100% barley brewing made possible

EDITING SLIDES IN THE NOVOZYMES TEMPLATEChoose Layout: Click Layout in the top menu Home. And choose between +30 different layouts.

Edit Header and footer: In the top left corner you find Slide no., Date and Header. Change settings in the menu: > Insert> Header and Footer

David MaradynNovozymes North AmericaFranklinton, NC, USA

100% Barley Brewing Made Possible

MBAA-RMD Technical SummitFort Collins, CO

Friday June 25, 2010

BULLETS IN PLANES 3Animation: The plane in the back is fixed. Plane (Today green) in front is animated: Fade on click.

Use: To highlight information when discussing past and future situations.

Agenda

Introduction and History Ondea Pro Concept Components of Ondea Pro Barley Considerations Amino Acid and FAN Filtration Milling Lautering Flavor North American Experience Conclusions Q&A

BASIC LAYOUTUse: This is the basic slide with no extra Novozymes graphics added.

Edit Layout: Click Layout in the top menu Home. And choose between +30 different layouts.


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 between barley and adjuncts)

where the resulting beer can be blended with other brews or be sold as is

This presentation is focused on the enzymatic solution behind Ondea® Pro




History Replacement of malted barley with enzymes and raw

barley in the brewing process is not new … Concept has been under investigation for over 50 years In the early 1960’s researchers at Labatt Breweries of

Canada studied the feasibility of adding protolytic and diastatic enzymes to raw cereal mash, such as unmalted barley or corn, to produce a brewers wort

Claimed to produce an acceptable beer both analytically and organoleptically

DENNIS. G.E . and QUITTENTON, R.C. U. S. Patent No. 3 ,081,172 (March 12, 1963)




History A subsequent publication revealed that the enzymatically

produced barley wort had issues compared to a traditional malted barley wort◦ Lower total nitrogen and fermentable sugars

Other issues◦ Difficulty in milling raw barley to produce a proper particle

size suitable for enzymatic hydrolysis◦ Higher final diacetyl levels were found at end of fermentation◦ Lower colloidal stability was found in the final product

Conclusion that idea was still in development and needs further research LATTIMER. R.A.; LAKSHMINARAYANA, K.; QUIT-TENTON, R. and DENNIS, G.E. Inst Brew (Aust Section) Proc 9 th

Conv. 1966, pp. 111-16.




The attenuation is ensured through a synergetic action between

Endogenous action of the barley beta-amylase

Exogenous added pullulanase and alpha-amylase

The fermentable amino acids are provided by a synergistic action of

Endogenous exo-peptidases An added protease

Good lautering with a clear wort is provided by a combination of well adjusted mills and

A filtration enzyme system including both beta-glucanase and xylanase components, and

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 temperature step infusion mashing profile

With a pH of 5.6-5.8

Time

54 °C / 30 min

64 °C / 45-75 min

78-82 °C / 10 min

Temperature

Mashing Profile

The pullulanase is the key for the attenuation performance of Ondea® Pro Starch is degraded by a synergetic

action of the added pullulanase and alpha-amylase and the beta-amylase from barley

The key is selection of the pullulanase

The main reason for the good performance of the chosen pullulanase is the temperature profile (stability) which fits the gelatinization temperature of barley well.

Lab mashing trials. Ondea Pro is compared with 2 commercial pullulanases at equal dose in enzyme

protein. DP 4+ measured by HPLC

Comparison of the amount of unfermentable dextrin (DP4+) of 3 pullulanases

Dose Ondea ProPullulanase

Commercial pullulanase A

Commercial Pullulanase B

0 30.7 30.7 30.7

17.5 g EP/T barley

17.1 20.1 27.5

Comparison of the temperature activity curves at pH 5 of three

pullulanases

20 30 40 50 60 70 800

20

40

60

80

100

120

Ondea Pro PullulanasePullulanase APullulanase B

% o

f max

act

ivity

oC

Unique pullulanase added

α-amylase added

Barley β-amylase

Starch Fermentable sugars

glucose

maltose

The attenuation components provide a typical RDF but with a very high maltose content Novozymes Ondea® Pro gives a

high maltose concentration A high RDF is ensured by a low

amount of dextrins

Ondea ® Pro at 2 kg/t barley gives around 70% RDF

Maltose dominates the sugar profile

Results from lab scale mashing trials

Mal

tose

con

c in

%

Sugar profileGlucose 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

Novozymes Ondea® Pro ensures processing also at higher gravities; but it takes a dosage adjustment or an extension of the 64°C saccharification rest High gravity reduces the level of

saccharification due to product inhibition of the enzymes

Ensuring the same level of saccharification at higher gravities

Either increase the Ondea® Pro dosage (demonstrated below), or

Extend the 64°C saccharification rest

Fermentable sugars (DP-1,2 and 3) measured after laboratory mashing trials with 2 kg Ondea® Pro per t barley and increasing gravity

The sugar profile is kept constant by increasing the amount of Ondea ® Pro. The increase in enzyme dose is plotted against gravity, defining the dose at gravity 1:5 as 100%




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

1516171819202122232425

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

Laboratory mashing. Different dosages of Ondea® Pro was applied at standard 2 hours mashing with 45 min saccharification rest. The unfermentable dextrins (DP 4+) were measured by HPLC

Ondea® Pro has been tested on close to 100 different barley samples collected around the world (as of April 2010)

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 beta-amylase content

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.

The effect of the synergy with endogenous enzymes is demonstrated by a comparison between a mashes at 50 °C with and without a 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

DOUBLE CONTENT HOLDERS WITH CLOVER ANIMATION (COLOR: PINK)Animation: Clover animation fades from left to right.

Edit: Click on the icons in Content Holder to insert, tables, charts, smart art, pictures, clipart and movies. Or add simple text.

Barley wort produced with Novozymes Ondea® Pro has significantly higher amount of fast absorption amino acids

The amino acids are often divided into 4 groups according to their absorption by the yeast cell

The amino acid profile from 100% barley wort differs from malt wort

The 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 found 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!

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

Average of 10 barley samples with the malt from the corresponding barley

Relative amino acid compositions of malt and barley worts according to the amino acid

fermentability groups

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

Pilot trial Industrial trial8 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%

Amino acid profile and FAN level of barley (mashed with 2 kg/t Ondea ® Pro) and malt wort in pilot (8 hl) and industrial scale (300 hl).

• Industrial barley trials showed a little higher proline content• 70% Group A and B FAA vs 54% Group A and B FAA Pilot barley vs malt worts• 63% Group A and B FAA vs 51% Group A and B FAA Industrial barley vs malt

worts

CONTENT HOLDER 2Edit: Click on the icons in Content Holder to insert, tables, charts, smart art, pictures, clipart and movies. Or add simple text.

Lab fermentation of 12% malt wort and barley wort (made with Novozymes Ondea® Pro). Fermentation temperature 12 °C, Yeast strain W34/70

100 % barley wort shows comparable fermentation performance with less FAN

• The FAN recommendations for malt brews are 120-220 mg/l (at 12 oP) or 10-18 mg/l/Plato• The FAN from 100% barley wort is lower; 108-170 ml/l (at 12 oP) or 9-14 mg/l/Plato• The barley worts have good fermentability• This leads to less unfermented FAN at the end of the fermentation




100% Barley Beer have a better amino acid profile for yeast growth that leads to less formation of Strecker aldehydes(aroma components in wort and beer)

Amino Acid Strecker Aldehydes FlavorMethionine Methional PotatoPhenylalanine Phenylacetaldehyde Sweet, green, floralLeucine 3-Methylbutanal Malty, burntIsoleucine 2-Methylbutanal Malty

Wort aroma components from trials in µg/kgPilot Plant (8 hl) Industrial Scale (300 hl)Barley Malt Barley Malt

3-Methylbutanal 31 115 99 2302-Methylbutanal 11 51 38 110Methional 13 12 19 93Phenylacetaldehyde 56 142 85 174Total Strecker aldehydes 109 325 245 607




Increasing the barley inclusion in mashing leads to an increased viscosity This cannot be solved by traditional filtration enzymes

The filtration system in Ondea® Pro keeps the viscosity low even for 100% barley, though a combination of

A beta-glucanase A viscosity reducing xylanase

The filtration enzyme also ensures a good extract yield Evaluated by low Extractable Extract in spent grain

The filtration and extract component ensures low wort viscosity and high extract yields

The wort viscosity from lab scale mashing trials was measured at different barley inclusion levels with and without addition of Novozymes Ondea® Pro

The turbidity reducing component ensures low turbidity and facilitates lautering

Barley wort (without use of the lipase in Ondea® Pro) has very high turbidity and is very difficult to lauter

The lipase in Ondea® Pro reduces the turbidity of the wort to well under the guideline of 80 NTU (20 EBC unit)

The lipase releases fatty acids similar to the level found in malt based mashing

Less lipid oxidation products are found in the barley beer, likely because there is a lower level of fatty acid in the beginning of mashing where the LOX is active and oxygen are present

The resulting free fatty acids from the lipase action in the mash are at the same level as malt with an similar or lower amount of lipid oxidation product

Laboratory mashing. The amount of free fatty acids before lautering was measured by HPLC

Lipase added to laboratory mashing. The turbidity was measured after filtration/lautering

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

The wort separation procedure needs no changes. Filtration speed and turbidity showed the same or improved performances when compared with all malt brews.

Lauter tun in combination with roller millA 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

The finest possible grist composition is needed 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 compositionBarley 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%)




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 pilsnerTaste panel executed by: Centre for Malting and Brewing Science at

K.U.Leuven

Intensity

Sulphury

EsteryFruity

Malty/Grainy *

0

5

Flavour Characteristics

Malt Barley

Overall Score *

Sweetness *

BitternessMouthfullness

Aftertaste *

0

5

10

Mouthfeel - Overall Score

Malt Barley




North American Experience Industrial trials in North America have been limited to date

since product Ondea Pro launch in fall 2009 One successful trial completed in US

High-maltose/low-glucose wort obtained No lautering issues

Low viscosity wort produced: similar beta-glucans, 80% lower arabinoxylans than in comparable 100% malt wort

Areas of attention Milling: further optimization should have been done, as the

grist we obtained was coarser than we desired Mashing: grist:water ratio, continuous stirring of the mash is

absolutely critical Lautering: bed is not as compact as when using 100% malt –

first wort ran off very quickly, no need to rake as deep Whirlpool: trub is not as compact as with 100% malt wort




North American Experience100% unmalted barley beer prototypes Two sets of Ondea Pro beers have been produced in North

America in 2010 and presented at two brewing conferences

Craft Brewers Conference, Chicago IL, April 2010 American Pale Ale

ASBC MBAA Brewing Summit, Providence RI, June 2010 Irish Stout IPA Helles

All beers were very well received by conference participants

Ondea Pro is one more tool at the brewmasters disposal for the creation of an interesting array of beers




Conclusions A enzyme solution – Ondea Pro - and process have been

developed to enable the brewing of great-tasting beer with 100% unmalted barley with existing brewing equipment

Ondea Pro works in synergy with the endogenous enzymes from barley

The special pullulanase found in Ondea Pro is essential for the attenuation performance

Ondea Pro yields a wort with a relatively high maltose content (> 60%)

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

The lauter tun performance 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

BULLETS IN PLANES 1Animation: The plane in the back is fixed. Plane (Today green) in front is animated: Fade on click.

Use: To highlight information when discussing past and future situations.

THANK-YOU for your

ATTENTION

Q&A

100% barley brewing made possible

Documents

raw barley

barley brewing

unmalted barley

basic slide

lower cost of barley

limited percentage of

menu home

ondea pro basic layoutuse