Enzyme Use in Beverage Production David J Maradyn Staff Scientist – Brewing Customer Solutions Novozymes North America Franklinton, NC

• About Novozymes

• Why are we selling enzymes?

• Basic enzymology

• Industrial enzyme production

• Enzymes used in the distilling industry

• Enzymes used in the wine industry

• Enzymes used in the juice industry

AGENDA

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Enzymes used in the brewing industry Cost effective cereal cooking Faster throughput, more extract Improved attenuation control Fermentation Cost effective adjunct and malt solutions Ondea Pro brewing

Summary – Exogenous enzyme use in brewing Q&A

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Our business is industrial enzymes, microorganisms, and biopharmaceutical ingredients. 47% global market share within industrial enzymes Novozymes’ solutions are used in the production of numerous products such as biofuels, detergents, feed and food. 14% of sales invested in R&D with more than 6,500 patents in place

World leader in bioinnovation

Our business is industrial enzymes, microorganisms, and biopharmaceutical ingredients.

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Our vision has never been more relevant

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Bioinnovation and sustainability

• Bioinnovation replaces chemical ingredients in processes and products

• Bioinnovation reduces the use of raw materials

• Bioinnovation optimizes processes to save energy and water

• Bioinnovation makes many food products healthier

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Practical examples of Bioinnovation across industries

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Improve process efficiency and the quality of the end product while reducing energy consumption with enzymatic process enhancements.

Household care

Agriculture

BioPharma

Leather

Pulp and Paper

Textiles

Wastewater Solutions

Bioenergy

Food and Beverages

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More than 700 products used in 130

countries

Sales and markets

Novozymes R&D A common STRONG technology platform combined with dedicated application teams

Screening Diversity

Characterization

Strain development

Culture collection Microbial screening Cloning Bioinformatics

Molecular modeling Rational protein design Molecular evolution

Assay development Automation HTS

Protein purification Enzyme kinetics Structural

characterization

Bacillus expression system Aspergillus

expression system Upscaling

Beverages

Biofuels

Food

Detergent

Technical

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Industrial enzyme production

What are enzymes and where do they come from ?

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Enzymes are catalysts

• Enzymes are biomolecules that catalyze (i.e., increase the rate of) chemical reactions

• Enzymes are proteins • Enzymes are not GMO … they are not organisms

• However, they may be derived from modified organisms to increase thermal stability, pH stability, co-factor dependence, ect

• In enzymatic reactions, the molecules at the beginning of the process are called substrates, and the enzyme converts them into different molecules, called products

• Almost all processes in a biological cell need enzymes to occur at significant rates

• Enzymes are selective for their substrates and speed up only a few reactions from among many possibilities

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C-domain

Calcium

A-domain

B-domain

Active site

Calcium

Calcium

Calcium

3D model of B. licheniformis α-amylase

Enzymes are proteins

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What keeps an enzyme folded?

Calcium

Sodium

•H-bonds

•van der Waals interactions

•Salt bridges

•S-S bridges (cys-cys)

•Hydrophobic interaction

•Metal ion binding (e.g. Ca2+)

•Ligand (incl. substrate or

substrate analog) binding

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Typical enzymes used in industrial processes

Class Industrial enzymes

Oxidoreductases Peroxidases (Catalases) Glucose oxidases Laccases

Transferases Fructosyltransferases Glucosyltransferases

Hydrolases

Amylases Cellulases Lipases Pectinases Proteases Pullulanases

Lyases Pectate lyases Acetolactate decarboxylases

Isomerases Glucose isomerases Ligases Not used at present

Phytases

Slide No. 15

Catalysts lower the activation energy of chemical reactions

Like all catalysts, enzymes work by lowering the activation energy (ΔG) for a reaction, thus dramatically increasing the rate of the reaction.

Uncatalyzed

Enzyme catalyzed

S: Substrate

ES: Enzyme-Substrate

EP: Enzyme-Product

P: Product

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How enzymes work

Binding specificity! Even when different substrate molecules are present, only those that have the specific complementary shape are able to bind with the enzyme's active site.

Active site

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Enzyme kinetics Enzyme reaction: E + S ↔ ES ↔ E + P

Michaelis-Menten equation:

V = Vmax * [S] Km + [S] Vmax is maximal rate,

i.e. amount of product formed per second Km is Michaelis-Menten constant (substrate concentration where the enzyme reaction rate is 50%

of the maximum rate) [S] = substrate concentration

Kd kcat

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Factors that influence the rate of an enzymatic reaction

1. Enzyme concentration 2. Substrate concentration 3. Temperature 4. pH 5. Inhibition

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Influence of substrate concentration

Saturation of enzyme active sites with substrate (ES complex).

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Inhibition

Competitive inhibition: two similar molecules fight for the same active site on the enzyme

Product inhibition: The reaction products of the enzyme reaction competes with the substrate for the active site

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How are enzymes deactivated? Denaturation due to high temperature, organic solvents,

unfavorable pH, chelating agents (removes e.g. calcium from enzyme)

(auto)-proteolysis by proteases Often seen in prepared industrial enzymes including a

protease and other enzymes or due to protease impurity Auto-proteolysis is a degradation by proteases

Chemical modification most often in the form of oxidation Deamidation

i.e. changes an asparagine to an aspartic acid or a glutamine to a glutamic acid residue

glycation (reaction between certain carbohydrates (such as glucose, maltose and starch) and amino-groups on the protein (lysine, arginine and N-terminal)

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Where and when do we meet enzymes?

The answer is

Everywhere

Anytime

There is no life without enzymes

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Novozymes enzymes

Where do they come from ?

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Production facility (Kalundborg, Denmark)

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Production facility (Franklinton, NC)

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Production organisms

Fungi E.g., Aspergillus niger

Bacteria E.g., Bacillus licheniformis

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Compressor

Mixing of nutrient medium

Water

Inoculation flask

Seed fermenter

Air Sterile filter Fermented broth for recovery of enzyme

Cooling water

Exhaust

Sterilisation

Raw materials

Ground grain/corn

Starch Glucose

Sugar

Soy bean meal

Gluten Corn steep liquor

Phosphates

Sulphates Ammonium salts

Carbohydrates:

Proteins:

Salts:

Fermentation 31

Fermenter

Culture broth

Cooling

Ultrafiltration

Stabilisation

Bacterial filtration

Bacterial

filtration

A

B

Drum filtration Filtration of

enzyme crystals

for granulation

Liquid concentrate

Liquid

product

Pretreatment

Filter aid

is added

Preservatives are added

Crystallisation

Enzyme recovery

Enzyme crystals for granulation

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Granulation

Raw materials

Enzyme crystals

or liquid

concentrate from

the recovery plant

Granulation mixer

Fluid-bed dryer Sifter

Sifter

Hopper

Fibre drums

Coating mixer

Fluid-bed cooler

Granulated final product

Processing aids are added

Big bag

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Production equipment

Fermentors

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Enzymes produced in large quantities

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Enzyme essentials Specific – Natural – Not alive

• Enzymes are specific A particular enzyme works only on a small class of substrates

• Enzymes are natural Enzymes are made by growing microorganisms during fermentation processes

• Enzymes are not alive Although derived from living organisms, enzymes are not alive

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Current main enzyme applications for beverages

Brewing ..

Improved processability

and utilization of raw materials New types of beer

Juice..

Increasing yield and

improving processing

Distilled spirits .

Increased fermentability

of the raw material

Wine ..

Improved processability

and wine quality

Enzymes used in Distilling

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Production diagram for distilled spirits

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Process Step Enzymes Goal

Viscosity Reduction Xylanases, Cellulases, Hemi-cellulases, Beta-glucanases

Significantly lower the viscosity of rye, barley or wheat mashes Increased output by processing at higher DS

Liquefaction Alpha-amylases Liquefaction of gelatinized starch Lower mash viscosity

Saccharification Gluco-amylases, Fungal-amylases

Conversion of dextrins to fermentable sugars Match the desired sugar profile

Fermentation enhancement

Proteases Increase yeast nutrition (protein degradation) Higher alcohol yield + quality

Use of industrial enzymes in the distilling process

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Enzymes used in the wine industry

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Winemaking process: Industrial production of an Australian Popular Premium white wine

6. Settling tanks

7. Fermentation tanks

Maturation

Filtration (enzymes)

Clarification (enzymes)

Extraction Maceration (enzymes)

Pict

ure

cred

its:

Wor

ld A

tlas

of W

ine

– M

. BEA

ZLE

Y.

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Fast and reliable wine filtration

… wine was allowed to clear on its own or was filtered without enzymes. Without filtration, refermentation and sedimentation could make the wine undrinkable. Without enzymes, filtered wine risked losing color and aroma during filtration.

In the old days…

With bioinnovation…

… wine filtration is problem free, smooth, and reliable. In addition, unique enzyme activity also maintains color and aroma that can otherwise be lost during filtration. Vinoflow® G

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Fruitier and richer wine

… white wine clarification and maturation took a long time. There was no way to release more aroma in the wine.

In the old days…

With bioinnovation…

… winemakers can offer consumers richer and fruitier white wine with enhanced aroma. Enzymes enhance aroma by liberating aroma precursors in the wine. They make the wine richer and fruitier by halving the maturation time.

Novarom® Blanc

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The chemistry behind wine color, flavor, and aroma

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. Carbohydrases release color, flavor, and aroma compounds trapped in the polysaccharide matrix.

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Two wine worlds are facing one another …

“New World”: 32% of total volume.

High Enzyme penetration.

Expanding production.

Large wineries dominate. High-end wine production

process. Branded and “Ready to drink

wines”.

“Old World”: 68% of total volume. Low Enzyme penetration. Restructuring and reduction of production. Small wineries dominate. Traditional wine making process. Wines typical from the region of production.

(

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Enzymes used in the juice industry

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Juice business is close to 100% penetrated – you need enzymes to run a profitable juice business

Target customers: - companies that manufacture juices from fruits

Customer needs: - produce storage stable juices at maximized juice yield, and provide maximized value to the consumer.

Markets served: - fruit crushers, drink formulators and service providers

20 fruit crushers produce 80% of the juices worldwide, consolidation

is going on right now and will continue

The juice industry is mature, juices are being traded like a

commodity

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Most of the targeted applications are fruits crushing & mashing and juice stability (depectinisation)

Major enzymes applications in:

Apples & Pears Berries Citrus Tropicals

Major fruits (table + juice, Mn tons, 2005-2006): Oranges: 62.5 Apples: 60.7 Peach: 15.8 Pineapple: 15.3 Pears: 14.3 Lemon: 13.7 Grapefruit: 5.9 Apricot: 2.7 Pomegranate: 1.6 Berries <1.0

80% of the enzyme market!

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Enzymatic essential oil recovery

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… essential citrus oils were recovered through centrifugation during the juice extraction process using water. This process involved large amounts of water, wear and tear on the centrifuge, and multiple cleaning cycles.

In the old days…

With bioinnovation…

… specific enzyme preparations increase the yield of essential oils.

Enzymatic oil recovery improves the performance of the centrifuge, cuts down the number of cleaning cycles, and reduces water consumption.

Citrozym®

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Apples is the key – berries, citrus and tropicals are growing niche markets

Fruit Application segment Benefits offered by enzymes Apples and pears 1st mash treatment Maximized juice yield and press capacity in 1st press

2nd mash treatment Liquefaction of the pomace

Pectin degradation Clear and stable juice

Starch degradation Clear and stable juice

Filtration Easier filtrability i.e. Increase throughput in filtration

Berries Mash treatment and pectin degradation

Maximized juice yield / press capacity + clear and stable juice

Citrus Clarification Clear and stable juice

Cloudy and peal treatment Achieve a stable cloudy juice

Essential oil extraction Facilitate extraction of oils of the peel

Tropical Mash treatment Maximize juice yield and press capacity

Others Sugar conversion

Increase sweetness of juices by converting saccharose into fructose and Glucose

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Enzymes used in the brewing industry

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Use of exogenous enzymes is today standard procedure in many breweries

because exogenous enzymes provide:

•More extraction from raw materials

•Faster processes

•Easier, simpler, and more consistent processes •More flexibility in the choice of raw materials

•More flexibility in the choice of processes

•Improved quality of final products

•More opportunities to create new products

Enzyme applications in use by breweries

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Cold block

Packaging

Attenuation control (Light beer)

Improve wort and beer filterability

Raw material utilization increase extract yield

Raw barley and wheat processing Adjunct processing

(rice and corn)

Brewhouse

Diacetyl control and tank use optimization

Attenuation control (Light beer)

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Guarantee

Innovation Extraction efficiency

Process efficiency

Quality

Fermentation α-acetolactate decarboxylase

Filtration β-glucanase

xylanase

Attenuation control amyloglucosidase, pullulanase,

α-amylase

Adjunct liquefaction thermostable α-amylase

Malt & cereal enhancement β-glucanase, xylanase, amylase protease, etc.

Ondea Brewing pullulanase, α-amylase, protease, β-glucanase, xylanase, lipase

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Get the best beer filtration

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… beer was filtered without enzymes. This meant that the brewer did not get the maximum benefit from the raw materials, even if they were of outstanding quality.

In the old days…

With bioinnovation…

... brewers can produce quality beers from available raw materials supplied with variations in quality.

Enzymatic filtration raises the benchmark for mash separation and beer filtration, providing process predictability and consistency for improved quality and cost-efficiency.

Ultraflo® Max

The chemistry behind enzymatic filtration

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. Breaking up complex carbohydrate structures into smaller, more soluble oligomers leads to lower viscosity and better filterability.

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Extract more, extract better

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... beer was traditionally produced from malted barley. The price of malt has risen tremendously, making it cost intensive to brew and prompting brewers to find alternative ways of brewing a great beer cost-effectively.

In the old days…

With bioinnovation…

... brewers are given great flexibility in their choice of raw materials for brewing. Enzymes make it possible to brew great-tasting, quality beers using nonconventional raw mate-rials as adjuncts like rice, corn, or sorghum. Now, even 100% barley brewing is possible.

Ceremix®

Cerezyme®

Ondea® Pro

Supplementing malt enzymes

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. If no or insufficient malt enzymes are present, carbohydrases and proteases will hydrolyze polysaccharides and proteins into oligomers for easy brewing of quality beer.

Making beer taste right 60

… diacetyl sometimes formed in beer, giving the beer an unappealing buttery taste. Diacetyl forms when beer is improperly fermented and matured.

In the old days…

With bioinnovation…

... there is no risk of diacetyl formation. Enzymes prevent the formation of diacetyl and make beer processing faster and more cost-effective.

Maturex®

The chemistry behind diacetyl removal

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. Acetolactate decarboxylase converts α-acetolactate into acetoin so that diacetyl formation can be avoided.

α-acetolactate

Acetolactate decarboxylase

Cost Effective Cereal Cooking

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Cost-effective cereal cooking Thermostable α-amylase

Termamyl SC Added at start of liquefaction

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Substrate: starch

Amylose: •α-1,4 glycosidic bond (~100%) •10–30% typical composition

Amylopectin: •α-1,4 glycosidic bond (94–95%) •α-1,6 glycosidic bond (5–6%) •70–90% typical composition

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What does α-amylase do?

α-amylase

Shorter dextrin chains

Large dextrin chains

DP6 DP4 DP3 DP2

α-amylase randomly cleaves starch (large dextrins) to form a mixture of smaller dextrin chains (polymers of glucose)

GOALS: Dextrinization Viscosity reduction

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Importance of enzyme thermostability in starch conversion

The activity of malt α-amylase starts to decline at 67 ºC

Thermostable α-amylases work better at 85–95 ºC

Barley, wheat, corn, rice gelatinization temperature range: 61–78 ºC

Starch gelatinization temperatures

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Raw material Starch content wt% “as is”

Gelatinisation temp., °C

Barley 54 - 65 53° - 63° Maize (Corn) 60 - 63 68° - 74° Maize (Corn) Grits 71 - 74 62° - 75° Maize (Corn) Starch 71 - 74 62° - 74° Manioc / Cassava 20 - 30 51° - 65° Manioc / Tapioca Meal 65 - 80 51° - 65° Oats 40 - 63 55° - 62° Potato 15 - 20 54° - 69° Potato Starch 65 - 85 56° - 69° Rice 65 - 70 65° - 75° Rice Grits 57 - 88 61° - 78° Rye 55 - 62 55° - 70° Sorghum (Milo) 55 - 65 70° - 78° Sorghum (Milo) Grits 70 - 74 68° - 75° Triticale * 63 - 69 55° - 70° Wheat 58 - 62 58° - 65° Wheat Starch 67 - 69 52° - 75° Barley Malt 35 - 56 61° - 65°

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When starch liquefaction is insufficient, there is a risk of producing retrograded starch

Retrogradation occurs when the amylose chains bind together in

helical and double helical coils Retrograded starch...

Does not give the typical blue iodine reaction Cannot be hydrolyzed enzymatically Remains in spent grains, resulting in loss of extract (up to 3%) Slows down mash filtration (viscosity) In the beer causes haze and filtration problems

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Fast and consistent liquefaction process

Lower adjunct mash viscosity means easier handling

No danger of resistant/retrogradated starch formation and

insufficient saccharification

Reduced processing costs through more efficient liquefaction and increased yield from adjuncts

Improved flexibility in using various types of adjuncts

Cost-effective cereal cooking – Main benefits of using thermostable α-amylases

Faster Throughput More Extract

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Faster throughput and more extract – β-glucanase, xylanase

Ultraflo Max added at start of mashing

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Substrate: cell walls

Barley cell walls stained with Calcofluor

Barley cell wall model of Bamforth et al.

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β-glucanases

β-glucan Long molecules of mixed linked 1,3–1,4-glucose

Endoglucanase (β-glucanase, cellulase) Endo-β-1,3(4)-glucanase

-1,4- -1,3 -1,4- -1,4- -1,4-

β-glucosidase Glucose

Cellobiohydrolase

The action of β-glucanases

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Xylose

Arabinoxylanases

Arabinose Ferulic acid Acetyl Glucoronic acid

Endoxylanase (GH11) Endoxylanase (GH10)

The action of xylanases

Arabinoxylan Long molecules of xylose backbones with arabinose

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Insufficient breakdown of cell walls creates problems in brewing

β-glucans and xylans: Make starch and proteins less available for

enzymatic hydrolysis Are big molecules leading to high wort and beer

viscosity Have high water-binding capacity

Therefore they:

Make wort and beer filtration processes much more difficult

May also prevent optimal extraction

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New generation of filtration enzymes

Showing unique wort viscosity reduction

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New benchmark for brewhouse performance has been formulated

No longer all-malt brew with well modified malt, but All-malt brew with well modified malt and exogenous enzymes

Higher extract yield – less loss

Shorter wort separation time

Longer beer filtration cycles

Consistent brewhouse performance with varying malt quality

Faster throughput and more extract – Main benefits of using enzymes

Improved Attenuation Control

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Improved Attenuation Control What is Attenuation in brewing?

A measure of the degree to which sugar in wort has been fermented into alcohol in beer

It is typically measured as ADF or RDF

ADF :Apparent Degree of Fermentation The original gravity (OG) of the wort prior to fermentation The apparent extract (AE) of the beer after fermentation is complete

AE is the final specific gravity of beer converted to degrees Plato. This measurement does not take into account the lower density of alcohol compared to water

RDF :Real Degree of Fermentation The original gravity (OG) of the wort prior to fermentation The real extract (RE) of the beer after fermentation is complete

RE is The final gravity of the beer, converted to degrees Plato and corrected to account for the lower density of alcohol compared to water

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Improved Attenuation Control Why attenuation control is important?

Degree of starch conversion has a direct impact on

several brewing parameters Beer specifications

alcohol content residual extract calories

Brewing process

Extract yield Breaking down more starch more

consistently

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Improved Attenuation Control The production of highly attenuated beers is typically

based on: Increasing the release of fermentable sugars from starch

(amylose and amylopectin) Diluting the excess of alcohol formed during

fermentation with water • As a result, highly attenuated beers will typically

have lower alcohol, residual extract, and calories compared to their regular beer versions

Amylose

Amylopectin

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What does glucoamylase do?

Glucoamylase generates simple sugars that the yeast can use in the fermentor

Long-chain sugars (dextrins)

Simple sugars

Improved Attenuation Control

Why is it so important to make highly attenuated beer / light beers?

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Improved Attenuation Control

•Following the consumer trend towards wellness requires highly attenuated beers with light and low calorie claims

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Facts on Attenuation Control Worts produced under normal brewing conditions generate

attenuation up to 75% Traditional brewing methods can not normally compensate for

the lack of enzymes necessary to attenuate wort over 75%

Approximately 25 % of the carbohydrate extract is present in the beer as non-fermentable short chain dextrin

Difficult to achieve predictable and targeted attenuation levels consistently due to variations in quality of raw materials

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First generation AMG 300L

Glucoamylase activity Fungyl alpha-amylase activity Non-GMO derived

Second generation Attenuzyme

Glucoamylase activity Fungyl alpha-amylase activity Derived from GMO More heat-stable, broader pH-activity range

Third generation Attenuzyme Flex

Glucoamylase activity Fungyl alpha-amylase activity Pullulanase activity Derived from GMO

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Improved Attenuation Control Product Solutions

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Improved attenuation control – Light beer – Brand building

Amyloglucosidase (glucoamylase), pullulanase, amylase

Attenuzyme Flex Added at the start of mashing or in special treatment tank

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Model of the effective breakdown of starch molecules by Attenuzyme® Flex components

Glucoamylase

Pullulanase

Starch or dextrin Glucose Maltose

α-amylase

The three enzyme components of Attenuzyme® Flex ensures a fast, effective breakdown of the starch molecules into fermentable sugars

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A broad range of attenuation targets can be achieved with Attenuzyme® Flex even at very short mashing times

Attenuzyme Flex delivers the highest attenuation targets even at very short conversion times. This makes Attenuzyme Flex a unique choice for production of a wide range of beer brands

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Possibility to make super-attenuated beers

Cost-effective way to make light beers

Cost-effective way to keep consistent fermentabilty with varying

malt quality, e.g., keep 70% RDF

Improved attenuation control – Main benefits of using enzymes

Fermentation

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Fermentation Diacetyl (2,3-butanedione) is arguably the most

important flavor characteristic of beer during maturation 2,3-Pentanedione acts in a similar way, although

with a much higher taste threshold These two compounds are considered together and

referred to as vicinal diketones (VDK’s) The breakdown of these compounds is considered

essential criteria for the state of beer maturation

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Fermentation 93

Fermentation

VDK reduction is accomplished by Increasing temperatures at the end of

fermentation Diacetyl rest

Extending maturation Depending on adjunct ratio, yeast type, physical

environment Time and temperature requirements for VDK

reduction are not easily predicted High barley ratios and use of sugars as adjunct can

lead to increased VDK formation by yeast

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Fermentation Alpha-acetolactate decarboxylase enzyme solution

by Novozymes Maturex® 2000L

Breaks down alpha-acetolactate directly into acetoin Avoids the formation of diacetyl Will not affect any diacetyl already formed

Added to cold wort at the beginning of fermentation Prior to pitching of yeast

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Fermentation

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Fermentation Maturex® allows brewers to

Shorten, or even bypass, rate-limiting warm maturation (diacetyl-rest)

Optimize vessel use Reduce energy consumption Maintain high quality index of final beer

Dosages can be adjusted to meet VDK specification at the same time as final attenuation is achieved

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Enzyme dosage response of Maturex® 2000L on the level of total diacetyl in beer

0 1 2 3 4 5 6 7 0.0

1.0

2.0

3.0

Diacetyl (mg/L)

0 1 2 3 4 5 6 7

Fermentation (days)

0 ADU/L

200 ADU/L 100 ADU/L

40 ADU/L

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Fermentation

0

0.1

0.2

0.3

0.4

0.5

0.6

0 20 40 60 80 100 120 140 160 180 200

Dia

cety

l (pp

m)

Time (hr)

Without Maturex ®

With Maturex ®

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Without Maturex® 2000 L

0 7 14 21

123456781

Tank

Days

Cleaningto FilterLagering -1°CCoolingLagering 7°C

With Maturex® 2000 L 0 7 14 21

1

3

5

7

1

3

Tank

Days

Cleaningto FilterLagering -1°CCoolingLagering 7°C

Output to filter plant:

8 CCV of 5,000 hl in 8 days

40,000 hl to beer filter

Output to filter plant:

8 + 3 CCV of 5,000 hl in 8 days

55,000 hl to beer filter Increase in capacity by 1/3

● Increased tank utlilization with Maturex®

Fermentation 100

Fermentation Dosage

0.75 to 1.50 g/hL pH dependence

Activity strongly pH dependent Activity at pH 5.0 3X higher than at pH 4.0

pH should be monitored during fermentation and controlled if needed Ensure diacetyl control at lowest treatment cost

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Fermentation Benefits

Cost savings Shorter production cycles Energy savings through cooler fermentations Enables increased adjunct ratios for raw material savings

without extending diacetyl rest Reduced capacity costs for new breweries through

improved utilization

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Fermentation Optimum throughput

Meet peak season capacity demands If capacity not an issue, apply time savings to extend cold

aging period for better cold break formation, and longer beer filtration cycle

Brand management Never release beer with detectable levels of diacetyl Increased flexibility to choose yeast types for maximum

flavor development

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Cost Effective Adjunct and Malt Solutions

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Cost-effective adjunct and malt solutions β-glucanase, xylanase, protease, amylase

Ultraflo Max, Neutrase®, Termamyl® SC, Attenuzyme® Flex

Alternatively Ceremix® Plus MG

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The enzyme blends can to a large extent substitute malt enzymes

Undermodified malt can perform similar to well modified malt

High flexibility in use of raw materials

High percentage of adjunct can be used

Cost-effective production can be achieved without jeopardizing

production efficiency and final product quality

Cost-effective adjunct and malt solutions – Main benefits of using enzymes

Ondea® Pro Brewing

Introduction Brewing with increased amount barley is primarily driven by the

lower cost of barley compared with malt Other drivers includes

Reduction of the CO2 emission Use of local raw materials

Barley is usually added by mashing-in of a limited percentage of barley, up to 30%

The Novozymes Ondea® Pro concept enables for brewing of 100% barley (or combinations of barley and adjuncts) Where the barley wort can be blended with a malt-wort to

produce beer or be sold as is

Or Barley and malt can be mixed together (varying ratios) in

the mash-tun to produce a barley/malt wort to produce beer

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109 Ondea Brewing β-glucanase, xylanase, protease, amylase,

pullulanase, lipase

Ondea® Pro

Barley enzymes and malt enzymes

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Barley enzymes and Ondea® Pro

111 So… Novozymes Ondea® Pro brewing can be regarded as a fusion of malting and mashing

Attenuation is ensured through a synergetic action between Endogenous barley beta-amylase Exogenous alpha-amylase and

pullulanase Wort amino acid profile resullts from

the synergistic action between Endogenous barley exo-

peptidases Exogenous added protease

Good lautering with a clear wort is ensured via a combination of well adjusted mills and … A filtration enzyme system

including both beta-glucanase and xylanase components

A lipase to ensure the wort clarity

Summary of the Ondea® Pro Enzymatic Solution for Brewing with 100% unmalted barley

The synergies between the enzymes are ensured through A three-step infusion mashing

profile (above) Wort pH of 5.6-5.8

Time

54 °C / 30 min

64 °C / 45-75 min

78-82 °C / 10 min

Temperature

Typical Mashing Profile

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The Attenuation Components Provide a Typical RDF … but with a Very High Maltose Content

High-maltose wort High RDF

Ondea ® Pro at 2 kg/t barley gives around 70% RDF Maltose dominates the sugar

profile

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0 1 2 3

RDF

Maltose and RDF as Function of the Enzyme Dose

Maltose % RDF%kg Ondea Proper ton barley

Mal

tose

con

c in

%

Sugar profile

Glucose 5.0 %

Fructose 1.5 %

Maltose > 60 %

Maltotriose < 15 %

Dextrins (DP4+) < 20 %

Low glucose level leafs the opportunity to add syrup and still create a “maltose” profile

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Different barley qualities requires different enzymes dosages

For each barley variety there is linear correlation between the enzyme dose and amount of non-fermentable dextrin’s

Different barley varieties do therefore appear as straight lines when then the amount of non-fermentable dextrin’s is plotted against the enzyme dose

1516171819202122232425

1 1.2 1.4 1.6 1.8 2

% DP4+

kg Ondea Pro per t grist

Effect on DP4+ of different Ondea Pro dosages applied at two barley varaities

Barley ABarley B

Ondea® Pro has been tested with over 100 different barley samples collected around the world (as of April 2011) It works on approx 95% of the

tested varieties The few barley where it fails are

not suited for malting

The observed variation in generated FAN is well correlated to the protein content We observe some variation in

needed enzyme dose to reach the target attenuation The reason for this variation is

under investigation It is not correlated to the β-

amylase

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Synergy with barley peptidases provides good yeast performance

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10111213

0 2 3 4

mg

FAN

/l/P

lato

Kg Ondea® Pro per ton barley

Without inactivation With preinactivation of barley enzymes

Novozymes Ondea® Pro’s proteolytic component works in synergy with the endogenous enzymes from barley This is demonstrated by a comparison between the effect of Ondea® Pro on

barley with and without inactivation of the endogenous enzymes.

NOTE: pre-inactivation of the endogenous proteolytic enzymes by a heat treatment at 75°C for 30 minutes

Barley wort needs only 9 mg FAN/l/Plato to secure a good fermentation

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Barley wort produced with Novozymes Ondea® Pro has significantly higher amount of fast absorption amino acids

Amino acids are divided into 4

groups according to their ease of absorption by the yeast cell

The amino acid profile from 100% barley-wort differs from malt-wort

Barley-wort contains relatively more of the easy fermentable amino acids (Groups A and B) and relatively less of the less fermentable amino acids (Groups C and D) - especially proline

This explains the good fermentability of the barley wort, despite the lower FAN than in malt wort

Having an amino acid profile more suitable for the yeast leads to less amino acid based Strecker aldehydes - and in turn improved flavor stability!

0%

20%

40%

60%

80%

100%

Malt wort Barley wort

DCBA

Group A Group B Group C Group D Fast Intermediate Slow Little or no absorption absorption absorption absorption Glutamic acid Valine Glycine Proline Aspartic acid Methionine Phenylalanine Asparagine Leucine Tyrosine Glutamine Isoleucine Tryptophan Serine Histidine Alanine Threonine Lysine Arginine

Relative Amino Acid Compositions of Malt and Barley worts

116

FAN level and FAA profile of barley and malt wort in pilot and industrial scale

Pilot Scale Trial Industrial Scale Trial

8 hl Barley MaltFAN (12 %) mg/l 114 174FAA CompositionGroup A 43.4% 32.7%Group B 27.0% 21.7%Group C 20.6% 18.2%Group D 9.0% 27.3%

300 hl Barley MaltFAN (12 %) mg/l 150 216FAA CompositionGroup A 38.8% 30.1%Group B 23.8% 20.9%Group C 23.6% 21.1%Group D 13.8% 27.8%

• Barley-wort versus malt-wort • Pilot: 70% Group A and B versus 54% Group A and B FAA • Industrial: 63% Group A and B versus 51% Group A and B FAA

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0

100

200

300

0 24 48 72 96

FAN

/ m

g/

l

Fermentation time /hr

FAN consumption during fermentation

100% Barley 100% Malt

100 % barley wort provides comparable fermentation performance with less FAN

020406080

0 24 48 72 96 120

% R

DF

Fermentation time/hr

RDF development during fermentation

100 % barley 100% malt

•The FAN recommendation for malt-wort is 20-220 mg/l (at 12 oP) or 10-18 mg/l/Plato • The total FAN from 100% barley-wort is lower than malt-wort

•108-170 ml/l (at 12 oP) or 9-14 mg/l/Plato • However, barley-wort has superior fermentability • This leads to less unfermented FAN at the end of the fermentation

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Barley-wort has a better amino acid profile for yeast growth, leading to formation of 1/3 less Strecker aldehydes

Amino Acid Strecker Aldehydes Flavor Methionine Methional Potato Phenylalanine Phenylacetaldehyde Sweet, green, floral Leucine 3-Methylbutanal Malty, burnt Isoleucine 2-Methylbutanal Malty

Wort aroma components from trials in µg/kg Strecker Aldehydes Pilot Plant (8 hl) Industrial (300 hl)

Barley Malt Barley Malt 3-Methylbutanal 31 115 99 230 2-Methylbutanal 11 51 38 110 Methional 13 12 19 93 Phenylacetaldehyde 56 142 85 174 Total Strecker aldehydes

109 325 245 607

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Increasing the barley inclusion in mashing leads to an increased viscosity, which cannot be solved by traditional filtration enzymes

The filtration components in Ondea® Pro keeps the viscosity low (even at 100% barley inclusion) and ensures a good extract yield, via Added beta-glucanase activity A viscosity reducing xylanase

1

1.5

2

2.5

3

3.5

50 60 70 80 90 100

mP

a*

s

% Barley

Wort viscosity as function of the barley inclusion

no enzyme

2 kg/t Novozymes Ondea® Pro

Filtration and extract components ensure low wort viscosity and high extract yields

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Barley milling in combination with lauter tun and mash filter

Mashfilter in combination with hammer-mill The fine grist of the

hammer mill is preferable for the enzyme system in Ondea Pro

The wort separation procedure needs no changes. Filtration speed and turbidity showed the same, or improved ,performance when compared with all malt brews.

Lauter tun in combination with roller mill A barley kernel is less friable, and the endosperm is more closely connected to the husk – compared to malt Milling barley with the same roller setup used for malt will yield a much more coarse grist composition Six and four roller mills are preferable

However, successful industrial trials have been completed with two roller mills

Optimized barley milling showed the following:

Optimized barley grist composition

Barley Malt

Sieve 1 25 -30% (18%)

Sieve 2 15-20% (8%)

Sieve 3 33-40% (33%)

Sieve 4 10-15% (21%)

Sieve 5 2-5% (10%)

Bottom 8-12% (11%)

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14% 13%

45%

14%

5%

8%

40%

15%

30%

8%

2%

6%

0%

5%

10%

15%

20%

25%

30%

35%

40%

45%

50%

0 1 2 3 4 5 6

Per

cen

tag

e o

f fr

acti

on

Sieve

Differences in grist composition of malt and barley for effective lautering

Malt Barley

122

Barley Milling

The concept has been tested in a number of pilot and production brews globally The majority of products produced were good-tasting beers – no defects The resulting sensory profiles have been different reflecting the brewmaster’s

optimization The barley beer taste can be optimized to resemble taste of a malt based beer

Flavor of 100% Barley Beer

Sensory panel comparison of a malt and a similar barley based pilsner

Taste panel executed by: Centre for Malting and Brewing Science at K.U.Leuven

0

1

2

3

4

5Intensity

Sulphury

EsteryFruity

Malty/Grainy *

Flavour Characteristics

Malt Barley

0123456

Overall Score *

Sweetness *

BitternessMouthfullness

Aftertaste *

Mouthfeel - Overall Score

Malt Barley

123

Beer quality

124

Ondea® Pro pilsner is similar to all-malt pilsner wrt Plato, RDF, gravity, alcohol, haze, pH, CO2, foam, etc. …..and most importantly…TASTE

Beer Made from Barley versus Malt: Sensory and Analytical Aspects

125

Beer Made from Barley versus Malt: Sensory and Analytical Aspects

126

Beer Made from Barley versus Malt: Sensory and Analytical Aspects Comments Beer made with barley (Novodog) quite similar to

beer made with malt (Bulldog) No flavor defects in either beer noted

Differences Novodog more astringent and drying than Bulldog Hop aroma more pronounced in Bulldog than Novodog Bulldog more malty than Novodog

127

GO DIRECTLY FROM BARLEY TO BEER USING

NOVOZYMES ONDEA® PRO

Save up to 3,000 t CO2 per year for every one million hl beer

produced from barley

128

8 % CO2 REDUCTION AT BREWERY 8 GRAM CO2 REDUCTION PER 33 CL

Barley

Malthouse

Brewery

Beer

Ondea® Pro Brewing - Positive impact on the environment

Conclusions A enzyme solution – Ondea® Pro - and process have been

developed to enable the brewing of great-tasting beer with up to 100% unmalted barley with existing brewing equipment

Ondea® Pro works in synergy with the endogenous enzymes from barley

The pullulanase component found in Ondea® Pro is essential for the attenuation performance, producing a high-maltose wort

The amino acid composition of 100% unmalted barley wort differs from malt based wort higher group A and B amino acids and lower proline content

Effective lautering and filtration is ensured through the enzyme system, milling adjustment and lauter tun management

Flavor of 100% unmalted barley beer is similar to 100% malted barley beer

129

130

Novozymes Ondea® Pro brewing

Any beer type can be made using Novozymes Ondea® Pro brewing – Just substitute pilsner malt with barley + Ondea® Pro

131

Summary – exogenous enzyme use in brewing

Enzymes are natural products, without which the brewing of beer would not be possible By adding exogenous enzymes, you facilitate the process, secure quality, make it shorter and more consistent

Enzymes can help your beer achieve quality targets you could not attain without

NOVOZYMES PRESENTATION 07/02/2012 132

Summary – exogenous enzyme use in brewing

Cost-effective cereal cooking Liquefy starch of adjuncts and allow homogeneous mash

Cost-effective adjuncts & malt solutions Use diversified adjuncts and malt qualities with consistent wort quality delivered

Faster throughput and more extract Maximize the extract and throughput time

Optimal fermentation & maturation time Skip ”warm maturation” step and save time & energy

Improved attenuation control Reach high level of fermentable sugars to allow light beer

production Ondea Brewing

Technological breakthrough of using up to 100% unmalted barley to produce a great tasting beer

CONTACT INFORMATION: DAVID MARADYN STAFF SCIENTIST, BREWING CUSTOMER

SOLUTIONS 77 PERRY CHAPEL CHURCH ROAD PO BOX 576 FRANKLINTON, NC 27587 PH: 919-494-3280 MOBILE: 919-339-6232 EMAIL: DVMY@NOVOZYMES.COM

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FOR YOUR ATTENTION A

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