AF duration was determined by glucose and fructose concentrations measurements. The yeast population was counted on TY and NS plates, and percentage of each survival species calculated after AF completion, and after 50 days of incubation, when it was aborted. In another experiment, the durability of the DMDC effect was tested by yeast re-inoculation few days after the DMDC addition. 2.5.2. Influence of DMDC on MLF Post-alcoholic wine [(12.2% v/v of ethanol, 2.02 g/L of L-malic acid, a pH of 3.55, content of 4 mg/L (H2SO4) of free SO2, and 16 mg/L (H2SO4) of total SO2, sterilized by filtration (0.22 lm))] was used to conduct the MLF with a co-culture of O. oeni (IOEBB0025) and B. bruxellensis (IOEBL0447) with different concentrations of DMDC. (0, 75, 150, 250, 500 and 1000 mg/L). DMDC was added three days after the microbial inoculation to be certain that the anti-microbial effect was the DMDC impact and not a failure of the inoculation. The O. oeni and B. bruxellensis were respectively inoculated at the level of 106 CFU/mL and 103 CFU/mL, which corresponds at the level commonly counted in wine inoculated with O. oeni commercial starter. Yeast and bacteria were counted on plates and the malic acid consumption rate was measured by enzymatic method (biopharm).
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2.5.3. Stabilization of finished wines by DMDC Post-fermentations wine, called finished wine, was filtered and inoculated with B. bruxellensis (IOEBL0447) at the initial concentration of 103 CFU/mL. After three days of incubation, 200 mg/L of DMDC were added. The B. bruxellensis population was monitored by plates counting in TY medium, with epifluorescence and by the speciesspecific PCR. Volatile phenols were measured. At the beginning of the assay the wine parameters were an ethanol content of 12.4% v/v, a pH of 3.58, a free SO2 content of 18 mg/L (H2SO4), and a total SO2 content of 48 mg/L (H2SO4). 2.5.4. Stabilization of lees with high level of microorganisms We followed lees wine containing a high and diverse intrinsic microflora after DMDC addition (200 mg/L). Microbial stabilization was estimated by the TY, NS, LAB and AAB population enumeration after DMDC addition (7 and 30 days after treatment). The lees chemical characteristics were an ethanol content of 13.6% v/v, a pH of 3.98, a free SO2 content of 14 mg/L (H2SO4), and a total SO2 content of 52 mg/L (H2SO4). The proportion of S. cerevisiae and B. bruxellensis among the total yeast species was determined by the Esteve-Zarzoso et al. (1999) identification protocol performed on colonies sampling using TY plates. 3. Results 3.1. DMDC effects during AF Results reported in Table 1 prove the DMDC efficacy against diverse oenological yeasts and bacterial species. A concentration of 600 mg/L of DMDC prevented the growth of all of species test but the most sensitive yeast species were inhibited starting 50 mg/L. Concerning yeasts, Pichia anomala and Rhodotorula mucilaginosa were the more resistant species. In general, bacteria appeared more resistant than yeast. Lactobacillus brevis and Lactobacillus collinoides were the more resistant bacterial species. For some of them, such as S. cerevisiae and O. oeni, the DMDC resistance was strains dependent. It was not the case for B. bruxellensis strains, which were all inhibited at 150 mg/L. Compared with the other species, B. bruxellensis strains appeared moderately resistant to DMDC in grape juice. The objective of the second experiment was to evaluate the effectiveness of DMDC against B. bruxellensis at the beginning of winemaking during the AF. We chose a resistant strain of S. cerevisiae (IOEBL0434) to perform co-cultures with a B. bruxellensis strain (IOEBL0411) and other species commonly detected in fresh must (H. uvarum IOEBL0401 and C. cantarelli IOEBL0404). For all assays, AF was aborted with 500 mg/L of DMDC. When B. bruxellensis was alone a concentration of 150 mg/L of DMDC was sufficient. A higher concentration was needed for S. cerevisiae single culture (250 mg/L). In all cases, the inhibitory concentration of DMDC was higher in a
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co-culture with single cultures. In the non-Saccharomyces mixture the DMDC inhibitory concentration was 250 mg/L. Moreover in the mixture, B. bruxellensis was the only species detected at the end. A similar phenomenon was observed for S. cerevisiae. 250 mg/L were sufficient to prevent S. cerevisiae growth in single culture. But when S. cerevisiae was inoculated with other yeast species, AF was delayed but not aborted at this DMDC concentration. The durability of the DMDC effect was tested in experiments shown in Fig. 1. AF was started by inoculating B. bruxellensis and S. cerevisiae. Quickly the S. cerevisiae population reached 108 CFU/mL and began the fermenta-
tion. When 250 mg/L of DMDC were added in the middle of the AF, AF was stopped. Consequently, cultivable population decreased below 1 CFU/mL. However the DMDC effect was not durable because when yeasts were re-introduced, a new growth phase started. Microbial growth as well as the fermentative activity did not seem be affected by the previous DMDC addition. S. cerevisiae was able to reach again to 107–108 CFU/mL and to ferment sugars were fermented. Fermentation kinetic of the second fermentation was similar to the first one, before DMDC addition. When S. cerevisiae and B. bruxellensis were both reinoculated, they both could grow, but S. cerevisiae growth
Fig. 1. AF evolution after DMDC addition. AF was started by inoculating a mixture of S. cerevisiae (IOEBL0434), and B. bruxellensis (IOEBL0447). After DMDC addition, wines were re-inoculated with the same yeasts at different concentrations and ratios at day 10 (: TY population, d: NS population and m: sugars concentrations).
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overcame B. bruxellensis growth in all cases. At the end of fermentation, B. bruxellensis populations were always inferior in the re-inoculation experiments than in the control ones using B. bruxellensis alone. 3.2. DMDC effect during MLF For the MLF assays (Fig. 2), 500 mg/L and 1000 mg/L of DMDC addition were able to stop MLF. For the two lowest DMDC concentrations (75 mg/L and 150 mg/L) the MLF kinetic was not affected. An intermediary DMDC concentration (250 mg/L) allowed MLF to start but stopped after 10 days. In this case, as shown in Fig. 3, B. bruxellensis population decreased just after inoculation, then grew back up to 4.1 · 103 CFU/mL when MLF began. The population immediately decreased after addition of DMDC, but was not completely eliminated. Finally, after equilibrium around 6 · 101 CFU/mL B. bruxellensis population increased until 103 CFU/mL. Concerning O. oeni, the initial 106 CFU/mL population grew up to 107 CFU/mL degrading actively malic acid. But as soon as DMDC was added (day 3) it decreased. At the day 10, it had dropped to less than 105 CFU/mL, simultaneously MLF stopped. 3.3. DMDC effects in finished wine In finished wine, results (Table 2) showed the DMDC activity to reduce B. bruxellensis population. After DMDC addition the B. bruxellensis population declined to less than 1 CFU/10 mL. At day 10, the volatile phenols production had not started in any wines tested. But after six months, the untreated wine contained higher concentrations of 4ethylphenol and 4-ethylguaiacol than the treated wine where no residual B. bruxellensis was detected. This result was always confirmed by negative PCR specific amplifica-
Fig. 3. Evolution of O. oeni and B. bruxellensis during assay with 250 mg/ L of DMDC added at the 3rd day of monitoring 5 (: O. oeni population and d: B. bruxellensis population).
tions. Non-contamination occurred, as shown by the non-inoculated control wines. 3.4. DMDC effects in lees DMDC in lees does not completely eliminate the populations by the DMDC treatment (Table 3). The total yeast population in the treated lot was lower one week after addition of DMDC (2.1 · 102 CFU/mL) than in the control (1.7 · 106 CFU/mL). B. bruxellensis percentage was lower than S. cerevisiae. After one month the results show that, despite the DMDC, yeasts could still actively grow. Their population has increased of more than 3 logs to nearly 106 CFU/mL in treated lees where B. bruxellensis and S. cerevisiae were in the same proportion. However, B. bruxellensis was dominant in the control suggesting a relative best resistance of indigenous Saccharomyces yeast. Concerning bacteria, the decrease of AAB could not be attributed to DMDC since has also decreased in non-treated lees. The LAB population was stable around 104 CFU/ mL in control but decreased to 102 CFU/mL in treated lees. These results shown that not all microorganisms could be destroyed by DMDC, presumably due to the high populations. Residual viable cells were able to grow again once the initial DMDC had reacted in the system. 4. Discussion
Fig. 2. DMDC impact on L-malic acid consumption during a O. oeni (IOEBB0025) and B. bruxellensis (IOEBL0447) co-culture in wine (: O. oeni, h: B. bruxellensis, : O. oeni and B. bruxellensis + DMDC = 0 mg/ L, j: O. oeni and B. bruxellensis + DMDC = 50 mg/L, d: O. oeni and B. bruxellensis + DMDC = 100 mg/L, : O. oeni and B. bruxellensis + DMDC = 250 mg/L, m: O. oeni and B. bruxellensis + DMDC = 500 mg/L, s: O. oeni and B. bruxellensis + DMDC = 1000 mg/L).
Previous investigations had demonstrated the high tolerance of B. bruxellensis against nutrient privation (Uscanga et al., 2000), ethanol (Medawar et al., 2003), initial cold maceration (Renouf, Perello, Strehaiano, & LonvaudFunel, 2006), thermal treatment (Couto, Neves, Campos, & Hoog, 2005) and winery disinfection (Renouf, Claisse et al., 2006). Therefore, B. bruxellensis can be considered as one of the best adapted species to cellar and wine environment. Tools for limiting its growth are limited. Thus, we assessed the DMDC addition as new treatment to prevent B. bruxellensis growth during and after fermentation.
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Table 2 B. bruxellensis and ethylphenol evolution in wines Control
Wine + B. bruxellensis without DMDC
Wine + B. bruxellensis + 200 mg/L of DMDC
3 days just before DMDC B. bruxellensis (log10 CFU/mL) 4-Ethylphenol (lg/L) 4-Ethylguaiacol (lg/L)
<1 208 ± 21 24 ± 4
1.4 ± 0.4 199 ± 11 27 ± 3
1.4 ± 0.7 196 ± 12 20 ± 6
7 days after DMDC B. bruxellensis (log10 CFU/mL) 4-Ethylphenol (lg/L) 4-Ethylguaiacol (lg/L)
<1 198 ± 11 19 ± 5
4.4 ± 1.7 238 ± 12 26 ± 2
<1 194 ± 24 21 ± 4
180 days B. bruxellensis (log10 CFU/mL) 4-Ethylphenol (lg/L) 4-Ethylguaiacol (lg/L)
<1 220 ± 16 24 ± 7
7.4 ± 1.9 940 ± 43 98 ± 32
<1 206 ± 34 20 ± 12
Effect of DMDC on finished wines inoculated with B. bruxellensis (IOEBL0447) and DMDC treated.
Table 3 Effect of DMDC on lees containing residual indigenous yeast and bacteria Lees (control) Before DMDC addition TY (log10 CFU/mL) NS (log10 CFU/mL) LAB (log10 CFU/mL) AAB (log10 CFU/mL) S. cerevisiae (%) B. bruxellensis (%)
Lees + DMDC (200 mg/L) 3.7 ± 1.4 1.2 ± 0.1 9.5 ± 0.1 6.3 ± 2.1 65% 25%
One week after TY (log10 CFU/mL) NS (log10 CFU/mL) LAB (log10 CFU/mL) AAB (log10 CFU/mL) S. cerevisiae (%) B. bruxellensis (%)
1.7 ± 1.1 1.2 ± 0.3 2.5 ± 0.9 8.3 ± 2.9 30% 65%
2.1 ± 1.0 1.0 ± 0.2 1.2 ± 0.9 6.4 ± 1.9 50% 40%
One month after TY (log10 CFU/mL) NS (log10 CFU/mL) LAB (log10 CFU/mL) AAB (log10 CFU/mL) S. cerevisiae (%) B. bruxellensis (%)
2.5 ± 1.1 2.3 ± 0.3 2.1 ± 0.3 1.2 ± 0.3 10% 90%
9.8 ± 0.2 4.9 ± 1.3 1.5 ± 0.5 1.0 ± 0.1 50% 50%
TY: total yeast, NS: non-Saccharomyces (yeast), LAB: lactic acid bacteria, AAB: acetic acid bacteria.
DMDC effect is not restricted to B. bruxellensis. Compared to species composing the grape surface microflora (Aureobasidium pullulans, Bulleromyces albus, R. mucilaginosa, and Cryptococcus albus) as well as the species known to intervene during the first stage of winemaking (P. anomala, Candida stellata, Metschnikowia fructicola, . . .), B. bruxellensis exhibited an intermediary DMDC resistance. During AF, non-Saccharomyces yeast (including B. bruxellensis), as well as S. cerevisiae were affected by DMDC. But the DMDC effect was transitory. Temple and Ough (1978) estimated that in an 12% v/v of ethanol alcoholic solution and for a pH range comprised between 2.0 and 6.0, four hours were enough for DMDC hydrolysis into carbon dioxide and methanol. Ough (1983) and Van der Riet, Botha, and Pinches (1989) reported variations of
the hydrolysis kinetic according to the temperature, the pH and the wine alcohol content. A maximum inhibitory effect occurs soon after the addition. Therefore DMDC should not be used as preventive agent but only as curative agent against unwanted populations already present in wine. This transitory effect could also be considered as an advantage. Hence, after a DMDC addition, it is possible to re-inoculate the wine with a commercial strain to achieve the fermentation. This could be used to prevent B. bruxellensis growth when it is growing simultaneously with indigenous S. cerevisiae strains at the beginning of the fermentation. After AF, the MLF period is crucial for B. bruxellensis development in wine (Renouf et al., 2005). But O. oeni is also affected and MLF could be delayed or totally prevented by DMDC addition. In other cases, such as in the lees, microbial populations were too high to be totally removed by DMDC additions within the tested concentrations. Daudt and Ough (1980) established a semi-log correlation between the initial viable yeast population and the amount of DMDC required. When the initial viable cells increased tenfold, an additional amount of 5 mg/L of DMDC was approximately required. For sweet wines, DMDC is often tested combined to of sulphur dioxide (Threlfall & Morris, 2001, 2002). In this case; DMDC can also help in lowering the amount of sulphur dioxide needed. Oher studies (Divol, Strehaiano, & Lonvaud-Funel, 2005; Ough, Kunkee, Vilas, Bordeu, & Huand, 1988; Threlfall & Morris, 2002) established a synergistic activity between DMDC and sulphur dioxide. From these cases, it was reported that 50 mg/L of DMDC with 25 mg/ L of free SO2 is sufficient to provide an efficient microbial control. However DMDC cannot replace SO2, which is necessary as an anti-oxidant in must and during ageing. Therefore a synergistic use of SO2 and DMDC can be proposed. SO2 should be added first in grape must, then at the end of the fermentation and all along the ageing period. Thus, DMDC could then be used when an accidental B. bruxellensis multiplication is observed. This would provide the winemaker with an efficient microbial control system. DMDC can also be used during the bottling as a final microbial pre-
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servative. It could be added just prior the filler bowl, in the bottling sequence, thus guarantying sterility. In this case, the amount of DMDC to add should be estimated after a microbiological analysis of the residual microflora. Acknowledgments We thank Marie-Claire Perello for her technical assistance and Pr. Gilles de Revel to allow the measurements of the volatile phenols concentrations in his laboratory. References Barbe, J. C., de Revel, G., Joyeux, A., Lonvaud-Funel, A., & Bertrand, A. (2000). Role of carbonyl compounds in binding phenomena in musts and wines from botrytised grapes. Journal of Agricultural and Food Chemistry, 48, 3413–3419. Brady, D., Stoll, A. D., Starke, L., & Duncan, J. R. (1994). Chemical and enzymatic extraction of heavy metal binding polymers from isolated cell walls of Saccharomyces cerevisiae. Biotechnology and Bioengineering, 44, 297–302. Cerrutti, P., & Alzamora, S. M. (1996). Inhibitory effects of vanillin on some food spoilage yeasts in laboratory media and fruit pure´es. International Journal of Food Microbiology, 29, 379–386. Chatonnet, P., & Boidron, P. (1988). Dosage de phe´nols volatils dans les vins par chromatographie en phase gazeuse. Sciences des Aliments, 8, 479–488. Clemente-Jimenez, J. M., Mingorance-Cazorla, L., Martinez-Rodriguez, S., Las-Heras Vazquez, F. J., & Rodriguez-Vico, F. (2004). Molecular characterization and oenological properties of wine yeasts isolated during spontaneous fermentation of six varieties of grape must. Food Microbiology, 21, 376–379. Conner, D. E., Beuchat, L. R., Worthington, R. E., & Hitchcock, H. L. (1984). Effects of essential oils and oleorestins of plants on ethanol production, respiration and sporulation of yeasts. International Journal of Food Microbiology, 1, 63–74. Couto, J. A., Neves, F., Campos, F., & Hoog, T. (2005). Thermal inactivation of the wine spoilage yeasts Dekkera/Brettanomyces. International Journal of Food Microbiology, 104, 337–344. Crowell, E. A., & Guymon, M. F. (1975). Wine constituents arising from sorbic acid addition and identification of 2-ethoxyhexa-3,5-diene as a source of geranium-like off odour. American Journal of Enology and Viticulture, 26, 97–102. Daeschel, M. A., Jung, D. S., & Watson, B. T. (1991). Controlling wine malolactic fermentation with nisin and nisin resistant strains of Leuconostoc oenos. Applied Environmental Microbiology, 57, 601–603. Daudt, C. E., & Ough, C. S. (1980). Action of dimethyldicarbonate on various yeasts. American Journal of Enology and Viticulture, 31, 21–23. Delaherche, A., Claisse, O., & Lonvaud-Funel, A. (2004). Detection and quantification of Brettanomyces bruxellensis and ‘rpoy’ Pediococcus damnosus’ strains in wine by real time polymerase chain reaction. Journal of Applied Microbiology, 97, 910–915. De Revel, G., Bloem, A., Augustin, M., Lonvaud-Funel, A., & Bertrand, A. (2005). Interaction of Oenococcus oeni and oak wood compounds. Food Microbiology, 22, 569–575. De Wulf, O., Thomart, P., Gaignage, P., Marlier, M., Paris, A., & Paquot, M. (1986). Bioconversion of vanillin to vanillin alcohol by Saccharomyces cerevisiae. Biotechnology and Bioengineering Symposium, 7, 605–616. Divol, B., Strehaiano, P., & Lonvaud-Funel, A. (2005). Effectiveness of dimethylcarbonate to stop alcoholic fermentation. Food Microbiology, 22, 169–178. Edlin, D. A. N., Narbad, A., Dickinson, R. J., & Lloyd, D. (1995). The biotransformation of simple phenolic compounds of Brettanomyces anomalus. FEMS Microbiology Letters, 125, 311–316.
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Renouf, V., Gindreau, E., Claisse, O., & Lonvaud-Funel, A. (2005). Microbial changes during malolactic fermentation in red wine elaboration. Journal International des Sciences de la Vigne et du Vin, 39, 179–190. Renouf, V., & Lonvaud-Funel, A. (2004). Racking are key stages for the microbial stabilization of wines. Journal International des Sciences de la Vigne et du Vin, 38, 219–230. Renouf, V., Perello, M. C., Strehaiano, P., & Lonvaud-Funel, A. (2006). Global survey of the microbial ecosystem during alcoholic fermentation in winemaking. Journal International des Sciences de la Vigne et du Vin, 40, 101–116. Renouf, V., Walling, E., Coulon, J., & Lonvaud-Funel, A. (2006). Conseils pratiques pour la mise en place d’un suivi microbiologique. Revue des Œnologues, 119, 39–42. Rico-Munoz, E., Bargiota, E. E., & Davidson, P. M. (1987). Effect of selected phenolic compounds on the membrane-bound adenosine triphosphatase of Staphylococcus aureus. Food Microbiology, 4, 239–249. Rodriguez, N., Gonc¸alves, G., Pereira-da-silva, S., Malfeito-Ferreira, M., & Loureiro, V. (2001). Development and use of a new medium to detect yeasts of the genera Dekkera/Bruxellensis sp.. Journal of Applied Microbiology, 90, 588–599. Roller, S., & Covill, N. (1999). The antifungal properties of chitosan in laboratory media and apple juice. International Journal of Food Microbiology, 47, 67–77. Stafford, P. A., & Ough, C. S. (1976). Formation of methanol and ethymethylcarbonate by dimethyldicarbonate in wine and model solutions. American Journal of Enology and Viticulture, 27, 7–11. Stevenson, D. D., & Simon, R. A. (1981). Sensitivity to ingested metabisulfites in asthmatic subjects. Journal of Allergy and Clinical Immunology, 68, 26–32.
Suarez, R., Surez-Lepe, J. A., Morata, A., & Claderon, F. (2007). The production of ethylphenols in wine by teasts of the genera Brettanomyces and Dekkera. A review. Food Chemistry, 102, 10–21. Sudraut, P., & Chauvet, S. (1985). Activite´ antilevure de l’anhydride sulfureux mole´culaire. Journal International des Sciences de la Vigne et du Vin, 19, 31–40. Temple, D. D., & Ough, C. S. (1978). The inactivation of yeast alcohol dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase by dimethyldicarbonate. Excerpts from M.S. Thesis, D. Temple. University of California, Davis, USA. Terrell, F. R., Morris, J. R., Johnson, M. G., Gbur, E. E., & Makus, D. J. (1993). Yeast inhibition in grape juice containing sulfur dioxide, sorbic acid and dimethlydicarbonate. Journal of Food Science, 58, 1132–1134. Threlfall, R. T., & Morris, J. R. (2001). Effectiveness of sulfur dioxide and dimethlydicarbonate on Saccharomyces bayanus growth at different pH levels in a juice and semi-sweet wine. In ASEV 52nd Annual Meeting, San Diego, CA, USA. Threlfall, R. T., & Morris, J. R. (2002). Using dimethyldicarbonate to minimize sulfur dioxide for prevention of fermentation from excessive yeast contamination on juice and semi-sweet wine. Journal of Food Science, 67, 2752–2758. Uscanga, M. G., Delia, M. L., & Strehaiano, P. (2000). Nutritional requirements of Brettanomyces bruxellensis: Growth and physiology in batch and chemostat cultures. Canadian Journal of Microbiology, 46, 1046–1050. Van der Riet, W. B., Botha, A., & Pinches, S. E. (1989). The effect of dimethylcarbonate on vegetative growth and ascopores of Byssichlamys fulva suspended in apple juice and strawberry nectar. International Journal of Food Microbiology, 8, 95–102.
Effectiveness of dimethlydicarbonate to prevent Brettanomyces bruxellensis growth in wine Vincent Renouf a
a,*
, Pierre Strehaiano b, Aline Lonvaud-Funel
a
UMR Œnologie 1219 Universite´ Bordeaux 2 Victor Se´galen, INRA, ISVV 351, cours de la libe´ration, 33405 Talence Cedex, France b Laboratoire de Ge´nie Chimique, UMR INP, CNRS, 5503, 5, rue Paulin Talabot, BP 1301, 31106 Toulouse Cedex, France Received 1 January 2007; received in revised form 17 March 2007; accepted 23 March 2007
Abstract The aim of this study was to investigate the anti-microbial properties of the dimethyldicarbonate (DMDC) towards the wine spoilage yeast Brettanomyces bruxellensis at different winemaking stages. DMDC anti-microbial activity was estimated in red must for diverse wine microorganisms including different strains of B. bruxellensis. DMDC effect before alcoholic fermentation, before malolactic fermentation, and in finished wine were investigated. DMDC was also tested on lees. Microbial monitoring was done by epifluorescence observation and plate numeration. The identification of yeast species and the specific detection of B. bruxellensis were performed with molecular tools. DMDC stopped B. bruxellensis growth at different winemaking stages. But it could also act on fermenting species like Saccharomyces cerevisiae and Oenococcus oeni. Therefore, its use before the end of fermentations should be avoided. On the other hand, the DMDC action was shown to be transitory. Therefore, single addition during ageing could be insufficient. Finally, DMDC could be used just before bottling as an ultimate anti-microbial tool. 2007 Elsevier Ltd. All rights reserved. Keywords: Brettanomyces; Wine; Stabilization; Dimethyldicarbonate; Volatiles phenols
1. Introduction Brettanomyces bruxellensis (B. bruxellensis) is considered a wine spoilage yeast species due to its ability to produce volatile phenols conferring off-odours and losses of fruity sensorial qualities in wines (Suarez, Surez-Lepe, Morata, & Claderon, 2007). Studies were focused on the development of efficient and sensitive tools of B. bruxellensis species (Ibeas, Lozano, Perdigones, & Jimenez, 1996)
Abbreviations: AAB, acetic acid bacteria; AF, alcoholic fermentation; DMDC, dimethyldicarbonate; LAB, lactic acid bacteria; MIC, Minimal Inhibitory concentration; MLF, malolactic fermentation; NS, non-Saccharomyces (yeast); TPI, total phenolic acids; TY, total yeast * Corresponding author. Present address: UMR EGV 1287 ENITAB, ISVV, 1 cours du ge´ne´ral de Gaulle, CS 40201, 33175 Gradignan Cedex, France. Tel.: +33 666214709. E-mail address: [email protected] (V. Renouf). 0956-7135/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2007.03.012
and strains identification (Miot-Sertier & Lonvaud-Funel, 2007) with also quantitative data by real time molecular analysis (Delaherche, Claisse, & Lonvaud-Funel, 2004). Other considered the possible routes for B. bruxellensis contamination (Renouf & Lonvaud-Funel, 2007). B. bruxellensis is a constant wine resident but it develops mainly at the end of fermentation, when other microbial species decline (Renouf, Gindreau, Claisse, & Lonvaud-Funel, 2005; Renouf, Falcou et al., 2006). It is a strong resistant species standing nutrients deprivation (Uscanga, Delia, & Strehaiano, 2000) and high ethanol degrees (Medawar, Strehaiano, & De´lia, 2003). Traditional practices such as racking (Renouf & Lonvaud-Funel, 2004) and fining (Murat & Dumeau, 2003) are partially on a transitorily efficient to limit B. bruxellensis growth. SO2 is generally used to control microbial spoilage. Its effectiveness depends on the pH and on the phenolic compounds level (Barbe, de Revel, Joyeux, Lonvaud-Funel, &
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Bertrand, 2000). Only molecular SO2 is active against microbial growth (Sudraut & Chauvet, 1985). Licker, Acree, and Henick-Kling (1999) required a minimum molecular SO2 concentration of 0.625 mg/L to have significant impact on B. bruxellensis. Hence, for high pH wines, a free SO2 concentration over than 50 mg/L is required (Renouf, Walling, Coulon, & Lonvaud-Funel, 2006). But high SO2 concentration confers off-odours and also causes allergic responses in some consumer (Stevenson & Simon, 1981). Therefore, the wine industry is seeking ways to minimize SO2 addition. Among the molecules tested, sorbic acid has a significant impact on cell viability (Terrell, Morris, Johnson, Gbur, & Makus, 1993) by disrupting homeostasis pH (Plumridge et al., 2004). But its degradation by certain lactic acid bacteria leads to the formation of 2-ethoxycarbonyl-3,5-hexadiene, which cause disagreeable ‘‘geranium’’ tastes (Crowell & Guymon, 1975). Chitosan (Roller & Covill, 1999) interacts with anionic groups on the yeast cell surface and limits the diffusion of essential solutes, such as sugars (Raltson, Tracey, & Wrench, 1964) and heavy metal cations (copper, cobalt, cadmium, . . .) (Brady, Stoll, Starke, & Duncan, 1994). However its effectiveness is strongly variable according to the pH. Vanillin is used against spoilage yeast in fruit juices and dairy products (Fitzgeral, Stratford, & Narbad, 2003). It inhibits enzymes implicated in cell energy production (Conner, Beuchat, Worthington, & Hitchcock, 1984). It also disrupts membrane functions (Rico-Munoz, Bargiota, & Davidson, 1987). However, some species are less sensitive to vanillin due to their ability to convert it to its alcohol and acid derivatives (De Wulf et al., 1986). It is notably the case for Brettanomyces sp. (Edlin, Narbad, Dickinson, & Lloyd, 1995). Moreover to insure an effective anti-microbial action, the level of vanillin required (30–100 mg/L) is one thousand higher (Cerrutti & Alzamora, 1996) than usual wine concentrations (De Revel, Bloem, Augustin, Lonvaud-Funel, & Bertrand, 2005). Its excessive use would lead to a modification of wine aroma. Nisin is a natural product of the bacteria Lactococcus lactis exhibiting anti-microbial activities towards a wide range of Gram positive bacteria (Ogden & Waites, 1986) by forming pores into the cytoplasmic membrane and allowing the efflux of essential cellular. But some strains present high tolerance due to nisinase activity (Daeschel, Jung, & Watson, 1991). A similar resistance phenomenon may occur for yeast for which the inhibitory effect of nisin is limited (Radler, 1990). Esters of pyrocarbonic acid are used as anti-microbial agent for sterilizing fermented beverages. The diethylpyrocarbonate (DEPC) is very efficient but it is banished due its ability to form ethyl carbamate, which may affect consumer’s safety (Larsen, 1974; Pound & Lawson, 1976). The dimethyldicarbonate (DMDC, Velcorin) is an alternative because its by-products in alcoholic beverages, methanol and methyl carbamate, are less formed (Peterson & Ough, 1979; Stafford & Ough, 1976). DMDC acts by inhibiting some glycolytic enzymes (Temple & Ough,
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1978) notably the alcohol-dehydrogenase and the glyceraldehyde-3-phosphate-dehydrogenase by methoxycarbonylation of the nucleophilic residues (imidazoles, amines, thiols) (Ough, 1983). The other hydroxyl-amino and thiols group of wine constituents may also be affected by the methoxylcarbonylation through reaction with DMDC, but the concentration levels of the resulting derivatives are however very low and DMDC yields no residual odours or flavours (Ough, 1983). In addition, its effects is not directly pH dependent (Threlfall & Morris, 2001). The aim of this work was to evaluate the effectiveness of DMDC towards B. bruxellensis in red wines. First, we evaluated the Minimal Inhibitory Concentration (MIC) of DMDC in red grape juice for several species and different strains of yeast and bacteria commonly found in wine. Then, DMDC was added (i) at several levels in grape juice before alcoholic fermentation (AF) containing mixtures of Saccharomyces cerevisiae, B. bruxellensis and other nonSaccharomyces species, (ii) in wine after AF and before malolactic fermentation (MLF) with mixture of Oenococcus oeni and B. bruxellensis and in post-fermented wines with only B. bruxellensis species (iii). Stabilization of lees containing high B. bruxellensis populations was also tested (iv). 2. Materials and methods 2.1. Strains and culture media Strains with their collection number are listed in Table 1. Yeast were cultivated on YPG plates (yeast extract 10 g/L, bactotryptone 10 g/L, glucose 20 g/L, agar 20 g/L, pH adjusted to 4.8 using orthophosphoric acid). For total yeast (TY) cultures the medium was supplemented with biphenyl (Fluka) (0.015% w/v) and chloramphenicol (Sigma– Aldrich) (0.01% w/v) to prevent mould and bacteria growth respectively. The addition of 0.01% (w/v) cycloheximide (Sigma–Aldrich) eliminated the Saccharomyces sp. and allowed the enumeration of non-Saccharomyces (NS) yeast population. Incubation was carried out at 25 C during 5 days for TY and 10 days for NS. Lactic acid bacteria (LAB) were cultivated on grape juice medium plates (red grape juice 250 mL/L, yeast extract 5 g/L, Tween 80 mL/L, pH adjusted to 4.8 with NaOH 5 N). During co-culture with B. bruxellensis, yeast growth was inhibited by adding 100 mg/L of pimaricine (Delvocid, DSM Food Specialities). The plates were incubated at 25 C for 7 days in anaerobic conditions, using an anaerobic system envelope with palladium catalyst (BBL). To count Acetic Acid Bacteria (AAB) the same medium was used but penicillin (15 mg/L) was added to prevent the Gram positive bacteria growth. AAB plates incubation lasted 5 days at 25 C in aerobic condition. Comparison between cultivable populations and cell viability was made by epifluorescence measurement according to Millet and Lonvaud-Funel (2000).
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Table 1 Minimum inhibitory concentration of DMDC for several oenological yeast and bacteria strains in red grape juice Microorganism
Species
Designationa
Minimum inhibitory concentration (mg/L)
Yeast
Aureobasidium pullulans Brettanomyces bruxellensis Brettanomyces bruxellensis Brettanomyces bruxellensis Brettanomyces bruxellensis Brettanomyces bruxellensis Brettanomyces bruxellensis Brettanomyces bruxellensis Brettanomyces bruxellensis Brettanomyces bruxellensis Brettanomyces bruxellensis Candida cantarelli Candida ethanolica Cryptococcus albus Hanseniaspora uvarum Lipomyces spencermartinsiae Metschnikowia fructicola Pichia anomala Pichia anomala Rhodotorula mucilaginosa Saccharomyces cerevisiae Saccharomyces cerevisiae Saccharomyces cerevisiaee Zygosaccharomyces bailii
IOEBL0448 IOEBL0308 IOEBL0407 IOEBL0411 IOEBL0447 IOEBL0453 IOEBL0463 IOEBL0465 IOEBL0512 IOEBL0542 CLIB300 IOEBL0404 IOEBL0504 CLIB373 IOEBL0401 IOEBL0450 IOEBL0530 IOEBL0426 CLIB284 CLIB370 IOEBL0434 IOEBL0437 IOEBL0440 IOEBL0310
50 150 150 150 150 150 150 150 150 150 150 50 100 250 50 50 250 400 400 400 200 150 100 250
Bacteria
Lactobacillus brevis Lactobacillus collinoides Lactobacillus hilgardii Lactobacillus plantarum Leuconostoc mesenteroides Oenococcus oeni Oenococcus oeni Oenococcus oeni Pediococcus damnosus Pediococcus dextrinicus Pediococcus parvulus Pediococcus pentosaceus
IOEBL0019 IOEBL0203 IOEBL0021 IOEBL0401 ATCC8293 ATCC23277 ATCC23279 IOEBL0025 ATCC25248 ATCC33087 ATCC19371 ATCC33316
600 600 250 250 100 150 150 250 250 250 400 400
a
IOEBL: Levures de l’Institut d’Oenologie de Bordeaux, Talence, France; IOEBB: Bacte´ries de l’Institut d’Oenologie de Bordeaux, Talence, France; CLIB: Collection de Levures d’Inte´reˆt Biotechnologique INRA, Thiverval Grignon, France, ATCC: American Type Culture Collection, Manassas, USA.
2.2. Yeast species identification The yeast species identification was performed by molecular tools supporting RFLP analysis of the 5.8S rRNA gene and the two ribosomal internal transcribed spacers (ITS1 and ITS2) using CfoI, HaeIII and HinfI endonucleases (Esteve-Zarzoso, Belloch, Uruburu, & Querol, 1999). Twenty colonies on plates carrying between 30 and 300 colonies were tested. This method allowed for the estimation of the percentage of each species (Clemente-Jimenez, Mingorance-Cazorla, Martinez-Rodriguez, Las-Heras Vazquez, & Rodriguez-Vico, 2004). To detect specifically B. bruxellensis, the species-specific PCR developed by Ibeas et al. (1996) was used. 2.3. Chemical analyses Conventional analyses: pH, alcohol content, free and total SO2 and total polyphenol index (TPI) were carried
out by the usual methods recommended by the international organization of the Vine and Wine (OIV). L-malic acid, glucose and fructose concentrations were measured by enzymatic methods (Biopharm). Volatile phenols were measured after dichloromethane extraction (Rodriguez, Gonc¸alves, Pereira-da-silva, Malfeito-Ferreira, & Loureiro, 2001) followed by gas chromatography (Chatonnet & Boidron, 1988). 2.4. Drug sensitivity assays The DMDC sensitivity was assayed by measuring the minimal inhibitory concentration (MIC) in a red grape juice liquid medium (pH = 4.0, glucose + fructose 180 g/ L, L-malic acid 2 g/L, TPI = 37) containing different drug concentrations comprised between 10 g/L and 10 mg/L (10, 5, 2.5, 1.25, 0.6, 0.4, 0.25, 0.2, 0.15, 0.100, 0.050, 0.025, 0.010 g/L). Viable yeast and bacteria were inoculated at the initial concentration of 103 cells/mL. The incu-
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bation was made in spectrophotometer tank previously sterilized by UV irradiation. In all cases, DMDC resistance was scored by measuring the optical density at 600 nm after 6 days of growth at 25 C and by comparison with a control without DMDC for each strain. For each concentration, assays were made on triplicate. 2.5. DMDC experiments All assays were carried out in triplicate. The wine storage temperature was 25 C. The flasks containing grape juice or wine sample were closed with healthy and sterilized caps. Each sampling was carried out in sterile conditions. Controls without microorganisms were also performed. 2.5.1. Influence of DMDC on grape must fermentation Grape juice was sterilized by filtration (0.22 lm). The sugar concentration (glucose + fructose) was standardized to 190 g/L. The pH was adjusted to 3.7. 200 mL of grape juice were inoculated with different single species and yeast mixtures S. cerevisiae (IOEBL0434), B. bruxellensis, (IOEBL0447), Candida cantarelli (IOEBL0404) and Hanseniaspora uvarum (IOEBL0401) at the initial concentration of 103 CFU/mL. Assays were conducted with different concentrations of DMDC (0, 75, 150, 250, 500 and 1000 mg/ L). Cell viability was estimated by epifluorescence measurement just after yeast inoculation, homogenisation before DMDC addition and 24 hours after DMDC addition. Volumes analysed by epilfluorescence were comprised between 0.1 mL and 10 mL and are chosen according to the viable cells population. The threshold of this technique was 102 ¢/mL. The AF duration was determined by glucose and fructose concentrations measurements. The yeast population was counted on TY and NS plates, and percentage of each survival species calculated after AF completion, and after 50 days of incubation, when it was aborted. In another experiment, the durability of the DMDC effect was tested by yeast re-inoculation few days after the DMDC addition. 2.5.2. Influence of DMDC on MLF Post-alcoholic wine [(12.2% v/v of ethanol, 2.02 g/L of L-malic acid, a pH of 3.55, content of 4 mg/L (H2SO4) of free SO2, and 16 mg/L (H2SO4) of total SO2, sterilized by filtration (0.22 lm))] was used to conduct the MLF with a co-culture of O. oeni (IOEBB0025) and B. bruxellensis (IOEBL0447) with different concentrations of DMDC. (0, 75, 150, 250, 500 and 1000 mg/L). DMDC was added three days after the microbial inoculation to be certain that the anti-microbial effect was the DMDC impact and not a failure of the inoculation. The O. oeni and B. bruxellensis were respectively inoculated at the level of 106 CFU/mL and 103 CFU/mL, which corresponds at the level commonly counted in wine inoculated with O. oeni commercial starter. Yeast and bacteria were counted on plates and the malic acid consumption rate was measured by enzymatic method (biopharm).
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2.5.3. Stabilization of finished wines by DMDC Post-fermentations wine, called finished wine, was filtered and inoculated with B. bruxellensis (IOEBL0447) at the initial concentration of 103 CFU/mL. After three days of incubation, 200 mg/L of DMDC were added. The B. bruxellensis population was monitored by plates counting in TY medium, with epifluorescence and by the speciesspecific PCR. Volatile phenols were measured. At the beginning of the assay the wine parameters were an ethanol content of 12.4% v/v, a pH of 3.58, a free SO2 content of 18 mg/L (H2SO4), and a total SO2 content of 48 mg/L (H2SO4). 2.5.4. Stabilization of lees with high level of microorganisms We followed lees wine containing a high and diverse intrinsic microflora after DMDC addition (200 mg/L). Microbial stabilization was estimated by the TY, NS, LAB and AAB population enumeration after DMDC addition (7 and 30 days after treatment). The lees chemical characteristics were an ethanol content of 13.6% v/v, a pH of 3.98, a free SO2 content of 14 mg/L (H2SO4), and a total SO2 content of 52 mg/L (H2SO4). The proportion of S. cerevisiae and B. bruxellensis among the total yeast species was determined by the Esteve-Zarzoso et al. (1999) identification protocol performed on colonies sampling using TY plates. 3. Results 3.1. DMDC effects during AF Results reported in Table 1 prove the DMDC efficacy against diverse oenological yeasts and bacterial species. A concentration of 600 mg/L of DMDC prevented the growth of all of species test but the most sensitive yeast species were inhibited starting 50 mg/L. Concerning yeasts, Pichia anomala and Rhodotorula mucilaginosa were the more resistant species. In general, bacteria appeared more resistant than yeast. Lactobacillus brevis and Lactobacillus collinoides were the more resistant bacterial species. For some of them, such as S. cerevisiae and O. oeni, the DMDC resistance was strains dependent. It was not the case for B. bruxellensis strains, which were all inhibited at 150 mg/L. Compared with the other species, B. bruxellensis strains appeared moderately resistant to DMDC in grape juice. The objective of the second experiment was to evaluate the effectiveness of DMDC against B. bruxellensis at the beginning of winemaking during the AF. We chose a resistant strain of S. cerevisiae (IOEBL0434) to perform co-cultures with a B. bruxellensis strain (IOEBL0411) and other species commonly detected in fresh must (H. uvarum IOEBL0401 and C. cantarelli IOEBL0404). For all assays, AF was aborted with 500 mg/L of DMDC. When B. bruxellensis was alone a concentration of 150 mg/L of DMDC was sufficient. A higher concentration was needed for S. cerevisiae single culture (250 mg/L). In all cases, the inhibitory concentration of DMDC was higher in a
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co-culture with single cultures. In the non-Saccharomyces mixture the DMDC inhibitory concentration was 250 mg/L. Moreover in the mixture, B. bruxellensis was the only species detected at the end. A similar phenomenon was observed for S. cerevisiae. 250 mg/L were sufficient to prevent S. cerevisiae growth in single culture. But when S. cerevisiae was inoculated with other yeast species, AF was delayed but not aborted at this DMDC concentration. The durability of the DMDC effect was tested in experiments shown in Fig. 1. AF was started by inoculating B. bruxellensis and S. cerevisiae. Quickly the S. cerevisiae population reached 108 CFU/mL and began the fermenta-
tion. When 250 mg/L of DMDC were added in the middle of the AF, AF was stopped. Consequently, cultivable population decreased below 1 CFU/mL. However the DMDC effect was not durable because when yeasts were re-introduced, a new growth phase started. Microbial growth as well as the fermentative activity did not seem be affected by the previous DMDC addition. S. cerevisiae was able to reach again to 107–108 CFU/mL and to ferment sugars were fermented. Fermentation kinetic of the second fermentation was similar to the first one, before DMDC addition. When S. cerevisiae and B. bruxellensis were both reinoculated, they both could grow, but S. cerevisiae growth
Fig. 1. AF evolution after DMDC addition. AF was started by inoculating a mixture of S. cerevisiae (IOEBL0434), and B. bruxellensis (IOEBL0447). After DMDC addition, wines were re-inoculated with the same yeasts at different concentrations and ratios at day 10 (: TY population, d: NS population and m: sugars concentrations).
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overcame B. bruxellensis growth in all cases. At the end of fermentation, B. bruxellensis populations were always inferior in the re-inoculation experiments than in the control ones using B. bruxellensis alone. 3.2. DMDC effect during MLF For the MLF assays (Fig. 2), 500 mg/L and 1000 mg/L of DMDC addition were able to stop MLF. For the two lowest DMDC concentrations (75 mg/L and 150 mg/L) the MLF kinetic was not affected. An intermediary DMDC concentration (250 mg/L) allowed MLF to start but stopped after 10 days. In this case, as shown in Fig. 3, B. bruxellensis population decreased just after inoculation, then grew back up to 4.1 · 103 CFU/mL when MLF began. The population immediately decreased after addition of DMDC, but was not completely eliminated. Finally, after equilibrium around 6 · 101 CFU/mL B. bruxellensis population increased until 103 CFU/mL. Concerning O. oeni, the initial 106 CFU/mL population grew up to 107 CFU/mL degrading actively malic acid. But as soon as DMDC was added (day 3) it decreased. At the day 10, it had dropped to less than 105 CFU/mL, simultaneously MLF stopped. 3.3. DMDC effects in finished wine In finished wine, results (Table 2) showed the DMDC activity to reduce B. bruxellensis population. After DMDC addition the B. bruxellensis population declined to less than 1 CFU/10 mL. At day 10, the volatile phenols production had not started in any wines tested. But after six months, the untreated wine contained higher concentrations of 4ethylphenol and 4-ethylguaiacol than the treated wine where no residual B. bruxellensis was detected. This result was always confirmed by negative PCR specific amplifica-
Fig. 3. Evolution of O. oeni and B. bruxellensis during assay with 250 mg/ L of DMDC added at the 3rd day of monitoring 5 (: O. oeni population and d: B. bruxellensis population).
tions. Non-contamination occurred, as shown by the non-inoculated control wines. 3.4. DMDC effects in lees DMDC in lees does not completely eliminate the populations by the DMDC treatment (Table 3). The total yeast population in the treated lot was lower one week after addition of DMDC (2.1 · 102 CFU/mL) than in the control (1.7 · 106 CFU/mL). B. bruxellensis percentage was lower than S. cerevisiae. After one month the results show that, despite the DMDC, yeasts could still actively grow. Their population has increased of more than 3 logs to nearly 106 CFU/mL in treated lees where B. bruxellensis and S. cerevisiae were in the same proportion. However, B. bruxellensis was dominant in the control suggesting a relative best resistance of indigenous Saccharomyces yeast. Concerning bacteria, the decrease of AAB could not be attributed to DMDC since has also decreased in non-treated lees. The LAB population was stable around 104 CFU/ mL in control but decreased to 102 CFU/mL in treated lees. These results shown that not all microorganisms could be destroyed by DMDC, presumably due to the high populations. Residual viable cells were able to grow again once the initial DMDC had reacted in the system. 4. Discussion
Fig. 2. DMDC impact on L-malic acid consumption during a O. oeni (IOEBB0025) and B. bruxellensis (IOEBL0447) co-culture in wine (: O. oeni, h: B. bruxellensis, : O. oeni and B. bruxellensis + DMDC = 0 mg/ L, j: O. oeni and B. bruxellensis + DMDC = 50 mg/L, d: O. oeni and B. bruxellensis + DMDC = 100 mg/L, : O. oeni and B. bruxellensis + DMDC = 250 mg/L, m: O. oeni and B. bruxellensis + DMDC = 500 mg/L, s: O. oeni and B. bruxellensis + DMDC = 1000 mg/L).
Previous investigations had demonstrated the high tolerance of B. bruxellensis against nutrient privation (Uscanga et al., 2000), ethanol (Medawar et al., 2003), initial cold maceration (Renouf, Perello, Strehaiano, & LonvaudFunel, 2006), thermal treatment (Couto, Neves, Campos, & Hoog, 2005) and winery disinfection (Renouf, Claisse et al., 2006). Therefore, B. bruxellensis can be considered as one of the best adapted species to cellar and wine environment. Tools for limiting its growth are limited. Thus, we assessed the DMDC addition as new treatment to prevent B. bruxellensis growth during and after fermentation.
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Table 2 B. bruxellensis and ethylphenol evolution in wines Control
Wine + B. bruxellensis without DMDC
Wine + B. bruxellensis + 200 mg/L of DMDC
3 days just before DMDC B. bruxellensis (log10 CFU/mL) 4-Ethylphenol (lg/L) 4-Ethylguaiacol (lg/L)
<1 208 ± 21 24 ± 4
1.4 ± 0.4 199 ± 11 27 ± 3
1.4 ± 0.7 196 ± 12 20 ± 6
7 days after DMDC B. bruxellensis (log10 CFU/mL) 4-Ethylphenol (lg/L) 4-Ethylguaiacol (lg/L)
<1 198 ± 11 19 ± 5
4.4 ± 1.7 238 ± 12 26 ± 2
<1 194 ± 24 21 ± 4
180 days B. bruxellensis (log10 CFU/mL) 4-Ethylphenol (lg/L) 4-Ethylguaiacol (lg/L)
<1 220 ± 16 24 ± 7
7.4 ± 1.9 940 ± 43 98 ± 32
<1 206 ± 34 20 ± 12
Effect of DMDC on finished wines inoculated with B. bruxellensis (IOEBL0447) and DMDC treated.
Table 3 Effect of DMDC on lees containing residual indigenous yeast and bacteria Lees (control) Before DMDC addition TY (log10 CFU/mL) NS (log10 CFU/mL) LAB (log10 CFU/mL) AAB (log10 CFU/mL) S. cerevisiae (%) B. bruxellensis (%)
Lees + DMDC (200 mg/L) 3.7 ± 1.4 1.2 ± 0.1 9.5 ± 0.1 6.3 ± 2.1 65% 25%
One week after TY (log10 CFU/mL) NS (log10 CFU/mL) LAB (log10 CFU/mL) AAB (log10 CFU/mL) S. cerevisiae (%) B. bruxellensis (%)
1.7 ± 1.1 1.2 ± 0.3 2.5 ± 0.9 8.3 ± 2.9 30% 65%
2.1 ± 1.0 1.0 ± 0.2 1.2 ± 0.9 6.4 ± 1.9 50% 40%
One month after TY (log10 CFU/mL) NS (log10 CFU/mL) LAB (log10 CFU/mL) AAB (log10 CFU/mL) S. cerevisiae (%) B. bruxellensis (%)
2.5 ± 1.1 2.3 ± 0.3 2.1 ± 0.3 1.2 ± 0.3 10% 90%
9.8 ± 0.2 4.9 ± 1.3 1.5 ± 0.5 1.0 ± 0.1 50% 50%
TY: total yeast, NS: non-Saccharomyces (yeast), LAB: lactic acid bacteria, AAB: acetic acid bacteria.
DMDC effect is not restricted to B. bruxellensis. Compared to species composing the grape surface microflora (Aureobasidium pullulans, Bulleromyces albus, R. mucilaginosa, and Cryptococcus albus) as well as the species known to intervene during the first stage of winemaking (P. anomala, Candida stellata, Metschnikowia fructicola, . . .), B. bruxellensis exhibited an intermediary DMDC resistance. During AF, non-Saccharomyces yeast (including B. bruxellensis), as well as S. cerevisiae were affected by DMDC. But the DMDC effect was transitory. Temple and Ough (1978) estimated that in an 12% v/v of ethanol alcoholic solution and for a pH range comprised between 2.0 and 6.0, four hours were enough for DMDC hydrolysis into carbon dioxide and methanol. Ough (1983) and Van der Riet, Botha, and Pinches (1989) reported variations of
the hydrolysis kinetic according to the temperature, the pH and the wine alcohol content. A maximum inhibitory effect occurs soon after the addition. Therefore DMDC should not be used as preventive agent but only as curative agent against unwanted populations already present in wine. This transitory effect could also be considered as an advantage. Hence, after a DMDC addition, it is possible to re-inoculate the wine with a commercial strain to achieve the fermentation. This could be used to prevent B. bruxellensis growth when it is growing simultaneously with indigenous S. cerevisiae strains at the beginning of the fermentation. After AF, the MLF period is crucial for B. bruxellensis development in wine (Renouf et al., 2005). But O. oeni is also affected and MLF could be delayed or totally prevented by DMDC addition. In other cases, such as in the lees, microbial populations were too high to be totally removed by DMDC additions within the tested concentrations. Daudt and Ough (1980) established a semi-log correlation between the initial viable yeast population and the amount of DMDC required. When the initial viable cells increased tenfold, an additional amount of 5 mg/L of DMDC was approximately required. For sweet wines, DMDC is often tested combined to of sulphur dioxide (Threlfall & Morris, 2001, 2002). In this case; DMDC can also help in lowering the amount of sulphur dioxide needed. Oher studies (Divol, Strehaiano, & Lonvaud-Funel, 2005; Ough, Kunkee, Vilas, Bordeu, & Huand, 1988; Threlfall & Morris, 2002) established a synergistic activity between DMDC and sulphur dioxide. From these cases, it was reported that 50 mg/L of DMDC with 25 mg/ L of free SO2 is sufficient to provide an efficient microbial control. However DMDC cannot replace SO2, which is necessary as an anti-oxidant in must and during ageing. Therefore a synergistic use of SO2 and DMDC can be proposed. SO2 should be added first in grape must, then at the end of the fermentation and all along the ageing period. Thus, DMDC could then be used when an accidental B. bruxellensis multiplication is observed. This would provide the winemaker with an efficient microbial control system. DMDC can also be used during the bottling as a final microbial pre-
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