A new acetolacte decarboxylase for diacetyl control

Transcription

1 A new acetolacte decarboxylase for diacetyl control JACOB F. CRAMER, LENE B. JENSEN AND TOVE BLADT DUPONT INDUSTRIAL BIOSCIENCE IBD CONVENTION 2016 SYDNEY, AUSTRALIA, 17 TH OF MARCH 2016

2 Agenda Vicinal di-ketones and enzymatic regulation of diacetyl A new alpha-acetolacte decarboxylase Reduction of diacetyl during beer fermentation Robustness and use of novel adjuncts Taste and beer quality Summary COPYRIGHT 2016 DUPONT. ALL RIGHTS RESERVED. 2

3 Vicinal Diketones VDKs are normal by-products of the yeast metabolism during brewery fermentation The two major VDKs of the concern to the brewer are diacetyl (2,3-butanedione) and 2,3 pentanedione They are referred to vicinal diketones due to adjacent ketone groups in the compound Diacetyl and pentanedione are the most important immature beer aromas and generally considered to be undesirable contributor to the flavor of beers introducing a butter-scotchlike and/or honey-like aroma (threshold for the total VDK content for a fully matured beer is 0.1mg/L) The breakdown reactions of these VDKs are parallel to other maturation processes during the beer conditioning and plays an important role as an indicator for the state of maturation of the beer O O Diacetyl (2,3-Butanedione) O O 2,3-Pentanedione COPYRIGHT 2016 DUPONT. ALL RIGHTS RESERVED. 3

4 Background for elevated diacetyl in traditionally fermented beers Type of yeast strain (e.g. flocculent/non-flocculent); Amount of pitched yeast (too little), elevated yeast pitching temperatures and/or exhausted yeast cells; Warm primary fermentations with forced maturations (no time given for VDK reduction); Oxygen exposure/uptake during primary fermentation, secondary fermentation, at transfers, or at packaging; VDKs may be caused by bacterial contamination, primarily by Pediococci species. Most research has focused on diacetyl formation and production and less well understood is reduction of diacetyl by the yeast. COPYRIGHT 2016 DUPONT. ALL RIGHTS RESERVED. 4

5 Production and breakdown of diacetyl and 2,3-pentadione Acetolactate-(butyrate) excretes from the yeast cell and spontaneously reacts under a oxidative decarboxylation (chemically, no enzyme involved) to diacetyl and 2,3-pentadione COPYRIGHT 2016 DUPONT. ALL RIGHTS RESERVED. 5

6 Formation and breakdown of diacetyl and 2,3-pentadione Acetolactate-(butyrate) excretes from the yeast cell and spontaneously reacts under a oxidative decarboxylation (chemically, no enzyme involved) to diacetyl and 2,3-pentadione Alphalase Advance 4000 is an alpha-acetolactate decarboxylase that bypasses the relatively slowly buildup of diacetyl and 2,3-pentadione. COPYRIGHT 2016 DUPONT. ALL RIGHTS RESERVED. 6

7 Diacetyl is tightly linked to yeast amino acids metabolism Acetolactate builds up and is secreted from the yeast as a consequence of a rate limiting step in the pyruvate pathway towards valine. Acetolactate is converted to diacetyl during fermentation Valine feedback inhibition AHAS Active Valine uptake The flux of the valine uptake may reduce diacetyl formation, however it is more complex regulated by FAN levels and the amino acid composition of the wort Lager Beer fermentation Haukeli and Lie et al., 1972 COPYRIGHT 2016 DUPONT. ALL RIGHTS RESERVED. Gibsen et al.,

8 Diacetyl control in brewing The enzyme is added to the fermentation tank and extends its action throughout fermentation, but most importantly when diacetyl is about to build up The highly concentrated enzyme may be dosed so the resulting effect is diacetyl levels below flavor threshold at the end of the main fermentation Thus the ALDC enzyme may serve an efficient tool to improve process efficiency and brewing more beer in shorter time Heat exchanger ALDC Yeast Piching (propagation) The enzymatic diacetyl control may ensure consistent throughput even during peak season Fermentation tank Maturation tank COPYRIGHT 2016 DUPONT. ALL RIGHTS RESERVED. 8

9 The alpha-acetolactate decarboxylase Lyase, alpha-acetolactate decarboxylase E.C Metalloprotein, 29kDa Zn +2 coordination of acetolactate to promote decarboxylation Two domain α/β structure assembled into a (functional) dimer ALDC dimer 1 Zinc essential for proposed decarboxylation mechanism Acetolactate Active site His-coordination Copyright 2016 DuPont. All rights reserved. 9 1 Marlow et al., 2013

10 Relative activity (%) Relative activity (%) Applying of Alphalase Advance 4000 in brewing Alpha-acetolactate decarboxylase Active over a broad ph range and applicable for various brew protocols Active throughout beer fermentation 100 ph optimum 100 Temperature optimum ph Temperature ( C) Copyright 2016 DuPont. All rights reserved. 10

11 Residual activity (%) Residual activity (%) Robustness of Alphalase Advance 4000 in the brewing application Robustness for the brewing application ph Temperature ( C) ph stability for Alphalase Advance 4000 in wort at 10 C for 2 days Temperature stability for Alphalase Advance 4000 in wort ph 5.0 for 2 days Copyright 2016 DuPont. All rights reserved. 11

12 Cooperation with 2 hl Research Brewery, Hopfenveredlung St. Johann GmbH, St. Johann, Hallertau Set up: Evaluation of Alphalase Advance 4000 at lab and pilot scale Mashing, adjunct cooking, lautering, mash filter separation, fermentation, filtration and filling of beer Application Brewing cooperates with other pilot plants at universities/suppliers Dupont Brabrand-Denmark IB R&D Brewing Center R&D IB Brewing team developing new enzymes for the global brewing industry. Capabilities to perform standard brewing analyses and highly advanced analyses. COPYRIGHT 2016 DUPONT. ALL RIGHTS RESERVED

13 Beer aplication; At the end of final attenuation, usually a maturation period is required to get diacetyl below its flavor threshold Diacetyl levels above flavor threshold at the end of final attenuation for control sample without enzyme addition COPYRIGHT 2016 DUPONT. ALL RIGHTS RESERVED. 13

14 Introduction and performance of Alphalase Advance 4000 Diacetyl levels during fermentation of 12 P all-malt wort and 14 C fermentation temperature Copyright 2016 DuPont. All rights reserved. 14

15 Influence on diacetyl production and conversion by yeast strains Diacetyl levels during fermentation of 12 P all-malt wort and 14 C fermentation temperature using different yeast strains Copyright 2016 DuPont. All rights reserved. 15

16 The effect of extract and fermentation temperature on diacetyl removal Enzymatic diacetyl reduction rate (at 14 C) is depending on the extract and may be regulated by enzyme dosage. Diacetyl production and conversion is greatly influenced by temperature. However, Alphalase Advance 4000 may ensure a fast and consistent processing with significant diacetyl reduction in challenged brewing protocols. Alphalase Advance 4000 is a robust solution for protocols with differing wort gravity and fermentation temperatures Alphalase Advance 4000 Maximum diacetyl level (mg/l) 12 P 16 P 14 C 18 C 0.0 g/hl (control) g/hl g/hl g/hl Copyright 2016 DuPont. All rights reserved. 16

17 Robustness of Alphalase Advance in novel adjuncts Maximum diacetyl levels compared to no enzyme are consistently reduced using different raw materials and brewing protocols at same application dosage. Copyright 2016 DuPont. All rights reserved. 17

18 Sensory evaluation of beer produced with Alphalase Advance 4000 Force choice study with trained sensory taste panel Triangle tests on beers produced with Alphalase Advance 4000; Benchmark and control sample without ALDC enzyme applied No perceived difference between beer with Alphalase Advance 4000 and control without Set up Triangle test Which one is Different? Random No different Significant different Control versus Alphalase Advance 4000 Alphalase Advance 4000 Versus Benchmark Control Versus Benchmark Data Copyright 2016 DuPont. All rights reserved. 18

19 Residual activity (%) Foam, side activities and inactivation of Alphalase Advance 4000 in novel adjuncts No detectable proteolytic side activities and no adverse effects on foam stability ,0 5,0 10,0 15,0 20,0 25,0 30,0 35 PU Stability in an all malt beer, 5.7 ABV at ph 4.3; 70 C/ 158 F The residual activity is highly dependent on the specific temperature, ph and substrate and should always be tested under local conditions Copyright 2016 DuPont. All rights reserved. 19

20 Summary Alphalase Advance 4000 for diacetyl control Alphalase Advance 4000 for brewers everywhere around the world who are looking into consistent maturation periods increased beer volumes high flexibility towards many brewing protocols and process conditions without compromising the quality of their beer COPYRIGHT 2016 DUPONT. ALL RIGHTS RESERVED. 20

21 Thanks for your attention! Acknowledgements to: Sang-Kuy Lee Lene B. Jensen Tove Bladt Tina K. Jensen Rikke T. Madsen Charlotte Poulsen Aart Mateboer Iliana Yanez COPYRIGHT 2016 DUPONT. ALL RIGHTS RESERVED. 21

22 Copyright 2016 DuPont or its affiliates. All rights reserved. The DuPont Oval Logo, DuPont TM Danisco, DuPont TM and all products denoted with TM or are registered trademarks or trademarks of DuPont or its affiliates. The information contained in this presentation is based on our own research and development work and to our knowledge is reliable. Users should, however, conduct their own tests to determine the suitability of our products for their own specific purposes and the legal status for their intended use of the product. Statements contained in this presentation should not be considered as, and do not constitute a warranty of any kind, expressed or implied, and no liability is accepted for the infringement of any patents. 1/17/