- Email: [email protected]
Influence of oak origin and ageing conditions on wine spoilage by Brettanomyces yeasts
Food Control 54 (2015) 176e180
Contents lists available at ScienceDirect
Food Control journal homepage: www.elsevier.com/locate/foodcont
Influence of oak origin and ageing conditions on wine spoilage by Brettanomyces yeasts pez, J. Martínez, A.R. Gutie rrez* P. Rubio, P. Garijo, P. Santamaría, R. Lo ~ o, Spain ICVV, Instituto de Ciencias de la Vid y el Vino (Universidad de La Rioja, Gobierno de La Rioja, CSIC), C/Madre de Dios 51, 26006 Logron
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 October 2014 Received in revised form 15 January 2015 Accepted 24 January 2015 Available online 3 February 2015
The use of oak wood is a common practice for the maturation of higher quality wines, but this practice also has been occasionally related with the spoilage of wines with ethylphenols produced by Brettanomyces/Dekkera yeasts. This work studied the presence of these yeasts and ethylphenol production in the same wine aged in different conditions: aerobic/anaerobic, sulfur dioxide additions, and oak origin of the casks used (American, French, Russian and Chinese), with the aim of characterizing factors affecting the development and production of volatile phenols by Brettanomyces. Results obtained indicated the spoilage risk exists when Brettanomyces cells are present, even at a low level, in wines subjected to ageing, both in the cask and the bottle. Brettanomyces presence (cfu/ml and strains) and ethylphenol production during ageing, is affected more by the ageing conditions (aerobic/anaerobic and sulfiting) than by the origin of the oak. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Wine ageing Brettanomyces Ethylphenols Oak origin
1. Introduction Nowadays, the use of oak wood in winemaking is a common practice for both the maturation of wine and even in the fermenleztation process of higher quality wines (Ortega-Heras, Gonza , & Gonza lez-Huerta, 2008) because the extractable comSanJose pounds of the casks induce positive changes in the composition and n and Ancínflavor of aged wine and spirits (Garde-Cerda Azpilicueta, 2006). Oak species normally used in the barrel making industry mostly belong to Quercus robur and Quercus petraea species coming from France, and Quercus alba from the United States (Ojeda, 2012). The use of oak from other regions has begun to appear in recent years for a variety of reasons: to find new sources of oak in order to preserve the current ones, lower price, or even to give wines new aromatic features. While the different species of oak have common characteristics, their structure and composition can be different, and consequently affect the wine aged in them. Studies of the influence of oak origin in ageing wines have been carried out comparing, in particular, the use of American and ndez de Simo n, & Cadahía, 2006; Ferna ndez de French wood (Ferna n, Cadahía, & Jalocha, 2003). Lately, however, casks made with Simo oak coming from Asia have begun to appear in enological markets.
* Corresponding author. Tel.: þ34 941299727; fax: þ34 941299721. rrez). E-mail address: [email protected] (A.R. Gutie http://dx.doi.org/10.1016/j.foodcont.2015.01.034 0956-7135/© 2015 Elsevier Ltd. All rights reserved.
Moreover, oak coming from Eastern European countries is increasingly being used for ageing quality wines, but studies about characteristics they bring to the wines are scarce (Chatonnet 1999, Vivas et al. 2000). However, the maturation of wine in oak casks has also been linked to wine spoilage caused by the presence of Brettanomyces (Silva et al. 2005). This yeast is considered to be the main cause of wine spoilage, and it has become a major worldwide enological concern in recent years. Yeasts belonging to the species Dekkera bruxellensis, or its anamorph Brettanomyces bruxellensis, have the capacity to spoil wines by producing ethylphenols (Loureiro & Malfeito-Ferreira, 2006), which are the compounds responsible for the off-flavors described as animal odors, farm, horse sweat, medicine and animal leather (Chatonnet, Dubordieu, & Boidron, 1993; Chatonnet, Dubourdieu, Boidron, & Pons, 1992). Wineries, and especially oak casks, are often considered as the main source of Brettanomyces contamination (Fugelsang & Zoecklein, 2003). The microporus structure of wood favors the deep penetration of microorganisms making it difficult to clean and sanitize the casks lez-Arenzana et al. 2013; Jiranek, Grbin, Yap, Barnes, & Bates, (Gonza 2008), and so increasing the risks of wine spoilage (Guzzon et al. 2011). The incidence of Brettanomyces spoilage problems has extended around the world, and so, various authors have concluded that controlling the growth of Brettanomyces is the most important challenge for modern winemaking (Wedral, Shewfelt, & Frank, 2010).
P. Rubio et al. / Food Control 54 (2015) 176e180
This work studied the presence and development of Brettanomyces yeasts in wines aged in different conditions of aerobiosis, sulfur dioxide addition and origin of oak used in casks, for the purpose of characterizing some factors affecting volatile phenol production by B. bruxellensis so as to better define preventive technological measures. 2. Material and methods 2.1. Conditions of assays The study was conducted on red wine with the following composition: ethanol 13.6%, pH 3.55, sugar 2.68 g/L, free and combined sulfur dioxide 29/69 mg/L respectively. This wine was distributed in homogenous batches which were aged for one year in three different conditions: A: 12 months in 750 cc bottles (anaerobic conditions), B: 12 months in 225 L new oak casks (aerobic conditions) of different origins and species: American (Q. alba), French (Q. petraea), Russian (Q. petraea) and Chinese (Quercus mongolicus), and C: 6 months in the same types of casks as in B, and the other six months in bottles (aerobic þ anaerobic conditions). The type B wines were racked and sulfited at six months and set in the same cleaned barrel. The type C wines were bottled at six months without adding sulfur dioxide. One year after the ageing started, the wines were analyzed. In this study, three new casks of each type of oak were employed, so all results were obtained in triplicate. All the casks were supplied by the same Company and were made in identical conditions: wood submitted to natural outdoor drying and medium degree of toasting. 2.2. Microbiological analysis of wines The wines were sown in duplicate quantities of 500 ml on Petri dishes with DBDM (0.7% Yeast Nitrogen Base; 0.7% ethanol; 0.001% cycloheximide; 0.01% ac. p-cumaric; 0.002% bromocresol green, 0.02% chloramphenicol, 20% agar, pH 5,4 adjusted with sorbic acid), a specific medium for detecting the presence of Brettanomyces/ Dekkera yeasts (Rodrígues, Gonçalves, Pereira-da-Silva, MalfeitoFerreira, & Loureiro, 2001). The yeast plates were incubated at 25 C for 21 days. Some diphenyl crystals (approximately 100 mg/plate) (Panreac Química SA, Barcelona, Spain), were added to the plates in order to impede the development of molds. Colonies were counted and cell populations (cfu) calculated from duplicate analyses. From each plate, ten colonies were randomly selected and stored for later analysis in LM medium (10% D-glucose, 5% mycopeptone, 3% yeast extract, 3% malt extract, 20% bacteriological agar). 2.3. Molecular characterization of yeasts The identification of yeasts species isolated from the DBDM medium was necessary because the growth of genera other than n Brettanomyces in the DBDM medium had been reported (Oco et al., 2013). This analysis was carried out using PCR-RFLP of the 5.8S-ITS region of the ribosomal DNA, following the method proposed by Esteve-Zarzoso, Belloch, Uruburu, and Querol (1999). The primers used to amplify the region were those described by White, Bruns, Lee, and Taylor (1990). The amplification reaction took place in the GeneAmp® PCR System 2700 thermocycler (Applied Biosystems). For the analysis of restriction of the product of PCR, 5 ml aliquots were subjected to digestion by the HinfI, HaeIII and CfoI enzymes. The identification of the isolated yeasts was performed through a comparison of the restriction profile of each yeast with those obtained in our laboratory in other works and with those
177
contained in the Yeast-id-database at www.yeast-id.com (Valencia University and CSIC, Spain). 2.4. Clonal characterization of Brettanomyces isolates Clonal characterization of Brettanomyces isolates was conducted by using the restriction endonuclease analysis pulse field gel electrophoresis method (REA-PFGE), described by Miot-Sertier and Lonvaud-Funel (2007) with some modifications in culture cell conditions, spheroplast obtention, cellular membrane rupture and plug washes. B. bruxellensis samples were prepared by growing strains for 24 h in a YPG medium under agitation (80 rpm) at 28 C. Instead of using lyticase, we used a mix of Zymolyase (1 mg/ml) from Arthrobacter luteus and Novozyme (1 mg/ml) from Trichoderma harzianum as suggested by Martorell (2006). Cellular membrane rupture was carried out with Proteinase K from Tritirachium album instead of Pronase E. The lasted two plug washes were carried out with TrisClH 10 mM instead of TE. Digested chromosomal DNA was separated using the Cheff-DRR III System (Bio Rad). Gels were stained with etidium bromide (0.5 mg/ml) and photographed under UV light using an Image Store 5000 (UVP) (Bio Rad). 2.5. Ethylphenol analysis Ethylphenol content was analyzed via gas Chromatography. Extraction of these compounds was carried out following the pez, Cacho, and Ferreira methodology described by Ortega, Lo (2001), modified according to Martínez, Ojeda, and Rubio (2011): 5 ml of centrifuged wine, 9.5 ml of saturated solution of ammonium sulphate, 15 ml of internal pattern (2-octanol and 3.4dimethylphenol) and 0.200 ml of dichloromethane were placed in a tube and shaken horizontally for 60 min at 400 rpm. Afterward, the tube was centrifuged at 0 C and 2500 rpm during 10 min. Finally organic phase from the bottom of the tube was recovered and analyzed. Gas Chromatography analysis was carried out on a 2 ml extract, in splitless (0.5 min) in a Hewlett Packard gas chromatograph (HP-6890 series II with FID detector). Compound separation was performed in a DB-WAX 50 m 0.20 mm capilar column, with 1 ml/min of nitrogen as a carrier gas. Chromatographic conditions were: injector temperature 250 C, detector temperature 240 C, initial oven temperature 75 C (5 min) increasing 3 C/min to 240 C (20 min). Compound identification and quantification were conducted by comparing retention time of pattern substances by means of calibration lines obtained with compound extracts in hydroalcoholic solutions. 2.6. Statistical analysis Statistical analysis of the data was performed using SPSS Version 20.0 statistical package for Windows (SPSS, Chicago, IL, USA). Data were analyzed using analysis of variance; differences between means were compared using the Tukey test at 0.05 probability level. 3. Results and discussion 3.1. Presence of Brettanomyces in wines No spoilage due to Brettanomyces had happened prior to the beginning of ageing because the levels of ethylphenols in the initial wine (Table 1) were low. The wine was well-protected with sulfur dioxide and the casks used could not contaminate it because they were new, and the toasting process eliminates contamination. So, no wine spoilage from this source was foreseeable from this cause.
178
P. Rubio et al. / Food Control 54 (2015) 176e180
However, after 12 months' ageing, wine spoilage was noted and also the presence of Brettanomyces and high levels of ethylphenols were detected in all the wines (Table 1). Spoilage and taint was also evident in the wine bottle-aged for 12 months, indicating that the source of Brettanomyces was the initial wine. Martorell et al. (2006) had showed that contamination of wines by Brettanomyces in most cases occurs before the wines are put into casks. Renouf and Lonvaud-Funel (2007) also indicated that despite the hygiene measures taken in wineries, the development of B. bruxellensis in wine at the beginning of the ageing period is still not prevented and that earlier phases in the work in cellars should be studied as a possible source of contamination. In a previous work carried out by our team (Garijo et al. 2014) we observed that almost 30% of red wines analyzed at the end of malolactic fermentation, contained Brettanomyces, most of them at a low level (1e10 cfu/ml) that will not produce wine spoilage. But if those values increase during the following months to reach high levels, taint will become evident. Molecular characterization of isolates from the DBDM medium showed that they all belonged to B. bruxellensis specie. All the wines analyzed after 12 months ageing contained these yeasts, showing clear differences between the wines which had spent 6 months in bottle and those that had only aged in casks (Table 1). The wines aged only in the cask had a Brettanomyces level between 0 cfu/ml (Russian oak) and 76 cfu/ml (Chinese oak), whereas those aged in both cask and bottle showed a higher level that was uncountable (higher than 300 cfu/ml). The lower level detected in wines aged 12 months in the cask was probably due to the addition of sulfur dioxide and racking carried out at 6 months. The antimicrobial potential of SO2 against Brettanomyces, and the effectiveness of physical treatment like racking to remove yeast cells from barrels have already been shown (Oelofse, Pretorius, & Du Toit, 2008; rez-Lepe, Morata, & Caldero n, 2007). In this study the Su arez, Sua inhibitory effect of these two actions (sulfiting and racking) overcame the stimulating effect of oxygen on Brettanomyces growth (Kheir, Salamech, Strehaiano, Brandam, & Lteif, 2013; Uscanga, lia, & Strehaiano, 2003). So, ageing only in aerobic conditions De (12 months cask) with racking and sulfur dioxide addition, showed lower Brettanomyces levels than the combined aerobic and anaerobic maturation (6 months in cask and 6 in bottle) with racking but without sulfur dioxide addition. These data confirm that SO2 is a crucial element inhibiting Brettanomyces (du Toit, Pretorius, & Lonvaud-Funel, 2005). Wines that were only aged in anaerobic conditions (12 months in the bottle) also showed a high Brettanomyces level, showing that in anaerobic conditions too, Brettanomyces can develop and spoil
Table 1 Brettanomyces and ethylphenol (4EG and 4EP) content in wines subjected to different ageing conditions for 12 months. Ageing time (months) Brettanomyces 4-EG (mg/L) 4EP (mg/L) (ufc/mL) Barrel Bottle Initial wine e Ageing conditions Bottle e Oak Americana 12 Oak Frencha 12 Oak Russiana 12 a Oak Chinese 12 Oak American 6 Oak French 6 Oak Russian 6 Oak Chinese 6
e 12 e e e e 6 6 6 6
e Uncountable 46 11 0 76 Uncountable Uncountable Uncountable Uncountable
7 199 180 175 179 180 181 177 196 172
the wine, even when they are present at a low level in the initial wine. So, winemakers must check these yeasts are absent in the wine at the time of bottling. Due to the impossibility of accurately counting the number of Brettanomyces colonies grown in the DBDM medium of wines aged six months in the cask and the other six in the bottle, it was impossible to establish differences due to oak origin in these ageing conditions. But it was possible in samples coming from wines caskaged for 12 months. The level of Brettanomyces at the end of 12 months maturation in oak casks was lower in French and Russian oak (both Q. petraea) than in American (Q. alba) and Chinese (Q. mongolicus) oak. This could be due to the characteristics of each ~ oz, Ferna ndez oak species and differences in porosity (Cadahía, Mun n, & García-Vallejo, 2001; Cadahía, Vareas, Ferna ndez de de Simo n, & García-Vallejo, 2001; Martínez, Rubio, & Rodríguez, 2011). Simo 3.2. Clonal diversity of B. bruxellensis In Table 2 it can be seen that the different kind of ageing favors the development of different clones of Brettanomyces. All these strains were probably present in the starting wine because ageing was conducted using new barrels. In wines aged in anaerobic conditions for one year in the bottle, only clone I has been detected. In wines aged in oak casks for 6 or 12 months clone I was not detected, and others developed. The majority clone in wines aged at the beginning of the period in aerobic conditions (6 or 12 months in oak barrel), was clone II. This clone was the only one detected in wines aged for 12 months in oak casks, and the majority one in wines which had spent the first 6 months in oak casks. In wines with combined ageing in barrel and bottle (aerobic plus anaerobic conditions), although clone II was the predominant one, other clones (III and IV) were identified, probably due to the absence of sulfur dioxide addition at the time of bottling, which favored other strains permanence and/or development. Barata et al. (2008) have showed different sulfur dioxide sensitivity among B. bruxellensis strains. These results would indicate that all these clones were present in the initial wine and the subsequent development of each different strain could depend on the greater or lesser adaptation to the ageing conditions of the wine. These data also suggest that oxygen availability at the beginning of the ageing process, during the yeast multiplication stage, can affect the presence and selection of different clones of this species. Clonal diversity found in B. bruxellensis was low, coinciding with that obtained by other authors (Albertín et al. 2014). Only 4 different strains were identified from all the colonies analyzed (Fig. 1) and one of them (strain II) was clearly predominant (76%).
Table 2 Percentage (%) of different strains of Brettanomyces found in the wines.
14 a a a a a a a a a
1576 1382 1270 1305 1298 1471 1451 1553 1237
a b b b b ab ab a b
a Racked and sulphited at 6 months. For each different ageing condition (12 months in the cask or 6 months' cask and six in the bottle), different letters indicate differences in origin of the oak (p 0.05).
Ageing time (Months)
Strains of Brettanomyces
Barrel
Bottle
I
II
III
IV
e
e
e
e
e
12 e e e e 6 6 6 6
100 e e e e e e e e
e 100 100 e 100 100 90 50 70
e e e e e e 10 e e
e e e e e e e 50 30
Initial wine e Ageing conditions Bottle e Oak Americana 12 Oak Frencha 12 Oak Russiana 12 a Oak Chinese 12 Oak American 6 Oak French 6 Oak Russian 6 Oak Chinese 6 a
Racked and sulphited at 6 months.
P. Rubio et al. / Food Control 54 (2015) 176e180
Conterno, Lucy Joseph, Arvik, Henick-Kling, and Bisson (2006) grouped 47 isolates in six clusters, Agnolucci et al. (2009) found seven haplotypes of B. bruxellensis in 84 isolates tested, and Campolongo, Rantsiou, Giordano, Gerbi, and Cocolin (2010) found 10 different strains in 196 isolates. Diversity level of this species is lower than that found in Saccharomyces cerevisiae. In this latter pez, Tenorio, and Gutie rrez (2005) species, Santamaría, Garijo, Lo found 38 different profiles out of 140 isolates studied, and Capece, Romaniello, Siesto, and Romano (2012) found 39 strains among 132 isolates, which means significant genetic variability. These results would indicate that the presence of Brettanomyces (cfu/ml and clones) during ageing is more a consequence of the ageing conditions (aerobiosis/anaerobiosis and sulfiting) than oak origin. 3.3. Wine ethylphenol content 4-ethylguaiacol and 4-ethyl phenol content were very low in the initial wine (Table 1). However, after 12 months and regardless of the ageing conditions, concentration of these two compounds was very high in all cases, widely exceeding the content level at which wines are generally rejected. The quality of wine may be negatively affected by concentrations above 425 mg/L (1:10 ratio of 4ethylguaiacol/4-ethyl-phenol in red wines), which is cited as the aroma threshold (Chatonnet, Boidron, & Pons, 1990). Wines aged only in anaerobic conditions had the highest level of both 4-ethylguaiacol and 4-ethyl phenol (Table 1). In Table 1 it can be seen that when ageing was carried out in aerobic conditions (12 months in the cask), no significant differences in ethylphenols content were found depending on the origin of the oak. However, when wines remained six months in the cask and another six in the
179
bottle, 4-ethyl phenol concentration was significantly different between wines stored in Russian and Chinese barrels. If ethyl phenol levels coming from wines aged 12 months in the cask are compared with those coming from wines that were 6 months in the cask and the other 6 in the bottle (statistical study not included), it was observed that the latter, despite the variability found, showed higher 4-ethyl phenol concentration (Table 1). This could be due to the racking and sulfiting carried out at six months in the former, leading to a reduction in the microbial population which could be the cause of the lower ethyl phenol accumulation in the wines. These differences can also be caused by the different strains of Brettanomyces found in the wines. Different studies have showed that the growth rate and ethylphenol production by Brettanomyces are strain-dependent characteristics (Farina, Boido, Carrau, & Dellacassa, 2007; Silva et al. 2005; Vigentini et al. 2008). Despite the significant differences, the similar levels of ethylphenols in all the samples were probably due to the predominance of strain II in all of them. The similar levels of ethylphenols in all three types of ageing also suggest that these compounds were most probably produced at the beginning of the ageing process, and the SO2 addition at six months killed the cells, but ethylphenols were already present in the wines. Therefore, high CFU numbers must have been attained, but were unreported because the number of cells and ethylphenols were determined only once at the end of 12 months. The influence of the kind of oak in the 4-ethylphenol content of wines depending on the ageing conditions showed that only Russian and French oak (both Q. petraea) levels are significantly higher in wines with 6 months' cask-ageing and 6 months in the bottle than those aged for 12 months in the cask. In the case of American and Chinese oak, although there were not significant differences, levels in wines with 6 months each spent ageing in cask and bottle were higher and lower respectively than those aged for 12 months in the cask. These data could be explained by the kind of oak (species and porosity), and also by the number and strains of Brettanomyces found in them.
4. Conclusions
Fig. 1. Pulse field gel electrophoresis of NotI digested chromosomal DNA from Brettanomyces bruxellensis strains I, II, III and IV. Lanes M: Lambda Ladder PFG Marker (Biolabs).
When Brettanomyces cells are present, even at a low level, in wines which undergo ageing, whether in the cask or in the bottle, there is a high risk of spoilage, even in well-protected wines. So, the periodic control of wines during cask-ageing to avoid the development of these spoiling yeasts, and to ensure that no Brettanomyces cells get inside the bottle is essential to avoid wine spoilage. Brettanomyces development (cfu/ml and strains) during caskageing, is affected more by ageing conditions than by the origin of the oak. The inhibitory effect of the two actions (sulfiting and racking) overcame the stimulating effect of oxygen on Brettanomyces growth: ageing only under aerobic conditions with racking and sulfur dioxide addition, showed lower Brettanomyces levels than the combined aerobic and anaerobic maturation with racking but without sulfur dioxide addition. These data confirm that SO2 is a crucial element inhibiting Brettanomyces. Four different B. bruxellensis clones were found in the assays, all of them coming from the initial wine. The development of different strains in different assays could depend on the greater or lesser adaptation to the ageing conditions of the wine. No big significant differences in ethyl phenol levels among wines aged in casks made from oak of different origin were found. However, wines aged in Chinese oak revealed different behavior to the other three in terms of the level of Brettanomyces and in the ethylphenol content, probably due to the fact that this oak has the highest porosity.
180
P. Rubio et al. / Food Control 54 (2015) 176e180
Acknowledgments This study has been undertaken with a grant from the Government of La Rioja, Spain (Project PR-11-11). References Agnolucci, M., Vigentini, I., Capurso, G., Merico, A., Tirelli, A., Compagno, C., et al. (2009). Genetic diversity and physiological traits of Brettanomyces bruxellensis strain isolated from Toscan Sangiovese wines. International Journal of Food Microbiology, 3, 238e244. Albertín, W., Panfili, A., Miot-Sertier, C., Goulielmakis, A., Delcamp, A., Salin, F., et al. (2014). Development of microsatellite markers for the rapid and reliable genotyping of Brettanomyces bruxellensis at strain level. Food Microbiology, 42, 188e195. Barata, A., Caldeira, J., Botheleiro, R., Pagliara, D., Malfeito-Ferreira, M., & Loureiro, V. (2008). Survival patterns of Dekkera bruxellensis in wines and inhibitory effect of sulphur dioxide. International Journal of Food Microbiology, 121, 201e207. ~ oz, L., Ferna ndez de Simo n, B., & García-Vallejo, M. C. (2001). Cadahía, E., Mun Changes in low molecular weight phenolics in Spanish, French and American oak during natural seasoning and toasting. Journal of Agricultural and Food Chemistry, 49, 1790e1798. n, B., & García-Vallejo, M. C. (2001). Cadahía, E., Vareas, S., Fern andez de Simo Evolution of Ellagitannins in Spanish, French and American oak wood during seasoning and toasting. Journal of Agricultural and Food Chemistry, 49, 3677e3684. Campolongo, S., Rantsiou, K., Giordano, M., Gerbi, V., & Cocolin, L. (2010). Prevalence and biodiversity of Brettanomyces bruxellensis in wine from Northwestern Italy. American Journal of Enology and Viticulture, 61(4), 486e491. Capece, A., Romaniello, R., Siesto, G., & Romano, P. (2012). Diversity of Saccharomyces cerevisiae yeasts associated to spontaneously fermenting grapes from an Italian “heroic vine-growing area”. Food Microbiology, 31, 159e166. ristiques et interest du bois de che ^ne de Russie Chatonnet, P. (1999). Caracte -Moreau: nouvelles acquisitions et perspectives. Revue Francaise Adyghe d'Oenologie, 175, 20e24. Chatonnet, P., Boidron, J. N., & Pons, M. (1990). Elevage des vins rouges en fûts de ne: evolution de certains compose s volatils et de leur impact aromatique. che Sciences des Aliments, 10, 565e587. Chatonnet, P., Dubordieu, D., & Boidron, J. (1993). Synthesis of volatile phenols by Saccharomyces cerevisiae in wines. Journal of the Science of Food and Agriculture, 62, 191e202. Chatonnet, P., Dubourdieu, D., Boidron, J. N., & Pons, M. (1992). The origin of ethylphenols in wines. Journal of the Science of Food and Agriculture, 60, 165e178. Conterno, L., Lucy Joseph, C. M., Arvik, T. J., Henick-Kling, T., & Bisson, L. F. (2006). Genetic and physiological characterization of Brettanomyces bruxellensis strains isolated from wines. American Journal of Enology and Viticulture, 57(2), 139e147. Esteve-Zarzoso, B., Belloch, C., Uruburu, F., & Querol, A. (1999). Identification of yeasts by RFLP analysis of the 5.8S rRNA gene and the two ribosomal internal transcribed spacers. International Journal of Systematic Bacteriology, 49, 329e337. Farina, L., Boido, E., Carrau, F., & Dellacassa, E. (2007). Determination of volatile phenols in red wines by dispersive liquid-liquid microextraction and gas chromatography-mass spectrometry detection. Journal of Chromatography A, 1157, 46e50. ndez de Simo n, B., & Cadahía, E. (2006). Evolution of volatile compounds in a Ferna Spanish red wine during two years bottled, after aging in barrels made of Spanish, French and American oak Wood. Analytica Chimica Acta, 563, 198e203. ndez de Simo n, B., Cadahía, E., & Jalocha, J. (2003). Volatile compounds in a Ferna Spanish red wine aged in barrels made of Spanish, French, and American oak wood. Journal of Agricultural and Food Chemistry, 51, 7671e7678. Fugelsang, K. C., & Zoecklein, C. G. (2003). Population dynamics and effects of Brettanomyces bruxellensis strains on Pinot noir (Vitis vinifera L.) wines. American Journal of Enology and Viticulture, 54, 294e300. n, T., & Ancín-Azpilicueta, C. (2006). Effect of oak barrel type on the Garde-Cerda volatile composition of wine: storage time optimization. LWT Food Science and Technology, 39, 199e205. pez-Alfaro, I., Garde-Cerda n, T., Lo pez, R., Garijo, P., Gonz alez-Arenzana, L., Lo Santamaría, P., et al. (2014). Analysis of grapes and the first stages of the vinification process in wine contamination with Brettanomyces bruxellensis. European Food Research and Technology. http://dx.doi.org/10.1007/s00217-014-2351-4. lez-Arenzana, L., Santamaría, P., Lo pez, R., Garijo, P., Gutie rrez, A. R., GardeGonza Cerd an, T., et al. (2013). Microwave technology as a new tool to improve microbiological control of oak barrels: a preliminary study. Food Control, 30, 536e539. Guzzon, R., Widman, G., Malacarne, M., Nardin, T., Nicolini, G., & Larcher, R. (2011). Survey of the yeast population inside wine barrels and the effects of certain techniques in preventing microbiological spoilage. European Food Research and Technology, 233, 285e291.
Jiranek, V., Grbin, P., Yap, A., Barnes, M., & Bates, D. (2008). High power ultrasonic as a novel tool offering new opportunities for managing wine microbiology. Biotechnology Letters, 30, 1e6. Kheir, J., Salamech, D., Strehaiano, P., Brandam, C., & Lteif, R. (2013). Impact of volatile phenols and their precursors on wine quality and control measures of Brettanomyces/Dekkera yeasts. European Food Research and Technology, 237, 655e671. Loureiro, V., & Malfeito-Ferreira, M. (2006). Food spoilage microorganisms. In C. Blackburn (Ed.), Spoilage activities of Dekkera/Brettanomyces spp (pp. 354e398). Cambridge, England: Woodhead Publishing Limited. tico del roble espan ~ ol Quercus Martínez, J., Ojeda, S., & Rubio, P. (2011). Aporte aroma pyrenaica durante la crianza en barrica de vinos de Tempranillo. In XI Congreso n Enolo gica (GIENOL) (pp. 555e558). Nacional de Investigacio Martínez, J., Rubio, P., & Rodríguez, R. (2011). Experiencias con robles de China y Rusia en la crianza de vinos de Rioja. Enoviticultura, 8, 20e27. pidos para la deteccio n de sistemas ra n, Martorell, P. (2006). Desarrollo y aplicacio n y caracterizacio n de levaduras alterantes en alimentos (Ph.D. thesis). identificacio ~ a: Universidad de Valencia. Espan ndez-Espinar, M. T., Loureiro, V., Martorell, P., Barata, A., Malfeito-Ferreira, M., Ferna & Querol, A. (2006). Molecular typing of the yeast species Dekkera bruxellensis and Pichia guilliermondii recovered from wine related sources. International Journal of Food Microbiology, 106, 789e884. Miot-Sertier, C., & Lonvaud-Funel, A. (2007). Development of a molecular method for the typing of Brettanomyces bruxellensis (Dekkera bruxellensis) at the strain level. Journal of Applied Microbiology, 102, 555e562. n, E., Garijo, P., Sanz, S., Olarte, C., Lo pez, R., Santamaría, P., et al. (2013). Analysis Oco of airborne yeast in one winery over a period of one year. Food Control, 30, 585e589. Oelofse, A., Pretorius, I. S., & Du Toit, M. (2008). Significance of Brettanomyces and Dekkera during winemaking: a synoptic review. South African Journal for Enology and Viticulture, 29(2), 128e144. Ojeda, S. (2012). Nuevos orígenes de la madera de roble para la crianza de vinos tintos de la D.O.Ca. Rioja (Ph.D. thesis). Spain: Universidad de La Rioja. lez-SanJose , M. L., & Gonza lez-Huerta, C. (2008). ConsidOrtega-Heras, M., Gonza eration of the influence of aging process, type of wine and oenological classic parameters on the levels of Wood volatile compounds present in red wines. Food Chemistry, 103, 1434e1448. pez, R., Cacho, J., & Ferreira, V. (2001). Fast analysis of important wine Ortega, C., Lo volatile compounds. Development and validation of a new method based on gas chromatographic-flame ionisation detection analysis of dichlorometane microextracts. Journal of Chromatography A, 923, 205e214. Renouf, V., & Lonvaud-Funel, A. (2007). Development of an enrichment medium to detect Dekkera/Brettanomyces bruxellensis, a spoilage wine yeast, on the surface of grape berries. Microbial Research, 162, 154e167. Rodrígues, N., Gonçalves, G., Pereira-da-Silva, S., Malfeito-Ferreira, M., & Loureiro, V. (2001). Development and use of a differential medium to detect yeast of the genera Dekkera/Brettanomyces. Journal of Applied Microbiology, 90, 588e599. pez, R., Tenorio, C., & Gutie rrez, A. R. (2005). Analysis of Santamaría, P., Garijo, P., Lo yeast population during spontaneous alcoholic fermentation: effect of the age of the cellar and the practice of inoculation. International Journal of Food Microbiology, 103, 49e56. Silva, L. R., Andrade, P. B., Valentao, P., Seabra, R. M., Trujillo, M. E., & Vel azquez, E. (2005). Analysis of non-coloured phenolics in red wine: effect of Dekkera bruxellensis yeasts. Food Chemistry, 89, 185e189. rez-Lepe, J. A., Morata, A., & Caldero n, F. (2007). The production of Su arez, R., Sua ethylphenols in wine by yeasts of the genera Brettanomyces and Dekkera. A review. Food Chemistry, 102, 10e21. du Toit, W. J., Pretorius, I. S., & Lonvaud-Funel, A. (2005). The effect of sulphur dioxide and oxygen on the viability and culturability of a strain of Acetobacter pasteurianus and a strain of Brettanomyces bruxellensis isolated from wine. Journal of Applied Microbiology, 98, 862e871. lia, M. L., & Strehaiano, P. (2003). Brettanomyces bruxellensis: Uscanga, M. G. A., De effect of oxygen on growth and acetic acid production. Applied Microbiology Biotechnology, 61, 157e162. Vigentini, I., Romano, A., Compagno, C., Merico, A., Molinari, F., Tirelli, A., et al. (2008). Physiological and oenological traits of different Dekkera/Brettanomyces bruxellensis strains under wine-model conditions. FEMS Yeast Research, 8, 1087e1096. Vivas, N., Absalon, C., Benoist, F., Vitry, Ch, Grazillier, S., De Revel, G., et al. (2000). ^nes europe ens Q. robur L. et Q. petraea (Matt.) Liebl.: Analyse des Les che s oenologiques des diffe rents massifs forestiers. In Ve Colloque des potentialite Sciences et Techniques de la Tonnellerie (pp. 31e37). Wedral, D., Shewfelt, R., & Frank, J. (2010). The challenge of Brettanomyces in wine. LWTeFood Sciences and Technology, 43, 1474e1479. White, T. J., Bruns, T., Lee, S., & Taylor, J. (1990). Amplification and direct sequencing of fungi ribosomal RNA genes for phylogenetics. In M. A. Innis, D. H. Gelfalnd, J. J. Sninsky, & T. T. White (Eds.), PCR protocols. A guide to methods and applications (pp. 315e320). San Diego: Academic Press.
Contents lists available at ScienceDirect
Food Control journal homepage: www.elsevier.com/locate/foodcont
Influence of oak origin and ageing conditions on wine spoilage by Brettanomyces yeasts pez, J. Martínez, A.R. Gutie rrez* P. Rubio, P. Garijo, P. Santamaría, R. Lo ~ o, Spain ICVV, Instituto de Ciencias de la Vid y el Vino (Universidad de La Rioja, Gobierno de La Rioja, CSIC), C/Madre de Dios 51, 26006 Logron
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 October 2014 Received in revised form 15 January 2015 Accepted 24 January 2015 Available online 3 February 2015
The use of oak wood is a common practice for the maturation of higher quality wines, but this practice also has been occasionally related with the spoilage of wines with ethylphenols produced by Brettanomyces/Dekkera yeasts. This work studied the presence of these yeasts and ethylphenol production in the same wine aged in different conditions: aerobic/anaerobic, sulfur dioxide additions, and oak origin of the casks used (American, French, Russian and Chinese), with the aim of characterizing factors affecting the development and production of volatile phenols by Brettanomyces. Results obtained indicated the spoilage risk exists when Brettanomyces cells are present, even at a low level, in wines subjected to ageing, both in the cask and the bottle. Brettanomyces presence (cfu/ml and strains) and ethylphenol production during ageing, is affected more by the ageing conditions (aerobic/anaerobic and sulfiting) than by the origin of the oak. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Wine ageing Brettanomyces Ethylphenols Oak origin
1. Introduction Nowadays, the use of oak wood in winemaking is a common practice for both the maturation of wine and even in the fermenleztation process of higher quality wines (Ortega-Heras, Gonza , & Gonza lez-Huerta, 2008) because the extractable comSanJose pounds of the casks induce positive changes in the composition and n and Ancínflavor of aged wine and spirits (Garde-Cerda Azpilicueta, 2006). Oak species normally used in the barrel making industry mostly belong to Quercus robur and Quercus petraea species coming from France, and Quercus alba from the United States (Ojeda, 2012). The use of oak from other regions has begun to appear in recent years for a variety of reasons: to find new sources of oak in order to preserve the current ones, lower price, or even to give wines new aromatic features. While the different species of oak have common characteristics, their structure and composition can be different, and consequently affect the wine aged in them. Studies of the influence of oak origin in ageing wines have been carried out comparing, in particular, the use of American and ndez de Simo n, & Cadahía, 2006; Ferna ndez de French wood (Ferna n, Cadahía, & Jalocha, 2003). Lately, however, casks made with Simo oak coming from Asia have begun to appear in enological markets.
* Corresponding author. Tel.: þ34 941299727; fax: þ34 941299721. rrez). E-mail address: [email protected] (A.R. Gutie http://dx.doi.org/10.1016/j.foodcont.2015.01.034 0956-7135/© 2015 Elsevier Ltd. All rights reserved.
Moreover, oak coming from Eastern European countries is increasingly being used for ageing quality wines, but studies about characteristics they bring to the wines are scarce (Chatonnet 1999, Vivas et al. 2000). However, the maturation of wine in oak casks has also been linked to wine spoilage caused by the presence of Brettanomyces (Silva et al. 2005). This yeast is considered to be the main cause of wine spoilage, and it has become a major worldwide enological concern in recent years. Yeasts belonging to the species Dekkera bruxellensis, or its anamorph Brettanomyces bruxellensis, have the capacity to spoil wines by producing ethylphenols (Loureiro & Malfeito-Ferreira, 2006), which are the compounds responsible for the off-flavors described as animal odors, farm, horse sweat, medicine and animal leather (Chatonnet, Dubordieu, & Boidron, 1993; Chatonnet, Dubourdieu, Boidron, & Pons, 1992). Wineries, and especially oak casks, are often considered as the main source of Brettanomyces contamination (Fugelsang & Zoecklein, 2003). The microporus structure of wood favors the deep penetration of microorganisms making it difficult to clean and sanitize the casks lez-Arenzana et al. 2013; Jiranek, Grbin, Yap, Barnes, & Bates, (Gonza 2008), and so increasing the risks of wine spoilage (Guzzon et al. 2011). The incidence of Brettanomyces spoilage problems has extended around the world, and so, various authors have concluded that controlling the growth of Brettanomyces is the most important challenge for modern winemaking (Wedral, Shewfelt, & Frank, 2010).
P. Rubio et al. / Food Control 54 (2015) 176e180
This work studied the presence and development of Brettanomyces yeasts in wines aged in different conditions of aerobiosis, sulfur dioxide addition and origin of oak used in casks, for the purpose of characterizing some factors affecting volatile phenol production by B. bruxellensis so as to better define preventive technological measures. 2. Material and methods 2.1. Conditions of assays The study was conducted on red wine with the following composition: ethanol 13.6%, pH 3.55, sugar 2.68 g/L, free and combined sulfur dioxide 29/69 mg/L respectively. This wine was distributed in homogenous batches which were aged for one year in three different conditions: A: 12 months in 750 cc bottles (anaerobic conditions), B: 12 months in 225 L new oak casks (aerobic conditions) of different origins and species: American (Q. alba), French (Q. petraea), Russian (Q. petraea) and Chinese (Quercus mongolicus), and C: 6 months in the same types of casks as in B, and the other six months in bottles (aerobic þ anaerobic conditions). The type B wines were racked and sulfited at six months and set in the same cleaned barrel. The type C wines were bottled at six months without adding sulfur dioxide. One year after the ageing started, the wines were analyzed. In this study, three new casks of each type of oak were employed, so all results were obtained in triplicate. All the casks were supplied by the same Company and were made in identical conditions: wood submitted to natural outdoor drying and medium degree of toasting. 2.2. Microbiological analysis of wines The wines were sown in duplicate quantities of 500 ml on Petri dishes with DBDM (0.7% Yeast Nitrogen Base; 0.7% ethanol; 0.001% cycloheximide; 0.01% ac. p-cumaric; 0.002% bromocresol green, 0.02% chloramphenicol, 20% agar, pH 5,4 adjusted with sorbic acid), a specific medium for detecting the presence of Brettanomyces/ Dekkera yeasts (Rodrígues, Gonçalves, Pereira-da-Silva, MalfeitoFerreira, & Loureiro, 2001). The yeast plates were incubated at 25 C for 21 days. Some diphenyl crystals (approximately 100 mg/plate) (Panreac Química SA, Barcelona, Spain), were added to the plates in order to impede the development of molds. Colonies were counted and cell populations (cfu) calculated from duplicate analyses. From each plate, ten colonies were randomly selected and stored for later analysis in LM medium (10% D-glucose, 5% mycopeptone, 3% yeast extract, 3% malt extract, 20% bacteriological agar). 2.3. Molecular characterization of yeasts The identification of yeasts species isolated from the DBDM medium was necessary because the growth of genera other than n Brettanomyces in the DBDM medium had been reported (Oco et al., 2013). This analysis was carried out using PCR-RFLP of the 5.8S-ITS region of the ribosomal DNA, following the method proposed by Esteve-Zarzoso, Belloch, Uruburu, and Querol (1999). The primers used to amplify the region were those described by White, Bruns, Lee, and Taylor (1990). The amplification reaction took place in the GeneAmp® PCR System 2700 thermocycler (Applied Biosystems). For the analysis of restriction of the product of PCR, 5 ml aliquots were subjected to digestion by the HinfI, HaeIII and CfoI enzymes. The identification of the isolated yeasts was performed through a comparison of the restriction profile of each yeast with those obtained in our laboratory in other works and with those
177
contained in the Yeast-id-database at www.yeast-id.com (Valencia University and CSIC, Spain). 2.4. Clonal characterization of Brettanomyces isolates Clonal characterization of Brettanomyces isolates was conducted by using the restriction endonuclease analysis pulse field gel electrophoresis method (REA-PFGE), described by Miot-Sertier and Lonvaud-Funel (2007) with some modifications in culture cell conditions, spheroplast obtention, cellular membrane rupture and plug washes. B. bruxellensis samples were prepared by growing strains for 24 h in a YPG medium under agitation (80 rpm) at 28 C. Instead of using lyticase, we used a mix of Zymolyase (1 mg/ml) from Arthrobacter luteus and Novozyme (1 mg/ml) from Trichoderma harzianum as suggested by Martorell (2006). Cellular membrane rupture was carried out with Proteinase K from Tritirachium album instead of Pronase E. The lasted two plug washes were carried out with TrisClH 10 mM instead of TE. Digested chromosomal DNA was separated using the Cheff-DRR III System (Bio Rad). Gels were stained with etidium bromide (0.5 mg/ml) and photographed under UV light using an Image Store 5000 (UVP) (Bio Rad). 2.5. Ethylphenol analysis Ethylphenol content was analyzed via gas Chromatography. Extraction of these compounds was carried out following the pez, Cacho, and Ferreira methodology described by Ortega, Lo (2001), modified according to Martínez, Ojeda, and Rubio (2011): 5 ml of centrifuged wine, 9.5 ml of saturated solution of ammonium sulphate, 15 ml of internal pattern (2-octanol and 3.4dimethylphenol) and 0.200 ml of dichloromethane were placed in a tube and shaken horizontally for 60 min at 400 rpm. Afterward, the tube was centrifuged at 0 C and 2500 rpm during 10 min. Finally organic phase from the bottom of the tube was recovered and analyzed. Gas Chromatography analysis was carried out on a 2 ml extract, in splitless (0.5 min) in a Hewlett Packard gas chromatograph (HP-6890 series II with FID detector). Compound separation was performed in a DB-WAX 50 m 0.20 mm capilar column, with 1 ml/min of nitrogen as a carrier gas. Chromatographic conditions were: injector temperature 250 C, detector temperature 240 C, initial oven temperature 75 C (5 min) increasing 3 C/min to 240 C (20 min). Compound identification and quantification were conducted by comparing retention time of pattern substances by means of calibration lines obtained with compound extracts in hydroalcoholic solutions. 2.6. Statistical analysis Statistical analysis of the data was performed using SPSS Version 20.0 statistical package for Windows (SPSS, Chicago, IL, USA). Data were analyzed using analysis of variance; differences between means were compared using the Tukey test at 0.05 probability level. 3. Results and discussion 3.1. Presence of Brettanomyces in wines No spoilage due to Brettanomyces had happened prior to the beginning of ageing because the levels of ethylphenols in the initial wine (Table 1) were low. The wine was well-protected with sulfur dioxide and the casks used could not contaminate it because they were new, and the toasting process eliminates contamination. So, no wine spoilage from this source was foreseeable from this cause.
178
P. Rubio et al. / Food Control 54 (2015) 176e180
However, after 12 months' ageing, wine spoilage was noted and also the presence of Brettanomyces and high levels of ethylphenols were detected in all the wines (Table 1). Spoilage and taint was also evident in the wine bottle-aged for 12 months, indicating that the source of Brettanomyces was the initial wine. Martorell et al. (2006) had showed that contamination of wines by Brettanomyces in most cases occurs before the wines are put into casks. Renouf and Lonvaud-Funel (2007) also indicated that despite the hygiene measures taken in wineries, the development of B. bruxellensis in wine at the beginning of the ageing period is still not prevented and that earlier phases in the work in cellars should be studied as a possible source of contamination. In a previous work carried out by our team (Garijo et al. 2014) we observed that almost 30% of red wines analyzed at the end of malolactic fermentation, contained Brettanomyces, most of them at a low level (1e10 cfu/ml) that will not produce wine spoilage. But if those values increase during the following months to reach high levels, taint will become evident. Molecular characterization of isolates from the DBDM medium showed that they all belonged to B. bruxellensis specie. All the wines analyzed after 12 months ageing contained these yeasts, showing clear differences between the wines which had spent 6 months in bottle and those that had only aged in casks (Table 1). The wines aged only in the cask had a Brettanomyces level between 0 cfu/ml (Russian oak) and 76 cfu/ml (Chinese oak), whereas those aged in both cask and bottle showed a higher level that was uncountable (higher than 300 cfu/ml). The lower level detected in wines aged 12 months in the cask was probably due to the addition of sulfur dioxide and racking carried out at 6 months. The antimicrobial potential of SO2 against Brettanomyces, and the effectiveness of physical treatment like racking to remove yeast cells from barrels have already been shown (Oelofse, Pretorius, & Du Toit, 2008; rez-Lepe, Morata, & Caldero n, 2007). In this study the Su arez, Sua inhibitory effect of these two actions (sulfiting and racking) overcame the stimulating effect of oxygen on Brettanomyces growth (Kheir, Salamech, Strehaiano, Brandam, & Lteif, 2013; Uscanga, lia, & Strehaiano, 2003). So, ageing only in aerobic conditions De (12 months cask) with racking and sulfur dioxide addition, showed lower Brettanomyces levels than the combined aerobic and anaerobic maturation (6 months in cask and 6 in bottle) with racking but without sulfur dioxide addition. These data confirm that SO2 is a crucial element inhibiting Brettanomyces (du Toit, Pretorius, & Lonvaud-Funel, 2005). Wines that were only aged in anaerobic conditions (12 months in the bottle) also showed a high Brettanomyces level, showing that in anaerobic conditions too, Brettanomyces can develop and spoil
Table 1 Brettanomyces and ethylphenol (4EG and 4EP) content in wines subjected to different ageing conditions for 12 months. Ageing time (months) Brettanomyces 4-EG (mg/L) 4EP (mg/L) (ufc/mL) Barrel Bottle Initial wine e Ageing conditions Bottle e Oak Americana 12 Oak Frencha 12 Oak Russiana 12 a Oak Chinese 12 Oak American 6 Oak French 6 Oak Russian 6 Oak Chinese 6
e 12 e e e e 6 6 6 6
e Uncountable 46 11 0 76 Uncountable Uncountable Uncountable Uncountable
7 199 180 175 179 180 181 177 196 172
the wine, even when they are present at a low level in the initial wine. So, winemakers must check these yeasts are absent in the wine at the time of bottling. Due to the impossibility of accurately counting the number of Brettanomyces colonies grown in the DBDM medium of wines aged six months in the cask and the other six in the bottle, it was impossible to establish differences due to oak origin in these ageing conditions. But it was possible in samples coming from wines caskaged for 12 months. The level of Brettanomyces at the end of 12 months maturation in oak casks was lower in French and Russian oak (both Q. petraea) than in American (Q. alba) and Chinese (Q. mongolicus) oak. This could be due to the characteristics of each ~ oz, Ferna ndez oak species and differences in porosity (Cadahía, Mun n, & García-Vallejo, 2001; Cadahía, Vareas, Ferna ndez de de Simo n, & García-Vallejo, 2001; Martínez, Rubio, & Rodríguez, 2011). Simo 3.2. Clonal diversity of B. bruxellensis In Table 2 it can be seen that the different kind of ageing favors the development of different clones of Brettanomyces. All these strains were probably present in the starting wine because ageing was conducted using new barrels. In wines aged in anaerobic conditions for one year in the bottle, only clone I has been detected. In wines aged in oak casks for 6 or 12 months clone I was not detected, and others developed. The majority clone in wines aged at the beginning of the period in aerobic conditions (6 or 12 months in oak barrel), was clone II. This clone was the only one detected in wines aged for 12 months in oak casks, and the majority one in wines which had spent the first 6 months in oak casks. In wines with combined ageing in barrel and bottle (aerobic plus anaerobic conditions), although clone II was the predominant one, other clones (III and IV) were identified, probably due to the absence of sulfur dioxide addition at the time of bottling, which favored other strains permanence and/or development. Barata et al. (2008) have showed different sulfur dioxide sensitivity among B. bruxellensis strains. These results would indicate that all these clones were present in the initial wine and the subsequent development of each different strain could depend on the greater or lesser adaptation to the ageing conditions of the wine. These data also suggest that oxygen availability at the beginning of the ageing process, during the yeast multiplication stage, can affect the presence and selection of different clones of this species. Clonal diversity found in B. bruxellensis was low, coinciding with that obtained by other authors (Albertín et al. 2014). Only 4 different strains were identified from all the colonies analyzed (Fig. 1) and one of them (strain II) was clearly predominant (76%).
Table 2 Percentage (%) of different strains of Brettanomyces found in the wines.
14 a a a a a a a a a
1576 1382 1270 1305 1298 1471 1451 1553 1237
a b b b b ab ab a b
a Racked and sulphited at 6 months. For each different ageing condition (12 months in the cask or 6 months' cask and six in the bottle), different letters indicate differences in origin of the oak (p 0.05).
Ageing time (Months)
Strains of Brettanomyces
Barrel
Bottle
I
II
III
IV
e
e
e
e
e
12 e e e e 6 6 6 6
100 e e e e e e e e
e 100 100 e 100 100 90 50 70
e e e e e e 10 e e
e e e e e e e 50 30
Initial wine e Ageing conditions Bottle e Oak Americana 12 Oak Frencha 12 Oak Russiana 12 a Oak Chinese 12 Oak American 6 Oak French 6 Oak Russian 6 Oak Chinese 6 a
Racked and sulphited at 6 months.
P. Rubio et al. / Food Control 54 (2015) 176e180
Conterno, Lucy Joseph, Arvik, Henick-Kling, and Bisson (2006) grouped 47 isolates in six clusters, Agnolucci et al. (2009) found seven haplotypes of B. bruxellensis in 84 isolates tested, and Campolongo, Rantsiou, Giordano, Gerbi, and Cocolin (2010) found 10 different strains in 196 isolates. Diversity level of this species is lower than that found in Saccharomyces cerevisiae. In this latter pez, Tenorio, and Gutie rrez (2005) species, Santamaría, Garijo, Lo found 38 different profiles out of 140 isolates studied, and Capece, Romaniello, Siesto, and Romano (2012) found 39 strains among 132 isolates, which means significant genetic variability. These results would indicate that the presence of Brettanomyces (cfu/ml and clones) during ageing is more a consequence of the ageing conditions (aerobiosis/anaerobiosis and sulfiting) than oak origin. 3.3. Wine ethylphenol content 4-ethylguaiacol and 4-ethyl phenol content were very low in the initial wine (Table 1). However, after 12 months and regardless of the ageing conditions, concentration of these two compounds was very high in all cases, widely exceeding the content level at which wines are generally rejected. The quality of wine may be negatively affected by concentrations above 425 mg/L (1:10 ratio of 4ethylguaiacol/4-ethyl-phenol in red wines), which is cited as the aroma threshold (Chatonnet, Boidron, & Pons, 1990). Wines aged only in anaerobic conditions had the highest level of both 4-ethylguaiacol and 4-ethyl phenol (Table 1). In Table 1 it can be seen that when ageing was carried out in aerobic conditions (12 months in the cask), no significant differences in ethylphenols content were found depending on the origin of the oak. However, when wines remained six months in the cask and another six in the
179
bottle, 4-ethyl phenol concentration was significantly different between wines stored in Russian and Chinese barrels. If ethyl phenol levels coming from wines aged 12 months in the cask are compared with those coming from wines that were 6 months in the cask and the other 6 in the bottle (statistical study not included), it was observed that the latter, despite the variability found, showed higher 4-ethyl phenol concentration (Table 1). This could be due to the racking and sulfiting carried out at six months in the former, leading to a reduction in the microbial population which could be the cause of the lower ethyl phenol accumulation in the wines. These differences can also be caused by the different strains of Brettanomyces found in the wines. Different studies have showed that the growth rate and ethylphenol production by Brettanomyces are strain-dependent characteristics (Farina, Boido, Carrau, & Dellacassa, 2007; Silva et al. 2005; Vigentini et al. 2008). Despite the significant differences, the similar levels of ethylphenols in all the samples were probably due to the predominance of strain II in all of them. The similar levels of ethylphenols in all three types of ageing also suggest that these compounds were most probably produced at the beginning of the ageing process, and the SO2 addition at six months killed the cells, but ethylphenols were already present in the wines. Therefore, high CFU numbers must have been attained, but were unreported because the number of cells and ethylphenols were determined only once at the end of 12 months. The influence of the kind of oak in the 4-ethylphenol content of wines depending on the ageing conditions showed that only Russian and French oak (both Q. petraea) levels are significantly higher in wines with 6 months' cask-ageing and 6 months in the bottle than those aged for 12 months in the cask. In the case of American and Chinese oak, although there were not significant differences, levels in wines with 6 months each spent ageing in cask and bottle were higher and lower respectively than those aged for 12 months in the cask. These data could be explained by the kind of oak (species and porosity), and also by the number and strains of Brettanomyces found in them.
4. Conclusions
Fig. 1. Pulse field gel electrophoresis of NotI digested chromosomal DNA from Brettanomyces bruxellensis strains I, II, III and IV. Lanes M: Lambda Ladder PFG Marker (Biolabs).
When Brettanomyces cells are present, even at a low level, in wines which undergo ageing, whether in the cask or in the bottle, there is a high risk of spoilage, even in well-protected wines. So, the periodic control of wines during cask-ageing to avoid the development of these spoiling yeasts, and to ensure that no Brettanomyces cells get inside the bottle is essential to avoid wine spoilage. Brettanomyces development (cfu/ml and strains) during caskageing, is affected more by ageing conditions than by the origin of the oak. The inhibitory effect of the two actions (sulfiting and racking) overcame the stimulating effect of oxygen on Brettanomyces growth: ageing only under aerobic conditions with racking and sulfur dioxide addition, showed lower Brettanomyces levels than the combined aerobic and anaerobic maturation with racking but without sulfur dioxide addition. These data confirm that SO2 is a crucial element inhibiting Brettanomyces. Four different B. bruxellensis clones were found in the assays, all of them coming from the initial wine. The development of different strains in different assays could depend on the greater or lesser adaptation to the ageing conditions of the wine. No big significant differences in ethyl phenol levels among wines aged in casks made from oak of different origin were found. However, wines aged in Chinese oak revealed different behavior to the other three in terms of the level of Brettanomyces and in the ethylphenol content, probably due to the fact that this oak has the highest porosity.
180
P. Rubio et al. / Food Control 54 (2015) 176e180
Acknowledgments This study has been undertaken with a grant from the Government of La Rioja, Spain (Project PR-11-11). References Agnolucci, M., Vigentini, I., Capurso, G., Merico, A., Tirelli, A., Compagno, C., et al. (2009). Genetic diversity and physiological traits of Brettanomyces bruxellensis strain isolated from Toscan Sangiovese wines. International Journal of Food Microbiology, 3, 238e244. Albertín, W., Panfili, A., Miot-Sertier, C., Goulielmakis, A., Delcamp, A., Salin, F., et al. (2014). Development of microsatellite markers for the rapid and reliable genotyping of Brettanomyces bruxellensis at strain level. Food Microbiology, 42, 188e195. Barata, A., Caldeira, J., Botheleiro, R., Pagliara, D., Malfeito-Ferreira, M., & Loureiro, V. (2008). Survival patterns of Dekkera bruxellensis in wines and inhibitory effect of sulphur dioxide. International Journal of Food Microbiology, 121, 201e207. ~ oz, L., Ferna ndez de Simo n, B., & García-Vallejo, M. C. (2001). Cadahía, E., Mun Changes in low molecular weight phenolics in Spanish, French and American oak during natural seasoning and toasting. Journal of Agricultural and Food Chemistry, 49, 1790e1798. n, B., & García-Vallejo, M. C. (2001). Cadahía, E., Vareas, S., Fern andez de Simo Evolution of Ellagitannins in Spanish, French and American oak wood during seasoning and toasting. Journal of Agricultural and Food Chemistry, 49, 3677e3684. Campolongo, S., Rantsiou, K., Giordano, M., Gerbi, V., & Cocolin, L. (2010). Prevalence and biodiversity of Brettanomyces bruxellensis in wine from Northwestern Italy. American Journal of Enology and Viticulture, 61(4), 486e491. Capece, A., Romaniello, R., Siesto, G., & Romano, P. (2012). Diversity of Saccharomyces cerevisiae yeasts associated to spontaneously fermenting grapes from an Italian “heroic vine-growing area”. Food Microbiology, 31, 159e166. ristiques et interest du bois de che ^ne de Russie Chatonnet, P. (1999). Caracte -Moreau: nouvelles acquisitions et perspectives. Revue Francaise Adyghe d'Oenologie, 175, 20e24. Chatonnet, P., Boidron, J. N., & Pons, M. (1990). Elevage des vins rouges en fûts de ne: evolution de certains compose s volatils et de leur impact aromatique. che Sciences des Aliments, 10, 565e587. Chatonnet, P., Dubordieu, D., & Boidron, J. (1993). Synthesis of volatile phenols by Saccharomyces cerevisiae in wines. Journal of the Science of Food and Agriculture, 62, 191e202. Chatonnet, P., Dubourdieu, D., Boidron, J. N., & Pons, M. (1992). The origin of ethylphenols in wines. Journal of the Science of Food and Agriculture, 60, 165e178. Conterno, L., Lucy Joseph, C. M., Arvik, T. J., Henick-Kling, T., & Bisson, L. F. (2006). Genetic and physiological characterization of Brettanomyces bruxellensis strains isolated from wines. American Journal of Enology and Viticulture, 57(2), 139e147. Esteve-Zarzoso, B., Belloch, C., Uruburu, F., & Querol, A. (1999). Identification of yeasts by RFLP analysis of the 5.8S rRNA gene and the two ribosomal internal transcribed spacers. International Journal of Systematic Bacteriology, 49, 329e337. Farina, L., Boido, E., Carrau, F., & Dellacassa, E. (2007). Determination of volatile phenols in red wines by dispersive liquid-liquid microextraction and gas chromatography-mass spectrometry detection. Journal of Chromatography A, 1157, 46e50. ndez de Simo n, B., & Cadahía, E. (2006). Evolution of volatile compounds in a Ferna Spanish red wine during two years bottled, after aging in barrels made of Spanish, French and American oak Wood. Analytica Chimica Acta, 563, 198e203. ndez de Simo n, B., Cadahía, E., & Jalocha, J. (2003). Volatile compounds in a Ferna Spanish red wine aged in barrels made of Spanish, French, and American oak wood. Journal of Agricultural and Food Chemistry, 51, 7671e7678. Fugelsang, K. C., & Zoecklein, C. G. (2003). Population dynamics and effects of Brettanomyces bruxellensis strains on Pinot noir (Vitis vinifera L.) wines. American Journal of Enology and Viticulture, 54, 294e300. n, T., & Ancín-Azpilicueta, C. (2006). Effect of oak barrel type on the Garde-Cerda volatile composition of wine: storage time optimization. LWT Food Science and Technology, 39, 199e205. pez-Alfaro, I., Garde-Cerda n, T., Lo pez, R., Garijo, P., Gonz alez-Arenzana, L., Lo Santamaría, P., et al. (2014). Analysis of grapes and the first stages of the vinification process in wine contamination with Brettanomyces bruxellensis. European Food Research and Technology. http://dx.doi.org/10.1007/s00217-014-2351-4. lez-Arenzana, L., Santamaría, P., Lo pez, R., Garijo, P., Gutie rrez, A. R., GardeGonza Cerd an, T., et al. (2013). Microwave technology as a new tool to improve microbiological control of oak barrels: a preliminary study. Food Control, 30, 536e539. Guzzon, R., Widman, G., Malacarne, M., Nardin, T., Nicolini, G., & Larcher, R. (2011). Survey of the yeast population inside wine barrels and the effects of certain techniques in preventing microbiological spoilage. European Food Research and Technology, 233, 285e291.
Jiranek, V., Grbin, P., Yap, A., Barnes, M., & Bates, D. (2008). High power ultrasonic as a novel tool offering new opportunities for managing wine microbiology. Biotechnology Letters, 30, 1e6. Kheir, J., Salamech, D., Strehaiano, P., Brandam, C., & Lteif, R. (2013). Impact of volatile phenols and their precursors on wine quality and control measures of Brettanomyces/Dekkera yeasts. European Food Research and Technology, 237, 655e671. Loureiro, V., & Malfeito-Ferreira, M. (2006). Food spoilage microorganisms. In C. Blackburn (Ed.), Spoilage activities of Dekkera/Brettanomyces spp (pp. 354e398). Cambridge, England: Woodhead Publishing Limited. tico del roble espan ~ ol Quercus Martínez, J., Ojeda, S., & Rubio, P. (2011). Aporte aroma pyrenaica durante la crianza en barrica de vinos de Tempranillo. In XI Congreso n Enolo gica (GIENOL) (pp. 555e558). Nacional de Investigacio Martínez, J., Rubio, P., & Rodríguez, R. (2011). Experiencias con robles de China y Rusia en la crianza de vinos de Rioja. Enoviticultura, 8, 20e27. pidos para la deteccio n de sistemas ra n, Martorell, P. (2006). Desarrollo y aplicacio n y caracterizacio n de levaduras alterantes en alimentos (Ph.D. thesis). identificacio ~ a: Universidad de Valencia. Espan ndez-Espinar, M. T., Loureiro, V., Martorell, P., Barata, A., Malfeito-Ferreira, M., Ferna & Querol, A. (2006). Molecular typing of the yeast species Dekkera bruxellensis and Pichia guilliermondii recovered from wine related sources. International Journal of Food Microbiology, 106, 789e884. Miot-Sertier, C., & Lonvaud-Funel, A. (2007). Development of a molecular method for the typing of Brettanomyces bruxellensis (Dekkera bruxellensis) at the strain level. Journal of Applied Microbiology, 102, 555e562. n, E., Garijo, P., Sanz, S., Olarte, C., Lo pez, R., Santamaría, P., et al. (2013). Analysis Oco of airborne yeast in one winery over a period of one year. Food Control, 30, 585e589. Oelofse, A., Pretorius, I. S., & Du Toit, M. (2008). Significance of Brettanomyces and Dekkera during winemaking: a synoptic review. South African Journal for Enology and Viticulture, 29(2), 128e144. Ojeda, S. (2012). Nuevos orígenes de la madera de roble para la crianza de vinos tintos de la D.O.Ca. Rioja (Ph.D. thesis). Spain: Universidad de La Rioja. lez-SanJose , M. L., & Gonza lez-Huerta, C. (2008). ConsidOrtega-Heras, M., Gonza eration of the influence of aging process, type of wine and oenological classic parameters on the levels of Wood volatile compounds present in red wines. Food Chemistry, 103, 1434e1448. pez, R., Cacho, J., & Ferreira, V. (2001). Fast analysis of important wine Ortega, C., Lo volatile compounds. Development and validation of a new method based on gas chromatographic-flame ionisation detection analysis of dichlorometane microextracts. Journal of Chromatography A, 923, 205e214. Renouf, V., & Lonvaud-Funel, A. (2007). Development of an enrichment medium to detect Dekkera/Brettanomyces bruxellensis, a spoilage wine yeast, on the surface of grape berries. Microbial Research, 162, 154e167. Rodrígues, N., Gonçalves, G., Pereira-da-Silva, S., Malfeito-Ferreira, M., & Loureiro, V. (2001). Development and use of a differential medium to detect yeast of the genera Dekkera/Brettanomyces. Journal of Applied Microbiology, 90, 588e599. pez, R., Tenorio, C., & Gutie rrez, A. R. (2005). Analysis of Santamaría, P., Garijo, P., Lo yeast population during spontaneous alcoholic fermentation: effect of the age of the cellar and the practice of inoculation. International Journal of Food Microbiology, 103, 49e56. Silva, L. R., Andrade, P. B., Valentao, P., Seabra, R. M., Trujillo, M. E., & Vel azquez, E. (2005). Analysis of non-coloured phenolics in red wine: effect of Dekkera bruxellensis yeasts. Food Chemistry, 89, 185e189. rez-Lepe, J. A., Morata, A., & Caldero n, F. (2007). The production of Su arez, R., Sua ethylphenols in wine by yeasts of the genera Brettanomyces and Dekkera. A review. Food Chemistry, 102, 10e21. du Toit, W. J., Pretorius, I. S., & Lonvaud-Funel, A. (2005). The effect of sulphur dioxide and oxygen on the viability and culturability of a strain of Acetobacter pasteurianus and a strain of Brettanomyces bruxellensis isolated from wine. Journal of Applied Microbiology, 98, 862e871. lia, M. L., & Strehaiano, P. (2003). Brettanomyces bruxellensis: Uscanga, M. G. A., De effect of oxygen on growth and acetic acid production. Applied Microbiology Biotechnology, 61, 157e162. Vigentini, I., Romano, A., Compagno, C., Merico, A., Molinari, F., Tirelli, A., et al. (2008). Physiological and oenological traits of different Dekkera/Brettanomyces bruxellensis strains under wine-model conditions. FEMS Yeast Research, 8, 1087e1096. Vivas, N., Absalon, C., Benoist, F., Vitry, Ch, Grazillier, S., De Revel, G., et al. (2000). ^nes europe ens Q. robur L. et Q. petraea (Matt.) Liebl.: Analyse des Les che s oenologiques des diffe rents massifs forestiers. In Ve Colloque des potentialite Sciences et Techniques de la Tonnellerie (pp. 31e37). Wedral, D., Shewfelt, R., & Frank, J. (2010). The challenge of Brettanomyces in wine. LWTeFood Sciences and Technology, 43, 1474e1479. White, T. J., Bruns, T., Lee, S., & Taylor, J. (1990). Amplification and direct sequencing of fungi ribosomal RNA genes for phylogenetics. In M. A. Innis, D. H. Gelfalnd, J. J. Sninsky, & T. T. White (Eds.), PCR protocols. A guide to methods and applications (pp. 315e320). San Diego: Academic Press.