Wort boiling

Wort boiling
Objective
To identify the (1) reasons for and (2) what happens during wort boiling.
Why boil wort?
Clarified wort from the mash separation system is collected in a wort Copper
for boiling. The purpose of wort boiling is to stabilise the wort composition and
to extract The amount of fermentable matter derived from the brewing
process. the desirable compounds from hops that gives beer its characteristic
aroma and flavour.
Boiling also removes some of the undesirable volatile compounds which come
from the raw materials. Wort boiling provides both flavour and good shelf life
in beer.
What happens during wort boiling?
Sterilisation of the wort & stopping enzymatic action.
Sterilising
Brewing raw materials such as malt Barley (and other cereals) which has
been germinated to release enzymes and subsequently dried to halt enzyme
production (and therefore further seed growth) and to develop typical malt
flavour., hops and occasionally brewing water itself are infected by micro-
organisms Small organisms such as bacteria, virus or
fungus.bacteria Microscopic single celled organisms, virus or fungus.. These
have to be killed during the brewing process The set of controlled
modifications that occur in a sequence to achieve a desired outcome. to
prevent wort and beer spoilage.
After wort boiling the wort is normally free from
microbial contamination Anything in a product which is not there by design
(e.g. in beer: glass, oil etc.). Some micro-organisms are able to form spores
and to withstand heat treatment, including wort boiling. If they are present in
the raw materials or the brewing water they may persist into the finished beer.
However, standard beer is a poor growth medium for these types of
organisms. The pH A measure of acidity measured from 1 to 14. Alkalis are
low pH (7-14). Acids are high pH (1-7). Pure water is neutral at 7. is too low.
They do not normally represent a product or health hazard, except possibly in
low alcohol beers.
Enzymes Proteins that catalyze (i.e. accelerate) chemical reactions. In
these reactions, the molecules at the beginning of the process are called

substrates, and the enzyme converts these into different molecules, the
products.
Above a certain temperature (usually in the range of 50-
800
C), enzyme Chemical compound that causes other chemicals to be
transformed rapidly from one form to another.structure is broken down and the
enzymes lose their activity. All the natural malt enzymes are denatured by the
time the mash temperature reaches 76 to 780
C. Thus enzyme activity will
cease by the end of a normal lager Beer traditionally fermented at low
temperature with a bottom fermenting yeast mash.
Some brewers add external enzymes, such as thermostable beta-glucanase
or alpha amylase, intended to help with wort filtration. These enzymes have a
higher heat stability and are active throughout mashing but will be de-
activated during wort boiling. It is important that they are destroyed otherwise
they would continue working. This would change the profile of the beer.
Concentration of Wort
During wort boiling water is driven off as steam. This concentrates the wort.
The amount of water removed during the boil is directly proportional to the
rate of evaporation Conversion of a liquid into a gas form (i.e. from water to
vapour) which then rises up into the atmosphere. (and hence the amount of
energy supplied). The efficiency will be affected by the design of the Copper,
particularly the surface area.
Isomerisation of Bitter Substances
The process of hop The hop (Humulus) is a small genus of flowering plants,
native to the temperate Northern Hemisphere. The female flowers, commonly
called hops, are used as flavouring and stabilisers during beer
brewing. isomerisation was covered in an earlier section. See Hops in this
module.
Isomerisation is a relatively rapid reaction with production of over 90% of the
wort bitterness occurring within the first 30 minutes of boil. Complete
extractable bitterness occurs within 60 to 70 minutes.
The following graph shows hop utilisation as a % of total utilisation against
time

The isomerisation reaction is faster at higher temperature. Results from high
temperature wort boiling show that the rate of isomerisation of alpha acid is
directly related to temperature. Higher bitterness levels are achieved within a
few minutes using continuous wort boiling systems at 1400
C compared to
conventional Copper boiling under atmospheric pressure.
Removal of Volatiles
During wort boiling undesirable volatile compounds are driven off with the
steam. Research at BRI identified a number of these volatiles from malt and
hops which have to be removed in order to produce beer with a satisfactory
flavour .
The following graph shows the effect of evaporation rate on volatile removal

Evaporation rates as low as 2% of the initial wort volume are sufficient to
produce good beers after 60 minute boil.
DMS
A principal malt derived volatile lost during wort boiling (particularly in lager
malt) is DMS Di Methyl Sulphide: Tastes and smells of cooked
vegetables/corn/cabbage or shellfish/seafood. Is acceptable in light lagers to a
degree. Produced by malt & bacterial infection. (dimethyl sulphide). This gives
lagers a taste described as "sweetcorn". It is produced by heat breakdown of
S-methyl-methionine (SMM).
The DMS released during boiling is rapidly lost through evaporation.
However, the breakdown of S-methyl methionine continues during the period
between the end of boiling and wort cooling. The DMS then released is not
lost and persists into the finished beer. It is, therefore, possible to control the
level of DMS by varying the duration of boil and whirlpool stage.

It is necessary to control DMS levels in beer and this is achieved by:
 Selecting malt with low S- methyl methionine content
 Extending wort boiling time to maximize the breakdown of DMS .
 Minimise whirlpool stand time to reduce the decomposition of DMS
precursor to DMS in the wort and beer.
 Cooling the wort rapidly from the whirlpool to reduce the
decomposition of DMS precursor to DMS in the wort and beer.
Hop oils
The principal hop volatiles lost during wort boiling are the hop oils. If these are
present at too high a concentration they will contribute a bitter, vegetable
grassy flavour to the beer. Most of the hop oil volatiles are lost during a
standard 60 to 90 minute boil. Where late hop character is required in beer, a
small amount (up to 20% of the total hop charge) of selected aroma hops can
be added to the Copper 5 to 15 minutes before the end of the boil.
The principal factors which will effect the evaporation of volatiles include:
 Temperature of wort
 Vigour of boil
 Surface tension Physical effect of liquids which forms a “skin” on the
surface. This skin is resistant to breaking or penetration.
 Condensation of volatiles in the vapour stack
 Duration of boil
The Copper design will have a major influence. It has been found that more
late hop character persists in worts with poorly agitated worts.
Increase in Colour
The colour of wort increases during the boil. The reactions responsible for
colour development fall into two broad categories :
 Maillard reaction between carbonyl and amino compounds.
 The oxidation of polyphenols.
Oxidation during wort boiling increases the colour. Mash and wort produced
with low oxidation produces wort and beer with lower colours and improved
flavour stability.
Reducing Wort pH
Control of pH throughout the brewing process, from brewing water to final
package, is fundamental for product consistency.
The effects of mineral ion composition particularly Ca2+
on pH was covered in
an earlier section. (See section on water). Wort pH continues to fall during
wort boiling. The principal fall in pH is due to the reaction of Ca2+
compounds

with phosphates and polypeptides. These form an insoluble compound
releasing H+
(hydrogen ions). This lowers pH.
These reactions continues throughout wort boiling
Some of the calcium A metal. Found in scale Deposits of minerals forming on
surfaces when water is heated. as an ion combined with other chemicals. is
precipitated as calcium oxalate. If oxalate is not precipitated during the boil it
can form crystals which can cause gushing in finished beer.
It is important to achieve the required decrease in pH (boiled wort pH is
generally around pH 5.0) as it affects wort and beer character, in particular:
 Lower pH improves Protein Coagulation
 Lower pH improves beer flavour stability in particular VDK (diacetyl)
reduction
 Lower pH encourages yeast A special type of Fungus that converts
sugar to alcohol. growth
 Lower pH inhibits the growth of many contaminating organisms
 Lower pH results in less colour formation
 Lower pH results in poorer hop utilisation
Reducing Wort Nitrogen Levels
During the brewing process it is necessary to decrease the level of high
molecular weight nitrogen A gaseous element Chemical atom that cannot be
reduced further but can form compounds with other elements.. When
combined with other atoms into molecules it is an essential precursor of
protein (and therefore essential to growth), which comes from the malt. If this
nitrogen is not removed it can affect:
 pH.
 Colloidal stability (chill haze Cloudy particles sometimes seen in
beer or other products, caused by long protein chains that have not
been removed at filtration. and permanent haze)
 Fining and clarifying properties
 Fermentation The conversion of sugar to alcohol giving off carbon
dioxide as a by product. and taste of the beer
The effect in reducing the amount of wort nitrogen (measured by the Kjeldahl
method ) for a standard boil at 1000
C are given below.
Nitrogen removal after different boiling times for a standard boil
Duration of boil (hours) % wort nitrogen reduction
0 0

0.5 5.4%
1 6.2%
1.5 7.7%
2 9.9%
3 10.4%
Table taken from Hough, Briggs and Stephen "Malting and Brewing Science"
The total % nitrogen removed appears to be relatively small, but using a more
specific test, (gel electrophoresis), it is possible to separate the nitrogen
compounds by their molecular weight. This shows that wort boiling is more
effective at removing the higher molecular weight fraction, which is also the
fraction responsible for colloidal instability in packaged beer.
Effect of boiling on the molecular weight distribution of
wort proteins Complex chains of molecules used to build muscle, tissues etc..
The graph shows (roughly) a halving of the high molecular weight protein
during boiling.
The process of protein coagulation increases the size of the molecules and
makes them less soluble. This creates suspended flocs. During the whirlpool
phase, these aggregates continue to form and sediment out as hot break.
Vigour is only one feature of importance for coagulation, since protein floc
formation is improved by intense vapour bubble formation. The actual wort

surface temperature, and the duration of the contact of the wort with the
heating surface is also important.
Criteria used for evaluating efficient wort boiling are:
 Temperature of boil (usually just above l000
C when boiling under
atmospheric pressure).
 Length of boil
 Evaporation % per hour
Traditionally conditions for wort boiling were between 90 and 120 minute boil
with a minimum of 10% evaporation per hour. In traditional boiling systems the
vigour or boiling intensity has been related to evaporation rate. However,
because of the need to reduce energy costs and to improve brewhouse
efficiencies shorter boiling times with lower evaporation rates are now used.
Typical modern Coppers operate with a 60 minute boil with between 5% and
8% evaporation.
Production of Reducing Compounds
Malt and wort contain a number of reducing compounds. These can protect
beer against ageing. It is important therefore not to add air to the boiling
process as reducing compounds react with oxygen Gas that makes up nearly
20% of the air that we breathe. Whilst essential for life, Oxygen in food
products causes them to taste bad and feeds the bacteria that makes them go
sour. and are lost.
DMS
DMS ou Dimethyl sulfides
d'après le Home Brewing Wiki
Le Sulfure de Dyméthyle (DMS) est un composé organosulfuré présent en niveauxinférieurs à son
seuil de perception dans la plupart des bières. Du fait de son seuil de perception relativement bas,
10-150 ppb, c'est un composant gustatif et aromatique principal contribuant de manière significative
au caractère de la bière, et tout particulièrement dans les bières de types Lager.
Dimethyl sulfide (DMS) is an organic sulfur compound present above its flavor threshold in most
beers. Because of its low flavor threshold, 10 - 150 ppb, it is a primary flavor and aroma compound
that makes a significant contribution to beer character, especially in lager beers. It has a
characteristic taste and aroma of cooked corn or creamed corn.
The level of S-Methyl methionine (SMM) in malt is responsible for the DMS level in wort. During
mashing the SMM, DMS and very soluble dimethyl sulfoxide (DMSO) are brought into solution. SMM
can be hydrolyzed to DMS during mashing however much of the DMS is driven off since it is very
volatile. Wort will always have some concentration of SMM, DMS, and DMSO - different grains and
mashing techniques can effect these concentrations. During fermentation little to no SMM is
converted to DMS, however DMSO can be reduced to DMS by yeast during fermentation.
SMM:(CH3)2S-CH2-CH2-CH(NH2)-COOH
DMS: (CH3)2S

DMSO: (CH3)2S=O
DMS in Beer Some detectable level of DMS is characteristic of manylager styles, and is especiallynoticeable in light
lagers.However,DMS is presentin mostbeers at some level.It is excessive DMS that gives some home brewed
ales a "cooked corn" character.The amountof DMS found in beer is lowestin British ales,10 - 20 ppb and highestin
German lagers and all-maltbeers,50 -175 ppb, while the United States' lagers generallycontain 40 - 100 ppb.Beers
with high adjunctratios or low gravities allow the DMS taste or off-taste to be more detectable,while German beers,
all-maltbeers,flavorful beers,especiallydark beers,make the taste of DMS less discernible even at higher levels.3
[edit] Causes ofDMS DMS is created whenever wort is heated,by the breakdown ofprecursors found in pale malts.
Under ordinary circumstances,mostofthe DMS that is created by heatis then evaporated during the boil. Some
DMS is also removed during vigorous ale fermentations,which is whyhigher levels are often found in lagers.
Covered boil Covering the brew kettle during the boil prevents DMS from evaporating,and results in high levels of
DMS in the finished beer.Slow cooling Because DMS is created at temperatures below boiling,cooling the worttoo
slowlymeans thatexcessive levels of DMS can be created which cannotbe evaporated once the boil has stopped.
The DMS produced during the hot wort stand will stay in solution even if the hot wort tank is vented. For every extra
hour of hot wort stand,a DMS increase ofapproximately30% will result.The level of DMS in the wort determines the
level of DMS in finished beer.In order to predictthe level of DMS in finished beer Table V shows the relationship
between SMM in maltand DMS in beer.
The major source ofDMS in finished beer is derived from its precursor,S-Methylmethionine (SMM), an amino acid,
which is formed during the germination and kilning process ofmalting barley.Barley does notcontain DMS or SMM.
However, both are formed by the biosynthesis occurring during germination.SMM, also known as DMS precursor
(DMSP), is heat-labile and decomposes on heating to form DMS during kilning,wortboiling and hot wort storage.
The mosteffective way to reduce SMM during germination is byslightlyunder- modifying malt,specificallyby
reducing the moisture contentof barley at steep-outto 40-42% and reducing the germination temperature to 55-60oF.
It has been shown thata reduced airflow during germination resulted in a 50% lower SMM level in the finished malt.4
Alkaline steeping liquor and use of potassium bromate and other factors which reduce the metabolic growth rate
during germination have been shown to significantlyreduce SMM and insuring DMS levels in finished malt.
Two row barley which has a normallylower nitrogen content than six row barley, has been shown to produce
significantlyless SMM during the malting process.European malthas less SMM and DMS than North American and
Canadian malt.1
Regardless ofvariety or growing conditions,the mostimportantfactor for reducing SMM and DMS occurs during
kilning.The SMM formed during germination is converted by the heatof kilning and air flow to DMS. The DMS formed
is either removed or volatilized in the kilning draft, oxidized to (Dimethyl sulfoxide) [DMSO], or remains in the finished
malt.Since DMS is easilyremoved during the kettle boil,it is importantthat the ratio of SMM to DMS be as low as
possible in the finished malt.
The conversion of SMM to DMS occurs at about1401F. Therefore,by increasing the withering temperature,
increasing the final kilning or curing temperature and extending the final curing time the level of SMM and DMS will
significantlybe reduced in the finished malt.The stabilityof SMM in maltis greater at higher moisture levels.
[edit] Preventing DMS The level of SMM in maltis responsible for the DMS level in wort. During mashing the SMM,
DMS and very soluble DMSO are broughtinto solution.No SMM is hydrolized to DMS at this time.

Kettle boiling hydrolizes SMM to DMS which is removed during evaporation.The half life or time needed to remove
half of the DMS is 40 minutes so thatthree-fourths is removed in 90 minutes.Narssis recommends a 100 minute boil
to reduce the level of SMM and DMS to acceptable levels in mostbeers.
The level of DMSO does notchange during the kettle boil.A small amountofDMS, 0.4 ppb,may be contributed by
hops,especiallyif added in large amounts late in the boil. As long as the wort is hot SMM will be converted to DMS. It
is importantto convert SMM to DMS in the kettle so that build up during the hot wort stand is minimized.
The following steps should insure low levels ofDMS in the finished beer:
Boil the entire wort 90 minutes or longer Ensure thatthe boil is vigorous - rolling Allow at least8% evaporation
Minimize the hot wort standing time Rapidlycool the wort [edit] Fermentation and DMS During fermentation,the
evolution of CO2 removes and reduces the level of DMS. At moderate DMS levels of 30-60 ppb a 30-35% reduction
can occur, while a 35-60% reduction can occur at higher initial DMS levels,60-150 ppb.
The yeast's metabolism does notconvert SMM to DMS but certain yeast can produce higher DMS levels by reducing
DMSO to DMS, especiallyin lager beer production at cooler temperatures.Certain wild yeasts and Enterobacter
agglomerans can produce DMS.5 Table VI shows the reactions thattake place in malting and wortboiling.
[edit] DMS After fermentation Purging and contamination occurs can can change the the DMS concentration in
beer.Water dilution ofhigh gravity beers may reduce the perceived threshold ofDMS due to dilution of other mashing
flavors. If DMS precursors,e.g.DMSO, reach the final productthey are reduced to DMS,incresing the DMS
concentration during the beer shelflife.
[edit] Creating DMS DMS is naturally presentin relatively high levels in many beers.There is no easy way to add
DMS character to a beer artificailly, but to increase levels during brewing,simplycover the wort for part of the boil,
taking care to avoid boilovers.
[edit] Beer Styles and DMS Types of beers in order of their perceived threshold ofDMS. Those with the lowest
thresholds are mostlikelyto have off tastes at excessive DMS levels.
Lagers (lowest):
Low adjunctbeers with low gravities or diluted flavor. High adjunctbeers with corn grits High adjunct beers with other
adjuncts Low to medium gravity(1.040-1.048) beers - all maltAll maltGerman or higher gravity, lightcolored -
flavored beers Amber - dark flavorful beers Ales (highest):
British lightales American or British amber or dark ales Stouts or strong flavored beers [edit]PROPERTIES of DMS
(CH3)2S H3C-S-CH3 Boiling Point:99oF Density:0.848 Flavor Threshold (Beer):Perceived Threshold:10-150 ppb
Depends on amountofflavor in beer Flavor: Cooked corn,creamed corn Aroma: Same - highly volatile Source:
Precursor in malt(SMM), hops (minor)
DecoctionMashing
A decoction mash is a type of mash in which at leastone mash resttemperature is reached byremoving part of the
mash,boiling itin a separate vessel,and then mixing it back in to raise the temp of the mash.It is traditional in many
continental European beer styles,especiallyin Germany and the Czech Republic.Butmostbrewries in these regions
have switches to the more economical directlyheated step infusion mashing.
Decoction mashing is notvery common among home brewers,since ithas a reputation as a time and labor-intensive
process.Buta decoction mash is basicallyjusta step infusion mash where some ofthe gristis heated and returned

instead ofinfusion water.While it does take some extra time and require some extra stirring,it is a procedure that can
be performed by mosthome brewers.
History of the Decoction Mash
Decoction mashing refers to removing a part of the mash,boiling itand returning it to the main mash to raise the
temperature to the next rest. This mashing procedure originates from a time when maltqualitywas not consistentand
temperatures could notbe measured.The long boiling ofthe grain makes the starches more accessible for the
enzymes. This is particularlyimportantfor undermodified malts where the cell walls are notas broken down as well
as they are in well modified or overmodified malts.The boiling ofa defined portion of the mash and returning itto the
main mash to raise the temperature also helped the consistencyin mashing temperatures before thermometers were
available.
Chemistry of the Decoction Mash
Today even mostEuropean malts are generallywell modified and can be used in infusion step mashes or even single
infusion mashes,thus removing the need for decoction mashing.Butdecoction mashing is still widelyused,
particularlyin smaller southern German breweries and for dark beers like Bocks and Dunkels.Many brewers believe
that the boiling of the mash gives the beer a flavor profile that cannot be achieved otherwise.Butespeciallyin the
home brewing community,there has been a hot debate aboutthe actual benefits of a mash as labor intensive as a
decoction mash.Many say that with the malts thatare available to the home brewer decoction mashing doesn'tmake
for a difference and if there is a difference it could also be achieved by the use of specialtymalts.But in the end every
brewer has to determine thatfor him or herself.
Decoction Mash Procedure
The basic procedure for performing a decoction mash is very simple.Water is added to the gristto reach the initial
mash temperature.Once the first temperature restis complete,a portion of the grain and water is scooped outof the
mash tun and into the kettle or another heated vessel,where it is broughtto a boil. The portion removed,which can
often be as much as a third of the grist,is called the decoction.
The decoction may require stirring during heating to avoid scorching the grain;this adds some extra work during the
mash.The decoction step also adds time to the mash process,since a decoction cannotbe heated as fastas
infusion water and it is usuallyboiled for 5 – 45 min.After boiling,the decoction is returned to the mash tun to achieve
the next temperature rest.
Sample Decoction Mash Schedules
This section contains discussions ofa number of sample schedules for decoction mashes,including single,double,
and triple decoctions.The brewer should bear in mind thatsome mash schedules are better suited for use with
modern,well-modified European malts than others.

The triple decoction is the grand father of all decoction mashes.This is how the firstPilsners were brewed in Pilzen
and how German beer was brewed for a long time and some are still brewed like this.
The triple decoction mash employs 3 main temperature rests:acid rest,protein restand saccharification rest.At each
of these rests a decoction is used to reach the next rest until the mash-outis reached.The acid restis a convenient
restto do mash pH adjustments.Notonly does itserve to lower the pH by simplyusing the phosphatase and other
acid forming enzymatic activity, but since there is no enzymatic activity that can have a detrimental affect on the final
result,there is no rush to move to the next rest.
There are several formulas outthere for calculating the decoction volume.Some of them are simple and others try to
accountfor factors such as the heat capacity of the grains and the mash-tun.The easiestway however is to estimate
the decoction volume with a simple formula like this:
decoction volume = total mash volume * (target temp - start temp) / (boil
temp - start temp)
and add about15 - 20%. The idea is to decoct more mash than necessary.When the decoction is added back to the
main mash,itis not all added at once. Instead it is added in steps while the temperature of the mash is constantly
checked.This requires a thorough mixing of the mash after each addition.Once the target temperature is reached the
remaining decoction is left to cool and added once its temperature is close to the mash temperature.By doing so one
can accountfor additional factors that effect the actually needed decoction volume such as:evaporation during the
boil,unexpected temperature drop in the main mash and others.
The thickness ofthe decoction depends on the thickness ofthe main mash.Though itis preferred to leave a lot of the
liquid back in the mash tun,the decoction should notbe too thick (grain should still be submerged in liquid) to make
stirring it easier and keep it from scorching easily.If the main mash can be kept at the preferred thickness of1.5 - 2
qts/lb (3-4 kg/l) the decoction should have a thickness of1-1.25 qts/lb (2-2.5 kg/l). At this thickness and with gentle
heating,only little stirring is necessaryto keep the mash from scorching.For lager grists (high gravity beers) the
thickness ofthe mash mayhowever be limited by the volume of the mash tun.
All the decoction schedules provided here assume a decoction rise temp of2-4 *F/min (1-2 *C/min).This is what is
generallyrecommended in the literature for heating the mash.In technical brewing this is a resultof the decoction
kettle design which cannotheatthe mash anyfaster. There is also a saccharification restat155 - 162 *F (68 - 72 *C).
The purpose ofthis restis to utilize the enzymatic power of the decoction before its enzymes are destroyed by further
heating.This is particularlyimportantwhen brewing beers with a large percentage of the enzymatic weaker dark base

malts.This restdoesn'thave to be held at the main saccharification temperature.It is sufficientto restin the alpha
amylase range where the conversion is also done much quicker.After the decoction is converted or almostconverted
(iodine test) the heating of the mash is resumed.To hold this restthe pot can be taken off the burner and wrapped in
blankets for insulation.
The decoction is then boiled for 10 - 40 minutes.Shorter boil times for lightcolored beers,longer boil times for dark
colored beers. If only gentle heat is applied during the boil,stirring should onlybe necessaryoccasionally.Similar to
wort boiling,excessive thermal loading ofthe decoction can resultin a burnt flavor of the beer. If the decoction is
boiled for an extended amount of time evaporation losses can be compensated with the addition of water (which can
also be added after the decoction has been pulled,where it helps in tinning itout and makes itmore manageable) or
by boiling with the lid on. Any trapped DMS will be boiled off during the wort boil anyway.
The decoction is then added to the main mash to reach the protein rest. The resttemperature and time before pulling
the next decoction should be based on the maltthat is used.Less modified malts benefitfrom a rest closer to 122 *F
(50 *C) which produces more amino acids,which is an essential yeastnutrient.In undermodified malts the protein
conversion has notbeen driven far enough during malting to allow for sufficientwort FAN (free amino nitrogen)
withoutthe use of a more intensive protein rest. If the maltis a well modified modern malt,the protein rest
temperature should be keptcloser to 133 *F (55 *C) and the next decoction should be pulled 5 - 10 minutes after the
resttemperature has been reached.This serves to protect more of the medium chained proteins thatare important
for body and head retention. Decoction schedules thatallows for a shorter protein restin general will be described
later.
A decoction is pulled again,rested for conversion and then broughtto a boil. This time to reach the saccharification
resttemperature.This temperature is similar to the saccharification resttemperature thatis used for a single infusion
mash,butthe same temperature thatwas used in a single infusion mash maynotgive the same fermentabilityin a
decoction mash.Boiling has destroyed more ofthe enzymes while it has made the starch also more easily
accessible.The former would lead to a less fermentable resultwhile the latter would shiftthe fermentabilitytowa rds a
more fermentable wort.This is only to illustrate thatexperimenting with the saccharification resttemperature mightbe
necessaryfor optimal results.The saccharification resttemperature thatwould have been used in a single infusion
mash is however a good starting point.
After holding the saccharification restfor about45 min or longer,if starch conversion is notcomplete after that time,
the final decoction is pulled.This decoction can also be thinner and doesn'thave to be rested for starch conversion
any more,since the starches have already been converted and enzymes protection is not as crucial anymore.

Single Decoction
n a single decoction mash onlyone decoction is used.This decoction can be used to reach any rest, but most
commonlyit is used to reach the mash-outtemperature.This can be a simple enhancementofa single infusion or
step mash.
The mash schedule shown above is well suited for European and continental lager malts.It features a shortprotein
restat the higher end of the temperature range for proteolytic activity, a single temperature saccharification restand a
decoction to get to mash-out.Calculate your strike water to aim for a protein rest between 53 and 55 *C (129 - 133
*F) at a mash consistencyof about2.5 l/kg (1.2 qts/lb). This temperature puts emphasis on the protein degrading
enzymes that produce the medium chained proteins which are good for head retention and mouth feel.The well -
modified modern malts alreadyhave enough shortproteins (amino acids) and a res tcloser to 50 *C (122 *F) is not
necessary.Dough-in and check the temperature.Plan on holding this temperature for 20 min.During this time bring
abouthalf of the amountof water, that was used for dough-in,to a boil.The pH of the mash should be checked and
corrected if it is not within the 5.3 - 5.6 range.When the protein restis over, use a heatresistantvessel to scoop the
boiling water into the mash.This is bestdone by holding the thermometer in the mash with one hand and scooping
the water or stirring with the other hand.It is importantto stir the mash well to even out its temperature.Add water
until the desired mash temp of65 - 68 *C (148 - 155 *F) is reached.You will notice that the thinned mash makes
stirring easier.This mash will have a consistencyofabout 3.5 - 4 l/kg (1.7 - 2.0 qts/lb) which is typical for German
style beers.Hold this restfor 45 min.
Check for conversion.Calculate the amountof decoction necessaryto get to the mash-outtemperature of74 - 76 *C
(165 - 170 *F) and pull this decoction.In order to prevent scorching getgood mix of liquid and grains.This decoction
should be broughtto a boil over the next 10 to 15 minutes.A gentle flame while keeping the pot covered will prevent
scorching even withoutthe need of constantstirring.Boil for 10 - 30 min.Shorter for lightworts and longer for darker
worts.

Double Decoction
Classic Double Decoction
The classic version ofthe double decoction is a shortened triple decoction.It omits the acid restand starts with the
protein rest. Because ofthis only 2 decoctions are needed to get to mash-out.One to get from the protein restto the
saccharification restand another one to get from the saccharification restto mash-out.Like the triple decoction,this
mash rests the main mash atthe protein restfor a long time. With well modified lager malts,this mayresultin overly
extensive protein degradation.The following two sections show mash schedules thatavoid this problem.
Enhanced Double Decoction
This is a mash schedule thatwas taken from German brewing literature [Narziss,2005].The greatthing aboutthis
mash is thatit is almostas intensive as a triple decoction,when it comes to the amountofmash that is boiled,buta
little bit shorter and withouta long protein rest. The basic idea of this mash is to pull a decoction that is large enough

to get the mash from acid restdirectly to the saccharification rest.Butwhen this decoction is returned to the main
mash,itis returned in 2 parts:first to reach the protein restand later to reach the saccharification rest.The second
decoction is done to reach mash-out.
This mash starts like the triple decoction.Dough-in is done to reach the acid rest where the mash pH is corrected if
necessary.Now a large decoction (about50 - 60% of the mash) is pulled and heated.Itis advisable to add some
water 5-10% of the decoction volume to compensate for boil-offand thin it out a little. Due to the size of the decoction
it may be rested for protein restand mustbe rested for the saccharification rest.The saccharification restis
necessaryto get the mostout of the enzymes in this mash as they will be destroyed once the decoction is broughtto
a boil.It also reduces the viscosity of the mash which mitigates the risk ofscorching itlater. This rest can be done at
155 - 162 *F (68 - 72 *C) where the conversion should onlytake 15 - 20 min.Taking the pot off the burner, closing it
with a lid and wrapping it in blankets for insulation works very well.Check temperature and conversion after 15 min.
When fully or almostconverted,return the pot to the burner and start heating it gently again to bring it to a boil.Watch
out for the foam-up of the decoction shortly before and after it comes to a boil.
The mash is now boiled from 10 - 30 min.After that a heatresistantvessel is used to scoop some ofthe decoction
back into the main mash.Stir well and check the temperature of the main mash.Continue adding the decoction and
mixing until the desired protein resttemperature is reached.After that the main mash is rested for 15 - 20 min while
the restof the decoction is still boiling.Once the protein restis over, continue adding the decoction,stir and check
temperature until the desired saccharification resttemperature is reached.Hopefullyenough decoction was pulled to
reach this rest. If some decoction is leftover once the resttemperature is reached,letit cool and add it when its
temperature is close to the temperature ofthe main mash.
Restthe mash for saccharification.Check for conversion and when the desired mash time resttime is up and
conversion was achieved,pull the 2nd decoction.Heat it to boiling,boil for 5-20 min and return it to the main mash to
reach mash-out.
Variation
If you wanta highly fermentable wortand truly boil and convert almostall of the starches in the decoction,this
variation should getyou there:
Dough-in in at the acid rest in your your boil kettle. Stir well to make sure you are dissolving the enzymes into the
mash water.While the mash is resting,preheatyour mash tun with some boiling water.This will prevent a significant
temperature drop later.
After the mash has been resting for 10 - 20 min and the pH is corrected, remove the top 40% of the mash (mostly
liquid) and place it into the mash-tun.This contains the enzymes thatwill be needed for the saccharification rest.The
kettle should contain a thick decoction that is similar to the one that a brewery would get when they pump the
decoction from the bottom of the mash tun into their boil kettle. Heat, convert and boil the decoction.After that return
a part of it to the mash tun for a protein rest (can be skipped) and the restlater later for a saccharification rest.
Note that the start of the saccharification resthas now more glucose chain ends available compared to a
conventional decoction mash or infusion mash since almostall ofthe starch was converted in the decoction.This will
provide lots of opportunity for the beta amylase to create maltose.If you want to have wort of norm al fermentability
you should hold this restcloser to 160Fwhere the beta amylase denatures more quickly.But if you want a very
fermentable wort,hold this restcloser to 140F where the b-amylase will be active for much longer.
Use a 2nd decoction to get to mash-out.

Hochkurz Double Decoction
This version of a double decoction mash is known as Hochkurz Mash in German brewing [Narziss,2005].It uses a 2
temperature saccharification rest.The first decoction is used to get from the 1st saccharification rest(maltose rest) to
the 2nd saccharification rest(dextrinization rest) and the 2nd decoction is used for mash-out.The dough-in can
happen with the protein rest, an intermediate restor the maltose rest. Hochkurz refers to the fact that these mashes
dough in high (hoch) and are short(kurz).
To optimize the use of the beta amylase and produce a wort with high levels of maltose,German brewers often use a
2 step saccharification scheme.With today's well modified malts the protein restis generallysipped. The firstrest,
usuallyheld at 140 - 146 *F (60 - 63 *C) gives the beta amylase time to convert the glucose chains (large dextrins)
into maltose.At this temperature there is already sufficientalpha amylase activity available to provide enough
glucose chain ends for the beta amylase.This is needed because the beta amylase can only clip maltose from the
non reducing end of a glucose chain.Due to the lower temperature,the beta amylase will be active for a longer time
as it would in a single saccharification restheld athigher temperatures.To reduce and eventually terminate the beta
amylase activity and to ensure thatall starch in the wort has been converted (especiallythe small starch granules
which have a higher gelatinization temperature),a dextrinization restis held at 158 - 162 *F (70 - 72 *C). At this
temperature the beta amylase is quicklydeactivated and only the alpha amylase works on the starches.The restis
held until the mash is iodine negative (no starch or long dextrines in the wort). Narziss [Narziss,2005]and Fix [Fix,
1999]suggest,thata rest at 158 - 162 *F (70 - 72 *C) benefits head retention and body of the beer though
glycoproteides thatare extracted from the maltbut not degraded by enzymatic activity. Because ofthat Narzis s
suggests holding this restup to 60 min.After that resta mash-outis performed at167-173F(75-78 C). The
temperature should notbe higher as this would deactivate all the alpha amylase activity and some alpha amylase
activity is still needed during lautering to convert any rouge starches,thatmightbe liberated during sparging,on their
way to the kettle.
The length as well as the temperature of the maltose restdetermines the fermentabilityof the wort. Shorter rests
and/or higher temperatures will resultin a less fermentable wortas the beta amylase gets less time for maltose
production.

The steps for the water infusions and decoctions necessaryfor this mash have already been covered with the other
mash schedule examples.This mash schedule can also be done w/o the use of decoctions through hotwater
infusions or directheatto the mash.The latter has become standard practice in mostGerman breweries.
Brewingprocess
ABSTRACT
The invention relates to a process for the production of wort, comprising the enzymatic treatment of grist
in up to 100% unmalted (grain) form, for further processing into high quality beverage products. By the
addition of a combination of exogenous enzymes (α-amylase, isoamylase/pullulanase, FAN generating
activity (proteases) and beta-glucanase activity) to the mash and by the simultaneously thermal activation
of the maltose-generating endogenous β-amylase, it is possible to obtain a wort based on up to even
100% barley. The invention further relates to a process for the production of a high quality beer or beer
product and to the high quality beer produced according to the process.
CLAIMS(1)
1. 1. A process for the production of a brewer's wort, comprising:
a. obtaining a mash by mashing a grist, of which at least 70wt% is unmalted cereal(s)
comprising β-amylase activity and of which less than 30wt% is malted cereals), at a
temperature at which exogenous (added) enzymes and the endogenous β-amylase
are active;
b. contacting the mash with exogenous enzymes comprising: i. an α-amylase activity,
ii. a pullulanase activity, iii. a proteolytic activity, and iv. a β-glucanase activity;
c. mashing-off and filtering the mash to obtain the wort.
2. The process according to claim 1 , where the grist comprises at least 75%wt,
preferably at least 80wt%, more preferably at least 90wt%, even more preferably
95wt%, and most preferably 100wt% unmalted cereal(s).
3. The process according to claim 1 or 2, wherein the unmalted cereal(s) are barley,
spelt, wheat, rye, corn, oat or rice or any mixture thereof.
4. The process according to any of claims 2 or 3 wherein the unmalted cereal is
barley.
5. The process according to any of the preceding claims, wherein the grist further
comprises other carbohydrate sources such as, brewing syrups.

6. The process according to any of the preceding claims, where the exogenous
enzymes of step b. in claim 1 further comprises a xylanase activity, preferable of family
GH 10.
enzymes of step b. in claim 1 further comprises a lipase activity.
enzymes of step b. in claim 1 further comprises a phytase activity.
9. The process according to any of the preceding claims, where the mashing
temperature is in a range optimizing the β-amylase activity.
10. A mashing process according to any of the preceding claims, wherein
A first step is carried out between 50 and 58 0C, A second step is carried out between
60 and 65 0C, and A third step is carried out between 70 and 80 0C.
1 1. The mashing process according to claim 10, wherein the mashing process is
completed within 160 minutes, preferable within 120 minutes
12. The process according to any of the preceding claims, where the α-amylase
activity is provided by an α-amylase having at least 50%, more preferably at least
60%, more preferably at least 70%, more preferably at least 80%, more preferably at
least 90%, more preferably at least 95%, more preferably at least 98%, and most
preferably at least 99% identity to the amino acid sequence shown in SEQ ID NO: 1.
13. The process according to any of the preceding claims, where the debranching
activity is provided by a pullulanase having at least 50%, more preferably at least 60%,
more preferably at least 70%, more preferably at least 80%, more preferably at least
90%, more preferably at least 95%, more preferably at least 98%, and most preferably
at least 99% identity to the amino acid sequence shown in SEQ ID NO: 8.
14. The process according to any of the proceeding claims where the pullulanase is
thermostable having a relative enzyme activity above 60% over a period of 30 min, at
65 0C and at pH level 5.
15. The process according to any of the preceding claims, where the protease activity
is provided by a proteolytic enzymes system, including endo-proteases, exopeptidaes
or any combination thereof, preferably a metallo-protease.

16. The process according to any of the preceding claims, where the protease activity
comprises an activity provided by a protease having at least 50%, more preferably at
least 60%, more preferably at least 70%, more preferably at least 80%, more
preferably at least 90%, more preferably at least 95%, more preferably at least 98%,
and most preferably at least 99% identity to the amino acid sequence shown in SEQ
ID NO: 3.
17. The process according to any of the claims 7-16, where the lipase activity is
provided by a lipase from Fusarium, Aspergillus or Rhizopus.
18. Use of a process according to any of the preceding claims for the production of
beer.
19. A wort produced according to any of claims 1-17.
20. Use of the wort according to claim 19 for the production of beers of any type.
21. The wort according claim 19 comprising one or more amino acids selected from a.
proline at a concentration at less than 2 mM, preferably less than 1 mM, and most
preferably less than 0.5 mM in the wort; b. serine at a concentration above 0.1 mM,
preferably above 0.125 mM, and most preferably above 0-15 mM; and c. methionine at
a concentration above 0.05 mM, preferably above 0.08 mM, and most preferably
above 0.10 mM.
22. The wort according to any of the preceding claims, where the maltose
concentration is above 40%, preferably above 50%, preferably above 60% of the total
concentration of carbohydrates.
23. The wort according to claim 22 where the glucose concentration is below 10%,
preferably below 6%, most preferably below 4%.
24. The wort according to any of the proceeding claims where the total of the glucose,
maltose and maltotriose concentration is above 60%, preferably above 70%, and most
preferably above 80% of the total concentration of carbohydrates.
25. An enzyme mixture comprising;
i. an α-amylase activity, ii. a pullulanase activity, wherein the pullulanase is
thermostable iii. a proteolytic activity, and iv. a β-glucanase activity;
26. An enzyme mixture comprising;

i. an α-amylase activity, ii. a pullulanase activity, iii. a proteolytic activity, iv. a β-
glucanase activity; and v. a xylanase activity.
27. The enzyme mixture according to claim 25 or 26 further comprising a lipase
activity.
DESCRIPTION
BREWING PROCESS
REFERENCE TO A SEQUENCE LISTING
This application contains a sequence listing in computer readable form. The computer readable form is
incorporated herein by reference.
TECHNICAL FIELD OF THE INVENTION
The invention relates to a process for the production of a brewer's wort, comprising an enzymatic
treatment of a grist comprising up to 100% unmalted (grain) form, and further relates to the wort
obtainable by the process. The invention further relates to the use of said wort for the further processing
into high quality beverage products and relates to a process for the production of a high quality beer or
beer product, and to the high quality beer produced according to the process. In addition the invention
relates to enzyme mixtures.
BACKGROUND OF THE INVENTION
Mashing is the process of converting starch from the milled barley malt and adjuncts into fermentable and
unfermentable sugars to produce wort of the desired composition. Traditional mashing involves mixing
milled barley malt and adjuncts with water at a set temperature and volume to continue the biochemical
changes initiated during the malting process. The mashing process is conducted over a period of time at
various temperatures in order to activate the endogenous enzymes responsible for the degradation of
proteins and carbohydrates. After the mashing process the mash is filtered to obtain the wort for the
fermentation to beer. Traditionally, beer has been brewed from malted barley, hops, yeast and water.
Malting cereals such as barley activate the endogenous enzymes necessary for degradation of the
starch. However, the malting process is energy and time-consuming and thereby rather costly. Thus, one
way to reduce costs is to substitute some of the malt with readily available adjuncts such as refined starch
or readily fermentable carbohydrates and/or substituting with unmalted cereals, such as barley corn, rice,
sorghum, and wheat,. However, unmalted cereals lack endogenous enzymes, which may result in
incomplete saccharification, increased mash/wort viscosity, lautering difficulties, poor fermentability, beer
filtration difficulties, colloidal instability and poor flavour. Exogenous enzymes such as alpha-amylase and
β-glucanase have previously been added to compensate for the lacking malt enzymes. The following prior

art describes the substitution of part of the malted cereals with unmalted cereals and exogenously added
enzymes. ZA9803237 describes a process for producing a beer by fermenting a wort obtained from partly
unmalted barley and an enzyme blend of alpha-amylase, β-glucanase and proteinase. Wieg et.al.
Process Biochemistry, 1970 also describes a process for brewing with a mixture of malted and unmalted
barley and an enzyme blend of alpha-amylase, β-glucanase and proteinase. Further WO04/01 1591
describes a process for producing a wort adding a protease and a cellulase to a mash from maltet and
unmalted barley. A resume of barley brewing is given by Wieg et.al. Brewing science, 1987.
Another way to produce wort is known from the Japanese Happoshu beers. In Japan, taxes on malt-
containing alcohol beverages are relatively high, which is why Happoshu beers are brewed with as less
as 25% malted barley. Usually, mash prepared on such a low content of malt is impossible to filter in
order to obtain the wort, as the mash is too thick for filtering. There are only few technical descriptions
available concerning the composition of the Hap- poshu mash. However, it is known that it is necessary to
add exogenous enzymes to the mash in order to obtain filterability, e.g. proteinases, β-glucanase and
amylases. The Happoshu beers have different flavor characteristics even compared to traditional beers of
the more plain lager type. JP 2004173533 describes the production of such a beer with use of pressed
barley and lesser amount of malt. Different enzymes are used to aid e.g. saccharification.
The wort obtained in the prior art references are based on grist comprising considerable amount of malt.
The enzyme composition in raw cereals is substantially different from malted cereals and the endogenous
and the exogenous enzymes involved in the degradation of starch are working together in a complex
manner during mashing and it is generally assumed that some malt should be present in the grist. Thus
even with exogenously added enzymes some of the above mentioned problems e.g. with filterability,
fermentability and turbidity of worts based on unmalted cereals still exists. Consequently, very few
attempts have been made to substitute larger amount or all of the malted cereals with unmalted cereals.
One example is Goode et.al. describing the production of a wort from 100 % raw barley substrate and an
enzyme blend of two different alpha-amylases and a beta-glucanase. Alpha amylase has a positive effect
on mash separation, but the speed of filtration dropped when high amounts of unmalted barley were
present. Also in US 3081172 producing a wort from unmalted raw material is suggested however nothing
is mentioned about FAN (Free Amino Nitrogen), the amount of fermentable sugars and other crucial
parameters of the resulting wort. Consequently, problems such as low fermentability, non optimal amino
acids composition and high viscosity and turbidity of the wort are not solved and these obstacles tend to
increase with increasingly amounts of unmalted cereals.
Another disadvantages with the prior art brewing with unmalted cereals is that prolonged mashing time
may be needed in order for the exogenous and endogenous enzymes in the mash produce a wort which
is comparable e.g. with regards to fermentability to a wort produced from malted cereals. The prolonged
mashing time is clearly uneconomic and may neu- tralize the economical advantages of substituting
malted with unmalted cereals.

Thus until now no enzyme blend has fully compensated the malt enzymes, such that it when adding
together with up to 100 % unmalted cereals could fully substitute for a grist based on malted cereals.
Thus even though producing wort from barley has been attempted since the late 1960 no real brewing
process based on raw material from high amount of unmalted cereals has been developed.
In the light of a desire to reduce the costs related to malting of cereals, and further to obtain a wort
suitable for producing a beer comparable in taste characteristics to traditional beers, there exists a need
for a method to obtain a mash based on up to 100% unmalted cereals. The process should be easily
adaptable to the brewing systems used in brewing based malted raw material. Thus the mash should be
filterable and in addition other parameters such as the amino acid composition and amount of
fermentable sugars should be comparable to mash based on the corresponding malted cereals even if
the cereal(s) is/are 100% unmalted cereal(s). Finally, the mashing time should be comparable to that of
mashing of malted raw material while still retaining the good characteristics e.g. the sugar profile of the
mash and the beer product.
Thus, it is an object of the invention to develop a process for producing a wort from a grist comprising
more than 70%, and even up to 100%, unmalted cereals. SUMMARY OF THE INVENTION
The inventors of the present invention have surprisingly found that by addition of a suitable combination
of exogenous enzymes to the mash , and by thermal activation/inactivation of endogenous enzymes, it
is now possible to obtain a wort based on up to 100% unmalted cereals, such as barley.
A process for the production of a brewer's wort, comprising:
a. obtaining a mash by mashing a grist, of which at least 70wt% is unmalted cereals) comprising β-
amylase activity and of which less than 30wt% is malted cereal(s), at a temperature at which exogenous
(added) enzymes and the endogenous β-amylase are active;
b. contacting the mash with exogenous enzymes comprising: i. an α-amylase activity, ii. a pullulanase
activity, iii. a proteolytic activity, and iv. a β-glucanase activity;
c. mashing-off and filtering the mash to obtain the wort.
In a preferred embodiment, the unmalted cereal(s) are of the tribe Triticeae, e.g. barley, spelt, wheat, rye.
In another embodiment the unmalted cereal(s) are any unmalted cereal(s), such as but not limited to
barley, spelt, wheat, rye, corn, oat or rice or any mixture thereof. Thus in another embodiment of the
invention the grist comprises a mixture of unmalted cereals, such as but not limited to a mixture of
unmalted barley and unmalted wheat, a mixture of unmalted rice and unmalted barley.

In one embodiment, the invention relates to a process, where the grist further comprises other
carbohydrate sources, such as brewing syrups or any mixture thereof.
In another embodiment, the exogenous enzymes of step b. above further comprise a xylanase activity , a
lipase activity, and/or a phytase activity.
In a preferred embodiment, the mashing temperature is in a range optimizing the β- amylase activity and
reducing the lipoxygenase activity. A preferred embodiment of the invention concerns a process where
the pullulanase is thermostable having a relative enzyme activity above 60% over a period of 30 min, at
65 0C and at pH level 5.
In a further aspect, the invention relates to a wort produced by the process of the invention. Furthermore,
the invention relates to the use of the wort for the production of beers of any type, e.g. light and dark lager
types, light and dark ale types, wheat beers, all porter, stout, ice concentrated (eg. eisbock), barley wine
types or happoushu.
In a further aspect, the wort produced according to the invention comprises one or more amino acids
selected from a. proline at a concentration at less than 2 mM, preferably less than 1 mM, and most
preferably less than 0.5 mM in the wort; b. serine at a concentration above 0.1 mM, preferably above
0.125 mM, and most preferably above 0.15 mM; and c. methionine at a concentration above 0.05 mM,
preferably above 0.08 mM, and most preferably above 0.10 mM.
The invention further concerns an enzyme mixture comprising;
i. an α-amylase activity, ii. a pullulanase activity, w herein the pullulanase is thermostable iii. a proteolytic activity, and iv. a β-
glucanase activity;
In a particular embodiment the enzyme mixture comprising;
i. an α-amylase activity, ii. a pullulanase activity, iii. a proteolytic activity, iv. a β-glucanase activity; and v. a xylanase activity.
In another embodiment the enzyme mixture further comprises lipase activity. FIGURES
Figure 1 show s the turbidity (NTU) of a w ort produced from increasingly amount of barley w hen only Ultraflo Max is
exogenously added.
Figure 2 show s the fermentability of a w ort produced from 100 % unmalted barley or 100 % malted barley.
DEFINITIONS
Throughout this disclosure, various terms generally understood by persons skilled in the art are used. Several terms are
used w ith specific meanings, how ever, and are meant as defined by the follow ing. The term "malting" is a process w hereby
grains are made to germinate and are then dried.

The term "malted grain" is understood as any cereal grain, in particular barley, w hich has been subjected to a malting
process.
The term "unmalted grain" is understood as any cereal grain, in particular barley, w hich has not been subjected to a malting
process. The terms unmalted and non-malted could be used interchangeably in the present context.
The term "grist" is understood as the starch-containing or sugar-containing material that is the basis for beer production. It
may include malted and unmalted cereal as w ell as adjunct.
The term "cereals" is understood as grains w hich are any starch containing material used as raw material e.g. for production
of beer such as, but not limited to, barley, w heat, sorghum, maize, rice, oat and rye. The cereals may be malted or
unmalted.
The term "adjuncts" is usually understood as raw material w hich may be added to the main ingredient of the grist, w hich
traditionally are malted cereals. Thus since the unmalted grains usually only comprises a small part of the grist, unmalted
cereals is typically defined as an adjunct together w ith liquid carbohydrates such as sugars and sirups. The adjuncts could
be either solid or liquid or both, w here the solid part may be unmalted cereals, such as barley, corn and rice w hereas the
liquid part may be readily fermentable carbohydrates such as sugar and syrups.
In this context how ever, w hat might be regarded as adjunct may be the main ingredient. Thus unmalted cereals w hich in a
traditional context are an adjunct may according to the present invention comprise 100 % of the raw material. Accordingly,
unmalted cereals is usually comprised in the term adjunct how ever since the unmalted cereals preferably comprise more
than 70 % of the raw material and the malted cereals preferably is less than 30 % of the raw material the terms are in this
contexts most easily understood as:
The grist may comprise malted and unmalted cereals and adjuncts. Adjuncts are in this context understood as the part of the
grist w hich is not malted or unmalted cereal. Thus the adjuncts according to the present invention are preferably the liquid
part such as brew ing syrups and sugars.
Whereas unmalted cereals is any cereal not malted, thus any starch containing grains such as, but not limited to, barley,
corn, rice, rye, oats, sorghum and w heat. Accordingly grist from 100 % unmalted grains may comprise unmalted barley and
other non barley unmalted cereals such as rice and w heat.
In another embodiment of the invention the grist comprises a mixture of unmalted cereals, such as but not limited to a
mixture of unmalted barley and unmalted w heat, a mixture of unmalted rice and unmalted barley. Thus the grist may
comprise 50 % unmalted barley and 50 % unmalted other cereals, such as w heat and rice.
In a specially preferred embodiment of the invention the unmalted cereal(s) comprises more than 70% of the grist and the
malted cereals comprise less than 30% of the grist.
The term "mash" is understood as a starch-containing slurry comprising crushed barley malt, other starch-containing
material, or a combination thereof, steeped in w ater to make w ort.
The term "mashing process" or mashing profile or simply mashing is understood as the process of combining grains w ith
w ater and heating the mixture up w ith rests at certain temperatures to allow the enzymes in the mash to break dow n the
starch in the grain into sugars, to create a w ort.

"mashing off" or mashing out is w hen the temperature of the mash is raised. This frees up about 2% more starch, and
makes the mash less viscous.
The term "w ort" is understood as the unfermented liquor run-off follow ing the extraction of the grist during mashing. The
terms brew ers w ort and w ort is used interchangeably through out the application.
The term "spent grains" is understood as the drained solids remaining w hen the grist has been extracted and the w ort
separated. The term "beer" is here understood as a fermented w ort.
The term "beer product" is here understood as comprising "mash", "w ort", "spent grains" and "beer".
The term "DPV means glucose. The term "DP2" means maltose. The term "DP3" means maltotriose.
The terms "DP4+" or "DP4/4+" mean dextrin, or maltooligosaccharides of a polymerization degree of 4 or higher.
The term "Fru" means fructose. The term "RDF" means real degree of fermentation. The term "FAN" means free amino
nitrogen.
The term "Plato" (0
P) means grams extract pr 100 g w ort (gram extract/100 g w ort).
DETAILED DESCRIPTION OF THE INVENTION
By the addition of a combination of exogenous enzymes, e.g. α-amylase, isoamylase/pullulanase, FAN generating activity
(proteases) and filterability promoting activities
(beta-glucanase and/or xylanase), to the mash and by the simultaneous thermal activation of the maltose-generating
endogenous β-amylase, it is possible to obtain a w ort based on up to even 100% unmalted cereal(s).
Thus, in a first aspect, the invention relates to a process for the production of a brew er's w ort, comprising:
a. obtaining a mash by mashing a grist, of w hich at least 70w t% is unmalted cereals) comprising β-amylase activity and of
w hich less than 30w t% is malted cereal(s), at a temperature at w hich exogenous (added) enzymes and the endogenous β-
amylase are active;
b. contacting the mash w ith exogenous enzymes comprising: i. an α-amylase activity, ii. a pullulanase activity, iii. a
proteolytic activity, and iv. a β-glucanase activity; c. mashing-off and filtering the mash to obtain the w ort.
In a preferred aspect, the invention relates to a process, w here the grist comprises at least 70w t% unmalted cereal(s), such
as at least 75w t%, more preferably at least 80w t%, more preferably at least 85w t%, more preferably at least 86w t%, more
preferably at least 87w t%, more preferably at least 88w t%, more preferably at least 89w t%, more preferably at least 90w t%,
more preferably at least 91w t%, more preferably at least 92w t%, more preferably at least 93w t%, more preferably at least
94w t%, more preferably at least 95w t%, more preferably at least 96w t%, more preferably at least 97w t%, more preferably at
least 98w t%, even more preferably 99w t%, and most preferably 100w t% unmalted cereal(s).
It is to be understood that the at least 70 w t % unmalted cereal(s) may be one or more cereal(s) w herein at least one of the
cereal(s) contain β-amylase activity.

In one aspect of the invention the grist comprises less than 30w t% malted cereals, more preferably less than 25w t%, more
preferably less than 20w t%, more preferably less than 15w t%, more preferably less than 10w t%, more preferably less than
5w t% and even more preferably less than 3w t%, and most preferably the grist comprises 0 w t% malted cereals.
In a preferred embodiment, the unmalted cereal(s) are of the tribe Triticeae. Preferred w ithin this tribe are barley, spelt,
w heat and rye. Triticeae is a tribe w ithin the Pooideae subfamily of grasses that includes genera w ith many domesticated
species, EA Kellogg, R Appels, RJ Mason-Gamer - SYSTEMATIC BOTANY, 1996. Major crop genera are found in this tribe
including w heat, barley, and rye. In another preferred embodiment the grist comprises unmalted cereals other than from the
Triticeae tribe, such as but not limited to rice, corn, oat, sorghum.
In another preferred embodiment the unmalted cereal(s) are selected from the group comprising barley, spelt, w heat, rye,
corn, oat or rice or any mixture thereof.
Thus in one embodiment, the invention relates to a process, w here the grist further comprises of one or more additional
unmalted cereal(s) such as corn grist, corn starch and rice. The grist may therefore comprise a mixture of unmalted cereals,
such as but not limited to a mixture of unmalted barley and unmalted w heat or a mixture of unmalted rice and unmalted
barley. In a particular preferred embodiment of the invention the unmalted cereal is barley.
In yet another aspect the grist further comprises 0- 50 w t% other carbohydrate sources, such as brew ing syrups or any
mixture thereof.
In another embodiment, the exogenous enzymes of step b. above further comprise a xylanase activity, preferably family
GH10 (glycosyl hydrolase family 10) w hich may improve the filtration of w ort and beer.
In another embodiment, the exogenous enzymes of step b. above further comprise a lipase activity, w hich may improve the
w ort filtration and reduce haze.
In another embodiment the exogenous enzymes of step b. above further comprise a phytase activity.
In another embodiment the exogenous enzymes of step b. above further comprise one or more of the follow ing activities; a
xylanase activity, a lipase activity, and/or a phytase activity.
In a preferred embodiment the mashing temperature, i.e. the temperature at w hich the exogenous (added) enzymes and the
endogenous β-amylase are active, is in a range optimizing each of the different enzymes activity, at each heating step. The
mashing process is preferably performed in three steps each optimized to the different enzymes. These steps may be
referred to as enzyme rests or enzyme steps.
Thus a special embodiment of the invention concerns the temperature profile of a mashing process for producing a brew ers
w ort, w herein
A first step is carried out betw een 50 and 58 0
C, A second step is carried out betw een 60 and 65 0
C, and A third step is
carried out betw een 70 and 80 0
C.
The different enzymes in the mashing process both exogenous and endogenous have different temperature optimum and
the mashing process may be run at different temperatures for a certain period of time in order to let the enzymes react.
These periods is often referred to as enzyme rests.

In the first step, w hich might be termed the proteolytic step, the temperature is preferably w ithin the range of optimising e.g.
the proteolytic enzyme, the temperature is preferably betw een 450
C and 580
C, such as preferably betw een 460
C and 570
C,
such as preferably betw een 470
C and 560
C and 550
C
and 540
C, such as preferably betw een 5O0
C and 540
C, such as preferably betw een 510
C and 540
C, such as preferably
betw een 520
C and 540
C, most preferably betw een 530
C and 540
C, such as 540
C,
In the second step the temperature is preferably w ithin the range of optimising e.g. the starch converting enzymes, such as
the β-amylase and pullulanase. This step is often referred to as the saccharification step and the temperature is preferably
betw een 6O0
C and 720
C, such as preferably betw een 6O0
C and 7O0
C and 680
C, such as
preferably betw een 630
C and 670
C and 660
C, and most preferably betw een 640
C and
650
C, such as 640
C.
In the third step, w hich also may be referred to as mashing off or mashing out, this frees up about 2% more starch, and
makes the mash less viscous, allow ing the lauter to process faster. The temperature of the mashing out is preferably
betw een 720
C and 820
C and 810
C and 8O0
C, such as
preferably betw een 750
C and 790
C and 780
C, most preferably the temperature is
betw een 78°C-80 0
C, such as 80 0
C.
Endogenous lipoxygenase is know n to be a source of off-flavour, and in a preferred embodiment, the mashing temperature,
in the first mashing step referred to above is in a range reducing the lipoxygenase activity w ith at least 50%, preferably 55%,
preferably 60%, preferably 65%, preferably 70%, preferably 75%, preferably 80%, preferably 85% most preferably 90%
relative to the activity at mashing at 540
C.
The invention further relates to an enzyme mixture comprising:
i. an α-amylase activity, ii. a pullulanase activity, w herein the pullulanase is thermostable iii. a proteolytic activity, and iv. a β-
glucanase activity;
or in another embodiment of the invention concerns an enzyme mixture comprising;
v. an α-amylase activity, vi. a pullulanase activity, vii. a proteolytic activity, viii. a β-glucanase activity; and ix. a xylanase
activity.
The enzyme mixtures may further comprise lipase activity.
The terms enzyme mixture and enzyme blend are used interchangeably in through out the application. The terms are to be
understood as a mixture or blend of different enzymes or enzyme activities. The enzymes in the mixture or blend may be
added in any order or together. The enzymes if not added together could be added in any order and is not necessarily
added in the order listed above.
The enzymes according to the invention could be added at anytime of the mashing or before mashing. Thus the enzymes
may be added to the mash ingredients, e.g., the w ater and/or the grist before, during or after forming the mash. The
enzymes may be added together or separately.
In a preferred aspect, the α-amylase activity is provided by an α-amylase of fungal origin, e.g. from Aspergillus niger, or
bacterial origin, e.g. Bacillus. Thus the α-amylase might be a bacterial α-amylase variant having increased thermo stability at
acidic pH and/or low Ca2+
concentration. Preferably, the α-amylase activity in the mash is 0.1-1.0 KNU(S)/g, more preferably
0.2-0.4 KNU(S)/g, and most preferably 0.25-0.35 KNU(S)/g dry w eight cereal(s). Preferably the α-amylase has at least 50%,

more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, preferably at leas t 85%, more
preferably at least 90%, preferably at least 91 %, preferably at least 92%, preferably at least 93%, preferably at least 94%,
more preferably at least 95%, preferably at least 96%, preferably at least 97%, more preferably at least 98%, and most
preferably at least 99% identity to the amino acid sequence show n in SEQ ID NO:1 (a variant of the B. stearothermophilus
α-amylase w ith the mutations 1181 *
G182*
N193F, described in WO99/19467 and available as Termamyl®
SC from
Novozymes A/S).
In a preferred embodiment of the invention, the starch debranching activity is provided by a pullulanase. In another
embodiment of the invention the debranching activity is provided by other debranching enzymes such as but not limited to
an isoamylase or limit dextrinase. In a certain embodiment of the invention the debranching activity is provided by a mixture
of debranching enzymes such as but not limited to a pullulanase and an isoamylase. Thus in a preferred embodiment of the
invention, a pullulanase (E. C. 3.2.1.41 ) enzyme activity is exogenously supplied and present in the mash. The pullulanase
may be added to the mash ingredients, e.g., the w ater and/or the grist before, during or after forming the mash.
The pullulanases according to the present invention is preferably pullulanase from e.g. Pyro- coccus or Bacillus, such as
Bacillus acidopullulyticus e.g. the one described in FEMS Microbiol. Letters 1 15: 97-106, or pullulanase is available from
Novozymes as Promozyme 400L and having the sequence show ed in SEQ ID NO: 2. The pullulanase may also be from
Bacil- lus naganoencis, or Bacillus deramificans e.g. such as derived from Bacillus deramificans (US Patent 5,736,375) and
having the sequence show ed in SEQ ID NO: 7. The pullulanase may also be an engineered pullulanases from, e.g. a
Bacillus strain.
Other pullulanases may be derived from Pyrococcus w oeseidescribed in PCT/DK91/00219, or the pullulanase may be
derived from Fervidobacterium sp. Ven 5 described in
PCT/DK92/00079, or the pullulanase may be derived from Thermococcus celer described in
PCT/DK95/00097, or the pullulanase may be derived from Pyrodictium abyssei described in
PCT/DK95/00211 , or the pullulanase may be derived from Fervidobacterium pennavorans described in PCT/DK95/00095,
or the pullulanase may be derived from Desulforococcus mu- cosus described in PCT/DK95/00098.
Most preferably the pullulanase is derived from Bacillus acidopullulyticus. A preferred pullulanase enzyme to be used in the
processes and/or compositions of the invention is a pullulanase having an amino acid sequence w hich is at least 50%, such
as at least 55%, such as at least 60%, such as at least 65%, such as at least 66%, such as at least 70%, such as at least
75%, such as at least 80%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as
at least 89%, such as at least 90%, such as at least 91 %, such as at least 92%, such as at least 93%, such as at least 94%,
such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% or even 100%
identical to the sequence show n in SEQ ID NO:8 (NS26062, PuIC, from Bacillus acidopullulyticus); in particular w hen
aligned using the Program Needle using Matrix: BLO- SUM62; Gap initiation penalty: 10.0; Gap extension penalty: 0.5;
Gapless Identity Matrix. The terms PuI C, NS26062 and pullulanase C is used interchangeably throughout the application.
The pullulanase is added in dosage of 0.1 to 3 PUN/g DM, such as 0.2 to 2,9, such as 0.3 to 2.8, such as 0.3 o 2.7 such as
0.3 o 2.6 such as 0.3 to 2.5 such as 0.3 to 2.4, such as 0.3 to 2.3, such as 0.3 to 2.2, such as 0.3 to 2.1 , such as 0.3 to 2.0,
such as 0.3 to 1.9, such as 0.3 to 1.8, such as 0.3 to 1.7, such as 0.3 to 1.6, most preferably pullulanase is added in dosage
such as 0.3 to 1.5, preferably 0.4 to 1.4, more preferably 0.5 to 1.3, more preferably 0.6 to 1.2, more preferably 0.7 to 1.1 ,
more preferably 0.8 to 1 .0, more preferably 0.9 to 1.0. In a particular embodiment of the invention the enzyme is added in
0.3 PUN/g DM, such as 0.4 PUN/g DM, such as 0.5 PUN/g DM in a particularly preferred embodiment of the invention the
enzymes dose is not larger than 1 PUN/g DM. Preferably the isoamylase or/and pullulanase activity in the mash is 0.1-2.0
PUN/g, more preferably 0.5-1.0 PUN/)/g dry w eight cereal(s).

The relative activity of the debranching enzymes, such as pullulanases, may vary considerably at different temperatures e.g.
as demonstrated in example 2 of the application. The debranching enzymes are w orking together w ith the other enzymes in
the mash, in particular the β-amylase, w hich is usually endogenous and the α-amylase w hich may be endogenous or
exogenously added. Thus a preferred debranching enzyme according to the invention is an enzyme having high relative
enzyme activity in the temperature range at w hich both the β- amylase and the α-amylase is active. The α-amylase is
usually active at a higher temperature than the β-amylase and the saccharification step of the mashing process, the step
w here the starch is converted into fermentable sugars by α-amylase, β-amylase and a debranching enzyme, is preferably
run at a high temperature, such as at least 63 C°. Thus the debranching enzyme according to the invention is preferably
thermostable and thermoactive. The terms thermostable and thermo active is used interchangeably through out the
application.
In this context a thermostable enzyme is an enzyme having a relative enzyme activity above 60% measured over a period of
30 min, at 65 0
C and at pH level 5.
The relative activity, w hich in this context is the relative enzyme activity, is calculated by setting the highest activity to 100%
(maximum) and setting the activities at other temperatures relative to the temperature maximum.
Thus preferably the debranching enzyme is a pullulanase and even more preferably the pullulanase activity is provided by a
pullulanase w hich is thermostable having a relative enzyme activity above 60% over a period of 30 min, at 65 0
C and at pH
level 5. An example of a thermostable pullulanase is given in example 2. In one embodiment the pullulanase relative
enzyme activity is above 60%, such as above 61%, such as above 62%, such as above 63%, such as above 64%, such as
above 65%, such as above 66%, such as above 67%, such as above 68%, such as above 69%, such as above 70%, such
as above 71%, such as above 72%, such as above 73%, such as above 74%, such as above 75%, such as above 76%,
such as above 77%, such as above 78%, such as above 79%, such as above 80%, such as above 81%, such as above
82%, such as above 83%, such as above 84%, such as above 85%, such as above 86%, such as above 87%, such as
above 88%, such as above 89%, such as above 90%, such as above 91%, such as above 92%, such as above 93%, such
as above 94%, such as above 95%, such as above 96%, such as above 97%, such as above 98%, such as above 99% and
even 100% at 65°C, w hen measured over a period of 30 minutes, at pH 5,0.
In a particular preferred embodiment of the invention a thermostable pullulanase has a relative enzyme activity above 80%
over a period of 30 min, at 650
C and at pH level 5.
In a certain embodiment the pullulanase has above 80%, such as above 85%, such as above 90% such as above 95%, or
even 100% remaining enzyme activity over a period of 30 min under mashing conditions w ith 12 0
P barley, at gelatinization
temperature of unmalted barley, and at pH in the range of 5.6-6.2, compared to the activity before incubation at the
gelatinization temperature of unmalted barley.
In another embodiment the protease activity is provided by a proteolytic enzymes system having a suitable FAN generation
activity including endo-proteases, exopeptidases or any combination hereof, preferably a metallo-protease. Preferably the
protease activity in the mash is 0.0005-0.002 AU/g, more preferably 0.001-0.0015 AU/g dry w eight cereal(s). Preferably, the
protease has at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more
preferably at least 85%, more preferably at least 90%, more preferably at least 91 %, more preferably at least 92%, more
preferably at least 93%, more preferably at least 94%, more preferably at least 95% more preferably at least 96%, more
preferably at least 97% more preferably at least 98%, and most pref erably at least 99% or even 100 % identity to the amino
acid sequence show n in SEQ ID NO:3 (a metallo- protease from Bacillus amyloliquefaciens, described in WO9967370,
available as Neutrase®
from Novozymes A/S).

In a further embodiment, β-glucanase (E.C3.2.1.4.) activity is added to the mash. Preferably the β-glucanase activity in the
mash is 0.1-1.5 FBG/g, such as 0.2-1.2 FBG/g, such as 0.4-1.0 FBG/g, such as 0.5-1.0 FBG/g dry w eight cereal(s). β-
glucanase is also termed cellulase and may be of fungal or bacterial origin. Such as from Aspergillus orzyae, Aspergillus
niger or from bacillus such as B subtilis. The added β-glucanase activity may also origin from malt. In one particular
preferred embodiment of the invention the β-glucanase is added together w ith xylanase in an enzyme blend termed Ultraflo
Max. Ultraflo Max is an enzyme blend of Xy- lanase and β-glucanase, the blend is described in the application
WO2005/059084 A1.
In another embodiment, the xylanase activity is provided by a xylanase from glycosyl hydrolase family 10. Preferably the
xylanase activity in the mash is 0.02-0.1 FXU-S/g, more preferably 0.04-0.08 FXU-S/g dry w eight cereal(s). Preferably, the
xylanase has at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more
preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more
preferably at least 93%, more preferably at least 94% more preferably at least 95%, more preferably at leas t 96%, more
preferably at least 97% more preferably at least 98%, and most preferably at least 99% or even 100% identity to the amino
acid sequence show n in SEQ ID NO:4 (described in WO 94/21785, available as Shearzyme®
from Novozymes A/S).
In another embodiment, the lipase activity is provided by a lipase having activity to triglycerides and/or galactolipids and/or
phospholipids. Preferably, the lipase activity is provided by a lipase from Fusarium (including F. oxysporum and F.
heterosporum), Aspergillus (including A. tubigensis), Rhizopus (including R. oryzae) or Thermomyces (including T. lanu-
ginosus) or a variant of these. An example is Lipopan X (Lipopan Xtra), a variant of the Thermomyces lanuginosus lipase
w ith the substitutions G91A +D96W +E99K +P256V +G263Q +L264A +I265T +G266D +T267A +L269N +270A +271 G
+272G +273F (+274S), described in WO2004099400A2. Preferably, the lipase has at least 50%, more preferably at least
60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least
94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97% more preferably at least
98%, and most preferably at least 99% or even 100 % identity to residues 1-316 or 1-273 of the amino acid sequence shown
in SEQ ID NO:5 (lipase/phospholipase from Fusarium oxysporum, described in EP 869167, available from Novozymes A/S
as Lipopan®
F). Preferably, the lipase activity in the mash is 0-50 LU/g, such as 0-40 LU/g, such as 0-30 LU/g, such as 0-20
LU/g dry w eight cereal(s). In a specially preferred embodiment of the invention the lipase is Li- pozyme TL or lipolase, this
lipase has a significantly good effect on filtration speed and haze reduction. Thus in a special preferred embodiment of the
invention the lipase has at least 50 %, more preferably at least 60%, more preferably at least 70%, more preferably at least
96%, more preferably at least 97% more preferably at least 98%, and most preferably at least 99% or even 100 % identity to
the amino acid sequence show n in SEQ ID NO 9. The lipase may also be Lipex, a variant of Li- pozyme having at least
95%, more preferably at least 98%, and most preferably at least 99% or even 100 % identity to the amino acid sequence
show n in SEQ ID NO: 10. The lipases degrade the lipid from barley e.g. the triglyceride into partial glycerides and free fatty
acids. This leads to a low er turbidity and much improved mash filtration and lautering properties.
In another embodiment, the phytase activity is provided by a phytase from Aspergillus niger, Peniophora or Citrobacter.
Preferably, the phytase activity in the mash is 0-5 FYT/ g, more preferably 0.5-1.5 FYT/g dry w eight cereal(s). Preferably,
the phytase has at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%,
more preferably at least 85%, more preferably at least 90%, more preferably at least 91 %, more preferably at least 92%,
more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%,
more preferably at least 97%, more preferably at least 98%, and most preferably at least 99% or even 100 % identity to the
amino acid sequence show n in SEQ ID NO:6 (a variant of Peniophora lycii phytase, described in WO 2003/066847).

In some embodiment of the invention Flavourzyme is added. Flavourzyme is an enzyme composition obtained from A.
oryzae strain NN000562 originally obtained as ATCC 20386. FLAVOURZYME contains alkaline and acid protease activities.
In a further aspect, the invention relates to a w ort produced by the process of the invention.
Furthermore, the invention relates to the use of the w ort for the production of beers of any type, e.g. light and dark lager
types, light and dark ale types, w heat beers, all porter, stout, ice concentrated (e.g. eisbock), barley w ine types or
happoushu.
The nitrogen containing components are important components of the w ort because they affect the character of the beer,
such as the taste and fermentation pattern. The nitrogen containing compounds are important nutrients for the yeast w ith the
exception of proline w hich is hardly assimilated by the yeast thus it is favourable to have a small amount of proline or no
proline in the w ort. Thus in a further aspect, the w ort comprises one or more amino acids selected from a. proline at a
concentration at less than 2 mM, preferably less than 1 mM, and most preferably less than 0.5 mM in the w ort; b. serine at a
concentration above 0.1 mM, preferably above 0.125 mM, and most preferably above 0-15 mM; and c. methionine at a
concentration above 0.05 mM, preferably above 0.08 mM, and most preferably above 0.10 mM.
Thus in one aspect the proline concentration is below 2 mM, such as below 1.5 mM, such as below 1 mM, such as below
0.5 mM, such as below 0.25 mM.
In another aspect the serine concentration is above 0.1 mM, such as 0.125 mM, such as 0.15 mM, such as 0.2 mM.
In another aspect the methionine concentration is above 0.005 mM, such as 0.008 mM, such as 0.1 mM, such as 0.125 mM,
such as 0.15 mM.
The inventors has surprisingly found that even w ith very high amounts e.g. above 80 % of unmalted cereals such as
unmalted barley a w ort could be produced w hich has a high amount of fermentable sugars, w hich in this context is DP1-DP3
(glucose, maltose and maltotriose) and in a particular preferred aspect of the invention the amount of maltose is high
compared to the amount of glucose, w hich is favorable because it prevents osmotic pressure on the yeast and regulates the
ester production and therefore the flavour and aroma profile of the final beer.
Thus one aspect if the invention concerns a w ort, w here the maltose concentration is above 45%, preferably above 50%,
preferably above 55%, preferably above 56%, preferably above 57%, preferably above 58%, preferably above 59%,
preferably above 60%, preferably above 61 %, preferably above 62%, preferably above 63%, preferably above 64%,
preferably above 65%, most preferably the maltose concentration is above 70% of the total concentration of carbohydrates.
In another aspect the invention concerns a w ort w here the glucose concentration is below 10%, preferably below 9%,
preferably below 8%, preferably below 7%, preferably below 6%, preferably below 5% most preferably below 4%.
Yet another aspect of the invention concerns a w ort according to any of the proceeding claims w here the total of the
glucose, maltose and maltotriose concentration is above 50%, preferably above 55%, preferably above 60%, preferably
above 61%, preferably above 62%, preferably above 63%, preferably above 64%, preferably above 65%, preferably above
66%, preferably above 67%, preferably above 68%, preferably above 69%, preferably above 70%, preferably above 71 %,
preferably above 72%, preferably above 73%, preferably above 74%, preferably above 75%, preferably above 76%,
preferably above 77%, preferably above 78%, preferably above 79% and preferably above 80% of the total concentration of
carbohydrates.

RDF (Real degree of fermentation) is calculated as RDF% = 100*
(OE%P - ER%) / OE%P w hereas OE means Original
Extract in %P and ER means Real Extract % P measured by a densitometer (Analytica EBC reference).
Thus in one aspect of the invention the RDF in the w ort is more than 60%, such as at least 65%, such as at least 70%, such
80%, such as at least 81 %, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such
91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as
at least 97%, such as at least 98%, such as at least 99%, such as at least 100%.
Some brew eries add brew ery syrup, e.g. high maltose, brew ing syrup to the w ort kettle w hich may increase the amount of
fermentable sugars. How ever, though brew ing syrup may be added according to the invention this is not necessary for
increasing the amount of fermentable sugars or RDF.
In another embodiment of the invention concerns a process, w herein the ratio of maltose:glucose in the w ort is higher than
5:1 , such as higher than 6:1 , such as higher than 7:1 , preferably higher than 8:1 , preferably higher than 9:1 , preferably
higher than 10:1 , preferably higher than 1 1 :1 in a particular preferred embodiment the ratio of maltose:glucose in the w ort
is higher than 12:1.
During the mashing process starch is degraded into fermentable and unfermentable sugars and the proteinous material is
converted the free amino acids w hich is used by the yeast. According to the invention the raw material used for mashing can
be up to 100 % unmalted cereals, such as unmalted barley w ithout reducing the fermentability of the w ort or reducing the
amount of amino acids available for the yeast. In addition, brew ing on unmalted cereals may give problems w ith filterability
due to excess of non converted starch and β-glucan or xylan, w hich may also cause haze of the beer. Adding filtration aiding
enzymes such as β-glucanase may increase the filterability of the w ort. How - ever, w hen unmalted cereal comprises main
part of the grist, β-glucanase alone is not enough provide filterable w ort.
The inventors have surprisingly found that adding exogenous enzymes according to the invention, comprising α-amylase
activity, pullulanase activity, proteolytic activity, lipase activity and β-glucanase activity; to the mash prepared from a grist
comprising at least 70 % unmalted cereal(s) produced a w ort w hich is comparable or even better w ith regards to e.g. FAN,
fermentable sugars (DP1-DP3) and w hich is filterable and also have an acceptable low turbidity w hen compared to a w ort
produced from a malted grist.
The lauter tun time, the time is takes to filter the mash in the lauter tun, if this is in a separate vessel, is influenced e.g. by
the turbidity. Thus in a certain aspect of the invention the w ort is filterable and has a low turbidity and in one embodiment of
the invention the turbidity is below 20 NTU (The units of turbidity from a calibrated nephelometer, Nephelometric Turbidity
Units), such below 19 NTU, such below 18 NTU, such below 17 NTU, such below 16 NTU, such below 15 NTU, such
below 14 NTU, such below 13 NTU, such below 12 NTU, such below 1 1 NTU, such below 10 NTU.
One w ay of increasing the amount of fermentable sugars is by increasing the mashing time e.g. by increasing the
saccharification step. How ever, in another important aspect of the invention the mashing time needed for producing a w ort
w hich is highly fermentable is not increased compared to the mashing time for producing an equally fermentable w ort based
on the same amount of malt.
This is surprising since generally longer mashing time is needed w hen the mash is based on high amount unmalted cereals
e.g. 70% barley to give the same fermentability and FAN as in a w ort produced on corresponding amounts (70%) of malt.

Thus in a particular embodiment of the invention the mashing process is completed w ithin 160 minutes, preferably w ithin
120 minutes. In one embodiment of the invention the mashing process comprising all the enzymes rests and all heating
steps, is completed w ithin 180 minutes, such as w ithin 170 minutes, such as w ithin 160 minutes, such as w ithin 155
minutes, such as w ithin 150 minutes, such as w ithin 145 minutes, such as w ithin 140 minutes, such as w ithin 135 minutes,
such as w ithin 130 minutes, such as w ithin 125 minutes, such as w ithin 120 minutes, such as w ithin 1 15 minutes, such as
w ithin 1 10 minutes, such as w ithin 105 minutes, such as w ithin 100 minutes, such as w ithin 95 minutes, such as w ithin 90
minutes, such as w ithin 85 minutes, such as w ithin 80 minutes, such as w ithin 75 minutes, such as w ithin 70 minutes, such
as w ithin 65 minutes, such as w ithin 60 minutes.
When malt is substituted w ith grains such as rice and corn the grist may need to be treated by decoction or decoction
mashing or adjunct decoction, w hich is process w here a proportion of the grains are boiled separately w ith thermostable α-
amylase and then returned to the mash. This process is often needed for these types of grains as the gelatinization
temperature is higher than for barley, malt, and e.g. w heat. Thus pregelatinization is needed to make the starch acc essible
for all the needed endogenous and added enzymes. The process may also be used to give a malty flavor to the beer.
Unmalted cereals, such as barley show s a general different behaviour in milling than malted cereals, as an example barley
has higher w ater content, is unmodified and is much harder than malt.
To run a lauter tun w ith malt and achieve an acceptable performance (yield and lauter time) a certain grist composition is
necessary, the grist composition can be measured by a sieving test.
The grist composition made by roller mills are mainly influenced by the gap betw een the roller pair(s) (tw o roller mill = one
pair, four roller mill = tw o pair). The first pair has alw ays a w ider gap than the second one. In order to obtain a lauter
performance compared to a grist made of malt the inventors has changed, these roller gap(s).
The inventors found that a four roller mill and a six roller mill (three pairs) could mill w ith adjusted roller gaps are w ell suited
to mill the barley into usable grist. This is important since a good lauter performances only can be achieved w ith an
optimized grist composition that is different to the optimal grist composition of malt. The sieving test w as performed
according to the sieving test described in Anger, H.: MEBAK Band Rohstoffe. 1. Auflage Brautechnische
Analysenmethoden. 2006, Freising: Selbstverlag der MEBAK.
Table 1
Milled barley compared to malt

The results show that for a successful100 % barley lauter performance more coarse grist w ith more focus on sieve 1-3
leads to a good lauter performance. It could also be seen that the barley grist is significantly different from the grist from
malt.
EXAMPLES:
MATERIALS AND METHODS
Enzymes
Alpha-amylase activity (KNU)
The amylolytic activity may be determined by using potato starch as substrate. This method is based on the break-dow n of
modified potato starch by the enzyme, and the reaction is follow ed by mixing samples of the starch/enzyme solution w ith an
iodine solution. Initially, a blackish-blue color is formed, how ever, during the break-dow n of the starch the blue color gets
w eaker and gradually turns into a reddish-brow n, w hich is compared to a colored glass standard.
One Kilo Novo alpha amylase Unit (KNU) equals 1000 NU. One KNU is defined as the amount of enzyme w hich, under
standard conditions (i.e. at 37°C +/- 0.05; 0.0003 M Ca2+; and pH 5.6) convert 5.26 g starch dry substance (Merck Amylum
solubile) into dextrins sufficiently small not to make a colour reaction w ith iodine Debranching activity (PUN)
Pullulanase activity may be determined relative to a pullulan substrate. Pullulan is a linear D-glucose polymer consisting
substantially of maltotriosyl units joined by 1 ,6- alpha - links. Endopullulanases hydrolyze the 1 ,6-α-links at random,
releasing maltotriose, 63
- alpha - maltotriosyl-maltotriose, 63
- alpha -(63
- alpha -maltotriosyl-maltotriosyl)-maltotriose, etc. the
number of links hydrolyzed is determined as reducing carbohydrate using a modified Somo- gyi-Nelson method.
One pullulanase unit (PUN) is the amount of enzyme w hich, under standard conditions (i.e. after 30 minutes reaction time at
400
C and pH 5.0; and w ith 0.2% pullulan as substrate) hydrolyzes pullulan, liberating reducing carbohydrate w ith a
reducing pow er equivalent to 1 micromol glucose per minute.
Proteolytic Activity (AU)
The proteolytic activity may be determined by using denatured hemoglobin as substrate. In the Anson-Hemoglobin method
for the determination of proteolytic activity, denatured hemoglobin is digested, and the undigested hemoglobin is precipitated
w ith trichloroacetic acid (TCA). The amount of the TCA soluble product is determined by using phenol reagent, w hich gives
a blue color w ith tyrosine and tryptophan.
One Anson Unit (AU) is defined as the amount of enzyme w hich under standard conditions (i.e. 25°C, pH 7.5 and 10 min.
reaction time) digests hemoglobin at an initial rate such that there is liberated an amount of TCA soluble product per minute
w hich gives the same colour w ith phenol reagent as one milliequivalent of tyrosine.
β-glucanase activity (FBG)
One fungal beta glucanase unit (FBG) is the amount of enzyme, w hich, according to the standard conditions outlined below ,
releases reducible oligosaccharides or reduces carbohydrate w ith a reduction capacity equivalent to 1 mol glucose per
minute.

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