Enzymes in brewing

Publiceret April 2008

Even for an old industry like beer brewing new industrial processes benefit from using enzymes developed from microbial sources. In the last years quality issues like flavour control, beer stability and general cost savings in the industry go hand in hand with efficient solutions of environmental problems. Future aspects focus on a wider application of enzymes to brew with high amounts of inexpensive raw materials like barley. Alternative beer processes for production of wort and beer with higher productivity and reduced amounts of waste and by-products are under development.

Introduction

Beer and wine are both alcoholic beverages which have been part of our social life for thousands of years. Both beverages are produced by yeast fermentation of sugars. Wine is based on grapes, and beer is traditionally based on barley. The matured grapes already contain the sugars needed for the fermentation, while barley contain starch that has to be broken down to fermentable sugars before the yeast can make alcohol. Therefore, traditional brewing contains and extra step compared with wine-making, namely malting in which enzymes needed for the degradation of starch into fermentable sugars are produced.

Germinating barley kernel
Figure 1: Germinating barley kernel

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

Enzyme production during malting
Figure 2. Enzyme production during malting, ref. Aastrup et al. (2004)

In former days, production of malt was an integrated part of every brewery, but to day most malt is produced outside the brewery in large malt factories, and malt has become a purchased raw material, like other raw materials. This means that the breweries to day are more flexible in the use raw materials, and for that matter for the source of enzymes.

The malt enzymes do have some limitations. They can only work at certain temperatures, pH values etc., and the activities might be too low to do a proper job in proper time. In contrast, commercial exogenous enzymes can be designed to work at preferred temperatures and pH values, to have more enzymatic power, or to express wanted enzyme activities that are not present in malt. Addition of exogenous enzymes at various steps during the brewing process can therefore make brewing easier, faster and more consistent. It gives the brewmasters extra flexibility in the choice of raw materials due to less dependence on malt enzymes, as well as providing opportunity to create new products, which is not possible to make with malt enzymes alone. Also the possibility to improve beer quality by avoiding off-flavours is possible with commercial enzymes. The increasing concern on resources and CO2- emission has also put the use of commercial enzymes within the brewing industry in focus. By the use of exogenous enzymes more can be extracted from the raw materials, more local raw materials can be used, and more unmalted grains can be used, saving significant amounts of energy and transport. 

The brewing process

Traditionally, beer is produced by mixing crushed barley malt and hot water in a mash copper to perform the mashing. Besides malt, other starchy cereals such as maize, sorghum, rice and barley, or pure starch itself, can be added to the mash. These are known as adjuncts.

The standard mashing for pilsner type beer consists of several temperature steps, each favouring different malt enzyme activities. The lowest temperature (45 ºC) is the optimal temperature for cell wall degrading enzymes, β-glucanases. The proteases works best at 52 ºC, the β-amylase best at 63 ºC and the α-amylase at 72°C. The last step in the mashing is inactivation of the enzymes at 78 ºC (Figure 3).

The traditional mashing temperature profile is determined by the temperature optima for the various malt enzymes
Figure 3: The traditional mashing temperature profile is determined by the temperature optima for the various malt enzymes. Larger version

If β-glucan and protein are properly broken down during malting, single temperature mashing at 65-71°C has shown to be sufficient, as in the case of traditional ale brewing.

During mashing the starch is degraded to dextrin and fermentable sugars. α-amylase liquefy the gelatinized starch by hydrolysis of the α-1,4 linkages at random. β-amylases are exo-enzymes which attack the liquefied starch chains resulting in successive removal of maltose units from the non-reducing end. 

After mashing, the mash is sieved in a lauter tun or on a mash filter. The resulting liquid, known as sweet wort, is then transferred to the copper, where it is boiled with hops. The hopped wort is cooled and transferred to the fermentation vessels, where yeast is added. In normal wort 2/3 of the carbohydrates are fermentable sugars. After fermentation, the so-called ‘green beer' is matured before final filtration and bottling. Fig. 4 shows a diagram of the brewing process and where external enzymes are used for process aids.

The processing steps in brewing where exogenous enzymes can be added
Figure 4: The processing steps in brewing where exogenous enzymes can be added. Larger version.

Commercial enzymes from exogenous sources

The traditional source of enzymes used for the conversion of cereals into beer is barley malt. If too little enzyme activity is present in the mash, there will be several undesirable consequences: the extract yield will be too low; wort separation will take too long; the fermentation process will be too slow; too little alcohol will be produced; the beer filtration rate will be reduced; and the flavour and stability of the beer will be inferior.

Exogenous enzymes are used to supplement the malt's own enzymes in order to prevent these problems. Furthermore, industrial enzymes are used to ensure better adjunct liquefaction, to produce low-carbohydrate beer (‘light beer'), to shorten the beer maturation time, and to produce beer from cheaper raw materials.

The various steps of the brewing operations, where microbial enzymes are occasionally added, are shown in table 1. Enzymes, enzymic action and their functions are summarized.

Enzymes at work

Quality and supply constraints on malt, and doubling of malt prices have given increased interest for enzyme solutions in 2007 and 2008. Many breweries has run programs within the last two years in order to increase efficiency and optimize raw material usage, and many of them have focused on commercial enzymes to shorten the production time, increase capacity, and to allow use of raw material alternative to malt. Three important examples are mentioned:

Exchanging part of the malt with barley has been popular because using barley in combination with commercial enzymes gives the same beer quality as with malt.

Introducing a higher content of starch hydrolysing enzymes offer the possibilities of producing "light beer" also called "low calorie beer".

An enzyme solution for diacetyl control after fermentation improves vessel utilization, save energy and ensures a high beer quality after a reduced maturation time.

Operation

Enzymes

Enzyme ac­tion

Function

Decoction ves­sel (cereal cooker)

α-amylase

Hydrolyse starch

Adjunct* lique­fac­tion.
Reduce vis­cosity

β-glucanase

Hydrolyse glucans.

Aid the filtra­tion.

Mashing

α-amylase

Hydrolyse starch.

Malt improve­ment.

Amyloglucosidase

Increase glucose content.

Increase % fermen­table sugar in “light” beer.

Debranching en­zyme

Hydrolyse α-1,6 branch points of starch.

Secures maximum fermentability of the wort.

Proteases

Increase soluble protein, and free amino- nitrogen (FAN).

Malt improve­ment
Improved yeast growth.

β-glucanase

Hydrolyse glucans.

Improve wort sepa­ration.

Pentosanase/xylanase

Hydrolyse pen­tosans of malt, barley, wheat.

Improve extraction and beer filtration.

Fermentation

Fungal α-amylase

Increase mal­tose and glucose con­tent.

Increase % fermen­table sugar in “light” beer.

β-glucanase

Hydrolyze glu­cans.

Reduce viscosity and aid filtration.

α-acetolactate- decarboxylase (ALDC)

Converts α-ace­tolactate to ace­toin directly.

Decrease fermenta­tion time by avoid­ing formation of diacetyl.

Conditioning tank

Protease

Modify protein-polyphenolic com­pounds.

Reduce the chill haze formed in beer.

* Adjunct is starchy cereals such as maize, rice, wheat, sorghum, barley or pure starch materials added to the mash.

Table 1. Steps of the brewing operations where microbial enzymes are used.

Brewing with barley

Traditionally, the use of barley has been limited to 10-20% of the grist when using high-quality malts. At higher levels of barley or using undermodified malts, processing becomes more difficult. In these cases the mash needs to be supplemented with extra enzyme activity if the brewer is to benefit from the advantages of using unmalted barley while still maintaining brewing performance.

Brewers can either add a malt-equivalent blend of α-amylase, β-glucanase and protease at the mashing-in stage or add the enzymes separately as required.

As an example of the production of 6000 litre pilsner type beer from malt, barley and maize grits, the following raw materials, liquefaction - and mashing enzymes can be used:

Raw materials:

Malt

475 kg

 

Barley

475 kg

 

Maize grits

400 kg

Liquefaction enzyme:

Termamyl®BrewQ

0.15 kg

Mashing enzymes:

Ceremix®Plus

0.50 kg

 

Ultraflo®Max

0.20 kg

Termamyl®BrewQ is an enzyme preparation containing a thermophilic α-amylase.

Ceremix®Plus is an enzyme preparation containing β-glucanase, xylanase, α-amylase and protease

Ultraflo®Max is an enzyme preparation containing β-glucanase and arabinoxylanase.

Mashing diagram for barley brewing (an example)
Figure 5. Mashing diagram for barley brewing (an example).

The mashing diagram is shown in figure 5. The maize grits are liquefied separately with help of the α-amylase Termamyl®BrewQ at 96°C for 30 minutes, through a short holding time at 70°C. It is stabilised by approximately 100 ppm Ca++ at a water-to-adjunct ratio of approximately 4:1. Milled malt and barley are mashed-in at a temperature of 50°C. After 30 minutes the adjunct mash from the decoction vessel is added to increase the temperature to 63-66°C. After 60 minutes the mash is heated to hold at 76-78°C until starch-negative (no blue colour is formed with iodine in potassium iodide). Hereafter the wort separation is made in the lauter tun.

Brewing with high amounts (>50%) of barley instead of malt is now possible thanks to the introduction of the new enzyme system Ultraflo®Max (2).

Enzymes to improve fermentation

Small adjustments in fermentability can be achieved by adding amyloglucosidase alone or in combination with debranching enzymes at mashing-in or a fungal α-amylase at the start of fermentation.

To describe to which extent the extracted sugars are fermentable brewers define degree of attenuation, which is synonymously with degree of fermentation or fermentability.

Total fermentable sugar production with different dosages of Attenuzyme® (kg per ton malt) and extended mashing at 63°C
Figure 6. Total fermentable sugar production with different dosages of Attenuzyme® (kg per ton malt) and extended mashing at 63°C

Beer types with very high attenuation ("light beer" or "low calorie beer") are most often produced using amyloglucosidase alone. Extended mashing at 63°C and high dosages of enzymes is necessary to produce extremely high attenuated beer (see figure 6).

Fungal α-amylases are used to produce mainly maltose and dextrins whereas amyloglucosidase produces glucose from both linear and branched dextrins.

Diacetyl control

An important question for brewers is "When exactly is a beer mature?", because this determines when they can "rack" the beer to make way for the next batch. The simple answer to the above question is when the diacetyl level drops below a certain limit (about 0.07 ppm). Diacetyl gives beer an off-flavour like buttermilk and one of the main reasons for maturing a beer is to allow the diacetyl to drop to a level where it can't be tasted.

Diacetyl is formed by the non-enzymatic oxidative decarboxylation of α-acetolactate, which is produced by the yeast during primary fermentation. The yeast removes the diacetyl again during the beer maturation stage by conversion to acetoin, which has a much higher flavour threshold value. In fact, acetoin is almost tasteless compared with diacetyl.

By adding the enzyme α-acetolactate decarboxylase (ALDC) (e.g. Novozymes' Ma­turex) at the beginning of the primary fermentation process, it is possible to bypass the diacetyl step (Figure 7) and convert α-acetolactate directly into acetoin. Most of the α-acetolactate is degraded before it has a chance to oxidise and less diacetyl is therefore formed. This makes it possible to shorten or completely eliminate the maturation period (3) and (4). The brewery enjoys greater fermentation and maturation capacity without investing in new equipment.

The removal of α-acetolactate during fermentation.
Figure 7. The removal of α-acetolactate during fermentation. 

Current enzyme solutions provided by Novozymes A/S

Effective Cereal Cooking

Heat stable enzyme preparations like the α-amylase Termamyl®BrewQ have 200-300 times more liquefaction power than malt. Just 0.25 kg of Termamyl can replace 100 kg of malt. The result is 0.5-2% more extract yield, shorter cooking cycles, no risk of residual starch being carried over into the mashing vessel and more flexibility to change adjunct ratios and types.

Effective Adjunct and Malt Solutions

Brewers who desire raw material cost savings or use of local raw materials may source under-modified malts or increase the ratio of adjunct. The limiting factor is to ensure an adequate complex of enzymatic activities for high-quality wort. The enzyme suppliers offers a range of blended products like Ceremix®Plus to ensure sufficient FAN, extract yield, filterability, and fermentability for high quality index final beers. Cost effective adjunct and malt solutions are made with β-glucanase, xylanase, α-amylase and protease.

Faster Throughput and More Extract

Even with good malts it is possible to achieve 40% longer beer filter cycle runs, 0.5% to 1% more extract, and 0.5% reduced beer losses for more brews per day. The experience with Ultraflo®Max has shown the benchmark for brewhouse and cellar performance should not be an all-malt brew with well-modified malt, but an all-malt brew with well-modified malt and exogenous enzymes like Ultraflo®Max. Faster throughput and more extract are made with β-glucanase and arabinoxylanase.

Optimal Fermentation and Maturation

Maturex® prevents the formation of diacetyl, one of the most common flavour defects in lager beers. Adding Maturex® at the start of the primary fermentation process allows brewers to bypass the rate limiting warm maturation or diacetyl stand after fermentation improving vessel utilization, energy savings and ensuring a high quality index of the final beer. Maturation time can be reduced several days up to 14 days.

Improved Attenuation Control

Controlling fermentability of the wort enables brewers to grow their business by taking advantage of changing consumer trends. Whether it is achieving a consistent attenuation or developing a brand extension with a highly attenuated beer using e.g. Attenuzyme®. Improved attenuation control is made with starch hydrolysing enzyme like α-amylase, amyloglucosidase, pullulanase (debranching enzyme).

Conclusion and future perspectives

To day several brewing groups use exogenous enzymes as a strategic tool to optimize the brewing process and the brewing capacity. More and more breweries also think in enzyme solutions for development of new products.

The role of enzymes in tomorrows brewing industry, we do not know, but a lot of new opportunities are now provided for the breweries. Maybe the future with enzymes will bring:

  • No aging of beer within one year: Exogenous enzymes might prevent development of aging components due to oxidation, keeping the taste of fresh beer for extended time regardless of the storage conditions.
  • Beer made from all-barley with the same taste as from all-malt: An "enzyme package" might completely substitute the endogenous enzymes produced during malting. Elimination of the malting process and less transportation can obviously save a lot of energy and CO2 emission.
  • Re-thinking the way of making beer: An efficient and inexpensive process for production of wort and beer was patented in 2004 (5). The mash liquefaction process was a jet-cooking and application of microbial enzymes. Wort was produced from de-hulled and de-germinated grist. The mash was liquefied using entirely microbial enzymes. The fermentation was made simultaneously with the saccharification. The amount of waste or by-products was reduced significantly.
  • Better waste water control: Water and wastewater management constitutes a practical problem for the brewing industry, and exogenous enzymes can play a significant role in waste water treatment. An overview of significant improvement and operation processes and economic reality was described by Fillaudeau et al. (6).
  • ...or something completely different!

References

(1) Aastrup, Sten, Noel Bautista, Elmar Janser and Kurt Dörreich. "Choice of enzyme solution should determine choice of raw materials and process". Presentation given at World Brewing Conference, San Diego, USA, 2004

(2) Aastrup, Sten, Claudio Visigalli, Jürg Obrecht, Marcel Mischler, Niels Elvig, Stefan Kreisz, "Rethink beer filtration with new xylanase", Paper submitted for publication in Brauwelt (2008) - special filtration issue.

(3) Aunstrup, K. and Olsen, F. Alpha-acetolactate decarboxylase enzyme and preparation thereof. U.S.Patent 4,617,273. (1986)

(4) Rostgaard Jensen, B., Svendsen, I., Ottesen, M. Isolation and characterization of α-acetolactate decarboxylase useful for accelerated beer maturation. Proceedings of the European Brewery Convention Congress, p. 393-400. (1987).

(5) Olsen, H. S. and Bisgaard-Franzen, H. „Beer mashing process", WO 2004/050820 A1.

(6) Fillaudeau, L, Pascal Blanpain-Avet, Georges Daufin, „Water, wastewater and waste management in brewing industries", Journal of Cleaner Production 14 (2006) 463-471.