Enzymes in brewing

Sten Aastrup and Hans Sejr Olsen, Novozymes. hso@novozymes.com

Even for an old industry like beer brewing new industrial processes benefit from
using enzymes developed from microbial sources. In the last years quality issue
s like flavour control, beer stability and general cost savings in the industry
go hand in hand with efficient solutions of environmental problems. Future aspec
ts focus on a wider application of enzymes to brew with high amounts of inexpens
ive 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 li
fe 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 ba
rley contain starch that has to be broken down to fermentable sugars before the
yeast can make alcohol. Therefore, traditional brewing contains and extra step c
ompared with wine-making, namely malting in which enzymes needed for the degrada
tion of starch into fermentable sugars are produced.
Malt is germinated barley or other cereals like wheat and sorghum: First the gra
ins are steeped bringing the water content from about 12% to 45%, then they are al
lowed to germinate for 4-6 days and finally the germination is stopped by heatin
g (kilning) reaching a final moisture content of about 4%. Some enzymes are alre
ady present in the barley, e.g. -amylases, but the majority of enzymes are produc
ed during the germination, e.g. a-amylases and proteases, and in the final malt
all the enzymes needed for the conversion of grains into a fermentable liquid (wor
t) is present (Figure 1 and 2)
Figure 1: Germinating barley kernel
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 ma
lt has become a purchased raw material, like other raw materials. This means tha
t 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 tempera
tures, 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 exp
ress wanted enzyme activities that are not present in malt. Addition of exogeno
us enzymes at various steps during the brewing process can therefore make brewin
g 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 w
ith malt enzymes alone. Also the possibility to improve beer quality by avoiding
off-flavours is possible with commercial enzymes. The increasing concern on res
ources and CO2- emission has also put the use of commercial enzymes within the b
rewing 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.

Click for a larger version

Figure 3: The traditional mashing temperature profile is determined by the tempe
rature optima for the various malt enzymes. Larger version here.
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 mas
h. 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 prot
eases works best at 52 C, the -amylase best at 63 C and the a-amylase at 72C. The la
st step in the mashing is inactivation of the enzymes at 78 C (Figure 3).
If -glucan and protein are properly broken down during malting, single temperatur
e 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. a-amyla
se liquefy the gelatinized starch by hydrolysis of the a-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 resul
ting 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 ferm
entable sugars. After fermentation, the so-called green beer is matured before fi
nal filtration and bottling. Fig. 4 shows a diagram of the brewing process and w
here external enzymes are used for process aids.
Click for a larger version
Figure 4: The processing steps in brewing where exogenous enzymes can be added.
Larger version here.
Commercial enzymes from exogenous sources
The traditional source of enzymes used for the conversion of cereals into beer i
s 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 sep
aration will take too long; the fermentation process will be too slow; too littl
e alcohol will be produced; the beer filtration rate will be reduced; and the fl
avour and stability of the beer will be inferior.
Exogenous enzymes are used to supplement the malt s own enzymes in order to preven
t these problems. Furthermore, industrial enzymes are used to ensure better adju
nct liquefaction, to produce low-carbohydrate beer ( light beer ), to shorten the be
er maturation time, and to produce beer from cheaper raw materials.
The various steps of the brewing operations, where microbial enzymes are occasio
nally added, are shown in table 1. Enzymes, enzymic action and their functions a
re summarized.
Enzymes at work
Quality and supply constraints on malt, and doubling of malt prices have given i
ncreased 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 short
en the production time, increase capacity, and to allow use of raw material alte
rnative 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 possibiliti
es of producing light beer also called low calorie beer .
An enzyme solution for diacetyl control after fermentation improves vessel utili
zation, save energy and ensures a high beer quality after a reduced maturation t
ime.
Operation
Enzymes
Enzyme action
Function
Decoction vessel (cereal cooker)
a-amylase
Hydrolyse starch
Adjunct* liquefaction.
Reduce viscosity
-glucanase
Hydrolyse glucans.
Aid the filtration.
Mashing
a-amylase
Hydrolyse starch.
Malt improvement.
Amyloglucosidase
Increase glucose content.
Increase % fermentable sugar in light

beer.

Debranching enzyme
Hydrolyse a-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 separation.
Pentosanase/xylanase
Hydrolyse pentosans of malt, barley, wheat.
Improve extraction and beer filtration.
Fermentation
Fungal a-amylase
Increase maltose and glucose content.
Increase % fermentable sugar in

light

beer.

-glucanase
Hydrolyze glucans.
Reduce viscosity and aid filtration.
a-acetolactate- decarboxylase (ALDC)
Converts a-acetolactate to acetoin directly.
Decrease fermentation time by avoiding formation of diacetyl.

Conditioning tank
Protease
Modify protein-polyphenolic compounds.
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 us
ing high-quality malts. At higher levels of barley or using undermodified malts,
processing becomes more difficult. In these cases the mash needs to be suppleme
nted 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 a-amylase, -glucanase and prote
ase 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, barle
y and maize grits, the following raw materials, liquefaction - and mashing enzym
es 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 a-amylase.
CeremixPlus is an enzyme preparation containing -glucanase, xylanase, a-amylase an
d 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 separate
ly with help of the a-amylase TermamylBrewQ at 96?C for 30 minutes, through a sho
rt holding time at 70?C. It is stabilised by approximately 100 ppm Ca++ at a wat
er-to-adjunct ratio of approximately 4:1. Milled malt and barley are mashed-in a
t a temperature of 50?C. After 30 minutes the adjunct mash from the decoction ve
ssel is added to increase the temperature to 63-66?C. After 60 minutes the mash
is heated to hold at 76-78?C until starch-negative (no blue colour is formed wit
h iodine in potassium iodide). Hereafter the wort separation is made in the laut
er tun.
Brewing with high amounts (>50%) of barley instead of malt is now possible thank
s to the introduction of the new enzyme system UltrafloMax (2).
Enzymes to improve fermentation
Small adjustments in fermentability can be achieved by adding amyloglucosidase a
lone or in combination with debranching enzymes at mashing-in or a fungal a-amyl
ase 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 ferm
entability.
Figure 6. Total fermentable sugar production with different dosages of Attenuzym
e (kg per ton malt) and extended mashing at 63 C
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 dos
ages of enzymes is necessary to produce extremely high attenuated beer (see figu
re 6).
Fungal a-amylases are used to produce mainly maltose and dextrins whereas amylog
lucosidase 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 li
mit (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 l
evel where it can t be tasted.

Diacetyl is formed by the non-enzymatic oxidative decarboxylation of a-acetolact

ate, which is produced by the yeast during primary fermentation. The yeast remov
es 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 tas
teless compared with diacetyl.
By adding the enzyme a-acetolactate decarboxylase (ALDC) (e.g. Novozymes Maturex)
at the beginning of the primary fermentation process, it is possible to bypass t
he diacetyl step (Figure 7) and convert a-acetolactate directly into acetoin. Mo
st of the a-acetolactate is degraded before it has a chance to oxidise and less
diacetyl is therefore formed. This makes it possible to shorten or completely el
iminate the maturation period (3) and (4). The brewery enjoys greater fermentati
on and maturation capacity without investing in new equipment.
Click for a larger version
Figure 7. The removal of a-acetolactate during fermentation. Larger version her
e.
Current enzyme solutions provided by Novozymes A/S
Effective Cereal Cooking
Heat stable enzyme preparations like the a-amylase TermamylBrewQ have 200-300 tim
es more liquefaction power than malt. Just 0.25 kg of Termamyl can replace 100 k
g of malt. The result is 0.5-2% more extract yield, shorter cooking cycles, no r
isk of residual starch being carried over into the mashing vessel and more flexi
bility 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 s
ource 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 ensu
re sufficient FAN, extract yield, filterability, and fermentability for high qua
lity index final beers. Cost effective adjunct and malt solutions are made with glucanase, xylanase, a-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-m
alt brew with well-modified malt and exogenous enzymes like UltrafloMax. Faster t
hroughput 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 defect
s 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 af
ter fermentation improving vessel utilization, energy savings and ensuring a hig
h quality index of the final beer. Maturation time can be reduced several days u
p 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 consist
ent attenuation or developing a brand extension with a highly attenuated beer us
ing e.g. Attenuzyme. Improved attenuation control is made with starch hydrolysing

enzyme like a-amylase, amyloglucosidase, pullulanase (debranching enzyme).

Conclusion and future perspectives
To day several brewing groups use exogenous enzymes as a strategic tool to optim
ize the brewing process and the brewing capacity. More and more breweries also t
hink 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 enzy
mes will bring:
* No aging of beer within one year: Exogenous enzymes might prevent developm
ent of aging components due to oxidation, keeping the taste of fresh beer for ex
tended time regardless of the storage conditions.
* Beer made from all-barley with the same taste as from all-malt: An enzyme p
ackage 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 f
or production of wort and beer was patented in 2004 (5). The mash liquefaction p
rocess 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 m
icrobial enzymes. The fermentation was made simultaneously with the saccharifica
tion. 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 sig
nificant role in waste water treatment. An overview of significant improvement a
nd operation processes and economic reality was described by Fillaudeau et al. (
6).
* ...or something completely different!