Wort boiling

Wort boiling

ObjectiveTo 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.

SterilisingBrewing 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 50-800C), 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 780C. 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 WortDuring 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 1400C compared to conventional Copper boiling under atmospheric pressure.

Removal of VolatilesDuring 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.

DMSA 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 oilsThe 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 ColourThe 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 pHControl 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 boilingSome 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 LevelsDuring 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 1000C are given below.

Nitrogen removal after different boiling times for a standard boil

Duration of boil (hours) % wort nitrogen reduction0 00.5 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 l000C 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 CompoundsMalt 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.

DMSDMS ou Dimethyl sulfides

d'après le Home Brewing Wiki

Le Sulfure de Dyméthyle (DMS) est un composé organosulfuré présent en niveaux inférieurs 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

http://fr.wikipedia.org/wiki/Compos%C3%A9_organosulfur%C3%A9

DMS in Beer Some detectable level of DMS is characteristic of many lager styles, and is especially noticeable in light lagers. However, DMS is present in most beers at some level. It is excessive DMS that gives some home brewed ales a "cooked corn" character. The amount of DMS found in beer is lowest in British ales, 10 - 20 ppb and highest in German lagers and all-malt beers, 50 -175 ppb, while the United States' lagers generally contain 40 - 100 ppb. Beers with high adjunct ratios or low gravities allow the DMS taste or off-taste to be more detectable, while German beers, all-malt beers, flavorful beers, especially dark beers, make the taste of DMS less discernible even at higher levels.3

[edit] Causes of DMS DMS is created whenever wort is heated, by the breakdown of precursors found in pale malts. Under ordinary circumstances, most of the DMS that is created by heat is then evaporated during the boil. Some DMS is also removed during vigorous ale fermentations, which is why higher 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 wort too slowly means that excessive levels of DMS can be created which cannot be 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 of approximately 30% will result. The level of DMS in the wort determines the level of DMS in finished beer. In order to predict the level of DMS in finished beer Table V shows the relationship between SMM in malt and DMS in beer.

The major source of DMS in finished beer is derived from its precursor, S-Methylmethionine (SMM), an amino acid, which is formed during the germination and kilning process of malting barley. Barley does not contain 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, wort boiling and hot wort storage.

The most effective way to reduce SMM during germination is by slightly under- modifying malt, specifically by reducing the moisture content of barley at steep-out to 40-42% and reducing the germination temperature to 55-60oF. It has been shown that a 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 significantly reduce SMM and insuring DMS levels in finished malt.

Two row barley which has a normally lower nitrogen content than six row barley, has been shown to produce significantly less SMM during the malting process. European malt has less SMM and DMS than North American and Canadian malt.1

Regardless of variety or growing conditions, the most important factor for reducing SMM and DMS occurs during kilning. The SMM formed during germination is converted by the heat of 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 easily removed during the kettle boil, it is important that the ratio of SMM to DMS be as low as possible in the finished malt.

The conversion of SMM to DMS occurs at about 1401F. 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 significantly be reduced in the finished malt. The stability of SMM in malt is greater at higher moisture levels.

[edit] Preventing DMS The level of SMM in malt is responsible for the DMS level in wort. During mashing the SMM, DMS and very soluble DMSO are brought into 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 that three-fourths is removed in 90 minutes. Narssis recommends a 100 minute boil to reduce the level of SMM and DMS to acceptable levels in most beers.

The level of DMSO does not change during the kettle boil. A small amount of DMS, 0.4 ppb, may be contributed by hops, especially if added in large amounts late in the boil. As long as the wort is hot SMM will be converted to DMS. It is important to 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 of DMS in the finished beer:

Boil the entire wort 90 minutes or longer Ensure that the boil is vigorous - rolling Allow at least 8% evaporation Minimize the hot wort standing time Rapidly cool 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 not convert SMM to DMS but certain yeast can produce higher DMS levels by reducing DMSO to DMS, especially in lager beer production at cooler temperatures. Certain wild yeasts and Enterobacter agglomerans can produce DMS.5 Table VI shows the reactions that take place in malting and wort boiling.

[edit] DMS After fermentation Purging and contamination occurs can can change the the DMS concentration in beer.Water dilution of high gravity beers may reduce the perceived threshold of DMS due to dilution of other mashing flavors. If DMS precursors,e.g. DMSO, reach the final product they are reduced to DMS,incresing the DMS concentration during the beer shelf life.

[edit] Creating DMS DMS is naturally present in 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, simply cover 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 of DMS. Those with the lowest thresholds are most likely to have off tastes at excessive DMS levels.

Lagers (lowest):

Low adjunct beers with low gravities or diluted flavor. High adjunct beers with corn grits High adjunct beers with other adjuncts Low to medium gravity (1.040-1.048) beers - all malt All malt German or higher gravity, light colored - flavored beers Amber - dark flavorful beers Ales (highest):

British light ales 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 amount of flavor in beer Flavor: Cooked corn, creamed corn Aroma: Same - highly volatile Source: Precursor in malt (SMM), hops (minor)

Decoction MashingA decoction mash is a type of mash in which at least one mash rest temperature is reached by removing part of the mash, boiling it in 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, especially in Germany and the Czech Republic. But most brewries in these regions have switches to the more economical directly heated step infusion mashing.

Decoction mashing is not very common among home brewers, since it has a reputation as a time and labor-intensive process. But a decoction mash is basically just a step infusion mash where some of the grist is heated and returned instead of infusion water. While it does take some extra time and require some extra stirring, it is a procedure that can be performed by most home brewers.

History of the Decoction Mash

Decoction mashing refers to removing a part of the mash, boiling it and returning it to the main mash to raise the temperature to the next rest. This mashing procedure originates from a time when malt quality was not consistent and temperatures could not be measured. The long boiling of the grain makes the starches more accessible for the enzymes. This is particularly important for undermodified malts where the cell walls are not as broken down as well as they are in well modified or overmodified malts. The boiling of a defined portion of the mash and returning it to the main mash to raise the temperature also helped the consistency in mashing temperatures before thermometers were available.

Chemistry of the Decoction MashToday even most European malts are generally well modified and can be used in infusion step mashes or even single infusion mashes, thus removing the need for decoction mashing. But decoction mashing is still widely used, particularly in 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. But especially in the home brewing community, there has been a hot debate about the actual benefits of a mash as labor intensive as a decoction mash. Many say that with the malts that are available to the home brewer decoction mashing doesn't make for a difference and if there is a difference it could also be achieved by the use of specialty malts. But in the end every brewer has to determine that for him or herself.

Decoction Mash ProcedureThe basic procedure for performing a decoction mash is very simple. Water is added to the grist to reach the initial mash temperature. Once the first temperature rest is complete, a portion of the grain and water is scooped out of the mash tun and into the kettle or another heated vessel, where it is brought to 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 cannot be heated as fast as infusion water and it is usually boiled for 5 – 45 min. After boiling, the decoction is returned to the mash tun to achieve the next temperature rest.

Sample Decoction Mash SchedulesThis section contains discussions of a number of sample schedules for decoction mashes, including single, double, and triple decoctions. The brewer should bear in mind that some 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 first Pilsners 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 rest and saccharification rest. At each of these rests a decoction is used to reach the next rest until the mash-out is reached. The acid rest is a convenient rest to do mash pH adjustments. Not only does it serve to lower the pH by simply using 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 out there for calculating the decoction volume. Some of them are simple and others try to account for factors such as the heat capacity of the grains and the mash-tun. The easiest way 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 about 15 - 20%. The idea is to decoct more mash than necessary. When the decoction is added back to the main mash, it is 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 account for 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 of the decoction depends on the thickness of the main mash. Though it is preferred to leave a lot of the liquid back in the mash tun, the decoction should not be 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 of 1.5 - 2 qts/lb (3-4 kg/l) the decoction should have a thickness of 1-1.25 qts/lb (2-2.5 kg/l). At this thickness and with gentle heating, only little stirring is necessary to keep the mash from scorching. For lager grists (high gravity beers) the thickness of the mash may however be limited by the volume of the mash tun.

All the decoction schedules provided here assume a decoction rise temp of 2-4 *F/min (1-2 *C/min). This is what is generally recommended in the literature for heating the mash. In technical brewing this is a result of the decoction kettle design which cannot heat the mash any faster. There is also a saccharification rest at 155 - 162 *F (68 - 72 *C). The purpose of this rest is to utilize the enzymatic power of the decoction before its enzymes are destroyed by further heating. This is particularly important when brewing beers with a large percentage of the enzymatic weaker dark base

malts. This rest doesn't have to be held at the main saccharification temperature. It is sufficient to rest in the alpha amylase range where the conversion is also done much quicker. After the decoction is converted or almost converted (iodine test) the heating of the mash is resumed. To hold this rest the 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 light colored beers, longer boil times for dark colored beers. If only gentle heat is applied during the boil, stirring should only be necessary occasionally. Similar to wort boiling, excessive thermal loading of the decoction can result in 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 it out and makes it more 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 rest temperature and time before pulling the next decoction should be based on the malt that is used. Less modified malts benefit from a rest closer to 122 *F (50 *C) which produces more amino acids, which is an essential yeast nutrient. In undermodified malts the protein conversion has not been driven far enough during malting to allow for sufficient wort FAN (free amino nitrogen) without the use of a more intensive protein rest. If the malt is a well modified modern malt, the protein rest temperature should be kept closer to 133 *F (55 *C) and the next decoction should be pulled 5 - 10 minutes after the rest temperature has been reached. This serves to protect more of the medium chained proteins that are important for body and head retention. Decoction schedules that allows for a shorter protein rest in general will be described later.

A decoction is pulled again, rested for conversion and then brought to a boil. This time to reach the saccharification rest temperature. This temperature is similar to the saccharification rest temperature that is used for a single infusion mash, but the same temperature that was used in a single infusion mash may not give the same fermentability in a decoction mash. Boiling has destroyed more of the enzymes while it has made the starch also more easily accessible. The former would lead to a less fermentable result while the latter would shift the fermentability towards a more fermentable wort. This is only to illustrate that experimenting with the saccharification rest temperature might be necessary for optimal results. The saccharification rest temperature that would have been used in a single infusion mash is however a good starting point.

After holding the saccharification rest for about 45 min or longer, if starch conversion is not complete after that time, the final decoction is pulled. This decoction can also be thinner and doesn't have 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 only one decoction is used. This decoction can be used to reach any rest, but most commonly it is used to reach the mash-out temperature. This can be a simple enhancement of a single infusion or step mash.

The mash schedule shown above is well suited for European and continental lager malts. It features a short protein rest at the higher end of the temperature range for proteolytic activity, a single temperature saccharification rest and 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 consistency of about 2.5 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 already have enough short proteins (amino acids) and a rest closer 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 about half of the amount of 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 rest is over, use a heat resistant vessel to scoop the boiling water into the mash. This is best done by holding the thermometer in the mash with one hand and scooping the water or stirring with the other hand. It is important to stir the mash well to even out its temperature. Add water until the desired mash temp of 65 - 68 *C (148 - 155 *F) is reached. You will notice that the thinned mash makes stirring easier. This mash will have a consistency of about 3.5 - 4 l/kg (1.7 - 2.0 qts/lb) which is typical for German style beers. Hold this rest for 45 min.

Check for conversion. Calculate the amount of decoction necessary to get to the mash-out temperature of 74 - 76 *C (165 - 170 *F) and pull this decoction. In order to prevent scorching get good mix of liquid and grains. This decoction should be brought to a boil over the next 10 to 15 minutes. A gentle flame while keeping the pot covered will prevent scorching even without the need of constant stirring. Boil for 10 - 30 min. Shorter for light worts and longer for darker worts.

Double DecoctionClassic Double Decoction

The classic version of the double decoction is a shortened triple decoction. It omits the acid rest and starts with the protein rest. Because of this only 2 decoctions are needed to get to mash-out. One to get from the protein rest to the saccharification rest and another one to get from the saccharification rest to mash-out. Like the triple decoction, this mash rests the main mash at the protein rest for a long time. With well modified lager malts, this may result in overly extensive protein degradation. The following two sections show mash schedules that avoid this problem.

Enhanced Double Decoction

This is a mash schedule that was taken from German brewing literature [Narziss,2005]. The great thing about this mash is that it is almost as intensive as a triple decoction, when it comes to the amount of mash that is boiled, but a little bit shorter and without a long protein rest. The basic idea of this mash is to pull a decoction that is large enough

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http://braukaiser.com/wiki/index.php?title=File:Mash_diagram_double_decoction_enhanced.gif

to get the mash from acid rest directly to the saccharification rest. But when this decoction is returned to the main mash, it is returned in 2 parts: first to reach the protein rest and 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 (about 50 - 60% of the mash) is pulled and heated. It is advisable to add some water 5-10% of the decoction volume to compensate for boil-off and thin it out a little. Due to the size of the decoction it may be rested for protein rest and must be rested for the saccharification rest. The saccharification rest is necessary to get the most out of the enzymes in this mash as they will be destroyed once the decoction is brought to a boil. It also reduces the viscosity of the mash which mitigates the risk of scorching it later. This rest can be done at 155 - 162 *F (68 - 72 *C) where the conversion should only take 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 almost converted, 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 heat resistant vessel is used to scoop some of the 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 rest temperature is reached. After that the main mash is rested for 15 - 20 min while the rest of the decoction is still boiling. Once the protein rest is over, continue adding the decoction, stir and check temperature until the desired saccharification rest temperature is reached. Hopefully enough decoction was pulled to reach this rest. If some decoction is left over once the rest temperature is reached, let it cool and add it when its temperature is close to the temperature of the main mash.

Rest the mash for saccharification. Check for conversion and when the desired mash time rest time 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 want a highly fermentable wort and truly boil and convert almost all of the starches in the decoction, this variation should get you 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, preheat your 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 that will 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 rest later later for a saccharification rest.

Note that the start of the saccharification rest has now more glucose chain ends available compared to a conventional decoction mash or infusion mash since almost all of the 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 normal fermentability you should hold this rest closer to 160F where the beta amylase denatures more quickly. But if you want a very fermentable wort, hold this rest closer 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 rest or 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 rest is generally sipped. The first rest, usually held 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 sufficient alpha 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 rest held at higher temperatures. To reduce and eventually terminate the beta amylase activity and to ensure that all starch in the wort has been converted (especially the small starch granules which have a higher gelatinization temperature), a dextrinization rest is held at 158 - 162 *F (70 - 72 *C). At this temperature the beta amylase is quickly deactivated and only the alpha amylase works on the starches. The rest is held until the mash is iodine negative (no starch or long dextrines in the wort). Narziss [Narziss, 2005] and Fix [Fix, 1999] suggest, that a rest at 158 - 162 *F (70 - 72 *C) benefits head retention and body of the beer though glycoproteides that are extracted from the malt but not degraded by enzymatic activity. Because of that Narziss suggests holding this rest up to 60 min. After that rest a mash-out is performed at 167-173F (75-78 C). The temperature should not be 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, that might be liberated during sparging, on their way to the kettle.

The length as well as the temperature of the maltose rest determines the fermentability of the wort. Shorter rests and/or higher temperatures will result in a less fermentable wort as the beta amylase gets less time for maltose production.

http://braukaiser.com/wiki/index.php?title=File:Mash_diagram_double_decoction_hochkurz.gif

The steps for the water infusions and decoctions necessary for 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 hot water infusions or direct heat to the mash. The latter has become standard practice in most German breweries.

Brewing process

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.

7. The process according to any of the preceding claims, where the exogenous enzymes of step b. in claim 1 further comprises a lipase activity.

8. The process according to any of the preceding claims, where the exogenous 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;


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 shows the turbidity (NTU) of a wort produced from increasingly amount of barley when only Ultraflo Max is exogenously added.

Figure 2 shows the fermentability of a wort 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 with specific meanings, however, and are meant as defined by the following. The term "malting" is a process whereby grains are made to germinate and are then dried.

The term "malted grain" is understood as any cereal grain, in particular barley, which has been subjected to a malting process.

The term "unmalted grain" is understood as any cereal grain, in particular barley, which 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 well as adjunct.

The term "cereals" is understood as grains which are any starch containing material used as raw material e.g. for production of beer such as, but not limited to, barley, wheat, sorghum, maize, rice, oat and rye. The cereals may be malted or unmalted.

The term "adjuncts" is usually understood as raw material which may be added to the main ingredient of the grist, which 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 with liquid carbohydrates such as sugars and sirups. The adjuncts could be either solid or liquid or both, where the solid part may be unmalted cereals, such as barley, corn and rice whereas the liquid part may be readily fermentable carbohydrates such as sugar and syrups.

In this context however, what might be regarded as adjunct may be the main ingredient. Thus unmalted cereals which 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 however 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 which is not malted or unmalted cereal. Thus the adjuncts according to the present invention are preferably the liquid part such as brewing 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 wheat. Accordingly grist from 100 % unmalted grains may comprise unmalted barley and other non barley unmalted cereals such as rice and wheat.

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. Thus the grist may comprise 50 % unmalted barley and 50 % unmalted other cereals, such as wheat 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 water to make wort.

The term "mashing process" or mashing profile or simply mashing is understood as the process of combining grains with water and heating the mixture up with rests at certain temperatures to allow the enzymes in the mash to break down the starch in the grain into sugars, to create a wort.

"mashing off" or mashing out is when the temperature of the mash is raised. This frees up about 2% more starch, and makes the mash less viscous.

The term "wort" is understood as the unfermented liquor run-off following the extraction of the grist during mashing. The terms brewers wort and wort is used interchangeably through out the application.

The term "spent grains" is understood as the drained solids remaining when the grist has been extracted and the wort separated. The term "beer" is here understood as a fermented wort.

The term "beer product" is here understood as comprising "mash", "wort", "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" (0P) means grams extract pr 100 g wort (gram extract/100 g wort).

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 wort 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 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 aspect, the invention relates to a process, where the grist comprises at least 70wt% unmalted cereal(s), such as at least 75wt%, more preferably at least 80wt%, more preferably at least 85wt%, more preferably at least 86wt%, more preferably at least 87wt%, more preferably at least 88wt%, more preferably at least 89wt%, more preferably at least 90wt%, more preferably at least 91wt%, more preferably at least 92wt%, more preferably at least 93wt%, more preferably at least 94wt%, more preferably at least 95wt%, more preferably at least 96wt%, more preferably at least 97wt%, more preferably at least 98wt%, even more preferably 99wt%, and most preferably 100wt% unmalted cereal(s).

It is to be understood that the at least 70 wt % unmalted cereal(s) may be one or more cereal(s) wherein at least one of the cereal(s) contain β-amylase activity.

In one aspect of the invention the grist comprises less than 30wt% malted cereals, more preferably less than 25wt%, more preferably less than 20wt%, more preferably less than 15wt%, more preferably less than 10wt%, more preferably less than 5wt% and even more preferably less than 3wt%, and most preferably the grist comprises 0 wt% malted cereals.

In a preferred embodiment, the unmalted cereal(s) are of the tribe Triticeae. Preferred within this tribe are barley, spelt, wheat and rye. Triticeae is a tribe within the Pooideae subfamily of grasses that includes genera with many domesticated species, EA Kellogg, R Appels, RJ Mason-Gamer - SYSTEMATIC BOTANY, 1996. Major crop genera are found in this tribe including wheat, 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, wheat, rye, corn, oat or rice or any mixture thereof.

Thus in one embodiment, the invention relates to a process, where 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 wheat 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 wt% 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, preferably family GH10 (glycosyl hydrolase family 10) which may improve the filtration of wort and beer.

In another embodiment, the exogenous enzymes of step b. above further comprise a lipase activity, which may improve the wort 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 following activities; a xylanase activity, a lipase activity, and/or a phytase activity.

In a preferred embodiment the mashing temperature, i.e. the temperature at which 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 brewers wort, 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.

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, which might be termed the proteolytic step, the temperature is preferably within the range of optimising e.g. the proteolytic enzyme, the temperature is preferably between 450C and 580C, such as preferably between 460C and 570C, such as preferably between 470C and 560C, such as preferably between 480C and 550C, such as preferably between 490C and 540C, such as preferably between 5O0C and 540C, such as preferably between 510C and 540C, such as preferably between 520C and 540C, most preferably between 530C and 540C, such as 540C,

In the second step the temperature is preferably within 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 between 6O0C and 720C, such as preferably between 6O0C and 7O0C, such as preferably between 620C and 680C, such as preferably between 630C and 670C, such as preferably between 640C and 660C, and most preferably between 640C and 650C, such as 640C.

In the third step, which also may be referred to as mashing off or mashing out, this frees up about 2% more starch, and makes the mash less viscous, allowing the lauter to process faster. The temperature of the mashing out is preferably between 720C and 820C, such as preferably between 730C and 810C, such as preferably between 740C and 8O0C, such as preferably between 750C and 790C, such as preferably between 760C and 780C, most preferably the temperature is between 78°C-80 0C, such as 80 0C.

Endogenous lipoxygenase is known 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 with 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 540C.

The invention further relates to an enzyme mixture comprising:


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 water 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 weight 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 least 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 shown in SEQ ID NO:1 (a variant of the B. stearothermophilus α-amylase with 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 water 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 showed 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 showed 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 woesei described 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 which 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 shown in SEQ ID NO:8 (NS26062, PuIC, from Bacillus acidopullulyticus); in particular when 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 weight 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 working together with the other enzymes in the mash, in particular the β-amylase, which is usually endogenous and the α-amylase which 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 which 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 where 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 0C and at pH level 5.

The relative activity, which 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 which is thermostable having a relative enzyme activity above 60% over a period of 30 min, at 65 0C 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, when 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 650C 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 with 12 0P 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 weight 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 preferably at least 99% or even 100 % identity to the amino acid sequence shown 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 weight 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 with 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 weight 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 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 shown 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. lanuginosus) or a variant of these. An example is Lipopan X (Lipopan Xtra), a variant of the Thermomyces lanuginosus lipase with 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 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 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 weight 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 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 shown in SEQ ID NO 9. The lipase may also be Lipex, a variant of Li- pozyme having at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 95%, more preferably at least 98%, and most preferably at least 99% or even 100 % identity to the amino acid sequence shown 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 lower 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 weight 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 shown 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 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 (e.g. eisbock), barley wine types or happoushu.

The nitrogen containing components are important components of the wort 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 with the exception of proline which is hardly assimilated by the yeast thus it is favourable to have a small amount of proline or no proline in the wort. Thus in a further aspect, the wort 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.

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 with very high amounts e.g. above 80 % of unmalted cereals such as unmalted barley a wort could be produced which has a high amount of fermentable sugars, which 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, which 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 wort, where 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 wort where 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 wort according to any of the proceeding claims where 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 whereas 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 wort is more than 60%, such as at least 65%, such as at least 70%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 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 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%, such as at least 100%.

Some breweries add brewery syrup, e.g. high maltose, brewing syrup to the wort kettle which may increase the amount of fermentable sugars. However, though brewing 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, wherein the ratio of maltose:glucose in the wort 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 wort 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 which 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 without reducing the fermentability of the wort or reducing the amount of amino acids available for the yeast. In addition, brewing on unmalted cereals may give problems with filterability due to excess of non converted starch and β-glucan or xylan, which may also cause haze of the beer. Adding filtration aiding enzymes such as β-glucanase may increase the filterability of the wort. How- ever, when unmalted cereal comprises main part of the grist, β-glucanase alone is not enough provide filterable wort.

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 wort which is comparable or even better with regards to e.g. FAN, fermentable sugars (DP1-DP3) and which is filterable and also have an acceptable low turbidity when compared to a wort 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 wort 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 way of increasing the amount of fermentable sugars is by increasing the mashing time e.g. by increasing the saccharification step. However, in another important aspect of the invention the mashing time needed for producing a wort which is highly fermentable is not increased compared to the mashing time for producing an equally fermentable wort based on the same amount of malt.

This is surprising since generally longer mashing time is needed when the mash is based on high amount unmalted cereals e.g. 70% barley to give the same fermentability and FAN as in a wort produced on corresponding amounts (70%) of malt.

Thus in a particular embodiment of the invention the mashing process is completed within 160 minutes, preferably within 120 minutes. In one embodiment of the invention the mashing process comprising all the enzymes rests and all heating steps, is completed within 180 minutes, such as within 170 minutes, such as within 160 minutes, such as within 155 minutes, such as within 150 minutes, such as within 145 minutes, such as within 140 minutes, such as within 135 minutes, such as within 130 minutes, such as within 125 minutes, such as within 120 minutes, such as within 1 15 minutes, such as within 1 10 minutes, such as within 105 minutes, such as within 100 minutes, such as within 95 minutes, such as within 90 minutes, such as within 85 minutes, such as within 80 minutes, such as within 75 minutes, such as within 70 minutes, such as within 65 minutes, such as within 60 minutes.

When malt is substituted with grains such as rice and corn the grist may need to be treated by decoction or decoction mashing or adjunct decoction, which is process where a proportion of the grains are boiled separately with 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. wheat. Thus pregelatinization is needed to make the starch accessible 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 shows a general different behaviour in milling than malted cereals, as an example barley has higher water content, is unmodified and is much harder than malt.

To run a lauter tun with 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 between the roller pair(s) (two roller mill = one pair, four roller mill = two pair). The first pair has always a wider 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 with adjusted roller gaps are well suited to mill the barley into usable grist. This is important since a good lauter performances only can be achieved with an optimized grist composition that is different to the optimal grist composition of malt. The sieving test was 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

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The results show that for a successful 100 % barley lauter performance more coarse grist with 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-down of modified potato starch by the enzyme, and the reaction is followed by mixing samples of the starch/enzyme solution with an iodine solution. Initially, a blackish-blue color is formed, however, during the break-down of the starch the blue color gets weaker and gradually turns into a reddish-brown, which 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 which, 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 with 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 which, under standard conditions (i.e. after 30 minutes reaction time at 400C and pH 5.0; and with 0.2% pullulan as substrate) hydrolyzes pullulan, liberating reducing carbohydrate with a reducing power 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 with trichloroacetic acid (TCA). The amount of the TCA soluble product is determined by using phenol reagent, which gives a blue color with tyrosine and tryptophan.

One Anson Unit (AU) is defined as the amount of enzyme which 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 which gives the same colour with phenol reagent as one milliequivalent of tyrosine.

β-glucanase activity (FBG)

One fungal beta glucanase unit (FBG) is the amount of enzyme, which, according to the standard conditions outlined below, releases reducible oligosaccharides or reduces carbohydrate with a reduction capacity equivalent to 1 mol glucose per minute.

Fungal beta glucanase reacts with beta glucan during the formation process to glucose or reducing carbohydrate which is determined as reducing sugar according to the So- mogyi Nelson method.

The sample should be diluted to give an activity between 0.02-0.10 FBG/ml. The standard reaction conditions are: Substrate: 0.5% barley beta glucan, temperature: 3O0C, pH: 5.0 and the reaction time 30 min.

However the cellulytic activity in the commercial product is measured in endo- glucanase units (EGU), which can be converted to FBG. For celluclast the EGU can be converted to FBG by multiplying the EGU by a factor 3.2. Xylanase (FXU(S) )

The xylanolytic activity can be expressed in FXU(S)-units, determined at pH 6.0 with remazol-xylan (4-O-methyl-D-glucurono-D-xylan dyed with Remazol Brilliant Blue R, Fluka) as substrate. An xylanase sample is incubated with the remazol-xylan substrate. The background of non-degraded dyed substrate is precipitated by ethanol. The remaining blue colour in the supernatant (as determined spectrophotometrically at 585 nm) is proportional to the xylanase activity, and the xylanase units are then determined relatively to an enzyme standard at standard reaction conditions, i.e. Substrate concentration 0.45% w/v, Enzyme concentration 0.04 - 0.14 FXU(S)/ml_ at 50.0 0C, pH 6.0, and in 30 minutes reaction time. Xylanase activity in FXU(S) is measured relative to a Novozymes FXU(S) enzyme standard (obtainable from No- vozymes), comprising the monocomponent xylanase preparation Shearzyme from Aspergillus aculeatus.

Lipase (LU) One Lipase Unit (LU) is the amount of enzyme which liberates 1 micromol of titrable butyric acid per minute at 30.00C; pH 7.0; with Gum Arabic as emulsifier and tributyrine as substrate.

Phvtase (FYT)

One phytase unit (FYT) is the amount of enzyme which liberates 1 micro-mol of inorganic ortho-phosphate per min. under the following conditions: pH 5.5; temperature 37°C; substrate: sodium phytate (C6H6O24PeNaI2) at a concentration of 0.0050 mol/l.

Leucine Amino Peptidase Unit (LAPU)

1 Leucine Amino Peptidase Unit (LAPU) is the amount of enzyme, which decomposes 1 micro-M substrate per minute at the following conditions: 26 mM of L-leucine-p- nitroanilide as substrate, 0.1 M Tris buffer (pH 8.0), 4O0C, 10 minutes reaction time.

Laboratory mashing method

Unless stated otherwise, the mashing method used in the examples was performed as follows: First, barley is milled to fine grist (Bϋhler Unirvisale gab 0.2 mm), then 50 g milled barley is added to a mashing cup and 200 g pre heated water (with calcium chloride) is added. The cup is placed in the mashing bath (Lochner LB 12 Electronic with 12 cups), the mashing diagram is set (e.g. mashing-in at 500C or 54°C, keep temperature for 20-30 minutes, increase by 1 °C/minute to 64°C, keep for 40-60 minutes, increase by 1 °C/minute 78 or 800C, keep for 10-20 minutes and reduce temperature to 200C). The enzyme solution is added at the start to the cups and the mashing is initiated giving a total mashing period of 140-160 minutes. After mashing water is added to a total of 300 g in the cup and the mash is filtered with a Whatman 597 1/2 (Schleicher & Schuell) folded filter to obtain the wort, where after the wort can be analyzed.

The wort sugar/dextrin (carbonhydrate) concentrations were analyzed in a Waters HPLC system (Novozymes method: 345-SM-2004.01/01 ) with pre-column (Cation H refill cat. 1250129) two column BoiRad Aminex HPX 87 H heated to 60°C and flow of 0.4 ml/minutes with Rl detection (Waters 2410 Rl detector).

RDF: Real Degree of Fermentation, was determind by the method described in MEBAK method: 2.9.2. Principal: Reduction of wort dry matter, in %, by fermentation to alcohol and CO2

NTU: Haze in wort was analyzed by MEBAK method 2.15.1

In general the enzyme doses are calculated as follows:

Enzyme dosage in the target dosage

Example 1

The purpose of this example was to select the best suitable pullulanase for the production of wort based on RDF and DP2 (maltose). A grist comprising 100% unmalted barley was prepared as described above.

All cups were then added 50 ppm Na2SO3 and the enzymes: α-amylase (Termamyl SC): 0.3 KNU(S)/g, β-glucanase and xylanase (Ultraflo MaxA/iscoflow XL): 300 ppm,

Protease (Neutrase 0.8 ) 0.002 AU/g,

Flavourzyme™ 1000 L: 0.1 LAPU/g, and

Pullulanase as described in Table 2:



The mashing was performed and the cups were added city water to a total of 300 g after mashing. The mash was filtered, and the results in Table 1 below were obtained by analyzing the wort:

Table 1 100% barley: RDF, % and wort sugar.

Enzyme dosage DP2 DP4+ RDF

PUN/g % of total % of total %

- 0.00 48.7 33.2 62.0

Promozyme 0.1 49.4 31.6 64.0

Promozyme 0.2 49.7 30.6 65.0

Promozyme 0.3 50.3* 29.6* 65.8*

Promozyme 0.5 51.6 27.6 67.0

- 0.00 48.7 33.2 62.0

NS26062 0.1 50.0 30.2 64.6

NS26062 0.2 50.8 28.4 66.1

NS26062 0.3 51.3* 27.3* 67.3*

NS26062 0.5 52.4 25.0 69.5

Estimated by linear regression The RDF is above 60 % with both pullulanases, however RDF is a higher when NS26062 (PUL C) is added. The amount of maltose (DP2) relative to the amount of dextrins (DP4) was also higher for both pullulanase but again the amount of maltose relative to dextrins are higher for NS26062 (PUL C). Thus in conclusion: NS26062 (pullulanase C or PuIC) showed the best performance, compared to Promozyme (promozyme 400 L) on PUN activity, on RDF% and maltose (DP2) generation. In this experiment the advantage of the thermostable NS26062 (pullulanase C or PULC) is clearly demonstrated.

Example 2

The following example demonstrates the different temperature optimum and the relative activity at different temperatures. The relative enzyme activity of three different pullulanases was analysed. The method of analyzing pullulanase activity is by detection of increased reducing sugar capacity (Somogyi-Nelson reacition) in the following conditions:

Substrate: 0.2% pullulan, pH 5.0, reaction time 30 minutes, stop of enzyme reaction by adding Somogyi copper reagent, followed by Nelson color reagent and boiling in 20 minutes.

Samples were incubated at 300C, 45°C, 55°C, 600C, 62.5°C, 65°C and 700C in 30 minutes. The samples were analyzed by spectrophotometer at OD520 nm, and the difference between sample and blank (increased by enzyme activity) were used in calculation of the results.

The highest activity was set to 100% (maximum) and activities at other temperatures set relative to the temperature maximum.

Table 2 Relative activity of different pullulanases at different temperatures

This example clearly demonstrates that PuI C is the most thermostable and thermoactive of the three pullulanase since it has a significant higher relative activity above 62.5 0C and since the highest activity measured over30 minutes at 65°C. The PuI C pullulanase have highest activity of all three pullulanase between 62.5°C and 65°C, which is the preferred temperature for mashing thus using PuI C as debranching enzyme in a mashing process, is clearly advantageous.

Example 3

The purpose of this experiment was to evaluate the effective dosage enzyme protein (EP) per g dm (gram dry matter) of 3 of different pullulanases (NS26062/PulC, Promozyme 400 L and Promozyme D2 (Optimax 1000 L) in saccharification of either 100% unmalted barley or 100% malted barley when applied in infusion mashing for 2 hours.

All cups were added enzymes blend 2 kg /1000 kg barley:

α-amylase (Termamyl SC) 0.3 KNU(S )/g, β-glucanase and xylanase (Ultraflo Max/Viscoflow XL) 300 ppm, Protease (Neutrase) 0.001 AU/g, Lipase (Lipozyme TL 20 LU/g)

Different pullulanases dosages were added.

Specific activity: NS26062: 57 PUN/mg EP. Promozyme 400 L: 136 PUN/mg EP Promozyme D2: 236 NPUN/mg EP

Iso-amylase from Hayashibara Co Ltd: Specific activity unknown

The specific activities were measured after the pullulanases were purified by standard chromatographic techniques


Mashing conditions: 54°C in 30 minutes, increase to 64°C in 10 minutes and maintain 45 minutes, increase to 800C in 16 minutes and maintain 10 minutes, producing wort with 12.6 Plato. Table 3: Pullulanase effect on degradation of dextrin in 100% unmalted barley mashing: showing % of non fermentable carbohydrate in wort (dextrin/DP4+) with different dosages (g (gram) EP (enzyme protein) / 1000 kg unmalted barley. Some experiments were done several times.

Table 3 shows that all three pullulanases but not the Hayashibara isoamylase could reduce the amount of non-fermentable sugars (dextrin DP4+) and thereby increase the amount of fermentable sugars. However, the best performing is clearly the NS26062 (PuI C) pullulanase, which reduced the amount of non-fermentable sugars relative to the amount of added enzymes much more than the Pullulanase 400 L and the pullulanase D2. This is a clear demonstration of the advantage of using the thermostable PuIC. It is furthermore demonstrated that a DP4+ of less than 20%, corresponding to more than 80%


glucose, maltose and maltotriose can be reached in 120 minutes of mashing. Thus the choice of pullulanase is important for controlling the amount of fermentable sugars and for probably reduction of the non fermentable DP4+ dextrins. This is important since a good sugar profile (many fermentable sugars compared to non fermentable sugars) promote a good fermentation of the wort.

Example 4

The purpose of this example was to evaluate the effect of the pullulanase NS26062 on the DP2 (maltose) formation in the wort. A grist comprising 100% unmalted barley was prepared as described above. All cups were then added 50 ppm Na2SO3+ 3.0 ml 1 M H3PO4 and enzymes: α-amylase (Termamyl SC): 0.3 KNU(S)/g, β-glucanase and xylanase (Ultraflo MaxA/iscoflow XL): 300 ppm ~ 0.23 EGU/g,

Protease (Neutrase 0.8 L): 0.002 AU/g,

Pullulanases as described in Table 4:


Table 4. 100% barley: RDF, % and wort sugar.

Enzyme dosage DP1 DP2 DP4+ RDF

PUN/g (NS26062)

- 0 3.8 47.5 34.0 61.2

NS26062 0.1 3.8 48.2 32.0 63.2

NS26062 0.3 3.8 49.8 28.8 65.7

NS26062 0.5 3.7 51.2 26.4 68.6

NS26062 1 3.7 52.6 23.9 71.0

NS26062 2 3.6 55.6 20.1 74.3

The maltose concentration (DP2) was increased by increasing the dosage of NS26062 (PuI C), and the increase in maltose % was followed by an increase in attenuation (RDF%). The dextrin fraction (HPLC analysis DP4/4+) was at the same time decreasing. Only barley β-amylase could produce maltose in this reaction, and NS26062 (PuI C) facilitated the action of the barley beta-amylase.

Thus the NS26062 (PuI C) was a suitable pullulanase, providing a wort with high RDF and low glucose.

Example 5

The purpose of this example was to evaluate the three proteases Neutrase 0.8 L, Al- calase and Flavourzyme for FAN development and maltose formation. A grist comprising 100% unmalted barley was prepared as described above. Then all cups (trials 1-3 below) were added enzymes as indicated in the tables 5-7 below. The mashing was performed and the cups were added city water to a total of 300 g after mashing. The mash was filtered, and the results in the tables 5-7 were obtained by analyzing the wort:

Trial 1 :

All samples were added: α-amylase (Termamyl SC): 0.3 KNU(S)/g, β-glucanase and xylanase (Ultraflo Max): 300 ppm (0.23 EGU/g) and different activities of the proteases Alcalase and Neutrase 0.8 L as indicated in table 5.

Table 5. FAN and % wort sugar at different dosages of Alcalase and Neutrase 0.8 L (enzyme activity per g dm mash)

Trial 2:

All samples were added: α-amylase (Termamyl SC): 0.3 KNU(S)/g, β-glucanase and xylanase (U ltraflo Max): 300 ppm (0.23 EG U/g), Flavourzyme: 0.1 LAPU/g,and the proteasesAlcalase and/or Neutrase 0.8 L as indicated in table 6.



Trial 3:

All samples were added: α-amylase (Termamyl SC): 0.3 KNU(S)/g, β-glucanase and xylanase (Ultraflo Max): 300 ppmθ.23 EGU/g, Flavourzyme: 0.1 LAPU/g, and the proteases Alcalase or Neutrase 0.8 L as indicated in table 7.



These examples clearly demonstates that addition of the proteases Alcalase and Neutrase but not Flavorzyme has a positive effect on the generation of particular free available amino nitrogen (FAN) and that neutrase had the most positive effect on FAN generation. Thus the choice of protease is a critical parameter for FAN generation.

Example 6

A grist comprising 0-90% unmalted barley was prepared as described above. Then all cups were added the enzymes β-glucanase and xylanase. The mashing was performed and the cups were added city water to a total of 300 g after mashing. The mash was filtered, and the results in the table 8 were obtained by analyzing the wort: The following experiment is to demonstrate the effect on the turbidity (NTU) with increasing amount of unmalted barley, when only filtration enzyme blend β-glucanase and xylanase (Ul- traflo Max 300 ppm) is added.

Table 8. NTU when increasing amount of malt barley is substituted with unmalted barley, from 0 % unmalted barley to 90 % unmalted barley.

%barley NTU

1 0 19.8

2 8 19.7

3 16 15.3

4 24 12.4

5 32 12

6 40 10.2


7 48 10.8

8 56 8.43

9 64 10.9

10 72 21

11 80 35.7

12 90 56.2

The result is also shown in Figure 1. It is evident from this experiment that mashing unmalted barley with simple enzyme blends (only filtration enzymes) becomes increasingly difficult with increasing amount of unmalted barley is substituted for malted barley and when the amount exceeds 80 % the turbidity is so high that the wort is difficult to filtrate. Thus when having high amount of unmalted barley adding filtration enzymes alone is not enough to get a wort which is filterable. Example 7

The purpose of this example was to evaluate the turbidity (NTU) and the filtration of wort from 100% barley infusion mashing with a different dosage of Lipopan F, Lipopan X and β- glucanase and xylanase (Ultraflo Max). The study comprised two independent trials for Lipopan F and Lipopan X, respectively, i.e. 2 x 12 cups as indicated in table 9 below. A grist comprising 100% unmalted barley was prepared as described previously. Then all cups were added enzymes.

To each cup was added:

- 3.0 ml 1 M H3PO4, - 0.3 KNU(S )/g α-amylase (Termamyl SC),

- 0.002 AU/g protease (Neutrase 0.8 L),

- 0.5 PUN/g pullulanase (NS26062), and the enzymes in table 9.


Table 9. Enzyme dose Activity/g DM in mashing

Both Lipases Lipopan F and Lipopan X markedly reduced the turbidity (NTU) of the wort. Lipopan X is the most efficient (on enzyme activity LU(g)) for reduction of the turbidity in wort, but Lipopan F can reduce the turbidity to a level within the specification of wort. The amount of filtration enzymes can be reduced to 100 ppm in the presence of lipase without reducing the filtration speed significantly. Example 8

The purpose of this example was to evaluate the effect of the protease Neutrase 0.8 L, phytase and the pullulanase NS26062 (PuI C) on the FAN generation and the wort sugar profile in a standard mashing. A grist comprising 100% unmalted barley was prepared as described above. Then all cups were added α-amylase (Termamyl SC) 0.3 KNU(S)/g, 300


ppm β-glucanase and xylanase (Ultraflo Max), adjusted to pH 5.3, and the the protease Neutrase 0.8 L, the phytase and the pullulanase NS26062 (PuI C) enzymes, where added as indicated in Tables 10A and 10B below, and the results obtained:

Table 10A. 100% barley: FAN generation in wort. The dosages is enzyme activity unit and ppm (100 ppm=100g/1000 kg unmalted barley). FYT is phytase unit, PUN is pullulanase activity and AU is proteolytic activity.

Enzyme dosages FAN mg/l/Plato

0 5.09

1.5 FYT 5.09

0.5 PUN 5.11

1.5 FYT + 0.5 PUN 5.22

0.002 AU 8.58

0.002 AU + 1.5 FYT 7.53

0.002 AU + 0.5 PUN 7.85

0.002 AU + 1.5 FYT + 0.5 PUN 7.91

0.002 AU + 0.5 PUN + 0.5 FYT 7.87

0.002 AU + 0.5 PUN + 5 FYT 7.77

0.001 AU + 0.5 PUN + 5 FYT 7.35

Table 1OB. 100% barley: wort sugar profile.

Enzyme dosage DP1 DP2 DP3 DP4/4+ Fm

Neutrase 0.8 L (AU) % % % % %

0 4.15 31.80 14.82 38.13 1.82

0.002 AU 3.81 46.61 12.79 34.97 1.82. 1.5 FYT 4.13 42.11 14.78 37.14 1.85

0.5 PUN 4.06 44.28 18.28 31.55 1.82

0 4.15 31.80 14.82 38.13 1.82

5.0 FYT + 0.5 PUN 4.14 47.01 18.17 28.82 1.86

5.0 FYT + 0.5 PUN +

0.001 AU 3.84 50.67 17.42 26.25 1.8iiO

5.0 FYT + 0.5 PUN +

0.002 AU 3.88 49.26 17.61 27.41 1.83

0 4.15 31.80 14.82 38.13 -

0.002 AU 3.81 46.61 12.79 34.97 -

0.002 AU + 0.5 PUN 3.81 47.78 17.74 28.87

"15

0.002 AU + 0.5 PUN +

0.5 FYT 3.88 49.26 17.61 27.41 -

0.002 AU + 0.5 PUN +

1.5 FYT 3.91 50.12 17.56 26.58 -

0.001 AU + 0.5 PUN +

5.0 FYT 3.84 50.67 17.42 26.25 .20

Table 10 A shows that the protease increase the FAN in the wort, and table 10 B shows that when adding phytase and pullulanase to the protease a comparable high amount of DP1-DP3 could be generated with protease concentrations of 0.001 and 0.002 AU respectively. Thus the protease concentration could be reduced in the production of maltose wort when phytase and pullulanase is present without reducing the amount of fermentable sugars (DP1-DP3).

Example 9

The purpose of this example was to elucidate some general parameters concerning wort prepared on 100% unmalted barley in order to identify critical issues compared to wort prepared on malted barley.

Malt mashing (100%) with no enzyme added and unmalted barley (100%) mashing, with enzyme blend. The wort was boiled, and beer fermentation performed with 100% (barley) malt and a 100% unmalted barley wort. Data:

Mashing:

Barley: Scarlet and malt from same batch of Scarlet.

Mash: 10 kg malt or barley, + 35 I mash liquor and spargings 25 I to a total of 60 I. Profile: 54°C 30 minutes, increase to 64°C (1 °C/minute) and maintain for 60 minutes, increase to 800C (1 °C/minute) and maintain for 10 minutes and transfer to lautering. Enzymes blend in 100% barley mash:

• α-amylase (Termamyl SC): 0.3 KNU(S)/g dm

• β-glucanase and xylanase (Ultraflo Max): 300 ppm • Protease (Neutrase 0.8 L): 0.0015 AU/g dm

• Pullulanase (NS26062, PuI C): 1.0 PUN/g dm

Wort amino acid composition (table 11 ) analyzed:

The free amino acid analyzed in the wort is organized according to the paper "Elucidation of the Role of Nitrogenous Wort Components in yeast Fermentation" (J. Inst. Brew. 1 13(1 ), 3-8, 2007)

Table 11. FAN in wort

Malted barley Unmalted wort barley wort

Group A, fast absorption: mM mM

Aspartic acid 0.076 0.151

Glutamic acid 0.244 0.210

Asparagine 0.310 0.273

Serine 0.007 0.187

Glutamine 0.074 0.048

Threonine 0.210 0.188

Arginine 0.149 0.265

Lysine 0.216 0.354

Sum 1.286 1.675 (130%)

Group B, intermediate absorption:

Valine 0.245 0.252

Methionine 0.047 0.111

Leucine 0.236 0.446 lsoleucine 0.114 0.163

Histidine 0.157 0.091

Sum 0.798 1.064 (133%)

Group C, slow absorption:

Glycine 0.149 0.158

Phenylalanine 0.196 0.206

Tyrosine 0.131 0.158

Tryptophan 0.087 0.062

Alanine 0.312 0.361

Sum 0.875 0.945 (108%)

Group D, little or no absorption

Proline 2.500 0.413 (16.5%)

Total - sum 5.458 4.098 Table 1 1 shows that when mashing with 100 % unmalted barley and an enzyme blend comprising α-amylase activity, β-glucanase activity, protease activity and a pullulanase activity a wort can be produced which has considerably less of the for the yeast unusable amino acid proline, which is clearly advantagous since the presence of this amino acid in a beer product gives an unpleasent taste. In addition, the amount of amino acids in the groups

A and B, which could be quickly metabolised by the yeast, is considerably increased when mashing unmalted barley and the enzyme blend comprising α-amylase activity, β-glucanase activity, protease activity and a pullulanase. Thus it is clear from this example that the proline concentration is less than 2 mM, and the serine and methionine concentration is above 0.1 mM and 0.05 mM respectively.

Example 10

The following experiment analyses the wort obtained from grist comprising 100 % unmalted barley and 100 % malt. The trials have been executed at Ziemann GmbH, Ludwigburg, Germany. All analysis has been done according to the Analytica EBC or MEBAK respectively. (van Erde, P., Analytica-EBC. 1998, Nϋrnberg: Verlag Hans Carl.; Anger, H., MEBAK Band Rohstoffe. 1. Auflage ed. Brautechnische Analysenmethoden. 2006, Freising: Selbstverlag der MEBAK).

The barley used was a two rowed spring barley from Germany harvest 2008. The enzymes added were:

• α-amylase (Termamyl SC): 0.3 KNU(S)/g cereal

• β-glucanase and xylanase (Ultraflo Max): 300 ppm

• Protease (Neutrase 0.8 L): 0.001 AU/g dm • Pullulanase (NS26062, PuI C): 2.0 PUN/g dm

• Lipase (Lipozyme TL 100): 20 LU/g dm

The mashing profile used was 54 0C at 30 min; increase the temperature 1 °C/min to 64°C and rest for 60 min increase the temperature 1 °C/min to 78 0C and rest for 30min, total mashing time 144 min.

Table 12 Wort composition of 100 % barley brews in comparison with all malt specifications

The results show 100 % unmalted barley in combination with the enzyme blend comprising α-amylase activity, β-glucanase activity, proteolytic activity and pullulanase debranching activity can fully match all malt specification in all key parameters like viscosity, turbidity, free amino nitrogen supply, yield and final attenuation are within all malt (100 % malt) specifications.

The lauter performance was also investigated. The turbidity in the lauter wort describes the quality of the lauter performance. The lipase component in the enzyme blend was able to reduce the normally significant higher haze level of unmalted barley approaches to haze levels below 80 NTU within a comparable lauter time.

Also the fermentation performance was tested: 8 hi of wort from 100 % malt and 8 hi of wort from 100 % unmalted barley was fermented. The result is demonstrated in figure 2.

Both worts have been fermented with bottom fermenting yeast strain W34, figure 2 shows the comparable extract drop of both brews. Furthermore, no differences in ethanol production have been found.

Finally the barley beer has been tasted by a professional taste panel at the institute for brewing technology 1 in Weihenstephan, Germany. The results show comparable result to a standard lager beer with indications of enhanced flavour stability.

Bitterness total 4.1 3.9 4.1 3.9




Total 4.03 3.61 3.91 3.56

General evaluation: DLG 5 = very good, 1 unacceptable.

Example 11

The following examples was to evaluate different important parameters when mashing on a grist comprising 30% corn grist or rice (unmalted) with 70% unmalted barley main mash and an enzyme blend comprising α-amylase activity, β-glucanase activity, protease activity and a pullulanase.

The process used were decoction mashing where the rice or corn grist part where boiled with thermostable α-amylase and then mixed with the mash comprising unmalted barley. The enzymes added to 70% barley mash + 30 % corn grist or rice:

• α-amylase (Termamyl SC): 0.3 KNU(S)/g dm

• β-glucanase and xylanase (Ultraflo Max): 300 ppm (0.23 EGU/g)

• Protease (Neutrase 0.8 L): 0.001 and 0,002 AU/g dm

Pullulanase was also added to the unmalted barley mash and the concentration was variated, se table 14 and 15, the pullulanase added is (NS26062, PuI C).

The mashing was performed as follows: The milled barley was added to mashing cup (40.0 g as is) and added 1 15 g water at 600C, CaCI (330 g/1000 kg barley) was added in addition with the enzyme blend indicated above. The mixture was maintain at 54°C for 30 minutes, then the decoction mash are added (15.4 g dm) and temperature maintained at 64°C in 60 minutes, increased to 800C and maintained 10 minutes, cooled and filtrated. All the mashes could be filtrated without any problem and no significant differences between the different enzymes dosage.

The preparation of the decoction mash was performed as follows: The rice or corn grist was mixed with water (5.66 part) at a temperature of 600C - 70°C, CaCI2 (220 g/1000 kg grist) was added in addition with an α-amylase (Termamyl SC 0.600 kg/1000 kg grist). The mixture was heated to 85°C and kept at that temperature for 20 minutes, the heat was increased 1000C (boiling) and kept at that temperature (boiling) for 15 minutes. The mixture was cooled to 800C and mixed with the barley containing mash. The mashing was performed and the cups were added city water to a total of 300 g after mashing. The mash was filtered, and the results in Table 14 below were obtained by analyzing the wort. In some examples the wort was boiled 10 minutes diluted 9.7 Plato and fermented by Forced Fermentation, Analytica - EBC nr. 8.6.

Table 14: FAN , RDF and sugar content of a wort from mash based on decoction of 30 % corn grist and 70 % unmalted barley mash which is mixed.

Table 15: FAN, RDF and sugar content of a wort from mash based on decoction of 30 % rice grist and 70 % unmalted barley mash which is mixed.

7 0.002 - 5.50 53.23 12.79 28.48 14.45 125 62.4

8 0 .002 0 .5 5 .55 57 .09 15 .28 22 .08 14 .45 124 69.0



9 0 .002 1 .0 5 .65 59 .17 16 .32 18 .86 14 .44 126 72.1

10 0 .002 1 .5 5 .68 60 .38 16 .95 16 .99 14 .45 127 73.9

11 0 .002 2 .0 5 .67 61 .37 17 .38 15 .58 14 .45 126 75.3

12 0 .002 2 .5 5 .72 62 .11 17 .56 14 .59 14 .39 127 76.7

Table 14 and 15 shows the sugar profile, Plato, FAN and RDF of the wort based on mashing a grist comprising 30 % corn or rice and 70 % unmalted barley (100 % unmalted grains). The result shows clearly that the amount of fermentable sugars (DP1-3) is very high (above 80 %), the RDF is above 60 % and increasing with increasing pullulanase concentration and the FAN is high and increasing with increasing protease concentration, when the wort is produced from 100 % unmalted grains comprising 70 % unmalted barley and 30 % unmalted corn grist or rice and an enzyme blend comprising α-amylase activity, β-glucanase activity, protease activity and a pullulanase. There were no significant difference between using rice or corn grist.

Example 12

The following examples were to evaluate different important parameters when mashing on a grist comprising 50% barley + 50% wheat mash and the following enzymes.

• α-amylase (Termamyl SC): 0.3 KNU(S)/g dm (dry matter)

• β-glucanase and xylanase (Ultraflo Max): 300 ppm/0.23 EGU/g dm

• Protease (Neutrase): 0.001 AU/g dm

• Lipase (Lipozyme TL): 20 LU/g dm

• Pullulanase (NS26062, PuI C): 0-3.0 PUN/g dm

In one example the pullulanase concentration was varied, see table 17, the pullulanase added is (NS26062, PuI C).

In the other two examples the enzyme blend concentration pr kg raw material was varied, see table 18.

The mash was prepared by mixing milled barely and wheat (25 g of each (total 50.0 g) with 200 g water added to mashing cups, then Ca2+ and the enzyme blend indicated above were added and mashing started. The mashing profile was as follows: The mash was heated to 54°C (1 °C/min.) and kept at that temperature for 30 minutes, the temperature was increased to 64°C within 10 minutes and maintain at that temperature for 45 minutes, the temperature was increased to 800C within 16 minutes and maintain at that temperature for 10 minutes. Water to 300 g total was added and the mash was filtered.

The wort was filtrated and in some examples the wort was boiled 10 minutes diluted 9.7 Plato and fermented by Forced Fermentation.

The results are showed in Figure 3 and in table 16 and 17.

Table 16: The enzyme blend was as described above Termamyl SC, Ultraflo Max, Neu- trase, and Lipozyme TL with different concentrations (PUN/g) of pullulanase (NS26062) added.

Table 16 shows that a wort made form a grist comprising 50 % wheat and 50 % barley, that is 100 % unmalted grains and mashed with an enzyme blend comprising α-amylase activity, β- glucanase activity, protease activity, lipase activity and a pullulanase is filterable and with low turbidity and importantly the RDF is high (above 65 %) and increasing with increasing amount of pullulanase NS26062 (PuI C). In table 17 the enzyme blend was as described above Termamyl SC, Ultraflo Max, Neutrase, and Lipozyme TL in the same relative amount, but in this example the amount of the blend relative to amount of raw material was varied. All blend were with 1.0 PUN of pullulanase (NS26062) added.

Table 17: Different doses of enzyme blend


With all tested amounts of enzyme blend comprising α-amylase activity, β-glucanase activity, protease activity, lipase activity and a pullulanase the wort produced had a very high amount of RDF above 70 %, it was slightly increasing with increasing amounts of enzyme blend. However, a high RDF and good filterability was obtained with 2 kg/1000 kg enzyme blend/ raw material, which corresponded to 100 % enzyme blend. Importantly the total mashing time was 2 hours.

Mashing process

ABSTRACT

The present invention provides processes for production of wort and beer from a malt and adjunct grist mashed-in at high temperature.

CLAIMS(1)

1. 1. A process for producing a brewers wort comprising forming a mash from a grist, and contacting said mash with a pullulanase, wherein said pullulanase has an amino acid sequence which a) is at least 50% identical to the amino acid sequence shown in SEQ ID NO:3; or b) is encoded by a nucleic acid sequence which hybridizes under low stringency conditions with i) a complementary strand of a nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO:3; or ii) a subsequence of (i) of at least 100 nucleotides.

2. The process according to any of the preceding claims wherein the pullulanase is derived from Bacillus acidopullulyticus.


3. The process according to any of the preceding claims further comprising contacting said mash with a glucoamylase and/or alpha-amylase.

4. The process according to any of the preceding claims wherein the glucoamylase and/or alpha-amylase is derived from Aspergillus niger or Talaromyces emersonii.

5. The process according to any of the preceding claims further comprising contacting said mash with an enzyme selected from the group consisting of cellulase, isoamylase, xylanase and protease.

6. The process according to any of the preceding claims, wherein the grist comprises malted and/or unmalted grain.

7. The process according to any of the preceding claims, wherein the unmalted grain and/or the malted grain is selected from the list consisting of barley, wheat, rye, sorghum, millet, corn and rice.

8. The process according to any of the preceding claims, wherein the malted grain comprises malted grain selected from malted barley, wheat, rye, sorghum, millet, corn, and rice.

9. The process according to any of the preceding claims, wherein the wort is concentrated and/or dried.

10. The process according to any of the preceding claims, further comprising fermenting the wort to obtain an alcoholic beverage.

1 1. The process according the preceding claim wherein the alcoholic beverage is a beer.

12. The process according to any of the preceding claims, wherein the beer is ale, strong ale, bitter, stout, porter, lager, export beer, malt liquor, barley wine, happoushu, high-alcohol beer, low- alcohol beer, low-calorie beer or light beer.

13. A wort produced by the process according to any of the preceding claims.

14. A concentrated and/or dried wort according to any of the preceding claims.

15. A beer produced from the wort according to any of the preceding claims.

16. A composition suitable for use in the process according to any of the preceding claims, said composition comprising a pullulanase. a glucoamylase and optionally an

alpha-amylase, wherein said pullulanase has an amino acid sequence which a) is at least 50% identical to the amino acid sequence shown in SEQ ID NO:3. or b) is encoded by a nucleic acid sequence which hybridizes under low stringency conditions with i) a complementary strand of a nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO:3. or ii) a subsequence of (i) of at least 100 nucleotides.

17. The composition according to the preceding claim wherein the glucoamylase and/or the alpha-amylase is derived from Aspergillus niger or Talaromyces emersonii.

DESCRIPTION

MASHING 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.

FIELD OF THE INVENTION

The present invention relates to an improved mashing process for production of a brewer's wort and for production of a beer.

BACKGROUND OF THE INVENTION In modern mashing processes enzymes are often added as a supplement when mashing malt is low in enzymes or to allow use of all adjunct grists. Enzymes may also be applied in mashing of well modified malts with high enzyme content in order to increase the extract recovery as well as the amount of fermentable sugars. It is thus well known to apply debranching enzymes, e.g. isoamylase or pullulanase to increase the yield fermentable sugars. Debranching enzymes may be applied in processes for production of low calorie beer. Such processes are the subject of

Willox, et al. (MBAA Technical Quarterly, 14, 105, 1977), US4528198, US4666718, GB2056484, GB2069527 and US4318927.

SUMMARY OF THE INVENTION

The present inventors have now surprisingly discovered that by using a certain pullulanase mashing, can be achieved using a smaller amount of enzyme protein.

Accordingly, in a first aspect the invention provides a process for producing a brewers wort comprising forming a mash from a grist, and contacting said mash with a pullulanase (E. C. 3.2.1.41 ), wherein said pullulanase has an amino acid sequence which a) is at least 50% identical to the amino acid sequence

shown in SEQ ID NO:3, or b) is encoded by a nucleic acid sequence which hybridizes under low stringency conditions with i) a complementary strand of a nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO:3, or ii) a subsequence of (i) of at least 100 nucleotides.

In a second aspect the invention provides a wort produced by the process of the first aspect.

In a third aspect the invention provides concentrated and/or dried wort produced by the process of the first aspect.

In a fourth aspect the invention provides beer produced from the wort of the second and third aspect. In a fifth aspect the invention provides a composition suitable for use in the process of the first aspect, said composition comprising pullulanase (E. C. 3.2.1.41 ), glucoamylase and optionally alpha-amylase, wherein the pullulanase has an amino acid sequence which a) is at least 50% identical to the amino acid sequence shown in SEQ ID NO:3, or b) is encoded by a nucleic acid sequence which hybridizes under low stringency conditions with i) a complementary strand of a nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO:3, or ii) a subsequence of (i) of at least 100 nucleotides.

DETAILED DESCRIPTION OF THE INVENTION

Brewing processes are well-known in the art, and generally involve the steps of malting, mashing, and fermentation. Mashing is the process of converting starch from the milled barley malt and solid 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. By far the most important change brought about in mashing is the conversion of starch molecules into fermentable sugars. The principal enzymes responsible for starch conversion in a traditional mashing process are alpha- and beta-amylases. Alpha-amylase very rapidly reduces insoluble and soluble starch by splitting starch molecules into many shorter chains that can be attacked by beta-amylase. The disaccharide produced is maltose. In addition to the maltose formed during mashing short branched glucose oligomers are produced. The short branched glucose oligomers are non fermentable sugars and add to the taste as well as the calories of the finished beer.

After mashing, when all the starch has been broken down, it is necessary to separate the liquid extract (the wort) from the solids (spent grains). Wort separation, lautering, is important because the solids contain large amounts of protein, poorly modified starch, fatty material, silicates, and polyphenols (tannins). Following the separation of the wort from the spent grains the wort may be fermented with brewers yeast to produce a beer.

Further information on conventional brewing processes may be found in "Technology Brewing and Malting" by Wolfgang Kunze of the Research and Teaching Institute of Brewing, Berlin (VLB), 2nd revised Edition 1999, ISBN 3-921690-39-0.

The short branched glucose oligomers formed during mashing may be further hydrolyzed by addition of exogenous enzymes (enzymes added in addition to the malt). Debranching enzymes such as pullulanase and isoamylase hydrolyses the branching alpha-1-6 glucosidic bonds in these oligomers, thereby releasing glucose or maltose and straight-chained oligomers which are subject to the action of endogenous (malt derived) and/or exogenous enzymes, e.g. alpha-amylases, beta-amylases and glucoamylases.

The present invention provides a new process suitable for producing a wort that is low in non- fermentable sugars. The process applies an expressly selected pullulanase activity.

Definitions

Throughout this disclosure, various terms that are generally understood by those of ordinary skill in the arts, are used. Several terms are used with specific meaning, as defined below.

As used herein the term "grist" is understood as the starch or sugar containing material that's the basis for beer production, e.g. the barley malt and the adjunct. Generally, the grist is does not contain any added water.

The term "malt" is understood as any malted cereal grain, in particular barley.

The term "adjunct" is understood as the part of the grist which is not barley malt. The adjunct may comprise any starch rich plant material, e.g. unmalted grain, such as barley, rice, corn, wheat, rye, sorghum and readily fermentable sugar and/or syrup. The term "mash" is understood as a starch containing slurry comprising grist steeped in water.

The term "wort" is understood as the unfermented liquor run-off following extracting the grist during mashing.

The term "spent grains" is understood as the drained solids remaining when the grist has been extracted and the wort separated. The term "beer" is understood as fermented wort, i.e. an alcoholic beverage brewed from barley malt, optionally adjunct and hops.

The term "homologous sequence" is used to characterize a sequence having an amino acid sequence that is at least 70%, preferably at least 75%, or at least 80%, or at least 85%, or 90%, or at least 95%, at least 96%, at least 97%, at least 98% at least 99% or even at least 100% identical to a known sequence. The relevant part of the amino acid sequence for the homology determination is the mature polypeptide,

i.e., without the signal peptide. The term "homologous sequence" is also used to characterize DNA sequences which hybridize at low stringency, medium stringency, medium/high stringency, high stringency, or even very high stringency with a known sequence. Suitable experimental conditions for determining hybridization at low, medium, or high stringency between a nucleotide probe and a homologous DNA or RNA sequence involves presoaking of the filter containing the DNA fragments or RNA to hybridize in 5 x SSC (Sodium chloride/Sodium citrate, Sambrook et al. 1989) for 10 min, and prehybridization of the filter in a solution of 5 x SSC, 5 x Denhardt's solution (Sambrook et al. 1989), 0.5% SDS and 100 micrograms/ml of denatured sonicated salmon sperm DNA (Sambrook et al. 1989), followed by hybridization in the same solution containing a concentration of 10ng/ml of a random-primed (Feinberg, A. P. and Vogelstein, B. (1983) Anal. Biochem. 132:6-13), 32P-dCTP-labeled (specific activity > 1 x 109 cpm/microgram) probe for 12 hours at about 45°C. The filter is then washed twice for 30 minutes in 2 x SSC, 0.5% SDS at about 55°C (low stringency), more preferably at about 60°C (medium stringency), still more preferably at about 65°C (medium/high stringency), even more preferably at about 70°C (high stringency), and even more preferably at about 75°C (very high stringency). Molecules to which the oligonucleotide probe hybridizes under these conditions are detected using an x-ray film. The term "identity" when used about polypeptide or DNA sequences and referred to in this disclosure is understood as the degree of identity between two sequences indicating a derivation of the first sequence from the second. The identity may suitably be determined by means of computer programs known in the art such as GAP provided in the GCG program package (Program Manual for the Wisconsin Package, Version 8, August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 5371 1 ) (Needleman, S. B. and Wunsch, CD., (1970), Journal of Molecular Biology, 48, 443-453. The following settings for polypeptide sequence comparison are used: GAP creation penalty of 3.0 and GAP extension penalty of 0.1. The degree of identity between an amino acid sequence of the present invention and a different amino acid sequence ("foreign sequence") is calculated as the number of exact matches in an alignment of the two sequences, divided by the length of the "invention sequence" or the length of the "foreign sequence", whichever is the shortest. The result is expressed in percent identity.

Wort production

In accordance with the first aspect the invention provides a process for producing a brewers wort comprising forming a mash from a grist, and contacting said mash with a pullulanase (E. C. 3.2.1.41 ), wherein said pullulanase has an amino acid sequence which a) is at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, or at least 85%, or 90%, or at least 95%, at least 96%, at least 97%, at least 98% or even at least 99% identical to the amino acid sequence shown in SEQ ID NO:3, or b) is encoded by a nucleic acid sequence which hybridizes under low stringency, medium stringency, medium/high stringency, high stringency, or even very high stringency with i) a complementary strand of a nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO:3, or ii) a subsequence of (i) of at least 100 nucleotides, at least 200 nucleotides, at least 300 nucleotides, at least 500 nucleotides, at least 1000 nucleotides, or even at least 1500 nucleotides. In a preferred embodiment, the pullulanase has an amino acid sequence which differs by no more than 100 amino acids, preferably by no more than 80 amino acids, more preferred by no more than 50 amino acids, more preferably by no more than 30 amino acids, even more preferably by no more than 20 amino acids, and most preferably by no more than 10 amino acids from the amino acid sequence of SEQ ID NO: 3.

The grist of the first aspect comprises starch containing malted grain and/or adjunct. The grist may preferably comprises from 0% to 100%, preferably from 20% to 1000%, preferably from 30% to 100%, more preferably from 40% to 100%, even more preferably from 50% to 100%, yet more preferably from 100% to 80%, most preferably from 100% to 80% adjunct, or even most preferably from 90% to 100% adjunct, unmalted grain and/or unmalted barley. In a particular embodiment the adjunct is composed of 100% unmalted barley. Furthermore, the grist preferably comprises from 0% to 100%, preferably from 20% to 100%, preferably from 30% to 100%, more preferably from 40% to 100%, even more preferably from 50% to 100%, yet more preferably from 60% to 100%, or most preferably from 70% to 100%, or even most preferably from 90% to 100% malted grain and/or malted barley. In a particular embodiment the grist comprises approximately 50% malted grain, e.g. malted barley, and approximately 50% adjunct, e.g. unmalted grain, such as unmalted barley. Malted grain used in the process of the first aspect may comprise any malted grain, and preferably malted grain selected from malted barley, wheat, rye, sorghum, millet, corn, and rice, and most preferably malted barley.

The adjunct used in the process of the first aspect may be obtained from tubers, roots, stems, leaves, legumes, cereals and/or whole grain. The adjunct may comprise raw and/or refined starch and/or sugar containing material derived from plants like wheat, rye, oat, corn, rice, milo, millet, sorghum, potato, sweet potato, cassava, tapioca, sago, banana, sugar beet and/or sugar cane. Preferably, the adjunct comprises unmalted grain, e.g. unmalted grain selected from the list consisting of barley, wheat, rye, sorghum, millet, corn, and rice, and most preferably unmalted barley. Adjunct comprising readily fermentable carbohydrates such as sugars or syrups may be added to the barley malt mash before, during or after mashing process of the invention but is preferably added after the mashing process.

According to 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 water and/or the grist before, during or after forming the mash. In a particularly preferred embodiment an alpha-amylase (E. C. 3.2.1.1 ) and/or a glucoamylase (E. C. 3.2.1.3), is added and present in the mash together with the pullulanase.

In another preferred embodiment a further enzyme is added to the mash, said enzyme being selected from the group consisting of isoamylase, protease, laccase, xylanase, lipase, phospholipolase, phytase, phytin and esterase. During the mashing process, starch extracted from the grist is gradually hydrolyzed into fermentable sugars and smaller dextrins. Preferably, the mash is starch negative to iodine testing, before extracting the wort.

The mashing process generally apply a controlled stepwise increase in temperature, where each step favors one enzymatic action over the other, eventually degrading proteins, cell walls and starch. Mashing temperature profiles are generally known in the art. In the present invention the saccharification (starch degradation) step in the mashing process is preferably performed between 60 0C and 66 ° C, more preferably between 61 0C and 65 0C, even more preferably between 62 0C and 640C, and most preferably between 63 0C and 64 0C. In a particular embodiment of the present invention the saccharification temperature is 64 0C.

Obtaining the wort from the mash typically includes straining the wort from the spent grains, i.e. the insoluble grain and husk material forming part of grist. Hot water may be run through the spent grains to rinse out, or sparge, any remaining extract from the grist. The application of a thermostable cellulase in the process of the present invention results in efficient reduction of beta- glucan level facilitating wort straining thus ensuring reduced cycle time and high extract recovery. Preferably the extract recovery is at least 80%, preferably at least 81 %, more preferably at least 82%, even more preferably at least 83%, such as at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, and most preferably at least 91 %.

Following the separation of the wort from the spent grains of the grist of any of the aforementioned embodiments of the first aspect the wort may be used as it is or it may be dewatered to provide a concentrated and/or dried wort e.g. The

concentrated and/or dried wort may be used as brewing extract, as malt extract flavoring, for non-alcoholic malt beverages, malt vinegar, breakfast cereals, for confectionary etc.

In a preferred embodiment the wort is fermented to produce an alcoholic beverage, preferably a beer, e.g., ale, strong ale, bitter, stout, porter, lager, export beer, malt liquor, barley wine, happoushu, high-alcohol beer, low-alcohol beer, low-calorie beer or light beer. Fermentation of the wort may include pitching the wort with a yeast slurry comprising fresh yeast, i.e. yeast not previously used for the invention or the yeast may be recycled yeast. The yeast applied may be any yeast suitable for beer brewing, especially yeasts selected from Saccharomyces spp. such as S. cerevisiae and S. uvarum, including natural or artificially produced variants of these organisms. The methods for fermentation of wort for production of beer are well known to the person skilled in the arts.

The process of the invention may include adding silica hydrogel to the fermented wort to increase the colloidal stability of the beer. The processes may further include adding kieselguhr to the fermented wort and filtering to render the beer bright. According to an aspect of the invention is provided beer produced from the wort of the second or third aspect, such as a beer produced by fermenting the wort to produce a beer. The beer may be any type of beer, e.g., ales, strong ales, stouts, porters, lagers, bitters, export beers, malt liquors, happoushu, high-alcohol beer, low-alcohol beer, low-calorie beer or light beer.

Enzymes

The enzymes to be applied in the present invention should be selected for their ability to retain sufficient activity at the process temperature of the processes of the invention, as well as under the pH regime in the mash and should be added in effective amounts. The enzymes may be derived from any source, preferably from a plant or an alga, and more preferably from a microorganism, such as from a bacterium or a fungus.

Pullulanase (E.C. 3.2.1.41 )

A preferred pullulanase enzyme to be used in the processes and/or compositions of the invention is a pullulanase having an amino acid sequence which is at least 50%, preferably 60% at least, more preferably at least 70%, even more preferably at least 80%, such as at least 90%, at least 95%, at least 98% or even 100% identical to the sequence shown in SEQ ID NO:3. Most preferably the pullulanase is derived from Bacillus acidopullulyticus. The pullulanase may have the amino acid sequence disclosed by Kelly et al., 1994 (FEMS Microbiol. Letters, 115, 97-106) or a homologous sequence.

Isoamylase (E.C. 3.2.1.68) Another enzyme applied in the processes and/or compositions of the invention may be an alternative debranching enzyme, such as an isoamylase (E.C. 3.2.1.68). lsoamylase hydrolyses alpha-1 ,6-D-glucosidic branch linkages in amylopectin and beta-limit dextrins and can be distinguished from pullulanases by the inability of isoamylase to attack pullulan, and by the limited action on alpha-limit dextrins. Isoamylase may be added in effective amounts well known to the person skilled in the art. Isoamylase may be added alone or together with a pullulanase.

Alpha-amylase (EC 3.2.1.1 )

A particular alpha-amylase enzyme to be used in the processes and/or compositions of the invention may be a Bacillus alpha-amylase. Well-known Bacillus alpha-amylases include alpha- amylase derived from a strain of B. licheniformis, B. amyloliquefaciens, and B. stearothermophilus. In the context of the present invention a contemplated Bacillus alpha-amylase is an alpha-amylase as defined in WO99/19467 on page 3, line 18 to page 6, line 27. A preferred alpha-amylase has an amino acid sequence having at least 90% identity to SEQ ID NO:4 in WO99/19467, such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or particularly at least 99%. Most preferred variants of the maltogenic alpha-amylase comprise the variants disclosed in WO99/43794. Contemplated variants and hybrids are described in WO96/23874,

WO97/41213, and WO99/19467. Specifically contemplated is an alpha-amylase (E. C. 3.2.1.1 ) from B. stearothermophilus having the amino acid sequence disclosed as SEQ ID NO:4 in WO99/19467 with the mutations: 1181 * + G182* + N193F.

Bacillus alpha-amylases may be added in the amounts of 1.0-1000 NU/kg DS, preferably from 2.0-500 NU/kg DS, preferably 10-200 NU/kg DS.

Another particular alpha-amylase to be used in the processes of the invention may be any fungal alpha-amylase, e.g. an alpha-amylase derived from a species within Aspergillus, and preferably from a strain of Aspergillus niger. Especially contemplated are fungal alpha-amylases which exhibit a high identity, i.e. at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or even at least 90% identity to the amino acid sequences shown SEQ ID NO:1 in WO 2002/038787. Fungal alpha-amylases may be added in an amount of 1-1000 AFAU/kg DS, preferably from 2-500 AFAU/kg DS, preferably 20-100 AFAU/kg DS.

Glucoamylases (E.C.3.2.1.3)

A further particular enzyme to be used in the processes and/or compositions of the invention may be a glucoamylase (E.C.3.2.1.3) derived from a microorganism or a plant. Preferred are glucoamylases of fungal or bacterial origin selected from the group consisting of Aspergillus glucoamylases, in particular A. niger GI or G2 glucoamylase (Boel et al. (1984), EMBO J. 3 (5), p.

1097-1102), or variants thereof, such as disclosed in WO92/00381 and WO00/04136; the A. awamori glucoamylase (WO84/02921 ), A. oryzae (Agric. Biol. Chem. (1991 ), 55 (4), p. 941-949), or variants or fragments thereof.

Other contemplated glucoamylases include Talaromyces glucoamylases, in particular derived from Talaromyces emersonii (WO99/28448), Talaromyces leycettanus (US patent no. Re. 32,153), Talaromyces duponti, Talaromyces thermophilus (US patent no. 4,587,215). Preferred glucoamylases include the glucoamylases derived from Aspergillus oryzae, such as a glucoamylase having at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or particularly at least 99% or even at least 90% identity to the amino acid sequence shown in SEQ ID NO:2 in WO00/04136. Other preferred glucoamylases include the glucoamylases derived from Talaromyces emersonii such as a glucoamylase having at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or particularly at least 99% or even at least 90% identity to the amino acid sequence shown in SEQ ID NO:2 in 60/738,448

Bacterial glucoamylases contemplated include glucoamylases from the genus Clostridium, in particular C. thermoamylolyticum (EP 135,138), and C. thermohydrosulfuricum (WO86/01831 ).

Also contemplated are the commercial products AMG 200L; AMG 300 L; SAN™ SUPER and AMG™ E (from Novozymes); OPTIDEX™ 300 (from Genencor Int.); AMIGASE™ and AMIGASE™ PLUS (from DSM); G-ZYME™ G900 (from Enzyme Bio-Systems); G-ZYME™ G990 ZR (A. niger glucoamylase and low protease content). Glucoamylases may be added in effective amounts well known to the person skilled in the art.

Protease

Suitable proteases include microbial proteases, such as fungal and bacterial proteases. Preferred proteases are acidic proteases, i.e., proteases characterized by the ability to hydrolyze proteins under acidic conditions below pH 7.

The proteases are responsible for reducing the overall length of high-molecular-weight proteins to low-molecular-weight proteins in the mash. The low-molecular-weight proteins are a necessity for yeast nutrition and the high-molecular-weight-proteins ensure foam stability. Thus it is well-known to the skilled person that protease should be added in a balanced

amount which at the same time allows amble free amino acids for the yeast and leaves enough high-molecular- weight-proteins to stabilize the foam. Proteases may be added in the amounts of 0.1-1000 AU/kg DS, preferably 1-100 AU/kg DS and most preferably 5-25 AU/kg DS.

Cellulase (E.C. 3.2.1.4) The cellulase may be of microbial origin, such as derivable from a strain of a filamentous fungus (e.g., Aspergillus, Trichoderma, Humicola, Fusarium). Specific examples of cellulases include the endo-glucanase (endo-glucanase I) obtainable from H. insolens and further defined by the amino acid sequence of fig. 14 in WO 91/17244 and the 43 kD H. insolens endo-glucanase described in WO 91/17243. A particular cellulase to be used in the processes of the invention may be an endo- glucanase, such as an endo-1 ,4-beta-glucanase. Especially contemplated is the beta-glucanase shown in SEQ.ID.NO:1 in WO 2003/062409 and homologous sequences. Commercially available cellulase preparations which may be used include CELLUCLAST®, CELLUZYME®, CEREFLO® and ULTRAFLO® (available from Novozymes A/S), LAMINEX™ and SPEZYME® CP (available from Genencor Int.) and ROHAMENT® 7069 W (available from Rohm, Germany).

Beta-glucanases may be added in the amounts of 1.0-10000 BGU/kg DS, preferably from 10-5000 BGU/kg DS, preferably from 50-1000 BGU/kg DS and most preferably from 100-500 BGU/kg DS.

MATERIALS AND METHODS Enzymes

Pullulanase 1 derived from Bacillus acidopullulyticus and having the sequence showed in SEQ ID NO:1. Pullulanase 1 is available from Novozymes as Promozyme 400L.

Pullulanase 2 derived from Bacillus deramificans (US Patent 5,736375) and having the sequence showed in SEQ ID NO:2. Pullulanase 2 is available from Novozymes as Promozyme D2. Pullulanase 3 derived from Bacillus acidopullulyticus and having the sequence showed in SEQ ID N0:3.

Acid fungal alpha-amylase derived from Aspergillus niger and having the sequence showed in SEQ ID N0:1 in WO 2002/038787 (SEQ ID NO: 1 is hereby incorporated by reference). Glucoamylase G1 derived from Aspergillus niger (Boel et al. supra)

Methods

Alpha-amylase activity (NU)

Alpha-amylase activity may be determined using potato starch as substrate. This method is based on the break-down of modified potato starch by the enzyme, and the reaction is followed by mixing samples of the starch/enzyme solution with an iodine solution. Initially, a blackish-blue color is formed, but during the break-down of the starch the blue color gets weaker and gradually turns into a reddish-brown, which 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 which, under standard conditions (i.e. at 37°C +/- 0.05; 0.0003 M Ca2+; and pH 5.6) degrades 5.26 g starch dry matter (Merck Amylum solubile).

A folder AF 9/6 describing this analytical method in more detail is available upon request to Novozymes A/S, Denmark, which folder is hereby included by reference.

Acid alpha-amylase activity (AFAU)

Acid alpha-amylase activity may be measured in AFAU (Acid Fungal Alpha-amylase Units), which are determined relative to an enzyme standard. 1 FAU is defined as the amount of enzyme which degrades 5.260 mg starch dry matter per hour under the below mentioned standard conditions.

Acid alpha-amylase, an endo-alpha-amylase (1 ,4-alpha-D-glucan-glucanohydrolase, E. C. 3.2.1.1 ) hydrolyzes alpha-1 ,4-glucosidic bonds in the inner regions of the starch molecule to form dextrins and oligosaccharides with different chain lengths. The intensity of color formed with iodine is directly proportional to the concentration of starch. Amylase activity is determined using reverse colorimetry as a reduction in the concentration of starch under the specified analytical conditions.

ALPHA -AMYLASE STARCH + IODINE 40 : , pH 2,5 > DEXTRINS + OLIGOSACCHARIDES λ = 590 nm blue/violet t = 23 sec. decoloration Standard conditions/reaction conditions:

Substrate: Soluble starch, approx. 0.17 g/L

Buffer: Citrate, approx. 0.03 M

Iodine (I2): 0.03 g/L

CaCI2: 1.85 mM pH: 2.50 ± 0.05

Incubation temperature: 4O0C

Reaction time: 23 seconds

Wavelength: 590nm

Enzyme concentration: 0.025 AFAU/mL

Enzyme working range: 0.01-0.04 AFAU/mL

A folder EB-SM-0259.02/01 describing this analytical method in more detail is available upon request to Novozymes A/S, Denmark, which folder is hereby included by reference.

Glucoamylase activity (AGU) The Novo Glucoamylase Unit (AGU) is defined as the amount of enzyme, which hydrolyzes 1 micromole maltose per minute at 37°C and pH 4.3.

The activity is determined as AGU/ml by a method modified after (AEL-SM-0131 , available on request from Novozymes) using the Glucose GOD-Perid kit from Boehringer Mannheim, 124036. Standard: AMG-standard, batch 7-1195, 195 AGU/ml. 375 microL substrate (1 % maltose in 50 mM Sodium acetate, pH 4.3) is incubated 5 minutes at 37°C. 25 microL enzyme diluted in sodium acetate is added. The reaction is stopped after 10 minutes by adding 100 microL 0.25 M NaOH. 20 microL is transferred to a 96 well microtitre plate and 200 microL GOD-Perid solution (124036, Boehringer Mannheim) is added. After 30 minutes at room temperature, the absorbance is measured at 650 nm and the activity calculated in AGU/ml from the AMG-standard. A detailed description of the analytical method (AEL-SM-0131 ) is available on request from Novozymes.

Example 1

In the example the ability of different pullulanases to reduce the amount of non-fermentable carbohydrates (dextrin/DP4/4+) in a wort was analysed.

100% well modified malt was mashed using a mashing temperature profile comprising 46°C for 26 minutes, followed by a 1°C/minute increase till 64°C after which the temperature was held constant. Samples were collected at 98, 128 and 158 minutes.

Enzymes were added at 0 minutes. Glucoamylase and alpha-amylase were added to all treatments in amounts of 1000 AGU/kg DS and 250 AFAU/kg DS respectively. Pullulanase was added according to table 1. The samples were boiled 10 minutes and filtered (Pore size 0.20μm). The samples were analyzed by HPLC and % non fermentable carbohydrate (DP4/4+) was calculated.

The data in table 1 was used to calculate, by regression, the enzyme dosages of pullulanase 1 and pullulanase 2 needed to get the same effect as 2.74 mg enzyme protein/kg of pullulanase 3. (see table 2).



From these results it can be seen that Pullulanase 3 is the most efficient enzyme. Consequently, less Pullulanase 3 enzyme protein is needed to reduce the amount of non-fermentable carbohydrates (dextrin/DP4/4+) and thereby increase attenuation of the wort. Example 2

The pH profile and temperature profile of different pullulanases was analyzed in the present example.

The pH and temperature profile investigations were based on relative enzyme activity analysis with the conditions described below.

Principals of the analytical method:

The alpha-1 ,6-glycosidic bounds in pullulan were hydrolyzed by a pullulanase enzyme and the increased reducing sugar capacity was detected by a modified Somogyi-Nelson method.

In the present experiment the activity is assessed as relative activity, where the most active sample is given as 100%. The assay conditions are as follows:

Buffer: citrate 0.1 M + 0.2 M phosphate (adjusted in the pH profile, pH 5 in temperature profile)

Substrate: 0.2 % pullulan Sigma (p-4516)

Temperature: 6O0C in pH profile, adjusted in temperature profile

Reaction time: 30 minutes The reducing sugars released by pullulanases were detected according to the principle described in Nelson, N. J. Biol. Chem (1944), 153, 375-380 and Somogyi, M. J. Biol. Chem (1945) 160, 61 - 68. In brief, the hydrolysis reaction is stopped by adding Somogyi's cobber reagent in a volume corresponding to the sample volume (e.g. 2 ml to a sample of 2 ml). The samples are boiled for 20 minutes and cooled down prior to the color reaction. This reaction is performed by adding Nelson's reagent corresponding to ΛA the volume of the sample (e.g. 2 ml to 4 ml sample÷

Somogyi's cobber reagent). The samples are mixed for 2 minutes followed by addition of water in the same amount as Nelson's reagent. The samples are incubated 30 minutes in the dark and measured in a spectrophotometer at 540 nm.

Reagents can be prepared as follows: Somogyi's cobber reagent:

Dissolve 70.2 g Na2HPO4x2H2O and 80.0 g KNAC4H4O6x4 H2O (kaliumsodiumtartrat) in 1000 ml H2O (heat slightly). Furthermore add 60 g NaOH; 16.0 g CuS04x5 H2O and 360.0 g Na2SO4 and fill to 2000 ml. Adjust pH to 10.8 with NaOH

Nelson's reagent: Dissolve 100.0 g (NH4)6Mo7O24x7 H2O in 1200 ml H2O. Add 84.0 ml H2SO4 carefully. Additionally, dissolve 12.00 g Na2HAs04x7 H2O (disodiumhydrogenarsenate) in 100 ml H2O, and add this solution slowly to the first solution and fill to 2000 ml. The pH and temperature profiles for the three pullulanases are given in table 3 and 4 below.

These results show that pullulanase 3 has a broad pH profile and activity at high temperatures when compared to the other two pullulanases. These properties make pullulanase 3 a very robust enzyme in brewing (mashing conditions), in particular for saccharification temperatures between 620C and 650C.

Mashing process

ABSTRACT

The present invention provides processes for production of wort and beer from a granular starch adjunct grist mashed-in at a temperature below the gelatinization temperature of said starch.

CLAIMS(1)



1. 1 A process for production of a Brewer's wort, comprising mashing a grist comprising malt and a granular starch adjunct in the presence of an exogenously supplied enzyme composition at a temperature at which the endogenous malt enzymes are active.

2 A process for production of a Brewer's wort, comprising; a) providing a mash comprising i) malt, ii) adjunct comprising granular starch, and iii) an exogenously supplied enzyme composition, b) mashing said mash at a temperature below the initial gelatinisation temperature of said granular starch, c) mashing off at a temperature above the initial gelatinisation temperature; and d) separating the spent grain from the mash and obtaining a wort.

3 The process according to any of the preceding claims, wherein the malted grain in the malt is selected from the list consisting of corn, barley, wheat, rye, sorghum, millet and rice.

4 The process according to any of the preceding claims, wherein the adjunct comprises at least one unmalted grain.

5 The process according to any of the preceding claims, wherein the adjunct comprises grain selected from corn, barley, wheat, rye, sorghum, millet and rice.

6 The process according to any of the preceding claims, wherein the adjunct comprises starch derived from corn, rice, barley, wheat, rye, sorghum, millet, cassava, sago palm.

7 The process according to any of the preceding claims, wherein the adjunct comprises corn grits or corn starch, preferably corn starch from a wet-milling process.

8 The process according to any of the preceding claims, wherein the grist of the mash comprises from 1 % to 100%, preferably from 5% to 90%, more preferably from 10% to 80%, and even more preferably from 50 to 70% malted grain.

9 The process according to any of the preceding claims, wherein the grist of the mash comprises from 1 % to 50%, preferably from 5% to 45%, more preferably from 10% to 40%, and even more preferably from 20 to 35% adjunct starch.

10 The process of any of the preceding claims, wherein the extract yield from the mash is at least 88%, preferably at least 89%, more preferably at least 90%, and most preferably at least 91 %.

1 1 The process according to any of the preceding claims, wherein the exogenously supplied enzyme composition comprises a glucoamylase and/or an alpha-amylase.

12 The process according to any of the preceding claims, wherein the exogenously supplied enzyme composition further comprises a cellulase, a pullulanase and/or a protease.

13 The process according to claim 1 1 or 12, wherein the alpha-amylase is a bacterial alpha- amylase.

14 The process according to claim 13, wherein the alpha-amylase is a bacterial alpha-amylase comprising a starch binding module.

15 The process according to any of the claims 1 1 to 14, wherein the alpha-amylase is an polypeptide having at least 50% identity to the amino acid sequence shown in SEQ ID NO:1.

16 The process according to any of the claims 1 1 to 14, wherein the alpha-amylase is an polypeptide having at least 50% identity to any amino acid sequence shown in SEQ ID NO:2.

17 The process according to claim 1 1 or 12, wherein the glucoamylase is derived from Aspergillus niger.

18 The process according to any of the preceding claims, further comprising concentrating and/or drying the wort.

19 The process according to any of the preceding claims, further comprising fermenting the wort to obtain an alcoholic beverage.

20 The process according the preceding claim, wherein the alcoholic beverage is a beer.

21 The process according to claim 19 or 20, wherein the beer is ale, strong ale, bitter, stout, porter, lager, export beer, malt liquor, barley wine, happoushu, high-alcohol beer, low- alcohol beer, low-calorie beer or light beer.

22 A wort produced by the process according to any of claims 1 to 18.

23 A beer produced by the process according to any of the claims 19 to 21.

DESCRIPTION

MASHING PROCESS

FIELD OF THE INVENTION

The present invention relates to an improved mashing process using grist comprising ungelatinized adjunct.

BACKGROUND OF THE INVENTION

Traditionally, beer has been brewed from just barley malt, hops and water. However, often part of the barley malt is substituted with adjuncts such as corn, rice, sorghum, and wheat, refined starch or readily fermentable carbohydrates such as sugar or syrups. Adjuncts are used mainly because they are readily available and provide carbohydrates at a lower cost than is available from barley malt. Other advantages may also be achieved, e.g. enhanced physical stability, superior chill-proof qualities, and greater brilliancy.

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. However, rice and corn starch which are often used as adjunct starch have a higher gelatinization temperature than the malt starch. Therefore, such adjuncts are cooked and gelatinized in a separate "cereal cooker" before being added to the malt mash. Thus, while the use of adjunct reduces the costs of raw material price it requires an additional investment in the cereal cooker as well as an additional cost for energy for heating the adjunct. A more simple mashing process allowing use of ungelatinized adjunct grist is thus desirable.

SUMMARY OF THE INVENTION The inventor of the present invention has surprisingly found that an adjunct starch can be added to the malt mash and be efficiently mashed without prior gelatinization. Thus an adjunct such as corn grits, corn starch or rice starch, can be mashed with the malt at temperatures where the endogenous malt enzymes are active. The liquefaction of the ungelatinized adjunct requires that the endogenous malt enzymes are supplemented by an exogenously supplied enzyme composition.

Accordingly in a first aspect the invention provides a process for production of a Brewer's wort, comprising mashing a grist comprising malt and a granular starch adjunct in the presence of an exogenously supplied enzyme composition at a temperature at which the endogenous malt enzymes are active. The invention further provides a process for production of a Brewer's wort, comprising; a) providing a mash comprising i) malt, ii) adjunct comprising granular starch, and iii) an exogenously supplied enzyme composition, b) mashing said mash at a temperature below the initial gelatinisation temperature of said granular starch, c) mashing off at a temperature above the initial gelatinisation

temperature, and d) separating the spent grain from the mash and obtaining a wort. In a second aspect the invention provides a wort produced by the process according to the first and second aspect.

In a third aspect the invention provides a beer produced by fermenting the wort of the third aspect.

DETAILED DESCRIPTION OF THE INVENTION It is the intention of the present invention to provide a more simple mashing process allowing use of ungelatinized adjunct grist in the process.

Definitions

Throughout this disclosure, various terms that are generally understood by those of ordinary skill in the arts are used. Several terms are used with specific meaning, however, and are meant as defined by the following.

As used herein the term "grist" is understood as the starch or sugar containing material that is the basis for beer production, e.g. the barley malt and the adjunct.

The term "malt" is understood as any malted cereal grain, in particular barley.

The term "adjunct" is understood as the part of the grist which is not barley malt. The adjunct may be any starch rich plant material such as, but not limited to, corn, rice, sorghum, and wheat. Preferred adjunct for the invention is corn grits.

The term "mash" is understood as a starch containing slurry comprising crushed barley malt, crushed unmalted grain, other starch containing material, or a combination hereof, steeped in water to make wort. The term "wort" is understood as the unfermented liquor run-off following extracting the grist during mashing.

The term "spent grains" is understood as the drained solids remaining when the grist has been extracted and the wort separated.

The term "beer" is here understood as a fermented wort, i.e. an alcoholic beverage brewed from barley malt, optionally adjunct and hops.

The term "granular starch" is understood as ungelatinized starch, or raw starch.

The term "initial gelatinization temperature" is understood as the lowest temperature at which gelatinization of the starch commences. Starch heated in water begins to gelatinize between 500C and 75°C; the exact temperature of gelatinization depends on the specific starch, and can readily be determined by the skilled person. Thus, the initial gelatinization temperature may vary according to the plant species, to the particular variety of the plant species as well as with the growth conditions. In the context of this invention the initial gelatinization temperature of a given starch is the temperature at which

birefringence is lost in 5% of the starch granules using the method described by Gorinstein. S. and Lii. C, Starch/Starke, Vol. 44 (12) pp. 461- 466 (1992). For corn starch the initial gelatinization temperature is approximately 62°C (midpoint: 67°C, completion: 72°C), and for rice starch the initial gelatinization temperature is approximately 68°C (midpoint: 74.5 °C, completion: 78°C) (Starch, 2nd ed. Industrial microscopy of starch by Eileen Maywald Snyder).

The term "extract recovery" in the wort is understood as the sum of soluble substances extracted from the grist (malt and adjuncts) expressed in percentage based on dry matter. The term "identity" when used about polypeptide or DNA sequences and referred to in this disclosure is understood as the degree of identity between two sequences indicating a derivation of the first sequence from the second. The identity may suitably be determined by means of computer programs known in the art such as GAP provided in the GCG program package (Program Manual for the Wisconsin Package, Version 8, August 1994, Genetics Com- puter Group, 575 Science Drive, Madison, Wisconsin, USA 5371 1 ) (Needleman, S. B. and Wunsch, CD., (1970), Journal of Molecular Biology, 48, 443-453. The following settings for polypeptide sequence comparison are used: GAP creation penalty of 3.0 and GAP extension penalty of 0.1.

In a first aspect the invention a process for production of a Brewer's wort is provided. The process comprises mashing a grist comprising malt and a granular starch adjunct in the presence of an exogenously supplied enzyme composition at a temperature at which the endogenous malt enzymes are active. The invention further provides a process for production of a Brewer's wort, comprising; a) providing a mash comprising i) malt, ii) adjunct comprising granular starch, and iii) an exogenously supplied enzyme composition, b) mashing said mash at a temperature below the initial gelatinisation temperature of said granular starch, c) mashing off at a temperature above the initial gelatinisation temperature, and d) separating the spent grain from the mash and obtaining a wort.

In accordance with the first aspect of the present invention a grist comprising malt and a granular starch adjunct is mashed in the presence of an exogenously supplied enzyme composition at a temperature at which the endogenous malt enzymes e.g. alpha-amylases, proteases and beta-amylases, that the traditional mashing processes rely on, are active.

The water may preferably, before being added to the grist, be preheated in order for the mash to attain the desired mash temperature at the moment of mash forming. If the temperature of the formed mash is below the desired mashing temperature additional heat is preferably supplied in order to attain the desired process temperature. Preferably, the desired mashing temperature is attained within 15 minutes, or more preferably within 10 minutes, such as within 9, 8, 7, 6, 5, 4, 3, 2 minutes or even more preferably within 1 minute after the mash forming, or most preferably the desired mashing temperature is attained at the mash forming. The temperature profile of the mashing process may be a profile from a conventional mashing process wherein the temperatures are set to achieve optimal degradation of the grist dry matter by the malt enzymes.

The malt is preferably derived from one or more of the grains selected from the list consisting of corn, barley, wheat, rye, sorghum, millet and rice. Preferably, the malt is barley malt.

The grist preferably comprises from 0.5% to 99%, preferably from 1 % to 95%, more preferably from 5% to 90%, even more preferably from 10% to 80%, and most preferably from 50% to 70% malted grain. In addition to malted grain, the grist may preferably comprise adjunct such as unmalted corn, or other unmalted grain, such as barley, wheat, rye, oat, corn, rice, milo, millet and/or sorghum, or raw and/or refined starch and/or sugar containing material derived from plants like wheat, rye, oat, corn, rice, milo, millet, sorghum, potato, sweet potato, cassava, tapioca, sago, banana, sugar beet and/or sugar cane. For the invention adjuncts may be obtained from tubers, roots, stems, leaves, legumes, cereals and/or whole grain. Preferred is adjunct obtained from corn and/or rice, more preferred the adjunct is rice starch, corm starch and/or corn grits. The mash preferably comprises from 1 % to 50%, preferably from 5% to 45%, more preferably from 10% to 40%, and even more preferably from 20 to 35% adjunct starch. Adjunct comprising readily fermentable carbohydrates such as sugars or syrups may be added to the malt mash before, during or after the mashing process of the invention but is preferably added after the mashing process.

Prior to forming the mash the malt and/or adjunct is preferably milled and most preferably dry or wet milled.

According to the invention an enzyme composition is exogenously supplied and may be added to the mash ingredients, e.g. the water or the grist before during or after forming the mash. In a particularly preferred embodiment the enzyme composition comprises an alpha- amylase (EC 3.2.1.1 ) and/or a glucoamylase (EC 3.2.1.3). The alpha-amylase is preferably a bacterial alpha-amylase or and/or a fungal alpha-amylase, e.g., an acid fungal alpha-amylase. The glucoamylase is preferably a fungal glucoamylase. By selecting the enzymes making up the enzyme composition the sugar profile of the resulting wort can be controlled. An enzyme composition comprising alpha-amylase, preferably a bacterial alpha-amylase, and little or no glucoamylase will result in a maltose rich wort similar to an all malt wort. An enzyme composition comprising glucoamylase will result in a glucose rich wort. In yet a preferred embodiment a further enzyme is added, said enzyme being selected from the group consisting of a cellulase, a pullulanase, a protease, a maltose generating enzyme, a laccase, a lipase, a phospholipolase, a phytase, a phytin esterase, and a xylanase.

During the mashing process, starch extracted from the grist is gradually hydrolyzed into fermentable sugars and smaller dextrins. Preferably the mash is starch negative to iodine testing, before extracting the wort.

The mashing is finalized by mashing-off at temperature of 700C or more, preferably at least 710C, at least 72°C, at least 73°C, at least 74°C, at least 75°C, at least 76°C at least 77°C, at least 78°C, least 79°C, at least 800C and more preferably at least 810C or even at least 82°C or more.

Obtaining the wort from the mash typically includes straining the wort from the spent grains, i.e. the insoluble grain and husk material forming part of grist. Hot water may be run through the spent grains to rinse out, or sparge, any remaining extract from the grist. Preferably, the extract recovery is at least 80%, preferably at least 85%, at least 90%, least 95%, at least 98% and more preferably at least 99% or even at least 100%. The wort may be used as it is, or it may be concentrated and/or dried.

In a preferred embodiment a grist comprising 60-80% barley malt and 20% to 40% corn starch and/or corn grits and/or rice starch is mashed in the presence of an alpha-amylase. Preferably, the alpha-amylase AMY1 or AMY2 described below is

used in amount of approximately 2 KNU/g DM, preferably in amount of 0.05 to 10 KNU/g DM, more preferable 0.1 to 8 KNU/g DM, even more preferable 0.5 to 5 KNU/g DM. Preferably, the starch is mashed using a temperature profile preferably starting at approximately 52°C, increasing to at approximately 64°C, and mashing off preferably at approximately 78°C or more. A second aspect of the invention is a wort produced by the method described above. In addition to the second aspect of the invention the wort produced by the process of the first aspect of the invention may be fermented to produce an alcoholic beverage, preferably a beer. Fermentation of the wort may include pitching the wort with a yeast slurry comprising fresh yeast, i.e. yeast not previously used for the invention or the yeast may be recycled yeast. The yeast applied may be any yeast suitable for beer brewing, especially yeasts selected from Sac- charomyces spp. such as S. cerevisiae and S. uvarum, including natural or artificially produced variants of these organisms. The methods for fermentation of wort for production of beer are well known to the person skilled in the arts.

The processes of the invention may include adding silica hydrogel to the fermented wort to increase the colloidal stability of the beer. The processes may further include adding kieselguhr to the fermented wort and filtering to render the beer bright.

A third aspect of the invention is a beer produced by the processes of the invention, such a beer may be any type of beer. Preferred beer types comprise ales, strong ales, stouts, porters, lagers, bitters, export beers, malt liquors, happoushu, high-alcohol beer, low-alcohol beer, low-calorie beer or light beer.

ENZYMES

The exogenous enzymes to be applied in the present invention should be selected for their ability to retain sufficient activity at the process temperature of the processes of the invention, as well as for their ability to retain sufficient activity under the moderately acid pH regime in the mash and should be added in effective amounts. The enzymes may be derived from any source, preferably from a plant or an algae, and more preferably from a microorganism, such as from a bacteria or a fungi.

Alpha-amylase (EC 3.2.1.1 )

A particular alpha-amylase enzyme to be used in the processes of the invention may be a Bacillus alpha-amylase, e.g., an alpha-amylase derived from a strain of B. licheniformis, B. amyloliquefaciens, and B. stearothermophilus. A preferred bacterial alpha-amylase is a recom- binant B. stearothermophilus alpha-amylase variant with the mutations; 1181* + G182* + N193F. The variant is shown in SEQ ID NO:2 (AMY1 ). Also preferred are alpha-amylases having an amino acid sequence with least 50% such as at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or particularly at least 99% identity to the amino acid sequence shown in SEQ ID NO:2. Even more preferred for the invention is a bacterial alpha-amylase comprising a starch binding module, preferably a starch binding module of family 20. Such an alpha-amylase may be derived from Bacillus flavothermus (syn. Anoxybacillus contaminans). Most preferred is an alpha-amylase having at least 50% such as at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or particularly at least 99% identity to the amino acid sequence shown in SEQ ID NO:1 (AMY2).

Bacillus alpha-amylases may be added in the amounts of 1.0-1000 NU/kg dm, preferably from 2.0-500 NU/kg dm, preferably 10-200 NU/kg dm.

Another particular alpha-amylase to be used in the processes of the invention may be any fungal alpha-amylase. Particularly preferred are acid fungal alpha-amylases. Especially contemplated are fungal alpha-amylases which exhibit a high identity, i.e. at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or even at least 90% identity to the amino acid sequence shown in SEQ ID NO: 10 in WO96/23874.

Fungal alpha-amylases may be added in an amount of 1-1000 AFAU/kg DM, preferably from 2-500 AFAU/kg DM, preferably 20-100 AFAU/kg DM. Glucoamylases

A further particular enzyme to be used in the processes of the invention may be a glucoamylase (E. C.3.2.1.3) derived from a microorganism or a plant. Preferred are glucoamylases of fungal or bacterial origin selected from the group consisting of Aspergillus glucoamylases, in particular A. niger G1 or G2 glucoamylase (Boel et al. (1984), EMBO J. 3 (5), p. 1097-1 102), or variants thereof, such as disclosed in WO92/00381 and WO00/04136; the A. awamori glucoamylase (WO84/02921 ), A. oryzae (Agric. Biol. Chem. (1991 ), 55 (4), p. 941-949), or variants or fragments thereof. Other contemplated Aspergillus glucoamylase variants include variants to enhance the thermal stability: G137A and G139A (Chen et al. (1996), Prot. Engng. 9, 499-505); D257E and D293E/Q (Chen et al. (1995), Prot. Engng. 8, 575-582); N 182 (Chen et al. (1994), Biochem. J. 301 , 275-281 ); disulphide bonds, A246C (Fierobe et al. (1996), Biochemistry, 35, 8698-8704; and introduction of Pro residues in position A435 and S436 (Li et al. (1997), Protein Engng. 10, 1 199-1204). Other contemplated glucoamylases include Talaromyces glucoamylases, in particular derived from Talaromyces emersonii (WO99/28448), Talaromyces leycettanus (US patent no. Re. 32,153), Talaromyces duponti, Talaromyces thermophilus (US 4,587,215). Bacterial glucoamylases contemplated include glucoamylases from the genus Clostridium, in particular C. thermoamylolyticum (EP 135,138), and C. thermohydrosulfuricum (WO86/01831 ). Preferred glucoamylases include the glucoamylases derived from Aspergillus oryzae, such as a glucoamylase having at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or particularly at least 99%. even at least 90% identity to the amino acid sequence shown in SEQ ID NO:2 in WO00/04136. Also contemplated are the commercial products AMG 200L; AMG 300 L; SAN™ SUPER and AMG™ E (from Novozymes); OPTIDEX™ 300 (from Genencor Int.); AMIGASE™ and AMIGASE™ PLUS (from DSM); G-ZYME™ G900 (from Enzyme Bio- Systems); G-ZYME™ G990 ZR (A. niger glucoamylase and low protease content). Glucoamylases may be added in effective amounts well known to the person skilled in the art.

Cellulase (E.C. 3.2.1.4) The cellulase may be of microbial origin, such as derivable from a strain of a filamentous fungus (e.g., Aspergillus, Trichoderma, Humicola, Fusarium). Specific examples of cellulases include the endo-glucanase (endo-glucanase I) obtainable from H. insolens and further defined by the amino acid sequence of fig. 14 in WO 91/17244 and the 43 kD H. insolens endo- glucanase described in WO 91/17243. A particular cellulase to be used in the processes of the invention may be an endo- glucanase, such as an endo-1 ,4-beta-glucanase. Contemplated are beta-glucanases having at least 90% identity to amino acid sequence disclosed as SEQ ID NO:1 in WO 2003/062409 such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or particularly at least 99%. Commercially available cellulase preparations which may be used include CELLU-

CLAST®, CELLUZYME®, CEREFLO® and ULTRAFLO® (available from Novozymes A/S), LAMINEX™ and SPEZYME® CP (available from Genencor Int.) and ROHAMENT® 7069 W (available from Rohm, Germany).

Beta-glucanases may be added in the amounts of 1.0-10000 BGU/kg dm, preferably from 10-5000 BGU/kg dm, preferably from 50-1000 BGU/kg dm and most preferably from 100- 500 BGU/kg dm. Debranching enzymes

Another enzyme applied in the process of the invention may be a debranching enzyme, such as an isoamylase (E. C. 3.2.1.68) or a pullulanases (E. C. 3.2.1.41 ). lsoamylase hydrolyses alpha-1 ,6-D-glucosidic branch linkages in amylopectin and beta-limit dextrins and can be distinguished from pullulanases by the inability of isoamylase to attack pullulan, and by the limited action on alpha-limit dextrins. Debranching enzyme may be added in effective amounts well known to the person skilled in the art.

Protease

Suitable proteases include microbial proteases, such as fungal and bacterial proteases. Preferred proteases are acidic proteases, i.e., proteases characterized by the ability to hydrolyze proteins under acidic conditions below pH 7.

Contemplated acid fungal proteases include fungal proteases derived from Aspergillus, Mucor, Rhizopus, Candida, Coriolus, Endothia, Enthomophtra, Irpex, Penicillium, Sclerotiumand Torulopsis. Especially contemplated are proteases derived from

Aspergillus niger (see, e.g., Koaze et al., (1964), Agr. Biol. Chem. Japan, 28, 216), Aspergillus saitoi (see, e.g., Yoshida, (1954) J. Agr. Chem. Soc. Japan, 28, 66), Aspergillus awamori (Hayashida et al., (1977) Agric. Biol. Chem., 42(5), 927-933, Aspergillus aculeatus (WO 95/02044), or Aspergillus oryzae, such as the pepA protease; and acidic proteases from Mucor pusillus or Mucor miehei.

Contemplated are also neutral or alkaline proteases, such as a protease derived from a strain of Bacillus. A particular protease contemplated for the invention is derived from Bacillus amyloliquefaciens and has the sequence obtainable at Swissprot as Accession No. P06832 as well as proteases having at least 90% identity to said amino acid sequence, such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or particularly at least 99%.

Further contemplated are the proteases having at least 90% identity to amino acid sequence disclosed as SEQ ID NO:1 in the Danish patent applications PA 2001 01821 and PA 2002 00005, such as at 92%, at least 95%, at least 96%, at least 97%, at least 98%, or particularly at least 99%.

Also contemplated are papain-like proteases such as proteases within E. C. 3.4.22.* (cysteine protease), such as EC 3.4.22.2 (papain), EC 3.4.22.6 (chymopapain), EC 3.4.22.7 (asclepain), EC 3.4.22.14 (actinidain), EC 3.4.22.15 (cathepsin L), EC 3.4.22.25 (glycyl endopeptidase) and EC 3.4.22.30 (caricain).

The proteases are responsible for reducing the overall length of high-molecular-weight proteins to low-molecular-weight proteins in the mash. The low-molecular-weight proteins are a necessity for yeast nutrition and the high-molecular-weight-proteins ensure foam stability. Thus it is well-known to the skilled person that protease should be added in a balanced amount which at the same time allows amble free amino acids for the yeast and leaves enough high- molecular-weight-proteins to stabilize the foam. Proteases may be added in the amounts of 0.1- 1000 AU/kg dm, preferably 1-100 AU/kg dm and most preferably 5-25 AU/kg dm. MATERIALS AND METHODS

Enzymes

AMY1 : An S.stearothermophilus alpha-amylase variant having the amino acid sequence shown in SEQ ID NO:4 in WO99/19467 with the mutations; 1181* + G182* + N193F. AMY2: An Anoxybacillus contaminans alpha-amylase having the amino acid sequence shown in SEQ ID NO:1.

Methods

Proteolytic Activity (AU)

The proteolytic activity may be determined with 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 with trichloroacetic acid (TCA). The amount of TCA soluble product is determined with phenol reagent, which gives a blue color with tyrosine and tryptophan.

One Anson Unit (AU) is defined as the amount of enzyme which under standard conditions (i.e. 250C, pH 7.5 and 10 min. reaction time) digests hemoglobin at an initial rate such that there is liberated per minute an amount of TCA soluble product which gives the same color with phenol reagent as one milliequivalent of tyrosine.

A folder AF 4/5 describing the analytical method in more detail is available upon request to Novo Nordisk A/S, Denmark, which folder is hereby included by reference. Alpha-amylase activity (NU)

The amylolytic activity may be determined using potato starch as substrate. This method is based on the break-down of modified potato starch by the enzyme, and the reaction is followed by mixing samples of the starch/enzyme solution with an iodine solution. Initially, a blackish-blue color is formed, but during the break-down of the starch the blue color gets weaker and gradually turns into a reddish-brown, which 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 which, under standard conditions (i.e. at 37°C +/- 0.05; 0.0003 M Ca2+; and pH 5.6) dextrinizes 5.26 g starch dry substance Merck Amylum solubile.

A folder AF 9/6 describing this analytical method in more detail is available upon request to Novozymes A/S, Denmark, which folder is hereby included by reference.

Glucoamylase activity (AGU)

The Novo Glucoamylase Unit (AGU) is defined as the amount of enzyme, which hydrolyzes 1 micromole maltose per minute at 37°C and pH 4.3.

The activity is determined as AGU/ml by a method modified after (AE L-S M-0131 , available on request from Novozymes) using the Glucose GOD-Perid kit from Boehringer Mannheim, 124036. Standard: AMG-standard, batch 7-1195, 195 AGU/ml. 375 microL substrate (1 % maltose in 50 mM Sodium acetate, pH 4.3) is incubated 5 minutes at 37°C. 25 microL enzyme diluted in sodium acetate is added. The reaction is stopped after 10 minutes by adding 100 microL 0.25 M NaOH. 20 microL is transferred to a 96 well microtitre plate and 200 microL GOD-Perid solution (124036, Boehringer Mannheim) is added. After 30 minutes at room temperature, the absorbance is measured at 650 nm and the activity calculated in AGU/ml from the AMG-standard. A detailed description of the analytical method (AEL-SM-0131 ) is available on request from Novozymes.

Beta-glucanase activity (BGU)

The cellulytic activity may be measured in beta-glucanase units (BGU). Beta-glucanase reacts with beta-glucan to form glucose or reducing carbohydrate which is determined as reducing sugar using the Somogyi-Nelson method. 1 beta-glucanase unit (BGU) is the amount of enzyme which, under standard conditions, releases glucose or reducing carbohydrate with a reduction capacity equivalent to 1 μmol glucose per minute. Standard conditions are 0.5% beta-glucan as substrate at pH 7.5 and 30°C for a reaction time of 30 minutes. A detailed description of the analytical method (EB-SM-0070.02/01 ) is available on request from Novozymes A/S.

Standard Congress mashing process

The standard Congress mashing process was performed according to the procedure of EBC: 4.5.1 Extract of Malt: Congress Mash. The temperature profile consisted of initial mashing temperature of 45°C for 30 minutes, increasing to 70°C with 1.0°C/min for 25 minutes, finalized by 70°C for 65 minutes giving a total mashing period of 2 hours.

Additional methods

Methods for analysis of raw products, wort, beer etc. can be found in Analytica-EBC, Analysis Committee of EBC, the European Brewing Convention (1998), Verlag Hans Carl Geranke-Fachverlag. For the present invention the methods applied for determination of the following parameters were:

Plato: refractometer.

Assimilable N: Based on EBC: 8.10 but with TNBS (2,4,6 trinitrobenzen sul- phonic acid) as reagent instead of ninhydrin. TNBS reacts in a solution of free amino groups or amino acids and peptides, which creates a yellow complex, which is measured spectrophotometric at 340nm. Beta-glucan: EBC: 8.13.2 (High Molecular weight beta-glucan content of wort:

Fluorimetric Method). Color: EBC: 4.7.2

Modification EBC: 4.14 Modification and Homogenity of malt, Calcoflour method Filterability: Volume of filtrate (ml) determination: According to EBC: 4.5.1 (Extract of Malt: Congress Mash) subsection 8.2. Filtration: Filtration volume is read after 1 hour of filtration through fluted filter paper, 320 mm diameter. Schleicher and Schϋll No.597 Y2, Machery, Nagel and Co. in funnels, 200 mm diameter, fitted in 500 ml flasks. pH: EBC: 8.17 (pH of Wort).

Kolbach Index: EBC: 4.9.1 (Soluble Nitrogen of Malt: Spectrophotometric Method) and EBC: 3.3.1 (Total Nitrogen of Barley: Kjeldahl Method (RM)).

Extract recovery: EBC: 4.5.1 (Extract of Malt: Congress Mash, Extract in dry, yield). The term extract recovery in the wort is defined as the sum of soluble substances (glucose, sucrose, maltose, maltotriose, dextrins, protein, gums, inorganic, other substances) extracted from the grist (malt and adjuncts) expressed in percentage based on dry matter. The remaining insoluble part is defined as spent grains. P(M + 800) a) E1 =

100 - P

100 b) E2 =

100 -M where;

E1 = the extract content of sample, in % (m/m)

E2 = the extract content of dry grist, in % (m/m)

P = the extract content in wort, in % Plato

M = the moisture content of the grist, in % (m/m)

800 = the amount of destilled water added into the mash to 100 g of grist

Mash preparation

Unless otherwise stated mashing was performed as follows. The mash was prepared according to EBC: 4.5.1 using malt grounded according to EBC: 1.1. Mashing trials were performed in 500 ml lidded vessels each containing a mash with 50 g grist and adjusted to a total weight of 250±0.2 g with water preheated to the initial mashing temperature + 1 °C. During mashing the vessels were incubated in water bath with stirring. After mashing and before filtration water was added to each vessel to a total of 300 g. After filtration the wort was boiled for 10 minute and diluted 1 :1 with water. To portions of 200 g wort was added 1.2 g yeast and fermentation was performed for 4 days. Examples

Example 1

A grist comprising 65% well modified malt and 35% corn starch was mashed in the presence of an alpha-amylase using the mashing temperature profile consisting of 34 min at 52°C, increase by 1 °C/min for 18 min, 60°C for 60 min, increase by 1 °C/min for 18 min, 78°C for 20 min followed by cooling to 20°C. The wort and young beer was analyzed by HPLC. Results are shown in table 1.

Table 1. Corn starch: Sugar profile, Plato and yield of diluted wort, alcohol of young beer.

Enzyme dosage DP1 DP2 DP3 Ferment. DP3+ Plato Yield Alcohol

KNU/g DM g/ι g/ι g/ι sugar g/l g/i % w/w

- 0,00 9,91 26,39 11 ,57 47,87 28,70 7,47 88,85 2,17

AMY1 0,50 10,21 29,34 15,26 53,81 25,99 7,79 93,29 2,44

AMY1 0,75 10,05 28,83 14,29 53,17 24,21 7,57 90,30 2,46

AMY1 1 ,00 10,26 29,31 14,72 54,28 23,55 7,60 90,68 2,51

AMY1 1 ,50 10,65 30,26 15,39 56,29 22,82 7,72 92,31 2,59

AMY1 2,00 10,71 30,33 15,74 56,78 22,00 7,71 92,28 2,61

AMY2 0,50 9,72 35,42 15,94 61 ,08 21 ,40 8,02 96,65 2,79

AMY2 0,75 9,21 35,32 15,82 60,35 18,56 7,72 92,38 2,78

AMY2 1 ,00 8,97 34,51 15,74 59,22 17,33 7,49 89,16 2,61

AMY2 1 ,50 9,14 36,11 16,76 62,01 16,66 7,70 92,10 2,85

AMY2 2,00 9,31 37,53 17,45 64,29 15,81 7,86 94,30 2,96

Example 2

A grist comprising 65% well modified malt and 35% rice starch was mashed in the presence of an alpha-amylase using the mashing temperature profile consisting of 34 min at 52°C, increase by 1 °C/min for 18 min, 64°C for 60 min, increase by 1 °C/min for 18 min, 78°C for 20 min followed by cooling to 20°C. The wort and young beer was analyzed by HPLC. Results are shown in table 2.

Table 2. Rice starch: Sugar profile, Plato and yield of diluted wort, alcohol of young beer.

Enzyme dosage DP1 DP2 DP3 Ferment. DP3+ Plato Yield Alcohol RDF RDF KNU g/DM g/l g/l g/l sugar g/l g/l % w/w

- 0,00 15, ,50 28,41 13, ,41 57, ,32 14,63 7,25 86, 1 1 2,57 82, ,8 67,2

AMY1 0,50 14, ,70 26,99 13, ,79 55, ,48 12,87 7,52 89,87 2,70 83, ,9 68, 1

AMY1 0,75 15, ,37 28,25 14, ,60 58, ,22 12,79 7,22 85,77 2,54 84, ,4 68,6

AMY1 1 ,00 14, ,33 26,65 13, ,94 54, ,93 1 1 ,69 7,31 86,94 2,56 84, ,9 68,9

AMY1 1 ,50 14, ,32 26,33 13, ,99 54, ,64 1 1 ,08 7,32 87, 14 2,56 85, ,2 69,2

AMY1 2,00 14, ,46 26,39 14, ,34 55, ,18 1 1 ,22 7,41 88,38 2,60 85, ,4 69,4

AMY2 0,50 14, ,23 28,36 14, ,61 57, ,20 1 1 ,01 7,61 91 ,22 2,70 86, ,1 69,9

AMY2 0,75 14, ,41 28,69 15, ,05 58, ,15 10,55 7,49 89,52 2,62 86, ,2 70,0

AMY2 1 ,00 14, ,16 28,67 15, ,13 57, ,96 9,94 7,34 87,38 2,60 87, ,0 70,6

AMY2 1 ,50 14, ,53 29,60 15, ,78 59, ,91 9,53 7,72 92,72 2,76 88, ,1 71 ,5

AMY2 2,00 14, ,76 29,77 16, ,12 60, ,65 9,51 7,55 90,32 2,70 87, ,3 70,8

Wort boiling

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