Basic Brewing Enzymology

A beginner’s primer on enzymes in beer

Presentation Summary

This seminar is a companion to the following presentation, Practical Enzymatic Brewing. This presentation is beginner level, covering:

• Background biology and chemistry of enzymes, proteins, and carbohydrates;

• Enzymes endogenous to malted grains (how and why they form);

• The typical process of brewing, as viewed through the lens of enzyme activity (from mash-in to clarification).

Alan B. WindhausenHead Brewer

Holidaily Brewing

Learning Objectives

• Understand the foundation of enzymology (biology and chemistry of enzymes);

• Review the biological and chemical nature of the components to beer;

• Know the sources of endogenous enzymes in malt;

• Understand the role of enzymes in a basic brewing process.

OutlineDefinitions and Background• What are Enzymes?

• What are Proteins?

• What are Carbohydrates and Starches?

• Endogenous vs. Exogenous

• Enzyme Naming Conventions

Endogenous Enzymes in Beer• Malting

• Background and basics• Germination and Modification• Kilning

• Mashing and Boiling• Enzymes, Functions, and Ranges• Adjuncts

• Fermentation• Dry Hop Creep

Definitions and Background

What Are Enzymes?

Glucosidase, breaking Maltose into two Glucose.

© Thomas Schafee, 2015, Wikimedia Commons

• They are proteins (usually) with a job.

• Specifically, made by a living organism to catalyze a biochemical reaction

• Not all enzymes are proteins, but the vast majority are.

What Are Enzymes? (cont’d)

A simplified graph of the breakdown of glucose.

© Mcy Jerry, 2005, Wikimedia Commons

• Biological catalysts.

• Catalysts reduces the amount of energy needed for the reaction.

• Thus, it will occur faster and with greater frequency.

• Catalysts are not consumed in the reaction!

“substrate:

a substance acted upon (as by an enzyme)”

– Merriam Webster

The molecules that enzymes act upon are called “substrates.”

Sometimes, more than one substrate is involved.

Enzymes are structured to only interact with a very specific substrate (or substrates)

What Are Enzymes? (cont’d)

Two substrates interacting with an enzymeFrom https://openstax.org/books/anatomy-and-physiology/pages/2-5-organic-compounds-essential-to-human-functioning

Lock and Key model of enzyme bonding© Saylor Academy, 2012

What Are Proteins?• Proteins are long-chain molecules

comprised of amino acids.

• 4 levels to their structure:• Primary – the sequence of the

amino acids.

• Secondary and Tertiary – the ‘folding’ and ‘pinning’ of the molecule into a specific shape.

• Quaternary – multiple separate chains forming a single structure.

Structure of ProteinsSchmid, Silvan. (2020). Immobilization of DNA and protein to polymerized SU-8 photoresist investigated with microarray assays.

What are Proteins? (cont’d)

Proteins only function when the entire structure is intact!

Factors that will break down that structure:

• pH’s outside their ‘functional’ range.

• High temperatures.

Relatively low temperatures are fine, but reactions slow down in cold.

A protein broken down to just primary structure is fully ‘denatured’.

Denatured proteins no longer function as intended. Denatured enzymes will not perform their task.

What are Proteins? (cont’d)

A graph of enzyme activity vs temp.© Thomas Shafee - Own work, CC BY 4.0, https://commons.wikimedia.org/w/index.php?curid=47436421

Carbohydrates and StarchesCarbohydrates: molecules built out of simple sugars.

Two sugars together are called disaccharides.

Long chains of sugars (polysaccharides) create larger carbohydrates.

Cellulose is made of β-glucose, while starches are made of α-glucose. Structure of mono- and di-saccharides

© Christine Dobrowolski, CC BY-SA 2.0

Carbs and Starches (cont’d)Starches: long chain-like molecules made up of many glucose.

Amylose - Straight chains of sugars

Amylopectin - Branched

Enzymes will interact with only one of type, or even a specific section. Amylose and Amylopectin, acted upon by

α-amylaseThe filled circles indicate glucose subunits with α-1,6, glycosidic linkages

Margaret E. Smith PhD DSc, Dion G. Morton MD DSc, in The Digestive System (Second Edition), 2010

Endogenous and ExogenousEndogenous: “internally originating”

• Created inside an organism to perform a task critical to that organism’s survival.

Exogenous: “externally originating”

• Added from an external source (i.e. by you)

Endogenous (in)

Endo- & Exogenous (cont’d)Commercial enzymes:

• From bacteria or fungal sources, sometimes yeast – sometimes GMO.

• Older sources: animal (α-amylases) or plant sources (papain).

Exogenous doesn’t always mean commercial:

• Traditional Chicha is chewed to convert maize starches.

A commercial Chicha (Dos Luces)Courtesy of Dos Luces Brewing

Enzyme Naming• Pre-1955, named for the substrate,

product, discoverer, or damn-near randomly.

• Today, enzymes common names are “substrate + ase.”• E.g. lactase breaks down lactose.

• Sometimes, the action being performed is added:• E.g. β-glucan solubilases solubilize

β-glucan.

Role of Solubilases in Cell Wall DegradationCourtesy of Canadian Malting Barley Technical Centre

Enzyme Naming (cont’d)• The common or ‘trivial’ name for an

enzyme is typically used.

• The ‘proper’ enzyme name is further broken down:• List all substrates• State exact reaction

• E.g., α-amylase is “4-α-D-glucan glucanohydrolase.”

• The Enzyme Code is a number used for research• α-amylase is 3.2.1.1

Enzyme Code (first numbers only):

EC 1 – oxidoreductases (perform oxidation-reduction reactions on substrates)

EC 2 – transferases (transfer molecular components between substrates)

EC 3 – hydrolases (breaks bonds on a substrate using hydrolysis, the cleaving of bonds with an H+

and OH- from water – amylases are hydrolases)

EC 4 – lyases (break apart bonds on a substrate without using hydrolysis)

EC 5 – isomerases (isomerize substrates – that is, they rearrange the components of a substrate)

EC 6 – ligases (join together substrates, while also using a molecular energy source called ATP)

EC 7 – translocases (added 2018 – transport ions or molecules across membranes in cells)

Endogenous Enzymes in Beer

Malting• The process of transforming grains

(or seeds) into transportable packets of starches, enzymes, and proteins / amino acids.

• I.e., the breaking down cell walls and proteins while preserving the starch granules and enzymes.

• I.e., maltsters trick grain into freeing their starches, and brewers trick that into becoming yeast nutrients, because yeast has tricked us all into feeding it. Modification of barley, two paths

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

Hydrating the grain kick-starts the process.

Allows chemicals to move freely.

Malting consists of:

Steeping: Germination: And Kilning:

Malting (cont’d)

Enzymes break down stored nutrients for the plant.

“Modification”: conversion to usability for the plant (and brewer).

Halts modification, creates flavor.

Germination

1 – The hydrated embryo eats sugars in its immediate vicinity.

2 – Once those are depleted, Gibberellins are activated.

3 – These hormones trigger the release or production of specific enzymes.

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)Well-modified malt:

• Cell walls and matrix broken down.

• Starch and amylases intact for the brewer to control.

Poorly modified malt

• Has remaining cellular matrix.

• Sticky long-chain molecules remain.

Cell Wall breakdown during modificationCourtesy of Canadian Malting Barley Technical Centre

Barley modification by dayGianinetti, Theory in Biosciences, 2008

KilningSuspends modification, leaves starches and amylases intact.

• Allow enzymes to attach to remaining matrix (preserves structure).

• High temperatures and longer times breaks down enzymes.

Caramel and crystal malts rehydrate ‘green’ malt prior to kilning / roasting.

• Sugars form, then are caramelized

Munich 20L Malt vs Caramel 20LDavid Richter, Briess Malt and Ingredients – blogpost, Jan. 2018

MashingFinally, the brewhouse!

Goal of Mashing:

• Solubilize the soluble malt matter.

• Gelatinize starches and convert into sugars.

• Release and solubilize other desirable malt components.

• All of this relies on the endogenous enzymes preserved from malting.

Triple mash processAEE Institute for Sustainable Technologies, technology wiki

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-glucanase 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)

Rough enzyme ranges in MashJohn Palmer – How to Brew

Amylasesβ-amylase:

• Lower temp

• Acts faster on larger molecules

• Only works on linear starches from non-reducing end.

• Produces maltose.

α-amylase:

• Cuts ‘randomly’

• Works on branched amylopectin (but not at branch points.

Enzyme activity on starchesKunze

Amylases (cont’d)

Enzyme activity on starchesKunze

Amylases (cont’d)

Brewer’s WindowJake McWhirter – Missionary Brewer Blog

Limit DextrinaseWill further break down limit-dextrins.

• HOWEVER – inhibited at a very early point in the mash.

• Inhibitors act to block an enzyme from performing its job (without denaturing it).

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

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

Proteases and Glucanases

Enzymes can break down the remaining cellular matrix (protein and glucan rests):

• Higher efficiency

• Lower mash / wort viscosity

• Reduce protein haze in the beer

• Increase FAN

However:

Kilning denatures many of these enzymes.

Prolonged rests increases 4-vinyl-guaiacol off flavor (ferulic acid converts in whirlpool).

Also can increase viscosity.

Proteases and Glucanases (cont’d)

β-glucan Reducing Mash (poorly modified malt):Biokemist

Enzyme Temperature Range

Denatures

α-amylase 150-160 oF (66-71 oC)

~170 oF (77 oC)

β-amylase 130-150 oF (54-66 oC)

~160 oF (71 oC)

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)

Limit-dextrinase

95-140 oF (35-60 oC) ~150 oF (65 oC)

Proteases and Glucanases (cont’d)

Alternate β-glucan Reducing Mash:

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

Enzyme Temperature Range

Denatures

α-amylase 150-160 oF (66-71 oC)

~170 oF (77 oC)

β-amylase 130-150 oF (54-66 oC)

~160 oF (71 oC)

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)

Limit-dextrinase

95-140 oF (35-60 oC) ~150 oF (65 oC)

oF

176

167

158

149

140

131

122

113

104

95

86

Adjuncts, Cereal Cookers, Decoction Mashing

In an infusion mash, barley malt carries the DP load.

• Can support adjuncts with no amylase activity, up to roughly 20%.

• Slightly more, if the adjunct is flaked.

• Malted adjunct has some DP.

Competitive inhibitorCourtesy of Canadian Malting Barley Technical Centre

Adjuncts, Cereal Cookers, Decoction Mashing (cont’d)Cereal Cookers:

• Gelatinize starches in adjuncts.

• Barley malt or mash is added for α-amylase, which rapidly denatures.

• A ‘lossy’ process for enzymes.

Decoction mashing:

• Solution to poor temp control.

• Portion of the mash is boiled, then added to larger mash. • Heated portion’s enzymes are

denatured.

• Again creating an enzymatically ‘lossy’ process.

Brew ProcessCourtesy of

Yuengling & Son, Inc

Fermentation:Dry Hop Creep (Thanks OSU!):

Hops contain enzymes.

High rates of dry-hopping + unfiltered beer = new sugars = re-ferment.

Results: increased ABV, carb levels, and potentially more diacetyl.

Check out the CBC 2020 presentation, and look for the new white paper! Excerpt from “The Brewer’s Guardian”

March 28, 1893, page 93

Questions?(ask below to be included in the recording)

“Have a keen understanding of the microbiology [of brewing], because it is very interesting.

No, not interesting – actually, it’s boring as shit. But it is important.”

- Professor Michael Lewis

Email:awindhausen@holidailybrewing.com