Abstract
The ability of commercial strains of Oenococcus oeni to degrade tartaric acid ester-bound hydroxycinnamic acids (TAE-HCAs) and their impact on the production of volatile phenols by Brettanomyces bruxellensis was investigated. Of 10 commercial O. oeni strains evaluated, only one strain, O. oeni Viniflora Oenos (VFO), was able to degrade TAE-HCAs during growth in a Pinot noir wine. This degradation resulted in an increase in the corresponding free forms of the hydroxycinnamic acids in the wine. As a result, growth of B. bruxellensis UCD-2049 in Pinot noir wine where malolactic fermentation (MLF) was conducted by VFO resulted in higher concentrations of 4-ethylphenol and 4-ethylguaiacol in this wine than in wine that did not undergo MLF or underwent MLF with O. oeni strains that did not degrade TAE-HCAs. While wineries must continue to use sound winemaking practices to prevent the growth of Brettanomyces spp. in their wines, minimizing the amount of free hydroxycinnamic acids in the wine will reduce the production of volatile phenols if growth of Brettanomyces spp. does occur. The use of an O. oeni strain that cannot degrade TAE-HCAs is a simple and practical strategy to help achieve this.
- Brettanomyces bruxellensis
- volatile phenols
- hydroxycinnamic acids
- malolactic fermentation
- Oenococcus oeni
Brettanomyces bruxellensis is regarded as the most important spoilage yeast encountered during winemaking as it survives in wine for long periods, requires minimal nutrients for growth, is difficult to control, and produces a number of sensorially potent spoilage compounds (Joseph et al. 2013, Puig et al. 2011, Loureiro and Malfeito-Ferreira 2006, Chatonnet et al. 1995). The spoilage compounds most commonly associated with B. bruxellensis wine contamination are the volatile phenols 4-ethylphenol (4-EP; Band Aid, medicinal, or barnyard aroma) and 4-ethylguaiacol (4-EG; smoky, clove, or leather aroma) (Romano et al. 2008, Chatonnet et al. 1995). 4-EP and 4-EG are produced in two-step enzymatic reactions from the hydroxycinnamic acids p-coumaric acid and ferulic acid, respectively (Chatonnet 1992). A hydroxycinnamic decarboxylase first converts each hydroxycinnamic acid to a vinylphenol, which a vinylphenol reductase reduces to an ethylphenol. Both p-coumaric and ferulic acids are naturally present in grapes and are typically found as esters of tartaric acid (as coutaric and fetaric acids, respectively). During winemaking, these tartaric acid esters may be hydrolyzed, resulting in the release of the free hydroxycinnamic acids (Nagel et al. 1979). However, this process generally occurs slowly during aging, so wines can still retain high amounts of tartaric acid ester-bound hydroxycinnamic acids (TAE-HCAs; Ginjom et al. 2011, Nagel et al. 1979). Because ester-bound forms of hydroxycinnamic acids represent a potential pool of precursor compounds for hydroxycinnamic acid production by B. bruxellensis, Schopp et al. (2013) investigated whether B. bruxellensis was able to utilize TAE-HCAs. The authors reported that B. bruxellensis was unable to metabolize TAE-HCAs and could convert free hydroxycinnamic acids only to volatile phenols. However, other wine microorganisms besides Brettanomyces spp. are present throughout the winemaking process. If other wine microorganisms can degrade TAE-HCAs, the subsequent release of free hydroxycinnamic acids could result in higher production of volatile phenols by Brettanomyces spp.
A number of wine microorganisms, such as some lactic acid bacteria (LAB) and Saccharomyces cerevisiae, can degrade free hydroxycinnamic acids (Couto et al. 2006, Chatonnet et al. 1993, Cavin et al. 1993), but little is known about the ability of wine LAB to also degrade TAE-HCAs. Although Hernandez et al. (2007) and Cabrita et al. (2008) noted an increase in free hydroxycinnamic acids after malolactic fermentation (MLF), the identity of the wine LAB responsible for the increase was unknown because MLF was either being conducted spontaneously (Hernandez et al. 2007) or involved a mixed culture of wine LAB (Cabrita et al. 2008). Burns and Osborne (2013) observed an increase in free hydroxycinnamic acids (i.e., of p-coumaric and caffeic acids) after MLF and noted that only one out of four Oenococcus oeni strains used in MLF caused this increase. However, the authors used only a small number of different O. oeni strains and did not report the effect on volatile phenol production by B. bruxellensis. Therefore, the objectives of the present study were to screen a number of commercial O. oeni strains for the ability to degrade TAE-HCAs in wine and to examine the impact of this degradation on volatile phenol production by B. bruxellensis.
Material and Methods
Microorganisms
Commercial O. oeni strains were purchased from several sources. O. oeni strains Viniflora Oenos (VFO) and Viniflora CH-35 were procured from Chr. Hansen (Hørsholm, Denmark); strains Lalvin VP41, Lalvin MBR31, Lalvin Inobacter, Lalvin Elios 1, Enoferm Alpha, and Enoferm Beta were from Lallemand (Montreal, Canada); and strains 350 PreAC and 450 PreAC were from Laffort (Petaluma, CA). After isolation from a single colony, the strains were maintained in de Man, Rogosa, and Sharpe (MRS) stabs (pH 4.5) and stored at 4°C. For use in cultures, they were transferred from stabs to acidic grape juice (AGJ) broth (250 mL/L white grape juice, 5 g/L yeast extract, 0.125 g/L magnesium sulfate, 0.0025 g/L manganese sulfate, and 1 mL/L 5% Tween solution, pH 3.5) and grown at 25°C for seven days. Cells were harvested by centrifugation (4,000 g for 20 min) and resuspended in 0.2 M phosphate buffer (pH 7.0) before inoculation.
The B. bruxellensis strain UCD-2049 was obtained from the UC Davis Department of Viticulture and Enology Culture Collection housed in the University of California Davis Department of Viticulture and Enology (Davis, CA). B. bruxellensis UCD-2049 was streaked and isolated on yeast–peptone–dextrose (YPD) agar (10 g/L yeast extract, 20 g/L peptone, 20 g/L dextrose, and 20 g/L agar, pH 6.5), and single colonies were grown in YPD broth (pH 6.5). After seven days of growth at 25°C, cells were stored in glycerol (15% v/v) at −80°C for future use. For use in cultures, B. bruxellensis UCD-2049 was prepared from frozen stocks by inoculation into AGJ broth (pH 3.5) and growth at 25°C for five days until stationary phase was reached. Cells were harvested by centrifugation (4,000 g for 20 min) and resuspended in 0.2 M phosphate buffer (pH 7.0) prior to inoculation.
Winemaking
Pinot noir wine was produced at the Oregon State University Research Winery from grapes harvested from Woodhall Vineyard (Alpine, OR) in 2011 and 2012. The time of harvest was determined by measuring soluble solid levels and by perceived fruit ripeness, assessed by the vineyard manager. Grapes were stored at 4°C for 24 hr and then hand-sorted and destemmed with a Velo DPC 40 destemmer/crusher (Altivole, Italy). Grapes were divided into three 100-L stainless steel tanks, each containing ~60 L of must. Thirty mg/L of SO2 (as potassium metabisulfite) was added to each tank, and the yeast nutrient Fermaid K (Lallemand) was added at 0.125 g/L. Each tank was inoculated with the commercial S. cerevisiae strain RC-212 (Lallemand) at a rate of 0.25 g/L after rehydration according to the manufacturer’s specification, and tanks were placed in a temperature-controlled room held at 27°C. Cap punch-downs were performed uniformly twice daily, and temperature and Brix were measured with an Anton-Paar DMA 35N density meter (Graz, Austria). Alcoholic fermentation in all three fermentations was completed (determined as a reducing sugar concentration of <0.2 g/100 mL) in 10 days as confirmed by testing with Bayer Clinitest tablets (Morristown, NJ).
After fermentation, the wines were pressed (Willmes Model 6048 pneumatic bladder press, Lorsch, Germany), pooled, and placed in a cold room at 4°C for 48 hr to cold settle. Thereafter, wines were racked and filtered through a plate and frame filter fitted with 20 cm × 20 cm Beco K-1 3.0-μm nominal filter sheets (Langenlonsheim, Germany). Wines were then successively filtered through a 1.0-μm nylon cartridge and a 0.45-μm sterile polyethersulfone cartridge (G.W. Kent, Ypsilanti, MI). The filtered wines were dispensed into sterilized 12-L carboys and stored at 4°C for future use. Basic wine parameters for the 2012 Pinot noir were 13.2% (v/v) alcohol, pH 3.53, and 0.67 g/100 mL titratable acidity (determined as grams of tartaric acid). Basic wine parameters for the 2011 Pinot noir were 12.6% (v/v) alcohol, pH 3.44, and 0.68 g/100 mL titratable acidity.
Degradation of TAE-HCA
The O. oeni strains were prepared from stabs as described above and were screened for TAE-HCA degradation in wine. After harvesting by centrifugation, the O. oeni strains were inoculated at ~1 × 105 cfu/mL into 20 mL of 2012 Pinot noir wine in sterile screwcapped test tubes and incubated at 25°C. The amount of inoculum was based on previous experiments that assessed bacterial counts by plating after growth for a set period of time and temperature. An uninoculated control was also prepared, and all treatments were performed in triplicate. MLF was followed by degradation of malic acid as assessed by an enzymatic test kit (R-Biopharm, Darmstadt, Germany), and all strains completed MLF in 14 days. After 21 days, samples were taken and stored at −80°C for later analysis of hydroxycinnamic acids by HPLC with diode array detection (DAD).
The timing of TAE-HCA degradation relative to the MLF was investigated by inoculating O. oeni strains VFO, VP41, and Alpha (prepared from stabs as described above) into 500 mL of 2012 Pinot noir in 500-mL Schott bottles at a rate of ~1 × 106 cfu/mL. An uninoculated control was also prepared, and all treatments were performed in triplicate and incubated at 25°C. Bacterial growth was assessed by plating on MRS agar (pH 4.5), while MLF was monitored by measuring malic acid levels as described above. Samples were taken regularly and frozen for storage at −80°C for later analysis of hydroxycinnamic acids via HPLC-DAD.
Volatile phenol production by B. bruxellensis UCD-2049
O. oeni strains VFO, VP41, and Alpha were prepared from stabs as previously described and inoculated into 500 mL of 2011 Pinot noir in 500-mL Schott bottles at a rate of ~1 × 105 cfu/mL. An uninoculated control was also prepared and all treatments were prepared in triplicate. Samples were incubated at 25°C until MLF was complete, confirmed as described above. The wines were then inoculated with B. bruxellensis strain UCD-2049, prepared from frozen cultures as described above, at a rate of ~1 × 104 cfu/mL. Growth of B. bruxellensis was followed by plating on YPD media and by taking samples for storage at −80°C for later hydroxycinnamic acid analysis as described above. After degradation of p-coumaric acid was completed, a 50-mL culture sample was taken and 4-EP and 4-EG were measured as outlined by Jensen et al. (2009). In brief, 4-EP and 4-EG were analyzed using a headspace–solid phase microextraction method with an 85-μm polyacrylate fiber (Supelco, Bellefonte, PA). The fiber was thermally desorbed at 280°C for 3 min at the injection port of a GC-MS-MS (Varian model 4000, Walnut Creek, CA). Separation was achieved with a DB-5MS capillary column (0.18 mm, i.d. 20 μm) with 0.18-mm film thickness (J&W/Agilent Technologies, Wilmington, DE). The carrier gas, helium, was held at a constant flow of 0.8 mL/min. The temperature program consisted of 40°C held for 2.0 min, an increase of 20°C/min to 160°C held for 0 min, followed by an increase of 50°C/min to 300°C held for 0.2 min. The volatile phenols were identified by retention times as well as by fragmentation patterns compared with those from chemical standards.
Hydroxycinnamic acid analysis
Hydroxycinnamic acids were quantified by HPLC-DAD as described by Burns and Osborne (2013). Prior to HPLC analysis, wine samples were centrifuged at 12,000 rpm for 10 min in an Allegra X-22 centrifuge (Beckman Coulter, Brea, CA). Wines were sampled in 20-μL aliquots, and hydroxycinnamic acids were detected by scanning wavelengths from 190 to 700 nm. Phenolic compounds were identified according to UV-vis spectra, and retention times of known standards (caftaric, caffeic, and p-coumaric acids) were obtained from Sigma-Aldrich (St. Louis, MO). When chemical standards were not available (i.e., for coutaric acid), retention times were based on those reported by Hernandez et al. (2007) and verified with UV-vis spectra from Baranowski and Nagel (1981). Calibration curves were prepared for caffeic and p-coumaric acids. Caftaric acid was quantified using the caffeic acid curve, and coutaric acid was quantified using the p-coumaric acid curve.
Statistical analysis
Statistical analysis was performed using The R Project for Statistical Computing (Auckland, New Zealand). Significant sources of variation were evaluated using one-way analysis of variance (ANOVA) followed by Tukey’s honest significance difference test for mean comparison at the p < 0.05 level.
Results
Ten commercial O. oeni strains were examined for their ability to degrade TAE-HCAs in Pinot noir wine. Although all strains completed MLF, only one strain, O. oeni VFO, degraded the TAE-HCAs caftaric and coutaric acids, resulting in an increase of caffeic and p-coumaric acids in the wine. An increase in ferulic acid was also noted in wines in which VFO grew, although we were unable to measure the corresponding bound form of this hydroxycinnamic acid, fetaric acid. For the remaining nine strains (CH-35, VP41, MBR31, Inobacter, Elios 1, Alpha, Beta, 350 PreAC, and 450 PreAC), no changes in the concentration of free or TAE-HCAs were detected (data not shown).
A time course study was performed to determine when O. oeni VFO started to degrade TAE-HCAs during cell growth and malic acid degradation. Although the initial population of VFO in the culture was lower than expected given the amount of inoculum used, VFO still grew well and reached a maximum population of >1 × 107 cfu/mL (Figure 1A). During the logarithmic growth phase (i.e., during days 15 to 30), malic acid was degraded along with coutaric acid. Malic acid was degraded to levels of <0.03 g/L after 18 days, and degradation of coutaric acid began after 21 days and was complete by day 30. As coutaric acid was degraded, a corresponding increase in p-coumaric acid concentration was observed (Figure 1A). In contrast, when Pinot noir wine was inoculated with O. oeni strain Alpha, coutaric and p-coumaric acid concentrations did not change, even though the Alpha strain grew well and completely degraded the malic acid (Figure 1B). Inoculating O. oeni VP41 into Pinot noir gave the same results as observed for Alpha (data not shown).
Growth of O.oeni strains VFO (A) and Alpha (B) in Pinot noir wine and changes in the concentrations of malic, coutaric, and p-coumaric acids. Values represent means, and error bars denote SD.
The impact of O. oeni on volatile phenol production by B. bruxellensis was investigated by inoculating B. bruxellensis UCD-2049 into Pinot noir wine in which either the O. oeni VFO, Alpha, or VP41 strain had conducted MLF. At the completion of the MLF and prior to inoculation of B. bruxellensis UCD-2049, wines were assessed for free hydroxycinnamic acids and TAE-HCAs (Table 1). Compared with the uninoculated control, no significant changes in free and TAE-HCAs were observed for wines that had undergone MLF with the Alpha or VP41 strain (Table 1). However, wine that had undergone MLF with the VFO strain had significantly lower concentrations of caftaric and coutaric acids and higher concentrations of caffeic and p-coumaric acids (Table 1). For example, at the end of MLF with the VFO strain, the wine contained 6.9 mg/L of p-coumaric acid, while the other wines contained between 1.6 and 1.2 mg/L p-coumaric acid. B. bruxellensis UCD-2049 grew well in all treatments (Figure 2), although we noted a lower maximum population when UCD-2049 grew in wine in which MLF had been conducted by the VP41 strain (Figure 2D). We observed a decrease in p-coumaric acid during growth of B. bruxellensis UCD-2049 and no change in TAE-HCAs in the control wine or the wines that underwent MLF with the Alpha or VP41 strain (Figure 2A, C, D). Because of the degradation of coutaric acid by strain VFO, wine in which this strain had conducted MLF initially had a higher p-coumaric acid concentration than the control wine (Figure 2B). The p-coumaric acid was subsequently degraded by B. bruxellensis UCD-2049 during growth. At the completion of the experiment (40 days after B. bruxellensis inoculation), a wine sample was analyzed for free and TAE-HCAs as well as for 4-EP and 4-EG. This analysis showed that p-coumaric acid concentration had declined to very low levels by this time (0.1 mg/L) (Table 1). The control wine and wines that underwent MLF with strain Alpha or VP41 had essentially the same concentrations of 4-EP and 4-EG. However, wine that had undergone MLF with the VFO strain had significantly higher amounts of both 4-EP and 4-EG (Table 1). For example, while the control wine had a 4-EP concentration of 260.1 μg/L, the 4-EP concentration was more than five-fold higher in wine in which VFO had conducted the MLF (1580.3 μg/L).
Concentrations (mg/L) of free and tartaric ester-bound hydroxycinnamic acids in Pinot noir wine at the completion of MLF and after 40 days growth of B. bruxellensis UCD-2049, and final 4-ethylphenol (4-EP) and 4-ethylguaiacol (4-EG) concentrations (μg/L) in the wine.
Growth of B. bruxellensis UCD-2049 and changes in the concentrations of caffeic, caftaric, p-coumaric, and coutaric acids in Pinot noir wine that had not undergone MLF (A) or in which MLF had been conducted by either O. oeni strain VFO (B), Alpha (C), or VP41 (D). Values represent means, and error bars denote SD.
Discussion
Wine spoilage by Brettanomyces spp. is primarily caused by production of 4-EP and 4-EG from metabolized hydroxycinnamic acids (Chatonnet 1992). In grapes and wines, a large proportion of hydroxycinnamic acids is present as tartaric acid–bound esters and represents a large pool of precursors available for conversion to volatile phenols. Because B. bruxellensis is unable to degrade TAE-HCAs (Schopp et al. 2013), the ability of other wine microorganisms such as O. oeni to break down TAE-HCAs could significantly impact volatile phenol production by Brettanomyces spp. In this study, only O. oeni strain VFO was able to degrade TAE-HCAs resulting in an increase in the corresponding free hydroxycinnamic acids. This finding agrees with observations by Hernández et al. (2007) and Cabrita et al. (2008) who also reported an increase in these free acids after MLF. However, in our work, we have identified a specific LAB responsible for the TAE-HCA conversion to the free acids; by contrast, Hernández et al. (2007) had studied MLF that conducted spontaneously and Cabrita et al. (2008) had used a mixed culture of wine LAB.
The increased free hydroxycinnamic acid content in wines that underwent MLF with O. oeni strain VFO significantly increased the production of 4-EP and 4-EG during growth of B. bruxellensis UCD-2049 compared with wines in which the MLF had been conducted by other O. oeni strains. Because of the relatively low amount of free hydroxycinnamic acids in the original wine, only low amounts of 4-EP and 4-EG were produced in the control wine and in wines in which strain Alpha or VP41 had conducted the MLF. In fact, the levels of volatile phenols in these wines were at or below the reported sensory threshold concentrations for 4-EP and 4-EG in Pinot noir of 230 μg/L and 47 μg/L, respectively, reported by Chatonnet et al. (1990). Chatonnet (1992) reported a combined detection threshold of 426 μg/L when 4-EP to 4-EG were present at a 10 to 1 ratio, whereas Goode (2005) noted that trained judges could identify “Brett characters” only when combined volatile phenol concentrations exceeded 668 μg/L. In the present study, despite growing well in all wines, B. bruxellensis produced less than 230 μg/L 4-EP in wines that underwent MLF with strain Alpha or VP41. These findings underscore that hydroxycinnamic acid precursor concentrations are important determinants of the impact that spoilage with Brettanomyces spp. may have on a wine’s sensory profile. The results also suggest an additional cause for the reported variability in the severity of adverse sensory profiles, which is often attributed to diverse genetic and physiological factors in Brettanomyces spp. wine spoilage (Joseph et al. 2013, Conterno et al. 2006).
The impact of TAE-HCA degradation on wine spoilage depends on the relative concentrations of the free and bound hydroxycinnamic acids in the wine. In the current study, both the 2011 and 2012 Oregon Pinot noir wines contained low concentrations of p-coumaric acid and relatively high concentrations of coutaric acid. The concentrations of p-coumaric acid were lower than has been reported for Pinot noir from the Pacific Northwest, which are on average closer to 2.4 mg/L (Goldberg et al. 1998). However, that study also noted significant differences among Pinot noir wines of different geographical origins. In addition, other factors, such as grape ripeness, cultivar, site, and the degree of sun exposure, may also affect both the concentration of hydroxycinnamic acids and the ratio of free to bound acids (Bubola et al. 2012, Pérez-Magariño and González-San José 2005, Romeyer et al. 1983, Ong and Nagel 1978). Because of the potential variability in the concentration and form of hydroxycinnamic acids, Brettanomyces spp. growth in wines from some cultivars and regions may result in higher levels of ethylphenols regardless of any TAE-HCA degradation during MLF. On the other hand, in wines that contain low levels of free hydroxycinnamic acids, the degradation of TAE-HCAs during wine spoilage by Brettanomyces spp. may have a greater impact. Additional research is required to better understand how viticultural practices and climatic factors affect the concentration and chemical form of volatile phenol precursors.
Strain VFO degraded TAE-HCAs during the logarithmic growth phase and near the end of malic acid degradation. Because of this timing, it would be difficult for a winemaker to conduct MLF with the VFO strain while preventing the degradation of the TAE-HCAs. Very close monitoring of malic acid levels would be required so that as soon as malic acid degradation is complete, addition of SO2 would prevent further metabolic activity of the VFO strain. The simplest approach to minimizing the risk for increased free hydroxycinnamic acids after an MLF would be to use a strain of O. oeni that cannot degrade TAE-HCAs. Performing spontaneous MLF increases the risk of spoilage if Brettanomyces spp. growth occurs in the wine because the identity of the wine LAB conducting the MLF would be unknown and so would be its ability to degrade TAE-HCA. For example, Hernandez et al. (2007) reported an increase in free hydroxycinnamic acids in wine that underwent a spontaneous MLF with an unidentified LAB.
Additional screening of wine LAB for their ability to degrade TAE-HCAs should include LAB isolated from wines undergoing spontaneous MLF and spoilage LAB such as Pediococcus and Lactobacillus. Results from these studies would help determine how prevalent the TAE-HCA degradation trait is among wine bacteria. For example, Guglielmetti et al. (2008) reported that some Lactobacillus species can degrade quinic acid–hydroxycinnamic acid esters, but their ability to degrade tartaric acid esters was unknown. Additional research to identify the genes encoding the O. oeni tartaric acid–hydroxycinnamic acid esterase would enable more efficient screening of wine microorganisms. It would aid in the selection of wine LAB strains for use as commercial cultures and in the characterization of strains in wineries that perform spontaneous MLF.
Conclusion
Whether growth of Brettanomyces spp. in a wine will result in spoilage depends on several factors. This research has demonstrated that the O. oeni strain conducting the MLF can be one such factor because some of these strains increase the amount of free hydroxycinnamic acids in the wine. In wines with low free hydroxycinnamic acid content, the degradation of TAE-HCAs by O. oeni during MLF may increase the risk for wine spoilage due to higher production of 4-EP and 4-EG by B. bruxellensis. Although only one O. oeni strain could degrade TAE-HCAs in the present study, additional screening of commercial O. oeni strains, as well as those isolated from spontaneous MLFs, may provide more accurate information about how widespread this trait is among O. oeni strains. Preventing the growth of B. bruxellensis through rigorous cleaning, sanitation, and adequate additions of SO2 is still the most effective method to prevent wine spoilage. However, minimizing the amount of free hydroxycinnamic acids in the wine will reduce the amount of volatile phenols produced if growth of Brettanomyces spp. does occur. Accordingly, use of O. oeni strains that degrade TAE-HCAs should be avoided in barrel-aged red wines that are most at risk for spoilage by Brettanomyces spp.
Acknowledgments
The authors gratefully acknowledge the Northwest Center for Small Fruits Research for providing financial support for this study. They also thank Charles Edwards (Washington State University) for assistance with the analysis of wine volatile phenols.
- Received September 2014.
- Revision received February 2015.
- Accepted March 2015.
- ©2015 by the American Society for Enology and Viticulture
Literature Cited
Vol 66 Issue 3
