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Physiological State of Reused Brewing Yeast

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In brewing, yeast may be reused many times. A number of yeast repitchings differs significantly among the breweries. Adjusting the number of times a strain may be serially repitched is of great importance for quality and consistency of final products. The fermentative and physiological characteristics of the yeast culture used in successive laboratory scale fermentations were determined. Yeast physiological state was assessed by the measurement of the levels of intracellular carbohydrates. In our investigation there were not any detectable changes in yeast capacity to ferment. No significant variation in the production of flavour compounds was found either. However, intracellular glycogen and trehalose contents were dependant on the yeast strain, generation number and wort gravity. Nevertheless, an alteration in the yeast physiological condition during serial repitchings occurred in a different mode than in previous studies confirming that the impact of serial repitchings is strain and medium dependent to a large extent.
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Vol. 31, 2013, No. 3: 264–269 Czech J. Food Sci.
Physiological State of Reused Brewing Yeast
E KORDIALIK-BOGACKA and A DIOWKSZ
Institute of Fermentation Technology and Microbiology, Faculty of Biotechnology
and Food Sciences, Lodz University of Technology, Lodz, Poland
Abstract
K-B E., D A. (2013): Physiological state of reused brewing yeast. Czech J.
Food Sci., 31: 264–269.
In brewing, yeast may be reused many times. A number of yeast repitchings differs significantly among the breweries.
Adjusting the number of times a strain may be serially repitched is of great importance for quality and consistency
of final products. The fermentative and physiological characteristics of the yeast culture used in successive labora-
tory scale fermentations were determined. Yeast physiological state was assessed by the measurement of the levels of
intracellular carbohydrates. In our investigation there were not any detectable changes in yeast capacity to ferment.
No significant variation in the production of flavour compounds was found either. However, intracellular glycogen
and trehalose contents were dependant on the yeast strain, generation number and wort gravity. Nevertheless, an
alteration in the yeast physiological condition during serial repitchings occurred in a different mode than in previous
studies confirming that the impact of serial repitchings is strain and medium dependent to a large extent.
Keywords: flavour compounds; glycogen; serial repitching; trehalose; yeast
On the completion of brewery fermentation yeast
is harvested from the fermentation vessel and after
a short period of time reinoculated into a fresh
wort batch. A number of times yeast can be reused
depends on a variety of factors, but mainly on the
individual strain, quality of the cropped yeast,
original wort gravity and company policy. There
is a big variation in a number of yeast repitchings
among the breweries. In some breweries a lager
brewing yeast culture is used 2–3 times while in
others even 7–9 times for fermentation of wort at
similar original gravity (O’C-C 1997).
It has also been reported that lager yeast can be
reused even up to 20 times (P et al. 2003;
S 2009).
Fermentation performance is affected by exter-
nal factors, such as wort gravity, wort oxygena-
tion and clarity, pitching rate and temperature.
The increased osmotic and hydrostatic pressure,
elevated alcohol concentration and modified nu-
trient balance have a profound influence on yeast
performance. And such conditions are often met
in modern brewing. With increasing wort gravity
the number of yeast generations (cycles) that can
be employed is reduced.
During the course of serial repitching yeast physi-
ological condition may be deteriorated and micro-
bial contamination can occur. Yeast physiological
state prior to pitching determines the consistency
of fermentation and product quality. Physiological
condition can be assessed by the determination
of specific yeast cell components important for
fermentation activity (A & O’C-C
1996). Two major intracellular storage carbohy-
drates – trehalose and glycogen ‒ are commonly
used physiological marker substances.
Trehalose protects the cell against stress induced
by osmotic pressure, ethanol, high and low tempera-
ture and desiccation (O et al. 1993; S
et al. 2006). It is an important stress indicator in
brewing yeast cultures. It also plays a role during
the initiation of the cell cycle, as it quickly supplies
a carbohydrate and serves as an energy source.
Glycogen is the major reserve energy storage
material in yeast cells. During the first 6–8 h of
wort fermentation there is a rapid utilisation of in-
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Czech J. Food Sci. Vol. 31, 2013, No. 3: 264–269
tracellular glycogen, which is directly proportional
to the synthesis of lipids and sterols, used by the
cells to produce membranes during cell division
(S  2009). Once nutrients are consumed
and cell division terminates, glycogen accumu-
lates (Q & T 1982; S 2009). It is
important that maximum levels of intracellular
glycogen are present in the yeast culture when it
is harvested for storage, prior to being repitched
into a subsequent wort fermentation. Depleted
glycogen levels lead to incomplete fermentation.
The good yeast quality is a requisite to obtain
consistent beer with correct flavour balance. A
wide range of esters and higher alcohols which are
produced as a result of yeast metabolic activity
contribute to beer flavour. They are considered
positive flavour attributes of beer, although their too
high concentration can impart an unpleasant aroma.
In this study the effect of serial repitching on
yeast physiological condition was examined.
MATERIAL AND METHODS
Yeast strains. Two bottom-fermenting yeast
strains of Saccharomyces pastorianus designated
308 and B4 (LOCK 0100 and 0075) were used.
Yeast was grown in 10°Plato wort for 48 h at 25°C
on an orbital shaker at 350 rpm. The cells were
harvested by centrifugation (2500 g, 5min) and
pitched into 500 ml cylinders containing 400 ml
of hopped malt wort at original gravity of 10°Plato
or 15°Plato collected from a commercial brewery.
The pitching rate was 2 × 107cells/ml. This initiated
the first fermentation cycle.
Serial repitching. Yeast strains were serially
repitched by reusing a proportion of biomass
removed at the end of each fermentation. Each
time yeast was pitched into fresh wort to a final
concentration 2 × 107cells/ml. Fermentations were
carried out at 9°C for 9 days (10°P wort) or 14 days
(15°P wort). All fermentations were performed at
the same conditions using the same wort. For each
strain ten successive fermentations were carried out
with 10°P wort and eight ones with 15°P wort. Each
fermentation was conducted in three replicates.
Beer was then centrifuged to remove yeast (2500g,
5 min, 9°C). One portion of yeast biomass was
used for inoculating the next batch of fresh wort
and the other, after chosen fermentations, for the
determination of yeast viability with methylene
blue as well as glycogen and trehalose content.
Fermentation performance of yeast slurries was
also assessed in terms of beer attenuation and
flavour profile of produced beer.
Glycogen and trehalose determination. Physio-
logical conditions of two yeast strains after propagation
and upon completion of successive fermentations
were studied. It was done by determining the content
of glycogen and trehalose in yeast cells. For the
measurement of glycogen and trehalose content
the method described by J et al. (2003)
was applied.
Volatile ester and higher alcohol determination.
After successive fermentations beer flavour was
evaluated using Agilent Technologies 6890N gas
chromatograph (Agilent Technologies, Inc., Santa
Clara, USA), equipped with a flame ionisation
detector (FID). Higher alcohols (propanol, 2-methyl-
1-propanol, 2-methyl-1-butanol, 3-methyl-1-bu-
tanol) and esters (ethyl acetate, ethyl butyrate,
isoamyl acetate, ethyl caproate and ethyl caprylate)
were determined (Institute of Brewing 1997).
Statistical analysis. Statistical significance of
the data was determined by Student’s t-test. A
P-value below 0.05 was considered statistically
significant.
RESULTS AND DISCUSSION
In brewing yeast may be reused for fermenta-
tion several times. However, after a certain time
deterioration of cropped yeast is observed and a
newly propagated yeast culture has to be applied.
Lower yeast slurry quality is directly related to
poorer fermentation performance and consequently
to lower quality of final beer. Alterations in fer-
mentation performance can already be assessed by
the measurement of attenuation, which provides
information on the extent to which wort sugars
can be used. It describes yeast ability to reach a
certain attenuation limit.
Yeast fermentation characteristics were evalu-
ated in ten successive fermentation cycles of
10°P wort and eight cycles of 15°P wort. It was
observed that attenuation profiles did not differ
significantly in successive fermentations. In the
case of fermentation of 15°P wort the final gravity
amounted to 3.5 ± 0.2°P (strain 308) and 5.1 ±
0.2°P (strain B4). When 10°P wort was fermented,
the final gravity was 3.0 ± 0.1°P (strain 308) or
4.0 ± 0.2°P (strain B4). Thus the yeast ability to
utilise sugars remained stable over time.
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The viability of yeast cells after successive fer-
mentations did not change significantly either. The
amount of viable cells was always higher than 98%.
Among many parameters cited in the literature to
assess the yeast physiological state, the measurement
of intracellular glycogen and trehalose content was
chosen in this study.
Glycogen content in repitched yeast
To examine the relative quality of slurries for each
strain, samples were collected after propagation im-
mediately before pitching into the first fermentation.
During propagation in the presence of oxygen
glycogen is utilised to synthesise sterols and lipids
for the membrane production. It is unusual that
this phenomenon was not observed for strain B4
(Figure 1). Glycogen content in yeast strain 308
after propagation was much lower than after fer-
mentation processes.
During serial repitching of strain 308 used for
fermentation of 10°P wort the glycogen level after
initial fermentation processes was lower than after
final ones (Figure 1a). There were not any differ-
ences in glycogen content among generations 5, 8,
and 9. However, the statistically significant decline
in glycogen content was observed for generation10
(P < 0.01), which can suggest deterioration of the
yeast physiological state.
There were not any statistically significant changes
in glycogen content during serial repitching of strain
B4 until generation 10. Glycogen content in yeast
collected upon completion of fermentation 10 was
significantly lower than in cropped yeast sample 5
(P < 0.05), when glycogen content was the highest.
In the case of fermentation of 15°P wort the low-
est glycogen content was in yeast cropped from
the last performed fermentation 8 for both strains
(Figure 1b). For strain B4 there was a statistically
significant change in glycogen level starting from
yeast sample 7 (P < 0.001). Earlier there were not
any statistically significant differences in glycogen
content after fermentations 1, 2 and 6. In analysed
samples for strain 308 the glycogen level was similar
until generation 8.
0
1
2
3
4
5
6
Prop. 1 2 3 5 8 9 10
Fermentation
Glycogen (% yeast DW)
308 B4
Figure 1. Glycogen content in yeast cells of strains 308 and B4 after propagation and successive fermentation of 10°P
(a) and 15°P (b) wort
0
1
2
3
4
5
Prop. 1 2 5 6 7 8
Fermentation
Glycogen (% yeast DW)
308 B4
(a) (b)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Prop. 1 2 5 6 7 8
Fermentation
Trehalose (% yeast DW)
308 B4
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Prop. 1 2 3 5 8 9 10
Fermentation
Trehalose (% yeast DW)
308 B4
Figure 2. Trehalose content in yeast cells of strains 308 and B4 after propagation and successive fermentation of 15°P
(a) and 10°P (b) wort
(a) (b)
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Czech J. Food Sci. Vol. 31, 2013, No. 3: 264–269
Trehalose content in repitched yeast
During serial repitching when 15°P wort was
fermented, a decline in trehalose content was
observed in strain 308 after initial fermentation
processes (Figure 2a). Trehalose content increased
in the last generations. All these changes were
statistically significant at P < 0.05. The accumula-
tion of trehalose in repeatedly used yeast was also
seen for strain B4, but not all changes were statis-
tically significant. The accumulation of trehalose
with increased generation number is in line with
observations of J et al. (2003). However,
the initial decrease in the first generations was
not previously reported.
The accumulation of trehalose in the last genera-
tions of both strains was also observed when 10°P
wort was fermented (Figure 2b). The trehalose
level remained invariable in the first generations
of strain B4. Similar data were not obtained in the
case of strain 308.
For strain 308 the trehalose level in cropped
slurries was much higher than in the propagation
sample (Figures 2). This tendency was not found
for strain B4.
Effect of serial repitching on yeast
physiological condition
The impact of yeast exposure to repeated cy-
cles of stress during its employment in successive
fermentations on slurry quality was previously
examined both for lager yeast (J et al. 2003;
K et al. 2007) and ale yeast (S &
W 1996; P & D 2007). In
these investigations the effect of serial repitching
on yeast flocculation and surface characteristics,
viability, membrane integrity, acidification power
test results, intracellular carbohydrate and isoa-
myl alcohol concentration was analysed. Despite
a lot of studies related to this issue, the influence
of extended reusing of the same yeast on yeast
quality, including physiological state, has not been
well elucidated.
In J et al. (2003) study the evaluation of
glycogen and trehalose content was conducted on
seven sequentially harvested yeast samples. The
progressive accumulation of trehalose in successive
yeast generations and the constant level of glycogen
starting from the 4th generation were observed. In
our investigation an increase in trehalose content
0
2
4
6
8
10
12
14
16
1
Fermentation
2 3 4 5678
Esters (mg/l)
Ethyl acetate (308) Isoamyl acetate (308) Total esters (308)
Ethyl acetate (B4) Isoamyl acetate (B4) Total esters (B4)
Figure 3. Effect of yeast generation number on ester content in green beer produced from 15°P (a) and 10°P (b) wort with
strain 308 and B4
0
1
2
3
4
5
6
7
8
1
Fermentation
2 3 4 5 6 7 8 9 10
Esters (mg/l)
(a)
(b)
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and a simultaneous decline in glycogen content in
aged culture were found out. However, a decline
in the glycogen level was observed only for the old
culture of the last evaluated generations. J
et al. (2003) did not report the original gravity of
wort used in their study and other fermentation
conditions and therefore a comparison of the results
has to be conducted with caution.
The number of serial repitchings which did not
cause any change in intracellular carbohydrate
content depended on original wort gravity to a
large extent. A drop in the glycogen level during
fermentation of 15°P wort was observed earlier
than during fermentation of 10°P wort. It was
connected with higher osmotic stress and ethanol
concentration generated when higher gravity wort
was fermented. It implies a faster impairment of
the yeast physiological state during fermentation
of higher gravity wort.
K et al. (2007) also characterised the
physiological state of recycled lager yeast by de-
termination of membrane potential and isoamyl
alcohol secretion. However, in their work yeast
was not used to ferment normal brewery wort
but yeast extract-dextrose medium, which only
imitated industrial low-malt wort. They observed
the most distinct changes in the growth rate and
isoamyl alcohol production and stated that yeast
harvested after the fourth fermentation should not
be reused any longer. In our study no significant
variation in the flavour of beer produced with suc-
cessive yeast generations was noticed (Figures3
and 4). In turn, a change in glycogen and trehalose
content in yeast was found out. But the decline in
glycogen content was not seen sooner than in the
7th generation. In yeast used for fermentation of
10°P wort, alterations occurred even in the ninth
generation. Yeast employed in our study was much
more resistant to repeated exposure to stress.
However, in our investigation apart from different
medium, different fermentation temperature was
also applied. Fermentations were performed at 9°C
instead of at 15°C as it was in K et al.
(2007) work. And the temperature of fermentation
as well as fermentation medium have a consider-
able influence on yeast quality and production of
flavour compounds.
Q et al. (2003) repitched the same yeast
seven times in succession and noted that aged
yeast cultures produced higher quantities of esters
such as ethyl acetate and isoamyl acetate. With
each repitching the amount of isoamyl acetate
increased steadily until the 5th repitching, after
which it began to fall. In our investigation the
initial increase in isoamyl acetate secretion was
not observed (Figure 3). But after the seventh re-
pitching a slight decrease was observed for strain
308 during fermentation of 10°P wort and after
0
10
20
30
40
50
60
70
80
1
Fermentation
2 3 4 5 6 7 8
Higher alcohols (mg/l)
Isoamyl alcohol (308) Total higher alcohols (308) Ratio ‘higher alcohols/esters’ (308)
Isoamyl alcohol (B4) Total higher alcohols (B4) Ratio ‘higher alcohols/esters’ (B4)
Figure 4. Effect of yeast generation number on higher alcohol content in green beer produced from 15°P (a) and 10°P
(b) wort with strain 308 and B4
0
10
20
30
40
50
60
1
Fermentation
2 3 4 5 6 7 8 9 10
Higher alcohols (mg/l)
(a)
(b)
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Czech J. Food Sci. Vol. 31, 2013, No. 3: 264–269
the fifth for 15°P wort. Ethyl acetate synthesis was
not a generation number dependent. In general,
there was not any tendency for the level of esters
to rise with the yeast generation number. Both
Q et al. (2003) and K et al. (2007)
demonstrated that the number of serial repitch-
ings might affect the secretion of higher alcohols
by yeast. Q et al. (2003) found out that
the amount of amyl alcohol increased steadily up
with each repitching until the 5th repitching, after
which it began to fall. These phenomena were not
observed in our research. Multiple yeast reusing
did not affect the total amount of higher alcohols
(Figure 4). Furthermore, no difference in the ‘higher
alcohols to esters’ ratio was noticed either. The
yeast ability to produce a correct ratio of flavour
compounds remained stable.
Breweries have to carefully consider how many
fermentations to pitch with the same yeast cultures.
Since the yeast physiological condition and conse-
quently beer quality depend on many factors, such
as strain, wort gravity, yeast handling procedure, it
is difficult to settle in general how many times to
reuse yeast in the brewery. In particular, because
researches are conducted in different conditions
and contradictory findings are often reported.
This study emphasised that there is no reason
for an extreme reduction of the number of yeast
repitchings without controlling the individual
strain susceptibility to extended reusing.
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Received for publication February 28, 2012
Accepted after corrections September 4, 2013
Corresponding author:
Dr E K-B, Lodz University of Technology, Institute of Fermentation Technology
and Microbiology, 171/173 Wolczanska Street, 90-924 Lodz, Poland; E-mail: edyta.kordialik-bogacka@p.lodz.pl
... In some breweries, a bottom brewing yeast culture is used 2-3 times, while in others, 7-9 fermentations of wort at similar original gravity are used. Lager yeast can even be reused up to 20 times (Powell et al. 2003;Kordialik-Bogacka & Diowksz 2013). ...
... In an earlier laboratory-scale study, Kordialik-Bogacka and Diowksz (2013) showed that both the multiplicity of the yeast usage and the successive accumulation of trehalose increased yeast viability. A similar tendency of change in the viability of yeast after successive uses was presented in a study by Stewart (2015). ...
... Research conducted by Kordialik-Bogacka and Diowksz (2013) showed that during repitching, the yeast strain was the most important factor that influenced the content of esters. One of the investigated strains produced constant amounts of ethyl acetate and isoamyl acetate. ...
Article
The aim of the study was to determine the effect of yeast generations on fermentation and maturation processes, the content of volatile compounds of beer and viability and vitality of yeast biomass on an industrial scale. The experiments with fermentation and maturation were performed in fermentation tanks. The wort was aerated with sterile air. Yeast (S. pastorianus) bottom fermentation was used in fermentation. For pitching four generations (passages) of yeast were used as follows: 1st, 2nd, 3rd and 4th generation. The processes of fermentation and maturation were carried out in the same technological conditions (temperature and pressure). During fermentation and maturation, the changes in the content of the extract, yeast growth and vitality and selected volatile compounds like esters, alcohols and carbonyl compounds were investigated. With the increase in the number of yeast generations, especially from the 2nd generation used in the fermentation process, the content of acetaldehyde and esters increased. Despite the slight differences between generations, the changes are statistically significant. The content of diacetyl is stable for the 1st, 2nd and 3rd generation and higher for the 4th generation. Diversified yeast generations used in the process of fermentation did not affect significantly the final quality of beer.
... The number of yeast re-pitching varies among breweries. Some breweries use a lager yeast culture up to 20 times of the same fermentation conditions and wort gravity (Stewart 2009;Kordialik-Bogacka and Diowksz 2013;Bühligen et al. 2013). Yeast cultures in typical brewery fermentation divide nearly two to three times times (Powell et al. 2003a). ...
... The effect of yeast exposure to repeated cycles of stress during fermentations on yeast slurry quality has been reported both for bottom-fermenting yeast (Kobayashi et al. 2007;Gabriel et al. 2008;Verbelen et al. 2009b;Kordialik-Bogacka and Diowksz 2013;Bühligen et al. 2013) and topfermenting yeast (Kobi et al. 2004;Powell and Diacetis 2007). Several but contrary results have been reported. ...
... Effect of re-pitching on yeast physiological state Serial re-pitching subjects a yeast culture to recurrent stress that may initiate reversible or irreversible injury, depending on the strain (Layfield and Sheppard 2015). Levated osmotic pressure, increased alcohol concentration, and modified nutrient balance may distort yeast metabolism (Pratt et al. 2003;Kordialik-Bogacka and Diowksz 2013). ...
Article
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Background Serial re-pitching is a term given to a practice whereby yeast harvested at the end of fermentation is re-used in subsequent fermentations. Purpose The purpose of this paper was to review and summarize existing literatures, research data, and case studies to illustrate the effect of re-pitching on the physiology and fermentation performance of brewing yeast and the resulting quality of beer. Methods Data related to biomarkers used to assess yeast physiology and fermentation performance and quality of beer were compared for various articles. Results And comparison of the results was done with caution as many of the studies were conducted using different yeast strains, wort gravity, pitching rate, and other fermentation conditions. Conclusion This study confirms that serial re-pitchings aggravate the effect of pitching rate, wort gravity, cell age, yeast oxygenation, and yeast strain on yeast cell physiology, fermentation performance, and quality of final beer. However, further empirical research at molecular level is crucial.
... While the negative effects of serial re-pitchings have been reported by researchers (e.g., petite generation and flocculation mutations), others have indicated little change in lager yeast serially re-pitched up to 135 times. [2][3][4][5] Also, it was noted that extents of deterioration can vary between yeast strains. [6] Where such introductions are necessary, they generally happen after approximately ten fermentation cycles. ...
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A novel high-density yeast propagation system has been developed, which produced yeast that performed as well as cropped yeast in commercial brewery trials. This process is capable of producing yeast concentrations 13 times greater than traditional yeast propagation approaches used in breweries to date. The system is based on a controlled fed-batch yeast fermentation, which can produce pitching yeast in as little as 24 h. A demonstrator plant was installed in a regional brewery and yeast produced from the test-platform was used to pitch commercial brews. Plant-scale trials have shown that the yeast propagated using this new system had the same fermentation profile compared to control fermentations that used cropped yeast. Volatile analysis showed no significant difference between the control and experimental beers. The experimental beers tasted true-to-type and were released to trade. The new process allows for smaller pitching volumes while maintaining overall beer quality. (PDF) Fed-Batch System for Propagation of Brewer’s Yeast. Available from: https://www.researchgate.net/publication/353366924_Fed-Batch_System_for_Propagation_of_Brewer%27s_Yeast [accessed Jul 21 2021].
... Many brewery operations reuse yeast harvested from fermentation vessels several times in subsequent batches, sometimes as many as 20 times(Kordialik-Bogacka and Diowksz 2013). Therefore, the health of yeast cells after fermentation is of great importance to brewers. ...
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High gravity (HG) brewing has become the most used strategy for maximizing fermenter productivity in commercial brewing. While HG brewing has many benefits, the additional stress placed on the yeast due to the higher concentration of fermentable sugars in the wort can negatively impact fermentation performance and flavor compound formation. A proper dissolved oxygen (DO) level is vital to guarantee adequate yeast performance during HG fermentations. Dissolved oxygen is vital to yeast viability throughout the fermentation process, as yeast requires oxygen to synthesize vital cell membrane components needed for continued anaerobic growth and cell division. Previous research has demonstrated the importance of DO in wort for regular gravity fermentation and flavor compound production. However, the impact of dissolved oxygen during HG brewing on fermentation performance and how this will impact the production of flavor compounds have not been fully researched. The objectives of this research were to analyze the impact of wort aeration timing and concentration on fermentation performance, flavor stability, and the formation of volatile flavor compounds, determined using gas chromatography. Gas chromatography analysis was modeled after the ASBC Method Beer-48. Flavor stability and staling was analyzed during aging under normal and accelerated conditions utilizing TBA analysis. Pre-pitch oxygen treatments at levels greater than 8 ppm dissolved oxygen significantly increased attenuation when compared to the unoxygenated controls. Post-pitch oxygenation significantly increased attenuation, with DO treatments at levels of 8 ppm showed the most significant decrease in wort specific gravity. Aldehyde, ester, and higher alcohol production were all significantly affected by DO concentration. Aldehyde production decreased with increased DO concentration. Ester production increased from 0 to 8 ppm DO treatment and decreased at DO treatments greater than 8 ppm. Higher alcohol production increased from 0 to 10 ppm and decreased with DO treatments greater than 10 ppm. Greater concentrations v of DO resulted in greater TBA index values after normal and accelerated aging, with accelerated aging producing greater TBA index values than normal aging.
... In terms of yeast volatile producing compounds (higher alcohols and esters), Verbelen et al., 2009b andKordialik-Bogacka andDiowksz, 2013 showed that their formation by S. pastorianus strains was not influenced until 10 serial repitching, while Sigler et al., 2009 observed slight variations, but only repitched S. pastorianus strain 3 times. On the other hand, Deželak et al., 2015c verified little influence on higher alcohols formation (2-and 3methylbutanol, 2-phenylethanol, 1-propanol, and isobutanol) during 11 serial repitchings of S. pastorianus, but higher rates of esters formation (ethyl acetate, isoamyl acetate, and 2phenylethyl acetate) from 1 st to 4 th repitching. ...
Chapter
Beer is the second most consumed beverage in the world, accounting ca. 35% of all recorded alcohol consumed in 2010. Brewing industry is extremely competitive, highlighting the importance of constant innovation. The success of global beer commercialization is its intrinsic quality that depends of a network of variables, namely raw materials, yeast strain and the brewing process itself. The biochemical performance of yeasts, usually Saccharomyces spp., is one of the parameters with significant importance in brewing once it limits the alcoholic content of final beer and produces different metabolites with crucial impact on beer aroma and flavor. It will be given focus to the different metabolic pathways with significant impact on beer’s aroma profile, namely the carbohydrates and nitrogen compounds metabolism; and also the formation of aldehydes, higher alcohols, vicinal diketones, esters, fatty acids, sulfur and terpenic compounds. Environmental changes can promote stress in yeasts along brewing. The stress factors will be systematized in this book chapter, as well as the yeasts cellular effects and their biological response. Furthermore, brewing companies tend to reused (or repitch) yeasts several times to minimize resources costs, while maintaining quality of the final product. Also, different approaches will be described in order to monitor yeast quality. Therefore, the impact of yeasts repitching along brewing will be systematized, considering its associated advantages and drawbacks. In summary, this book chapter aims to elucidate about the raw materials and the brewing process, and also to give an overview of the Saccharomyces spp. metabolism (special attention will be done on its impact on beer aroma profile), as well as to understand the effects of serial repitching on yeasts’ behavior.
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Individual cells of the yeast Saccharomyces cerevisiae exhibit a finite replicative lifespan, which is widely believed to be a function of the number of divisions undertaken. As a consequence of ageing, yeast cells undergo constant modifications in terms of physiology, morphology and gene expression. Such characteristics play an important role in the performance of yeast during alcoholic beverage production, influencing sugar uptake, alcohol and flavour production and also the flocculation properties of the yeast strain. However, although yeast fermentation performance is strongly influenced by the condition of the yeast culture employed, until recently cell age has not been considered to be important to the process. In order to ascertain the effect of replicative cell age on fermentation performance, age synchronised populations of a lager strain were prepared using sedimentation through sucrose gradients. Each age fraction was analysed for the ability to utilise fermentable sugars and the capacity to flocculate. In addition cell wall properties associated with flocculation were determined for cells within each age fraction. Aged cells were observed to ferment more efficiently and at a higher rate than mixed aged or virgin cell cultures. Additionally, the flocculation potential and cell surface hydrophobicity of cells was observed to increase in conjunction with cell age. The mechanism of ageing and senescence in brewing yeast is a complex process, however here we demonstrate the impact of yeast cell ageing on fermentation performance.
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The term serial repitching is given to the harvesting of the yeast biomass upon completion of fermentation and its use in subsequent fermentations. Although it is generally accepted that serial repitching causes deterioration in yeast quality, the impact of repeated exposure to yeast handling stress on the physiological state has not been previously reported. In this study, lager yeast viability and vitality was examined as a function of generation number. It was observed that the impact of serial repitching on the physiological state was not universal but a strain-dependant phenomenon. Repeated exposure to stress resulted in the accumulation of both reversible and irreversible damage. In addition, evidence for the activation of the global stress (stress response element [STRE]) response during brewing yeast handling is presented.
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On completion of a brewery fermentation, the yeast crop is removed from green beer, stored, and repitched. The effect of serial repitching on yeast inoculum condition and its subsequent fermentation performance is important but not well characterized. The physical properties of the brewing yeast cell surface influence flocculation and may be used to ascertain the physiological state of the cell. Cropped yeast from production and laboratory-scale fermentations have been examined. The effect of serial repitching on the physical properties of a production ale strain was monitored using dye retention, latex microsphere attachment, electron microscopy, and fluorescent dye techniques. The flocculation of each generation was determined. It was observed that a correlation between cell surface condition and fermentation performance existed. It is suggested that cell surface condition may be used to predict the fermentation performance of the subsequent pitch.
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Isoamyl acetate is a natural flavour ester, widely used as a source of banana flavour by the food industry. Fusel alcohols such as amyl alcohol are produced in significant quantities as a waste product, sometimes referred to as “lees oil” or “fusel oil”, of the alcohol distilling industry. By manipulation of brewing yeast fermentation conditions, a significant portion of added amyl alcohol was shown to be converted to isoamyl acetate. This was achieved by the addition of L-leucine and amyl alcohol in fermentations carried out by a high ester-producing brewing yeast strain of Saccharomyces cerevisiae and by the use of alkaline fermentation conditions coupled with high gravity media. Mutant strains selected on 5,5,5 trifluoro-DL-leucine produced substantially high levels of isoamyl acetate. The adjustment of fermentation conditions outlined in this paper may act as a stepping stone for the potential use of Saccharomyces cerevisiae and other yeasts to produce high levels of natural flavour esters.
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J. Inst. Brew. 115(1), 3–29, 2009 Horace Brown spent fifty years conducting brewing research in Burton-on-Trent, Dublin and London. His contributions were remarkable and his focus was to solve practical brewing prob-lems by employing and developing fundamental scientific prin-ciples. He studied all aspects of the brewing process including raw materials, wort preparation, fermentation, yeast and beer stability. As a number of previous presenters of the Horace Brown Lecture have discussed Brown's achievements in detail, the focus of this paper is a review of the brewing research that has been conducted by the author and his colleagues during the past forty years. Similar to Horace Brown, fundamental research has been employed to solve brewing problems. Research studies that are discussed in this review paper include reasons for pre-mature flocculation of ale strains resulting in wort underattenua-tion including mechanisms of co-flocculation and pure strain flocculation, storage procedures for yeast cultures prior to prop-agation, studies on the genetic manipulation of brewer's yeast strains with an emphasis on the FLO1 gene, spheroplast fusion and the respiratory deficient (petite) mutation, the uptake and metabolism of wort sugars and amino acids, the influence of wort density on fermentation characteristics and beer flavour and stability, and finally, the contribution that high gravity brewing has on brewing capacity, fermentation efficiency and beer quality and stability.
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J. Inst. Brew. 112(2), 134–147, 2006 This study focused on the implementation of fluorescence opti-cal methods and laser scanning confocal microscopy for moni-toring brewing yeast performance. Physiological parameters and cell compounds in yeast cells (glycogen, neutral lipids, treha-lose, bud scars, DNA and intracellular proteinases) have been successfully visualised with the aid of highly specific fluoro-chromes. The expression and sub cellular localisation of pro-teinase A during fermentation has been studied employing a Saccharomyces cerevisiae green fluorescent protein clone. This novel approach to monitoring brewing yeast performance pro-vides new insights into physiological events that occur during wort fermentation.
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J. Inst. Brew. 113(1), 67–74, 2007 Two brewing yeast strains, one employed for the production of an ale type product and the other used solely for bottle condi-tioning, were serially repitched over a period of one year. Subse-quently each yeast culture was compared to the original stocks for a variety of phenotypic and genotypic characteristics. Fer-mentation performance was assessed in terms of flocculation capacity and the time required to achieve attenuation in 150 barrel fermentation vessels, while the propensity for the popu-lation to accumulate variants was assessed by analysing giant colony morphology. Changes to the genome were monitored by DNA fingerprinting of each yeast culture using RAPD-PCR and RFLP. Although some colony morphology variation was ob-served between fresh and old ale yeast cultures, there were no detectable genetic changes or alterations in fermentation charac-teristics to either yeast strain over the course of serial repitching. It is suggested that although some brewing yeast strains are sus-ceptible to genetic drift, others are more resilient and can remain stable over extended periods of time. The propensity to produce variants may therefore play a significant role in determining the number of times a strain may be serially repitched, or its suit-ability for beer fermentations.
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The effects of heat and ethanol shock on fatty acid composition and intracellular trehalose concentration of lager and ale brewing yeasts were examined. Exposure of cells to heat shock at 37C or 10% (v/v) ethanol for 60 min resulted in a significant increase in the ratio of the total unsaturated to saturated fatty acyl residues and the intracellular trehalose concentration of cells. A similar increase in the amount of unsaturated fatty acids was observed in cells after 24 h of fermentation of 16P (degree Plato) or 25P wort, at which time more than 2% (v/v) ethanol was present in the growth medium. These results suggest that unsaturated fatty acids and high concentrations of intracellular trehalose may protect the cells from the inhibitory effects of heat and ethanol shock.