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Received 5 September 2009; received in revised form 17 March 2010; accepted 21 April 2010.
Abstract
Lager beers cover the largest part of the beer market, hence the increase interest for the lager brewing yeast. The
most widely used lager brewing yeast is represented by the WS34/70 population. Previous analysis revealed that the
WS34/70 population is actually made of a number of variant strains instead of being a pure culture. Thus, one could
find within mixture: variant a 47%, variant b 714%, variant c 4277%, variant d 218%, variant e 17%, variant f
1%, and so on: from samples taken from different locations (breweries as well as agar slant from Weihestephan and
Saflager dried yeast). The present study focuses on the fermentation behaviour displayed by two of the selected
variants and the initial mixture, represented by the commercial brewers lager yeast WS34/70, applying two different
fermentation temperatures: 10C and 15C. The two variants used in this study have disparities as far as the nuber of
chromosomes is concerned, the main difference is the lack of one chromosome for one of the variants, while the
other possesses a highlighted chromosome band in the corresponding location. The evolution of the attenuation
degree, yeast cell multiplication, pH value, fermentation rate, free amino nitrogen (FAN) values, and the diacetyl and
2,3 pentandione production were analysed during the trials, as well as the flavour compounds profile for each strain
and each temperature. The results indicate the higher temperature as an accelerator for the extract reduction, pH,
number of cells and vicinal diketone reduction for all three strains. Variant a is slightly faster in terms of
fermentation rate at 15C and diacetyl reduction both at 10C and 15C. The acetate ester production was higher at
15C, while the acetaldehyde production was favoured by lower fermentation temperatures.
Keywords: brewing yeast, fermentation temperature, fermentation rate, attenuation degree, extract reduction, free
amino nitrogen, diacetyl, esters, flavour compounds, yeast cell multiplication, chromosome, strain.
1. Introduction
Lager yeast cultures that are mixtures of very
closely related strains are usually employed in the
breweries. Each yeast strain may perform differently
under a given set of fermentation conditions. The
choice of a yeast strain depends on characteristics
considered important: attenuation limit, fermentation
* Corresponding author: Tel.: +40 744 363190,
E-mail: maria.turtoi@asiar.ro
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Romanian Journal of Food Science 2011, 1(1): 2638
Food Microbiology
characterization
WS34/70.
of
the
lager
brewing
yeast
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n=
ln N ln N 0
ln 2
(1)
where:
N is the peak cell count/mL;
N0 the initial cell count/mL.
The values for this parameter were higher for all
the variants during the fermentation at 15C, as
shown in Figure 3 than for 10C: with 26% higher
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Romanian Journal of Food Science 2011, 1(1): 2638
Attenuation degree, % .
80
70
60
50
40
WS34/70 (10C)
variant a (10C)
30
WS34/70 (15C)
variant a (15C)
variant b (10C)
variant b (15C)
20
10
0
0
24
48
72
96
120
144
168
192
216
Fermentation time, h
Figure 1. Attenuation degree during fermentation at 10C and 15C. Mean values of the duplicates.
3.00
10C
2.00
2.44
2.31
1.83
1.83
2.32
1.83
1.50
1.00
0.50
0.00
WS34/70
Variant a
Variant b
Figure 2. Fermentation rate of the two variants and the control strain.
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2.50
15C
Number of generations
3.0
2.5
10C
2.25
15C
2.48
2.44
1.81
2.02
2.0
1.5
1.0
0.5
0.0
WS34/70
Variant a
Variant b
Figure 3. Number of generations calculated for the two variants and the control strain.
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Romanian Journal of Food Science 2011, 1(1): 2638
FAN, mg/L .
200
Variant a (10C)
Variant a (15C)
Variant b (10C)
Variant b (15C)
150
100
50
0
0
24
48
72
96
120
144
168
192
216
Fermentation time, h
Figure 4. Free amino nitrogen utilization during fermentation at 10C (filled markers) and 15C (empty markers).
Values are means of the duplicates at 10C and 15C respectively.
5.0
WS34/70 (10C)
Variant a (10C)
Variant b (10C)
WS34/70 (15C)
Variant a (15C)
Variant b (15C)
pH
4.5
3.5
0
24
48
72
96
120
144
168
192
216
Fermentation time, h
Figure 5. pH evolution during fermentation at 10C and 15C respectively of the two variants and the control strain.
Values are means of the duplicates at 10C and 15C.
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4.0
35
Acetealdehyde (mg/L) .
30
Variant a (10C)
WS34/70 (15C)
Variant b (15C)
25
20
15
10
5
0
0
24
48
72
96
120
144
168
192
216
240
Figure 6. Acetaldehyde production during fermentation at 10C and 15C of three lager yeast strains.
Values are means of the duplicates performed at 10C and 15C.
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Romanian Journal of Food Science 2011, 1(1): 2638
Isoamylacetate (mg/L) .
3.0
2.5
2.0
1.5
1.0
0.5
0.0
0
24
48
72
WS34/70 (10C)
WS34/70 (15C)
Taste threshold
Poly. (Variant a (10C) )
Poly. (Variant b (15C) )
96
120
144
Fermentation time, h
168
Variant a (10C)
Variant a (15C)
Poly. (Variant b (10C) )
Poly. (WS34/70 (15C) )
192
216
240
Variant b (10C)
Variant b (15C)
Poly. (WS34/70 (10C) )
Poly. (Variant a (15C) )
Figure 7. Isoamyl acetate production during fermentation at 10C and 15C of three lager yeast strains.
Values are means of the duplicates at 10C and 15C.
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30
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Ethylacetate (mg/L) .
25
20
15
10
WS34/70 (10C)
Variant b (10C)
Variant a (15C)
Taste threshold
Variant a (10C)
WS34/70 (15C)
Variant b (15C)
0
0
24
48
72
96
120
144
168
192
216
240
Fermentation time, h
Figure 8. Ethyl acetate production during fermentation at 10C and 15C of three lager yeast strains.
Values are means of the duplicates at 10C and 15C.
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Romanian Journal of Food Science 2011, 1(1): 2638
In both cases, the immediate precursors are 2oxo-acids (-keto acids). In the anabolic route these
acids derive from pyruvate or acetyl-CoA as part of
amino acid biosynthetic pathways. In the catabolic
route or Ehrlich pathway of fusel alcohol formation,
the -keto acid is formed by transamination of an
amino-acid. The -keto acid is successively
decarboxylated and reduced to a fusel alcohol. By
starting with leucine as the amino acid, isoamyl
alcohol is thus formed. (Boulton and Quain, 2001;
Renger et al., 1992) Generally, when low levels of
amino acids are available, the anabolic route
predominates, and when high concentrations of
amino acids are present, the catabolic pathway is
favoured (Priest and Stewart, 2006).
Table 2. Ratio R = ethylacetate / isoamyl acetate of the
mean values for each strain at 10C and 15C
Ethylacetate
, mean values,
Iso amylacetate
as a function of temperature t, C
10C
15C
14.18
9.40
14.27
9.60
13.60
10.20
R=
Strain
Control sample
Variant a
Variant b
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Romanian Journal of Food Science 2011, 1(1): 2638
200
150
100
50
WS34/70 (10C)
Varianta a (10C)
Variant b (10C)
WS34/70 (15C)
Variant b (15C)
Varianta d (15C)
0
0
24
48
72
96
120
144
Fermentation time, h
168
192
216
240
Figure 9. Total higher alcohol production during fermentation at 10C and 15C of three lager yeast strains.
Values are means of the duplicates at 10C and 15C.
600
500
WS34/70 (10C)
Variant b (10C)
Variant a (10C)
WS34/70 (15C)
Variant a (15C)
Taste threshold
Variant b (15C)
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Diacetyl, ppb .
400
300
200
100
0
0
24
48
72
96
120
144
Fermentation time, h
168
192
216
240
Figure 10. Diacetyl evolution during fermentation at 10C and 15C of three lager yeast strains.
Values are means of the duplicates.
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Romanian Journal of Food Science 2011, 1(1): 2638
Moreover, at 10C, the precursor of diacetyl (acetolactate) is slowly degraded into diacetyl, hence
the ratio between the amount of diacetyl reduced by
the cells and the newly formed amount is lower than
at 15C. To overcome this problem, a high
temperature period is used in industry called
diacetyl rest.
In parallel, the amount of 2,3-pentandione was
considered and measured during both fermentations
at 10C and 15C. The evolution of this compound
is shown in Figure 11 and resembles the diacetyl
profile at the mentioned temperatures. This
compound (2,3-pentandione) is produced below its
threshold of 900 ppb in all cases.
The peaks registered in the first part of the
cultivation period are the result of low amounts of
isoleucine available for yeast this being forced to
produce it. Similar to the diacetyl reduction, at 10C
it can be observed a slow decrease of this compound
due to the uptake and release ratio mentioned above.
4. Conclusions
Fermentation temperature represents one of the
few factors a brewer can use to influence the process
inside an industrial fermenter. Other tools are
dissolved oxygen via wort aeration, pitching rate as
the intial amount of yeast added to the fermenter and
the amount of zinc and overpressure. High
temperatures promote the decrease in extract, the
absorption of nitrogen substances, the activation of
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700
2,3-pentanedione, ppb .
600
WS34/70 (10C)
Variant a (10C)
Variant b (10C)
WS34/70 (15C)
Variant a (15C)
Variant b (15C)
500
400
300
200
100
0
0
24
48
72
96
120
144
168
192
216
240
Fermentation time, h
Figure 11. 2,3-pentandione production during fermentation at 10C and 15C of three lager yeast strains.
Values are means of duplicates.
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Acknowledgments
The authors wish to thank Heineken Supply Chain
for their support of this work.
References
Babb M. 2008. Brewing Fermentation
Presentation to Science Caf, 9.08.2008.
Flavours.
Food Microbiology
Abbreviations
DNA deoxyribonucleic acid
FAN Free amino nitrogen
GC/ECD Gas chromatography / electron capture
detector
GC/FID Gas chromatography / flame ionization
detection
n number of generations
ppb part per billion
ppm part per million
VDK Vicinal diketones
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