Enzymes in brewing

Even for an old industry like beer brewing new industrial processes benefit from using enzymes
developed from microbial sources. In the last years quality issues like flavour control, beer stability
and general cost savings in the industry go hand in hand with efficient solutions of environmental
problems. Future aspects focus on a wider application of enzymes to brew with high amounts of
inexpensive raw materials like barley. Alternative beer processes for production of wort and beer
with higher productivity and reduced amounts of waste and by-products are under development.
Introduction
Beer and wine are both alcoholic beverages which have been part of our social life for thousands of
years. Both beverages are produced by yeast fermentation of sugars. Wine is based on grapes,
and beer is traditionally based on barley. The matured grapes already contain the sugars needed
for the fermentation, while barley contain starch that has to be broken down to fermentable sugars
before the yeast can make alcohol. Therefore, traditional brewing contains and extra step
compared with wine-making, namely malting in which enzymes needed for the degradation of
starch into fermentable sugars are produced.

Figure 1: Germinating barley kernel
Malt is germinated barley or other cereals like wheat and sorghum: First the grains are "steeped"
bringing the water content from about 12% to 45%, then they are allowed to germinate for 4-6
days and finally the germination is stopped by heating (kilning) reaching a final moisture content
of about 4%. Some enzymes are already present in the barley, e.g. -amylases, but the majority
of enzymes are produced during the germination, e.g. -amylases and proteases, and in the final
malt all the enzymes needed for the conversion of "grains" into a fermentable liquid (wort) is
present (Figure 1 and 2)

Figure 2. Enzyme production during malting, ref. Aastrup et al. (2004)
In former days, production of malt was an integrated part of every brewery, but to day most malt
is produced outside the brewery in large malt factories, and malt has become a purchased raw
material, like other raw materials. This means that the breweries to day are more flexible in the
use raw materials, and for that matter for the source of enzymes.
The malt enzymes do have some limitations. They can only work at certain temperatures, pH
values etc., and the activities might be too low to do a proper job in proper time. In contrast,
commercial exogenous enzymes can be designed to work at preferred temperatures and pH
values, to have more enzymatic power, or to express wanted enzyme activities that are not
present in malt. Addition of exogenous enzymes at various steps during the brewing process can
therefore make brewing easier, faster and more consistent. It gives the brewmasters extra
flexibility in the choice of raw materials due to less dependence on malt enzymes, as well as
providing opportunity to create new products, which is not possible to make with malt enzymes
alone. Also the possibility to improve beer quality by avoiding off-flavours is possible with
commercial enzymes. The increasing concern on resources and CO2- emission has also put the use
of commercial enzymes within the brewing industry in focus. By the use of exogenous enzymes
more can be extracted from the raw materials, more local raw materials can be used, and more
unmalted grains can be used, saving significant amounts of energy and transport.
The brewing process
Traditionally, beer is produced by mixing crushed barley malt and hot water in a mash copper to
perform the mashing. Besides malt, other starchy cereals such as maize, sorghum, rice and
barley, or pure starch itself, can be added to the mash. These are known as adjuncts.
The standard mashing for pilsner type beer consists of several temperature steps, each favouring
different malt enzyme activities. The lowest temperature (45 C) is the optimal temperature for
cell wall degrading enzymes, -glucanases. The proteases works best at 52 C, the -amylase best
at 63 C and the -amylase at 72C. The last step in the mashing is inactivation of the enzymes at
78 C (Figure 3).

Figure 3: The traditional mashing temperature profile is determined by the temperature optima for the various malt
enzymes. Larger version
If -glucan and protein are properly broken down during malting, single temperature mashing at
65-71C has shown to be sufficient, as in the case of traditional ale brewing.
During mashing the starch is degraded to dextrin and fermentable sugars. -amylase liquefy the
gelatinized starch by hydrolysis of the -1,4 linkages at random. -amylases are exo-enzymes
which attack the liquefied starch chains resulting in successive removal of maltose units from the
non-reducing end.
After mashing, the mash is sieved in a lauter tun or on a mash filter. The resulting liquid, known
as sweet wort, is then transferred to the copper, where it is boiled with hops. The hopped wort is
cooled and transferred to the fermentation vessels, where yeast is added. In normal wort 2/3 of
the carbohydrates are fermentable sugars. After fermentation, the so-called green beer' is
matured before final filtration and bottling. Fig. 4 shows a diagram of the brewing process and
where external enzymes are used for process aids.

Figure 4: The processing steps in brewing where exogenous enzymes can be added. Larger version.
Commercial enzymes from exogenous sources
The traditional source of enzymes used for the conversion of cereals into beer is barley malt. If too
little enzyme activity is present in the mash, there will be several undesirable consequences: the
extract yield will be too low; wort separation will take too long; the fermentation process will be
too slow; too little alcohol will be produced; the beer filtration rate will be reduced; and the flavour
and stability of the beer will be inferior.
Exogenous enzymes are used to supplement the malt's own enzymes in order to prevent these
problems. Furthermore, industrial enzymes are used to ensure better adjunct liquefaction, to
produce low-carbohydrate beer (light beer'), to shorten the beer maturation time, and to produce
beer from cheaper raw materials.
The various steps of the brewing operations, where microbial enzymes are occasionally added, are
shown in table 1. Enzymes, enzymic action and their functions are summarized.
Enzymes at work
Quality and supply constraints on malt, and doubling of malt prices have given increased interest
for enzyme solutions in 2007 and 2008. Many breweries has run programs within the last two
years in order to increase efficiency and optimize raw material usage, and many of them have
focused on commercial enzymes to shorten the production time, increase capacity, and to allow
use of raw material alternative to malt. Three important examples are mentioned:
Exchanging part of the malt with barley has been popular because using barley in combination
with commercial enzymes gives the same beer quality as with malt.
Introducing a higher content of starch hydrolysing enzymes offer the possibilities of producing
"light beer" also called "low calorie beer".
An enzyme solution for diacetyl control after fermentation improves vessel utilization, save energy
and ensures a high beer quality after a reduced maturation time.
Operation Enzymes Enzyme action Function
Decoction vessel
(cereal cooker)
-amylase Hydrolyse starch Adjunct* liquefac-
tion.
Reduce viscosity
-glucanase Hydrolyse glucans. Aid the filtration.
Mashing -amylase Hydrolyse starch. Malt improvement.
Amyloglucosidase Increase glucose
content.
Increase % fermen-
table sugar in light
beer.
Debranching enzyme Hydrolyse -1,6
branch points of
starch.
Secures maximum
fermentability of the
wort.
Proteases Increase soluble
protein, and free
amino- nitrogen
(FAN).
Malt improvement
Improved yeast
growth.
-glucanase Hydrolyse glucans. Improve wort sepa-
ration.
Pentosanase/xylanase Hydrolyse pen-
tosans of malt,
barley, wheat.
Improve extraction
and beer filtration.
Fermentation Fungal -amylase Increase maltose
and glucose con-
tent.
Increase % fermen-
table sugar in light
beer.
-glucanase Hydrolyze glucans. Reduce viscosity and
aid filtration.
-acetolactate- decarboxylase
(ALDC)
Converts -ace-
tolactate to ace-
toin directly.
Decrease fermenta-
tion time by avoiding
formation of
diacetyl.
Conditioning tank Protease Modify protein-
polyphenolic com-
pounds.
Reduce the chill haze
formed in beer.
* Adjunct is starchy cereals such as maize, rice, wheat, sorghum, barley or pure starch materials
added to the mash.
Table 1. Steps of the brewing operations where microbial enzymes are used.
Brewing with barley
Traditionally, the use of barley has been limited to 10-20% of the grist when using high-quality
malts. At higher levels of barley or using undermodified malts, processing becomes more difficult.
In these cases the mash needs to be supplemented with extra enzyme activity if the brewer is to
benefit from the advantages of using unmalted barley while still maintaining brewing performance.
Brewers can either add a malt-equivalent blend of -amylase, -glucanase and protease at the
mashing-in stage or add the enzymes separately as required.
As an example of the production of 6000 litre pilsner type beer from malt, barley and maize grits,
the following raw materials, liquefaction - and mashing enzymes can be used:
Raw materials: Malt 475 kg
Barley 475 kg
Maize grits 400 kg
Liquefaction enzyme: TermamylBrewQ 0.15 kg
Mashing enzymes: CeremixPlus 0.50 kg
UltrafloMax 0.20 kg
TermamylBrewQ is an enzyme preparation containing a thermophilic -amylase.
CeremixPlus is an enzyme preparation containing -glucanase, xylanase, -amylase and
protease
UltrafloMax is an enzyme preparation containing -glucanase and arabinoxylanase.

Figure 5. Mashing diagram for barley brewing (an example).
The mashing diagram is shown in figure 5. The maize grits are liquefied separately with help of the
-amylase TermamylBrewQ at 96C for 30 minutes, through a short holding time at 70C. It is
stabilised by approximately 100 ppm Ca++ at a water-to-adjunct ratio of approximately 4:1.
Milled malt and barley are mashed-in at a temperature of 50C. After 30 minutes the adjunct
mash from the decoction vessel is added to increase the temperature to 63-66C. After 60 minutes
the mash is heated to hold at 76-78C until starch-negative (no blue colour is formed with iodine
in potassium iodide). Hereafter the wort separation is made in the lauter tun.
Brewing with high amounts (>50%) of barley instead of malt is now possible thanks to the
introduction of the new enzyme system UltrafloMax (2).
Enzymes to improve fermentation
Small adjustments in fermentability can be achieved by adding amyloglucosidase alone or in
combination with debranching enzymes at mashing-in or a fungal -amylase at the start of
fermentation.
To describe to which extent the extracted sugars are fermentable brewers define degree of
attenuation, which is synonymously with degree of fermentation or fermentability.

Figure 6. Total fermentable sugar production with different dosages of Attenuzyme (kg per ton malt) and extended
mashing at 63C
Beer types with very high attenuation ("light beer" or "low calorie beer") are most often produced
using amyloglucosidase alone. Extended mashing at 63C and high dosages of enzymes is
necessary to produce extremely high attenuated beer (see figure 6).
Fungal -amylases are used to produce mainly maltose and dextrins whereas amyloglucosidase
produces glucose from both linear and branched dextrins.
Diacetyl control
An important question for brewers is "When exactly is a beer mature?", because this determines
when they can "rack" the beer to make way for the next batch. The simple answer to the above
question is when the diacetyl level drops below a certain limit (about 0.07 ppm). Diacetyl gives
beer an off-flavour like buttermilk and one of the main reasons for maturing a beer is to allow the
diacetyl to drop to a level where it can't be tasted.
Diacetyl is formed by the non-enzymatic oxidative decarboxylation of -acetolactate, which is
produced by the yeast during primary fermentation. The yeast removes the diacetyl again during
the beer maturation stage by conversion to acetoin, which has a much higher flavour threshold
value. In fact, acetoin is almost tasteless compared with diacetyl.
By adding the enzyme -acetolactate decarboxylase (ALDC) (e.g. Novozymes' Maturex) at the
beginning of the primary fermentation process, it is possible to bypass the diacetyl step (Figure 7)
and convert -acetolactate directly into acetoin. Most of the -acetolactate is degraded before it
has a chance to oxidise and less diacetyl is therefore formed. This makes it possible to shorten or
completely eliminate the maturation period (3) and (4). The brewery enjoys greater fermentation
and maturation capacity without investing in new equipment.

Figure 7. The removal of -acetolactate during fermentation.
Current enzyme solutions provided by Novozymes A/S
Effective Cereal Cooking
Heat stable enzyme preparations like the -amylase TermamylBrewQ have 200-300 times more
liquefaction power than malt. Just 0.25 kg of Termamyl can replace 100 kg of malt. The result is
0.5-2% more extract yield, shorter cooking cycles, no risk of residual starch being carried over
into the mashing vessel and more flexibility to change adjunct ratios and types.
Effective Adjunct and Malt Solutions
Brewers who desire raw material cost savings or use of local raw materials may source under-
modified malts or increase the ratio of adjunct. The limiting factor is to ensure an adequate
complex of enzymatic activities for high-quality wort. The enzyme suppliers offers a range of
blended products like CeremixPlus to ensure sufficient FAN, extract yield, filterability, and
fermentability for high quality index final beers. Cost effective adjunct and malt solutions are made
with -glucanase, xylanase, -amylase and protease.
Faster Throughput and More Extract
Even with good malts it is possible to achieve 40% longer beer filter cycle runs, 0.5% to 1% more
extract, and 0.5% reduced beer losses for more brews per day. The experience with UltrafloMax
has shown the benchmark for brewhouse and cellar performance should not be an all-malt brew
with well-modified malt, but an all-malt brew with well-modified malt and exogenous enzymes like
UltrafloMax. Faster throughput and more extract are made with -glucanase and
arabinoxylanase.
Optimal Fermentation and Maturation
Maturex prevents the formation of diacetyl, one of the most common flavour defects in lager
beers. Adding Maturex at the start of the primary fermentation process allows brewers to bypass
the rate limiting warm maturation or diacetyl stand after fermentation improving vessel utilization,
energy savings and ensuring a high quality index of the final beer. Maturation time can be reduced
several days up to 14 days.
Improved Attenuation Control
Controlling fermentability of the wort enables brewers to grow their business by taking advantage
of changing consumer trends. Whether it is achieving a consistent attenuation or developing a
brand extension with a highly attenuated beer using e.g. Attenuzyme. Improved attenuation
control is made with starch hydrolysing enzyme like -amylase, amyloglucosidase, pullulanase
(debranching enzyme).
Conclusion and future perspectives
To day several brewing groups use exogenous enzymes as a strategic tool to optimize the brewing
process and the brewing capacity. More and more breweries also think in enzyme solutions for
development of new products.
The role of enzymes in tomorrows brewing industry, we do not know, but a lot of new
opportunities are now provided for the breweries. Maybe the future with enzymes will bring:
No aging of beer within one year: Exogenous enzymes might prevent development of aging
components due to oxidation, keeping the taste of fresh beer for extended time regardless of
the storage conditions.
Beer made from all-barley with the same taste as from all-malt: An "enzyme package" might
completely substitute the endogenous enzymes produced during malting. Elimination of the
malting process and less transportation can obviously save a lot of energy and CO2
emission.
Re-thinking the way of making beer: An efficient and inexpensive process for production of
wort and beer was patented in 2004 (5). The mash liquefaction process was a jet-cooking
and application of microbial enzymes. Wort was produced from de-hulled and de-germinated
grist. The mash was liquefied using entirely microbial enzymes. The fermentation was made
simultaneously with the saccharification. The amount of waste or by-products was reduced
significantly.
Better waste water control: Water and wastewater management constitutes a practical
problem for the brewing industry, and exogenous enzymes can play a significant role in
waste water treatment. An overview of significant improvement and operation processes and
economic reality was described by Fillaudeau et al. (6).
...or something completely different!
References
(1) Aastrup, Sten, Noel Bautista, Elmar Janser and Kurt Drreich. "Choice of enzyme solution
should determine choice of raw materials and process". Presentation given at World Brewing
Conference, San Diego, USA, 2004
(2) Aastrup, Sten, Claudio Visigalli, Jrg Obrecht, Marcel Mischler, Niels Elvig, Stefan Kreisz,
"Rethink beer filtration with new xylanase", Paper submitted for publication in Brauwelt (2008) -
special filtration issue.
(3) Aunstrup, K. and Olsen, F. Alpha-acetolactate decarboxylase enzyme and preparation thereof.
U.S.Patent 4,617,273. (1986)
(4) Rostgaard Jensen, B., Svendsen, I., Ottesen, M. Isolation and characterization of -
acetolactate decarboxylase useful for accelerated beer maturation. Proceedings of the European
Brewery Convention Congress, p. 393-400. (1987).
(5) Olsen, H. S. and Bisgaard-Franzen, H. Beer mashing process", WO 2004/050820 A1.
(6) Fillaudeau, L, Pascal Blanpain-Avet, Georges Daufin, Water, wastewater and waste
management in brewing industries", Journal of Cleaner Production 14 (2006) 463-471.