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Influence of assimilable nitrogen on volatile acidity production by Saccharomyces cerevisiae during high sugar fermentation
JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 96, No. 6, 507–512. 2003
Influence of Assimilable Nitrogen on Volatile Acidity Production by Saccharomyces cerevisiae during High Sugar Fermentation MARINA BELY,1* ALESSANDRA RINALDI,1 AND DENIS DUBOURDIEU1 Faculté d’Œnologie, Université Victor Segalen Bordeaux 2, 351 Cours de la Libération, 33405 Talence, France1 Received 19 March 2003/Accepted 2 September 2003
We analyzed the variability of volatile acidity and glycerol production by Saccharomyces cerevisiae on a large sample of high sugar musts. The production of volatile acidity was inversely correlated with the maximum cell population and the assimilable nitrogen concentration. The higher the nitrogen concentration, the less volatile acidity was produced. An approach to minimize volatile acidity production during high sugar fermentations by adjustment of assimilable nitrogen in musts was investigated in terms of both quantity and addition time. It was found that the optimal nitrogen concentration in the must is 190 mgN×l–1. The best moment for nitrogen addition was at the beginning of fermentation. Addition at the end of the growth phase had less effect on volatile acidity reduction. We suggest that by stimulating cell growth, nitrogen addition provides NADH in the redox-equilibrating process, which in turn reduces volatile acidity formation. [Key words: Saccharomyces cerevisiae, high sugar fermentation, nitrogen, volatile acidity, enology]
volatile acidity concentration from 0.56 to 1.46 g × l–1 in wines made from botrytized grapes. The high sugar content of the musts and substances secreted by Botrytis cinerea inhibit yeast growth (12) and contribute to the formation of large quantities of volatile acidity. The overproduction of volatile acidity (principally acetate) is due to the expression of certain genes (13, 14). During the anaerobic fermentation of high gravity must, S. cerevisiae accumulates solutes such as glycerol to prevent osmotic stress (15, 16). In this work, we tested a large number of high sugar musts such as botrytized musts to demonstrate the variability in volatile acidity and glycerol production. Furthermore, we assessed the impact of the assimilable nitrogen content on volatile acidity production by yeast and reduced the volatile acidity levels by the controlled addition of ammonium sulfate.
Volatile acidity, principally acetate, can play a significant role in wine aroma and an excessive concentration of this alcoholic fermentation by-product is highly detrimental to wine quality. The amount of volatile acidity produced is usually low (0.25 to 0.50 g ×l–1) but may be higher under certain fermentation conditions. In particular, during fermentation of high gravity musts such as botrytized musts, the volatile acidity content may be 1.8 g × l–1 or even higher, which is over the EEC legal limit of 1.5 g × l–1 expressed in acetic acid. Several authors have studied the origins of volatile acidity production by Saccharomyces cerevisiae under usual winemaking conditions with initial sugar concentrations around 200 g × l–1. They describe the impact of the physiological conditions and quantity of the inoculum on volatile acidity concentrations (1, 2). Moreover, volatile acidity is formed at the beginning of cell growth (3, 4). This production is affected by the yeast strain (5–7), the medium composition, vitamins, initial sugar concentration (8, 9) and fermentation conditions such as variations in temperature (8). Other studies have demonstrated the stimulating effect of fermentation by insoluble materials, which reduce the production of volatile acidity by providing saturated and unsaturated fatty acids to the yeasts (3, 10; Lavigne, Doctoral thesis, Université Bordeaux II, France, 1996). On the other hand, little is known about the control of volatile acidity production in conditions of high sugar fermentation. Lafon-Lafourcade et al. (11) showed that raising the initial sugar content from 189 to 391 g × l–1 increased the
MATERIALS AND METHODS Yeast strain The wine yeast used was the commercial S. cerevisiae strain Zymaflore ST (Laffort Œnologie, Floirac, France), which belongs to the laboratory collection. The strain was isolated from the native microflora of the spontaneous fermentation of a botrytized must owing to its low capacity for volatile acidity production (17). Must The musts were obtained from noble rot Sauvignon and Semillon grapes harvested in the vineyards of the Sauternes and Barsac appellations (2000–2001–2002 vintage). They were collected in the cellar after settling. The initial sugar concentration varied from 320 to 370 g ×l–1. Nitrogen addition The nitrogen enrichment of musts was obtained by addition of ammonium sulfate [(NH4)2SO4]. The maximal dose authorized by European and French legislation is 300
* Corresponding author. e-mail: [email protected] phone: +33-5-4000-6489 fax: +33-5-4000-6468 507
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mg × l–1, corresponding to 63 mgN × l–1. The supplementation was done generally before the inoculation except for one experiment mentioned. Fermentation conditions In the laboratory Forty fermentations were carried out in 375 ml sterile bottles (adding 330 ml of must in each bottle) in a room regulated at 23°C. Fermentations were monitored by CO2 release. The amount of CO2 released was determined by measuring weight loss every 24 h. Each fermentation was performed in triplicate. In the winery Five fermentations were carried out in the winery. Musts were placed in identical 225-l barrels filled to 90%. Alcoholic fermentations were controlled daily by measuring the density. The fermentation temperature was not controlled; the temperature ranged from 17°C to 25°C. Every experiment was performed twice. In these laboratory and winery conditions, when the required alcohol concentration of 14% vol. was reached, fermentation was stopped by addition of sulfur dioxide (300 mg × l–1). The residual sugars ranged between 80 to 120 g× l–1 depending on the initial concentration. Cell population determination The musts were inoculated with 200 mg ×l–1 active dry yeast previously rehydrated and grown for 24 h in a diluted half must. Cell population was determined by measuring the absorbance at 610 nm, one absorbance unit corresponding to 107 CFU/ml. After 72 h of fermentation, a sample of each replicate was taken. Yeast strain implantation was verified by comparing the initial industrial yeast karyotype with the biomass using the pulsed field electrophoresis method (18). All the inoculated yeasts were well implanted. Analytical methods Assimilable nitrogen Assimilable nitrogen concentration in musts, including ammonium salts and a-amino acids, was estimated by the Sörensen method (19, 20). This method is based on the reaction of formaldehyde with amino functions. The coefficient of variation was <5%. Wine analysis Ethanol concentration (%vol) was measured by the infrared reflectance method (Infra Analyser 450; Technicon, Plaisir, France). Volatile acidity expressed in g × l–1 acetic acid was determined chemically after distillation by colorimetry (A460 nm) in continuous flux (Sedimap, Montauban, France). Glycerol concentration was determined by the enzymatic method described in the Diffchamb (Västra Frölunda, Sweden) test kit, reference 1.002.809.
RESULTS AND DISCUSSION Production of volatile acidity and glycerol Forty fermentations were carried out at 23°C in the laboratory in the same conditions with different musts supplemented or not with sugar. The volatile acidity concentration in the wine varied considerably, ranging from 0.56 to 1.5 g × l–1 acetic acid (mean value 0.96) depending on the musts and particularly on the initial sugar concentrations, as observed by Lafon-Lafourcade et al. (11). As shown in Fig. 1, the maximum cell population and the volatile acidity production are closely correlated. The formation of volatile acidity was stimulated when yeast growth was inhibited. Concerning the glycerol production, the variation among the musts was smaller but remained significant, ranging from 11.9 to 18.4 g ×l–1 (mean value 15.9 g ×l–1). These concentrations were high compared to typical values in dry table wines (4–10 g ×l–1) and corresponded to an overproduction by S. cerevisiae in response to high osmotic pressure (15,
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FIG. 1. Relationship between volatile acidity (VA) and maximum cell population in laboratory conditions at 23°C. VA= -0.076 A610max +1.80 (correlation coefficient = 0.72).
FIG. 2. The kinetics of glycerol production (filled circles), volatile acidity production (filled squares), and cell growth (open circles) in laboratory conditions at 23°C. Assimilable nitrogen concentration: 190 mgN × l–1. Initial sugar concentration: 350 g × l–1.
16). Glycerol production was not correlated with maximum cell population or volatile acidity production, as already observed by Prior et al. (21) in dry wines. Six of the 40 fermentations in the laboratory were performed, to assess the kinetics of volatile acidity and glycerol production throughout fermentation. The volatile acidity and glycerol were produced essentially during cell growth, as shown in the example in Fig. 2. When the final populations were reached, at least 80% of the maximum volatile acidity was already released. This is consistent with previous results obtained in standard winemaking conditions by Alexandre et al. (3) and Coote et al. (4). On the other hand, no decrease in volatile acidity content at the end of the fermentation was observed, as described by Peynaud (22). Importance of assimilable nitrogen Variability of assimilable nitrogen content The assimilable nitrogen content of 20 botrytized must samples from the 2000 to 2002 vintages was assayed in the laboratory using the formaldehyde method. Concentrations varied from 25 to 157 mgN × l–1 depending on the origin of the must and the extent to which the fungus (B. cinerea) had developed, with an average of 84 mgN ×l–1. This average is low compared to the average concentrations in dry white must from
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FIG. 3. Relationship between maximum cell population (A610 max) and assimilable nitrogen concentration (natural or supplemented at t= 0) (N) in laboratory conditions at 23°C. A610 max= 0.027 N +6.4 (correlation coefficient = 0.62).
FIG. 4. Relationship between glycerol concentration (Gly) and assimilable nitrogen concentration (N) (natural or supplemented at t = 0) in laboratory conditions at 23°C.
the Bordeaux area of 180 mgN×l–1 (23) and the deficiency threshold of 140 mgN ×l–1 under standard fermentation conditions, as proposed by Agenbach (24) and Bely et al. (25). Influence of assimilable nitrogen concentration Assimilable nitrogen has often been reported to be an important factor of cell growth. In our laboratory conditions, in high sugar musts, the maximum cell population was also influenced by the initial nitrogen concentration (Fig. 3). On the other hand, the nitrogen content did not affect the final glycerol content (Fig. 4). To study the influence of nitrogen on the volatile acidity production, 12 high sugar musts not supplemented with nitrogen were fermented under laboratory conditions. The initial assimilable nitrogen concentration affected the volatile acidity production (Fig. 5). This production was inversely correlated with the assimilable nitrogen concentrations. We observed that the initial nitrogen concentration available in natural musts was not always sufficient to obtain fermentation with a low volatile acidity level. Therefore, it appears essential to add nitrogen to this specific high gravity must. The impact of adding nitrogen on volatile acidity production had not previously been investigated either in dry or sweet wines.
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FIG. 5. Effects of assimilable nitrogen (natural) concentration (N) in must on volatile acidity production (VA) in laboratory conditions at 23°C. VA = -0.351 Ln(N)+ 2.6 (correlation coefficient = 0.68).
FIG. 6. Impact of nitrogen [(NH4)2SO4] addition at t = 0 on volatile acidity production. Initial assimilable nitrogen: 90 mgN ×l–1. Initial sugar: 350 g × l–1. Filled circles, Laboratory experiments at 23°C; open circles, winery experiments, non-controlled temperature. Means of duplicate (winery) or triplicate (laboratory) fermentations, max. SD ± 0.03.
Effectiveness of nitrogen addition on volatile acidity production Influence of the initial content An experiment was carried out simultaneously in the laboratory and in the winery using nitrogen-deficient must to investigate the effect of adding up to 300 mgN × l–1 ammonium sulfate on volatile acidity formation. This experiment was also performed to compare the volatile acidity production in different environments, i.e., the laboratory and the winery. It is noteworthy that these fermentations were carried out simultaneously, the temperature being controlled in the laboratory conditions at 23°C; on the other hand, the winery temperature ranged from 17°C to 25°C. The results (Fig. 6) indicate that the alcoholic fermentation process in the laboratory satisfactorily reproduced the behavior of industrial winemaking barrels and that the temperature seems to affect volatile acidity production, in accordance with previous reports concerning standard winemaking (8). The assimilable nitrogen concentration influenced volatile acidity production, thereby confirming the previous results. Adding up to 210 mgN ×l–1 ammonium sulfate to this must reduced the formation of volatile acidity by 40% compared to untreated must, a value which was significant. Nevertheless, at a higher final concentration (300 mgN × l–1), the volatile acidity concentration
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TABLE 1. Impact of nitrogen addition (NH4)2SO4 at t= 0 on fermentation duration and on maximum cell population in laboratory conditions at 23°C Max Assimilable Fermentation cell population nitrogen duration (A610 max) (mgN × l–1) (h) 92 408 9.4 130 384 11.6 170 360 12.4 210 264 15 300 216 15.6 –1 Initial assimilable nitrogen concentration: 92 mgN × l . Initial sugar concentration: 350 g × l–1.
was not reduced to the same extent. Compared to non-supplemented musts (Table 1), nitrogen addition significantly reduced fermentation time and increased cell populations, except at the high nitrogen level. These results are in accordance with previous studies (24, 25). In order to propose an optimal value of nitrogen concentration with regard to volatile acidity production, other experiments were carried out in the laboratory. For this, we used 26 musts with an initial sugar concentration adjusted to 350 g ×l–1, in order to preclude the impact of the sugar concentration on volatile acidity production. As shown in Fig. 7, the initial nitrogen concentration of musts (natural or supplemented with ammonium sulfate) affected the volatile acidity production at the end of alcoholic fermentation. When the initial nitrogen was low, the addition of ammonium salts considerably reduced the volatile acidity concentration. On the other hand, when the initial content exceeded 200 mgN× l–1, addition increased the volatile acidity production, as described above. Therefore, an optimal nitrogen concentration in the must of 190 mgN ×l–1 is proposed (Fig. 7). Influence of timing of addition Since nitrogen addition before the inoculation had an impact on volatile acidity production, we verified whether the results obtained with nitrogen supplements were modified positively or negatively depending on the timing of the addition. Table 2 summarizes fermentation durations, volatile acidity productions and cell populations observed at 23°C using three different musts with different nitrogen and sugar concentrations, for additions made at two fermentation times. The first one at the beginning of the fermentation and the second when the maximum cell population had been reached, i.e., after 4 d of fermentation, which corresponded to less than 50 g×l–1 of sugar consumed. As we already observed, early addition always reduced volatile acidity production in the wine by
FIG. 7. Effects of assimilable nitrogen concentration (natural and supplemented at t= 0) on volatile acidity production in laboratory conditions at 23°C. Initial sugar: 350 g × l–1. Arrow indicates the optimal nitrogen concentration.
18% (must A), 27% (must B), and 22% (must C). On the contrary, later addition had the drawback of considerably increasing the final content beyond the control fermentation level. Regardless of addition time, a reduction in fermentation duration was always observed but the effectiveness of the addition was not always the same. This observation differs from previous results obtained by Bely et al. (25) who found that reduction time by nitrogen addition in classic winemaking conditions was the same provided that the addition was made before the halfway point in the fermentation. The timing of nitrogen addition also affected the maximum cell population. An addition made at the beginning of cell growth resulted in the maximum cell population. On the other hand, addition during the stationary phase had less impact on the cell population level but did significantly reduce the duration of fermentation by increasing the cell activity, as already observed by Salmon (26). All these results demonstrate the impact of the assimilable nitrogen concentration in the must on the production of volatile acidity (principally acetate). This observation is due to the fact that nitrogen is a key factor for yeast growth, thus affecting the growth rate and final cell population. Apparently, this growth stimulation restricts volatile acidity production. The indirect role of nitrogen on volatile acidity production may be understood in terms of the mechanism of adaptation of yeast in high sugar medium. S. cerevisiae responds to increased external osmolarity by enhanced production and intercellular accumulation of glycerol to counterbalance the osmotic pressure (15, 16, 27). This regula-
TABLE 2. Influence of timing of nitrogen [(NH4)2SO4] addition on maximum cell population, on volatile acidity and on fermentation duration in laboratory conditions at 23°C Max cell population (A610 max) Volatile aciditya (g × l–1 acetic acid) Fermentation duration (h) Experiment no. 1 2 3 1 2 3 1 2 3 Must A 7.4 11.2 8.2 1.14 0.94 ± 0.02 1.45 432 220 310 Must B 8.2 12.6 9 0.97 0.71 ± 0.02 1.12 408 360 380 Must C 8 10.4 8.6 1.38 1.07 ± 0.03 1.37 528 276 324 Initial sugar concentration (g ×l–1): must A, 340; must B, 350; must C, 360. Initial assimilable nitrogen concentration (mgN ×l–1): must A, 55; must B, 92; must C, 78. Nitrogen concentration after addition: 190 mgN ×l–1. Experiments: 1, no addition; 2, addition made at t = 0; 3, addition made at the beginning of the stationary growth phase. a Mean of triplicate fermentations ± SD.
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tion mechanism is due to the expression of certain genes. Several genes have been shown to be induced by laboratory strains of S. cerevisiae exposed to hyperosmotic stress exerted by sugar. The osmoresponsive genes include glycerol 3-phosphate dehydrogenase which is encoded by GPD1 (14, 28), and also two types of aldehyde dehydrogenase encoded by ALD2 and ADL3 (13). These two cytosolic ALDH both use NAD+ as coenzyme while the ADLH encoded by ALD6 gene under usual winemaking conditions is preferentially NADP+ (13). Glycerol is synthesized by the reduction of dihydroxyacetone phosphate to glycerol 3-phosphate by glycerol 3-phosphate dehydrogenase (NAD+), followed by dephosphorylation of glycerol 3-phosphate to glycerol. In order to maintain the intracellular redox balance, yeast cells regenerate an equimolar amount of cytoplasmic NADH. This requirement seems to be partially met by a decreased reduction of acetaldehyde to ethanol, on the one hand, and an increased oxidation to acetate, on the other (27). Thus, it is clear that an overproduction of acetate cannot be avoided in high sugar fermentation. Nevertheless, one way to decrease the acetate level could be to provide more NADH in the redox-equilibrated process. This may be obtained by stimulating the growth yeast, thereby generating a net production of NADH during the conversion of glucose and nutrients into biomass. Nitrogen plays an important role in this stimulation. It has been estimated that some 60–80% of NADH generated in biosynthetic reactions originates from the synthesis of amino acids (29, 30). Therefore, it seems essential to control the nitrogen content in high density musts in order to reduce volatile acidity production. An optimal nitrogen concentration of 190 mgN × l–1 is proposed. Early addition of nitrogen at the beginning of the cell growth results in maximum yeast production and therefore a decrease in volatile acidity production. Later addition, at the beginning of the stationary phase, has little effect on cell population level and dramatically raises the volatile acidity level. In the latter condition, nitrogen addition increases the fermentation rate by reactivating the hexose transport system (31) as well as the volatile acidity production rate, without any compensation of the NADH surplus from biomass formation. A similar mechanism is observed when the initial nitrogen concentration is supplemented above the optimal nitrogen concentration, i.e., 190 mgN × l–1. The excess of nitrogen compounds increases the fermentation rate but the biomass formation is not stimulated to the same extent, particularly at high nitrogen concentrations. The direct consequence is an increase in the production of volatile acidity. Nitrogen has an important impact on volatile acidity production by S. cerevisiae, which is especially high in conditions of high sugar fermentation. This production can be reduced by controlled nitrogen addition. In the present work, a 40% reduction was obtained during addition up to 210 mgN ×l–1 at the beginning of fermentation in a must with low assimilable nitrogen content (92 mgN× l–1). To limit the production of volatile acidity by S. cerevisiae, an optimal nitrogen concentration of 190 mgN× l–1 in the must is found. The yeast used for these experiments, S. cerevisiae strain Zymaflore ST, was selected for its low potential to produce volatile acidity during high sugar fermentation. Other yeast
strains were also tested to verify the advantage of adjusting nitrogen content to reduce volatile acidity (unpublished data).
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ACKNOWLEDGMENTS The authors thank Château Rayne Vigneau for its collaboration and acknowledge SARCO for technical assistance.
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Influence of Assimilable Nitrogen on Volatile Acidity Production by Saccharomyces cerevisiae during High Sugar Fermentation MARINA BELY,1* ALESSANDRA RINALDI,1 AND DENIS DUBOURDIEU1 Faculté d’Œnologie, Université Victor Segalen Bordeaux 2, 351 Cours de la Libération, 33405 Talence, France1 Received 19 March 2003/Accepted 2 September 2003
We analyzed the variability of volatile acidity and glycerol production by Saccharomyces cerevisiae on a large sample of high sugar musts. The production of volatile acidity was inversely correlated with the maximum cell population and the assimilable nitrogen concentration. The higher the nitrogen concentration, the less volatile acidity was produced. An approach to minimize volatile acidity production during high sugar fermentations by adjustment of assimilable nitrogen in musts was investigated in terms of both quantity and addition time. It was found that the optimal nitrogen concentration in the must is 190 mgN×l–1. The best moment for nitrogen addition was at the beginning of fermentation. Addition at the end of the growth phase had less effect on volatile acidity reduction. We suggest that by stimulating cell growth, nitrogen addition provides NADH in the redox-equilibrating process, which in turn reduces volatile acidity formation. [Key words: Saccharomyces cerevisiae, high sugar fermentation, nitrogen, volatile acidity, enology]
volatile acidity concentration from 0.56 to 1.46 g × l–1 in wines made from botrytized grapes. The high sugar content of the musts and substances secreted by Botrytis cinerea inhibit yeast growth (12) and contribute to the formation of large quantities of volatile acidity. The overproduction of volatile acidity (principally acetate) is due to the expression of certain genes (13, 14). During the anaerobic fermentation of high gravity must, S. cerevisiae accumulates solutes such as glycerol to prevent osmotic stress (15, 16). In this work, we tested a large number of high sugar musts such as botrytized musts to demonstrate the variability in volatile acidity and glycerol production. Furthermore, we assessed the impact of the assimilable nitrogen content on volatile acidity production by yeast and reduced the volatile acidity levels by the controlled addition of ammonium sulfate.
Volatile acidity, principally acetate, can play a significant role in wine aroma and an excessive concentration of this alcoholic fermentation by-product is highly detrimental to wine quality. The amount of volatile acidity produced is usually low (0.25 to 0.50 g ×l–1) but may be higher under certain fermentation conditions. In particular, during fermentation of high gravity musts such as botrytized musts, the volatile acidity content may be 1.8 g × l–1 or even higher, which is over the EEC legal limit of 1.5 g × l–1 expressed in acetic acid. Several authors have studied the origins of volatile acidity production by Saccharomyces cerevisiae under usual winemaking conditions with initial sugar concentrations around 200 g × l–1. They describe the impact of the physiological conditions and quantity of the inoculum on volatile acidity concentrations (1, 2). Moreover, volatile acidity is formed at the beginning of cell growth (3, 4). This production is affected by the yeast strain (5–7), the medium composition, vitamins, initial sugar concentration (8, 9) and fermentation conditions such as variations in temperature (8). Other studies have demonstrated the stimulating effect of fermentation by insoluble materials, which reduce the production of volatile acidity by providing saturated and unsaturated fatty acids to the yeasts (3, 10; Lavigne, Doctoral thesis, Université Bordeaux II, France, 1996). On the other hand, little is known about the control of volatile acidity production in conditions of high sugar fermentation. Lafon-Lafourcade et al. (11) showed that raising the initial sugar content from 189 to 391 g × l–1 increased the
MATERIALS AND METHODS Yeast strain The wine yeast used was the commercial S. cerevisiae strain Zymaflore ST (Laffort Œnologie, Floirac, France), which belongs to the laboratory collection. The strain was isolated from the native microflora of the spontaneous fermentation of a botrytized must owing to its low capacity for volatile acidity production (17). Must The musts were obtained from noble rot Sauvignon and Semillon grapes harvested in the vineyards of the Sauternes and Barsac appellations (2000–2001–2002 vintage). They were collected in the cellar after settling. The initial sugar concentration varied from 320 to 370 g ×l–1. Nitrogen addition The nitrogen enrichment of musts was obtained by addition of ammonium sulfate [(NH4)2SO4]. The maximal dose authorized by European and French legislation is 300
* Corresponding author. e-mail: [email protected] phone: +33-5-4000-6489 fax: +33-5-4000-6468 507
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mg × l–1, corresponding to 63 mgN × l–1. The supplementation was done generally before the inoculation except for one experiment mentioned. Fermentation conditions In the laboratory Forty fermentations were carried out in 375 ml sterile bottles (adding 330 ml of must in each bottle) in a room regulated at 23°C. Fermentations were monitored by CO2 release. The amount of CO2 released was determined by measuring weight loss every 24 h. Each fermentation was performed in triplicate. In the winery Five fermentations were carried out in the winery. Musts were placed in identical 225-l barrels filled to 90%. Alcoholic fermentations were controlled daily by measuring the density. The fermentation temperature was not controlled; the temperature ranged from 17°C to 25°C. Every experiment was performed twice. In these laboratory and winery conditions, when the required alcohol concentration of 14% vol. was reached, fermentation was stopped by addition of sulfur dioxide (300 mg × l–1). The residual sugars ranged between 80 to 120 g× l–1 depending on the initial concentration. Cell population determination The musts were inoculated with 200 mg ×l–1 active dry yeast previously rehydrated and grown for 24 h in a diluted half must. Cell population was determined by measuring the absorbance at 610 nm, one absorbance unit corresponding to 107 CFU/ml. After 72 h of fermentation, a sample of each replicate was taken. Yeast strain implantation was verified by comparing the initial industrial yeast karyotype with the biomass using the pulsed field electrophoresis method (18). All the inoculated yeasts were well implanted. Analytical methods Assimilable nitrogen Assimilable nitrogen concentration in musts, including ammonium salts and a-amino acids, was estimated by the Sörensen method (19, 20). This method is based on the reaction of formaldehyde with amino functions. The coefficient of variation was <5%. Wine analysis Ethanol concentration (%vol) was measured by the infrared reflectance method (Infra Analyser 450; Technicon, Plaisir, France). Volatile acidity expressed in g × l–1 acetic acid was determined chemically after distillation by colorimetry (A460 nm) in continuous flux (Sedimap, Montauban, France). Glycerol concentration was determined by the enzymatic method described in the Diffchamb (Västra Frölunda, Sweden) test kit, reference 1.002.809.
RESULTS AND DISCUSSION Production of volatile acidity and glycerol Forty fermentations were carried out at 23°C in the laboratory in the same conditions with different musts supplemented or not with sugar. The volatile acidity concentration in the wine varied considerably, ranging from 0.56 to 1.5 g × l–1 acetic acid (mean value 0.96) depending on the musts and particularly on the initial sugar concentrations, as observed by Lafon-Lafourcade et al. (11). As shown in Fig. 1, the maximum cell population and the volatile acidity production are closely correlated. The formation of volatile acidity was stimulated when yeast growth was inhibited. Concerning the glycerol production, the variation among the musts was smaller but remained significant, ranging from 11.9 to 18.4 g ×l–1 (mean value 15.9 g ×l–1). These concentrations were high compared to typical values in dry table wines (4–10 g ×l–1) and corresponded to an overproduction by S. cerevisiae in response to high osmotic pressure (15,
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FIG. 1. Relationship between volatile acidity (VA) and maximum cell population in laboratory conditions at 23°C. VA= -0.076 A610max +1.80 (correlation coefficient = 0.72).
FIG. 2. The kinetics of glycerol production (filled circles), volatile acidity production (filled squares), and cell growth (open circles) in laboratory conditions at 23°C. Assimilable nitrogen concentration: 190 mgN × l–1. Initial sugar concentration: 350 g × l–1.
16). Glycerol production was not correlated with maximum cell population or volatile acidity production, as already observed by Prior et al. (21) in dry wines. Six of the 40 fermentations in the laboratory were performed, to assess the kinetics of volatile acidity and glycerol production throughout fermentation. The volatile acidity and glycerol were produced essentially during cell growth, as shown in the example in Fig. 2. When the final populations were reached, at least 80% of the maximum volatile acidity was already released. This is consistent with previous results obtained in standard winemaking conditions by Alexandre et al. (3) and Coote et al. (4). On the other hand, no decrease in volatile acidity content at the end of the fermentation was observed, as described by Peynaud (22). Importance of assimilable nitrogen Variability of assimilable nitrogen content The assimilable nitrogen content of 20 botrytized must samples from the 2000 to 2002 vintages was assayed in the laboratory using the formaldehyde method. Concentrations varied from 25 to 157 mgN × l–1 depending on the origin of the must and the extent to which the fungus (B. cinerea) had developed, with an average of 84 mgN ×l–1. This average is low compared to the average concentrations in dry white must from
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FIG. 3. Relationship between maximum cell population (A610 max) and assimilable nitrogen concentration (natural or supplemented at t= 0) (N) in laboratory conditions at 23°C. A610 max= 0.027 N +6.4 (correlation coefficient = 0.62).
FIG. 4. Relationship between glycerol concentration (Gly) and assimilable nitrogen concentration (N) (natural or supplemented at t = 0) in laboratory conditions at 23°C.
the Bordeaux area of 180 mgN×l–1 (23) and the deficiency threshold of 140 mgN ×l–1 under standard fermentation conditions, as proposed by Agenbach (24) and Bely et al. (25). Influence of assimilable nitrogen concentration Assimilable nitrogen has often been reported to be an important factor of cell growth. In our laboratory conditions, in high sugar musts, the maximum cell population was also influenced by the initial nitrogen concentration (Fig. 3). On the other hand, the nitrogen content did not affect the final glycerol content (Fig. 4). To study the influence of nitrogen on the volatile acidity production, 12 high sugar musts not supplemented with nitrogen were fermented under laboratory conditions. The initial assimilable nitrogen concentration affected the volatile acidity production (Fig. 5). This production was inversely correlated with the assimilable nitrogen concentrations. We observed that the initial nitrogen concentration available in natural musts was not always sufficient to obtain fermentation with a low volatile acidity level. Therefore, it appears essential to add nitrogen to this specific high gravity must. The impact of adding nitrogen on volatile acidity production had not previously been investigated either in dry or sweet wines.
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FIG. 5. Effects of assimilable nitrogen (natural) concentration (N) in must on volatile acidity production (VA) in laboratory conditions at 23°C. VA = -0.351 Ln(N)+ 2.6 (correlation coefficient = 0.68).
FIG. 6. Impact of nitrogen [(NH4)2SO4] addition at t = 0 on volatile acidity production. Initial assimilable nitrogen: 90 mgN ×l–1. Initial sugar: 350 g × l–1. Filled circles, Laboratory experiments at 23°C; open circles, winery experiments, non-controlled temperature. Means of duplicate (winery) or triplicate (laboratory) fermentations, max. SD ± 0.03.
Effectiveness of nitrogen addition on volatile acidity production Influence of the initial content An experiment was carried out simultaneously in the laboratory and in the winery using nitrogen-deficient must to investigate the effect of adding up to 300 mgN × l–1 ammonium sulfate on volatile acidity formation. This experiment was also performed to compare the volatile acidity production in different environments, i.e., the laboratory and the winery. It is noteworthy that these fermentations were carried out simultaneously, the temperature being controlled in the laboratory conditions at 23°C; on the other hand, the winery temperature ranged from 17°C to 25°C. The results (Fig. 6) indicate that the alcoholic fermentation process in the laboratory satisfactorily reproduced the behavior of industrial winemaking barrels and that the temperature seems to affect volatile acidity production, in accordance with previous reports concerning standard winemaking (8). The assimilable nitrogen concentration influenced volatile acidity production, thereby confirming the previous results. Adding up to 210 mgN ×l–1 ammonium sulfate to this must reduced the formation of volatile acidity by 40% compared to untreated must, a value which was significant. Nevertheless, at a higher final concentration (300 mgN × l–1), the volatile acidity concentration
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TABLE 1. Impact of nitrogen addition (NH4)2SO4 at t= 0 on fermentation duration and on maximum cell population in laboratory conditions at 23°C Max Assimilable Fermentation cell population nitrogen duration (A610 max) (mgN × l–1) (h) 92 408 9.4 130 384 11.6 170 360 12.4 210 264 15 300 216 15.6 –1 Initial assimilable nitrogen concentration: 92 mgN × l . Initial sugar concentration: 350 g × l–1.
was not reduced to the same extent. Compared to non-supplemented musts (Table 1), nitrogen addition significantly reduced fermentation time and increased cell populations, except at the high nitrogen level. These results are in accordance with previous studies (24, 25). In order to propose an optimal value of nitrogen concentration with regard to volatile acidity production, other experiments were carried out in the laboratory. For this, we used 26 musts with an initial sugar concentration adjusted to 350 g ×l–1, in order to preclude the impact of the sugar concentration on volatile acidity production. As shown in Fig. 7, the initial nitrogen concentration of musts (natural or supplemented with ammonium sulfate) affected the volatile acidity production at the end of alcoholic fermentation. When the initial nitrogen was low, the addition of ammonium salts considerably reduced the volatile acidity concentration. On the other hand, when the initial content exceeded 200 mgN× l–1, addition increased the volatile acidity production, as described above. Therefore, an optimal nitrogen concentration in the must of 190 mgN ×l–1 is proposed (Fig. 7). Influence of timing of addition Since nitrogen addition before the inoculation had an impact on volatile acidity production, we verified whether the results obtained with nitrogen supplements were modified positively or negatively depending on the timing of the addition. Table 2 summarizes fermentation durations, volatile acidity productions and cell populations observed at 23°C using three different musts with different nitrogen and sugar concentrations, for additions made at two fermentation times. The first one at the beginning of the fermentation and the second when the maximum cell population had been reached, i.e., after 4 d of fermentation, which corresponded to less than 50 g×l–1 of sugar consumed. As we already observed, early addition always reduced volatile acidity production in the wine by
FIG. 7. Effects of assimilable nitrogen concentration (natural and supplemented at t= 0) on volatile acidity production in laboratory conditions at 23°C. Initial sugar: 350 g × l–1. Arrow indicates the optimal nitrogen concentration.
18% (must A), 27% (must B), and 22% (must C). On the contrary, later addition had the drawback of considerably increasing the final content beyond the control fermentation level. Regardless of addition time, a reduction in fermentation duration was always observed but the effectiveness of the addition was not always the same. This observation differs from previous results obtained by Bely et al. (25) who found that reduction time by nitrogen addition in classic winemaking conditions was the same provided that the addition was made before the halfway point in the fermentation. The timing of nitrogen addition also affected the maximum cell population. An addition made at the beginning of cell growth resulted in the maximum cell population. On the other hand, addition during the stationary phase had less impact on the cell population level but did significantly reduce the duration of fermentation by increasing the cell activity, as already observed by Salmon (26). All these results demonstrate the impact of the assimilable nitrogen concentration in the must on the production of volatile acidity (principally acetate). This observation is due to the fact that nitrogen is a key factor for yeast growth, thus affecting the growth rate and final cell population. Apparently, this growth stimulation restricts volatile acidity production. The indirect role of nitrogen on volatile acidity production may be understood in terms of the mechanism of adaptation of yeast in high sugar medium. S. cerevisiae responds to increased external osmolarity by enhanced production and intercellular accumulation of glycerol to counterbalance the osmotic pressure (15, 16, 27). This regula-
TABLE 2. Influence of timing of nitrogen [(NH4)2SO4] addition on maximum cell population, on volatile acidity and on fermentation duration in laboratory conditions at 23°C Max cell population (A610 max) Volatile aciditya (g × l–1 acetic acid) Fermentation duration (h) Experiment no. 1 2 3 1 2 3 1 2 3 Must A 7.4 11.2 8.2 1.14 0.94 ± 0.02 1.45 432 220 310 Must B 8.2 12.6 9 0.97 0.71 ± 0.02 1.12 408 360 380 Must C 8 10.4 8.6 1.38 1.07 ± 0.03 1.37 528 276 324 Initial sugar concentration (g ×l–1): must A, 340; must B, 350; must C, 360. Initial assimilable nitrogen concentration (mgN ×l–1): must A, 55; must B, 92; must C, 78. Nitrogen concentration after addition: 190 mgN ×l–1. Experiments: 1, no addition; 2, addition made at t = 0; 3, addition made at the beginning of the stationary growth phase. a Mean of triplicate fermentations ± SD.
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tion mechanism is due to the expression of certain genes. Several genes have been shown to be induced by laboratory strains of S. cerevisiae exposed to hyperosmotic stress exerted by sugar. The osmoresponsive genes include glycerol 3-phosphate dehydrogenase which is encoded by GPD1 (14, 28), and also two types of aldehyde dehydrogenase encoded by ALD2 and ADL3 (13). These two cytosolic ALDH both use NAD+ as coenzyme while the ADLH encoded by ALD6 gene under usual winemaking conditions is preferentially NADP+ (13). Glycerol is synthesized by the reduction of dihydroxyacetone phosphate to glycerol 3-phosphate by glycerol 3-phosphate dehydrogenase (NAD+), followed by dephosphorylation of glycerol 3-phosphate to glycerol. In order to maintain the intracellular redox balance, yeast cells regenerate an equimolar amount of cytoplasmic NADH. This requirement seems to be partially met by a decreased reduction of acetaldehyde to ethanol, on the one hand, and an increased oxidation to acetate, on the other (27). Thus, it is clear that an overproduction of acetate cannot be avoided in high sugar fermentation. Nevertheless, one way to decrease the acetate level could be to provide more NADH in the redox-equilibrated process. This may be obtained by stimulating the growth yeast, thereby generating a net production of NADH during the conversion of glucose and nutrients into biomass. Nitrogen plays an important role in this stimulation. It has been estimated that some 60–80% of NADH generated in biosynthetic reactions originates from the synthesis of amino acids (29, 30). Therefore, it seems essential to control the nitrogen content in high density musts in order to reduce volatile acidity production. An optimal nitrogen concentration of 190 mgN × l–1 is proposed. Early addition of nitrogen at the beginning of the cell growth results in maximum yeast production and therefore a decrease in volatile acidity production. Later addition, at the beginning of the stationary phase, has little effect on cell population level and dramatically raises the volatile acidity level. In the latter condition, nitrogen addition increases the fermentation rate by reactivating the hexose transport system (31) as well as the volatile acidity production rate, without any compensation of the NADH surplus from biomass formation. A similar mechanism is observed when the initial nitrogen concentration is supplemented above the optimal nitrogen concentration, i.e., 190 mgN × l–1. The excess of nitrogen compounds increases the fermentation rate but the biomass formation is not stimulated to the same extent, particularly at high nitrogen concentrations. The direct consequence is an increase in the production of volatile acidity. Nitrogen has an important impact on volatile acidity production by S. cerevisiae, which is especially high in conditions of high sugar fermentation. This production can be reduced by controlled nitrogen addition. In the present work, a 40% reduction was obtained during addition up to 210 mgN ×l–1 at the beginning of fermentation in a must with low assimilable nitrogen content (92 mgN× l–1). To limit the production of volatile acidity by S. cerevisiae, an optimal nitrogen concentration of 190 mgN× l–1 in the must is found. The yeast used for these experiments, S. cerevisiae strain Zymaflore ST, was selected for its low potential to produce volatile acidity during high sugar fermentation. Other yeast
strains were also tested to verify the advantage of adjusting nitrogen content to reduce volatile acidity (unpublished data).
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ACKNOWLEDGMENTS The authors thank Château Rayne Vigneau for its collaboration and acknowledge SARCO for technical assistance.
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