``Exploring Brewing Enzymes``

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DSM Food Specialties ACBC 2016, Australian Craft Brewers Conference Brsibane ``Exploring Brewing Enzymes`` Siaw Yon Miaw, Enzymes Solutions / Food & Diary 21/July/ 2016

Contents :- 1) About Enzymes in General. 2) Brewing Enzymes - Endogenous & Exogenous. 3) Exogenous Enzymes - Application & Advantages - Recently launched products - Future trend & development 4) Summary & Way Forward.

About Enzymes in General Enzymes are :- proteins with catalytic activity or biological catalyst natural products, present in all living organisms keys in nature to build and degrade organic material. A well known enzyme : amylase from saliva, important for a good «pre-digestion» of starch containing foodstuff specific : catalyse one type of reaction dependent on temperature and ph conditions inactivated by extreme conditions of temperature

3-D structure of an Enzyme

How do Enzymes work? 5 Modified substrate Enzyme Enzyme Enzyme

one enzyme = one substrate theory

Brewing Enzymes :- Endogenous enzymes in malt - # produced during barley germination. # released from the aleurone layer of the barley grain. # done during the malting process.

German Purity Law for brewing :- ``Reinheitsgebot`` ``Only barley ( malted ), hops and water could be used in the brewing of beer`` Established in 1516 This year 2016, is 500 th year jubilee

Barley to Malt Spraying the barley Germination Box & Kiln Empty Germination Vessel Germination Box

Barley to Malt Before Steeping After germination After Steeping 10

Malting Process : KILNING Kilning Green malt is transferred to kilns for warm air drying to arrest the germination process and reduce the moisture content of the grain to 3-4%. The kilning process takes around 18-24 hours. At this stage the malt takes on its distinctive flavour and colour. 11

Malted barley

The key malt enzymes

Unmodified Barley endosperm Modified Malt endosperm

Malt vs Barley brewing :- Differences in physiochemical characteristics Enzymatic activities Friability Moisture MALT α- Amylase β- Amylase Limit Dextrinase Peptidases Endo-proteases β- glucanase Good 3-4% BARLEY β- Amylase Peptidases Poor 12-14%

This is the most important step in the Brewhouse :- Mashing Regime The enzymes present in the barley malt contain all that is required to: 1) Convert the starch to fermentable sugars mainly maltose temp. range α -amylase 60C to 74C β -amylase 58C to 69C 2) Hydrolyse proteins into peptides and amino acids temp. range carboxy & amino peptidases; endoproteases <52C 3) Breakdown the hemi-celulytic material which makes up the cell walls temp. range β-glucanases <45C 16

Exogenous or Industrial enzymes :- Exogenous enzymes are :- # external enzymes produced by any living organism. # added to the brewing process.

«Industrial»/ Exogenous enzymes are produced from different sources :- 1) Fungal Sources Trichoderma longibrachiatum Aspergillus oryzae Aspergillus niger 2)Bacterial Sources Bacillus amyloliquefaciens 3) Plant Sources. Papaya 4) Animal Sources Pancreatic enzymes

Food enzyme production flow diagram

Enzyme Fermentation Growth factors # ph # O2 Feed composition # Carbon source # Nitrogen source # Trace elements Growth of desired micr.organism producing desired enzyme

Use of Exogenous enzymes in brewing :- Advantages :- 1) Reduce production cost and have process flexibility by adopting high % of adjunct in the brewhouse grist recipe ( rice, corn, barley, sorghum etc ) Feasible recipe ( proven successful) :- # 60% rice # 30-100% barley brewing. # 100% sorghum

Barley brewing with Brewers Compass Dose rate recommendations Barley (%) Malt (%) Dose rate (kg/t grains) 100 0 2.0-4.0 80 20 1.3 3.0 60 40 0.5 1.0 40 60 0.3 0.6 30 70 0.2 0.4 Brewers Compass is a natural mix of enzyme activities, complementing and working synergistically with barley enzymes Brewers Compass added at mash-in Total mash profile time can be reduced as the percentage of malt in the grist increases Dose rate dependent on mash cycle time available, raw material quality (malt and barley) and economics

2) Increase Brewhouse efficiency and yield :- # could smoothen process variation whenever there is raw materials quality fluctuation. # more robust in term of thermal stability, ph tolerance and hence better performance than malt enzymes.

3) Could improve wort filtration and finished beer filtration by adding thermal stable beta glucanse + xylanase at the mashing stage. Resulting :- # better brewhouse and cellar yield # reduced cost of production # reduced filter aids usage and reduced waste disposal # improved production efficiency # reduced carbon footprint

Green Technology 4) Safe the environment by reducing the carbon footprint in the brewing process. Since, # lesser malt are used # better brewhouse yield # better plant efficiency # lesser energy used

Emission (kg CO2-eq / Hl) Summary of LCA results kg CO 2 equivalent CO2-eq emission per Hl beer (from field to BBT) 20 Avg efficiency State of art eff. 16 12 8 4 0 100 % malt, 100% malt, 100% barley, 100% barley, conventional stab PSEP stab conventional stab PSEP stab

LCA Output summary, 100 % malt, stabilised with Brewers Clarex yeast, COD, etc beer, 1 Hl 1.4 kg CO 2 eq. 16.8 kg CO 2 eq. -5.7 kg CO 2 eq. wort 79.2 kg 5.0 kg CO 2 eq. Beer stabilizer Brewers Clarex 0.013 kg CO 2 eq. electricity 22.9 MJ 4.3 kg CO 2 eq. heat 86.1 MJ 6.1 kg CO 2 eq. mash, 56 kg 10.7 kg CO 2 eq. Electricity lower for PSEP compared to SHG + PVPP feed 13.7 kg -6.1 kg CO 2 eq. grist, 16 kg 0.116 kg CO 2 eq. 10.4 kg CO 2 eq. -0.4 kg CO 2 eq. malt, 16 kg 10 kg CO 2 eq. 1.24 kg CO 2 eq. electricity 7.4 MJ 1.4 kg CO 2 eq. heat 34.4 MJ 2.44 kg CO 2 eq. Others barley 18.9 kg 6.5 kg CO 2 eq. Barley Beer stabilizer Energy Heat

LCA Output summary, 100 % barley brewed with Brewers Compass, stabilised with SHG + PVPP yeast, COD, etc beer, 1 Hl 1.4 kg CO 2 eq. 14.3 kg CO 2 eq.(-15%) feed 13.7 kg -6.1 kg CO 2 eq. wort, 79.2 kg 1.4 kg CO 2 eq. Beer stabilizer SHG + PVPP 0.16 kg CO 2 eq. electricity 27.7 MJ 5.2 kg CO 2 eq. heat 86.1 MJ 6.1 kg CO 2 eq. enzyme complex 0.05 kg 0.3 kg CO 2 eq. mash, 56 kg 7.1 kg CO 2 eq. grist, 17.6 kg 0.116 kg CO 2 eq. 6.6 kg CO 2 eq. X malt, convt 16 kg 10 kg CO 2 eq. electricity 1.07 MJ 0.179 kg CO 2 eq. Others barley 17.6 kg 6.2 kg CO 2 eq. Barley Beer stabilizer Energy Heat

LCA Output summary, 100 % barley brewed with Brewers Compass, stabilised with Brewers Clarex yeast, COD, etc beer, 1 Hl 1.4 kg CO 2 eq. 13.2 kg CO 2 eq.(- 21% ) feed 13.7 kg -6.1 kg CO 2 eq. wort, 79.2 kg 1.4 kg CO 2 eq. Beer stabilizer PSEP 0.013 kg CO 2 eq. electricity 22.9 MJ 4.3 kg CO 2 eq. heat 86.1 MJ 6.1 kg CO 2 eq. enzyme complex 0.05 kg 0.3 kg CO 2 eq. mash, 56 kg 7.1 kg CO 2 eq. Electricity lower for PSEP compared to SHG + PVPP grist, 17.6 kg 0.116 kg CO 2 eq. 6.6 kg CO 2 eq. malt, X convt 16 kg 10 kg CO 2 eq. electricity 1.07 MJ 0.179 kg CO 2 eq. Others barley 17.6 kg 6.2 kg CO 2 eq. Barley Beer stabilizer Energy Heat

Producing Specialty beers :- 5) Option of producing Specialty beers such as Dry beers or Low Calorie beers may need some specific enzymatic activities. Such as glucoamylase Dry beer & Low Calorie beers :- with a low carbohydrate level ( dextrins ). Could be achieved by a controlled hydrolysis of dextrins by glucoamylase This is not naturally present in the malt.

Potential benefits of Using Exogenous Enzymes in Brewing 80 % tested malts are significantly improvable. Starch is often responsible for filtration delays and beer hazes. Wort viscosity may be decreased by a maximum of 10 % to 15 %. Wort filterability often improved by more than 30%. Beer filtration sessions are longer (sometimes + 50 % to + 60 %), with savings in water, steam and filter aids. Extract is significantly improved (average of + 1 %). High brilliance of the wort, guarantee of a good fermentation. 36

Recently launched Exogenous Enzymes :- 1) Brewers Clarex for colloidal haze stabilization :- # a proline-specific endo-protease # hydrolyses only the haze active proteins ( proline) where bonding with the polyphenols occurs, hence preventing the complex formation of chilled haze.

Computer simulation of interaction between proteins and polyphenols :- 38 Proline in chain 1 Proline in chain 2

39 Computer simulation of colloidal haze formation :-

Brewers Clarex stabilizes beer by specifically degrading haze sensitive proteins (because these are high in Proline content) protein / polypeptide =any amino acid =Proline DSM Brewers Clarex specific hydrolysis (only Proline rich proteins) Hydrolyzed peptides Haze sensitive proteins High in Proline content These complex with polyphenols to form colloidal beer haze Short fragments of Haze sensitive proteins Other (foam positive) proteins are not touched!

Degraded protein will not form visible aggregates Hydrolyzed peptides Poly phenol + Poly phenol Poly phenol Peptide-polyphenol aggregates are too small to be visible Poly phenol P

Advantages of using Brewers Clarex :- # removes the deep cooling step in stabilization, significant maturation capacity increase. # increase cold stabilization temperature (up to 7ºC tested) significant energy savings. # does not have to be filtered out and no rinsing is needed, resulting reduced beer loss and filtration cycle increased. # is a liquid technology, no production waste as compared with other powder stabilizers. # reduced carbon footprint a green technology. # is easy to apply, low dosage ( 2-3 g/hl ), needs no big CAPEX investment. # has no impact on beer (taste or foam) quality.

2) Produced Gluten Free beer using Brewers Clarex

Euromonitor: Gluten-free audience is far beyond coeliacs :-

Gluten-free touches close to 1 in 10 consumers in some European markets Source: Mintel, 2015

A record number of gluten-free beers launched in 2015 Global gluten-free beer launches 70 60 +62% 63 Launches 50 40 30 25 31 39 20 10 13 0 2011 2012 2013 2014 2015 Source: Mintel GNPD, January 2016 46

Go Gluten-free Enzyme solution for gluten-free beers

Future trends :- on Acceptance of Exogenous Enzymes Higher acceptance in food application due to :- Clean, Sustainable process less chemicals, lower temperatures Pressure on raw material costs / increasing population need to optimize the use of agro-resources Waste valorization

New Exogenous Enzymes Development - Future Trend Focus on :- # Eco /Environment friendly # Simplified production process # Reduced energy consumption # Minimum CAPEX # Improved yield and process efficiency # Minimize waste production, reuse by-product # Non GMO

Summary and Way Forward :- Considering the many challenges facing the world such as resources scarcity, continuous population growth, environment destruction, technology advances and on the other hand, the advantages of using Exogenous Enzymes, so are you still hesitating of using them? -- The Decision is Yours!

Thank you for your attention