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Impact of available nitrogen and sugar concentration in musts on alcoholic fermentation and subsequent wine spoilage by Brettanomyces bruxellensis
Food Microbiology 46 (2015) 604e609
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Impact of available nitrogen and sugar concentration in musts on alcoholic fermentation and subsequent wine spoilage by Brettanomyces bruxellensis Bradford C. Childs a, Jeffri C. Bohlscheid b, Charles G. Edwards a, * a b
School of Food Science, Washington State University, Pullman, WA 99164e6376, USA J.R. Simplot Company, Boise, ID 83605, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 June 2014 Received in revised form 17 October 2014 Accepted 20 October 2014 Available online 29 October 2014
The level of yeast assimilable nitrogen (YAN) supplementation required for Saccharomyces cerevisiae to complete fermentation of high sugar musts in addition to the impact of nonemetabolized nitrogen on postealcoholic spoilage by Brettanomyces bruxellensis was studied. A 2 3 factorial design was employed using a synthetic grape juice medium with YAN (150 or 250 mg N/L) and equal proportions of glucose/ fructose (230, 250, or 270 g/L) as variables. S. cerevisiae ECA5 (low nitrogen requirement) or Uvaferm 228 (high nitrogen requirement) were inoculated at 105 cfu/mL while B. bruxellensis E1 or B2 were added once alcoholic fermentation ceased. Regardless of YAN concentration, musts that contained 230 or 250 g/ L glucose/fructose at either nitrogen level attained dryness (mean ¼ 0.32 g/L fructose) while those containing 270 g/L generally did not (mean ¼ 2.5 g/L fructose). Higher concentrations of YAN present in musts yielded wines with higher amounts of aeamino acids and ammonium but very little (6 mg N/L) was needed by B. bruxellensis to attain populations 107 cfu/mL. While adding nitrogen to high sugar musts does not necessarily ensure completion of alcoholic fermentation, residual YAN did not affect B. bruxellensis growth as much as ethanol concentration. © 2014 Published by Elsevier Ltd.
Keywords: Saccharomyces cerevisiae Brettanomyces bruxellensis Nitrogen Sugar Fermentation
1. Introduction In many wineeproducing regions, sugar concentrations in commercial grape musts at harvest have generally increased over the last decades. As evidence, soluble solids contents of Cabernet Sauvignon berries at the time of harvest in California have risen from an average of 22.8 Brix (% w/w) in 1980 to upwards of 25.0 Brix by 2005 (Alston et al., 2011). Similarly, Petrie and Sadras (2008) reported that Brix levels of Shiraz and Cabernet Sauvignon grown in Australia rose by up to ~0.3 Brix per year from 1993 to 2006. Proposed explanations for these observations include warmer vineyard temperatures (Sadras and Moran, 2012) and/or consumer preferences for fullebodied, higheethanol wines with ~ a, 2010; Alston et al., 2011). ripe fruit flavors (Mira de Ordun Some researchers have suggested that higher sugar concentrations in grape musts may result in increased requirements of yeast assimilable nitrogen (YAN) in Saccharomyces cerevisiae. Assuming a 21 Brix grape must required 200 mg N/L YAN to complete fermentation, Bisson and Butzke (2000) recommended an * Corresponding author. Tel.: þ1 509 335 6612. E-mail address: [email protected] (C.G. Edwards). http://dx.doi.org/10.1016/j.fm.2014.10.006 0740-0020/© 2014 Published by Elsevier Ltd.
additional 25 mg N/L for every 1 Brix increase. In agreement, Jiranek et al. (1995) reported an extra 22 mg N/L was utilized by S. cerevisiae with an increase of 50 g/L glucose beyond a minimal nitrogen concentration. However, Wang et al. (2003) and Ugliano et al. (2009) noted that musts containing approximately 250 g/L glucose/fructose achieved dryness (2 g/L residual sugar) yet contained 100 mg N/L, a concentration far lower than the amount recommended by Bisson and Butzke (2000). In fact, some authors have suggested 140 mg N/L to be the minimal amount of nitrogen for most fermentations to be successful (Agenbach, 1977; Jiranek et al., 1995; Cramer et al., 2002; MartínezeMoreno et al., 2012). Though several studies have been published, a systematic approach to better understand nitrogen requirements of yeast during fermentation of higher sugar musts has been lacking. For instance, some studies utilized media containing only glucose (Jiranek et al., 1995; MartínezeMoreno et al., 2012), a sugar greatly preferred over fructose by S. cerevisiae (Schütz and Gafner, 1995) even though both sugars are found in grape musts. Still others relied on dilution of grape juices to alter nitrogen composition (Cramer et al., 2002), an approach that also lowered the concentrations of other nutrients that affect nitrogen metabolism (Wang et al., 2003). Finally, many studies have relied on a single strain of
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S. cerevisiae (Cramer et al., 2002; MendeseFerreira et al., 2004; MartínezeMoreno et al., 2012) and therefore did not account for differences in nitrogen utilization between isolates (Julien et al., 2000; MendeseFerreira et al., 2004). While having adequate nitrogen available for primary fermentation is crucial, nutrients not metabolized by S. cerevisiae during alcoholic fermentation could theoretically be available for spoilage microbes during the later stages of wine processing (Bisson and Butzke, 2000; Bell and Henschke, 2005). One such postealcoholic spoilage threat is Brettanomyces bruxellensis, a yeast which produces a variety of unpleasant aromas described as ‘medicinal,’ ‘mousey,’ ‘rancid,’ ‘sweat,’ ‘smoke,’ ‘horsey,’ ‘leather,’ or ‘BandeAid®’ (Oelofse et al., 2008). While B. bruxellensis can metabolize a variety of nitrogen and carbon sources, nutritional requirements for growth in wine are not well understood (AguilareUscanga et al., 2000; Conterno et al., 2006). The objectives of this study were to (a) investigate the relationship between available nitrogen and the sugar content of musts using strains of S. cerevisiae which differ in their nitrogen requirements and (b) determine the impact of nitrogen supplementation of musts on subsequent growth of B. bruxellensis in wines.
composition of Cabernet Sauvignon grapes from Washington State (Spayd and AnderseneBagge, 1996). A 2 3 factorial design was employed with YAN (150 or 250 mg N/L) and equal proportions of glucose/fructose (230, 250, or 270 mg/L) as variables. Enough DAP was added to the base medium to yield 250 mg N/L YAN. Tween 80, dissolved in water and sterilized at 121 C for 15 min, and pecoumaric acid, dissolved in absolute ethanol, were also added at concentrations of 5 mg/L. Once prepared, all media were sterile filtered through 0.22 mm PES Express™Plus bottle top filters (Millipore, Bedford, MA, USA) into 3 L Celstir® bioreactors (Wheaton Science Products, Millville, NJ, USA) with stainless steel headpieces (B. Braun Biotech, Allentown, PA, USA) and an aseptic system that avoids incorporation of air during sampling. A suspension of microcrystalline cellulose (Sigmacell Type 20, SigmaeAldrich, St. Louis, MO, USA) was sterilized at 121 C for 15 min and added at 1 g/L prior to inoculation of S. cerevisiae strain ECA5 or Uvaferm 228 at 105 cfu/mL. All fermentations were conducted with triplicate replication without shaking but were stirred for 2 min at 75 rpm before sampling.
2. Materials and methods
Synthetic wines (200 mL) was centrifuged 1000 g for 30 min, sterile filtered (0.22 mm), and divided into two sterile milk dilution bottles (100 mL). A sterile filtered (0.22 mm) vitamin solution was added so that wines contained 25 mg/L myoeinositol, 1 mg/L pyrodoxineHCl, 10 mg/L nicotinic acid, 1 mg/L thiamineHCl, and 30 mg/L biotin. A suspension of powdered cellulose (Sigmacell Type 20, SigmaeAldrich, St. Louis, MO, USA) was sterilized at 121 C for 15 min and added to wines to yield a concentration of 1 g/L. Starter cultures of B. bruxellensis were used to inoculate the wines at initial populations of 105 cfu/mL. Bottles were incubated at 20 C and shaken prior to sampling. Minimum nitrogen needs for B. bruxellensis were determined using the same composition as the base synthetic grape juice medium but with fructose (2 g/L), ethanol (13% v/v), and pecoumaric acid (5 mg/L). A stock solution with the same amino acid/DAP composition as the base medium was diluted to produce media containing 0, 6, 12, 24, 48, or 96 mg N/L YAN. All media were acidified to pH 3.5 with phosphoric acid and sterile filtered (0.22 mm) before inoculation with B. bruxellensis E1 or B2 at approximately 105 cfu/mL. Treatments were conducted in triplicate at 20 C and sampled once per week.
2.1. Microorganisms S. cerevisiae strains ECA5 and Uvaferm 228 were received as al, Quebec, Canada). active dry cultures (Lallemand Inc., Montre These strains were selected based on their relative nitrogen requirements, with ECA5 having a low requirement compared to Uvaferm 228 (G. Specht, personal communication, 2011). B. bruxellensis strains E1 and B2 were isolated from Washington state wines as described by Jensen et al. (2009). All strains were isolated and maintained by repeated streaking on Wallerstein Laboratory Nutrient (WLN) agar (Oxoid, Hampshire, England) and incubated at 25 C. 2.2. Preparation of starter cultures One colony of S. cerevisiae was aseptically transferred into 10 mL YM broth (Difco, Sparks, MD, USA) acidified to pH 3.5 with phosphoric acid prior to incubation for 24 h at 25 C. Cultures were then transferred into 500 mL YM broth and incubated with shaking at 130 rpm until approximately 107 cfu/mL was reached. Cells were harvested by centrifugation at 1000 g for 30 min, washed with 0.2 M phosphate buffer, centrifuged, and suspended in phosphate buffer before inoculation into synthetic grape juice media at 105 cfu/mL. Single colonies of B. bruxellensis were aseptically transferred into 10 mL of YM broth and incubated at 25 C for approximately one week. One mL of this culture was transferred into 50 mL YM containing 10% (v/v) ethanol. Cells were incubated, harvested, and inoculated as previously described for S. cerevisiae but with different centrifugation conditions (2000 g for 15 min). 2.3. Synthetic grape must fermentations Fermentations were conducted using the synthetic grape juice medium described by Wang et al. (2003). The base medium contained 43 mg/L Ala, 132 mg/L Arg, 14 mg/L Asp, 55 mg/L Glu, 4 mg/L Gly, 30 mg/L His, 18 mg/L Ile, 22 mg/L Leu, 26 mg/L Lys, 8 mg/L Met, 18 mg/L Phe, 31 mg/L Ser, 28 mg/L Thr, 12 mg/L Trp, 14 mg/L Tyr, 120 mg/L Val, and 248 mg/L diammonium phosphate (DAP). These values represent approximately 120 mg N/L amino acids and 30 mg N/L DAP, concentrations that simulate the nitrogen
2.4. Inoculations of B. bruxellensis
2.5. Analyses Fermentation progress was evaluated by measuring the concentration of soluble solids (Ingledew and Kunkee, 1985) while final glucose and fructose concentrations were determined enzymatically (ReBiopharm AG, Darmstadt, Germany). Ethanol content was , France). determined using an ebulliometer (Alla France, Chemille Finally, the concentration of amino nitrogen in the wines was determined using the NOPA method (Dukes and Butzke, 1998) while an ioneselective electrode measured ammonia (Denver Instrument, Orville, NY, USA). Statistical analyses were performed with XLSTAT (Addinsoft, New York, NY, USA) with ANOVA and Tukey's HSD test for mean separation. 3. Results 3.1. Alcoholic fermentations Altering the concentration of YAN in the synthetic grape juice medium (150 or 250 mg N/L) did not affect fermentation
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completion of synthetic musts (Fig. 1). Here, musts inoculated with ECA5 exhibited longer fermentations as sugar concentrations increased from 230 up to 270 g/L. However, few differences existed between musts of the same sugar concentration but differed in YAN. Fermentations conducted by Uvaferm 228 required approximately 200 h longer for the same initial sugar levels than those inoculated with ECA5. An exception was 270 g/L ferments conducted by Uvaferm 228 where soluble solids differed by >20 g/L between the two nitrogen treatments at times, a difference which decreased to 4 g/L by 900 h. Differences in residual fructose, ethanol, and YAN were observed in resultant wines (Table 1). While glucose concentrations were generally <0.03 g/L, those musts containing higher amounts of sugar also produced wines with increased residual fructose. For example, musts that contained 230 or 250 g/L glucose/fructose at either YAN level attained dryness or 2 g/L residual fructose (mean ¼ 0.32 g/L) while those containing 270 g/L generally did not (mean ¼ 2.5 g/L) regardless of YAN concentration. Comparing yeast strains for a given nitrogen level, residual fructose concentrations were not significantly different. Similarly, ethanol contents of musts fermented with 150 or 250 mg N/L YAN were not significantly different for a given sugar level. Specifically, musts that had 230, 250, or 270 g/L sugar contained 13.5e14.1%, 14.7e14.9%, or 15.6e15.9% (v/v) ethanol, respectively. Averaging data from both yeast strains, wines from musts with 250 mg N/L YAN had more residual aeamino acids (86.8 mg N/L) and ammonium (49.7 mg N/ L) than those from musts containing 150 mg N/L (47.5 and 2.0 mg N/ L, respectively). Differences in residual ammonium between the yeast strains were also observed where wines fermented by
Uvaferm 228 contained 30e50 mg N/L while those by ECA5 had 60 mg N/L. 3.2. Growth of B. bruxellensis Inoculation of synthetic wines with B. bruxellensis yielded initial cell concentrations of 105 cfu/mL for both strains (Fig. 2). Subsequently, populations exceeded of 106 cfu/mL in wines made from musts that contained 230 or 250 g/L sugar but became undetectable (<60 cfu/mL) in those initially containing 270 g/L sugar. In addition, longer lag times for strains E1 and B2 were apparent as higher sugar in musts yielded higher amounts of ethanol. For example, populations reached >106 cfu/mL by day 15 in wines containing 13.5e14.1% (v/v) ethanol (musts with 230 g/L sugar). In wines with 14.7e14.9% (v/v) ethanol (musts with 250 g/L sugar), populations initially decreased approximately 0.5 log yet required >20 d to achieve similar populations. Finally, B. bruxellensis declined to undetectable populations by day 20 in wines containing 15.6e15.9% ethanol (musts with 270 g/L sugar). Few differences were noted between wines from musts initially containing 150 or 250 mg N/L for either strain of S. cerevisiae, even though total residual YAN ranged between 46 and 148 mg N/L depending on must composition (Table 1). To determine minimal amounts of nitrogen required for growth, B. bruxellensis were inoculated into synthetic wine media containing 0e96 mg N/L (Fig. 3). In media containing no added nitrogen, populations remained at 105 cfu/mL until day 15 (E1) or 23 (B2) when slight increases (0.5 log) were observed. By day 45, these ferments reached 5 105 cfu/mL. In contrast, populations exceeded 106 cfu/mL in all media containing 6 mg N/L. Here, strain E1 peaked approached 107 cfu/mL by day 30 in all media fermented by ECA5. More differences were noted in wines fermented by Uvaferm 228 where those wines obtained from musts with 48 or 96 mg N/L achieved higher populations sooner than those with 6, 12, or 24 mg N/L but remained at approximately 5 106 cfu/mL. 4. Discussion
Fig. 1. Decline of soluble solids during fermentation of synthetic musts containing 150 (B, , ) or 250 (C, :, -) mg N/L yeast assimilable nitrogen and 230 (B, C), 250 ( , :), or 270 ( , -) g/L glucose/fructose by S. cerevisiae strain ECA5 (A) or Uvaferm 228 (B).
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Availability of nitrogen (150 or 250 mg N/L) did not greatly affect fermentation of musts with varying sugar concentrations (230, 250, or 270 g/L) by either of the strains of S. cerevisiae studied, ECA5 (low nitrogen requirement) or Uvaferm 228 (high nitrogen requirement). Moreover, fermentations of synthetic grape musts containing very high amounts of sugar (295 g/L) did not complete even when supplemented to 580 mg N/L (data not shown), in agreement with MartínezeMoreno et al. (2012) who reported incomplete fermentations that contained 280 g/L glucose and 300 mg N/L. Overall, these findings were in contrast to the recommendations of Bisson and Butzke (2000) concerning the need to add extra nitrogen to high sugar musts in order to complete fermentation. Rather, these results support the contention that increasing nitrogen concentration of grape musts above a threshold (e.g., 140e150 mg N/L) will not necessarily yield successful fermentation of high sugar musts even taking into account nitrogen utilization requirements by different strains of S. cerevisiae. Similarly, Cramer et al. (2002) reported a proportional increase in cell concentration with nitrogen additions up to 140 mg N/L but beyond this amount, nitrogen was not the limiting factor for the yeast strain e). While Erasmus et al. (2003) noted that studied (Premier Cuve elevated sugar concentrations increased the expression of numerous genes with a simultaneous decrease of others by S. cerevisiae, it was unclear if net protein synthesis and, therefore, overall nitrogen requirements were also altered due to higher sugar amounts. In fact, ethanol accumulation may be the more important
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Table 1 Composition of wines made from synthetic grape musts that contained variable concentrations of YAN/fermentable sugar and were fermented by S. cerevisiae ECA5 or Uvaferm 228. Must Yeast strain ECA5
Wine YAN (mg N/L) 150
250
Uvaferm 228
150
250
Total sugar (g/L) 230 250 270 230 250 270 230 250 270 230 250 270
Glucose (g/L) <0.03 <0.03 0.071 <0.03 <0.03 <0.03 <0.03 <0.03 <0.03 <0.03 <0.03 <0.03
Fructose (g/L) b
0.11 0.30b 2.6a 0.19b 0.33b 2.7a 0.25b 0.43b 1.9a 0.32b 0.61b 2.2a
Ethanol (% v/v) c
14.0 14.8b 15.9a 14.1c 14.8b 15.9a 13.8bc 14.9a 15.6a 13.5c 14.7ab 15.8a
aeAmino acids (mg N/L) b
50 48bc 45c 85a 84a 86a 45b 44b 53b 90a 87a 89a
Ammonium (mg N/L) 1.0b 0.78b 1.3b 61a 60a 62a 3.5c 2.4c 3.1c 31b 30b 54a
aed
Means within columns followed by different letters differ significantly at p 0.05.
did not support the contention that higher concentrations of residual nitrogen enhances the growth of B. bruxellensis. Earlier work by AguilareUscanga et al. (2000) suggested that the yeast may have minimal nitrogen requirements since increased concentrations of ammonia only “weakly stimulated” B. bruxellensis. In fact, B. bruxellensis growth was more affected by ethanol content as opposed to available nitrogen. Increasing must sugar resulted in wines of higher ethanol content with more delayed growth of B. bruxellensis if not slow death. Previous studies have suggested that increasing ethanol concentrations resulted in prolonged lag phases, where concentrations up to 15% ethanol prevented growth (Dias et al., 2003; Medawar et al., 2003; Barata et al., 2008). However, the ability to survive high ethanol concentrations in wine is dependent on the strain of B. bruxellensis. For example, while the maximum concentration of ethanol tolerated by strains E1 and B2 was between 14.9 and 15.6% (v/v), Barata et al. (2008) demonstrated that most strains of B. bruxellensis tolerate 14.5%.
cause of fermentation problems associated with high sugar musts (Nishino et al., 1985; Mauricio and Salmon, 1992). Adding excessive amounts of nitrogen to grape musts yielded resultant wines with higher residual YAN, in agreement with others (Beltran et al., 2005; Taillandier et al., 2007; Sturgeon et al., 2013). Of concern is the possibility that higher concentrations of nitrogen in wines could support the growth of unwanted spoilage microorganisms during storage or in the bottle (Bisson and Butzke, 2000; Bell and Henschke, 2005). However, growth of B. bruxellensis was not influenced by the amount of residual nitrogen in the synthetic wines. As evidence, B. bruxellensis strain E1 or B2 reached similar populations in wines containing 46e143 mg N/L for those wines from initial must sugar concentrations of 230 or 250 g/L. In fact, both strains grew to populations of >106 cfu/mL in synthetic wine containing as little as 6 mg N/L, a value much lower than the 122 mg N/L reported by AguilareUscanga et al. (2000) and Conterno et al. (2006). Given this low concentration, these results
Fig. 2. Growth of B. bruxellensis strains E1 (A, B) or B2 (C, D) in synthetic wines fermented by S. cerevisiae strains ECA5 (A, C) or Uvaferm 228 (B, D). Musts originally contained 150 (B, , ) or 250 (C, :, -) mg N/L yeast assimilable nitrogen and 230 (B, C), 250 ( , :), or 270 ( , -) g/L glucose/fructose. Dashed lines indicate points that were below the detection limit (<60 cfu/mL) while error bars indicate standard error.
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Acknowledgments This research was conducted at Washington State University as part of the Master of Science thesis of Bradford C. Childs. The aual, thors would like to thank G. Specht (Lallemand Inc., Montre Quebec, Canada) for supplying yeast cultures and to the Northwest Center for Small Fruits Research (grant 11D-3057-3948; Corvallis, OR, USA) and Washington State University (Pullman, WA, USA) for financial support. References
Fig. 3. Growth of B. bruxellensis strain E1 (A) or B2 (B) in synthetic wine media that contained 0 (B), 6 (C), 12 ( ), 24 (:), 48 ( ) or 96 (-) mg N/L yeast assimilable nitrogen. Error bars indicate standard error.
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This suggests that the strains isolated from commercial Washington wines may be more ethanol tolerant than others. Although the strains of B. bruxellensis studied were adapted to ethanol prior to inoculation to increase tolerance (Vigentini et al., 2008), other factors such as media composition, pH, temperature, and SO2 levels also influence survivability (Barata et al., 2008; Zuehlke and Edwards, 2013). Given that the strains of B. bruxellensis used in the present study grew to high populations in synthetic grape medium (wine) containing low concentrations of nitrogen, this suggests that many commercial wines would likely contain enough residual nitrogen to support growth. Another source of nitrogen for B. bruxellensis would be S. cerevisiae which releases amino acids during the later stages of alcoholic fermentation (Ough et al., 1991). Finally, proline, an amino acid omitted from synthetic grape must fermentations but is abundant in finished wines, may serve as a sole nitrogen source for B. bruxellensis if enough oxygen is available (Conterno et al., 2006; Blomqvist et al., 2012). 5. Conclusions In general, 150 mg N/L YAN was sufficient for fermentations to reach dryness in musts containing 230 or 250 g/L glucose/fructose although higher levels (250 mg N/L) did not necessarily result in completion of fermentation of high sugar musts (270 or 295 g/L). Higher nitrogen supplementation also resulted in higher concentrations of residual nitrogen in the final wines, which theoretically could be metabolized by spoilage microorganisms. While adding high levels of nitrogen to musts does not necessarily ensure completion of alcoholic fermentation, residual YAN did not affect B. bruxellensis growth as much as ethanol concentration. Thus, it is not necessary for winemakers to add high amounts of nitrogen in order to complete alcoholic fermentation.
Agenbach, W.A., 1977. A study of must nitrogen content in relation to incomplete fermentations, yeast production and fermentation activity. In: Beukman, E.F. (Ed.), Proceedings of the South AfricaN Society for Enology and Viticulture, Stellenbosch, South Africa, pp. 66e88. AguilareUscanga, M., Delia, M.L., Strehaiano, P., 2000. Nutritional requirements of Brettanomyces bruxellensis: growth and physiology in batch culture and chemostat cultures. Can. J. Microbiol. 46, 1046e1050. Alston, J.M., Fuller, K.B., Lapsley, J.T., Soleas, G.S., 2011. Too much of a good thing? Causes and consequences of increases in sugar content of California wine grapes. J. Wine Econ. 6, 135e159. Barata, A., Caldiera, J., Botelheiro, R., Pagliara, D., MalfeitoeFerreira, M., Loureiro, V., 2008. Survival patterns of Dekkera bruxellensis in wines and inhibitory effect of sulphur dioxide. Int. J. Food Microbiol. 121, 201e207. Bell, S.J., Henschke, P.A., 2005. Implications of nitrogen nutrition for grapes, fermentation and wine. Aust. J. Grape Wine Res. 11, 242e295. s, N., Mas, A., Guillamo n, J.M., 2005. Influence of Beltran, G., EsteveeZarzoso, B., Roze the timing of nitrogen additions during synthetic grape must fermentations on fermentation kinetics and nitrogen consumption. J. Agric. Food Chem. 53, 996e1002. Bisson, L.F., Butzke, C.E., 2000. Diagnosis and rectification of stuck and sluggish fermentations. Am. J. Enol. Vitic. 51, 168e177. , V.S., GorwaeGrauslund, M., Passoth, V., 2012. Physiological Blomqvist, J., Nogue requirements for growth and competitiveness of Dekkera bruxellensis under oxygen limited or anaerobic conditions. Yeast 29, 265e274. Conterno, L., Joseph, C., Arvik, T.J., HenickeKling, T., Bisson, L.F., 2006. Genetic and physiological characterization of Brettanomyces bruxellensis strains isolated from wines. Am. J. Enol. Vitic. 57, 139e147. Cramer, A.C., Vlassides, S., Block, D.E., 2002. Kinetic model for nitrogenelimited wine fermentations. Biotechnol. Bioeng. 77, 49e60. Dias, L., PereiraedaeSilva, S., Tavares, M., MalfeitoeFerreira, M., Loureiro, V., 2003. Factors affecting the production of 4eethyl phenol by the yeast Dekkera bruxellensis in enological conditions. Food Microbiol. 20, 377e384. Dukes, B.C., Butzke, C.E., 1998. Rapid determination of primary amino acids in grape juice using an oephthaldialdehyde/NeacetyleLecysteine spectrophotometric assay. Am. J. Enol. Vitic. 49, 125e134. Erasmus, D.J., van der Merwe, G.K., van Vuuren, H.J.J., 2003. Genomeewide expression analyses: metabolic adaptation of Saccharomyces cerevisiae to high sugar stress. FEMS Yeast Res. 3, 375e399. Ingledew, W.M., Kunkee, R.E., 1985. Factors influencing sluggish fermentations of grape juice. Am. J. Enol. Vitic. 36, 65e76. Jensen, S.L., Umiker, N.L., Arneborg, N., Edwards, C.G., 2009. Identification and characterization of Dekkera bruxellensis, Candida pararugosa, and Pichia guillermondii isolated from commercial red wines. Food Microbiol. 26, 915e921. Jiranek, V., Langridge, P., Henschke, P.A., 1995. Amino acid and ammonium utilization by Saccharomyces cerevisiae wine yeasts from a chemically defined medium. Am. J. Enol. Vitic. 46, 75e83. Julien, A., Roustan, J.L., Dulau, L., Sablayrolles, J.M., 2000. Comparison of nitrogen and oxygen demands of enological yeasts: technological consequences. Am. J. Enol. Vitic. 46, 215e222. MartínezeMoreno, R., Morales, P., Gonzalez, R., Mas, A., Beltran, G., 2012. Biomass production and alcoholic fermentation performance of Saccharomyces cerevisiae as a function of nitrogen source. FEMS Yeast Res. 12, 477e485. Mauricio, J.C., Salmon, J.M., 1992. Apparent loss of sugar transport activity in Saccharomyces cerevisiae may mainly account for maximum ethanol production during alcoholic fermentation. Biotechnol. Lett. 14, 577e582. Medawar, W., Strehaiano, P., Delia, M., 2003. Yeast growth: lag phase modeling in alcoholic media. Food Microbiol. 20, 527e532. ~o, C., 2004. Growth and fermentation MendeseFerreira, A., MendeseFaia, A., Lea patterns of Saccharomyces cerevisiae under different ammonium concentrations and its implications in winemaking industry. J. Appl. Microbiol. 97, 540e545. ~ a, R., 2010. Climate change associated effects on grape and wine Mira de Ordun quality and production. Food Res. Int. 43, 1844e1855. Nishino, H., Miyazaki, S., Tohjo, K., 1985. Effect of osmotic pressure on the growth rate and fermentation activity of wine yeasts. Am. J. Enol. Vitic. 36, 170e174. Oelofse, A., Pretorius, I.S., du Toit, M., 2008. Significance of Brettanomyces and Dekkera during winemaking: a synoptic review. South Afr. J. Enol. Vitic. 29, 128e144.
B.C. Childs et al. / Food Microbiology 46 (2015) 604e609 Ough, C.S., Huang, Z., An, D., Stevens, D., 1991. Amino acid uptake by four commercial yeasts at two different temperatures of growth and fermentation: effects on urea excretion and readsorption. Am. J. Enol. Vitic. 41, 26e40. Petrie, P.R., Sadras, V.O., 2008. Advancement of grapevine maturity in Australia between 1993 and 2006: putative causes, magnitude of trends and viticultural consequences. Aust. J. Grape Wine Res. 14, 33e45. Sadras, V.O., Moran, M.A., 2012. Elevated temperature decouples anthocyanins and sugars in berries of Shiraz and Cabernet Franc. Aust. J. Grape Wine Res. 18, 115e122. Schütz, M., Gafner, J., 1995. Lower fructose uptake capacity of genetically characterized strains of Saccharomyces bayanus compared to strains of Saccharomyces cerevisiae: a likely cause of reduced alcoholic fermentation activity. Am. J. Enol. Vitic. 46, 175e180. Spayd, S.E., AnderseneBagge, J., 1996. Free amino acid composition of grape juice from 12 Vitis vinifera cultivars in Washington. Am. J. Enol. Vitic. 47, 389e402. Sturgeon, J.Q., Bohlscheid, J.C., Edwards, C.G., 2013. The effect of nitrogen source on yeast metabolism and H2S formation. J. Wine Res. 24, 182e194.
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Taillandier, P., Ramon Portugal, F., Fuster, A., Strehaiano, P., 2007. Effect of ammonium concentration on alcoholic fermentation kinetics by wine yeasts for high sugar content. Food Microbiol. 24, 95e100. Ugliano, M., Fredrizzi, B., Siebert, T., Travis, B., Magno, F., Versini, G., Henschke, P.A., 2009. Effect of nitrogen supplementation and Saccharomyces species on hydrogen sulfide and other volatile sulfur compounds in Shiraz fermentation and wine. J. Agric. Food Chem. 57, 4948e4955. Vigentini, I., Romano, A., Compagno, C., Merico, A., Molinari, F., Tirelli, A., Foschino, R., Volonterio, G., 2008. Physiological and oenological traits of different Dekkera/Brettanomyces bruxellensis strains under wineemodel conditions. FEMS Yeast Res. 8, 1087e1096. Wang, X.D., Bohlscheid, J.C., Edwards, C.G., 2003. Fermentative activity and production of volatile compounds by Saccharomyces grown in synthetic grape juice media deficient in assimilable nitrogen and/or pantothenic acid. J. Appl. Microbiol. 94, 349e359. Zuehlke, J.M., Edwards, C.G., 2013. Impact of sulfur dioxide and temperature on culturability and viability of Brettanomyces bruxellensis in wine. J. Food Prot. 76, 2024e2030.
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Impact of available nitrogen and sugar concentration in musts on alcoholic fermentation and subsequent wine spoilage by Brettanomyces bruxellensis Bradford C. Childs a, Jeffri C. Bohlscheid b, Charles G. Edwards a, * a b
School of Food Science, Washington State University, Pullman, WA 99164e6376, USA J.R. Simplot Company, Boise, ID 83605, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 June 2014 Received in revised form 17 October 2014 Accepted 20 October 2014 Available online 29 October 2014
The level of yeast assimilable nitrogen (YAN) supplementation required for Saccharomyces cerevisiae to complete fermentation of high sugar musts in addition to the impact of nonemetabolized nitrogen on postealcoholic spoilage by Brettanomyces bruxellensis was studied. A 2 3 factorial design was employed using a synthetic grape juice medium with YAN (150 or 250 mg N/L) and equal proportions of glucose/ fructose (230, 250, or 270 g/L) as variables. S. cerevisiae ECA5 (low nitrogen requirement) or Uvaferm 228 (high nitrogen requirement) were inoculated at 105 cfu/mL while B. bruxellensis E1 or B2 were added once alcoholic fermentation ceased. Regardless of YAN concentration, musts that contained 230 or 250 g/ L glucose/fructose at either nitrogen level attained dryness (mean ¼ 0.32 g/L fructose) while those containing 270 g/L generally did not (mean ¼ 2.5 g/L fructose). Higher concentrations of YAN present in musts yielded wines with higher amounts of aeamino acids and ammonium but very little (6 mg N/L) was needed by B. bruxellensis to attain populations 107 cfu/mL. While adding nitrogen to high sugar musts does not necessarily ensure completion of alcoholic fermentation, residual YAN did not affect B. bruxellensis growth as much as ethanol concentration. © 2014 Published by Elsevier Ltd.
Keywords: Saccharomyces cerevisiae Brettanomyces bruxellensis Nitrogen Sugar Fermentation
1. Introduction In many wineeproducing regions, sugar concentrations in commercial grape musts at harvest have generally increased over the last decades. As evidence, soluble solids contents of Cabernet Sauvignon berries at the time of harvest in California have risen from an average of 22.8 Brix (% w/w) in 1980 to upwards of 25.0 Brix by 2005 (Alston et al., 2011). Similarly, Petrie and Sadras (2008) reported that Brix levels of Shiraz and Cabernet Sauvignon grown in Australia rose by up to ~0.3 Brix per year from 1993 to 2006. Proposed explanations for these observations include warmer vineyard temperatures (Sadras and Moran, 2012) and/or consumer preferences for fullebodied, higheethanol wines with ~ a, 2010; Alston et al., 2011). ripe fruit flavors (Mira de Ordun Some researchers have suggested that higher sugar concentrations in grape musts may result in increased requirements of yeast assimilable nitrogen (YAN) in Saccharomyces cerevisiae. Assuming a 21 Brix grape must required 200 mg N/L YAN to complete fermentation, Bisson and Butzke (2000) recommended an * Corresponding author. Tel.: þ1 509 335 6612. E-mail address: [email protected] (C.G. Edwards). http://dx.doi.org/10.1016/j.fm.2014.10.006 0740-0020/© 2014 Published by Elsevier Ltd.
additional 25 mg N/L for every 1 Brix increase. In agreement, Jiranek et al. (1995) reported an extra 22 mg N/L was utilized by S. cerevisiae with an increase of 50 g/L glucose beyond a minimal nitrogen concentration. However, Wang et al. (2003) and Ugliano et al. (2009) noted that musts containing approximately 250 g/L glucose/fructose achieved dryness (2 g/L residual sugar) yet contained 100 mg N/L, a concentration far lower than the amount recommended by Bisson and Butzke (2000). In fact, some authors have suggested 140 mg N/L to be the minimal amount of nitrogen for most fermentations to be successful (Agenbach, 1977; Jiranek et al., 1995; Cramer et al., 2002; MartínezeMoreno et al., 2012). Though several studies have been published, a systematic approach to better understand nitrogen requirements of yeast during fermentation of higher sugar musts has been lacking. For instance, some studies utilized media containing only glucose (Jiranek et al., 1995; MartínezeMoreno et al., 2012), a sugar greatly preferred over fructose by S. cerevisiae (Schütz and Gafner, 1995) even though both sugars are found in grape musts. Still others relied on dilution of grape juices to alter nitrogen composition (Cramer et al., 2002), an approach that also lowered the concentrations of other nutrients that affect nitrogen metabolism (Wang et al., 2003). Finally, many studies have relied on a single strain of
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S. cerevisiae (Cramer et al., 2002; MendeseFerreira et al., 2004; MartínezeMoreno et al., 2012) and therefore did not account for differences in nitrogen utilization between isolates (Julien et al., 2000; MendeseFerreira et al., 2004). While having adequate nitrogen available for primary fermentation is crucial, nutrients not metabolized by S. cerevisiae during alcoholic fermentation could theoretically be available for spoilage microbes during the later stages of wine processing (Bisson and Butzke, 2000; Bell and Henschke, 2005). One such postealcoholic spoilage threat is Brettanomyces bruxellensis, a yeast which produces a variety of unpleasant aromas described as ‘medicinal,’ ‘mousey,’ ‘rancid,’ ‘sweat,’ ‘smoke,’ ‘horsey,’ ‘leather,’ or ‘BandeAid®’ (Oelofse et al., 2008). While B. bruxellensis can metabolize a variety of nitrogen and carbon sources, nutritional requirements for growth in wine are not well understood (AguilareUscanga et al., 2000; Conterno et al., 2006). The objectives of this study were to (a) investigate the relationship between available nitrogen and the sugar content of musts using strains of S. cerevisiae which differ in their nitrogen requirements and (b) determine the impact of nitrogen supplementation of musts on subsequent growth of B. bruxellensis in wines.
composition of Cabernet Sauvignon grapes from Washington State (Spayd and AnderseneBagge, 1996). A 2 3 factorial design was employed with YAN (150 or 250 mg N/L) and equal proportions of glucose/fructose (230, 250, or 270 mg/L) as variables. Enough DAP was added to the base medium to yield 250 mg N/L YAN. Tween 80, dissolved in water and sterilized at 121 C for 15 min, and pecoumaric acid, dissolved in absolute ethanol, were also added at concentrations of 5 mg/L. Once prepared, all media were sterile filtered through 0.22 mm PES Express™Plus bottle top filters (Millipore, Bedford, MA, USA) into 3 L Celstir® bioreactors (Wheaton Science Products, Millville, NJ, USA) with stainless steel headpieces (B. Braun Biotech, Allentown, PA, USA) and an aseptic system that avoids incorporation of air during sampling. A suspension of microcrystalline cellulose (Sigmacell Type 20, SigmaeAldrich, St. Louis, MO, USA) was sterilized at 121 C for 15 min and added at 1 g/L prior to inoculation of S. cerevisiae strain ECA5 or Uvaferm 228 at 105 cfu/mL. All fermentations were conducted with triplicate replication without shaking but were stirred for 2 min at 75 rpm before sampling.
2. Materials and methods
Synthetic wines (200 mL) was centrifuged 1000 g for 30 min, sterile filtered (0.22 mm), and divided into two sterile milk dilution bottles (100 mL). A sterile filtered (0.22 mm) vitamin solution was added so that wines contained 25 mg/L myoeinositol, 1 mg/L pyrodoxineHCl, 10 mg/L nicotinic acid, 1 mg/L thiamineHCl, and 30 mg/L biotin. A suspension of powdered cellulose (Sigmacell Type 20, SigmaeAldrich, St. Louis, MO, USA) was sterilized at 121 C for 15 min and added to wines to yield a concentration of 1 g/L. Starter cultures of B. bruxellensis were used to inoculate the wines at initial populations of 105 cfu/mL. Bottles were incubated at 20 C and shaken prior to sampling. Minimum nitrogen needs for B. bruxellensis were determined using the same composition as the base synthetic grape juice medium but with fructose (2 g/L), ethanol (13% v/v), and pecoumaric acid (5 mg/L). A stock solution with the same amino acid/DAP composition as the base medium was diluted to produce media containing 0, 6, 12, 24, 48, or 96 mg N/L YAN. All media were acidified to pH 3.5 with phosphoric acid and sterile filtered (0.22 mm) before inoculation with B. bruxellensis E1 or B2 at approximately 105 cfu/mL. Treatments were conducted in triplicate at 20 C and sampled once per week.
2.1. Microorganisms S. cerevisiae strains ECA5 and Uvaferm 228 were received as al, Quebec, Canada). active dry cultures (Lallemand Inc., Montre These strains were selected based on their relative nitrogen requirements, with ECA5 having a low requirement compared to Uvaferm 228 (G. Specht, personal communication, 2011). B. bruxellensis strains E1 and B2 were isolated from Washington state wines as described by Jensen et al. (2009). All strains were isolated and maintained by repeated streaking on Wallerstein Laboratory Nutrient (WLN) agar (Oxoid, Hampshire, England) and incubated at 25 C. 2.2. Preparation of starter cultures One colony of S. cerevisiae was aseptically transferred into 10 mL YM broth (Difco, Sparks, MD, USA) acidified to pH 3.5 with phosphoric acid prior to incubation for 24 h at 25 C. Cultures were then transferred into 500 mL YM broth and incubated with shaking at 130 rpm until approximately 107 cfu/mL was reached. Cells were harvested by centrifugation at 1000 g for 30 min, washed with 0.2 M phosphate buffer, centrifuged, and suspended in phosphate buffer before inoculation into synthetic grape juice media at 105 cfu/mL. Single colonies of B. bruxellensis were aseptically transferred into 10 mL of YM broth and incubated at 25 C for approximately one week. One mL of this culture was transferred into 50 mL YM containing 10% (v/v) ethanol. Cells were incubated, harvested, and inoculated as previously described for S. cerevisiae but with different centrifugation conditions (2000 g for 15 min). 2.3. Synthetic grape must fermentations Fermentations were conducted using the synthetic grape juice medium described by Wang et al. (2003). The base medium contained 43 mg/L Ala, 132 mg/L Arg, 14 mg/L Asp, 55 mg/L Glu, 4 mg/L Gly, 30 mg/L His, 18 mg/L Ile, 22 mg/L Leu, 26 mg/L Lys, 8 mg/L Met, 18 mg/L Phe, 31 mg/L Ser, 28 mg/L Thr, 12 mg/L Trp, 14 mg/L Tyr, 120 mg/L Val, and 248 mg/L diammonium phosphate (DAP). These values represent approximately 120 mg N/L amino acids and 30 mg N/L DAP, concentrations that simulate the nitrogen
2.4. Inoculations of B. bruxellensis
2.5. Analyses Fermentation progress was evaluated by measuring the concentration of soluble solids (Ingledew and Kunkee, 1985) while final glucose and fructose concentrations were determined enzymatically (ReBiopharm AG, Darmstadt, Germany). Ethanol content was , France). determined using an ebulliometer (Alla France, Chemille Finally, the concentration of amino nitrogen in the wines was determined using the NOPA method (Dukes and Butzke, 1998) while an ioneselective electrode measured ammonia (Denver Instrument, Orville, NY, USA). Statistical analyses were performed with XLSTAT (Addinsoft, New York, NY, USA) with ANOVA and Tukey's HSD test for mean separation. 3. Results 3.1. Alcoholic fermentations Altering the concentration of YAN in the synthetic grape juice medium (150 or 250 mg N/L) did not affect fermentation
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completion of synthetic musts (Fig. 1). Here, musts inoculated with ECA5 exhibited longer fermentations as sugar concentrations increased from 230 up to 270 g/L. However, few differences existed between musts of the same sugar concentration but differed in YAN. Fermentations conducted by Uvaferm 228 required approximately 200 h longer for the same initial sugar levels than those inoculated with ECA5. An exception was 270 g/L ferments conducted by Uvaferm 228 where soluble solids differed by >20 g/L between the two nitrogen treatments at times, a difference which decreased to 4 g/L by 900 h. Differences in residual fructose, ethanol, and YAN were observed in resultant wines (Table 1). While glucose concentrations were generally <0.03 g/L, those musts containing higher amounts of sugar also produced wines with increased residual fructose. For example, musts that contained 230 or 250 g/L glucose/fructose at either YAN level attained dryness or 2 g/L residual fructose (mean ¼ 0.32 g/L) while those containing 270 g/L generally did not (mean ¼ 2.5 g/L) regardless of YAN concentration. Comparing yeast strains for a given nitrogen level, residual fructose concentrations were not significantly different. Similarly, ethanol contents of musts fermented with 150 or 250 mg N/L YAN were not significantly different for a given sugar level. Specifically, musts that had 230, 250, or 270 g/L sugar contained 13.5e14.1%, 14.7e14.9%, or 15.6e15.9% (v/v) ethanol, respectively. Averaging data from both yeast strains, wines from musts with 250 mg N/L YAN had more residual aeamino acids (86.8 mg N/L) and ammonium (49.7 mg N/ L) than those from musts containing 150 mg N/L (47.5 and 2.0 mg N/ L, respectively). Differences in residual ammonium between the yeast strains were also observed where wines fermented by
Uvaferm 228 contained 30e50 mg N/L while those by ECA5 had 60 mg N/L. 3.2. Growth of B. bruxellensis Inoculation of synthetic wines with B. bruxellensis yielded initial cell concentrations of 105 cfu/mL for both strains (Fig. 2). Subsequently, populations exceeded of 106 cfu/mL in wines made from musts that contained 230 or 250 g/L sugar but became undetectable (<60 cfu/mL) in those initially containing 270 g/L sugar. In addition, longer lag times for strains E1 and B2 were apparent as higher sugar in musts yielded higher amounts of ethanol. For example, populations reached >106 cfu/mL by day 15 in wines containing 13.5e14.1% (v/v) ethanol (musts with 230 g/L sugar). In wines with 14.7e14.9% (v/v) ethanol (musts with 250 g/L sugar), populations initially decreased approximately 0.5 log yet required >20 d to achieve similar populations. Finally, B. bruxellensis declined to undetectable populations by day 20 in wines containing 15.6e15.9% ethanol (musts with 270 g/L sugar). Few differences were noted between wines from musts initially containing 150 or 250 mg N/L for either strain of S. cerevisiae, even though total residual YAN ranged between 46 and 148 mg N/L depending on must composition (Table 1). To determine minimal amounts of nitrogen required for growth, B. bruxellensis were inoculated into synthetic wine media containing 0e96 mg N/L (Fig. 3). In media containing no added nitrogen, populations remained at 105 cfu/mL until day 15 (E1) or 23 (B2) when slight increases (0.5 log) were observed. By day 45, these ferments reached 5 105 cfu/mL. In contrast, populations exceeded 106 cfu/mL in all media containing 6 mg N/L. Here, strain E1 peaked approached 107 cfu/mL by day 30 in all media fermented by ECA5. More differences were noted in wines fermented by Uvaferm 228 where those wines obtained from musts with 48 or 96 mg N/L achieved higher populations sooner than those with 6, 12, or 24 mg N/L but remained at approximately 5 106 cfu/mL. 4. Discussion
Fig. 1. Decline of soluble solids during fermentation of synthetic musts containing 150 (B, , ) or 250 (C, :, -) mg N/L yeast assimilable nitrogen and 230 (B, C), 250 ( , :), or 270 ( , -) g/L glucose/fructose by S. cerevisiae strain ECA5 (A) or Uvaferm 228 (B).
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Availability of nitrogen (150 or 250 mg N/L) did not greatly affect fermentation of musts with varying sugar concentrations (230, 250, or 270 g/L) by either of the strains of S. cerevisiae studied, ECA5 (low nitrogen requirement) or Uvaferm 228 (high nitrogen requirement). Moreover, fermentations of synthetic grape musts containing very high amounts of sugar (295 g/L) did not complete even when supplemented to 580 mg N/L (data not shown), in agreement with MartínezeMoreno et al. (2012) who reported incomplete fermentations that contained 280 g/L glucose and 300 mg N/L. Overall, these findings were in contrast to the recommendations of Bisson and Butzke (2000) concerning the need to add extra nitrogen to high sugar musts in order to complete fermentation. Rather, these results support the contention that increasing nitrogen concentration of grape musts above a threshold (e.g., 140e150 mg N/L) will not necessarily yield successful fermentation of high sugar musts even taking into account nitrogen utilization requirements by different strains of S. cerevisiae. Similarly, Cramer et al. (2002) reported a proportional increase in cell concentration with nitrogen additions up to 140 mg N/L but beyond this amount, nitrogen was not the limiting factor for the yeast strain e). While Erasmus et al. (2003) noted that studied (Premier Cuve elevated sugar concentrations increased the expression of numerous genes with a simultaneous decrease of others by S. cerevisiae, it was unclear if net protein synthesis and, therefore, overall nitrogen requirements were also altered due to higher sugar amounts. In fact, ethanol accumulation may be the more important
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Table 1 Composition of wines made from synthetic grape musts that contained variable concentrations of YAN/fermentable sugar and were fermented by S. cerevisiae ECA5 or Uvaferm 228. Must Yeast strain ECA5
Wine YAN (mg N/L) 150
250
Uvaferm 228
150
250
Total sugar (g/L) 230 250 270 230 250 270 230 250 270 230 250 270
Glucose (g/L) <0.03 <0.03 0.071 <0.03 <0.03 <0.03 <0.03 <0.03 <0.03 <0.03 <0.03 <0.03
Fructose (g/L) b
0.11 0.30b 2.6a 0.19b 0.33b 2.7a 0.25b 0.43b 1.9a 0.32b 0.61b 2.2a
Ethanol (% v/v) c
14.0 14.8b 15.9a 14.1c 14.8b 15.9a 13.8bc 14.9a 15.6a 13.5c 14.7ab 15.8a
aeAmino acids (mg N/L) b
50 48bc 45c 85a 84a 86a 45b 44b 53b 90a 87a 89a
Ammonium (mg N/L) 1.0b 0.78b 1.3b 61a 60a 62a 3.5c 2.4c 3.1c 31b 30b 54a
aed
Means within columns followed by different letters differ significantly at p 0.05.
did not support the contention that higher concentrations of residual nitrogen enhances the growth of B. bruxellensis. Earlier work by AguilareUscanga et al. (2000) suggested that the yeast may have minimal nitrogen requirements since increased concentrations of ammonia only “weakly stimulated” B. bruxellensis. In fact, B. bruxellensis growth was more affected by ethanol content as opposed to available nitrogen. Increasing must sugar resulted in wines of higher ethanol content with more delayed growth of B. bruxellensis if not slow death. Previous studies have suggested that increasing ethanol concentrations resulted in prolonged lag phases, where concentrations up to 15% ethanol prevented growth (Dias et al., 2003; Medawar et al., 2003; Barata et al., 2008). However, the ability to survive high ethanol concentrations in wine is dependent on the strain of B. bruxellensis. For example, while the maximum concentration of ethanol tolerated by strains E1 and B2 was between 14.9 and 15.6% (v/v), Barata et al. (2008) demonstrated that most strains of B. bruxellensis tolerate 14.5%.
cause of fermentation problems associated with high sugar musts (Nishino et al., 1985; Mauricio and Salmon, 1992). Adding excessive amounts of nitrogen to grape musts yielded resultant wines with higher residual YAN, in agreement with others (Beltran et al., 2005; Taillandier et al., 2007; Sturgeon et al., 2013). Of concern is the possibility that higher concentrations of nitrogen in wines could support the growth of unwanted spoilage microorganisms during storage or in the bottle (Bisson and Butzke, 2000; Bell and Henschke, 2005). However, growth of B. bruxellensis was not influenced by the amount of residual nitrogen in the synthetic wines. As evidence, B. bruxellensis strain E1 or B2 reached similar populations in wines containing 46e143 mg N/L for those wines from initial must sugar concentrations of 230 or 250 g/L. In fact, both strains grew to populations of >106 cfu/mL in synthetic wine containing as little as 6 mg N/L, a value much lower than the 122 mg N/L reported by AguilareUscanga et al. (2000) and Conterno et al. (2006). Given this low concentration, these results
Fig. 2. Growth of B. bruxellensis strains E1 (A, B) or B2 (C, D) in synthetic wines fermented by S. cerevisiae strains ECA5 (A, C) or Uvaferm 228 (B, D). Musts originally contained 150 (B, , ) or 250 (C, :, -) mg N/L yeast assimilable nitrogen and 230 (B, C), 250 ( , :), or 270 ( , -) g/L glucose/fructose. Dashed lines indicate points that were below the detection limit (<60 cfu/mL) while error bars indicate standard error.
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Acknowledgments This research was conducted at Washington State University as part of the Master of Science thesis of Bradford C. Childs. The aual, thors would like to thank G. Specht (Lallemand Inc., Montre Quebec, Canada) for supplying yeast cultures and to the Northwest Center for Small Fruits Research (grant 11D-3057-3948; Corvallis, OR, USA) and Washington State University (Pullman, WA, USA) for financial support. References
Fig. 3. Growth of B. bruxellensis strain E1 (A) or B2 (B) in synthetic wine media that contained 0 (B), 6 (C), 12 ( ), 24 (:), 48 ( ) or 96 (-) mg N/L yeast assimilable nitrogen. Error bars indicate standard error.
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This suggests that the strains isolated from commercial Washington wines may be more ethanol tolerant than others. Although the strains of B. bruxellensis studied were adapted to ethanol prior to inoculation to increase tolerance (Vigentini et al., 2008), other factors such as media composition, pH, temperature, and SO2 levels also influence survivability (Barata et al., 2008; Zuehlke and Edwards, 2013). Given that the strains of B. bruxellensis used in the present study grew to high populations in synthetic grape medium (wine) containing low concentrations of nitrogen, this suggests that many commercial wines would likely contain enough residual nitrogen to support growth. Another source of nitrogen for B. bruxellensis would be S. cerevisiae which releases amino acids during the later stages of alcoholic fermentation (Ough et al., 1991). Finally, proline, an amino acid omitted from synthetic grape must fermentations but is abundant in finished wines, may serve as a sole nitrogen source for B. bruxellensis if enough oxygen is available (Conterno et al., 2006; Blomqvist et al., 2012). 5. Conclusions In general, 150 mg N/L YAN was sufficient for fermentations to reach dryness in musts containing 230 or 250 g/L glucose/fructose although higher levels (250 mg N/L) did not necessarily result in completion of fermentation of high sugar musts (270 or 295 g/L). Higher nitrogen supplementation also resulted in higher concentrations of residual nitrogen in the final wines, which theoretically could be metabolized by spoilage microorganisms. While adding high levels of nitrogen to musts does not necessarily ensure completion of alcoholic fermentation, residual YAN did not affect B. bruxellensis growth as much as ethanol concentration. Thus, it is not necessary for winemakers to add high amounts of nitrogen in order to complete alcoholic fermentation.
Agenbach, W.A., 1977. A study of must nitrogen content in relation to incomplete fermentations, yeast production and fermentation activity. In: Beukman, E.F. (Ed.), Proceedings of the South AfricaN Society for Enology and Viticulture, Stellenbosch, South Africa, pp. 66e88. AguilareUscanga, M., Delia, M.L., Strehaiano, P., 2000. Nutritional requirements of Brettanomyces bruxellensis: growth and physiology in batch culture and chemostat cultures. Can. J. Microbiol. 46, 1046e1050. Alston, J.M., Fuller, K.B., Lapsley, J.T., Soleas, G.S., 2011. Too much of a good thing? Causes and consequences of increases in sugar content of California wine grapes. J. Wine Econ. 6, 135e159. Barata, A., Caldiera, J., Botelheiro, R., Pagliara, D., MalfeitoeFerreira, M., Loureiro, V., 2008. Survival patterns of Dekkera bruxellensis in wines and inhibitory effect of sulphur dioxide. Int. J. Food Microbiol. 121, 201e207. Bell, S.J., Henschke, P.A., 2005. Implications of nitrogen nutrition for grapes, fermentation and wine. Aust. J. Grape Wine Res. 11, 242e295. s, N., Mas, A., Guillamo n, J.M., 2005. Influence of Beltran, G., EsteveeZarzoso, B., Roze the timing of nitrogen additions during synthetic grape must fermentations on fermentation kinetics and nitrogen consumption. J. Agric. Food Chem. 53, 996e1002. Bisson, L.F., Butzke, C.E., 2000. Diagnosis and rectification of stuck and sluggish fermentations. Am. J. Enol. Vitic. 51, 168e177. , V.S., GorwaeGrauslund, M., Passoth, V., 2012. Physiological Blomqvist, J., Nogue requirements for growth and competitiveness of Dekkera bruxellensis under oxygen limited or anaerobic conditions. Yeast 29, 265e274. Conterno, L., Joseph, C., Arvik, T.J., HenickeKling, T., Bisson, L.F., 2006. Genetic and physiological characterization of Brettanomyces bruxellensis strains isolated from wines. Am. J. Enol. Vitic. 57, 139e147. Cramer, A.C., Vlassides, S., Block, D.E., 2002. Kinetic model for nitrogenelimited wine fermentations. Biotechnol. Bioeng. 77, 49e60. Dias, L., PereiraedaeSilva, S., Tavares, M., MalfeitoeFerreira, M., Loureiro, V., 2003. Factors affecting the production of 4eethyl phenol by the yeast Dekkera bruxellensis in enological conditions. Food Microbiol. 20, 377e384. Dukes, B.C., Butzke, C.E., 1998. Rapid determination of primary amino acids in grape juice using an oephthaldialdehyde/NeacetyleLecysteine spectrophotometric assay. Am. J. Enol. Vitic. 49, 125e134. Erasmus, D.J., van der Merwe, G.K., van Vuuren, H.J.J., 2003. Genomeewide expression analyses: metabolic adaptation of Saccharomyces cerevisiae to high sugar stress. FEMS Yeast Res. 3, 375e399. Ingledew, W.M., Kunkee, R.E., 1985. Factors influencing sluggish fermentations of grape juice. Am. J. Enol. Vitic. 36, 65e76. Jensen, S.L., Umiker, N.L., Arneborg, N., Edwards, C.G., 2009. Identification and characterization of Dekkera bruxellensis, Candida pararugosa, and Pichia guillermondii isolated from commercial red wines. Food Microbiol. 26, 915e921. Jiranek, V., Langridge, P., Henschke, P.A., 1995. Amino acid and ammonium utilization by Saccharomyces cerevisiae wine yeasts from a chemically defined medium. Am. J. Enol. Vitic. 46, 75e83. Julien, A., Roustan, J.L., Dulau, L., Sablayrolles, J.M., 2000. Comparison of nitrogen and oxygen demands of enological yeasts: technological consequences. Am. J. Enol. Vitic. 46, 215e222. MartínezeMoreno, R., Morales, P., Gonzalez, R., Mas, A., Beltran, G., 2012. Biomass production and alcoholic fermentation performance of Saccharomyces cerevisiae as a function of nitrogen source. FEMS Yeast Res. 12, 477e485. Mauricio, J.C., Salmon, J.M., 1992. Apparent loss of sugar transport activity in Saccharomyces cerevisiae may mainly account for maximum ethanol production during alcoholic fermentation. Biotechnol. Lett. 14, 577e582. Medawar, W., Strehaiano, P., Delia, M., 2003. Yeast growth: lag phase modeling in alcoholic media. Food Microbiol. 20, 527e532. ~o, C., 2004. Growth and fermentation MendeseFerreira, A., MendeseFaia, A., Lea patterns of Saccharomyces cerevisiae under different ammonium concentrations and its implications in winemaking industry. J. Appl. Microbiol. 97, 540e545. ~ a, R., 2010. Climate change associated effects on grape and wine Mira de Ordun quality and production. Food Res. Int. 43, 1844e1855. Nishino, H., Miyazaki, S., Tohjo, K., 1985. Effect of osmotic pressure on the growth rate and fermentation activity of wine yeasts. Am. J. Enol. Vitic. 36, 170e174. Oelofse, A., Pretorius, I.S., du Toit, M., 2008. Significance of Brettanomyces and Dekkera during winemaking: a synoptic review. South Afr. J. Enol. Vitic. 29, 128e144.
B.C. Childs et al. / Food Microbiology 46 (2015) 604e609 Ough, C.S., Huang, Z., An, D., Stevens, D., 1991. Amino acid uptake by four commercial yeasts at two different temperatures of growth and fermentation: effects on urea excretion and readsorption. Am. J. Enol. Vitic. 41, 26e40. Petrie, P.R., Sadras, V.O., 2008. Advancement of grapevine maturity in Australia between 1993 and 2006: putative causes, magnitude of trends and viticultural consequences. Aust. J. Grape Wine Res. 14, 33e45. Sadras, V.O., Moran, M.A., 2012. Elevated temperature decouples anthocyanins and sugars in berries of Shiraz and Cabernet Franc. Aust. J. Grape Wine Res. 18, 115e122. Schütz, M., Gafner, J., 1995. Lower fructose uptake capacity of genetically characterized strains of Saccharomyces bayanus compared to strains of Saccharomyces cerevisiae: a likely cause of reduced alcoholic fermentation activity. Am. J. Enol. Vitic. 46, 175e180. Spayd, S.E., AnderseneBagge, J., 1996. Free amino acid composition of grape juice from 12 Vitis vinifera cultivars in Washington. Am. J. Enol. Vitic. 47, 389e402. Sturgeon, J.Q., Bohlscheid, J.C., Edwards, C.G., 2013. The effect of nitrogen source on yeast metabolism and H2S formation. J. Wine Res. 24, 182e194.
609
Taillandier, P., Ramon Portugal, F., Fuster, A., Strehaiano, P., 2007. Effect of ammonium concentration on alcoholic fermentation kinetics by wine yeasts for high sugar content. Food Microbiol. 24, 95e100. Ugliano, M., Fredrizzi, B., Siebert, T., Travis, B., Magno, F., Versini, G., Henschke, P.A., 2009. Effect of nitrogen supplementation and Saccharomyces species on hydrogen sulfide and other volatile sulfur compounds in Shiraz fermentation and wine. J. Agric. Food Chem. 57, 4948e4955. Vigentini, I., Romano, A., Compagno, C., Merico, A., Molinari, F., Tirelli, A., Foschino, R., Volonterio, G., 2008. Physiological and oenological traits of different Dekkera/Brettanomyces bruxellensis strains under wineemodel conditions. FEMS Yeast Res. 8, 1087e1096. Wang, X.D., Bohlscheid, J.C., Edwards, C.G., 2003. Fermentative activity and production of volatile compounds by Saccharomyces grown in synthetic grape juice media deficient in assimilable nitrogen and/or pantothenic acid. J. Appl. Microbiol. 94, 349e359. Zuehlke, J.M., Edwards, C.G., 2013. Impact of sulfur dioxide and temperature on culturability and viability of Brettanomyces bruxellensis in wine. J. Food Prot. 76, 2024e2030.