INTERNATIONAL
TECHNICAL FEATURE
The science and understanding of the
flavour stability of beer: a critical
assessment
C. W. Bamforth, Davis, California/USA
uch is not the case for flavour life.
This should hardly come as a sur-
prise, for the simple fact that the
chemistry of flavour change is vastly more
complex than is that of haze formation.
Only a relatively few polymeric species are
involved in the precipitation reactions in
beer, these substances are pre-formed in
beer before storage and they are to be
found in significant and readily measurable
quantities. By contrast, the species respon-
sible for flavour are often present in such
low amounts that they can be difficult to
detect, let alone measure accurately. They
may be transient, in many cases not being
present in freshly packaged beer and only
developing (and perhaps disappearing)
when the beer is in trade. Even something
as fundamental as the nature of the gross
changes which take place in beer flavour
over time is incompletely understood: it is
generally believed that different beers will
age in different ways.
Whichever way a beer ages, it is generally
accepted by the brewer that the change will
be disadvantageous to the product. This
may be a naïve assumption. For instance,
the brewer is alarmed at any prospect of a
cardboard flavour developing in a beer,
whereas the fact remains that such a note
seems to be an inherent feature of many
beers in certain marketplaces and may ac-
tually be regarded as part of the character
of a given brand. If the brewer were to “im-
prove” the product by eliminating this fea-
ture, then there is every likelihood that this
will result in customer complaints.
Author: Charlie Bamforth is the Anheuser-
Busch Endowed Professor of Brewing
Science in the Department of Food Science
& Technology at the University of
California, Davis, CA 95616-8598, USA
98 BRAUWELT INTERNATIONAL 1999/II
Huge attention has been
paid over the years to the
ambition of achieving
prolonged shelf life in beer.
In the context of haze life,
this can be achieved,
provided a brewer is
conscientious and willing
to invest some money in
achieving the goal.
The aim must be consistency: providing
the customer with the product that they
expect. However, in this paper the author is
making the assumption that any flavour
change in a beer after packaging is unac-
ceptable and that the brewer’s aim is to
establish the target flavour profile on the
product as it leaves the packaging hall. The
paper seeks to review the current state of
the literature on flavour stability, providing
a view on best practice to achieve pro-
longed shelf life.
At the end of this review the reader will
have been reminded that the topic of fla-
vour change in beer is extremely complex
and that a great many questions remain to
be answered. It is small wonder that major
brewers have recognised a need to limit the
lifetime of their products in the trade and to
provide the customer with an image of
freshness through stressing the “youth” of
the beer.
■ The nature of flavour changes
occurring in beer
The number of reports delineating the
precise changes in flavour perception in a
beer throughout storage are surprisingly
few in number (see, for example, 1 – 6). It
certainly seems to the author that a good
starting point for any brewer would be for
them to understand which notes increase
or decrease in their own beers in order that
they may have a better focus on the ap-
proaches which they may take to improving
shelf life. The solution to one type of flavour
change may be different from that for an-
other. The “starting” flavour of a beer will
be significant, not least for its “depth”:
changes in character caused by the forma-
tion or loss of materials in trace quantities
will be more perceptible in a bland beer
than in one which is of fuller flavour. People
often refer to the greater flavour stability of
darker beers, suggesting that they contain
higher levels of anti-staling protectants. In
reality it may actually be the case that
changes in flavour are less apparent against
a more complex taste and aroma back-
ground.
Any generalised description of flavour
changes must of necessity be an oversim-
plification. Initially, and perhaps within a
1 – 3 month time frame, a beer will have
started to decline in bitterness, will per-
haps become more harsh, and will also
have declined in fruity/estery notes and flo-
ral features. Furthermore a “ribes” (black-
currant leaves, tomcat) character may
have become evident. Papery or cardboard
characters will have started to be detected.
Progressively, the beer will become bready
and either sweeter or toffee-like and per-
haps honey. Possibly metallic, earthy or
straw notes will develop. Finally, a beer will
become woody and winey (sherry-like).
The changes of flavour in beer are a result
of chemical reactions. As for all such reac-
tions, their rate depends greatly on temper-
ature, a 10°C increase in temperature lead-
ing to an approximate doubling of rate. Ac-
cordingly the single most significant step a
brewer (or retailer, or customer) can take to
slow down flavour changes in beer is to
maintain the coldest practical temperature
during storage in the trade. Refrigeration is,
of course, expensive, but it is incumbent
upon the brewer to give serious considera-
tion to the decision of whether such invest-
ment is justified. For instance, some compa-
nies ship their packaged beer considerable
 INTERNATIONAL
TECHNICAL FEATURE

Charles Bamforth Appointed Anheuser-Busch Endowed

Professor of Malting and Brewing Science at UC Davis
Dr. Charles W. Bamforth was selected as the first Anheuser-
Busch Endowed Professor following an international search.
He comes to this position very well qualified. Dr. Bamforth
earned his B. S. in Biochemistry as well as his Ph.D. from the
University of Hull in England. In 1993 his alma mater awarded
him a prestigious D.Sc. degree. This honor is a most significant
recognition of one’s lifetime accomplishments and mastery of
an area, in Dr. Bamforth’s case, brewing science.
The Anheuser-Busch Foundation has provided an endow-
ment to establish the Anheuser-Busch Professorship in Malt-
ing and Brewing Sciences. The purpose of the endowed profes-
sorship is to provide a permanent source of funding for teach-
ing and research in malting and brewing sciences. The an-
nouncement of the Foundation’s endowment was made by
Doug Muhleman, Anheuser-Busch Vice President of Brewing
Operations. Doug has stated that the individual selected to fill
the position should have “a demonstrated passion for the
brewer’s art and a scientific background in microbiology, bio-
chemistry or food science.”
Following a postdoctoral position at the University of Shef-
field, Dr. Bamforth joined the Brewing Research Foundation in
the UK in 1978. This organization was comprised of approxi-
mately 80 employees, primarily scientists, pursuing a shared
portfolio of non-competitive research, dissemination of infor-
mation, and training programs on behalf of companies sub-
scribing through membership fees. During his five years there,
he was promoted several times, attaining the ranks of Head of
Biochemistry and Physics and Head of Malt and Wort Produc-
tion.
In 1983, Dr. Bamforth was recruited by Bass PLC, then the
largest brewer in the UK, to direct a research team focused on
product quality issues. After ten months, his duties increased
to Research Manager, with responsibility for all research, in-
cluding raw materials, product quality research, and new prod-
uct development. From 1988 to 1991, he gained significant
practical experience as Quality Assurance Manager of a major
brewery in the Bass organization.
Dr. Bamforth rejoined the Brewing Research Foundation,
which was soon to become Brewing Research International
(BRI), in 1991 as Director of Research. The research activities
he directed included programs an barley and malt, hops, yeast
and fermentation, microbiology, product quality, process inno-
vation, analysis and instrumentation. His particular responsi-
bilities included research strategie planning, progress moni-
toring and management for all publications and presentations.
In 1997 Dr. Bamforth assumed the position of Director of Mem-
bership Services in BRI, with the goal of attracting new mem-
bers and retaining existing members. At the same time, he
Dr. Charles W. Bamforth (left) and Doug Muhleman, Vice
President of Brewing Operations at Anheuser-Busch, Inc.
retained responsibility for core research programs on product
quality and raw materials as well as sensory and analytical
service and for driving the contract research activities of the
organization. He also became Deputy Director-General of BRI.
In addition to his career in the brewing industry and research
sectors, for several years Dr. Bamforth has been a Visiting Pro-
fessor at the International Center for Brewing and Distilling at
Heriot-Watt University in Edinburgh, Scotland. In this capacity
he has presented lectures in Edinburgh and conducted brew-
ing courses overseas, most recently in China and India.
Also he has published a book, “Beer: tap into the art and
science of brewing”, which is written for a non-technical audi-
ence. It provides an easy-to-read blend of biochemistry, micro-
biology, and physical chemistry along with a sense of the histo-
ry of brewing. The prestigious international journal “Nature”
said that the book is “a triumph of user-friendly exposition.”
New Scientist said “if you will only ever read one book on
brewing in your life, this should probably be it.”
Dr. Bamforth is a research leader in the area of brewing
science. His research has been widespread, and has included
detailed investigations on the enzymology of malting and
mashing, on foam, and on flavor and flavor stability. In recogni-
tion of his scholarly accomplishments he has been named a
Fellow in both the Institute of Brewing and the Institute of
Biology. He is widely sought as a speaker, having lectured inter-
nationally in over 20 countries.
As the Anheuser-Busch Professor at Davis Dr. Bamforth will
teach undergraduate courses in Malting and Brewing Science,
and possibly enzymology. He will also mentor graduate stu-
dents and maintain a research program in the key areas for the
brewing industry of product quality. ■

distances across and between continents,
such beer encountering extremes of tem-
perature. It is not unusual for beer to be
subjected to temperatures of 35°C through
50oC for days or even weeks. According to
the Arrhenius equation, flavour changes
will be occurring over 30-fold faster at the
latter temperature than at 0°C, or in other
words a flavour deterioration only noticea-
ble after more than 6 months of cool stor-
age will be detectable within a week in the
beer stressed by heat. It has been amply
described by many researchers that when
beer is held at 0 – 4°C it fails to display signs
of oxidation even after months of storage.
There is evidence that the nature of fla-
vour changes ocurring at different tempera-
tures varies (6). In particular it was found
that there was an increase in carbonyl com-
pounds when beer was aged at 45°C, but
actually a decrease in overall carbonyls
was seen at room temperature, together
with a lowering of ester levels. Kaneda et al
(7) say that a beer aged at 25°C tends to
develop a predominantly caramel charac-
ter, whereas at 30 or 37°C more cardboard
notes are dominant. This has significance
for forcing tests, which are necessary in or-
der that research can be done over realistic
time frames. In contradiction of the findings
of Bright (6) and Kaneda (7), Greenhoff (8)
claimed that the nature of flavour changes,
in particular the type of cardboard charac-
ter, displayed at 60°C was similar to that at
18 or 37°C. We will come back to predictive
tests later.
1999/II BRAUWELT INTERNATIONAL 99

Table 1 Some carbonyl compounds found in beer
Saturated aldehyde Mono-unsaturated
aldehyde*
Pentanal (grassy)
Hexanal (vinous, bitter) 2-hexenal (astringent)
Heptanal (vinous, bitter)
Octanal (vinous, bitter) 2-octenal (bitter, stale)
Nonanal (astringent) 2-nonenal (cardboard)
Decanal (bitter) 2-decenal (bitter, rancid)
*Alkenals +Alkadienals
■ Potential flavour substances which
change in level during beer storage
In terms of bitterness drop, then it is well
understood that the iso-alpha-acids decay
relatively quickly in beer (9), although the
products formed are not clearly known.
However there are a great many other
changes in concentrations of compounds
occurring. Indeed, Lustig (10) identified 30
substances by multi-dimensional gas chro-
matography whose level either increases or
decreases during staling. Classes include
esters, O-heterocyclics and carbonyls. It
would be a surprise if this represented a
final list.
It is the carbonyls which have attracted
most attention whenever there is consider-
ation of flavour instability. However, con-
sideration of the flavour changes described
above should make it apparent that the
chemistry of staling is rather more complex
than carbonyl compounds alone. Lustig
(10) lists ten substances as the principal
determinants of staling and only some of
them are carbonyls: 2-methyl-butanal, 2-
furfural, 5-methyl-2-furfural, benzaldehyde,
2-phenylethanal, diethylsuccinate, ethyl
phenylacetate, 2-acetyl furan, 2-propionyl
furan and gamma-nonalactone. Com-
pounds such as furfural are generally held
to be good markers of oxidation in beer
without themselves being present in suffi-
cient quantity to be detectable. Others in
this category include acetaldehyde (11),
hydroxymethylfurfural (2), gamma-hexa-
lactone, 3-methylbutanal and the ethyl es-
ter of nicotinic acid (12) and 2-furfurylethyl
ether and furfuryl acetate (13).
There is no argument that carbonyl com-
pounds are important: consider, for in-
stance, Hashimoto’s elegant demonstration
that carbonyl scavengers such as hydroxy-
lamine will strip the oxidised flavour from
beer in moments (14). However we must be
careful to recognise that “oxidised” charac-
ter is only one aspect of “staleness”: the
myriad of flavour notes changing during
staling are due to a much broader range of
chemical entities.
100 BRAUWELT INTERNATIONAL 1999/II
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TECHNICAL FEATURE
Di-unsaturated
aldehyde+
2,4-nonadienal (rancid)
2,6-nonadienal (cucumber)
2,4-decadienal (oily, rancid)
Some other carbonyl compounds found
in beer are listed in Table 1.
Only three of the substances listed (2-
nonenal, 2,6-nonadienal and 2,4 decadien-
al) are present in beer in concentrations in
excess of their flavour thresholds, however
it is understood that mixtures of carbonyls
can cumulatively cause a perceived flavour
(15), presumably because they bind at a
common sensory site on the palate.
Of all these aldehydes, it is trans-2-nonenal
(now referred to as (E)-2-nonenal) which is
most frequently cited as the cause of a card-
board character in beer. This is on account
of it being present in levels in excess of its
flavour threshold and because it has been
reported to increase in level in ageing beer.
However this observation is by no means
universal (see, for instance, 15). It is likely
that nonenal is just one of a related group of
substances responsible for this flavour fea-
ture. They may or may not have their origins
in the same pathway, adding to the immense
complexity of this whole topic. Certainly
Drost and his colleagues felt that a combina-
tion of nonenal with 5-methylfurfural gave a
much more significant (but by no means
complete) representation of cardboard
character than did nonenal on its own (16).
One view is that nonenal may be a feature
of beer subjected to accelerated ageing,
whereas it is of less significance in naturally
stored beer (2).
We are reminded by Evans (17) that the
compounds responsible for stale flavours
may already be present in fresh beer and
that relatively few compounds arise de
novo during storage. Flavour deterioration
is not so simple as to be a time-dependent
production of the chemical species respon-
sible for “off” flavours, but rather a shift in
the balance of pre-formed entities, mani-
festing itself in overall shifts in perceived
flavours.
One point is quite clear, though: the
amounts of these materials that we are talk-
ing about are very small when considered
in relation to the substances which have
been put forward as their precursors (next
section). Take, for instance, trans-2-none-
nal, which has a flavour threshold of ap-
proximately 0.1 ppb. It is generally held that
a substance will certainly be detectable in
its own right if it is present at 5 flavour units,
such a unit representing the concentration
of the material divided by its flavour thresh-
old. If we assume for the present purposes
that the origins of trans-2-nonenal lie in un-
saturated fatty acids (and this is by no
means certain) and we suppose that some
0.5 kg of linoleic acid per hectolitre of wort
at 10°P is contributed by an all-malt grist
(18), then we can calculate that the efficien-
cy of conversion of linoleic acid into none-
nal need only be some 2 x 10 – 5% for the
required number of flavour units to be gen-
erated. In other words, the extent of conver-
sion of precursors does not have to be par-
ticularly great for there to be sufficient for-
mation of a substance like nonenal as to be
detectable by the nose. Equally, there does
not have to be very much of a precursor
present in beer for detectable levels to be
developed. As we shall see, all of the poten-
tial precursors of staling carbonyls in wort
and beer are present in relatively large
amounts.
■ Pathways involved in the synthesis
of staling substances
In the light of the above, it should come
as no surprise to the reader that several
routes contribute to the changes which
take place in the flavour chemistry of beer.
Without exception there is controversy
about all of these pathways in respect of
their relative importance in this context.
Each has their champions in the literature,
and their detractors.
In part the problem has arisen because of
the complexity of beer (and its production
process) as a vehicle for experimental in-
vestigation. This has pushed people to use
simpler model systems and one is seldom
entirely convinced that the observations
translate to beer per se.
Melanoidin-catalysed oxidation of
higher alcohols
Alcohols in beer can be converted to
their equivalent aldehydes through the
mediacy of melanoidins, with the oxidised
carbonyl groups on the latter acting as elec-
tron acceptors (14). In model systems it
was shown that the reaction was promoted
by increased temperatures, high levels of
oxygen and low pH. Devreux et al (19) cite a
requirement for light and an inhibition by
the polyphenols in beer as reasons why this
pathway is unlikely to be significant. Fur-
thermore, Irwin et al (20) observed a very
low efficiency (0.2%) of conversion of 2-
 INTERNATIONAL
TECHNICAL FEATURE

nonen-1-ol to nonenal in model systems. As

beer contains very little nine carbon alco-
hol (< 0.5 ppb), Irwin and his colleagues
would argue that this particular pathway is
irrelevant.

Oxidation of iso-alpha-acids

Hashimoto’s observation (21) that un-

hopped beers seldom develop oxidised fla-
vour indicated a possible role for iso-alpha-
acids as precursors of staling compounds
(or as pro-oxidants). Again in model sys-
tems it was shown that volatile carbonyls
(alkenals and alkadienals with chain
lengths of between 6 and 12 carbon atoms)
could be produced from a solution of bitter
substances, higher alcohols and melanoi-
dins, with rather more oxidation of the iso-
alpha-acids than the higher alcohols occur-
ring.

It was claimed by Barker et al (22) that

none of the iso-alpha-acids has a side chain
capable of generating nonenal. Similar argu-
ments have been applied to justify scepti-
cism about the Strecker route (see later).
The criticisms are valid, but it has long
since been realised that nonenal itself may
not have the prime significance it once was
thought to have as a contributor to stale
flavour.

The reduced side-chain iso-alpha-acids

used to impart light resistance to beers do
not breakdown to staling carbonyls (23)
and, as a result, beers bittered with such
agents may be more resistant to oxidation.

Strecker degradation of amino acids

The Strecker degradation involves a reac-

tion between an amino acid and an alpha-
dicarbonyl compound, such as the inter-
mediates in browning reactions. The amino
acid is converted into an aldehyde with one
fewer carbon atom. It is a plausible route by
which certain aldehydes may be produced
in beer (24, 25). For example, in model sys-
tems, it can be shown that isobutyraldehyde
is produced from valine and isovaleralde-
hyde from alanine (24). Polyphenols may
have a catalytic role to play (26). Thum et al
(27), however, doubt that the levels of amino
acid found in most beers are sufficient to
allow significant levels of carbonyls to be
formed by this route. Whilst these com-
pounds may only be developed at levels
substantially below their flavour thresh-
olds, we should once more be reminded that
cumulatively these and many other carbon-
yls may contribute to perceived staling.

Aldol condensations

The aldol condensation reaction between

separate aldehydes or ketones is a plausi-
ble route through which trans-2-nonenal
1999/II BRAUWELT INTERNATIONAL 101

may be produced, by a reaction between
acetaldehyde and heptanal (25). Diverse
other carbonyls may be generated in this
way, with the imino acid proline (abundant
in beer, 28) as a necessary catalyst.
It is feasible that longer chain staling al-
dehydes are built up in this way from the
products of Strecker degradation, higher
alcohol oxidation or bitter substance
breakdown. Whether the concentrations of
the various protagonists achieve sufficient
concentrations in the soup which is beer
such as to generate quantities of staling
substance which even in their entirety are
sufficient to be detectable as off flavour in
beer is debatable.
Oxidation of unsaturated fatty acids
The oxidative breakdown of lipids lead-
ing to rancidity is a well understood path-
way, extensively studied for its relevance in
industries as diverse as oil, rubber and fatty
foods (29). Small wonder, then, that in the
study of beer staling, too, it has attracted
more attention than any other route of car-
bonyl formation.
Despite the plethora of papers on lipid
oxidation in the context of staling, there re-
mains no absolute proof of the greater sig-
nificance of this pathway over any other in
beer deterioration. Thus, for example, one
might fundamentally anticipate that the
simple act of increasing the lipid content of
beer or wort would lead to a perceptible
increase in staling: in fact quite the oppo-
site has been observed (30, 31).
Despite this, there is unquestionably a
theoretical probability that the oxidative
degradation of polyunsaturated fatty acids
has a role to play in the formation of none-
nal and other potential staling carbonyls.
And this may be through either or both of
two routes: non-enzymic oxidation (which
is the major pathway in most systems, such
as dairy foods) and enzymic oxidation.
(a) Lipoxygenase-catalysed oxidation
of unsaturated fatty acids
Barley develops two lipoxygenase en-
zymes, LOX-1 and LOX-2 in its embryonic
tissues (32). The former is present in raw
barley in small quantities, and increases
during germination, whilst the latter is only
developed during germination (33). Barley
varieties vary to some extent in their ability
to develop the two isoforms of lipoxygen-
ase – indeed the finished malt from one va-
riety, Robust, was shown to contain only
LOX-2 (34). Both enzymes are extremely
heat-sensitive and are extensively lost dur-
ing most kilning regimes (33), with LOX-1
being the more resistant (33). Many work-
ers may have underestimated the amounts
102 BRAUWELT INTERNATIONAL 1999/II
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TECHNICAL FEATURE
of lipoxygenase in malt, because detergents
need to be employed to achieve full extrac-
tion of the enzyme which, understandably,
is closely associated with lipid-rich materi-
als in the grain (35). Furthermore, extracts
will contain variable levels of endogenous
inhibitors (chiefly polyphenols – see later),
which need to be dialysed away before full
activity can be realised (36). The level of
lipoxygenase present in malt declines dur-
ing storage (37), and it has been hypothe-
sised that this is the reason why malt
should be stored for a few weeks prior to
use: the enzyme is involved in oxidative re-
actions which contribute to the formation
of the oberteig layer in the grains bed that
impedes wort separation and when the lev-
el of the enzyme reduces, so too does the
ease of wort separation.
Both enzymes are capable of acting on
polyunsaturated fatty acids (those fatty ac-
ids with 2 or more carbon-carbon double
bonds). In effect, the only substrates of sig-
nificance arising from malted barley are li-
noleic acid and linolenic acid, which have 2
and 3 double bonds respectively, and the
former is much more important.
LOX-1 converts linoleic acid into 9-hy-
droxy-trans-10, cis-12-octadecadienoic acid,
generally referred to as a 9-hydroperoxy
acid. LOX-2 makes the equivalent 13-hy-
droperoxy acid (38). The first of these acids
can degrade to trans-2-nonenal, ergo it is
generally the case that LOX-1 is considered
to be the more important isozyme of lipox-
ygenase. This may be an unwarranted fo-
cus, if the degradation products arising
from the product of LOX-2 also contribute
to off flavour development – i.e. the argu-
ment, again, about the potentially exagger-
ated status of nonenal as the prime determi-
nant of staling.
Lipoxygenase represents but one stage in
the pathway from lipid to staling carbonyls.
LOX-1 is incapable of oxidising linoleic or
linolenic acid when they are esterified as
they are for the most part in the glycerides,
phospholipids etc. of barley and malt (39).
LOX-2 is, however, capable of this reaction,
but it has a higher reactivity with free fatty
acid (39). Therefore, as a necessary first
stage to facilitate at least some of the lipox-
ygenase activity, glycerides need to be hy-
drolysed through the mediacy of lipases
(40). At least two lipases develop in barley
during germination, the most important of
these being located in the embryo (40).
Both are somewhat insoluble, indeed sight
should not be lost of the fact that much of
any “damage” arising as a result of lipid deg-
radation will largely take place in the parti-
cles of a mash rather than in solution.
A further enzyme, produced during ger-
mination of barley, hydroperoxy acid iso-
merase (41, 42), converts the product of
lipoxygenase action to ketols, which are
non-enzymically converted to mono-, di-
and tri-hydroxy acids, which in turn can
break down non-enzymically to carbonyl
compounds. Although there has been a
championing of various hydroxy acids as
the main precursors of staling aldehydes
(for instance, 9,10,13-trihydroxy-11-trans-
octadecanoic acid, ref 43), in truth it is like-
ly that there are several such compounds
which may be important.
In view of the heat sensitivity of lipoxyge-
nase, the extent to which it may be signifi-
cant in mashing has been questioned (44).
However there is evidence for the enzymic
production of hydroxy fatty acids in small-
scale mashes, particularly at lower temper-
atures (e.g. 52°C), with preferential forma-
tion of the 9-hydroperoxide, indicating the
greater significance of LOX-1 (45, 46). Malts
with higher levels of lipoxygenase produce
more hydroperoxide, though it also worthy
of note that only about one third of the
lipoxygenase activity in malt may be ex-
tracted into a mash (34). In acidified mash-
es, taken from pH 5.5. to 5.0, lipoxygenase
levels were halved (Doderer et al (36)
showed that LOX-1 had a broad pH-activity
profile, but with activity falling by some
60% at pH 5, whilst there was essentially no
LOX-2 at this pH, the latter isozyme having
a much narrower pH-activity profile cen-
tred on pH 6.5.) and so too was hydroperox-
ide production greatly lowered (46).
The demonstration by Kobayashi et al
(46) that a specific inhibitor of lipoxygen-
ase inhibits hydroperoxide formation lends
further support to the notion that lipoxyge-
nase can act in mashes, especially at the
lower temperatures which may be used in
protein rests. As hydroperoxide isomerase
is more stable than lipoxygenase, it can be
rationalised that the ingredients needed to
commence a staling process from unsatu-
rated fatty acids are present. Indeed, Koba-
yashi et al (46) found that the hydroperox-
ides produced during mashing were also
lost again before the wort was finished, sug-
gesting an onward conversion to some oth-
er intermediate or even end product. One
cautionary note relevant to the work de-
scribed in this paragraph and in other plac-
es in this paper is that the observations
were made with small scale mashes, in
which opportunities for oxygen ingress will
be considerably greater than in commercial
operations. Kobayashi et al (46) demon-
strated that CO2 bubbling as an exercise in
lowering oxygen levels in a mash reduced
the development of hydroperoxides and it
is certainly true that in most full-scale
mashes the opportunity for oxygen ingress
will be much less than in the high surface-
mash ratios typical of laboratory scale ex-
ercises.

It has even been claimed that nonenal is
produced during mashing and that it is con-
verted to either 3-hydroxynonanal (by hy-
dration, 47), nonenoic acid (by oxidation)
or into a Schiff base with amino compounds
(48). It is suggested that, in each case, the
product may carry through into beer, pro-
gressively to be converted back into none-
nal.
The question has been asked though: as
lipid and lipoxygenase are localised in the
embryo of grain, to what extent can oxida-
tion of linoleic and linolenic acid occur dur-
ing germination? The increase in lipoxygen-
ase activity during germination is not exact-
ly mirrored by the increase in level of hy-
droperoxide (49), but it is certainly the case
that we can demonstrate the synthesis of
this chemical class during steeping and ger-
mination, with higher levels in those bar-
leys which produce more lipoxygenase.
Equally, however, it is possible to demon-
strate that hydroperoxide levels decline
late in germination and to undetectable lev-
els in finished malts, whether kilned to a
lager or ale specification. This may reflect
the action of the isomerase, but at this
stage there is no true understanding of the
levels of carbonyl precursor substances/
lipid degradation products in malt. Even so,
Tressl and co-workers have found over thir-
ty aldehydes in a series of malts, amongst
them trans-2-nonenal at 200 – 400 ppb (50).
Such pre-formed volatiles would be expect-
ed to be lost with the flue gases during boil-
ing and fermentation (unless they are “sta-
bilised” in some way – see later), indeed
there may even be arguments in favour of
promoting oxidation during malting and
wort production, provided that end-prod-
uct carbonyls were driven off (or reduced
in fermentation – see later) and no interme-
diates survived through to the final beer
where they could break down. Kretschmer
et al suggest that certain staling substances
are produced during malt production and
that these are partly expelled during wort
boiling (51).
The microflora inhabiting malt may also
contribute to lipoxygenase load (52).
(b) Non-enzymic oxidation of
unsaturated fatty acids
The non-enzymic pathway for break-
down of unsaturated fatty acids is well-es-
tablished and it leads to hydroperoxide
species analogous to those emerging from
the lipoxygenase route. To trigger this “au-
toxidation” process it requires a highly ac-
tive species able to abstract a hydrogen
from the carbon atom adjacent to the dou-
ble bond. The peroxy radical formed in the
pathway is capable of effecting this abstrac-
tion, hence the autocatalytic nature of the
process, but only a limited number of radi-
INTERNATIONAL
TECHNICAL FEATURE
cal species have this capability. Two of
them are radical forms of oxygen, superox-
ide (in its protonated state, perhydroxyl)
and hydroxyl (53).
The production of radical forms of oxy-
gen is promoted by light and certain en-
zyme systems. However of particular im-
portance are transition metal ions, for in-
stance iron and copper, hence the precau-
tions which most Brewers take to eliminate
these substances from their process and
product.
Radical formation is promoted by the
metal ions when they are in their lower va-
lence (more reduced) state. Thus the path-
way of radical production involves initial
reduction of the metal ion (M) by a suitable
reducing agent (such as polyphenols with
5’ hydroxy substitutions, e.g. delphinidin
(ref 20), RH2 equation 1) followed by
successive action of the metal ion in the
Haber-Weiss reactions (equations 2 – 4):
2M(n+1)+ + RH2 → 2Mn+ + R (1)
Mn+ + O2 → M(n+1)+ + O2– (2)
Mn+ + O2– + 2H+ → M(n+1)+ + H2O2 (3)
Mn+ + H2O2 → M(n+1)+ + OH– + OH• (4)
where Mn+ may be Cu+ or Fe2+ and M(n+1)+
may be Cu2+ or Fe3+.
The reactivity (or lack of it) of oxygen and
its activation by acquisition of electrons
was described by Bamforth et al (49). As
oxygen passes successively through super-
oxide, peroxide and hydroxyl it becomes
increasingly reactive. Hydroxyl is one of the
most reactive species so far identified, and
it will react instantly (i.e. with very high rate
constants) with a great many types of mole-
cule. If it is generated in wort or beer, then
the chances are that it will find a molecule
to react with (e.g. sugar or ethanol) before
it has the opportunity to “find” an unsatu-
rated fatty acid. Superoxide, on the other
hand, is less reactive and will have a greater
tendency to migrate and encounter an un-
saturated fatty acid molecule. It may be ra-
tionalised, therefore, that it is superoxide
which presents the greater danger.
In fact this can only be after protonation,
for HO2• is much more reactive than O2–. As
the pKa for this acid-base equilibrium is 4.8,
it will be realised that superoxide is likely
to be a much more problematic species in
beer than in wort. It has long been suggest-
ed (54) that beers stale more quickly at low-
er pH. Kaneda et al (55) noted that beers
displayed an increased chemilumines-
cence as pH was lowered from 4.3 to 3.8,
which was interpreted as an increase in the
level of radical species capable of oxidizing
iso-alpha-acids and polyphenols. They fur-
ther invoke the importance of a shift in the
1999/II BRAUWELT INTERNATIONAL 103

Table 2 Degradation products predicted from the breakdown of unsaturated fatty acids by
lipoxygenase and hydroperoxide isomerase (58)
Acid Products from
LOX Isomerase
Linoleic 9-hydro-peroxy cis-3-nonenal*
acid linoleic acid*
13-hydro-peroxy hexanal+
linoleic acid+
Linolenic 9-hydro-peroxy cis-3,cis-6-
acid linolenic acid* nonadienal*
13-hydro-peroxy cis-3- hexenal+
linolenic acid+
* products from initial action of LOX-1 + products from initial action of LOX-2
superoxide-perhydroxyl equilibrium in fa-
vour of the latter. Kaneda et al note that this
was associated with a downturn in flavour
stability of the beer, albeit a modest change.
It was recently claimed that quantitatively
the most important radical in beer (detect-
able by electron spin resonance – see later)
is 1-hydroxyethyl, formed by the reaction
between hydroxyl and ethanol, which is
perhaps unsurprising as ethanol is quanti-
tatively the second most important constit-
uent of beer and thus kinetically very likely
to encounter hydroxyl radicals (56). It is
further proposed that the 1-hydroxyethyl
radicals react with ground state oxygen to
yield the perhydroxyl radical, hence this
appears to be a logical mechanism respon-
sible for oxidative damage in beer.
Recently fundamental questions were
asked about the significance of lipids as
precursors of oxidised flavours with the
observation that, whilst free fatty acids
are extensively produced from glycerides
and phospholipids during germination, the
products are complexed, are not extracted
into wort and are not oxidised (57).
Should, however, the oxidation of linoleic
and linolenic acids actually have some rele-
vance in the present context, it can be
shown that a range of products may flow
from this route (Table 2). Comparison of
Tables 1 and 2 clearly shows that the prod-
ucts predicted to be formed by the oxida-
tion of the unsaturated fatty acids are in-
deed found in beer.
■ The central role of oxygen in staling
Whilst there is no real consensus of opin-
ion on the relative significance of the above
pathways as the route for flavour deteriora-
tion in beer, there are no arguments about
the pivotal significance of oxygen in this
regard. It has long been recognised that
oxygen levels in final pack should be main-
tained as low as possible and it remains
true to this day that the top priority for any
Brewer wishing to have long shelf life in a
104 BRAUWELT INTERNATIONAL 1999/II
INTERNATIONAL
TECHNICAL FEATURE
Chemical degradation Reduction by yeast
trans-2-nonenal* trans- and cis-2-nonen-1-ol*
trans-2-hexenal+ hexen-1-ol and
trans-2-hexen-1-ol+
trans-2,cis-6- trans-2,cis-6-
nonadienal* nonadien-1-ol*
& cis-3,cis-6- nonadiene-1-ol*
trans-2-hexenal+ trans-2-hexen-1-ol/
cis-3-hexen-1-ol+
product must be to package with the lowest
practical oxygen levels. Modern filling
equipment can achieve total oxygen levels
in pack of less than 0.1 ppm.
Increasingly, though, it is accepted that
even beers packaged under the most miser-
ly of oxygen regimes still deteriorate in fla-
vour. This has convinced people that oxy-
gen control is needed throughout the brew-
ing process – and perhaps even in the malt-
ings (59). Although it is notoriously difficult
to make accurate measurements of how
much oxygen may be consumed during
wort production, it has variously been esti-
mated to be as much as 50 – 100 mg per litre
(60, 61). Certainly there are a number of
reports of improvements to flavour life
caused by minimisation of oxygen ingress
in the brewhouse (see, for example, 62 –
64), but equally it should be understood
that the magnitude of the improvements
observed was frequently small and, with
commercial scale trials, sometimes non-ex-
istent (65, 66).
There are several possible effects of oxy-
gen in the brewhouse, amongst which are:
1. Direct involvement in the lipoxygenase
reaction.
2. Source of oxygen radicals for the autoxi-
dation of unsaturated fatty acids.
3. Oxidation of sulphydryl bonds in pro-
teins, leading to cross-linking and retar-
dation of lautering (67 – 69) but also to
the production of hydrogen peroxide
(70), which is a substrate for peroxidas-
es, of which there are several very active
ones in malt (71).
4. Reaction (directly, or via H2O2 formation)
with polyphenols, catalysed by peroxi-
dases in the mash but non-enzymically in
the wort boil. (Some people have sug-
gested that polyphenol oxidase may
have a role to play in promoting wort ox-
idation, however this enzyme is substan-
tially lost during germination of malt and
is virtually entirely destroyed during
kilning (72, 73), which means that there
is only a very narrow window of opportu-
nity for it to act at the start of mashing).
The last of these effects may be of espe-
cial significance, for the polyphenols are
the prime antioxidant materials native to
malt, hops and, subsequently, beer. If they
are lost by oxidation at this stage (they pol-
ymerise on oxidation and precipitate with
protein), then the subsequent beer will be
lessened in antioxidant capacity. It is now
over 30 years since Owades’ and Jakavac’s
classic report of the distribution of 18O2 in-
troduced into beer (74): they found that 5%
of the label entered into iso-alpha-acids,
35% into volatile carbonyls, but the greater
part went into the polyphenol fraction,
highlighting the affinity of this class of com-
pounds for oxygen.
■ Endogenous antioxidant systems
For as many systems as are native to beer
and its raw materials which promote the
formation of staling carbonyls, there are
other which directly or indirectly counter
this, either by scavenging the carbonyls or
by interfering in some way with their pro-
duction.
Polyphenols
The observations of Owades cited above
are the clearest evidence for the ability of
polyphenols to mop up oxygen in beer. As a
class of substances, however, the polyphe-
nols are of course diverse. As noted by Irwin
et al (20), some classes of polyphenol (for
example catechin and others which have
hydroxyl groups in the 3’ and 4’ positions on
the flavan ring) are antioxidant due to their
ability to scavenge free radicals. Others,
however, are pro-oxidants because they re-
duce transition metal ions to the lower va-
lence state in which they are involved in the
Haber-Weiss Reaction (see earlier). In the
latter case it is those flavanoids with an ex-
tra 5’-OH group, e.g. delphinidin.
In fact, polyphenols may be antioxidants
in brewing systems for at least three rea-
sons:
a. their ability to scavenge oxygen free rad-
icals, superoxide (75) and hydroxyl (76)
and also the peroxy radicals formed in
the autocatalytic oxidation of unsaturat-
ed fatty acids (77)
b. their capability as inhibitors of lipoxyge-
nase (78)
c. their capacity as chelating agents, there-
by sequestering transition metal ions
such as iron and copper.
Only a and c are relevant to the role of
polyphenols in beer whereas all three
mechanisms may be important for a protec-
tive role in wort production.

Walters and co-workers have made the
most comprehensive analysis of polyphe-
nols (together the related phenolic acid, fer-
ulic acid) in respect of antioxidant capability
in beer (9, 79 80).
The study served to illustrate how vastly
complex the topic is, insofar as agents such
as catechin were found to be pro-oxidant
at low concentrations through an ability
to promote hydroxyl formation, but anti-
oxidant at higher levels. Ferulic acid, too,
was found to have potentially deleterious
effect in that the levels of several carbonyl
compounds were actually promoted in its
presence. Above all, it was shown that
there was merit in promoting the level
of polyphenolic antioxidants in finished
beer as a device for protecting against
oxidation occurring in the beer per se,
but that beers had already developed an
inherent staling capability (presumably
due to oxidation and other reaction mecha-
nisms occurring in the production process)
and that enhancing the polyphenol level
in the finished beer was useless to prevent
staling thus caused. It seems, therefore,
that it may be particularly important to
have significant polyphenol levels in the
wort production process, to act as pro-
tectants by the mechanisms described
above.
INTERNATIONAL
TECHNICAL FEATURE
Melanoidins
Melanoidins, too, are capable of scav-
enging superoxide, peroxide and hydroxyl
(81) and Hashimoto claims that they sup-
press the oxidation of iso-alpha-acids and
unsaturated fatty acids (25). However, as
we have seen, melanoidins are also impli-
cated in promoting the oxidation of higher
alcohols.
At this stage it is important for the reader
to appreciate the complexity of the sys-
tems we are dealing with: the various po-
tential antioxidants in beer (and wort)
should not be considered as species in iso-
lation. As well as reactions between anti-
oxidants and oxygen species, a myriad of
potential electron transfer reactions can
also occur between the antioxidants them-
selves. The extent to which these occur
and the overall balance of concentrations
of the various species in their oxidised and
reduced forms will depend inter-alia on the
redox potentials of the species and the rel-
ative overall concentrations of the antioxi-
dant types.
The concept of a chain of redox agents
involved in the oxidative reactions of wort
and beer has long since been recognised (2,
82).
Sulphur dioxide
SO2 is also a radical scavenger and it
has been claimed that this is its main anti-
oxidative function in beer (83). However
SO2 can also form adducts with carbonyl
compounds, rendering complexes which
are not flavour active (22). The proposal is
that carbonyl compounds produced early
in the brewing process bind with the SO2
which is a natural product of yeast metabo-
lism (84). These adducts pass into beer,
where they progressively break down to
free the carbonyls which render the beer
stale. The bisulphite complexes are in equi-
librium with the free carbonyl and SO2.
Thus by maintaining as high as possible a
free SO2 level in beer, perhaps by adding
metabisulphite (within legal constraints),
the equilibrium is maintained in favour of
the binding of carbonyl.
The problem is that SO2 is progressively
lost from beer in reaction(s) which have not
yet been identified (85), thereby shifting
the balance in favour of carbonyl release.
Loss of SO2 is brand sensitive and, in partic-
ular, is promoted by increased temperature
(another clear justification for keeping beer
cold): at 40°C half of the SO2 in beer is lost in
27 days, whereas the half life is approxi-
mately 3 years at 0°C (86).
1999/II BRAUWELT INTERNATIONAL 105

The only endogenous source of SO2 in
beer is through production by yeast, from
the reduction of sulphate in water and grist
materials. Factors influencing the extent of
SO2 production by yeast will be reviewed
later.
Yeast
Perhaps the most powerful “reducing
agent” involved in brewing is yeast. Apart
from producing sulphur dioxide, it is also
capable of reducing carbonyl compounds
(other than those in adduct form with SO2)
to their equivalent alcohols (87). That is,
yeast is capable of rectifying the end-
products of oxidation (and other reactions
leading to carbonyls) which may have oc-
curred in the brewhouse.
Assuming that the reduced products of
yeast action are not re-convertible to stal-
ing aldehydes, it could be said that this is
another argument in favour of promoting
upstream oxidation: any carbonyls not vol-
atilised in the kettle boil will either be re-
duced by yeast or driven off with the fer-
mentation CO2, unless, that is, they have
first become associated with SO2.
Several enzymes in yeast seem to be in-
volved in the reduction of carbonyl com-
pounds (88 – 92), amongst them being alco-
hol dehydrogenase, aldose reductase and
two 3-methylbutanal reductases. It seems
that it is the rate of uptake of carbonyls
which is the rate limiting step in this reduc-
tion (88).
Chelation
Apart from the polyphenols, there are a
myriad of other species in wort and beer
which have a strong ability to chelate metal
ions, notable amongst them being amino
acids, phytic acid (93) and, of course, mela-
noidins (94).
In beer, there will be an equilibrium for
any pool of metal ions between a propor-
tion which is free (hydrated), and propor-
tions which are bound with individual
chelating agents. The nature of this “specia-
tion” will depend on the affinities of the var-
ious agents for the metal ion (binding con-
stant) and their accessibility to it. The fact
is that transition metal ions bound with
chelators sometimes have less capability to
promote oxygen radical formation, but
sometimes more (95). This may differ be-
tween chelators.
In the absence of any clear understand-
ing of the implications of this in a system
such as beer, the only realistic expedient is
to minimise the opportunity for ions such
as copper and iron to gain access to the
product at any stage in the process.
106 BRAUWELT INTERNATIONAL 1999/II
INTERNATIONAL
TECHNICAL FEATURE
■ The brewing process in relation to
flavour stability
Grist
Malt clearly comprises a complex mix of
(possibly) pre-formed staling precursors,
substrates for staling reactions, enzymes
capable of promoting flavour deterioration
(lipoxygenases, hydroperoxide isomerase),
enzymes capable of scavenging oxygen and
oxygen radicals (peroxidases (72) and su-
peroxide dismutases (96)), other substanc-
es which can react with oxygen (e.g. sulphy-
dryl groups in proteins) and various anti-
oxidants, amongst which are polyphenols,
phenolic acids and melanoidins.
Assuming that lipoxygenase activity is
unwanted in malt, then it is clear that malts
kilned to relatively intense regimes will con-
tain pro rata less lipoxygenase. Of course,
the malt must meet all other specifications
laid down by the brewer, and the desire for
low lipoxygenase should be balanced
against the risks of reduced levels of other
heat-labile enzymes (such as beta-gluca-
nase), excess colour and insufficient S-
methylmethionine (for those brewers seek-
ing to achieve a finite level of dimethyl sul-
phide in their lagers). It has been suggested
that barleys and malts may be selected on
the basis of their lipoxygenase level, but
reports studied by this author suggest that
the variations to be observed between vari-
eties and growth locations are limited.
The question remains (see earlier) con-
cerning the extent to which lipoxygenase
action during germination establishes a pre-
formed staling potential in malt. If it does, it
would be expected that any technique
which reduces embryo growth during malt-
ing would be to the benefit of flavour stabil-
ity: for example, use of agents such as potas-
sium bromate (where permitted), employ-
ment of high hydrostatic pressures etc. (97).
More highly kilned malts (and especially
roasted malts) have increased antioxidant
levels (98). When standardised for colour
delivery, roasted malts still possess a some-
what higher antioxidant activity, and this
can be separated on a size basis from col-
our (99). Notwithstanding this, Boivin et al
observed from measurements throughout
the malting process that the level of reduc-
ing compounds increased most extensively
during germination and that, if anything,
kilning was to the detriment of reducing
power (33). In relation to this, Lustig ob-
serves that malts with increased Kolbach
Index (and higher colour) gave increased
levels of staling substances in ensuing beer
(10). Furthermore as the final kilning tem-
perature was increased from 70, through 80,
to 90°C, so did the level of staling aldehydes
increase. Such observations highlight that
factors such as increased temperature kiln-
ing regimes could, on the one hand, be ben-
eficial in respect of elevated levels of anti-
oxidants and reduced levels of lipoxygen-
ase, but detrimental because of direct gen-
eration of staling substances. Lustig (10)
advocates the avoidance of thermal load in
order to avoid the development of staling
substances and their precursors by Mail-
lard reactions and Strecker degradation.
Certain adjuncts (for example sugars)
will dilute both the pro- and anti-oxidant
components contributed by malt. For other
types of adjunct it is no simple matter to
predict their effect. In one of the few studies
of the impact of adjuncts, Peppard et al
(100) concluded that (in pilot scale trials
with 30% malt replacement) rice and maize
grits decreased flavour stability, whereas
wheat flour, barley grits and maize syrup
enhanced shelf life.
Brewhouse
Considerable quantities of oxygen may
be consumed in the brewhouse and, whilst
it is proving difficult to categorically estab-
lish a major adverse effect on staling, equal-
ly it is difficult to rationalise how this could
be of any benefit whatsoever to flavour life
of beer (although oxidation in the brew-
house unquestionably enhances haze life,
101). Add in the risk to colour formation
and interference with rates of wort separa-
tion, then it is a worthwhile objective to
minimise the access of air to beer through-
out wort production.
The question remains as to the extent of
the investment which is warranted. Thus,
trials using the blanketing of all brewhouse
vessels with inert gas (nitrogen or carbon
dioxide) have shown clear benefits on small
scales (62, 63, 69) but this has not always
translated to the commercial scale, where
its application would in any event present
a formidable technological challenge. It is
a similar case with precautions such as
sparging the grist case with nitrogen or
deaerating sparge liquor. Even so, there are
reports of some commercial adjustments
which have been established, for example
the use of nitrogen injection into the
pressure relief system across the plates in
a lauter tun (102). O’Rourke and his col-
leagues (103) indicate that the benefits of
minimising oxidation in the brewhouse are
more substantial in respect of the taste of
fresh beer than in terms of extending shelf
life.
Various straightforward precautions can
be taken to lessen the ingress of air in the
brewhouse. These include use of a pre-
masher (c.f. Steel’s masher) to enable inti-
mate mixing of grist and water under low air
conditions; filling of vessels by bottom en-
 INTERNATIONAL
TECHNICAL FEATURE

try; not switching on agitators until they are has recently advocated the use of a trub dioxide and its ability to reduce carbonyl
covered and then only using them where separator to enhance shelf life (109). compounds.
absolutely necessary to ensure equilibra-
tion of vessel contents and temperature; Fermentation In respect of the latter then it is im-
good housekeeping in respect of pipe and portant to ensure the vigour of a fermen-
pump integrity; switching off pumps as Clearly oxygen is needed by yeast to pro- tation using established procedures for
soon as vessel contents have been trans- mote efficient fermentation: this should be having yeast of high viability and vital-
ferred, to avoid the risk of sucking air into introduced on the cold side of the heat ex- ity, pitched in the appropriate quanti-
the system; closing lids on kettles; use of changer and only as much as is needed by ties.
nitrogen as a motor gas. the strain in use. In the context of flavour
stability, direct oxygenation of yeast can Concerning the former, SO2 levels will
Assuming that lipoxygenase is disadvan- only be helpful as it precludes any opportu- be increased if the sulphate supply to
tageous for flavour stability, then its action nity for wort oxidation (110). the yeast is increased, wort clarity is in-
will be reduced by lowering the level of one creased, wort oxygenation and pitching
of its substrates, oxygen, but also by mash- Yeast has at least two direct impacts on rate are lowered and fermentation tem-
ing-in at the highest temperature commen- flavour life: its ability to produce sulphur perature is reduced (84).
surate with other mashing requirements. It
has recently been suggested that flavour
stability will be increased by restricting the
extent to which lipoxygenase can be ex-
tracted into the mash, notably by applying
milling regimes which leave the embryo in-
tact (104). Additionally, reduction of the
mashing pH to 5.1 has been proposed as a
further mechanism for reducing lipoxygen-
ase action (105).

Concerning autoxidation reactions, then

it could be rationalised that beer intended
for prolonged shelf life should not be
brewed using copper vessels, for these will
provide a sizeable charge of ions capable of
activating oxygen to its damaging radical
forms. Indeed both van Gheluwe (106) and
Narziss (63) have observed reduced flavour
stability in beers produced in copper-lined
equipment. However much beer of excel-
lent shelf life has been produced through
copper-based brewhouses. The reader is
again reminded of the phenomenon of spe-
ciation, viz the form in which a metal ion
such as copper is to be found in the mash. If
it is chelated with certain species, it may
not be accessible to oxygen or even be in a
state capable of causing radical formation.

Conflicting influences may be at play in

the kettle boil: on the one hand there is the
opportunity for loss of reducing power if
there is significant air ingress, but converse-
ly there is the opportunity for driving off
pre-formed staling substances. Thus Buckee
and Barrett (107) found that more stable
beer is produced with a 1h as opposed to
0.5h boil, with the length of boil being more
important than the degree of evaporation
(provided the latter was at least 2%).

Lustig and his colleagues (108) have sug-

gested that thermal degradation reactions
at the whirlpool stage generate undesirable
flavour compounds and that application of
a vacuum (0.5 bar) after this stage is advan-
tageous.

As discussed previously, there is no firm

evidence that enhancing the clarity of wort
has any benefit for flavour life, although Eils
1999/II BRAUWELT INTERNATIONAL 107

Yeast strains capable of increased sul-
phite production have been developed
(111, 112).
Downstream processing and
packaging
Downstream of the fermenter the over-
riding priority is to minimize oxygen pick-up.
The practical opportunities for this include
use of nitrogen or carbon dioxide as a motor
gas; blanketing vessels with either of these
gases prior to filling; rigorous deaeration of
water (to less than 30 ppb O2) used for any
purpose (other than cleaning) downstream:
(e.g. dilution, slurrying of filter aids, make
up of additions); precautions with pumps
and pumping and leaks (as described earlier
for brewhouse). A realistic target for bright
beer prior to packaging should be 50 ppb.
Cold beer reacts only slowly with oxygen
and therefore any high O2 levels can be cor-
rected at this stage (it is not possible to rec-
tify O2 ingress into hot streams in the same
way) by sparging with inert gas or, prefera-
bly, using hydrophobic membrane technol-
ogy of the type described by Gill (113).
Once the gas content of bright beer has
been brought into specification it is critical
that further ingress of oxygen should be
minimised. Good housekeeping precau-
tions have already been described, but at
the packaging stage it is crucial for “miserly
oxygen packaging”: that filler bowls are
counterpressured with inert gas and that
the bowl is continuously purged with the
gas; that containers are pre-evacuated pref-
erably twice, separated by a CO2 purge; that
the beer is fobbed prior to capping (bot-
tles) or seaming (cans), e.g. through use of
high pressure water jetting for bottles or
undercover gassing for cans.
Exogenous antioxidants and
protectants
Various antioxidants may be used in beer
(depending on regional legislation), with
the most prominent of these being sulphur
dioxide and ascorbic acid. The former is
demonstrably the more effective, either
through its role as a carbonyl binder or as a
radical scavenger. In the latter instance
(and this applies to other antioxidants of
this type, including ascorbic acid) it must
be remembered that these agents can only
protect against new oxidation occurring in
the beer: they will not rectify any damage
which has occurred upstream (79, 114).
However one intriguing suggestion is the
inclusion of enzymes in beer capable of re-
ducing pre-formed aldehydes to their
equivalent carboxylic acids, which do not
contribute to flavour (115).
Thus such antioxidants will to a limited
extent counter the adverse effects of oxy-
108 BRAUWELT INTERNATIONAL 1999/II
INTERNATIONAL
TECHNICAL FEATURE
gen during filling and pasteurisation. They
will certainly be helpful in protecting
against oxygen leaking into bottled beer
through the crown cork-bottle seal, which
is now established as a serious problem
(116). Thus there has been the advent of
oxygen barrier and oxygen scavenging bot-
tle closures (117, 118). Some of these have
been demonstrably beneficial, but others
have caused concerns for their adverse ef-
fect on flavour (118).
Whilst oxygen ingress into cans in stor-
age and trade should not be a problem, it
certainly has been a problem limiting the
development of plastic bottles as a main-
stream packaging medium for beer. Howev-
er, the advent of plastics with enhanced
barrier properties (119) has already led to
some mainstream brands being filled into
lightweight, robust and attractive packages
and this type of container seems certain to
occupy a much larger proportion of the
beer market in the future.
■ Predictive tests
As per normal in brewing, there is an ob-
session with having tests which can be ap-
plied to beer to predict how long its flavour
life will hold up. This author contends for
this (and for other issues of product stabil-
ity) that such tests are as often as not a
waste of time. The only merit in having tests
for shelf life is if they enable the brewer to
respond in some way such that stability is
enhanced, there being absolutely no bene-
fit from knowing that a product in package
is going to have a low flavour life (unless the
brewer is prepared to withdraw such beer
from trade or even refuse release to trade,
which would be a prohibitively expensive
option). Worthwhile procedures might be
applied in two ways:
a. those which can be applied in a process
research context in order to establish
raw materials, process conditions etc.
which will lead to enhanced product
stability;
b. those which could be applied in a QA/QC
set-up to indicate raw material, process
and product status to enable the brewer
to make adjustments in order to favour
shelf life.
To expedite the study of what is inherent-
ly (and preferably!) a long term phenome-
non, several researchers have developed
forced ageing procedures.
Greenhoff and Wheeler (8) for instance,
advocate holding beer at either 60°C for 22
hours or 37°C for 3 weeks prior to evalua-
tion by tasting, claiming that both are a
good mimic for 6 months at 18°C. Lustig’s
procedure (10) involves shaking beer (to
simulate transport – a factor seldom con-
sidered in the context of flavour life) fol-
lowed by 4 days of storage at 40°C. He
claims that this equates to 3 – 4 months at
20°C.
As an alternative to tasting, some have
advocated the chemical monitoring of spe-
cies produced in forced ageing techniques.
Grigsby (120) and later Parsons and Cope
(121) used thiobarbituric acid (TBA) to
measure carbonyl species produced on
forced ageing, but TBA is not a very specific
agent and preferentially reacts with
malondialdehyde, which is but one of the
breakdown products from unsaturated fat-
ty acids. Another breakdown product is
ethylene, which has also been cited as an
indicator of staling potential (122)
Drost and his colleagues (123) developed
the concept of “nonenal potential” for the
assessment of oxidation in the brewhouse.
Worts are heated to release nonenal which
is measured: higher levels of nonenal are
said to relate to more extensive oxidation
during wort production and worts with
high nonenal potential are believed to pro-
ceed to beers with a greater propensity to
staling.
Other species are sometimes measured
directly as indices of staling. Notable
amongst these is furfural, which is not be-
lieved to directly contribute to staling per
se but is felt to be a good yardstick for oxi-
dation (124). Bright and his colleagues
found that, whereas furfural correlated
well with oxidation in beers aged at 22°C or
45°C, trans-2-nonenal only predicted staling
when measured after forcing at the higher
temperature (6). A species such as 10-un-
decanal correlated positively with oxida-
tion after ageing at 45°C, but negatively at
22°C. Bright was able to develop a model
for predicting shelf life based on the meas-
urement of several compounds by solid
phase extraction and direct desorption
onto a gas chromatography column – fur-
fural and phenylethylacetate inter alia
were in the model but, interestingly, none-
nal was not.
Particular interest has been paid in re-
cent years to the direct measurement of
radicals in beer and its process stream.
This is rationalised on the basis that it is the
radical forms of oxygen which are key to
flavour instability (31), therefore tools to
measure the level of radicals in beer should
give the best possible indication of staling
potential.
Kaneda and his colleagues at Sapporo
have championed the measurement of
chemiluminescence, both directly and after
reaction of beer with the radical scavenger
2-methyl-6-phenyl-3,7-dihydroimidazo (1,2-
a) pyrazin-3-one (CLA) (125 – 128). Their

recommendation is to ally the use of chemi-
luminescence to the use of 1,1-diphenyl-2-
picryl-hydrazyl (DPPH) as a measure of re-
ducing power in beer (DPPH being claimed
to be a preferred alternative to 2,6-dichlo-
rophenol-indophenol and the Indicator
Time Test). It seems that DPPH correlates
with polyphenol species, though not with
SO2 and that the value increases through
brewhouse operations, but declines during
fermentation.
Ono’s team at Suntory, by contrast, advo-
cate the measurement of radicals by the
application of electron spin resonance
technology (ESR) (129, 130). They have de-
veloped the concept of an EA (Endogenous
Antioxidant) value, which is the time taken
before an ESR signal is developed in their
ageing test: the longer the lag, the greater
the antioxidant potential of the sample.
Amongst the interesting observations
made using this approach, it was shown
that the EA value is especially developed
during fermentation (due to SO2 produc-
tion?) and that temperature of storage
seemed to have a much bigger effect than
level of oxygen on the development of sig-
nals due to radical species (129).
This finding should be compared with
that of Walters et al (9) that raising tem-
perature from 0°C, through 25°C, to 40°C
has a disproportionate effect inter alia on
the loss of iso-alpha-acids from beer and
on the levels of furfural developed.
Lustig, too, says that the most important
factor determining stale flavour develop-
ment is temperature, albeit not all com-
pounds increase in level with increased
temperature (10).
Such observations are the clearest of in-
dications of the risks involved in any type of
forced ageing study. Takaoka et al (131)
found that EA values benefited greatly from
any opportunity to minimise the extent of
heating in the brewhouse.
A somewhat less expensive technique,
albeit one which is not yet satisfied by hav-
ing robust instrumentation commercially
available, is the measurement of redox po-
tential (132, 133). Such values give an over-
all indication of the oxidation-reduction
status of a sample and would be expected,
for instance, to give a more meaningful indi-
cation of oxidative damage when applied to
beer than would the measurement of oxy-
gen, because oxygen will be rapidly con-
sumed in package, particularly during pas-
teurisation.
Simpler, colorimetric techniques for as-
sessing oxidative damage include the use of
iodine staining (134) or the measurement of
free thiol groups using 5,5’-dithiobis (2-ni-
trobenzoic acid) (DTNB) (69).
INTERNATIONAL
TECHNICAL FEATURE
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TECHNICAL FEATURE

1999/II BRAUWELT INTERNATIONAL 111