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WINE FERMENTATION WITH A NEW, 100% ORGANIC NUTRIENT

Céline RAYNAL1, Caroline BONNEFOND2, Françoise RAGINEL1, Daniel GRANÈS2 and Anne ORTIZ-JULIEN1

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Lallemand SAS-Blagnac, 19, rue des Briquetiers, Blagnac Cedex, 31702 France Institut Coopératif du Vin, La Jasse de Maurin, 34970 Lattes France

Introduction

For winemakers, nitrogen, which stimulates the fermentation of wine yeasts, is an essential nutrient for a smooth alcoholic fermentation (AF). Over the past 20 years, numerous studies have shown that nitrogen has a positive impact on the growth and fermentation activity of yeast (Bell et al. 1979, Ough and Lee 1981, Bezenger and Navarro 1987). The existence of a correlation among the maximum speed of fermentation, the growth of yeast and the initial concentration of nitrogen in the must has also been shown (Sablayrolles et al. 2001). Deficiencies in yeast-available nitrogen (YAN) in the must increase considerably the risk of sluggish or stuck fermentations. In fact, a nitrogen deficiency in the must lowers the efficacy of yeast growth and, consequently, slows the rate of fermentation (Bely et al. 1990). The lower the nitrogen concentration in the must is, the greater the risk of slow fermentation. The concentration of nitrogen in the must can vary from 80 to 400 mg/L. We consider a must with an initial sugar level of about 200 g/L to be deficient when its nitrogen concentration is about 150 mg/L (Henschke and Jiranek 1993). A nitrogen deficiency in the must can arrest the protein synthesis in the yeast cells, with a major inhibitory effect on the transport of sugar and, consequently, an increase in the risk of a stuck fermentation (Basturia and Lagunas 1986). Before a sluggish or stuck fermentation occurs, the YAN deficiency in the must can also cause the yeast to increase the production of H2S (Henschke and Jiranek 1991).

Nitrogen Sources and Their Impact

The two types of nitrogen available to yeast are ammonium and -amino nitrogen (free amino acids without the proline, which cannot be assimilated by the yeast). Both of these types of YAN ­ the ammonium (inorganic nitrogen appreciated by the yeast and very quickly assimilated) and the amino acids (organic nitrogen that is assimilated more slowly, but with greater regularity) ­ naturally cohabit in the must. Several transport systems are involved in the assimilation of the amino acids, including the transporters specific to one or more individual amino acids, as well as the general amino acid permease (GAP) transporter, which acts as a conveyor for a set of amino acids. The GAP is inhibited by the presence of ammonium. In fact, it begins to act only when the must no longer contains ammonium. To ensure its growth, the yeast has to synthesize all the proteins it needs, even when it cannot manage to assimilate all the sources of nitrogen present in the must. The nitrogen sources regularly utilized for must nutrition are ammonium salts (DAP/DAS) ­ a source of 100% inorganic nitrogen ­ or a complex preparation composed of ammonium salts and inactivated yeast fractions rich in -amino organic nitrogen, which combines the positive effects of inorganic nitrogen with organic nitrogen for the growth and fermentation of yeasts. Each of these two sources of nitrogen has a different impact on the yeasts and their fermentation. Ammonium (mineral nitrogen), is "preferred" by the yeast and very rapidly assimilated, and has a direct influence on the biomass by producing significant growth in the yeast population during the growth phase. The impact of complex preparations on the rate of fermentation and growth is more moderate, but such preparations help ensure the steady operation of the AF, a final result that is just as interesting. The sensory impact of nitrogen supplementation on the must varies considerably according to the nitrogen source selected. Complex preparations ensure more complete nutrition. They contain inactivated yeast fractions that are rich in amino acids, which limit the excessive production of undesirable compounds (e.g., ethyl acetate and hexanol) and enhance the expression of positive aromatic compounds (e.g., phenylethanol, phenylacetate, terpenes and esters) (O. Pillet, 1

internal communication), while producing a clear reduction of sulphur odours in the must, whatever the variety (D. Granès, internal communication). The first part of our study focused on the comparative evaluation of the efficacy of different sources of YAN on fermentation kinetics when added to the must in equivalent quantities. In the second part of the study, we evaluated the sensory impact of these nitrogen sources. For the first part, two sources of nitrogen were compared in this study: 100% ammonium salts (DAP) and a complex preparation based on inactivated yeast fractions with a known concentration of YAN.

1. The Impact of Different Nitrogen Sources on Fermentation Kinetics and the Fermentation Activity of Yeasts 1.1 Trials carried out in collaboration with the INRA Pech-Rouge for the vinification of white must

The purpose of these trials is to compare the efficacy of adding ammonium and of adding an organic nitrogen preparation (made from inactivated yeast). · Fermented must submitted for trial: Viognier · Analysis of the initial content of the must: · Sugars: 215 g/L · Total acidity: 2.5 g H2SO4/L · pH: 3.65 · YAN: 150 mg/L · Turbidity: 42 uTN = addition of 0.5% of frozen Maccabeu grape solids to obtain a final turbidity of > 100 uTN Protocol The fermentations were then conducted at a constant temperature of 20°C in fermenters with a capacity of 1 hL, allowing the rates of fermentation to be monitored online. The yeast utilized was Lalvin EC1118, inoculated into the must a rate of 25 g/hL after rehydration in water at 37°C. In both cases, 16 mg/L of YAN was added. The addition protocols are described below. C Ammonium salts (DAP): Add 8 g/hL in two doses (4 g/hL at the start of fermentation and 4 g/hL at the one-third point of fermentation). C Organic nitrogen (Fermaid O, a specific preparation based on inactivated yeast fractions): Add 40 g/hL in two doses (20 g/hL at the start of fermentation and 20 g/hL at the one-third point of fermentation). C Control fermentation with no added nitrogen.

TABLE 1 . Experiment plan

Concentration of YAN in 30 g/hL DAP Fermaid O Control 69 mg/L 12 mg/L N/A

YAN to be added in the INRA trials 16 mg/L 16 mg/L N/A

Quantity of nitrogen source to obtain 16 mg/L of YAN 8 g/hL 40 g/hL N/A

Nitrogen source

100% ammonium 100% amino acids present in the yeast N/A

The fermentation kinetics are represented in Figure 1. They were obtained by the online monitoring of the rate of CO2 release in g/hL (Sablayrolles et al., Pech-Rouge experimental unit).

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FIGURE 1. Fermentation kinetics representing the fermentation activity of yeasts according to the three protocols (expressed in g/L.h-1 of CO2 released)

1.2 Fermentation rate (gL.h-1) 1 0.8 0.6

Second addition

Control Fermaid O DAP

0.4 First addition 0.2 0

0

50

100

150

200 Time (h)

250

300

350

0.3 Fermentation rate (gL.h-1) 0.25 0.2 0.15 0.1 0.05 0 200

Control Fermaid O DAP

220

240

260

280

300

320

340

Time (h)

FIGURE 2. Residual sugars at 240 hours of fermentation

10 9 8 Residual sugars (g/L) 7 6 5 4 3 2 1 0 Control DAP FERMAID O

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By observing the combined data of Figures 1 and 2, the difference between the effects of nitrogen from ammonium and organic nitrogen stand out in terms of their efficacy, as both sources of nitrogen contain an equivalent quantity of YAN. With the addition of 16 mg/L of YAN in organic form, fermentation was complete in 10 days. No difference was observed between the fermentation rates of the must to which 16 mg/L of YAN in ammonia form was added and the must with no nitrogen addition. The must to which inorganic nitrogen (ammonium) was added had a slow end to fermentation. The figure also shows that the level of residual sugars is higher in the wine where DAP was utilized, compared to that with Fermaid O. YAN is an essential nutrient that contributes to the smooth operation of AF by stimulating the growth of yeast, and by facilitating the protein syntheses that transform sugar into alcohol. These trials have confirmed the importance of the choice of the source of yeast-available nitrogen. At identical doses of added YAN, the preparation based on amino acids from yeast was shown to be more effective than 100% ammonium nitrogen.

1.2 Trials carried out in collaboration with the INRA Pech-Rouge for the vinification of red must

The purpose of these trials is to compare the efficacy in red winemaking of adding ammonium and of adding organic nitrogen (from inactivated yeast) presenting identical concentrations of YAN (16 mg/L). Fermented must submitted for trial: Portan (destemmed and crushed grapes) Analysis of the initial content of the must: · Sugars: 247 g/L · Total acidity: 3.4 g H2SO4/L · pH: 3.38 · YAN: 150 mg/L · Turbidity: 42 uTN = addition of 0.5% of frozen Maccabeu grape solids to obtain a final turbidity of > 100 uTN Protocol The must was sulphited at the rate of 3 g/100 kg and inoculated at the rate of 25 g/100kg with Lalvin EC1118 yeast. The temperature was maintained at 24°C and pigeage samples were taken daily. At the end of the AF, the juice was pressed and a blend of free-flowing and press juices was put in a 50 L barrel and then inoculated with a malolactic starter culture. At the end of the malolactic fermentation, the wine was racked into 30 L barrels, sulphited and placed in a cold room. The wine was then bottled and analyzed. For the comparison, we utilized the same protocols as for the Viognier trials. C Ammonium salts (DAP): Add 8 g/hL in two doses (4 g/hL at the start of fermentation and 4 g/hL at the one-third point of fermentation) to have 16 mg/L of YAN. C Organic nitrogen (Fermaid O): Add 40 g/hL in two doses (20 g/hL at the start of fermentation and 20 g/hL at the one-third point of fermentation) to have 16 mg/L of YAN. C Control fermentation with no added nitrogen.

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FIGURE 3. Residual sugars at the end of fermentation

10 14 12 Residual sugars (g/L) 10 8 6 4 2 0 160 hours 184 hours Décuvage 208 hours Control DAP FERMAID O

As shown in Figure 3, although no significant difference was observed between the results of the procedure when compared at the final stage, a tendency towards greater efficacy in the sugar uptake emerged when nitrogen in an organic form was added for the purpose of facilitating the fermentation.

1.3 Trials carried out by the ICV in the ICV experimental winery

Another series of trials was carried out to compare the efficacy of adding the nitrogen sources described below. C DAS: Add 22 g/hL at the one-third point of fermentation (following the protocol described by Sablayrolles et al. 1996). C Fermaid O: Add 40 g/hL at the one-third point of fermentation. C Fermaid E®: Add 30 g/hL at the one-third point of fermentation. The additions were made at the same time, which explains the considerable differences among the levels of added YAN (Table 2).

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TABLE 2. Experiment plan

Concentration of YAN in 30 g/hL DAS Fermaid E 69 mg/L 42 mg/L

YAN to be added in the ICV trials 50 mg/L 42 mg/L

Nitrogen source 100% ammonium 25% amino acids present in the yeast and 75% ammonium 100% amino acids present in the yeast

Fermaid O

12 mg/L

15 mg/L

The trials were carried out on three different raw materials (see Table 3). Despite the deficiencies of varying size and the differences among the quantities of added YAN, the data from the fermentation kinetics associated with the nitrogen sources studied did not present any notable differences. With only 15 mg/L of YAN added, the 40 g/hL of organic nitrogen is as effective as the 50 mg/L of YAN from the diammoniacal sulphate (Figure 4).

TABLE 3. Description of the musts

Sauvignon Potential degree Initial YAN 12.4 141 mg/L

Chardonnay 14.2 225 mg/L

Grenache Rosé 12.6 111 mg/L

FIGURE 4. Duration of alcoholic fermentation associated with three nutrition protocols applied to three different raw materials

25 DAS FERMAID E FERMAID O

Duration of AF in days

20

15

10

5

0 Sauvignon Chardonnay Grenache Rosé

Lastly, a trial carried out in an industrial situation (200 hL tanks) on a must with a high potential alcohol (15.5° v/v) showed the positive impact on the fermentation kinetics and therefore on the fermentation activity of the yeast. Compared to the control, receiving a mix of DAP, DAS and a yeast source at the rate of 30 g/hL at the one-third point of fermentation, the addition of organic nitrogen helped the yeast (ICV D80® in this case) consume the sugar under particular difficult winemaking conditions for red wines. The addition of a "classic" nitrogen nutrient in the same Grenache Noir must, at 15.5° v/v, did not permit the completion of the fermentation. When the fermentation stopped, the must contained 5 g/L of residual sugars.

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The addition of 30 g/hL of a 100% organic nutrient, with a level of about 16 mg/L of YAN, facilitated the completion of the fermentation. The results in Figure 5 show the positive effect of this nutrient on the fermentation metabolism of the yeast.

FIGURE 5. Fermentation kinetics with the addition of a nitrogen source at the one-third point of alcoholic fermentation in a 200 hL tank of Grenache Noir

1110

1090 Control 1070 Density Nitrogen nutrient (30 g/hL of Fermaid O) at the one-third point of AF

1050

1030

1010

990 0 5 Days 10 15

2. The Impact of Different Nitrogen Sources on the Sensory Profiles of Wines

We based our comparative evaluation of the impact of various nitrogen sources on the aroma and taste profiles of wines on the tasting experiences described below.

2.1 On a Sauvignon Blanc wine

The first wine tasted was a Sauvignon Blanc, fermented with ICV Opale® yeast according to the standard procedure, but with different nutritional additions. Sensory analysis was carried out in a blind tasting on the bottled wine, three months after bottling, by a jury trained with references. The jury utilized a structured intensity scale of four values. The charts below give the averages of the scores given by each taster (Delteil 2000).

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FIGURES 6 AND 7: Olfactory and gustatory profiles of a Sauvignon Blanc wine

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DAS FERMAID E FERMAID O

3

Intensity

2

1

0 Sulphur Mineral Tropical fruit Candied fruit

Volume 4 3 2 Bitterness 1 0

DAS FERMAID E FERMAID O

Roughness

Dryness

Astringency

The results show that the sensory profile was described as sulphur, but also candied fruit with the DAS, compared to a dominant exotic fruit note for the organic nitrogen sources. On the palate, the inorganic nitrogen emphasizes the aggressiveness with mouthfeel and volume inferior to the other sources, and with significantly higher levels of dryness and bitterness. In oenological terms, it could be said that the DAS "hardens" the mouthfeel while organic nitrogen "softens" it. 8

2.2 On a Syrah wine

The second wine submitted for tasting was a red Syrah fermented with ICV GRE® yeast. Sensory analysis was made under the same conditions and according to the same method as the first wine. However, the aroma descriptors were different. What emerges from this tasting is that the addition of organic or complex nitrogen intensifies the aroma sensations generally judged positive: red berries, jammy and spicy perfume. In the wine treated with DAS, sulphur and red berries dominate. On the palate, the differences are even clearer. The volume of the wine in the organic nitrogen nutrient sample is correlated to the softening of the final aggressive sensations. The DAS scores non-conformity points for a "Premium"-type red wine: sulphur odours, final dryness and bitterness.

FIGURES 8 AND 9: Olfactory and gustatory profiles of a Syrah wine

4 DAS FERMAID E FERMAID O 3

Intensity

2

1

0 Sulphur Red berries Jammy DAS FERMAID E FERMAID O Spicy

Volume 4 3 2 Bitterness 1 0

Tannin intensity

Dryness

Astringency

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Overall, the main tendencies that emerge from the results of the tastings carried out as part of this comparative study are as follows: organic nitrogen enhances the intensity of the berry notes and increases "softness" in the mouth, while mineral nitrogen often emphasizes the sulphur notes and the final aggressiveness.

Conclusion

The results of the study described herein show not only the importance of nitrogen supplementation during alcoholic fermentation, but also the importance of the choice of the form of nitrogen added. For musts threatened by nitrogen deficiency, it is better to aim for a complete nutritional supplement (i.e., a nutrient based on -amino acids) to effectively carry out fermentation. Indeed, this type of nitrogen, which is assimilated more slowly but more regularly, encourages aromatic expression while reducing the aggressive notes. In the inflationist context related to inorganic forms, this recommendation appears even more relevant. What's more, this new approach to yeast nutrition is well suited to winemakers who wish to limit the inorganic input in the winemaking process and focus on applying organic winemaking techniques to their products.

References and Bibliography

Agenbach, W. A. 1977. A study of must nitrogen content in relation to incomplete fermentations, yeast production and fermentation activity. Proc. South African Soc. Enol. Vitic., Cape Town, South Africa. Stellenbosh SA, 66-87. Bely, M., J. M. Sablayrolles, and P. Barre. 1991. Automatic detection of assimilable nitrogen deficiencies during alcoholic fermentation in enological conditions. J. Ferm. Bioeng. 70:246-252. Bezenger, M., and J. M. Navarro. 1987. Influence de l'azote sur la fermentation alcoolique en milieu modèle simulant les conditions de l'oenologie. Sciences des aliments 7:41-60. Busturia, A. and R. Lagunas. 1986. Catabolic inactivation of the glucose transport system in Saccharomyces cerevisiae. J. Gen. Microbiol. 132:379-385. Delteil, D. 2000. Exemple de mise au point de méthodes d'analyse sensorielle ­ 1re partie. Revue des OEnologues 97:36-40. Henschke, P. and V. Jiranek. 1993. Yeast metabolism of nitrogen compounds. Wine Microbiology and Biotechnology. G. H. Fleet (ed). Taylor & Francis Ltd 77-165. Henschke, P. and V. Jiranek. 1991. Hydrogen sulfide formation during fermentation: Effect of nitrogen composition in model grape must. Proceedings of the International Symposium on Nitrogen in Grapes and Wines, J. Rantz (ed.) 172-184. Jiranek, V., P. Langridge, and P. Henschke. 1991. Yeast nitrogen demand: Selection criterion for wine yeasts for fermenting low nitrogen musts. Proceedings of the International Symposium on Nitrogen in Grapes and Wines. J. Rantz (ed.) 266-269. Julien, A., J. L. Roustan, L. Dulau, and J. M. Sablayrolles. 2000. Comparison of nitrogen and oxygen demands of enological yeasts: Technological consequences. Am. J. Enol. Vitic. 51(3):215-222. Sablayrolles, J. M., C. Dubois, C. Manginot, J. L. Roustan, and P. Barre. 1996. Effectiveness of combined ammoniacal nitrogen and oxygen additions for completion of sluggish and stuck wine fermentations. J. Ferm. Bioeng. 82:377-381.

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