Enzyme Use in Beverage

Production
David J Maradyn
Staff Scientist – Brewing Customer Solutions
Novozymes North America
Franklinton, NC
2

AGENDA
• 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

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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
4

World leader in bioinnovation

Our business is industrial enzymes, microorganisms, and

biopharmaceutical ingredients.

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
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Our vision has never been more relevant

6

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
.
 7

Practical examples of
Bioinnovation across industries

Food and Beverages Household care

Improve process
efficiency and the
Bioenergy
 . of the end
quality
Agriculture
product while
reducing energy
consumption with
enzymatic process
enhancements.
Wastewater BioPharma
Solutions

Textiles Leather

Pulp and Paper

Sales and markets

 More than 700 products used in 130

countries
9

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

Diversity Screening

Beverages

 Culture collection  Molecular modeling  Bacillus expression Biofuels

 Microbial screening  Rational protein design system
 Cloning  Molecular evolution  Aspergillus
 Bioinformatics expression system
 Upscaling Food
Characterization

Strain development
Detergent
 Protein purification
 Enzyme kinetics
 Structural
characterization Technical
 Assay development
 Automation
 HTS
10

Industrial enzyme
production

What are enzymes and

where do they come from ?
11

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
12

Enzymes are proteins

C-domain
Calcium
Calcium

A-domain
Active site

Calcium

B-domain Calcium

3D model of B. licheniformis α-amylase

13

What keeps an enzyme folded?

•H-bonds
•van der Waals interactions
•Salt bridges
•S-S bridges (cys-cys)
•Hydrophobic interaction
Calcium
•Metal ion binding (e.g. Ca2+)
•Ligand (incl. substrate or
substrate analog) binding

Sodium
Slide
No. 15

Typical enzymes used in industrial processes

Class Industrial enzymes

Peroxidases (Catalases)
Oxidoreductases Glucose oxidases
Laccases
Fructosyltransferases
Transferases
Glucosyltransferases
Amylases
Cellulases
Hydrolases Lipases
Pectinases
Proteases
Pullulanases
Phytases

Lyases Pectate lyases

Acetolactate decarboxylases

Isomerases Glucose isomerases

Ligases Not used at present
16

Catalysts lower the activation energy

of chemical reactions

Like all catalysts,

enzymes work by Uncatalyzed
lowering the activation
energy (ΔG) for a
reaction, thus
dramatically increasing Enzyme catalyzed
the rate of the
reaction.
S: Substrate
ES: Enzyme-Substrate
EP: Enzyme-Product
P: Product
17

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
18

Enzyme kinetics

 Enzyme reaction: E + S ↔ ES ↔ E + P
Kd kcat

 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

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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
20

Influence of substrate concentration

Saturation of enzyme active sites

with substrate (ES complex).
21
22
23
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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
25

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)
26

Where and when do we meet enzymes?

 The answer is

Everywhere
Anytime
There is no life without enzymes
27

Novozymes enzymes

Where do they come from ?

28

Production facility (Kalundborg, Denmark)

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Production facility (Franklinton, NC)

30

Production organisms

Fungi Bacteria
E.g., Aspergillus niger E.g., Bacillus licheniformis
31

Fermentation
Water
Raw materials
Mixing of
nutrient medium
Carbohydrates: Exhaust
Ground grain/corn
Sterilisation
Starch
Glucose
Sugar
Cooling water
Proteins:
Soy bean meal
Gluten
Corn steep liquor

Salts:
Inoculation flask
Phosphates
Sulphates
Ammonium salts
Seed fermenter

Air Sterile filter Fermented broth for

recovery of enzyme
Compressor
32

Enzyme recovery
Fermenter Pretreatment
Preservatives
Filter aid are added
is added

Liquid
product
Bacterial
Culture filtration
Ultrafiltration
broth
Stabilisation
Liquid
concentrate
Cooling Crystallisation for granulation

A
B
Enzyme crystals for
granulation
Drum filtration Filtration of
Bacterial filtration
enzyme crystals
33

Granulation
Raw materials

Enzyme crystals
or liquid
concentrate from Granulation mixer Processing aids
the recovery plant are added
Fluid-bed dryer
Sifter

Hopper

Coating mixer

Fluid-bed cooler Fibre drums

Big bag
Sifter

Granulated final product

34

Production equipment

Fermentors
35

Enzymes produced in large quantities

36

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
Current main enzyme applications for
beverages

Distilled spirits . Wine .. Juice.. Brewing ..

 Increased fermentability  Improved processability  Increasing yield and  Improved processability

of the raw material and wine quality improving processing and utilization of raw
materials
 New types of beer
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Enzymes used in
Distilling
39

Production diagram for distilled spirits

40

Use of industrial enzymes in the distilling

process

Process Step Enzymes Goal

Viscosity Reduction Xylanases, Cellulases,  Significantly lower the viscosity of

Hemi-cellulases, rye, barley or wheat mashes
Beta-glucanases  Increased output by processing at
higher DS
Liquefaction Alpha-amylases  Liquefaction of gelatinized starch
 Lower mash viscosity

Saccharification Gluco-amylases,  Conversion of dextrins to

Fungal-amylases fermentable sugars
 Match the desired sugar profile
Fermentation Proteases  Increase yeast nutrition (protein
enhancement degradation)
Higher alcohol yield + quality
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Enzymes used in the

wine industry
 42

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

Clarification
(enzymes)

Extraction
Maceration
(enzymes)
6. Settling tanks

Maturation

Picture credits: World Atlas of Wine – M. BEAZLEY.

Filtration
(enzymes)

7. Fermentation
tanks
43

Fast and reliable wine filtration

In the old days…

… 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.

With bioinnovation…
… wine filtration is problem free, smooth, and
reliable.

In addition, unique enzyme activity also

maintains color and aroma that can otherwise
Vinoflow® G be lost during filtration.
44

Fruitier and richer wine

In the old days…

… white wine clarification and maturation took a
long time.

There was no way to release more aroma in the

wine.

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
Novarom® Blanc richer and fruitier by halving the maturation
time.
 45

The chemistry behind wine color, flavor, and aroma

 .

 .
Carbohydrases release
color, flavor, and aroma
compounds trapped in
the polysaccharide
matrix.
 46
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
48

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 fruits (table + juice,

 Major enzymes Mn tons, 2005-2006):
applications in:  Oranges: 62.5
 Apples & Pears  Apples: 60.7  80% of
 Peach: 15.8 the enzyme
 Berries market!
 Pineapple: 15.3
 Citrus  Pears: 14.3
 Tropicals  Lemon: 13.7
 Grapefruit: 5.9
 Apricot: 2.7
 Pomegranate: 1.6
 Berries <1.0
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Enzymatic essential oil recovery

In the old days…

… 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.

With bioinnovation…
… specific enzyme preparations increase the
yield of essential oils.

Enzymatic oil recovery improves the

performance of the centrifuge, cuts down the
Citrozym® number of cleaning cycles, and reduces water
consumption.
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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  Maximized juice yield / press capacity + clear and stable
degradation 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
53

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

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Enzyme applications in use by breweries

Brewhouse
Raw barley and wheat
Adjunct processing processing
(rice and corn)
Raw material utilization
increase extract yield

Attenuation control Improve wort and

(Light beer) beer filterability
Cold block

Diacetyl control and

tank use optimization

Attenuation control
(Light beer)

 Packaging
Guarantee Process efficiency
55
Fermentation
α-acetolactate
decarboxylase

Filtration
β-glucanase
xylanase

Quality
Attenuation control
amyloglucosidase, pullulanase,
α-amylase

Malt & cereal enhancement

β-glucanase, xylanase, amylase protease, etc.
Ondea Brewing
pullulanase, α-amylase, protease,
β-glucanase, xylanase, lipase
Adjunct liquefaction
thermostable α-amylase

Innovation Extraction efficiency

56

Get the best beer filtration

In the old days…

… 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.

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
Ultraflo® Max process predictability and consistency for
improved quality and cost-efficiency.
57

The chemistry behind enzymatic filtration

 .

 .
Breaking up complex
carbohydrate structures
into smaller, more
soluble oligomers leads
to lower viscosity and
better filterability.
58

Extract more, extract better

In the old days…

... 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.

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-
Ceremix® rials as adjuncts like rice, corn, or sorghum.
Cerezyme®
Ondea® Pro Now, even 100% barley brewing is possible.
 59

Supplementing malt enzymes

 .
If no or insufficient malt
 . are present,
enzymes
carbohydrases and
proteases will hydrolyze
polysaccharides and
proteins into oligomers
for easy brewing of
quality beer.
60

Making beer taste right

In the old days…

… diacetyl sometimes formed in beer, giving the
beer an unappealing buttery taste.

Diacetyl forms when beer is improperly

fermented and matured.

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®
 61

The chemistry behind diacetyl removal

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

Cooking
63

Cost-effective cereal cooking

Thermostable α-amylase
Termamyl SC
Added at start of liquefaction
64

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
 65

What does α-amylase do?

α-amylase randomly cleaves

starch (large dextrins) to form a GOALS:
mixture of smaller dextrin chains  Dextrinization
(polymers of glucose)
 Viscosity reduction
Large
dextrin chains

Shorter dextrin chains

α-amylase
DP6
DP4
DP3
DP2
66

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
67

Starch gelatinization temperatures

Raw material Starch content Gelatinisation
wt% “as is” 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°
68

 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
69

Cost-effective cereal cooking

– Main benefits of using thermostable α-amylases

 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

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Faster Throughput
More Extract
71

Faster throughput and more extract

– β-glucanase, xylanase

Ultraflo Max
added at start of mashing
72

Substrate: cell walls

Barley cell walls Barley cell wall model of Bamforth et al.

stained with Calcofluor
73

The action of β-glucanases

β-glucanases

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

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

β-glucan
Long molecules of mixed linked
1,3–1,4-glucose
74

The action of xylanases

Arabinoxylanases

Endoxylanase (GH11) Endoxylanase (GH10)

Xylose Arabinose Ferulic acid Acetyl Glucoronic acid

Arabinoxylan
Long molecules of xylose
backbones with arabinose
75

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
76

New generation of filtration enzymes

Showing unique wort viscosity reduction

77

Faster throughput and more extract

– Main benefits of using enzymes

 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

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Improved Attenuation
Control
79

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
80

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
81

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
82

What does glucoamylase do?

Simple
Long-chain sugars sugars
(dextrins)

Glucoamylase generates simple

sugars that the yeast can use in
the fermentor
83

Improved Attenuation Control

Why is it so important to make highly

attenuated beer / light beers?
84

Improved Attenuation Control

•Following the consumer trend towards wellness requires highly

attenuated beers with light and low calorie claims
85

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
 86

Improved Attenuation Control Product Solutions

 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
 Improved attenuation control – Light beer – Brand
87 building
Amyloglucosidase (glucoamylase), pullulanase,
amylase

Added at the start of mashing

Attenuzyme Flex
or in special treatment tank
88

Model of the effective breakdown of starch

molecules by Attenuzyme® Flex components

α-amylase

Pullulanase

Glucoamylase

Starch or dextrin Glucose Maltose

The three enzyme components of Attenuzyme® Flex ensures a fast,

effective breakdown of the starch molecules into fermentable sugars
89

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
 90

Improved attenuation control

– Main benefits of using enzymes

 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
91

Fermentation
92

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
93

Fermentation
94

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
95

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
96

Fermentation
97

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
98

Enzyme dosage response of Maturex® 2000L

on the level of total diacetyl in beer

Diacetyl
(mg/L)
3.0

2.0

1.0 0 ADU/L
40 ADU/L
0.0 100 ADU/L
200 ADU/L
0 1 2 3 4 5 6 7
Fermentation (days)
99

Fermentation
0.6

Without Maturex ®
0.5

0.4
Diacetyl (ppm)

0.3

With Maturex ®
0.2

0.1

0
0 20 40 60 80 100 120 140 160 180 200
Time (hr)
 Fermentation
100

● Increased tank utlilization with Maturex®

Without Maturex® 2000 L With Maturex® 2000 L

0 7 Days 14 21 0 7 Days 14 21

1 Cleaning 1 Cleaning
to Filter to Filter
2 Lagering -1°C Lagering -1°C
Cooling
3
Cooling
3 Lagering 7°C Lagering 7°C
4 5
Tank 5 Tank
7
6
7 1
8
3
1

Output to filter plant:

Output to filter plant:
8 + 3 CCV of 5,000 hl in 8 days
8 CCV of 5,000 hl in 8 days
40,000 hl to beer filter 55,000 hl to beer filter
Increase in capacity by 1/3
101

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
102

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
103

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
Cost Effective Adjunct and
Malt Solutions
 105

Cost-effective adjunct and malt solutions

β-glucanase, xylanase, protease, amylase

Ultraflo Max, Neutrase®, Termamyl® SC,

Attenuzyme® Flex

Alternatively Ceremix® Plus MG

 106

Cost-effective adjunct and malt solutions

– Main benefits of using enzymes

 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
Ondea® Pro Brewing
108

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

Ondea® Pro
110

Barley enzymes and malt enzymes

So… 111

Novozymes Ondea® Pro brewing can be regarded

as a fusion
Barley of malting
enzymes and
and Ondea mashing
® Pro
112

Summary of the Ondea® Pro Enzymatic Solution for

Brewing with 100% unmalted barley
 Attenuation is ensured through a
synergetic action between Typical Mashing Profile
 Endogenous barley beta-amylase
Temperature
 Exogenous alpha-amylase and
pullulanase 78-82 °C / 10 min
 Wort amino acid profile resullts from
the synergistic action between 64 °C / 45-75 min
 Endogenous barley exo-
54 °C / 30
peptidases min
Time
 Exogenous added protease
 Good lautering with a clear wort is
ensured via a combination of well
adjusted mills and …  The synergies between the
 A filtration enzyme system
enzymes are ensured through
including both beta-glucanase  A three-step infusion mashing
and xylanase components profile (above)
 A lipase to ensure the wort clarity  Wort pH of 5.6-5.8
113

The Attenuation Components Provide a Typical RDF … but with a

Very High Maltose Content

 High-maltose wort  Ondea ® Pro at 2 kg/t barley gives

around 70% RDF
 High RDF
 Maltose dominates the sugar
profile
Sugar profile
Maltose and RDF
as Function of the Enzyme Dose Glucose 5.0 %
70 76
68 74 Fructose 1.5 %
72
Maltose conc in %

66
70
64 68
Maltose > 60 %
62 66
RDF
60 64 Maltotriose < 15 %
58 62
60 Dextrins (DP4+) < 20 %
56
58
54 56
52 54 Low glucose level leafs the opportunity to add
0 1 2 3 syrup and still create a “maltose” profile
kg Ondea Pro
Maltose % RDF% per ton barley
114

Different barley qualities requires different

enzymes dosages
 Ondea® Pro has been tested  For each barley variety there is linear
with over 100 different barley correlation between the enzyme dose and
samples collected around the amount of non-fermentable dextrin’s
world (as of April 2011)  Different barley varieties do therefore
 It works on approx 95% of the appear as straight lines when then the
tested varieties amount of non-fermentable dextrin’s is
plotted against the enzyme dose
 The few barley where it fails are
not suited for malting
Effect on DP4+ of different Ondea Pro
 The observed variation in dosages applied at two barley varaities
generated FAN is well correlated % DP4+
25
to the protein content 24 Barley A
23 Barley B
 We observe some variation in 22
needed enzyme dose to reach 21
the target attenuation 20
19
 The reason for this variation is 18
under investigation 17 kg Ondea Pro per t grist
16
 It is not correlated to the β- 15
amylase 1 1.2 1.4 1.6 1.8 2
115

Synergy with barley peptidases provides good yeast

performance
 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.

13
12
11 Barley wort needs
10 only
9 9 mg FAN/l/Plato
mg FAN/l/Plato

8 to secure a good
7
fermentation
6
5
4
3
2
1 NOTE: pre-inactivation of
the endogenous
0
proteolytic enzymes by a
0 2 3 4 heat treatment at 75°C
Kg Ondea® Pro per ton barley for 30 minutes
Without inactivation With preinactivation of barley enzymes
Barley wort produced with Novozymes Ondea® Pro has
116

significantly higher amount of fast absorption amino

acids
Relative Amino Acid Compositions of
 Amino acids are divided into 4 Malt and Barley worts
groups according to their ease of
absorption by the yeast cell 100%

 The amino acid profile from 100% 80%

barley-wort differs from malt-wort D
60%
C
 Barley-wort contains relatively 40%
more of the easy fermentable B
amino acids (Groups A and B) and 20%
relatively less of the less A
fermentable amino acids (Groups C 0%
and D) - especially proline Malt wort Barley wort

 This explains the good

fermentability of the barley wort, Group A Group B Group C Group D
despite the lower FAN than in malt Fast Intermediate Slow Little or no
wort absorption absorption absorption absorption
Glutamic acid Valine Glycine Proline
Aspartic acid Methionine Phenylalanine
 Having an amino acid profile more Asparagine Leucine Tyrosine
suitable for the yeast leads to less
amino acid based Strecker Glutamine Isoleucine Tryptophan
aldehydes - and in turn improved Serine Histidine Alanine
flavor stability! Threonine
Lysine
Arginine
117

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

Pilot Scale Trial Industrial Scale Trial

8 hl Barley Malt 300 hl Barley Malt
FAN (12 %) mg/l 114 174 FAN (12 %) mg/l 150 216
FAA Composition FAA Composition
Group A 43.4% 32.7% Group A 38.8% 30.1%
Group B 27.0% 21.7% Group B 23.8% 20.9%
Group C 20.6% 18.2% Group C 23.6% 21.1%
Group D 9.0% 27.3% 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
118

100 % barley wort provides comparable fermentation

performance with less FAN
•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

RDF development during FAN consumption during

fermentation fermentation
80 300

FAN / mg/l
60 200
% RDF

40
20 100
0 0
0 24 48 72 96 120 0 24 48 72 96
Fermentation time/hr Fermentation time /hr
100 % barley 100% malt 100% Barley 100% Malt
119

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 109 325 245 607
aldehydes
120

Filtration and extract components ensure low

wort viscosity and high extract yields
 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

Wort viscosity as function of the barley inclusion

3.5

3
mPa*s

2.5
no enzyme
2
2 kg/t Novozymes
Ondea® Pro
1.5

1
50 60 70 80 90 100 % Barley
121

Barley milling in combination with lauter tun and

mash filter

 Mashfilter in combination  Lauter tun in combination with roller mill

with hammer-mill  A barley kernel is less friable, and the endosperm is more
 The fine grist of the closely connected to the husk – compared to malt
hammer mill is preferable  Milling barley with the same roller setup used for malt will
for the enzyme system in yield a much more coarse grist composition
Ondea Pro  Six and four roller mills are preferable
 The wort separation  However, successful industrial trials have been
procedure needs no completed with two roller mills
changes. Filtration speed  Optimized barley milling showed the following:
and turbidity showed the
same, or improved Optimized barley grist composition

,performance when Barley Malt

compared with all malt Sieve 1 25 -30% (18%)
brews. Sieve 2 15-20% (8%)
Sieve 3 33-40% (33%)
Sieve 4 10-15% (21%)
Sieve 5 2-5% (10%)
Bottom 8-12% (11%)
122

Barley Milling

Differences in grist composition of malt and barley

for effective lautering
50%

45% 45%

40%
40%
Percentage of fraction

35%

30%
30%
25%

20%
15%
15% 14% 14%
13%
10% 8%
8%
5% 6%
5%
2%
0%
0 1 2 3 4 5 6
Sieve

Malt Barley
123

Flavor of 100% Barley Beer

 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
Flavour Characteristics

Mouthfeel - Overall Score

Intensity
5
Overall Score *
4 6
3 5
4
2
Malty/Grainy * Sulphury 3
1 Aftertaste * 2 Sweetness *
0 1
0

Fruity Estery Mouthfullness Bitterness

Malt Barley Malt Barley

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
124

Beer quality
 Ondea® Pro pilsner is similar to all-malt pilsner wrt
 Plato, RDF, gravity, alcohol, haze, pH, CO2, foam, etc.
 …..and most importantly…TASTE
125

Beer Made from Barley versus Malt: Sensory

and Analytical Aspects
126

Beer Made from Barley versus Malt: Sensory and

Analytical Aspects
127

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
128

Ondea® Pro Brewing

- Positive impact on the environment

8 % CO2 Barley
REDUCTION AT BREWERY

8 GRAM CO2 REDUCTION Malthouse

PER 33 CL

Brewery

Beer

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
129

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
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
07/02/
2012 132 NOVOZYMES PRESENTATION

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
133

THANK-YOU

A FOR YOUR ATTENTION

Questions

Answers
CONTACT INFORMATION:
DAVID MARADYN

Comments STAFF SCIENTIST, BREWING CUSTOMER

SOLUTIONS
77 PERRY CHAPEL CHURCH ROAD
PO BOX 576
FRANKLINTON, NC
27587

Discussion PH: 919-494-3280

MOBILE: 919-339-6232
EMAIL: DVMY@NOVOZYMES.COM