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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. WindhausenHead 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

OutlineEndogenous Enzymes in Beer (Review)• Origins, Malting

Exogenous Enzymes• Origins• Available Enzymes

Process Optimization and Other Enzyme Uses• Mash and Lauter• Adjuncts• Kettle• Fermentation• 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 pathsMichael 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 maltingHans Sejr Olsen. Enzymes in brewing. Biokemisk Forening. 2008

Break down proteins, the grain uses throughout the process for various purposes.

Creates Free Amino Nitrogen, critical for yeast health.

The types of enzymes produced are:

Proteases:Cellulose digesting enzymes:

And Starch digesting enzymes:

Germination (cont’d)

Break down the walls that enclose the starch granules.

Principal enzymes are β-glucanases (for β-glucans), and pentosanases (such as xylanases).

(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 modificationCourtesy of Canadian Malting Barley Technical Centre

Barley modification by dayGianinetti, 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.

Adjuncts may increase the amount of limit dextrins (different starch ratios).

Exogenously, pullulanases will serve this same function without inhibition.

Competitive inhibitorKevin Ahern & Indira Rajagopal, Biochemistry Free & Easy, 2019

Non-Competitive inhibitorKevin 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 EnzymesEnzymes added to the brewing process.

Sources:

• Bacteria or fungi.• Yeast is common source.• Could be endogenous enzymes. • Could also be GMO (tailor-made

enzymes).

• Heat-resistant fungi are popular.

• Certain strains of E. coli are commonly used as well.

Historically:

• α-amylase originally produced from cattle and pigs.

• Barley and malt are still used (β-amylase, e.g.)

• Fruit still provides commercial proteases.

Exogenous Enzymes (cont’d)

Partial list of enzymes availableFrom 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(Ca2+

stabilized)

Cuts larger starches randomly

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

Proteases (peptidase)

122-138 oF (50-59 oC)113-128 oF (45-53 oC)

~155 oF (68 oC)~145 oF (63 oC)

4.6-5.3 Break down proteins (increase FAN)

β-glucanaseet. al.

95-131 oF (35-55 oC) ~140 oF (60 oC) 4.5-5.5 Breaks down cell-wall materials

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 MashJohn Palmer – How to Brew

Mash - Attenuation Control

Starch-reducing enzymescourtesy of Dupont, annotated by author

Mash - Attenuation Control (cont’d)

Enzyme activity on starchesKunze

Mash - Attenuation Control (cont’d)

Brewer’s WindowJake 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 ProcessCourtesy 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’

Starch-reducing enzymescourtesy of Dupont, annotated by author

“Giving yeast glucose before maltose is like giving a kid French fries – how on earth will you get it to eat the Brussel 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 enzymescourtesy of Dupont, annotated by author

Mash Filtration – Cellulases

Enzyme Temperature Range Denatures pH Range Function

Proteases (peptidase)

122-138 oF (50-59 oC)113-128 oF (45-53 oC)

~155 oF (68 oC)~145 oF (63 oC)

4.6-5.3 Break down proteins (increase FAN)

β-glucanaseet. al.

95-131 oF (35-55 oC) ~140 oF (60 oC) 4.5-5.5 Breaks down cell-wall materials

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

Triple mash processAEE Institute for Sustainable Technologies, technology wiki

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 (peptidase)

122-138 oF (50-59 oC)113-128 oF (45-53 oC)

~155 oF (68 oC)~145 oF (63 oC)

β-glucanase 95-131 oF (35-55 oC) ~140 oF (60 oC)

Mash – Protein Optimization

β-glucan Reducing Mash (poorly modified malt):

1: Thin Mash // 2: Thick mash (3/4) 3: Solubilase Rest // 4: Cold Water AdditionKunze

Alternate β-glucan Reducing MashBiokemist

Mash - Proteases and GlucanasesOptimizations 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

Typical sparge / final mash temps denature endogenous enzymes.

Boil definitely denatures them all.

No-boil brews: some enzymes could carry through to fermenter!

Enzyme Denatures

α-amylase ~170 oF (77 oC)

β-amylase ~160 oF (71 oC)

Proteases (peptidase)

~155 oF (68 oC)~145 oF (63 oC)

β-glucanaseet. al.

~140 oF (60 oC)

Limit-dextrinase ~150 oF (65 oC)

FermentationDiacetyl (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 yeastMichael 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

Hydrolysis of glycosidic bonds from hopsLallemand, Best Practices: Biotransformation, 2017

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)!

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 formationLewis 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)

Parting PossibilitiesAll – reduce variability in ingredients, use in combinations for adjuncts

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.

Cellulases:

• Reduce filtration issues and time, in mash and downstream

• Aid in adjunct breakdown

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.Throw the entire breadth of knowledge up on the board, all of Brewing.”

- Professor Michael Lewis

Email:awindhausen@holidailybrewing.com