Practical Enzymatic

Brewing
An intermediate exploration of Brewing Enzymes
 Presentation Summary
This seminar is a companion to a previous presentation, Basic Enzymology for
Brewing. This presentation is focused on:
• A review of the sources and historic/typical roles of endogenous enzymes
• An overview of exogenous enzymes available
• How to most effectively use both types of enzymes in the brewing process
Alan B. Windhausen
Head Brewer || Quality Trainer
Holidaily Brewing || Brewers Association
 Learning Objectives

• Know the origins of both endogenous and exogenous enzymes

• Be able to push endogenous enzymes and increase their effect
• Have a greater understanding of sources and varieties of exogenous enzymes
• Have an appreciation of the limitations of exogenous enzymes, and their
potential downsides
• Gain a sense of new products made possible by smart enzyme usage
 Outline
Endogenous Enzymes in Beer Process Optimization and Other
(Review) Enzyme Uses
• Origins, Malting • Mash and Lauter
• Adjuncts
Exogenous Enzymes • Kettle
• Origins • Fermentation
• Available Enzymes • Filtration
Endogenous Enzymes in Beer

Review with focus on optimization – for full

examination of process, please watch
Basic Enzymology for Brewing
Malting

Consists of:
• Steeping
• Hydrating the grain, starts the
process of growth.
• Germination
• Endogenous enzymes break
down stored nutrients
• And kilning
• Halts modification, creates
flavor.

Modification of barley, two paths

Michael Lewis and Tom Young, Brewing (2nd edition), 2002)
Germination

1 – The hydrated embryo eats sugars in

its immediate vicinity.

2 – Gibberellins (hormones that start

modification) are released from the
Scutellum and

3 – Specific enzymes get released or

produced to break down the
endosperm.
Enzyme production during malting
Hans Sejr Olsen. Enzymes in brewing. Biokemisk Forening. 2008
 Germination (cont’d)
The types of enzymes
produced are:
Proteases:
Break down proteins, the
grain uses throughout the
process for various
purposes.
Creates Free Amino
Nitrogen, critical for yeast
health.
Cellulose digesting
enzymes:
Break down the walls that
enclose the starch granules.
Principal enzymes are β-
glucanases (for β-glucans),
and pentosanases (such as
xylanases).
And Starch
digesting enzymes:
(α- and β-amylase) – break
down starches into sugars.
Brewers need these enzymes
to be created but to not break
down the starches yet.
Limit-dextrinase and other de-
branching enzymes are also
created.

Check out the CBC 2020 presentation on Malt COA’s!

Germination (cont’d)
 Germination (cont’d)

Solubilization:
- Xylanase
- Acetyl xylan esterase
- Feruloyl esterase
- Arabinofuranosidase
- Carboxypeptidase

Hydrolysis (breakdown)
- Endo- and Exoglucanases
- Glucosidases
- Xylosidase
 Germination (cont’d)

Protein matrix breakdown:

(Proteases)
- Endopeptidases
- Exopeptidases
 Germination (cont’d)

Cell Wall breakdown during

modification
Courtesy of Canadian Malting Barley Technical Centre

Barley modification by day

Gianinetti, Theory in Biosciences, 2008
Germination (cont’d)
Limit Dextrinase:
• Present and active in mash for only
short time:
• An inhibitor is rapidly solubilized
into the mash.
Competitive inhibitor
Kevin Ahern & Indira Rajagopal, Biochemistry Free & Easy, 2019
Adjuncts may increase the amount of
limit dextrins (different starch ratios).

Exogenously, pullulanases will serve

this same function without inhibition.
Non-Competitive inhibitor
Kevin Ahern & Indira Rajagopal, Biochemistry Free & Easy, 2019
Kilning

Goal is to suspend modification, leaving

starches and amylases intact.

Caramel and crystal malts are mini-

mashes inside the kernel.
• Sugars form, then are caramelized

Exogenous enzymes that break down

sugars may impact fermentability of
malts / adjuncts! Munich 20L Malt vs Caramel 20L
David Richter, Briess Malt and Ingredients – blogpost, Jan. 2018
Exogenous Enzymes

Overview of origins and available enzymes

Exogenous Enzymes
Enzymes added to the brewing process.

Sources:
Historically:
• Bacteria or fungi.
• Yeast is common source. • α-amylase originally produced from
cattle and pigs.
• Could be endogenous enzymes.
• Could also be GMO (tailor-made • Barley and malt are still used (β-
enzymes). amylase, e.g.)
• Heat-resistant fungi are popular. • Fruit still provides commercial
proteases.
• Certain strains of E. coli are
commonly used as well.
 Exogenous Enzymes
(cont’d)

Partial list of enzymes available

From Biokemi, BioZoom, issue 522
 Exogenous Enzymes (cont’d)

“There is a pervasive resistance of the brewers in North America to

use enzymes. It is not the norm [unless] there is a special target not
achievable by any other means.”

“Enzymes are a sensible way to improve beer and to equalize

differences from batch to batch. [However], when enzyme companies
are saying, ‘Hey you can do things in a totally different way, you can
use for example raw barley.’ Well you won’t get the same beer.”

- Dr. Charlie Bamforth

Exogenous Enzymes
Principal use of exogenous enzymes is
in addressing fluctuations in ingredients.
• Batch of malt has low FAN.
• Variability in filtration times due to
malt variation.
• Diacetyl rests and concentrations
vary with yeast
However, if used on known brands,
triangle test!
Alternate use (and more common in US
craft) is novel product design.
• Brut IPA is prime example
• Low-cal, low carb products
• Extremely high / 100% adjunct
mashes
• Faster production times
• Unusual mash regimes
Process Optimization and
Other Enzyme Uses

Mash, Adjuncts, and Lauter

 Mashing

Goals of Mashing:
• Solubilize the 15-25% of malt matter that is readily soluble.

• Gelatinize starches and convert into sugars usable by yeast in desired ratio
for style and gravity.

• Release and solubilize other desirable malt components (proteins, amino

acids, yeast nutrients, etc.)
 Mashing (cont’d)
Enzyme Temperature Range Denatures pH Range Function

α-amylase 150-160 oF (66-71 oC) ~170 oF (77 oC) 5.3-5.7 Cuts larger starches
(Ca2+ randomly
stabilized)

β-amylase 130-150 oF (54-66 oC) ~160 oF (71 oC) 5.0-5.5 Breaks down starch
chains, linearly, into
maltose

Proteases 122-138 oF (50-59 oC) ~155 oF (68 oC) 4.6-5.3 Break down proteins
113-128 oF (45-53 oC) ~145 oF (63 oC) (increase FAN)
(peptidase)

β-glucanase 95-131 oF (35-55 oC) ~140 oF (60 oC) 4.5-5.5 Breaks down cell-wall
materials
et. al.
Limit-dextrinase 95-140 oF (35-60 oC) ~150 oF (65 oC) 5.0-5.8 Breaks down sugars
left behind by
amylases, can be
inhibited
 Mashing (cont’d)

From Biokemi, BioZoom, issue 522

 Mashing (cont’d)

Rough enzyme ranges in Mash

John Palmer – How to Brew
 Mash - Attenuation Control

Starch-reducing enzymes
courtesy of Dupont, annotated by author
Mash - Attenuation
Control (cont’d)

Enzyme activity on starches

Kunze
 Mash - Attenuation Control (cont’d)

Brewer’s Window
Jake McWhirter – Missionary Brewer Blog
 Mash - Attenuation Control (cont’d)

Amylase optimizations / uses:

• Exogenous α-amylase to mash at β-amylase optimum (~142oF, 62oC)
• Adding α-amylase and fungal α-amylase to low DP malt / increase
fermentability
• Adjunct additions to mash (~20% endo, up to 100% exo)
• Reduce cost of running a cereal cooker / make kettle available as such
(malt liquor, anyone?)
 Mash - Attenuation Control (cont’d)

Brew Process
Courtesy of
Yuengling & Son, Inc
Mash – Attenuation (cont’d)
Amyloglucosidase (glucoamylase):
• Creates glucose, not maltose.
• Increases fermentability in standard
mash & Real Degree of Fermentation
• Can create lower-cal / lower-carb
products
Concern:
• Stalled Fermentation
• ‘Glucose suppression/repression’
“Giving yeast glucose before maltose is
like giving a kid French fries – how on Starch-reducing enzymes
earth will you get it to eat the Brussel courtesy of Dupont, annotated by author

sprouts?” – Professor Michael Lewis

Mash – Attenuation (cont’d)

Pullulanse (limit-dextrinase):
• Cuts at branch points, reduces
unfermentable dextrins
• Increases fermentablility (RDF above
88% w/ other exo.)

If used without other exo. enzymes:

• Increase in RDF, fermentable ratios
stay roughly the same!
• No glucose suppression/repression! Starch-reducing enzymes
courtesy of Dupont, annotated by author
 Mash Filtration – Cellulases

Enzyme Temperature Range Denatures pH Range Function

Proteases 122-138 oF (50-59 oC) ~155 oF (68 oC) 4.6-5.3 Break down proteins
113-128 oF (45-53 oC) ~145 oF (63 oC) (increase FAN)
(peptidase)

β-glucanase 95-131 oF (35-55 oC) ~140 oF (60 oC) 4.5-5.5 Breaks down cell-wall
materials
et. al.
 Mash Filtration – Cellulases (cont’d)

Commercial enzymes might be marketed as:

• β-glucanase (should be pure β-glucanase)
• Xylanase (should be pure xylanase)
• Pentosanases (often pure xylanase)
• Cellulases (β-glucanase + xylanase, most likely)
• Combination products (cellulases + protein/starch enzymes)
Cellulases (cont’d)

Cell wall materials clog mashes and

filters (downstream).
Small variations in modification have
outsized impact!
β-glucanase:
• Reduces variation (on larger scale,
offsets own cost)
• Aids in lauter and filtration, and
increase fermentability of adjuncts
Xylanase:
• Works in conjunction with β-
glucanase, has similar impact.
• Can be used on own to aid
downstream filtration
• Caution: can lead to 4-vinyl-guiacol.
Mash Filtration – Cellulases (cont’d)
Mash – Protein Optimization

Enzymes can break down the remaining

cellular matrix to achieve:
• Higher efficiency
• Lower mash / wort viscosity
• Reduce protein haze in the beer
• Increase FAN

Proteases 122-138 oF (50-59 oC) ~155 oF (68 oC)

113-128 oF (45-53 oC) ~145 oF (63 oC)
(peptidase)
Triple mash process
β-glucanase 95-131 oF (35-55 oC) ~140 oF (60 oC)
AEE Institute for Sustainable Technologies, technology wiki
 Mash – Protein Optimization

β-glucan Reducing Mash (poorly modified malt): Alternate β-glucan Reducing Mash
1: Thin Mash // 2: Thick mash (3/4) Biokemist
3: Solubilase Rest // 4: Cold Water Addition
Kunze
 Mash - Proteases and Glucanases
Optimizations and Uses

Endogenous optimization (decoction, step, protein / glucancase rests) really

not needed for typical North American Malt.

• Can help when using adjuncts (allows use of raw barley, e.g.)
• Lower temp cereal cooking (lower gelatinization temp)
• Shorter / unstuck lauters (wheat / rye beers, oats)
• Even-out down stream filter times
Sparge / Boil Enzyme Denatures

α-amylase ~170 oF (77 oC)

Typical sparge / final mash temps ~160 oF (71 oC)

β-amylase
denature endogenous enzymes.

Proteases ~155 oF (68 oC)

Boil definitely denatures them all. ~145 oF (63 oC)
(peptidase)

β-glucanase ~140 oF (60 oC)

No-boil brews: some enzymes could
et. al.
carry through to fermenter!
Limit-dextrinase ~150 oF (65 oC)
Fermentation
Diacetyl (VDK’s):
• Yeast creates VDK precursors during
amino acids production.

• These autoconvert to diacetyl (2,3-

butanedione) and 2,3-pentanedione
outside the cell

• Commercial α-acetolactate
decarboxylase prevents diacetyl and
pentadione from forming.
• Added during pitching, can effect
later yeast generations. Diacetyl’s life cycle in yeast
Michael Lewis and Tom Young, Brewing (2nd edition), 2002)
Fermentation (cont’d)
Hops contain a small amount of both
amylases, as well as traces of limit-
dextrinase and amyloglucosidase.

High rates of dry-hopping in unfiltered

beer provides yeast new sugars to re-
ferment.

Results: increased ABV, carb levels,

and potentially more diacetyl.

Check out the CBC 2020 presentation,

and look for the new technical brief! Excerpt from “The Brewer’s Guardian”
March 28, 1893, page 93
Fermentation Flavor Uses

Winemakers use a commercial α and β-

glycosidase, “to breakdown
glycosylated aroma precursors.”

Known to impact hop creep (endo.)

Yeast makers market β-glycosidase

activity.

More research needed, but likely the

cause of ‘juicy’ mid-ferm hop flavors
(biotransformation)! Hydrolysis of glycosidic bonds from hops
Lallemand, Best Practices: Biotransformation, 2017
Fermentation (cont’d)

Chill-haze reduction (proteases /

peptidases):
Papain & Bromelain, early exo. enzymes
• Foam degradation issue, requires
pasteurization.
Proline-specific endo-peptidases cut
specific regions.
• ‘Targeted,’ reduced impact of head
retention.

Seibert model for haze formation

Lewis and Bamforth, Essays in Brewing Science, 2006
 Fermentation (cont’d)
“Gluten-reduced beer” using endopeptidases:
• Beer claiming ‘gluten-reduced’ or ‘gluten-removed’ needs TTB
warning:
“Product fermented from grains containing gluten and [processed or
treated or crafted] to remove gluten. The gluten content of this
product cannot be verified, and this product may contain gluten.”

• Gluten is not being removed.

• ‘Gluten’ over different 300 proteins, not all are effected.

• Gluten reactions tend to worsen over time for people.

 Fermentation (cont’d)
“Gluten-reduced beer” using endopeptidases (cont’d):
• ‘Gluten-reduced’ beers under review by the FDA and TTB.
• Dr. Michelle Colgrave: passes R5-ELISA assay, but still dangerous for
patients. BA Seminar - Against the Traditional Grain-Gluten-Free-Beer

• R5 ELISA assay (accepted test by TTB) does not work on finished beer.
• The TTB does not allow for beer to be tested
• You cannot test beer

• Best practice – be fully upfront with customers about the beer, use for
clarifying purposes (not GR)
 Cellulases:
Parting Possibilities
• Reduce filtration issues and time, in
All – reduce variability in ingredients,
mash and downstream
use in combinations for adjuncts
• Aid in adjunct breakdown

Starch degradation enzymes:

Brut IPAs:
• Use in mash, or in fermenter (post
consumption of maltose).
• Known process, transferrable to other
styles?
Similarly, starch reduction creates
lower-carb and calorie beers.
Proteases:
• Clearer beer!
Other enzyme possibilities:
• β-glycosidase with cooler whirlpool
addition? In fermenter pre-dry hop?
Look to the macro brewers and wineries
for new ideas!
 Questions?
(ask below to be included in the recording)

“Think broadly and widely

when problem solving.
Email: Throw the entire breadth of
awindhausen@holidailybrewing.com knowledge up on the board,
all of Brewing.”

- Professor Michael Lewis