Introduction
-Maximum biomass levels, as estimated by O.D. 600 nm increase with increasing nitrogen availability,
Nitrogen is essential for yeast metabolism
and growth and is considered to be a
limiting factor during the fermentation
process. Nitrogen availability and uptake
also affects the aromatic profile in wine
(Bell & Henschke, 2005). For these
reasons, nitrogen requirements of wine
yeast during fermentation would be an
important control parameter for industrial
fermentations. In this work, a new
methodology to determine nitrogen
requirements of yeast depending on sugar
content in must was developed and these
results were tested in different
fermentation processes.
Materials & Methods
- The active dry wine yeast (ADWY) strain S.
cerevisiae EC1118 (Lallemand Inc, Canada)
was used throughout this study.
-Yeast was grown in different MS-300 based
synthetic musts. Different combinations of
sugar (200 g/L, 240 g/L and 280 g/L) and
nitrogen (from 0 to 240 mg N/L) content
were used. Four nitrogen sources were
tested (Table 1).
-In order to determine nitrogen
requirements and to study the effects of
nitrogen addition, yeast growth was
monitored in a microwell plate-reader
spectrophotometer (Omega PolarStar, BMG
Labtech).
-BacTrac 4300 microbiological analyzer (SY-
LAB Instruments, Austria) was used to test
cell vitality during fermentation process.
- Volatile compounds were analyzed by
organic solvent extraction followed by gas
chromatography.
Table 1. Nitrogen sources tested.
Nitrogen sources Composition
Inorganic (INS)
Simple organic
(SONS)
Complex organic
Leu/Val/Ile/Phe/Thr
(CONS)
NH4Cl/19 amino acids
Control (CNC)
(not Asn)
Conclusions
-A new predictive methodology based on
microwell plate-reader spectrophotomer
system to determine nitrogen requirements
has been developed in this study. The results
were satisfactorily tested in bottle
fermentations.
- EC1118 was not able to completely ferment
280 g/L of sugar with synthetic must recipe,
independently of the nature or the amount of
nitrogen source.
-Fermentation of must prepared with the
CONS nitrogen source show a slow kinetic
during the first hours after inoculation,
compensated by a faster kinetics thereafter.
Viability and vitality of cells fermenting CONS
containing must is also higher than with
other nitrogen sources.
-Nitrogen addition at inoculation time was
more efficient than at exponential growth
phase to ensure high biomass levels. The
highest biomass production was observed for
the addition of a nitrogen source with the
same proportions as that in control must.
E-mails:
Rubén Martínez1 - ruben.martinez@icvv.es; Pilar Morales1 – pilar.morales@icvv.es; Ramón González1 – rgonzalez@icvv.es
Gemma Beltrán2 – gemma.beltran@urv.cat; Albert Mas2 – albert.mas08@gmail.com
Results
up to a maximum that depends on the initial sugar content of the must (Figure 1). The lowest
nitrogen content required to reach biomass values close to the maximum was considered as the
minimum nitrogen requirement to reach a sufficient biomass level and to ensure complete
fermentation. These values were independent of the nature of the nitrogen source. The actual values
are 140, 160 and 180 mg N/L, for 200, 240 and 280 g/L of sugar, respectively.
-Using these nitrogen amounts, fermentations in
laboratory flasks were complete in two to three
weeks for 200 and 240 g/L sugar. However, musts
with 280 g/L of sugar never reached total sugar
consumption, independently of the nitrogen source
employed (Table 2). This cannot be attributed to low
nitrogen availability, since fermentations with excess
nitrogen (300 mg/L) did not finish either (Figure 2).
Table 2. Residual sugar of fermentations.
240 g/L Sugar (160 mg N/L) 280 g/L Sugar (180 mg N/L) Figure 1. Microwell spectrophotometer profile at 600 nm.
N. S. INS SONS CNC CONS INS SONS CNC CONS
-A particular density profile was observed in
T 300 300 257 257 688 688 688 688
fermentations where CONS was used as
R.S. 3,89 3,82 3,27 3,32 66,4 54,5 42,3 33,7
nitrogen source, as show figure 2.
N.S. = Nitrogen source; T = Time (H); R.S. = Residual Sugar (g/L)
Fermentation kinetics is slowed down for the
first few hours, and then accelerated to reach
higher sugar consumption that with other
nitrogen sources. In addition, higher cell
viability was found by the end of the
fermentation. After 270 h of fermentation
vitality of cells grown with CONS as nitrogen
source was again higher than that of cells
grown in the other synthetic musts (Table 3).
These were indeed the only cells able to induce
a 40 % decrease in impedance in the vitality
test.
-On the other hand, a characteristic profile of
Figure 2. Density evolution of 280 g/L sugar fermentation with volatile compounds was found in the
the four nitrogen sources and at 300 mg N/L. fermentation products of musts containing
CONS as nitrogen source, as compared to
Table 3. Vitality test (Tukey test -p<0,05-).
other nitrogen sources. This profile is
N Impedance (28% sugar - 180 mg N/L – 270 h
source fermentation) significantly enriched for branched-chain and
aromatic alcohols (Figure 3), probably derived
-10% -20% -40%
NH4Cl Time (H) SD Time (H) SD Time (H) SD from the catabolism of branched chain and
INS 10,67b 0,98 18,29b 1,45 aromatic amino acids in CONS.
Arg SONS 6,32a 0,71 16,39b 1,23
CNC 8,77a,b 0,77 18,66b 0,77
CONS 5,24a 0,52 9,41a 1,01 12,04 1,35
-Three different addition times were studied for the
correction of low nitrogen content must, inoculation
time, early exponential growth phase and late
exponential growth phase. The four nitrogen
sources were tested. Our results indicate that
nitrogen addition at the inoculation time results in
a higher final biomass content that addition during
exponential growth phase (Figure 4). Concerning
the specific nitrogen source, the highest biomass Figure 3. Aroma profile of final fermentation products.
Me = Methyl and Phe = Phenyl.
increase was observed for CNC additions (Figure 5)
Figure 4. Nitrogen supply to low nitrogen content must Figure 5. Nitrogen supply to low nitrogen content must
(60 mg N/L). Effect of time addition. (60 mg N/L). Effect of nitrogen source addition.
Acknowledgments References
This work was supported by a predoctoral fellowship from JAE Program Bell, S. J. and P. A. Henschke (2005). "Implications of
(CSIC) and the DEMETER project (Ingenio2010-CENIT). The nitrogen nutrition for grapes, fermentation
authors acknowledge to Cristina Juez, Laura López and Baraulio and wine." Australian Journal of Grape
Esteve for an excellent technical assistance and to Zoel and Wine Research 11(3): 242-295.
Salvadó, Marta Sancho and Manuel Quirós for their valuable
advices.