Romanian Journal of Food Science
EDITURA ACADEMIEI ROMÂNE
http://www.ear.ro
Official Journal of the Romanian Association
of Food Professionals
http://www.asiar.ro
Temperature response of new lager brewing strains isolated from
WS34/70
Irina BOLAT 1, Maria TURTOI 2,* and Michael C. WALSH 1
1
2
Heineken Supply Chain, Burgemeester Smeetsweg 1, 2382 PH Zoeterwoude, The Netherlands
Galati Dunarea de Jos University, 47 Domneasca St., 800008 Galati, Romania
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 4–7%, variant b 7–14%, variant c 42–77%, variant d 2–18%, variant e 1–7%, 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: 10°C and 15°C. 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 15°C and diacetyl reduction both at 10°C and 15°C. The acetate ester production was higher at
15°C, 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: [email protected]
rate, and oxygen requirements etc. (Priest and
Stewart, 2006) the extent of transformations during
the fermentation process depends on yeast ability to
adapt to the new environment (Vesely, 2004).
The desire to increase the capacity of breweries
without further investments calls for different
measures like high-gravity brewing or accelerated
fermentation. Higher temperature represents the
most attractive mean to accelerate fermentation by
stimulating yeast nitrogen uptake, cell growth and
budding but also causes an increase in the formation
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Romanian Journal of Food Science –2011, 1(1): 26–38
Temperature response of new lager brewing strains isolated from WS34/70
Heyse and Piendl (1974) studied the influence of
different fermentation temperatures on the activity
of some enzymes, grouping them as follows:
enzymes whose activities are increased with a rise in
fermentation temperature: maltotriase, maltase,
phosphofructokinase, pyruvate kinase, lactate
dehydrogenase, pyruvate decarboxylase, alcohol
dehydrogenase,
citrate
synthase,
isocitrate
dehydrogenase, enzymes whose activities decrease
with a rise in fermentation temperature: hexokinase,
glycerokinase, fumarase and enzymes with
intermediate activities at low temperatures and
increase
activity
at
high
temperatures:
phosphoglyceraldehyde dehydrogenase and glucose
6-phosphatedehydrogenase.
An increase in fermentation temperature is
assumed to induce higher concentrations of fusel
alcohols and esters.
The balance of flavour metabolites is largely a
consequence of the combination of raw material
quality, yeast strain and wort composition. The
essential character of a beer is determined by yeast
metabolism and the plethora of yeast metabolic byproducts
that
arise
during
fermentation
(Masschelein, 1981; Boulton and Quain, 2001;
Vesely et al., 2004). The flavour-active compounds
produced by yeast include: fusel alcohols
(8–10 carbon atoms aliphatic alcohols), glycerol,
esters, organic and fatty acids, sulphur compounds,
aldehydes and ketones, phenols, amines and a
number of miscellaneous compounds (Boulton and
Quain, 2001; Vesely et al., 2004; Babb, 2008; Priest
and Stewart, 2006).
Saerens et al. (2008 b), monitoring the influence
of fermentation temperature on flavour formation by
analysis of gene expression levels in brewing yeast,
noticed that there is good correlation between
flavour production and the expression level of
specific genes involved in the biosynthesis of aroma
compounds.
Yeast contains several enzyme systems with
different substrate specificities that positively impact
flavour stability by reducing the level of carbonyl
compounds present in wort. These attributes vary
among the yeast strain according to their genetic
background and sensitivity to fermentation
conditions (Boulton and Quain, 2001; Vesely et al.,
2004).
Vesely and his team presented the results of their
experiments on yeast reductase activity at different
temperatures, observing that a 10°C fermentation
delays the maximum activity of the yeast aldehyde
reductase to 4 days, whereas a 15°C fermentation
determines these enzymes to reach their maximum
within 2 days (Vesely et al., 2004).
The most important flavour-active compounds in
beer are the esters. They impart characteristic
flowery and fruit-like flavours and aromas to beer.
They have relatively low taste thresholds that are
often attained. Their presence is desirable at
appropriate concentrations; however, in higher
amounts, they can destroy the flavour balance.
(Verstrepen et al., 2003; Hammond, 1986) The
flavour-active esters, whose concentrations in beer
are considered crucial to product quality, include
ethyl acetate (solvent/fruity like), isoamyl acetate
(apple, banana like), isobutyl acetate (banana,
fruity), ethyl caproate (sour apple) phenyl ethyl
acetate (flowery, roses, honey) (Verstrepen et al.,
2003; Stewart, 2005; Piendl and Geiger, 1980) The
fact that most esters are present in concentrations
around the threshold value, implies that minor
changes in concentration may have dramatic effects
on beer flavour (Verstrepen et al., 2003; Renger et
al., 1992).
There are two main groups of flavour-active
esters in fermented beverages: the first group
contains the acetate esters (such as isoamyl acetate),
the second group is the ethyl esters (such as ethyl
hexanoate). There are many factors that affect the
ester profile, among them the yeast strain and the
fermentation temperature has strong effects on the
perception of the flavour of the beer produced
(Saerens et al., 2008 a; Stewart, 2005).
Fermentation temperatures and ester formation
are directly related due to the fact that higher
temperatures could make cell membranes more fluid
and this could possibly modulate the activity of the
membrane-bound alcohol acetyltransferase or
simply increase diffusion rates of esters from cells
into the beer (Boulton and Quain, 2001).
This paper focuses on the influence of two
different temperatures, 10°C and 15°C respectively,
upon the behaviour of new lager yeast strains
isolated from WS34/70 population against the initial
mixture.
The study follows the fermentation kinetics, yeast
behaviour and flavour compounds. This paper
represents a part of a larger project concerning the
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Romanian Journal of Food Science – 2011, 1(1): 26–38
Food Microbiology
of certain flavour active fermentation by-products,
producing a beer which is often quite different in
flavour from that produced by the same yeast strain
from a normal process (Takahashi et al., 1997;
Lewis, 1974). Yeast plasma membrane seems to be
the probable site of difference between yeasts grown
at various temperatures (Lewis, 1974).
Irina BOLAT, Maria TURTOI and Michael C. WALSH
characterization
WS34/70.
of
the
lager
brewing
yeast
2. Materials and methods
Samples were taken at certain intervals; the yeast
cells and the fermenting wort were separated by in a
refrigerated centrifuge at 2°C for 5 min at 3000 rpm.
Free amino nitrogen was determined by the EBC
ninhydrin method.
2.1. Yeast variants
The two variants used in this trial were isolated
and selected from the WS34/70 lager yeast
purchased from Weihenstephan (Germany) as yeast
agar slant. The WS34/70 population, as well as the
variants a and b, selected from WS34/70 as natural
variants, were preserved as glycerol stock at –80°C.
The identification of the variants within the initial
population was done using pulsed field gel
electrophoresis, a technique that allows separation of
large deoxyribonucleic acid (DNA) molecules,
typically ranging in size from 50 to 10 000 kb
(Johnston, 1994).
Variant a posses a highlighted chromosome band
in the location where variant b lacks the
chromosome. Furthermore, variant a is a medium
flocculent strain, while variant b is a flocculent
strain.
Food Microbiology
2.2. Fermentation conditions
Fermentations were performed on 16°Plato all
malt wort, collected from a brewery. Zinc was added
into the wort (0.5 ppm final concentration of Zn2+).
The fermentations were run in duplicate in 50 mL
glass bottles with 30 mL wort, at 10ºC and 15ºC
respectively, under continuous agitation. The
pitching rates are shown in Table 1. Fermentations
were stopped after 8 days (at around 80% apparent
degree of fermentation).
Table 1. Pitching rate used within the trial
Strain
Control
Variant a
Variant b
Pitching rate (×106 cell/mL)
10°C
15°C
24.65 ± 0.49
24.20 ± 0.14
31.25 ± 3.89
27.05 ± 2.19
21.90 ± 0.42
27.35 ± 2.05
2.3. Analysis of different parameters
The extract evolution was analysed using the
Anton Paar portable density meter. Yeast cell
concentration and yeast viability were determined
using the NucleoCounter YC-100 System with its
NucleoCassettes containing propidium iodine.
Gas chromatography analysis was used with
flame ionization detection (GC/FID) for esters and
higher alcohols and an electron capture detector
(GC/ECD) for diacetyl and 2,3-pentandione.
3. Results and discussion
3.1. Degree of fermentation, fermentation rate, cell
multiplication, free amino nitrogen profile
during fermentation, pH evolution
The two variants used in this trial were referred
to as variant a and variant b. The initial mixture out
of which these two yeast strains were selected, was
represented by WS34/70 population and referred to
as control strain. The fermentation behaviour of
these three lager yeast strains was monitored during
fermentation in all malt wort at 10°C and 15°C, at
laboratory scale. From the degree of fermentation
profiles, as seen in Figure 1 for the trial performed at
10°C and the trial performed at 15°C, a faster
fermentation is seen at 15°C, as expected. At 10°C,
all analysed variants reach a degree of fermentation
within 70–80% only after 6 days of fermentation,
while at 15°C after 4 days (Figure 1).
Among the yeasts studied at 10°C, variant b
stands out as the strain that reached the highest
attenuation rate at low temperature. At high
temperature, after 8 days, the degree of fermentation
overpasses 80%, while at the low temperature it
barely gets to 75%. These results are supported by
the fermentation speed values in Figure 2. Thus, the
fermentation rate was faster at 15°C than at 10°C,
with 0.48°Plato/day for the control strain,
0.61°Plato/day for variant a and 0.49°Plato/day for
variant b at high temperature. Moreover, the two
variants and the control strain analysed, showed the
same value for the fermentation rate at 10°C, while
small differences among the two variants and the
control mixture of strains had been noticed at 15°C
with a slightly faster rate for variant a (Figure 2).
Yeast cell growth during the two trials was
expressed as number of generations (n):
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 15°C, as
shown in Figure 3 than for 10°C: with 26% higher
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Romanian Journal of Food Science – 2011, 1(1): 26–38
Temperature response of new lager brewing strains isolated from WS34/70
100
90
Attenuation degree, % .
80
70
60
50
40
WS34/70 (10°C)
variant a (10°C)
30
WS34/70 (15°C)
variant a (15°C)
variant b (10°C)
variant b (15°C)
Attn. degree = 70%
Attn. degree = 80%
20
10
0
0
24
48
72
96
120
144
168
192
216
Fermentation time, h
Figure 1. Attenuation degree during fermentation at 10°C and 15°C. Mean values of the duplicates.
3.00
10°C
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.
for variant a, 20% for the control sample and 18.5%
for variant b.
A strong growth rate entails a greater utilisation
of ATP, its inhibiting effect is thus diminished and
the enzymes activity is gradually increasing (Heyse
and Piendl, 1974).
The values presented in Figure 3 indicate a
higher yeast yield of the control mixture both at
10°C and 15°C. Thus, one can speculate that the
control mixture probably contains other strains with
higher yeast yield than the two variants studied
herein.
Free amino nitrogen or FAN refers to the free
α-amino nitrogen and includes all of the amino acids
minus proline. Proline, the most plentiful aminoacid
in wort, is not an α-aminoacid and is not utilizable
by Saccharomyces under anaerobic conditions.
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Romanian Journal of Food Science – 2011, 1(1): 26–38
Food Microbiology
Fermentation rate (Plato/zi)
2.50
15°C
Irina BOLAT, Maria TURTOI and Michael C. WALSH
3.5
2.82
Number of generations
3.0
2.5
10°C
2.25
15°C
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.
Food Microbiology
However, under aerobic laboratory condition,
proline is assimilated after exhaustion of the other
aminoacids (Priest and Stewart, 2006). FAN is
expressed as milligrams N per liter assimilable
nitrogen and affects other fermentation factors, such
as cell growth, biomass, viability, pH and
attenuation rate.
FAN was reduced with a faster rate at high
temperature, the decrease being much more
significant at 15°C than at 10°C (Figure 4). The
amino acids rapid uptake at 15°C is reflected in the
higher rate of yeast growth expressed as number of
generations in Figure 3. During the fermentation at
higher temperatures there is no evident difference
among the two variants and the control yeast
regarding the FAN uptake profile, while at 10°C
variant b stands out with a low uptake rate and a
high free amino nitrogen amount after 8 days of
fermentation.
There is a good correlation between FAN
evolution during the two experiments and the pH
profile (Figure 5). The pH characteristically drops
during fermentation due to uptake of wort
aminoacids and proton extrusion by actively
growing and fermenting yeast. A higher amount of
amino-acids in wort (the main pH buffering capacity
in wort) will lead to higher pH values. High residual
quantities of amino nitrogen in beer can cause poor
flavour stability and an increase disposition to beer
spoiling microorganisms. After 144 hours of
fermentation, the pH in the beer fermented at 15°C
starts to raise, this is due to cell lysis characteristic-
ally within higher temperatures when the stress on
the yeast is the most severe.
3.2. Flavour compounds profile during
fermentation: acetaldehyde, isoamyl acetate,
ethyl acetate, total higher alcohols
Acetaldehyde is an indicator of the fermentation
quality, higher content is less desirable.
Acetaldehyde formation in beer occurs during the
period of active yeast growth (Boulton and Quain,
2001) as seen in Figure 6, with an increase in the
beginning and a decrease towards the end of
fermentation.
The maximum concentration of this compound
was registered within 30–55% degree of attenuation.
The control strain and variant a show a significant
decrease in acetaldehyde levels after 48 hours of
fermentation, while variant b after 72 hours of
fermentation at 10°C. During the fermentation at
15°C of the control strain and variant a a pronounced decrease in the acetaldehyde amount started
earlier, after only 24 hours of fermentation, and after
48 hours for variant b.
After 7 fermentation days, the amount of
acetaldehyde within the beers produced at 10°C was
higher than in the beers produced at 15°C;
nevertheless, after 8 fermentation days the situation
was reversed, with acetaldehyde values lower at
10°C than at 15°C. Vesely et al. (2004) noticed that
longer fermentation time at 10°C resulted in slightly
lower levels of carbonyl compounds.
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Romanian Journal of Food Science – 2011, 1(1): 26–38
Temperature response of new lager brewing strains isolated from WS34/70
250
WS34/70 (10°C)
WS34/70 (15°C)
FAN, mg/L .
200
Variant a (10°C)
Variant a (15°C)
Variant b (10°C)
Variant b (15°C)
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 10°C (filled markers) and 15°C (empty markers).
Values are means of the duplicates at 10°C and 15°C respectively.
5.0
WS34/70 (10°C)
Variant a (10°C)
Variant b (10°C)
WS34/70 (15°C)
Variant a (15°C)
Variant b (15°C)
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 10°C and 15°C respectively of the two variants and the control strain.
Values are means of the duplicates at 10°C and 15°C.
Among the flavour-active compounds in beer,
two very important acetate esters, i.e., isoamyl
acetate and ethyl acetate were analysed during this
trial.
– Ethyl acetate: 33 mg/L (Renger et al., 1992),
21–30 mg/L (Verstrespen et al., 2003), 25 mg/L
(Hammond, 1986), 30 mg/L (Piendl and Geiger,
1980).
The taste thresholds of these two esters are
mentioned in different papers:
– Isoamyl acetate: 1.6 mg/L (Renger et al., 1992;
Piendl and Geiger, 1980), 0.6–1.2 mg/L
(Verstrespen et al., 2003), 2mg/L (Hammond,
1986), 1.4 mg/L (Babb, 2008);
A certain ester level is necessary for the normal
flavour of beer, thus beer lacks in fruitiness if the
isoamyl acetate level is below 1 mg/L, while it is too
high if the level is above 3 mg/L (Piendl and Geiger,
1980).
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Romanian Journal of Food Science – 2011, 1(1): 26–38
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4.0
Irina BOLAT, Maria TURTOI and Michael C. WALSH
40
WS34/70 (10°C)
Variant b (10°C)
Variant a (15°C)
Taste threshold
35
Acetealdehyde (mg/L) .
30
Variant a (10°C)
WS34/70 (15°C)
Variant b (15°C)
25
20
15
10
5
0
0
24
48
72
96
120
144
168
192
216
240
Fermentation time (hr)
Figure 6. Acetaldehyde production during fermentation at 10°C and 15°C of three lager yeast strains.
Values are means of the duplicates performed at 10°C and 15°C.
Isoamyl acetate is produced from a reaction
between amyl alcohol and acetyl coenzyme A
catalyzed by the enzyme isoamyl alcohol acetyl
transferase (Quilter et al., 2003).
Food Microbiology
Within this trial both isoamyl acetate and ethyl
acetate were produced in higher amounts during the
fermentation performed at 15°C than at 10°C
(Figures 7 and 8).
In the case of isoamyl acetate, the mean value of
this ester during fermentation at 15°C was higher
with 50% for the control strain, with 40% for variant
a and 35% for variant b than at 10°C.
Smaller differences were noticed for the mean
value of the ethyl acetate generated during the
fermentation performed at 15°C as opposed to 10°C:
20% higher for the control strain, 14% for variant a
and 15% for variant b.
At the beginning of fermentation, when the active
lipid synthesis required for cell growth is taking
place, the specific rate of ester synthesis is relatively
low (Figures 7 and 8).
Later, as growth becomes restricted by the lack of
sterols and unsaturated fatty acids, the specific rate
of ester synthesis increases. After 6 days of
fermentation, the amount of isoamyl acetate in beer
starts to decrease both at 10°C and at 15°C
fermentation temperature.
Engang and Aubert (1977) also found an increase
in the concentration of these compounds with
temperature when they used 8°C, 10°C and 12°C,
both at full scale and laboratory scale. They
mentioned through the work of Drews and his team,
who found an increase in the isoamyl acetate content
when the temperature changed from 9°C to 14°C,
but no difference between 14°C and 19°C.
The differences in terms of ethyl acetate
production at 10°C and 15°C are rather limited.
The lower the ratio “ethyl acetate / isoamyl
acetate” the better the quality of the product. There
is a significant difference of this ratio for the two
temperatures (Table 2) with better values obtained
when 15°C fermentation temperature was applied.
In terms of differences among the two variants
and the control strain, variant b seems to produce a
beer with the most balanced flavour at 10°C, while
at 15°C the control strain showed the lowest ratio.
The main contribution of higher alcohols to beer
flavour is by a general intensification of alcoholic
taste and aroma and by imparting a warming
character. A second very important role of fusel
alcohols is to provide precursors for ester synthesis
(Boulton and Quain, 2001). Their formation is
linked to amino-acid and carbohydrate metabolism
particularly during growth.
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Temperature response of new lager brewing strains isolated from WS34/70
3.5
Isoamylacetate (mg/L) .
3.0
2.5
2.0
1.5
1.0
0.5
0.0
0
24
48
72
WS34/70 (10°C)
WS34/70 (15°C)
Taste threshold
Poly. (Variant a (10°C) )
Poly. (Variant b (15°C) )
96
120
144
Fermentation time, h
168
Variant a (10°C)
Variant a (15°C)
Poly. (Variant b (10°C) )
Poly. (WS34/70 (15°C) )
192
216
240
Variant b (10°C)
Variant b (15°C)
Poly. (WS34/70 (10°C) )
Poly. (Variant a (15°C) )
Figure 7. Isoamyl acetate production during fermentation at 10°C and 15°C of three lager yeast strains.
Values are means of the duplicates at 10°C and 15°C.
35
30
Food Microbiology
Ethylacetate (mg/L) .
25
20
15
10
WS34/70 (10°C)
Variant b (10°C)
Variant a (15°C)
Taste threshold
5
Variant a (10°C)
WS34/70 (15°C)
Variant b (15°C)
0
0
24
48
72
96
120
144
168
192
216
240
Fermentation time, h
Figure 8. Ethyl acetate production during fermentation at 10°C and 15°C of three lager yeast strains.
Values are means of the duplicates at 10°C and 15°C.
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Irina BOLAT, Maria TURTOI and Michael C. WALSH
In both cases, the immediate precursors are 2oxo-acids (α-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 α-keto acid is formed by transamination of an
amino-acid. The α-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 10°C and 15°C
Ethylacetate
, mean values,
Iso − amylacetate
as a function of temperature t, °C
10°C
15°C
14.18
9.40
14.27
9.60
13.60
10.20
R=
Strain
Control sample
Variant a
Variant b
Food Microbiology
High temperatures promote yeast cell growth and
thus higher alcohol production. The more free amino
nitrogen and sugars are taken up, the more higher
alcohol are produced (Mussche and Mussche, 2009;
Priest and Stewart, 2008). Narziss et al. (1981)
showed that excessive amounts of amino acids are
directly transformed into the relevant alcohols (by
the Ehrilch mechanism).
All the yeast strains analysed in the present paper
showed increased rates of higher alcohols when
fermented at 15°C than the same strains fermented at
10°C. Variant b produced the lowest amount of total
higher alcohols at 10°C and variant a at 15°C as
seen in Figure 9. This is supported by the low
capacity of these two variants to take up the free
amino nitrogen from the wort in comparison with
the other two yeasts analysed (Figure 4).
Engang and Aubert (1977) found during their
experiments that an increase in temperature from
8°C to 10°C is accompanied by an increase in total
higher alcohols concentration, but a further increase
of the temperature to 12°C gave a decrease in
concentration. They mention Ayrapaa’s observation
that the effect of temperature largely depends on the
amount and type of nitrogenous nutrients in the
medium.
2.3. Vicinal diketones profile during fermentation
and their correlation with free amino nitrogen
content
The two most important members of vicinal
diketones (VDK) group with respect to beer are
diacetyl and 2,3-pentanedione, which impart a
characteristic aroma and taste to beer, described as
“buttery”. These compounds arise as an indirect
result of yeast metabolism, being part of the normal
fermentation process. Their precursors are α-acetohydroxy acids, which are intermediates in the
biosynthesis of valine (acetolactate) and isoleucine
(acetobutyrate) and are being exported into the wort
from the yeast during fermentation. Outside the
yeast cell they undergo spontaneous oxidative
decarboxylation to form diacetyl and 2,3-pentanedione. From middle to late fermentation
extracellular VDK is converted by yeast cell
reductase in acetoin and 2,3-butanediol from
diacetyl and 2,3-pentanediol from 2,3-pentanedione.
In Figure 10 the diacetyl profile during
fermentation of the two variants and the control
strain at 10°C and 15°C is shown. The peak values
were between 241–369 ppb at 10°C, while at 15°C
higher values, between 429–495 ppb, were
registered (mean values of the duplicates). These
results are in accordance with Masschelein’s (1986)
remark that higher starting temperatures increase the
initial demand in nitrogenous nutrients leading to
beers with high α-acetolactate and diacetyl levels.
The peak values for diacetyl were reached at 10°C
after 48h of fermentation for the control yeast and
variant a and 96h for variant b. A different pattern
was noticed at 15°C with the diacetyl peak value
after 72h of fermentation for the control strain and
variant b and 96h for variant a.
From all the strains studied at 10°C variant a
produced the lowest amount of diacetyl and reduced
it below the taste threshold within 7 days. The same
strain synthesised higher amounts of diacetyl at
15°C, but was able to reduce it below the taste
threshold within the same period of time. This
behaviour is supported by its flocculation
characteristic as a medium flocculent strain.
During fermentation at 15°C the diacetyl
reduction is faster than at 10°C. At 15°C, after the
FAN value dropped, the diacetyl level started to
increase. It could be possible that an extra need of
amino acids was necessary for the cells that started
to synthesis them. The 10°C temperature slowed the
FAN uptake and the cells compensated their need of
amino acids by synthesising them.
34
Romanian Journal of Food Science – 2011, 1(1): 26–38
Temperature response of new lager brewing strains isolated from WS34/70
250
Total higher alcohols (mg/L)
200
150
100
50
WS34/70 (10°C)
Varianta a (10°C)
Variant b (10°C)
WS34/70 (15°C)
Variant b (15°C)
Varianta d (15°C)
0
0
24
48
72
96
120
144
Fermentation time, h
168
192
216
240
Figure 9. Total higher alcohol production during fermentation at 10°C and 15°C of three lager yeast strains.
Values are means of the duplicates at 10°C and 15°C.
600
500
WS34/70 (10°C)
Variant b (10°C)
Variant a (10°C)
WS34/70 (15°C)
Variant a (15°C)
Taste threshold
Variant b (15°C)
Food Microbiology
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 10°C and 15°C of three lager yeast strains.
Values are means of the duplicates.
35
Romanian Journal of Food Science – 2011, 1(1): 26–38
Irina BOLAT, Maria TURTOI and Michael C. WALSH
Moreover, at 10°C, the precursor of diacetyl (αacetolactate) 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 15°C. 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 10°C and 15°C. 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 10°C
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
various enzymes (due to the increase in the
intracellular level of NADH + H+ and other
substances), stimulate the formation of higher
alcohols and esters, while low temperatures result in
less intense degradation of the substrate, reduced
yeast growth and by-products formation.
Yeast strain may also influence the temperature
effect, as well as other factors like wort composition
and the scale of fermentation.
The yeast strain itself is a major contributor to the
flavour character of beer and many suitable strains
are available to the brewer, while some strains of
yeast are unacceptable for brewing because of the
poor balance of flavour compounds produced.
(Priest and Stewart, 2006).
The concentration of the most important esters
(ethyl acetate and isoamyl acetate) is influenced by
the fermentation method.
High-gravity fermentation and high temperatures
produce beers with an estery character. Less estery
beers are obtained with fermentations carried out
under pressure (Piendl and Geiger, 1980).
As WS34/70 population was proved to be a
mixture of very closely related strains, differences
among the control and the two variants in terms of
fermentation speed when using low temperature
could not be seen. At high temperature, a small
difference was noticed with the highest fermentation
performance assigned to variant a.
Food Microbiology
700
2,3-pentanedione, ppb .
600
WS34/70 (10°C)
Variant a (10°C)
Variant b (10°C)
WS34/70 (15°C)
Variant a (15°C)
Variant b (15°C)
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 10°C and 15°C of three lager yeast strains.
Values are means of duplicates.
36
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Temperature response of new lager brewing strains isolated from WS34/70
An increase in fermentation temperature has been
found to lower the level of acetaldehyde in beer.
Nevertheless, at the end of 8 days of fermentation,
the amount of acetaldehyde in beers produced at
15°C was slightly higher than at 10°C.
Both control sample and variant a started to
reduce acetaldehyde faster than variant b at 10°C, as
well as at 15°C.
Regarding the esters formation, different esters
show different temperature dependencies and the
balance between various esters is altered when the
fermentation temperature is varied (Engang and
Aubert, 1977). The esters possess a pleasant fruity
flavour by themselves; however, increased amounts
of ethyl acetate in beer result in a bitterness
associated with hop character, while additional
isoamylacetate causes a penetrating fruity ester
flavour.
An increase in temperature favours the
production of isoamyl acetate and ethyl acetate. The
isoamyl acetate produced at 15°C was 35–50%
higher than at 10°C and the ethyl acetate amount
was 14–20% higher than at 10°C. After 8 days of
fermentation at 10°C the beer produced with variant
b had the highest level of isoamyl acetate. All the
beers at 15°C displayed comparable amounts of
isoamyl acetate at the end of fermentation
In terms of total higher alcohols their
concentration increased with temperature in all three
cases. Variant b produced the lowest amount of total
higher alcohol at 10°C and variant a at 15°C, while
the control strain produced the highest amount of
total higher alcohols both at 10°C and 15°C. The
differences in concentration at 15°C were not
significant.
As expected, higher concentrations of diacetyl
were produced at 15°C than at 10°C. The most
significant difference was noticed for variant b,
which produced during the fermentation at 15°C
more than double the amount synthesised at 10°C.
Both at 10°C and 15°C, variant a was the fastest
in reducing the diacetyl, reaching 136 µg/L and
140 µg/L respectively, after 6 days of fermentation.
Acknowledgments
The authors wish to thank Heineken Supply Chain
for their support of this work.
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GC/ECD – Gas chromatography / electron capture
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GC/FID – Gas chromatography / flame ionization
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