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Effect of Saccharomyces cerevisiae inoculum size on wine fermentation aroma compounds and its relation with assimilable nitrogen content
International Journal of Food Microbiology 143 (2010) 81–85
Contents lists available at ScienceDirect
International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o
Short Communication
Effect of Saccharomyces cerevisiae inoculum size on wine fermentation aroma compounds and its relation with assimilable nitrogen content Francisco Carrau a,⁎, Karina Medina a, Laura Fariña a, Eduardo Boido a, Eduardo Dellacassa b a b
Enology Section, Department of Food Science and Technology, Facultad de Química, Universidad de la República, 11800 Montevideo, Uruguay Cátedra de Farmacognosia y Productos Naturales, Department of Organic Chemistry, Facultad de Química, Universidad de la República, Montevideo, Uruguay
a r t i c l e
i n f o
Article history: Received 19 March 2010 Received in revised form 14 June 2010 Accepted 16 July 2010 Keywords: Inoculum size Saccharomyces Wine fermentation Grape aroma compounds Yeast assimilable nitrogen
a b s t r a c t Different commercial Saccharomyces cerevisiae strains have been applied at the winemaking level, trying to establish a dominant population of selected strains from the start of fermentation and ensuring the complete consumption of sugars. Although a large population of active yeast cells can be introduced in the inoculated wines, resulting in a complete fermentation, this does not necessarily mean an improvement of the sensory characteristics of the wines. The impact of the size of the inocula in wine quality parameters has been very little studied, and in no case the nutrient balance of the grape must utilized was taken into account. In this work we present results obtained for wine aroma compounds at three inoculum levels (104, 105 and 106 cells/mL), and two different yeast assimilable nitrogen (YAN) in a white grape must, using two S. cerevisiae strains commonly used for winemaking. A significant effect in the final concentrations of higher alcohols, esters, fatty acids, free monoterpenes and lactones was attributed to the size of inoculum in both strains but not in an easily predictable way. However, a consistent increase of desired aroma compounds (esters, lactones and free monoterpenes), and a decrease of less desired compounds for white wine (higher alcohols and medium chain fatty acids), was shown at inoculum sizes of 105 cells/mL for both strains in real winemaking conditions. In a discriminant analysis six aroma compounds discriminate the three inoculum sizes for all wine samples: 1,8-terpine, hodiol I (trans-3,7-dimethyl-1,5-octadiene-3,7-diol), isobutyl alcohol, iso C4 acid, ethyl C6 ester and C8 acid. © 2010 Elsevier B.V. All rights reserved.
1. Introduction It is well known that Saccharomyces cerevisiae produces different concentrations of aroma compounds as a function of fermentation conditions and must treatments, including temperature, grape variety, micronutrients, vitamins and nitrogen composition of the must. Additionally, sluggish and stuck fermentations often have been related to nitrogen deficiency (Bisson, 1999) and to the small size of the inocula of S. cerevisiae (Cuinier, 1983). In the late seventies, these fermentation problems were partially solved by the addition of ammonium salts to deficient musts, increasing the fermentation rate and the sensory desirability of the wines (Fleet, 2003; Romano et al., 2003; Swiegers et al., 2005). The size of the inocula is a well known key process parameter in microbial fermentation (Medina et al., 1997; Papagianni and Moo-Young, 2002; SreenivasRao et al., 2004), paradoxically, the impact of it in wine quality parameters has been studied in a very limited fashion and in no case the nutrient balance of the grape must utilized has been taken into account (Cuinier, 1983; Erten et al., 2006; Mateo et al., 2001; Monk and Storer, 1986; Radler
⁎ Corresponding author: Tel.: + 598 2 9248194; fax: + 598 2 9241906. E-mail address: [email protected] (F. Carrau). 0168-1605/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2010.07.024
and Schütz, 1982). Some of these studies showed that a higher level of inoculum resulted in higher fermentation rates, although in sparkling wines this effect was not seen over 4 × 106 cells/mL (Monk and Storer, 1986). Higher inoculum size resulted in higher yields of glycerol and ethyl alcohol (Radler and Schütz, 1982). Furthermore, the influence of inoculum size in aroma compounds was little studied in wines (Erten et al., 2006; Mateo et al., 2001). An increase of higher alcohols in relation to increase the inocula size was shown in these reports. However, in general differences were attributed to strain and not to the size of the inocula. Moreover, in recent studies related to beer aromas (Verbelen et al., 2009) the influence of this parameter on aroma composition was seen as rather limited. However these studies were performed at a higher size of the inocula compared to spontaneous or inoculated wine fermentations. The aim of the present study was to characterise the effect of inoculum volume of S. cerevisiae on the production of key yeast aroma compounds (esters, alcohols, acids, free monoterpenes and lactones) in a grape white must. We have investigated the yeast derived aroma compounds in wines prepared under two defined yeast assimilable nitrogen (YAN) contents, similar to winemaking conditions (Carrau et al., 2005; Henschke and Jiranek, 1993), to better understand their relation with the nitrogen level of the medium. The different behavior of these yeast strains at wider initial nitrogen concentrations was
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examined previously in our model system (Carrau et al., 2008). Understanding the importance of the size of the inocula on aroma compositions will allow the improvement of data models for the application of metabolic aroma footprinting methods in yeast strain characterization (Kell et al., 2005). 2. Materials and methods 2.1. Yeast strains S. cerevisiae strains utilized were: Montrachet UCD 522 (University of California, Davis) referred to as M522 in this work, and KU1 (Uruguayan selected strain) (Carrau et al., 1993). Both strains are used in the commercial production of wine. Inocula were prepared in the same media used for fermentation, the grape juice by incubation for 12 h in a rotary shaker at 150 rpm and 25 °C. Inoculum concentrations were 1 × 104, 1 × 105 and 1 × 106 cells/mL of medium for both strains. The two yeast strains used were selected from a large group of strains as both showed a similar nitrogen consumption when tested under different nitrogen conditions, but produced different wines at low nitrogen concentration (75 mg N/L) from sensory and chemical points of view. 2.2. Fermentation conditions A grape white must of Muscat Alexandria was used as the fermentation medium sterilized with 0.45 μm pore filter membrane. The initial YAN content was 148 mg N/L and adjustments of YAN were made increasing the basic concentration by supplementation with 300 mg/L of diammonium phosphate (DAP, approximately 63 mg N/L of YAN increase) where indicated. The total sugar was 210 g/L and pH was 3.5. Fermentations were carried out in 125 mL of medium contained in 250 mL Erlenmeyer flasks, closed with Muller valves filled with pure sulfuric acid. Inoculum size and the addition of YAN were chosen as the two variables for this investigation since it was previously found that under similar experimental conditions this factor significantly affected production of fermentation aroma compounds by these yeasts (Carrau et al., 2008). Static batch fermentation conditions were conducted at 20 °C in duplicate, simulating wine making conditions. Fermentation activity was measured as CO2 weight loss and expressed in grams per 100 mL, and total residual sugars were analyzed (Zoecklein et al., 1995). Once a day samples were taken to measure cell growth in an improved Neubauer chamber. Samples of 100 ml of the finished wines for GC– MS analysis were taken 2 days after the end of fermentation, filtered through 0.45 μm pore membranes and SO2 was added as 50 mg/L of sodium metabisulphite. 2.3. GC and GC–MS analysis 2.3.1. Aroma volatile compounds Extraction of aroma compounds was performed by adsorption and separate elution from an Isolute ENV + cartridge (IST Ltd., Mid Glamorgan, U.K.) packed with 1 g of highly cross-linked styrenedivinyl benzene (SDVB) polymer. Treatment of samples and GC analysis were performed as described previously (Boido et al., 2003). 2.3.2. Identification and quantification The components of wine aromas were identified by comparison of their Linear Retention Indices, with pure standards or data reported in the literature. Comparison of mass spectral fragmentation patterns with those stored on databases was also performed. A sample of 50 mL of wine diluted with 50 mL of distilled water and containing 0.1 mL of internal standard was used for each analysis (1-heptanol at 230 ppm in a 50% hydroalcoholic solution). GC–FID and GC–MS
instrumental procedures were applied for quantitative purposes as described previously (Boido et al., 2003). 2.4. Statistical analysis A stepwise discriminant analysis was carried out with the aroma compounds analyzed of the twelve wines produced with both strains in triplicate in a basic fermentation medium containing an initial YAN of 148 mg N/L, with and without an extra YAN addition of 63 mg N/L as DAP. ANOVA for size of inoculum, initial YAN concentration and yeast strain were performed for each aroma compound determined using Statistica 5.1. Differences of free volatile compounds were evaluated; mean rating and least significant differences for initial YAN concentrations and the size of the inoculum for each strain were calculated from the analysis of variance. 3. Results and discussion Maximum yeast cell number in the culture and net yeast growth (difference between the maximum cell number per unit of volume and the cell number added as inoculum), were calculated for both strains in order to understand the effect of different inoculum sizes in cell growth. The results showed a direct correlation between total yeast cell number and inoculum size level, however a negative correlation of these two parameters was found with net yeast cell growth. For example, total cell number increases 2 times between 105 and 106 cells/mL of inoculum for both strains, while the net yeast growth decreases 5 times for the same two inocula sizes. Briefly, the higher the inocula, the less growth will occur in the culture, which was in agreement with previous studies in high gravity beer fermentations (Nguyen and Viet Man, 2009; Peddie, 1990). On the other hand, the higher YAN level resulted in higher total cell growth and obviously in higher net growth for all the inoculum size levels. Interestingly, this increase in growth with YAN addition was more significant at higher inoculum levels (at 106 cells/mL average increase 25%) than at lower levels (at 105 cells/mL average increase 10%). In Figs. 1 and 2 we show the size of inoculum effect on the volatile compounds at two different YAN concentrations and the three inoculum sizes for strains M522 and KU1. Some of these compound families are significantly affected by inoculum sizes, and it can be observed that YAN levels also affect the results. For M522, esters, free monoterpenes and γ-butyrolactone are produced in higher amounts at 105 cells/mL at both YAN levels (Fig. 1). Interestingly, a similar behavior can be seen for KU1 at the low YAN level, where 105 cells/mL produced higher amounts of the same volatiles. However, at higher levels of YAN KU1 shows a positive relation between inoculum size and these aroma compounds. The different behavior of KU1 and M522 at the high YAN level may be related to the different capacities of each strain to administrate the intracellular nitrogen. In relation to the volatile compounds produced, it was shown previously (Carrau et al., 2008) that M522 presents a less efficient use of YAN compared to KU1. As it is known that esters, free monoterpenes and γ-butyrolactone are positively correlated to YAN level (Carrau et al., 2008), it can be argued that the higher the inoculum, the more cells are demanding the YAN present in the medium at the initial stages. This situation could limit the formation of these compounds at the inocula of 106 cells/mL, and this could explain the behavior of M522 and of both strains at the low YAN level. Furthermore, as it was shown at 106 cells/ mL inoculum size the increase of growth (25%) due to YAN addition was higher than at 105 (10%). Growth is the cellular metabolism activity that demands acetyl-CoA, and this compound would be more available for ester synthesis with less net growth rates (Peddie, 1990). This could explain an increased demand of acetyl-CoA at 106 cells/mL of inoculum that could result in a decrease of some of these aroma compounds.
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Fig. 1. Inoculum size effect on aroma compound formation for model strains M522 (■) and KU1 (w) at two YAN levels. Inoculum sizes are indicated (1 × 104, 105 and 106 cells/mL) on a Muscat Alexandria grape must with a YAN level of 148 mg N/L increased with DAP to 211 mg N/L. Letters at each data point indicate the level of significant difference (p b 0.05) according to a LSD test of ANOVA calculated for each strain. Fermentations were in duplicate and bars indicate standard deviations. Esters (sum of isoamyl acetate, β-phenyl acetate, ethyl hexanoate, ethyl octanoate, ethyl decanoate, 4-hydroxybutyrolactate, diethyl malate and ethyl lactate); monoterpenes (hodiol I (trans 3,7-dimethyl-1,5-octadiene 3,7-diol), hodiol II (3,7-dimethyl-1,7-octadiene-3,6-diol), Ox.B (linalool cis-furanic oxide), Endiol (3,7-dimethyl-1-octen-3,7 diol), Linalool, ho-trienol, and α-terpineol) and γ-butyrolactone.
On the other hand, as it was discussed previously, the higher the inocula, the less net growth will occur during the fermentation process. This explanation can be applied to the positive correlation between the size of the inocula and ester production at a high YAN level for strain KU1 (see Fig. 1). Very limited studies about γ-butyrolactone (one of the main lactones of wine contributing with positive aromas related to caramel and coconut descriptors) are found in the literature. At low YAN level this compound behaves similar to esters and monoterpenes for both strains. Interestingly at the high YAN level there is a direct correlation with increased inocula sizes. Higher alcohols, iso-acids and fatty acids in general are not desirable compounds from the sensory point of view of wine over certain
concentration levels (Carrau et al., 2008). Fig. 2 shows that the lower conditions for production of higher alcohols and fatty acids for strain M522 were at 105 cells/mL, an opposite behavior compared to esters, monoterpenes and lactones considered desirable aroma compounds. In the case of strain KU1 Fig. 2 shows a positive correlation with inoculum sizes at a higher YAN level. Previous studies in grape wine fermentation are not in agreement with these results (Erten et al., 2006; Mateo et al., 2001), except with the behavior of KU1 at a high YAN level. But as it was stated these published results had not reported data about the YAN levels of the grape musts utilized and furthermore they had worked with higher sizes of inoculum levels (107 cells/mL and above). Fig. 2 also shows that iso-acids have an opposite behavior than fatty acids, except for M522 at high YAN concentration. These results
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Fig. 2. The same experimental and fermentation conditions of Fig. 1, showing the aroma compounds that could be considered less desirable. Alcohols (sum of 2-methyl-1-propanol, 2-methyl-1-butanol, β-phenyl ethyl alcohol, butanol, 2-ethyl hexanol and octanol), isocids (sum of isobutanoic) (isoC4) and isovaleric (isoC5) and medium chain fatty acids (sum of C4, C6, C8 and C10).
are in agreement with the opposite behavior found for these two families of compounds at a wider range of YAN levels (Carrau et al., 2008). To have a better understanding of how the key aroma compounds are affected by inoculum size, a discriminant analysis for 33 compounds of the two strains at both YAN levels was carried out (Fig. 3). The three inoculum sizes were discriminated and the six main discriminant compounds in this analysis are indicated with vectors in the figure. The compounds considered are 1,8-terpine, hodiol I (trans3,7-dimethyl-1,5-octadiene-3,7-diol), isobutyl alcohol, iso C4 acid, ethyl C6 ester and C8 acid. Other compounds that have influenced in this discrimination analysis but have less significance are the βphenyl ethyl alcohol, ethyl pyruvate, iso C5 acid and C10 acid. These results show that the inoculum size affects aroma compounds irrespective of the YAN level in both model strains.
Interestingly, relative to previous published work in wine, it was suggested that there are no significant differences between size of inoculum and aroma compounds concentration, except for higher alcohols and ethyl acetate (Erten et al., 2006; Mateo et al., 2001). However, in these studies it is difficult to draw firm conclusions concerning the relationship of inoculum size and the different aroma compounds produced by the yeast strains studied, due to the lack of information about the YAN levels of the fermentation media. Mateo et al. (2001) suggested that differences in the volatile fraction were mainly due to the yeast strain inoculated and not the size of inocula. Although Erten et al. (2006) did not suggest an ideal inoculum size level for the production of desirable aroma compounds, and that the YAN level was not reported for the white grape must utilized in that study, results shown in that paper are in agreement with our results. In that report a clear increase of esters (excluding ethyl acetate) and a
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size on wine quality are limited, but the presented results indicate the importance of defining this process parameter for key metabolic data models in aroma footprinting methods for discrimination and selection of S. cerevisiae wine yeasts.
Acknowledgements We would like to thank the critical comments made for this manuscript by Prof. Patrick Moyna. We would also thank the financial support of this project by PDT, ANII, INIA and CSIC (UdelaR, Facultad de Quimica), Uruguay.
References
Fig. 3. Discriminant analysis with 33 compounds produced from grape must for the three inoculum sizes: low 104; medium 105; and high 106 cells/mL, for both model strains and at two YAN levels. The main six compounds that discriminated the three cases are shown as vectors.
decreased concentration of higher alcohols and acetaldehyde at 105 cells/mL of inoculum could be seen. In the brewery industry there is also limited information on inocula size effects on beer aroma (Ayrapaa, 1968; Nordstrom, 1964; Peddie, 1990; Suihko et al., 1993), and it was recently stated that the influence of pitching rate on aroma compound production is rather limited, with the exception of total diacetyl levels, which strongly increase with inoculum size (Verbelen et al., 2009; Nguyen and Viet Man, 2009). However, the sizes of the inocula studied were very high compared to normal levels at wine fermentation processes (around 106 cells/mL). On the other hand, Verbelen et al. (2009) utilized a worth with more than 400 mg N/L of YAN, a value that was previously stated as too high for observing significantly variations in some key aroma compounds due to inoculum levels (Ayrapaa, 1968). Peddie (1990) hypothesized that a higher inoculum size produced higher levels of esters, and explained this behavior as connected to a lower level of growth demanding less acetyl-CoA in the cells, resulting in a higher availability of this intermediate for ester synthesis. However, this statement is only in agreement to the KU1 strain case reported by us at a high YAN level (see Fig. 1). On the other hand, Nordstrom (1964) proposed the opposite situation, where increased growth augments ester formation, an hypothesis that is partially in agreement with the results presented here at low YAN level except for the size of the inocula of 104 cells/mL. Our results suggest that the significant effect of inoculum size on many important wine aroma compounds is related to the YAN levels of the grape must. Although these effects are not easily predictable, we show here that initial cell numbers, net cell growth and fermentation rates could explain the behavior of our model strains. Further studies with artificial grape juice medium, must be performed in order to improve the understanding of this fermentation system, however our results already gave interesting practical consequences for quality wines. 4. Conclusions A consistent increase of desired aroma compounds (esters, lactones and free monoterpenes), and a decrease of less desired compounds (higher alcohols and medium chain fatty acids), was shown at inoculum sizes of 105 cells/mL for both strains in real winemaking conditions. YAN concentration levels of the grape must affect the production of different aroma compounds and this could be related to the quantity of cells at initial stages of the fermentation, rapid removal of nitrogen and net cell growth. Studies on the influence of inoculum
Ayrapaa, T., 1968. Formation of higher alcohols by various yeasts. Journal of the Institute of Brewing 74, 169–179. Bisson, L.F., 1999. Stuck and sluggish fermentations. American Journal of Enology and Viticulture 50, 107–119. Boido, E., Lloret, A., Medina, K., Farina, L., Carrau, F., Versini, G., Dellacassa, E., 2003. Aroma composition of Vitis vinifera cv. Tannat: the typical red wine from Uruguay. Journal of Agricultural and Food Chemistry 51, 5408–5413. Carrau, F.M., Neirotti, E., Gioia, O., 1993. Stuck wine fermentations: effect of killer/ sensitive yeast interactions. Journal of Fermentation and Bioengineering 76, 67–69. Carrau, F.M., Medina, K., Boido, E., Farina, L., Gaggero, C., Dellacassa, E., Versini, G., Henschke, P.A., 2005. De novo synthesis of monoterpenes by Saccharomyces cerevisiae wine yeasts. FEMS Microbiology Letters 243, 107–115. Carrau, F.M., Medina, K., Farina, L., Boido, E., Henschke, P.A., Dellacassa, E., 2008. Production of fermentation aroma compounds by Saccharomyces cerevisiae wine yeasts: effects of yeast assimilable nitrogen on two model strains. FEMS Yeast Research 8, 1196–1207. Cuinier, C., 1983. Reflections on yeast inocula in oenology. Vignes et Vins 322, 12–18. Erten, H., Tanguler, H., Cabaroglu, T., Canbas, A., 2006. The influence of inoculum level on fermentation and flavour compounds of white wines made from cv. Emir. Journal of the Institute of Brewing 112, 232–236. Fleet, G.H., 2003. Yeast interactions and wine flavour. International Journal of Food Microbiology 86, 11–22. Henschke, P.A., Jiranek, V., 1993. Yeast: metabolism of nitrogen compounds. In: Fleet, Graham H. (Ed.), Wine Microbiology and Biotechnology. Harwood Academic Publishers, pp. 77–164. Kell, D.B., Brown, M., Davey, H.M., Dunn, W.B., Spasic, I., Oliver, S.G., 2005. Metabolic footprinting and systems biology: the medium is the message. Nature Reviews in Microbiology 3, 557–565. Mateo, J.J., Jimenez, M., Pastor, A., Huerta, T., 2001. Yeast starter cultures affecting wine fermentation and volatiles. Food Research International 34, 307–314. Medina, K., Carrau, F.M., Gioia, O., Bracesco, N., 1997. Nitrogen availability of grape juice limits killer yeast growth and fermentation activity during mixed-culture fermentation with sensitive commercial yeast strains. Applied and Environmental Microbiology 63, 2821–2825. Monk, P.R., Storer, R.J., 1986. The kinetics of yeast growth and sugar utilization in tirage: the influence of different methods of starter culture preparation and inoculation levels. American Journal of Enology and Viticulture 37, 72–76. Nguyen, T.H., Viet Man, L.V., 2009. Using high pitching rate for improvement of yeast fermentation performance in high gravity brewing. International Food Research Journal 16, 547–554. Nordstrom, K., 1964. Studies on the formation of volatile esters in fermentation with brewer's yeast. Svensk Kemisk Tidskrift 76, 510–543. Papagianni, M., Moo-Young, M., 2002. Protease secretion in glucoamylase producer Aspergillus niger cultures: fungal morphology and inoculum effects. Process Biochemistry 37, 1271–1278. Peddie, H.A.B., 1990. Ester formation in brewery fermentations. Journal of the Institute of Brewing 96, 327–331. Radler, F., Schütz, H., 1982. Glycerol production of various strains of Saccharomyces. American Journal of Enology and Viticulture 33, 36–40. Romano, P., Fiore, C., Paraggio, M., Caruso, M., Capece, A., 2003. Function of yeast species and strains in wine flavour. International Journal of Food Microbiology 86, 169–180. SreenivasRao, R., Prakasham, R.S., Prasad, K.K., Rajesham, S., Sarma, P.N., Rao, L.V., 2004. Xylitol production by Candida sp.: parameter optimization using Taguchi approach. Process Biochemistry 39, 951–956. Suihko, M.-L., Vilpola, A., Linko, M., 1993. Pitching rate in high gravity brewing. Journal of the Insitute of Brewing 99, 341–346. Swiegers, J.H., Bartowsky, P.A., Henschke, P.A., Pretorius, I.S., 2005. Yeast and bacterial modulation of wine aroma and flavour. Australian Journal of Grape and Wine Research 11, 139–173. Verbelen, P.J., Dekoninck, T.M.L., Saerens, S.M.G., Mulders, S.E.V., Thevelein, J.M., Delvaux, F.R., 2009. Impact of pitching rate on yeast fermentation performance and beer flavour. Applied Microbiology and Biotechnology 82, 155–167. Zoecklein, B., Fugelsang, K., Gump, B., Nury, F., 1995. Wine Analysis and Production. Chapman & Hall, New York.
Contents lists available at ScienceDirect
International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o
Short Communication
Effect of Saccharomyces cerevisiae inoculum size on wine fermentation aroma compounds and its relation with assimilable nitrogen content Francisco Carrau a,⁎, Karina Medina a, Laura Fariña a, Eduardo Boido a, Eduardo Dellacassa b a b
Enology Section, Department of Food Science and Technology, Facultad de Química, Universidad de la República, 11800 Montevideo, Uruguay Cátedra de Farmacognosia y Productos Naturales, Department of Organic Chemistry, Facultad de Química, Universidad de la República, Montevideo, Uruguay
a r t i c l e
i n f o
Article history: Received 19 March 2010 Received in revised form 14 June 2010 Accepted 16 July 2010 Keywords: Inoculum size Saccharomyces Wine fermentation Grape aroma compounds Yeast assimilable nitrogen
a b s t r a c t Different commercial Saccharomyces cerevisiae strains have been applied at the winemaking level, trying to establish a dominant population of selected strains from the start of fermentation and ensuring the complete consumption of sugars. Although a large population of active yeast cells can be introduced in the inoculated wines, resulting in a complete fermentation, this does not necessarily mean an improvement of the sensory characteristics of the wines. The impact of the size of the inocula in wine quality parameters has been very little studied, and in no case the nutrient balance of the grape must utilized was taken into account. In this work we present results obtained for wine aroma compounds at three inoculum levels (104, 105 and 106 cells/mL), and two different yeast assimilable nitrogen (YAN) in a white grape must, using two S. cerevisiae strains commonly used for winemaking. A significant effect in the final concentrations of higher alcohols, esters, fatty acids, free monoterpenes and lactones was attributed to the size of inoculum in both strains but not in an easily predictable way. However, a consistent increase of desired aroma compounds (esters, lactones and free monoterpenes), and a decrease of less desired compounds for white wine (higher alcohols and medium chain fatty acids), was shown at inoculum sizes of 105 cells/mL for both strains in real winemaking conditions. In a discriminant analysis six aroma compounds discriminate the three inoculum sizes for all wine samples: 1,8-terpine, hodiol I (trans-3,7-dimethyl-1,5-octadiene-3,7-diol), isobutyl alcohol, iso C4 acid, ethyl C6 ester and C8 acid. © 2010 Elsevier B.V. All rights reserved.
1. Introduction It is well known that Saccharomyces cerevisiae produces different concentrations of aroma compounds as a function of fermentation conditions and must treatments, including temperature, grape variety, micronutrients, vitamins and nitrogen composition of the must. Additionally, sluggish and stuck fermentations often have been related to nitrogen deficiency (Bisson, 1999) and to the small size of the inocula of S. cerevisiae (Cuinier, 1983). In the late seventies, these fermentation problems were partially solved by the addition of ammonium salts to deficient musts, increasing the fermentation rate and the sensory desirability of the wines (Fleet, 2003; Romano et al., 2003; Swiegers et al., 2005). The size of the inocula is a well known key process parameter in microbial fermentation (Medina et al., 1997; Papagianni and Moo-Young, 2002; SreenivasRao et al., 2004), paradoxically, the impact of it in wine quality parameters has been studied in a very limited fashion and in no case the nutrient balance of the grape must utilized has been taken into account (Cuinier, 1983; Erten et al., 2006; Mateo et al., 2001; Monk and Storer, 1986; Radler
⁎ Corresponding author: Tel.: + 598 2 9248194; fax: + 598 2 9241906. E-mail address: [email protected] (F. Carrau). 0168-1605/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2010.07.024
and Schütz, 1982). Some of these studies showed that a higher level of inoculum resulted in higher fermentation rates, although in sparkling wines this effect was not seen over 4 × 106 cells/mL (Monk and Storer, 1986). Higher inoculum size resulted in higher yields of glycerol and ethyl alcohol (Radler and Schütz, 1982). Furthermore, the influence of inoculum size in aroma compounds was little studied in wines (Erten et al., 2006; Mateo et al., 2001). An increase of higher alcohols in relation to increase the inocula size was shown in these reports. However, in general differences were attributed to strain and not to the size of the inocula. Moreover, in recent studies related to beer aromas (Verbelen et al., 2009) the influence of this parameter on aroma composition was seen as rather limited. However these studies were performed at a higher size of the inocula compared to spontaneous or inoculated wine fermentations. The aim of the present study was to characterise the effect of inoculum volume of S. cerevisiae on the production of key yeast aroma compounds (esters, alcohols, acids, free monoterpenes and lactones) in a grape white must. We have investigated the yeast derived aroma compounds in wines prepared under two defined yeast assimilable nitrogen (YAN) contents, similar to winemaking conditions (Carrau et al., 2005; Henschke and Jiranek, 1993), to better understand their relation with the nitrogen level of the medium. The different behavior of these yeast strains at wider initial nitrogen concentrations was
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examined previously in our model system (Carrau et al., 2008). Understanding the importance of the size of the inocula on aroma compositions will allow the improvement of data models for the application of metabolic aroma footprinting methods in yeast strain characterization (Kell et al., 2005). 2. Materials and methods 2.1. Yeast strains S. cerevisiae strains utilized were: Montrachet UCD 522 (University of California, Davis) referred to as M522 in this work, and KU1 (Uruguayan selected strain) (Carrau et al., 1993). Both strains are used in the commercial production of wine. Inocula were prepared in the same media used for fermentation, the grape juice by incubation for 12 h in a rotary shaker at 150 rpm and 25 °C. Inoculum concentrations were 1 × 104, 1 × 105 and 1 × 106 cells/mL of medium for both strains. The two yeast strains used were selected from a large group of strains as both showed a similar nitrogen consumption when tested under different nitrogen conditions, but produced different wines at low nitrogen concentration (75 mg N/L) from sensory and chemical points of view. 2.2. Fermentation conditions A grape white must of Muscat Alexandria was used as the fermentation medium sterilized with 0.45 μm pore filter membrane. The initial YAN content was 148 mg N/L and adjustments of YAN were made increasing the basic concentration by supplementation with 300 mg/L of diammonium phosphate (DAP, approximately 63 mg N/L of YAN increase) where indicated. The total sugar was 210 g/L and pH was 3.5. Fermentations were carried out in 125 mL of medium contained in 250 mL Erlenmeyer flasks, closed with Muller valves filled with pure sulfuric acid. Inoculum size and the addition of YAN were chosen as the two variables for this investigation since it was previously found that under similar experimental conditions this factor significantly affected production of fermentation aroma compounds by these yeasts (Carrau et al., 2008). Static batch fermentation conditions were conducted at 20 °C in duplicate, simulating wine making conditions. Fermentation activity was measured as CO2 weight loss and expressed in grams per 100 mL, and total residual sugars were analyzed (Zoecklein et al., 1995). Once a day samples were taken to measure cell growth in an improved Neubauer chamber. Samples of 100 ml of the finished wines for GC– MS analysis were taken 2 days after the end of fermentation, filtered through 0.45 μm pore membranes and SO2 was added as 50 mg/L of sodium metabisulphite. 2.3. GC and GC–MS analysis 2.3.1. Aroma volatile compounds Extraction of aroma compounds was performed by adsorption and separate elution from an Isolute ENV + cartridge (IST Ltd., Mid Glamorgan, U.K.) packed with 1 g of highly cross-linked styrenedivinyl benzene (SDVB) polymer. Treatment of samples and GC analysis were performed as described previously (Boido et al., 2003). 2.3.2. Identification and quantification The components of wine aromas were identified by comparison of their Linear Retention Indices, with pure standards or data reported in the literature. Comparison of mass spectral fragmentation patterns with those stored on databases was also performed. A sample of 50 mL of wine diluted with 50 mL of distilled water and containing 0.1 mL of internal standard was used for each analysis (1-heptanol at 230 ppm in a 50% hydroalcoholic solution). GC–FID and GC–MS
instrumental procedures were applied for quantitative purposes as described previously (Boido et al., 2003). 2.4. Statistical analysis A stepwise discriminant analysis was carried out with the aroma compounds analyzed of the twelve wines produced with both strains in triplicate in a basic fermentation medium containing an initial YAN of 148 mg N/L, with and without an extra YAN addition of 63 mg N/L as DAP. ANOVA for size of inoculum, initial YAN concentration and yeast strain were performed for each aroma compound determined using Statistica 5.1. Differences of free volatile compounds were evaluated; mean rating and least significant differences for initial YAN concentrations and the size of the inoculum for each strain were calculated from the analysis of variance. 3. Results and discussion Maximum yeast cell number in the culture and net yeast growth (difference between the maximum cell number per unit of volume and the cell number added as inoculum), were calculated for both strains in order to understand the effect of different inoculum sizes in cell growth. The results showed a direct correlation between total yeast cell number and inoculum size level, however a negative correlation of these two parameters was found with net yeast cell growth. For example, total cell number increases 2 times between 105 and 106 cells/mL of inoculum for both strains, while the net yeast growth decreases 5 times for the same two inocula sizes. Briefly, the higher the inocula, the less growth will occur in the culture, which was in agreement with previous studies in high gravity beer fermentations (Nguyen and Viet Man, 2009; Peddie, 1990). On the other hand, the higher YAN level resulted in higher total cell growth and obviously in higher net growth for all the inoculum size levels. Interestingly, this increase in growth with YAN addition was more significant at higher inoculum levels (at 106 cells/mL average increase 25%) than at lower levels (at 105 cells/mL average increase 10%). In Figs. 1 and 2 we show the size of inoculum effect on the volatile compounds at two different YAN concentrations and the three inoculum sizes for strains M522 and KU1. Some of these compound families are significantly affected by inoculum sizes, and it can be observed that YAN levels also affect the results. For M522, esters, free monoterpenes and γ-butyrolactone are produced in higher amounts at 105 cells/mL at both YAN levels (Fig. 1). Interestingly, a similar behavior can be seen for KU1 at the low YAN level, where 105 cells/mL produced higher amounts of the same volatiles. However, at higher levels of YAN KU1 shows a positive relation between inoculum size and these aroma compounds. The different behavior of KU1 and M522 at the high YAN level may be related to the different capacities of each strain to administrate the intracellular nitrogen. In relation to the volatile compounds produced, it was shown previously (Carrau et al., 2008) that M522 presents a less efficient use of YAN compared to KU1. As it is known that esters, free monoterpenes and γ-butyrolactone are positively correlated to YAN level (Carrau et al., 2008), it can be argued that the higher the inoculum, the more cells are demanding the YAN present in the medium at the initial stages. This situation could limit the formation of these compounds at the inocula of 106 cells/mL, and this could explain the behavior of M522 and of both strains at the low YAN level. Furthermore, as it was shown at 106 cells/ mL inoculum size the increase of growth (25%) due to YAN addition was higher than at 105 (10%). Growth is the cellular metabolism activity that demands acetyl-CoA, and this compound would be more available for ester synthesis with less net growth rates (Peddie, 1990). This could explain an increased demand of acetyl-CoA at 106 cells/mL of inoculum that could result in a decrease of some of these aroma compounds.
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Fig. 1. Inoculum size effect on aroma compound formation for model strains M522 (■) and KU1 (w) at two YAN levels. Inoculum sizes are indicated (1 × 104, 105 and 106 cells/mL) on a Muscat Alexandria grape must with a YAN level of 148 mg N/L increased with DAP to 211 mg N/L. Letters at each data point indicate the level of significant difference (p b 0.05) according to a LSD test of ANOVA calculated for each strain. Fermentations were in duplicate and bars indicate standard deviations. Esters (sum of isoamyl acetate, β-phenyl acetate, ethyl hexanoate, ethyl octanoate, ethyl decanoate, 4-hydroxybutyrolactate, diethyl malate and ethyl lactate); monoterpenes (hodiol I (trans 3,7-dimethyl-1,5-octadiene 3,7-diol), hodiol II (3,7-dimethyl-1,7-octadiene-3,6-diol), Ox.B (linalool cis-furanic oxide), Endiol (3,7-dimethyl-1-octen-3,7 diol), Linalool, ho-trienol, and α-terpineol) and γ-butyrolactone.
On the other hand, as it was discussed previously, the higher the inocula, the less net growth will occur during the fermentation process. This explanation can be applied to the positive correlation between the size of the inocula and ester production at a high YAN level for strain KU1 (see Fig. 1). Very limited studies about γ-butyrolactone (one of the main lactones of wine contributing with positive aromas related to caramel and coconut descriptors) are found in the literature. At low YAN level this compound behaves similar to esters and monoterpenes for both strains. Interestingly at the high YAN level there is a direct correlation with increased inocula sizes. Higher alcohols, iso-acids and fatty acids in general are not desirable compounds from the sensory point of view of wine over certain
concentration levels (Carrau et al., 2008). Fig. 2 shows that the lower conditions for production of higher alcohols and fatty acids for strain M522 were at 105 cells/mL, an opposite behavior compared to esters, monoterpenes and lactones considered desirable aroma compounds. In the case of strain KU1 Fig. 2 shows a positive correlation with inoculum sizes at a higher YAN level. Previous studies in grape wine fermentation are not in agreement with these results (Erten et al., 2006; Mateo et al., 2001), except with the behavior of KU1 at a high YAN level. But as it was stated these published results had not reported data about the YAN levels of the grape musts utilized and furthermore they had worked with higher sizes of inoculum levels (107 cells/mL and above). Fig. 2 also shows that iso-acids have an opposite behavior than fatty acids, except for M522 at high YAN concentration. These results
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Fig. 2. The same experimental and fermentation conditions of Fig. 1, showing the aroma compounds that could be considered less desirable. Alcohols (sum of 2-methyl-1-propanol, 2-methyl-1-butanol, β-phenyl ethyl alcohol, butanol, 2-ethyl hexanol and octanol), isocids (sum of isobutanoic) (isoC4) and isovaleric (isoC5) and medium chain fatty acids (sum of C4, C6, C8 and C10).
are in agreement with the opposite behavior found for these two families of compounds at a wider range of YAN levels (Carrau et al., 2008). To have a better understanding of how the key aroma compounds are affected by inoculum size, a discriminant analysis for 33 compounds of the two strains at both YAN levels was carried out (Fig. 3). The three inoculum sizes were discriminated and the six main discriminant compounds in this analysis are indicated with vectors in the figure. The compounds considered are 1,8-terpine, hodiol I (trans3,7-dimethyl-1,5-octadiene-3,7-diol), isobutyl alcohol, iso C4 acid, ethyl C6 ester and C8 acid. Other compounds that have influenced in this discrimination analysis but have less significance are the βphenyl ethyl alcohol, ethyl pyruvate, iso C5 acid and C10 acid. These results show that the inoculum size affects aroma compounds irrespective of the YAN level in both model strains.
Interestingly, relative to previous published work in wine, it was suggested that there are no significant differences between size of inoculum and aroma compounds concentration, except for higher alcohols and ethyl acetate (Erten et al., 2006; Mateo et al., 2001). However, in these studies it is difficult to draw firm conclusions concerning the relationship of inoculum size and the different aroma compounds produced by the yeast strains studied, due to the lack of information about the YAN levels of the fermentation media. Mateo et al. (2001) suggested that differences in the volatile fraction were mainly due to the yeast strain inoculated and not the size of inocula. Although Erten et al. (2006) did not suggest an ideal inoculum size level for the production of desirable aroma compounds, and that the YAN level was not reported for the white grape must utilized in that study, results shown in that paper are in agreement with our results. In that report a clear increase of esters (excluding ethyl acetate) and a
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size on wine quality are limited, but the presented results indicate the importance of defining this process parameter for key metabolic data models in aroma footprinting methods for discrimination and selection of S. cerevisiae wine yeasts.
Acknowledgements We would like to thank the critical comments made for this manuscript by Prof. Patrick Moyna. We would also thank the financial support of this project by PDT, ANII, INIA and CSIC (UdelaR, Facultad de Quimica), Uruguay.
References
Fig. 3. Discriminant analysis with 33 compounds produced from grape must for the three inoculum sizes: low 104; medium 105; and high 106 cells/mL, for both model strains and at two YAN levels. The main six compounds that discriminated the three cases are shown as vectors.
decreased concentration of higher alcohols and acetaldehyde at 105 cells/mL of inoculum could be seen. In the brewery industry there is also limited information on inocula size effects on beer aroma (Ayrapaa, 1968; Nordstrom, 1964; Peddie, 1990; Suihko et al., 1993), and it was recently stated that the influence of pitching rate on aroma compound production is rather limited, with the exception of total diacetyl levels, which strongly increase with inoculum size (Verbelen et al., 2009; Nguyen and Viet Man, 2009). However, the sizes of the inocula studied were very high compared to normal levels at wine fermentation processes (around 106 cells/mL). On the other hand, Verbelen et al. (2009) utilized a worth with more than 400 mg N/L of YAN, a value that was previously stated as too high for observing significantly variations in some key aroma compounds due to inoculum levels (Ayrapaa, 1968). Peddie (1990) hypothesized that a higher inoculum size produced higher levels of esters, and explained this behavior as connected to a lower level of growth demanding less acetyl-CoA in the cells, resulting in a higher availability of this intermediate for ester synthesis. However, this statement is only in agreement to the KU1 strain case reported by us at a high YAN level (see Fig. 1). On the other hand, Nordstrom (1964) proposed the opposite situation, where increased growth augments ester formation, an hypothesis that is partially in agreement with the results presented here at low YAN level except for the size of the inocula of 104 cells/mL. Our results suggest that the significant effect of inoculum size on many important wine aroma compounds is related to the YAN levels of the grape must. Although these effects are not easily predictable, we show here that initial cell numbers, net cell growth and fermentation rates could explain the behavior of our model strains. Further studies with artificial grape juice medium, must be performed in order to improve the understanding of this fermentation system, however our results already gave interesting practical consequences for quality wines. 4. Conclusions A consistent increase of desired aroma compounds (esters, lactones and free monoterpenes), and a decrease of less desired compounds (higher alcohols and medium chain fatty acids), was shown at inoculum sizes of 105 cells/mL for both strains in real winemaking conditions. YAN concentration levels of the grape must affect the production of different aroma compounds and this could be related to the quantity of cells at initial stages of the fermentation, rapid removal of nitrogen and net cell growth. Studies on the influence of inoculum
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