Review article Institute of Brewing & Distilling

Received: 14 April 2014 Accepted: 9 May 2014 Published online in Wiley Online Library: 10 June 2014

(wileyonlinelibrary.com) DOI 10.1002/jib.145

Wort composition and its impact on the

flavour-active higher alcohol and ester
formation of beer – a review
Yang He, Jianjun Dong, Hua Yin, Yuxiang Zhao, Rong Chen, Xiujuan Wan,
Peng Chen, Xiaoping Hou, Jia Liu and Lu Chen*
The most significant factors influencing beer quality are the variety of aroma flavours that stem from a complex system of
interactions between many hundreds of compounds. With increasing demand for flavour control and enhanced productivity,
the presence of consistent and balanced amounts of higher alcohols and esters are critical aspects of process control.
Extensive research has focused on the formation of flavour compounds by the brewing yeast and the factors that influence
their synthesis. Fermenting wort is a complex medium from which the brewing yeast utilizes nutrients for living and growth
and to where it places its metabolic by-products. Thus, changes in wort composition will greatly influence final beer aroma.
The current paper reviews up-to-date knowledge on the contribution of wort composition to the flavour quality of the final
product, in particular higher alcohols and esters. Different wort constituents involved in the biosynthesis of these aromatic
substances, and which therefore require control during brewery fermentations, are reviewed. Copyright © 2014 The Institute
of Brewing & Distilling

Keywords: brewing yeast; wort composition; flavour compounds; higher alcohols; esters; high-gravity brewing

Introduction that are generally characterized by their fruity–flowery aromas

Beer is a complex chemical matrix that is the result of numerous
metabolic and chemical reactions. Various biosynthetic path-
ways in the living character of brewing yeast are interactive
during the fermentation process. An overview of metabolism,
with a focus on the most important metabolites inside the
brewing yeast cell during fermentation, is shown in Fig. 1. The
basic concern of a brewing scientist with industrial yeast strains
is how to make use of the biochemical and genetic properties of
brewing yeasts in order to improve their overall fermentation
performance, such as stress tolerance and ethanol production.
More important for the brewers is the consistent production of
fermentation by-products from batch to batch, in order to
produce beer of satisfactory quality and consistent characteristics,
which is particularly challenging in the case of brewers with
multisite brewing operations.
The quality of beer is mostly judged based on its flavour and
aroma profile arising from a broad spectrum of flavour-active
compounds, whose production is influenced by every process
in the malting and brewing supply chains. The major volatile
products of yeast metabolism are ethanol, glycerol and carbon
dioxide, which make a relatively weak but nonetheless funda-
mental contribution to beer flavour. Important aroma com-
pounds that characterize the quality and palatability of beer,
that is, esters, higher alcohols, sulphur compounds, organic acids
and carbonyl compounds, originate from yeast metabolism dur-
ing primary fermentation. Of all the yeast secondary metabolites
responsible for the principal flavour of finished beer, higher
alcohols and esters are deemed to be a highly significant group
of flavour-active metabolites and their biochemical formation
pathways are essentially known. Esters are flavour compounds
in beer. While volatile esters often only exist in trace amounts
in beer, they can have a synergistic effect with other flavour
compounds (1). Esters are formed intracellularly by an enzyme-
catalysed condensation reaction between an active acyl-
coenzyme A (acyl-CoA) and a higher alcohol (2). Alcohol acetyl
transferases (AATase) are well known, with enzymes responsible
for the formation of acetate esters and the total AATase activity
stemming from the expression of the ATF1 and ATF2 genes,
encoding for AATase I and II, respectively (3,4). Moreover, the
transcriptional activity of the ATF genes in brewer’s yeast is the
limiting parameter for acetate ester synthesis (5). In addition,
EHT1 and EEB1 encode two acyl-CoA ethanol O-acyltransferases
(AEATases) enzymes, which play a central role both in the
synthesis of ethyl esters and in the hydrolysis activity of esters
(6). The hydrolysing esterase designated IAH1 is also important
for the net rate of ester accumulation (7).
Higher alcohols contribute to beer flavour by intensifying
alcoholic perception and imparting a warm mouthfeel. Several
genes are involved in the biosynthesis of these compounds (8).
The biosynthesis of higher alcohols is directly related to amino
acid metabolism via the Ehrlich pathway (9,10). A rate-limiting
step in this process is the uptake of three branched-chain
amino acids by a BAP2-encoded branched-chain amino acid
* Correspondence to: Lu Chen, State Key Laboratory of Biological Fermenta-
tion Engineering of Beer, Tsingtao Brewing Ltd, Qingdao, 266061, People’s
Republic of China. E-mail: chenlu@tsingtao.com.cn
State Key Laboratory of Biological Fermentation Engineering of Beer,
Tsingtao Brewing Ltd, Qingdao, 266061, People’s Republic of China
157

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Institute of Brewing & Distilling
Figure 1. A schematic overview of the main metabolic routes inside a brewing yeast cell.
permease (11). Two branched-chain amino acid aminotransfer-
ases, Bat1 and Bat2, mediate the transfer of amino groups from
branched-chain amino acids to α-keto acids, which are precur-
sors for the synthesis of higher alcohols (12). Unlike higher
alcohols and esters, which are desirable volatile compounds
for a pleasant and drinkable beer, vicinal diketones (VDKs) are
often considered as off-flavours for the majority of beers.
According to Meilgaard (1), VDKs have the aroma of butterscotch,
which arises as an indirect result of yeast metabolism.
All flavour-active components in beer must be controlled
within specific limits. Their relative concentration can confer
beneficial or undesirable flavour traits, and even an insignificant
shift in their concentrations may result in an entirely different
flavour for the beer (13). Therefore, the main purposes of
fermentation management to maintain a high quality beer are:
(a) ensure the concentrations of negative flavour constituents
are less than their threshold levels; and (b) in the meantime,
ensure that the presence of well-adjusted amounts of higher
alcohols and esters, and other compounds, imparts a positive
contribution to the final product (14). Otherwise, the desired
harmonious flavour balance resulting from various tastes and
sensations in beer can be destroyed. For each beer style or
brand, customized descriptions in terms of ingredients, process
and the final features (especially product flavour) are specified
as a reference for the brewers.
The ability to control flavour production of beer during
fermentation is of paramount importance. It has been widely doc-
umented that brewing yeast produces different concentrations of
flavour-active compounds as a function of wort composition
(5,15–18), fermentation conditions (5,18,19) and particularly the
yeast strain employed (20). Individual ester and higher alcohol
profiles produced in a similar medium differ dramatically from
strain to strain, with ale strains generally producing more esters
and higher alcohols than lager strains (21). Fermentation process
variables such as wort gravity, temperature, pressure, oxygen
and pitching rate are all known to influence the levels of
flavour production (13,22–26). Wort is a complex yeast growth
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Y. He et al.
and fermentation medium that consists of carbohydrates (glucose,
fructose, sucrose, maltose, maltotriose and nonfermentable
dextrins), nitrogenous materials (amino acids, peptides, ammonia,
proteins, nucleic acids), vitamins, minerals, inorganic ions, fatty
acids, trace elements and other constituents too numerous to
mention (27). Brewery fermentations require a balanced supply
of nutrients, providing sufficient levels as a growth medium to
achieve adequate yeast growth, while at the same time acting as
a fermentation medium in the production of consistent levels of
ethanol, carbon dioxide and flavour-active secondary by-products.
It is not difficult to comprehend that changes in nutrient
content or fermentation parameters will trigger different yeast
responses, with the responsiveness of the yeast culture to changes
in the wort environment being critical to maintaining product
quality and consistency. Therefore, the control of raw ingredients
and wort preparation, followed by appropriate fermentation
conditions, will ensure beer quality and a stable flavour profile.
In order to obtain more control over flavour production, inten-
sive research has been carried out to elucidate the factors
influencing flavour formation. This review will limit the discus-
sion to the effect of wort composition on the production of
higher alcohols and volatile esters during the course of fermen-
tation. Different wort specific gravity, carbohydrate, nitrogen,
lipid profiles, certain vitamins and inorganic ions can lead to
differences in the yeast’s fermentation performance, which can
be reflected in the levels of esters and higher alcohols produced
(15,17,20,28–32). A better understanding of the impact of certain
wort compounds on the flavour formation may lead to improved
flavour control during a brewery fermentations. The objective of
this review is to enhance our knowledge of the role that wort
composition plays in influencing yeast growth and in achieving
stable product profiles with respect to aroma-active compounds.
Wort sugar profile
Standard brewery wort contains approximately 90% carbohy-
drate, which consists of sucrose, fructose, glucose, maltose and
J. Inst. Brew. 2014; 120: 157–163
Wort composition and impact on higher alcohol and ester formation
maltotriose, together with dextrin material. The wort sugar
profile depends on the raw ingredients (mainly barley malt)
and wort preparation procedures, and also relies on the type
and the quantity of adjuncts employed. When inoculated into
brewer’s wort, yeast utilizes the principal wort sugars in a
sequential order (Fig. 2). Maltose and maltotriose are the most
abundant sugars and will typically only be taken up after deple-
tion of the monosaccharides owing to the carbon catabolite
repression of metabolic pathways involved in the uptake and
utilization of alternative sugars (33). Although brewing fermenta-
tion is a process in which carbohydrates are utilized by yeast for
its proliferation and maintenance, through the energy provided
from the conversion of sugars into ethanol and carbon dioxide,
many more compounds are produced simultaneously during
this process with organoleptic properties, including esters and
higher alcohols.
The carbohydrate composition of wort and the manner in
which it is utilized by yeast has a direct influence on fermenta-
tion efficiency and yeast metabolism, as well as the organoleptic
profile of the final product (34). Previous work has shown that
significant differences in volatile profiles were obtained when
different carbohydrates were fermented individually (15,35).
Unfermentable dextrins, which cannot be metabolized by lager
yeast and most ale yeast strains, are also wort carbohydrates
and therefore remain in the final beer. They contribute to beer
flavour by imparting body, mouthfeel, palate fullness and drink-
ability to the beer (36). Beyond that, certain levels of maltose,
and especially maltotriose, are sometimes left unfermented at
the end of primary fermentation. This lowers the efficiency of
the process and potentially has an impact on beer quality by
influencing the relative production of flavour by-products.
Elevated production of higher alcohols by increasing the
levels of sucrose in the wort was reported by Jenard and
Devreux (37). There were no significant differences in the levels
of esters and higher alcohols produced during the fermentation
when glucose or fructose was metabolized as the sole carbohy-
drate source, when examined using several brewing yeast
strains (ale and lager) (15). However, the metabolism of maltose
results in considerably lower levels of esters and higher alcohols
compared with the easier assimilable glucose and fructose (15).
In addition to different carbohydrates affecting the concentra-
tion of the flavour compounds, different malt–adjunct ratio
worts also have an effect on the formation of a number of
Figure 2. Changes in different wort sugar compositions during generation 0
fermentation with the industrial lager yeast strain TT-21.
J. Inst. Brew. 2014; 120: 157–163 Copyright © 2014 The Institute of Brewing & Distilling
Institute of Brewing & Distilling
flavour components. Different flavour compounds are affected
in diverse ways when part of the wort is replaced by a carbohy-
drate solution, with fructose generally resulting in an increased
production of higher alcohols compared with other sugars,
except for n-propanol (35). This implies that, when beers are
produced with the addition of syrup, one should take the sugar
constituents of the syrup into consideration in order to keep the
final beer flavour consistent compared with standard worts.
Furthermore, when Younis and Stewart (20) reinforced fermenta-
tions with high-maltose syrups, they found that it led to signifi-
cant decreases in acetate ester production, particularly with
high-gravity worts.
It is not clear why the metabolism of a medium containing
glucose or maltose produces different levels of volatiles. Several
hypotheses have been proposed. It is possible that the fermen-
tation of maltose inhibits the transport of volatiles out of the cell,
perhaps by altering plasma membrane structure (15). However,
Stewart and Younis suggested that the idea of suppression of
volatiles’ transportation is unlikely in light of the increased viabil-
ity and vitality of the maltose-grown cells (38). Another possibil-
ity is that the metabolism of maltose produces lower levels of
acetyl-CoA, which is the main substrate for acetate ester synthe-
sis, resulting in fewer esters owing to the lack of this intermedi-
ary metabolite (39). Furthermore, Verstrepen et al. (40) reported
that a high glucose concentration strongly increased the
production of aroma-active esters through activation of the
Ras/cAMP/PKA pathway, which results in a stronger expression
of the ester synthase gene ATF1. Thus ester-synthesizing activities
could be rapidly induced by the addition of glucose.
Wort nitrogen compounds
The nitrogenous compounds available for consumption by yeast
are known as assimilable nitrogen, or free amino nitrogen (FAN),
which can be defined as the sum of the individual wort amino
acids, ammonium ions and small peptides (41). The relative
amounts of the above components can vary owing to differ-
ences in malting and mashing processes and/or raw materials.
There are also differences between lager and ale yeast strains
with respect to wort assimilable nitrogen uptake characteristics
(42). FAN, as a general measure of yeast nutrients, has been
regarded as an important index to predict yeast fermentation
and beer quality (43,44). It is not only used as an essential yeast
nutrient for yeast growth, but also for the biosynthesis of a
number of fermentation by-products that affect the flavour
and stability of beer (45). Worts with more FAN (12.0°P with
115, 160, and 230 mg/L, respectively) showed higher production
rates and final concentrations of isoamyl acetate (44). In addi-
tion, treating wort with proteases increased the levels of FAN,
leading to elevated concentrations of higher alcohols and esters
during either high-gravity brewing or normal-gravity brewing by
lager brewing yeast strains (46). Moreover, the wort FAN content
can affect the transcription of both ATF1 and BAT1 genes, which
are reported to strongly correlate with ester and higher alcohol
synthesis, respectively (47,48). This suggests that the adjustment
of FAN is a factor that can influence beer flavour.
It is not only the absolute amount of assimilable nitrogen that
influences beer aroma, but also the type of amino acid that can
lead to different responses by the yeast – and ultimately to final
beer aromatic profiles. Wort amino acids constitute an important
fraction of brewer’s wort and their determination is therefore of
considerable interest in experimental work and during brewing
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(49). As with wort sugars, the uptake of amino acids follows a
distinct order (50), with different amino acids being removed
at various points in the fermentation cycle (Fig. 3). The amino
acid composition of wort is considered to be an important part
of the complex system regulating the biosynthesis of flavour-
active compounds formed by the yeast (51). Therefore, the
content of amino acids in the wort, and their utilization by yeast
during brewer’s wort fermentation influences both the fermenta-
tion performance and the flavour profile of the finished product.
The addition of branched-chain amino acids, such as valine,
leucine and isoleucine, to the fermenting wort increases the for-
mation of their respective higher alcohols [i.e. isobutanol,
isoamyl alcohol and amyl alcohol (52,53)]. The constitutive ex-
pression of the BAP2 gene, which encodes a branched-chain
amino acid permease in brewer’s yeast, resulted in accelerated
assimilation rates of leucine, valine and isoleucine (54). Recently,
a report has implicated histidine as a key amino acid for lager
yeast strains during brewing, with histidine supplementation
having a large impact on the flavour by increasing the formation
of higher alcohols and esters (55). Isoamyl acetate production in
beer can be affected by the leucine concentration in the wort.
Leucine addition increased the rate of production and final
concentration of isoamyl acetate, while higher levels of valine
delayed the uptake of leucine and lowered isoamyl acetate
production (44).
Proline is the most abundant amino acid in wort but it is uti-
lized poorly, or not at all, during a typical brewing fermentation
and it is often classified as a nonpreferred amino acid (56).
Proline has long been neglected with regards to its significance
in fermentation, as its presence in wort is considered unimpor-
tant for yeast performance during brewing fermentations (16).
However, it has been shown by different working groups that
proline is taken up by yeast during the growth phase and is
excreted at the end of the fermentation (57,58). It was confirmed
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Figure 3. Assimilation of different amino acids in wort by the industrial lager yeast strain TT-21.
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Y. He et al.
by Procopio et al. (59) that, when using high proline concentra-
tions in a synthetic medium, an increased production of ethyl
acetate was observed by both lager and ale brewing strains,
demonstrating that proline is potentially an important
variable in influencing aroma compound profiles during beer
fermentation. Since proline cannot be converted into a
higher alcohol via the Ehrlich pathway, its significance in
aroma formation could be explained by the synthesis of
glutamate from proline (59).
Although the quality and stability of beer is related to the
levels of certain amino acids present in wort (60), deficiencies
in fermentation performance may not be caused by the lack of
any particular amino acid group, but by the efficiency of nitro-
gen utilization (56). Differences in the amino acid assimilation
by yeast were observed during generation 0 and 1 fermenta-
tions (61), which could explain the common differences of the
resultant beer flavour found between beer fermented with
propagated yeasts and repitched yeasts.
Finally, with regards to oligopeptides in the wort, some of
these may be assimilated by the yeast, while others remain
in the final beer, contributing to its flavour and stability,
respectively (62).
Lipids
The lipid content of wort can affect the ester concentration in
the final product. It has been shown that incorporation of
elevated levels of unsaturated fatty acids (UFAs) into the wort,
namely oleic, linoleic and linolenic acids, results in a decrease
in ester synthesis (29). This is due to a direct effect upon the
synthetic enzyme, whereby the expression of ATF1 and ATF2 is
directly inhibited by the addition of UFAs (63). In addition to
acetate esters, it has also been shown that higher levels of UFAs
J. Inst. Brew. 2014; 120: 157–163
Wort composition and impact on higher alcohol and ester formation
in the fermenting medium result in a general decrease in ethyl
ester production by yeast (18).
Vitamins and inorganic ions
Brewing yeasts require vitamins and a wide range of inorganic
ions for efficient proliferation and fermentation performance.
The major function of these wort components is to allow both
enzymes and coenzymes in yeast to function properly (64). A
lack of vitamins and inorganic ions can lead to many fermenta-
tion problems owing to malfunctions in yeast metabolic activi-
ties. Changes in the levels of these compounds result in
altered yeast growth profiles and metabolism (64). Thus, the
content of vitamins and inorganic ions in the wort might affect
the final flavour of beer by altering their metabolic activities.
High-gravity brewing as a general influence on beer flavour
From beer brewing practice, high-gravity brewing has been
adopted in many breweries worldwide. High-gravity brewing is
a process that employs worts of higher than normal sugar
concentrations (typically 16–18°P), which allows the brewery to
enhance its volumetric productivity and save labour and energy
costs without a large new investment. Nevertheless, increasing
the initial wort gravity can also modify the flavour and aroma
compound profile of the final beer, making it difficult to match
to flavour of beers produced from normal-gravity worts (5,65).
The most common and particularly pronounced negative effects
associated with high-gravity brewing are: (a) elevated levels of
acetate esters concurrent with (b) a much less marked degree
of higher alcohol stimulation (66–68), resulting in beers with
overly fruity and solvent-like aromas. This leads to an unbal-
anced beer flavour caused by the disproportionate levels of
volatiles produced, if no other process adjustments are made.
Anderson and Kirsop (66) observed up to an 8-fold increase in
acetate ester production when the specific gravity of wort was
doubled. In our previous work, elevated concentrations of both
higher alcohols and volatile esters were found using high-gravity
brewing (16°P) compared with a normal-gravity fermentation
(13°P), while the increment of volatile esters (78%) was more
than twice that of the higher alcohols (31%). In addition,
transcription levels for all of the higher alcohol and volatile ester
biosynthesis-related genes were enhanced during our high-
gravity brewing trials, with the exception of BAT2 originating from
both from Saccharomyces cerevisiae (Sc-type) and S. bayanus
(Sb-type) genomes (data unpublished).
When sugar adjuncts are added, the type of sugar syrup used
must be taken into consideration. Previous work using synthetic
media has shown that the metabolism of maltose results in the
production of lower levels of esters and higher alcohols (20).
Thus, increasing wort gravity by adding maltose syrup, as
opposed to glucose syrup, resulted in a more balanced fermen-
tation performance and a more favourable flavour profile of the
final beer (69). Fermentations using worts enriched with maltose
syrup, as opposed to glucose syrup, produced more favourable
flavour profiles, with less acetate ester formation, in very high-
gravity (21°P and 24 °P) fermentation trials by Piddocke et al.
(69). This facilitates the design of high-gravity worts resulting
in better flavour matching to beer produced from an all-malt
normal-gravity wort.
Another important factor whereby high-gravity brewing can
alter the aroma profile of beer is the carbon to assimilable
J. Inst. Brew. 2014; 120: 157–163 Copyright © 2014 The Institute of Brewing & Distilling
Institute of Brewing & Distilling
nitrogen (C:N) ratio. In an all-malt wort fermentation, where
the C:N ratio is low, nitrogenous compounds are still abundant
when yeast growth has ceased. Nitrogenous metabolites, includ-
ing acetyl-CoA, are still formed, which stimulates ester synthesis.
The addition of sugary syrups is a common practice to increase
wort specific gravity in high-gravity brewing. However as syrups
generally lack nitrogen, this normally reduces the total FAN
content of the wort, thereby modifying the nutrient balance in
addition to diluting the nitrogen concentration. Therefore, when
adjuncts are used, the C:N ratio is higher, which in turn may lead
to nitrogen being a growth-limiting factor (5,18,48,55). Hence,
when growth ceases there is no excess of nitrogen to form
acetyl-CoA, resulting in less ester synthesis (29).
As higher-gravity worts have reduced oxygen solubility
compared with standard gravity worts (70), under-oxygenation
can lead to suboptimal revitalization of the yeast, growth defects
and low subsequent fermentation rates (71). Moonjai et al. (72)
demonstrated the potential of UFA lipid supplements to wort
as an alternative to wort oxygenation (without losing fermenta-
tion potency or organoleptic quality). Therefore, UFA-treated
yeast may be particularly beneficial in high-gravity brewing.
Conclusions
Brewers have a broad range of different ways at their disposal to
control flavour production during beer fermentation. To a great
extent, the flavour balance of the overall metabolite concentra-
tion is largely a consequence of a combination of yeast strain
and wort composition. The selection of an appropriate brewers’
yeast strain with individual aroma profiles is crucial to fulfil the
consumer’s demand for flavour diversity. However, for a specific
beer production process with a designated yeast strain, a
considered adaptation of wort composition allows brewers to
assume command of the overall flavour character of their beers.
It is obvious considering the variety of ingredients and processes
in wort preparation that the types and levels of carbohydrates,
nitrogenous components and other trace nutrients in worts
can vary. Control of raw ingredients and wort preparation,
followed by appropriate fermentation conditions, will ensure
beer quality and flavour. Presented with a constant wort compo-
sition, a given yeast will metabolize medium nutrients in a
predictable and reproducible fashion, with flavour-active higher
alcohol and ester formation occurring in a consistent manner.
Knowledge regarding the roles of wort components in order
to meet yeast requirements and flavour formation has increased
substantially. Additionally, while genetic and proteomic research
has built up further information to explain the production of
different levels of volatiles in worts and defined media of differ-
ent composition, there is still much to be discovered in this
field. The optimization of wort nutritional content is a very
complicated issue owing to the many complex chemical
constituents in the medium.
Acknowledgements
This work was supported by grants from the National Basic
Research Program of China (973 Program, no. 2012CB723707)
and the National High Technology Research and Development
Program of China (863 Program, no. 2013AA102109). Gregory
Casey is gratefully acknowledged for reviewing the manuscript.
Mei Li is especially thanked for her technical support.
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