Professional Documents
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1957a, b; Varela et al., 2004; Hernandez-Orte et al., ent yeast strains are well known among wine yeast suppli-
2005). Nitrogen is often the limiting factor in grape juice ers, and commercial strains are categorized according to
fermentation (Varela et al., 2004; Bell & Henschke, 2005; the amount of nitrogen they require. Other aspects such
Carrau et al., 2008; Hazelwood et al., 2008). Low nitrogen as enological practices (pressing, macerations, etc.) alter
content in the must may result in sluggish or stuck fer- nitrogen availability and the nitrogen concentration in
mentations (Bisson & Butzke, 2000). To prevent these the must and thus are also considered when determining
problems, a common practice is to supplement the must the quality and concentration of the nitrogen source. Fur-
with nitrogen, preferably at early stages of alcoholic fer- thermore, although a nitrogen deficit can result in stuck
mentation (Jiranek et al., 1995). Several studies have been or sluggish fermentation, excess nitrogen can lead to
performed to find the optimal nitrogen concentration in other problems, including the production of unwanted
must to guarantee complete fermentation (Cantarelli, metabolites such as ethyl carbamate or biogenic amines
1957a, b; Bely et al., 1990; Mendes-Ferreira et al., 2004). (Ribéreau-Gayon et al., 2004). Thus, the amount of nitro-
The absolute minimum amount of nitrogen required for gen supplied to the must needs to be sufficient for fer-
alcoholic fermentation is very difficult to determine. mentation, but not in excess. Consequently, it is highly
In fact, the available references to date report ranges recommended that the wine producers understand the
from 120 to 140 mg yeast available nitrogen (YAN) L 1 nitrogen requirements of each yeast strain and routinely
(Bely et al., 1990) and from 200 to 267 mg YAN L 1 evaluate the YAN in the must.
FEMS Yeast Res 12 (2012) 477–485 ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
478 R. Martı́nez-Moreno et al.
Another aspect to be considered is that the nitrogen and 60 mg L 1 potassium disulfite (Merck). The media
requirements are also related to must sugar concentra- were filtered with 0.22 lm pore size nitrocellulose filters
tion. Until recently, the differences in sugar concentration and adjusted to pH 3.5 with 10 N KOH. All media prep-
for a given grape variety in a certain location were lim- arations were derived from MS300 as described by Bely
ited, with a rather constant concentration for every vin- et al. (1990).
tage. However, during the last decades and because of The carbon source used was a 50 : 50 mixture of glu-
global warming, larger differences in sugar concentration cose and fructose. The concentrations tested were 200,
have been observed with a trend toward increased sugar 240, and 280 g sugar L 1.
content. Although grapes can be harvested earlier, if Four different nitrogen sources were used: (1) ammo-
appropriate phenolic ripeness is sought, sugar concentra- nium chloride as an inorganic nitrogen source (I), (2)
tions will be higher and thus change the nitrogen require- arginine as a simple organic nitrogen source (O), (3) a
ª 2012 Federation of European Microbiological Societies FEMS Yeast Res 12 (2012) 477–485
Published by Blackwell Publishing Ltd. All rights reserved
Nitrogen availability and alcoholic fermentation 479
Data from the microwell spectrophotometer software and inoculated at an OD at 600 nm of 0.500 ± 0.014 into
were transformed with the polynomial curve a glass vial containing 5 mL of MF Medium. The vial
y = 0.1736*x3 0.205*x2 + 1.1086*x 0.0107 for cor- containing the sample was placed into the measuring cell
recting the nonlinearity of the optical recording at higher with 2 mL of a solution of 0.2% KOH solution, and the
cell densities. This polynomial curve was obtained from measuring cell was immediately tightly sealed and incu-
previous studies using serial dilutions of yeast suspensions bated. The BacTrac measured the impedance level every
as described by Warringer & Blomberg (2003). 10 min and drew a curve to show the percentage decrease
in impedance over time. The time taken to reach
decreases of 10% and 40% in impedance at 20 °C was
Fermentation conditions
selected as threshold value for 140-h samples (10% and
Fermentations were performed in 250-mL borosilicate 20% for the 270-h sample with 280 g sugar L 1 condi-
FEMS Yeast Res 12 (2012) 477–485 ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
480 R. Martı́nez-Moreno et al.
OD 600 nm
parameters and vitality test data obtained with the vari- 3.0
ous nitrogen sources. The means were compared by the
Tukey test (P < 0.05). 2.0
1.0
Results
0.0
OD 600 nm
ter the NRV. It was calculated by studying cell growth in
a range of nitrogen concentrations (5–220 mg L 1) and 3.0
ª 2012 Federation of European Microbiological Societies FEMS Yeast Res 12 (2012) 477–485
Published by Blackwell Publishing Ltd. All rights reserved
Nitrogen availability and alcoholic fermentation 481
Table 1. Nitrogen reference values (NRV, calculated as slope < 0.02), between the two nitrogen concentrations tested when
and maximum biomass shown as OD600 nm obtained for each ammonium was used as the nitrogen source, whereas in
combination of sugar concentration and nitrogen source
the other nitrogen conditions, the ethanol concentration
M O C I Signif. was generally one percentage point higher when
200 g L 1 300 mg L 1 of nitrogen was used, because of a higher
NRV 140 140 160 120 A consumption of residual sugars on those fermentations.
OD600 nm 3.91 ± 3.70 ± 4.79 ± 3.32 ± The analysis of ethanol yields did not show significant
0.10 0.09 0.11 0.09 differences among the nitrogen conditions. The different
1
240 g L levels of ethanol and residual sugars could be easily
NRV 160 160 160 160 B related to the amount of viable cells. In fact, no viable
OD600 nm 4.72 ± 4.25 ± 5.2 ± 3.90 ±
cells were recovered at the end of fermentation except
0.12 0.11 0.13 0.10
FEMS Yeast Res 12 (2012) 477–485 ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
482
Table 2. Main parameters of the laboratory fermentations performed with different nitrogen sources at three different sugar concentrations. (a) Fermentations with 200 and 240 g sugar L 1;
(b) Fermentations using 280 g sugar L 1 with 180 and 300 mg N L 1
M O C I M O C I
1 1
a 200 g sugar L (140 mg N L 1) 240 g sugar L (160 mg N L 1)
Residual sugar (g L 1) 2.53 ± 0.64a 3.83 ± 0.58a 2.6 ± 0.17a 3.73 ± 0.55a 2.02 ± 1.05a 3.76 ± 0.45a 3.23 ± 0.75a 3.8 ± 0.15a
Residual sugar (g L 1) 33.7 ± 0.96a 54.50 ± 0.79b 52.3 ± 1.2b 60.40 ± 1.54c 12.7 ± 0.25 37 ± 0.6 25 ± 0.5 60 ± 1.2
Ethanol (g L 1) 105.81 ± 1.77c 96.24 ± 1.99b 98.13 ± 1.41b 90.20 ± 0.93a 112.57 ± 1.37 105.74 ± 1.39 102.84 ± 1.41 90.90 ± 1.44
Glycerol (g L 1) 9.5 ± 0.08a 9.7 ± 0.07a 9.8 ± 0.11a 8.7 ± 0.10a 10.5 ± 0.05 10.9 ± 0.02 11.0 ± 0.06 11.4 ± 0.03
Ethanol yield (g sugar g etanol 1) 0.43 ± 0.02a 0.43 ± 0.02a 0.43 ± 0.02a 0.41 ± 0.01a 0.42 ± 0.01 0.44 ± 0.01 0.40 ± 0.01 0.41 ± 0.02
Total cells L 1 1.56E+08 8.08E+07 8.38E+07 3.48E+07 1.08E+08 9.10E+07 1.37E+08 3.55E+07
Viable cells L 1 5.33E+00 0.00E+00 0.00E+00 0.00E+00 5.20E+02 0.00E+00 0.00E+00 0.00E+00
% survival 3.42E 06 0 0 0 4.81E 04 0 0 0
MSFR (g L 1 day 1) 46.54 ± 1.25a 88.59 ± 5.82b 86.38 ± 6.03b 85.38 ± 2.38b 34.05 ± 1.92 60.40 ± 0.16 63.50 ± 0.10 62.15 ± 0.08
MSFR, major sugar fermentation rate; T100, time taken by cells to complete the fermentation process.
a,b mean statistically significant differences set at P < 0.05.
R. Martı́nez-Moreno et al.
FEMS Yeast Res 12 (2012) 477–485 ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
484 R. Martı́nez-Moreno et al.
MSFR or sugar consumption). In contrast, fermentations differences in yeast vitality, that is, the difference in the
with 280 g sugar L 1 were stuck. However, the stuck fer- ability of the yeast cells to modify their environment
mentations were not caused by inadequate nitrogen (Ribeiro et al., 2003). One way to analyze yeast vitality is
content because fermentations with the same sugar con- to determine the changes in the impedance of the med-
centration and moderate but not limiting nitrogen avail- ium, which is directly dependent on the metabolic
ability (Bell & Henschke, 2005) did not finish either, activity of the yeast cells. Different studies have been per-
regardless of the nitrogen source used. The reason for the formed to determine yeast vitality using methodologies
stuck fermentations is most likely a combination of dif- such as the BacTrac system (Redon et al., 2008; Rodri-
ferent factors, such as high osmotic pressure, ethanol con- guez-Porrata et al., 2008) and/or flow cytometry (Attfield
centration, medium chain fatty acids production, and the et al., 2000). These works have mainly focused on the
presence of potassium sulfite in the medium at the begin- study of vitality in the ADWY after rehydration. In this
ª 2012 Federation of European Microbiological Societies FEMS Yeast Res 12 (2012) 477–485
Published by Blackwell Publishing Ltd. All rights reserved
Nitrogen availability and alcoholic fermentation 485
project (Ingenio2010-CENIT) and by grant AGL2009- Hazelwood LA, Daran JM, van Maris AJA, Pronk JT &
07331 from the Ministerio de Educación y Ciencia, Spain. Dickinson JR (2008) The ehrlich pathway for fusel alcohol
The authors acknowledge Cristina Juez, Laura López, and production: a century of research on Saccharomyces cerevisiae
Braulio Esteve for excellent technical assistance and to metabolism. Appl Environ Microbiol 74: 2259–2266.
Zoel Salvadó, Marta Sancho and Manuel Quirós for their Hernandez-Orte P, Ibarz MJ, Cacho J & Ferreira V (2005)
valuable advice. Effect of the addition of ammonium and amino acids to
musts of Airen variety on aromatic composition and
sensory properties of the obtained wine. Food Chem 89:
163–174.
References
Jimenez-Marti E, Aranda A, Mendes-Ferreira A, Mendes-Faia
Albers E, Larsson C, Liden G, Niklasson C & Gustafsson L A & del Olmo ML (2007) The nature of the nitrogen source
(1996) Influence of the nitrogen source on Saccharomyces added to nitrogen depleted vinifications conducted by a
FEMS Yeast Res 12 (2012) 477–485 ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved