Enzyme Engineering XXII: Emerging Topics in Enzyme Engineering
Enzyme Use in Beverage Production - Enology and...
Transcript of Enzyme Use in Beverage Production - Enology and...
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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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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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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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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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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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
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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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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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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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
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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
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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 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 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 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
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 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 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
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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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
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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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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
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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
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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
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Beer quality
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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 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
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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
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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
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Novozymes Ondea® Pro brewing
Any beer type can be made using Novozymes Ondea® Pro brewing – Just substitute pilsner malt with barley + Ondea® Pro
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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: [email protected]
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FOR YOUR ATTENTION A
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