Strategies To Reduce The Iron Intake During The Brewing Process With Respect To Flavour Stability

Strategies to Reduce the Iron Intake during the Brewing
Process with respect to Flavour Stability
duur Mertens
SƵƉĞƌǀŝƐŽƌƐ͗ ŝƐƐĞƌƚĂƚŝŽŶƉƌĞƐĞŶƚĞĚŝŶƉĂƌƚŝĂůĨƵůĮůŵĞŶƚŽĨƚŚĞƌĞƋƵŝƌĞŵĞŶƚƐĨŽƌƚŚĞ
Prof. B. Gibson ĚĞŐƌĞĞŽĨŽŬƚŽƌĚĞƌ/ŶŐĞŶŝĞƵƌǁŝƐƐĞŶƐĐŚĂĨƚĞŶĨƌŽŵdhĞƌůŝŶĂŶĚ
Prof. G. Aerts ŽĐƚŽƌŝŶŶŐŝŶĞĞƌŝŶŐdĞĐŚŶŽůŽŐǇĨƌŽŵ<hLeuven
EŽǀĞŵďĞƌ2023
/ŶƐƟƚƵƚĞŽĨ&ŽŽĚdĞĐŚŶŽůŽŐǇĂŶĚ&ŽŽĚŚĞŵŝƐƚƌǇ Faculty of Engineering Technology

Technische Universität Berlin Katholieke Universiteit Leuven
ƌĞǁŝŶŐĂŶĚĞǀĞƌĂŐĞdĞĐŚŶŽůŽŐǇ ŶǌǇŵĞ͕&ĞƌŵĞŶƚĂƟŽŶĂŶĚƌĞǁŝŶŐdĞĐŚŶŽůŽŐǇ
Strategies to Reduce the Iron Intake during the Brewing Process with
respect to Flavour Stability
vorgelegt von
Ing.
Tuur Mertens
ORCID: 0000-0002-3143-6099
an der Fakultät III – Prozesswissenschaften

der Technischen Universität Berlin
und der
Katholieke Universiteit Leuven in Gent, Belgien
(im Rahmen des Doppel-Promotionsabkommens)
zur Erlangung des akademischen Grades
Dr. der Ingenieurwissenschaften

- Dr.-Ing. -
genehmigte Dissertation
Promotionsausschuss:
Vorsitzender: Prof. Dr. Hajo Haase, Technische Universität Berlin

Gutachter: Prof. Dr. Brian Gibson, Technische Universität Berlin
Gutachter: Prof. Dr. Guido Aerts, Katholieke Universiteit Leuven
Gutachter: Prof. Dr. Mogens Andersen, Københavns Universitet
Gutachter: Prof. Dr. Florian Weiland, Katholieke Universiteit Leuven
Tag der wissenschaftlichen Aussprache: Berlin, 6 November 2023

an der Technische Universität Berlin, Berlin
Berlin 2023
Strategies to Reduce the Iron Intake during the Brewing Process with
respect to Flavour Stability
Tuur Mertens
Supervisors:
Prof. Brian Gibson, Technische Universität Berlin
Prof. Guido Aerts, Katholieke Universiteit Leuven
Co-Supervisors:
Dipl.-Ing. Thomas Kunz, Technische Universität Berlin
Members of the Examination Committee:

Prof. Maarten Vergauwen, Katholieke Universiteit Leuven (Chair)
Prof. Bart Lievens, Katholieke Universiteit Leuven Dissertation presented in
Prof. Mogens Andersen, Københavns Universitet partial fulfilment of the
Dr.-Ing. Philip Wietstock, Technische Universität Berlin requirements for the
Dr. Ing. Gert De Rouck, Katholieke Universiteit Leuven degree of Doctor in
Dr. Boris Gadzov, FlavorActiV Limited Engineering Technology
Belgium, 26 September 2023

“Where there is beer, there is life.”
IV Abstract
Abstract
Flavour stability is a key aspect of beer quality, but remains a major challenge for brewing science and
the brewing industry. The gradual deterioration of the organoleptic qualities of beer during ageing
significantly diminishes its value in several respects, including market appeal, palatability and overall
drinkability. While beer is inherently unstable due to its complex composition, strategic measures can
be implemented to enhance the initial freshness of the product and slow the rate of flavour
deterioration. The main objective of this thesis was to address this issue by specifically targeting the
detrimental effects of transition metals (iron, copper and manganese), which act as catalysts for the
generation of damaging radicals through Fenton and Haber-Weiss reactions. By sequestering and
subsequently removing these metal ions using chelating agents, their deleterious effects can be
avoided from the mashing stage onwards. A total of nineteen chelating agents were investigated for
their ability to form filterable complexes with transition metals in brewing relevant setups. These
included EDTA, citric acid, tartaric acid, quercetin, chlorogenic acid, ferulic acid, gallic acid, phytic acid
and tannic acid, as well as extracts derived from green tea, pomegranate, grapeseed, reishi, cinnamon,
curcuma, milk thistle, ginkgo, grapefruit seed and raspberry.
In the initial model study, it was revealed that a typical wort pH of 5.60 provided a more favourable
environment for the removal of transition metals through complex formation than a beer pH of 4.30.
Of the first nine chelators listed, tannic acid (a high molecular weight polyphenol) showed the greatest
efficacy. At wort pH, it resulted in significant reductions in iron and copper levels in solution after 0.2
µm microfiltration (-94.0 % FeII, -96.8 % FeIII, -98.3 % CuII) compared to chelator-free controls. Other
chelators also exhibited noticeable effects at pH 5.60, albeit to a lesser extent, namely quercetin (-34.6
% FeII, -96.2 % FeIII), chlorogenic acid (-90.2 % FeIII), gallic acid (-72.6 % FeIII, -39.9 % CuII) and ferulic acid
(-9.4 % FeII, -11.6 % FeIII). Phytic acid demonstrated an undesirable property in chelating zinc (-70.3 %
ZnII). Despite forming complexes, EDTA, citric acid and tartaric acid did not lead to reductions in metal
concentrations, as the chelates were able to pass through the microfilter.
A subsequent study, evaluating the performance of all nineteen chelators in wort during laboratory
scale mashing, reaffirmed the significant impact of pH on chelator efficacy and wort metal load.
Acidified mashing led to worts with higher levels of iron, manganese and zinc; with increases of 230 %,
320 % and 150 %, respectively, when the mash pH was lowered from 6.0 to 5.0. The effectiveness of
tannic acid, as observed in the wort buffer solution, was confirmed in wort for iron, but not for copper.
While none of the other chelators from the initial study retained their ability to substantially reduce
transition metal levels in lautered wort, two novel chelating agents, green tea and pomegranate
extract, were discovered among the ten alternative extracts tested in this study. In particular,
pomegranate extract (containing 90 % ellagic acid) exhibited remarkable capability in decreasing iron
content and suppressing radical formation in the wort. With a mash addition of 60 mg/L, it achieved
approximately 80 % reductions in both variables compared to chelator-free controls.
The final study investigated the effects of incorporating tannic acid, green tea and pomegranate
extract into pilot scale mashes and found that all three additives were effective in the removal of iron
during brewing. Among the twelve trials conducted, additions of pomegranate extract again
demonstrated the highest efficacy, resulting in a reduction of almost 90 % in iron level and 80 % in
radical concentration in the final wort. On average, the inclusion of these chelators led to a 40-60 %
reduction in total post-boil aldehydes. Overall, the findings suggest that natural chelators have the
potential to improve beer quality and flavour stability by reducing radical formation during brewing
and lowering the concentration of transition metals and aldehydes in the final product.
Zusammenfassung V
Zusammenfassung
Die Geschmacksstabilität ist ein Schlüsselaspekt der Bierqualität, stellt aber nach wie vor eine große
Herausforderung für die Brauwissenschaft und die Brauindustrie dar. Die allmähliche
Verschlechterung der organoleptischen Eigenschaften von Bier während der Alterung mindert seinen
Wert in vielerlei Hinsicht, einschließlich seiner Marktattraktivität, Schmackhaftigkeit und allgemeinen
Trinkbarkeit. Obwohl Bier aufgrund seiner komplexen Zusammensetzung von Natur aus instabil ist,
können strategische Maßnahmen ergriffen werden, um die anfängliche Frische des Produkts zu
verbessern und die Geschwindigkeit der geschmacklichen Verschlechterung zu verlangsamen. Das
Hauptziel dieser Arbeit war es, dieses Problem zu lösen, indem die schädlichen Auswirkungen von
Übergangsmetallen (Eisen, Kupfer und Mangan), die als Katalysatoren bei der Bildung von schädlichen
Radikalen durch Fenton- und Haber-Weiss-Reaktionen wirken, angegangen werden. Durch die
Sequestrierung und anschließende Entfernung dieser Metallionen mit Hilfe von Chelatbildnern können
ihre schädlichen Auswirkungen bereits in der Maischephase vermieden werden. Insgesamt wurden
neunzehn Chelatbildner auf ihre Fähigkeit, mit Übergangsmetallen filtrierbare Komplexe zu bilden, in
braurelevanten Versuchsanordnungen untersucht. Dazu gehörten EDTA, Zitronensäure, Weinsäure,
Quercetin, Chlorogensäure, Ferulasäure, Gallussäure, Phytinsäure, Tanninsäure sowie Extrakte aus
grünem Tee, Granatapfel, Traubenkernen, Reishi, Zimt, Kurkuma, Mariendistel, Ginkgo,
Grapefruitkernen und Himbeeren.
In der ersten Modellstudie wurde festgestellt, dass ein für Würze typischer pH-Wert von 5.6 ein
günstigeres Umfeld für die Entfernung von Übergangsmetallen durch Komplexbildung bietet, als ein
Bier-pH-Wert von 4.30. Von den ersten neun aufgeführten Chelatbildnern zeigte Tanninsäure (ein
Polyphenol mit hohem Molekulargewicht) die größte Wirksamkeit. Bei einem Würze-pH-Wert
entfernte diese nach 0.2 µm Mikrofiltration signifikant Eisen und Kupfer aus der Lösung (-94.0 % FeII, -
96.8 % FeIII, -98.3 % CuII) im Vergleich zu chelatorfreien Kontrollen. Andere Chelatbildner zeigten bei
pH 5.60 ebenfalls signifikante Effekte, wenn auch in geringerem Ausmaß, nämlich Quercetin (-34.6 %
FeII, -96.2 % FeIII), Chlorogensäure (-90.2 % FeIII), Gallussäure (-72.6 % FeIII, -39.9 % CuII) und Ferulasäure
(-9.4 % FeII, -11.6 % FeIII). Phytinsäure zeigte eine unerwünschte Eigenschaft durch Chelatbildung mit
Zink (-70.3 % ZnII). EDTA, Zitronensäure und Weinsäure führten trotz Komplexbildung nicht zu einer
Verringerung der Metallkonzentrationen, da die Chelate den Mikrofilter passieren konnten.
Eine nachfolgende Studie, in der die Leistung aller neunzehn Chelatbildner in Würze während des
Maischens im Labormaßstab bewertet wurde, bestätigte erneut den signifikanten Einfluss des pH-
Werts auf die Wirksamkeit der Chelatbildner und die Metallbelastung der Würze. Die angesäuerte
Würze führte zu Würzen mit höheren Gehalten an Eisen, Mangan und Zink, die um 230 %, 320 % bzw.
150 % zunahmen, wenn der pH-Wert der Würze von 6.0 auf 5.0 gesenkt wurde. Die in der
Würzepufferlösung beobachtete Wirksamkeit der Tanninsäure wurde in der Würze für Eisen, aber
nicht für Kupfer bestätigt. Während keiner der anderen Chelatbildner aus der ersten Studie seine
Fähigkeit, den Gehalt an Übergangsmetallen in der Läuterwürze signifikant zu senken, beibehielt,
wurden unter den zehn alternativen Extrakten, die in dieser Studie getestet wurden, zwei neue
Chelatbildner entdeckt: Grüner Tee und Granatapfelextrakt. Insbesondere der Granatapfelextrakt (der
zu 90 % aus Ellagsäure besteht) zeigte eine bemerkenswerte Fähigkeit, den Eisengehalt zu senken und
die Radikalbildung in der Würze zu unterdrücken. Bei einer Würzezugabe von 60 mg/L wurden beide
Größen im Vergleich zu chelatorfreien Kontrollen um ca. 80 % reduziert.
Die letzte Studie untersuchte die Auswirkungen der Zugabe von Tanninsäure, grünem Tee und
Granatapfelextrakt zu Maischen im Pilotmaßstab und stellte fest, dass alle drei Zusatzstoffe bei der
VI Zusammenfassung
Entfernung von Eisen während des Brauens wirksam waren. Von den zwölf durchgeführten Versuchen
erwies sich wiederum die Zugabe von Granatapfelextrakt als am effektivsten und führte zu einer
Reduktion des Eisengehalts um fast 90 % und der Radikalenkonzentration in der Endwürze um 80 %.
Im Durchschnitt führte die Zugabe dieser Chelatoren zu einer Verringerung der
Gesamtaldehydkonzentration nach dem Kochen um 40-60 %. Insgesamt deuten die Ergebnisse darauf
hin, dass natürliche Chelatoren das Potenzial haben, die Bierqualität und Geschmacksstabilität zu
verbessern, indem sie die Bildung von Radikalen während des Brauprozesses reduzieren und die
Konzentration von Übergangsmetallen und Aldehyden im Endprodukt senken.
Samenvatting VII
Samenvatting
Smaakstabiliteit is een belangrijk aspect van bierkwaliteit, maar blijft een grote uitdaging voor de
brouwwetenschap en de brouwerijsector. De geleidelijke achteruitgang van de organoleptische
kwaliteiten van bier tijdens het verouderen vermindert diens waarde aanzienlijk in verscheidene
opzichten, waaronder de marktaantrekkelijkheid, smakelijkheid en algemene drinkbaarheid. Hoewel
bier van nature instabiel is vanwege zijn complexe samenstelling, kunnen strategische maatregelen
worden genomen om de initiële versheid van het product te verbeteren en de snelheid van de
smaakafbraak te vertragen. Het hoofddoel van dit proefschrift was om dit probleem tegen te gaan
door specifiek de nadelige effecten van overgangsmetalen (ijzer, koper en mangaan) aan te pakken,
aangezien deze fungeren als katalysatoren bij het genereren van schadelijke radicalen via Fenton- en
Haber-Weiss-reacties. Door deze metaalionen te sequestreren en vervolgens te verwijderen met
chelatoren, kunnen hun schadelijke effecten vanaf het maischen worden vermeden. In totaal werden
negentien chelatoren onderzocht op hun vermogen om filtreerbare complexen te vormen met
overgangsmetalen in brouwrelevante omstandigheden. Deze omvatten EDTA, citroenzuur,
wijnsteenzuur, quercetine, chlorogeenzuur, ferulinezuur, galluszuur, fytinezuur, looizuur, evenals
extracten van groene thee, granaatappel, druivenpit, reishi, kaneel, kurkuma, mariadistel, ginkgo,
pompelmoespit en framboos.
In de initiële modelstudie bleek dat een voor wort typische pH van 5.60 een gunstigere omgeving bood
voor de verwijdering van overgangsmetalen door complexvorming dan een bier-pH van 4.30. Van de
eerste negen opgelijste chelatoren bleek looizuur (een polyfenol met hoog moleculair gewicht) het
meest effectief. Bij wort-pH verwijderde het ijzer en koper aanzienlijk uit de oplossing na 0.2 µm
microfiltratie (-94.0 % FeII, -96.8 % FeIII, -98.3 % CuII) in vergelijking met chelatorvrije controlestalen.
Ook andere chelatoren vertoonden merkbare effecten bij pH 5.60, zij het in mindere mate, namelijk
quercetine (-34.6 % FeII, -96.2 % FeIII), chlorogeenzuur (-90.2 % FeIII), galluszuur (-72.6 % FeIII, -39.9 %
CuII) en ferulinezuur (-9.4 % FeII, -11.6 % FeIII). Fytinezuur vertoonde een ongewenste eigenschap in het
cheleren van zink (-70.3 % ZnII). Ondanks het vormen van complexen leidde EDTA, citroenzuur en
wijnsteenzuur niet tot een verlaging van de metaalconcentraties, omdat de chelaten de microfilter
doordrongen.
Een vervolgstudie, waarin de werking van alle negentien chelatoren werd geëvalueerd in wort tijdens
het maischen op laboratoriumschaal, bevestigde opnieuw de significante invloed van pH op de
werkzaamheid van chelatoren en de metaalbelasting van het wort. Aangezuurd maischen leidde tot
wort met hogere gehaltes aan ijzer, mangaan en zink; met toenames van respectievelijk 230 %, 320 %
en 150 % wanneer de pH van het beslag werd verlaagd van 6.0 naar 5.0. De effectiviteit van looizuur,
zoals waargenomen in de wort-bufferoplossing, werd bevestigd in wort voor ijzer, maar niet voor
koper. Terwijl geen van de overige chelatoren uit het initiële onderzoek hun vermogen behielden om
het gehalte aan overgangsmetalen in het geklaarde wort substantieel te verlagen, werden er twee
nieuwe chelatoren, groene thee en granaatappelextract, ontdekt onder de tien alternatieve extracten
die in dit onderzoek werden getest. Vooral granaatappelextract (met 90 % ellaginezuur) bleek
opmerkelijk in staat het ijzergehalte te verlagen en de vorming van radicalen in het wort te
onderdrukken. Met een beslagtoevoeging van 60 mg/L zorgde het voor een circa 80 % reductie in beide
variabelen, vergeleken met chelatorvrije controles.
Het laatste onderzoek onderzocht de effecten van het toevoegen van looizuur, groene thee en
granaatappelextract aan pilootschaal-maischen en ontdekte dat alle drie de toevoegingen effectief
waren in het verwijderen van ijzer tijdens het brouwen. Van de twaalf uitgevoerde proeven toonden
VIII Samenvatting
de addities van granaatappelextract opnieuw de hoogste effectiviteit, met een reductie van bijna 90
% van het ijzergehalte en 80 % van de radicaalconcentratie in het finale wort. Gemiddeld leidde de
toevoeging van deze chelatoren tot een vermindering van 40-60 % van de totale aldehyden na het
koken. In het algemeen suggereren de bevindingen dat natuurlijke chelatoren de bierkwaliteit en
smaakstabiliteit kunnen verbeteren door de vorming van radicalen tijdens het brouwen te
verminderen en de concentratie van overgangsmetalen en aldehyden in het eindproduct te verlagen.
List of Contents IX
List of Contents
ABSTRACT ---------------------------------------------------------------------------------------------------------------------------------- IV
ZUSAMMENFASSUNG -------------------------------------------------------------------------------------------------------------------- V
SAMENVATTING ------------------------------------------------------------------------------------------------------------------------- VII
LIST OF CONTENTS ----------------------------------------------------------------------------------------------------------------------- IX
DECLARATION OF ORIGINALITY ------------------------------------------------------------------------------------------------------- X
ACKNOWLEDGEMENTS ----------------------------------------------------------------------------------------------------------------- XI
LIST OF ABBREVIATIONS -------------------------------------------------------------------------------------------------------------- XII
LIST OF PUBLICATIONS ---------------------------------------------------------------------------------------------------------------- XIV
AUTHOR CONTRIBUTIONS ------------------------------------------------------------------------------------------------------------------------ XIV
1. THESIS INTRODUCTION ----------------------------------------------------------------------------------------------------------- 1
2. LITERATURE REVIEW -------------------------------------------------------------------------------------------------------------- 2
2.1. PREAMBLE --------------------------------------------------------------------------------------------------------------------------------2
2.2. BREWING, OXIDATIVE STABILITY AND BEER STALING ------------------------------------------------------------------------------------ 4
2.3. CHELATION IN THE BEVERAGE INDUSTRY ---------------------------------------------------------------------------------------------- 11
3. RESEARCH QUESTIONS & THESIS OVERVIEW----------------------------------------------------------------------------- 16
4. MATERIALS & METHODS ------------------------------------------------------------------------------------------------------- 19
4.1. CHEMICALS, CONSUMABLES, EQUIPMENT AND SOFTWARE --------------------------------------------------------------------------- 20
4.2. SAMPLE PREPARATION ----------------------------------------------------------------------------------------------------------------- 25
4.3. ANALYTICAL METHODS ----------------------------------------------------------------------------------------------------------------- 27
4.4. STANDARD BEER ANALYSES ------------------------------------------------------------------------------------------------------------ 28
4.5. DATA ANALYSIS------------------------------------------------------------------------------------------------------------------------- 28
5. RESULTS ---------------------------------------------------------------------------------------------------------------------------- 29
5.1. ASSESSMENT OF CHELATORS IN WORT AND BEER MODEL SOLUTIONS ---------------------------------------------------------------- 29
5.2. COMPLEXATION OF TRANSITION METALS BY CHELATORS ADDED DURING MASHING AND IMPACT ON BEER STABILITY-------------- 32
5.3. EFFECTS OF MASH CHELATOR ADDITION ON TRANSITION METAL CONTENT AND OXIDATIVE STABILITY OF BREWER’S WORT ------- 38
5.4. APPLICATION OF PUNICALAGIN/ELLAGIC ACID TO IMPROVE OXIDATIVE AND COLLOIDAL STABILITY OF BEVERAGES (ESP. BEER)---- 42
6. SUMMARISING DISCUSSION -------------------------------------------------------------------------------------------------- 45
7. CONCLUSION & OUTLOOK ----------------------------------------------------------------------------------------------------- 50
8. REFERENCES ----------------------------------------------------------------------------------------------------------------------- 52
9. THESES ------------------------------------------------------------------------------------------------------------------------------ 64
PUBLICATIONS --------------------------------------------------------------------------------------------------------------------------- 65
TRANSITION METALS IN BREWING AND THEIR ROLE IN WORT AND BEER OXIDATIVE STABILITY: A REVIEW -------------------------------------- 67
ASSESSMENT OF CHELATORS IN WORT AND BEER MODEL SOLUTIONS ------------------------------------------------------------------------- 89
COMPLEXATION OF TRANSITION METALS BY CHELATORS ADDED DURING MASHING AND IMPACT ON BEER STABILITY ----------------------- 101
EFFECTS OF MASH CHELATOR ADDITION ON TRANSITION METAL CONTENT AND OXIDATIVE STABILITY OF BREWER’S WORT ------------ 125
APPLICATION OF PUNICALAGIN/ELLAGIC ACID TO IMPROVE OXIDATIVE AND COLLOIDAL STABILITY OF BEVERAGES (ESP. BEER) ------------- 143
APPENDIX -------------------------------------------------------------------------------------------------------------------------------- 187
I. OTHER CONTRIBUTIONS-------------------------------------------------------------------------------------------------------------- 187
X Declaration of Originality
Declaration of Originality
The work presented was carried out at the Chair of Brewing and Beverage Technology, Institute of
Food Technology and Food Chemistry (TU Berlin, Berlin, Germany) and the Laboratory of Enzyme,
Fermentation and Brewing Technology, Department of Microbial and Molecular Systems, Faculty of
Engineering Technology (KU Leuven, Ghent, Belgium).
I hereby declare that this thesis and the work reported herein are entirely my original work. I am fully
aware of the regulations of both universities regarding plagiarism and the potential disciplinary
consequences that may arise from any act of plagiarism. Any information derived from the published
or unpublished work of others is appropriately acknowledged in the text and complete references are
provided in the reference list.
Berlin, 2023
Tuur Mertens
Acknowledgements XI
Acknowledgements
Completing this PhD was more than just an academic challenge. Above all, it has been a deeply
personal and transformative one. Not only did it take me across many European borders, introducing
me to numerous countries and cultures, it also pushed me beyond my own borders and allowed me
to discover new aspects of myself.
First and foremost, I would like to offer my felicitations to Dr. hab. Aleksander Poreda and the entire
EJD consortium for their remarkable achievement in bringing the European Joint Doctorate in Food
Science project to fruition. Special thanks go to the Universities of Berlin, Leuven, Kraków,
Copenhagen, Ghent and Nottingham, as well as the institutions VLB, FlavorActiV, Carlsberg and
Boortmalt for providing us with exceptional courses and training.
None of this would have been possible without the generous support of the European Union's Horizon
2020 research and innovation programme, which funded this project under the Marie Skłodowska-
Curie grant agreement No. 722166. While the project’s catchwords ‘young, brave and ambitious’ never
failed to amuse, their significance was not lost on us. In the same spirit, I would also like to express my
gratitude towards Prof. Frank-Jürgen Methner for believing in me and granting me the opportunity to
be a part of the EJD adventure.
What made the EJD feel like a family, rather than a project focused solely—or soullessly—on reaching
the next milestone, were the fellow doctoral students who embarked on this journey with me. Celina,
Jonas, Maciej, Magda, Marcus and Weronika, I extend my heartfelt thanks to each of you. Really. Your
presence together made everything so much more meaningful and I could not imagine a better team.
Dear Prof. De Cooman, dear Luc. I greatly regret that our time together was cut short by your untimely
passing. Know that the impression you made on me was deep and lasting, and I hope you would have
enjoyed this work. To my current supervisors, Prof. Brian Gibson and Prof. Guido Aerts, I am sincerely
grateful for your guidance, freedom and support whenever I sought it. My warmest thanks go to Philip
Wietstock, Thomas Kunz and Gert De Rouck for their expert insights and for always being there to offer
an educated opinion—often over a few beers—whenever I felt stuck (quite often), needed advice or
simply someone to vent to (also quite often).
To my colleagues and the students at the Laboratory of Enzyme, Fermentation and Brewing
Technology (KU Leuven) and the Chair of Brewing and Beverage Technology (TU Berlin), thank you for
your assistance, camaraderie and the countless beers, laughs and stories shared. To Jeroen, Jasper,
Wouter, Katharina, Agata, Paula, Markus, Giovanni, Natalia, Frederico, Mariana, Matthias, Torsten,
Christian, and many others, I am grateful for our time together.
Last, but certainly not least, I want to express my deepest appreciation to my partner, Nina. Thank you
for standing by me all these years. I know it hasn't always been easy, but I'm positive that together,
despite the difficulties, we have grown stronger and more resilient. I hope that makes up for the
abundance of patience you have shown. Also, lots of love to my friends and family for always being
there to support me and for providing me with a sense of grounding.
Cheers!
Tuur
XII List of Abbreviations
List of Abbreviations
(n/µ/m/h)-L/g/m/M (nano/micro/milli/hecto)-litre/gram/metre/mol per litre
(R)SD (relative) standard deviation
°C degrees Celsius
°P degrees Plato
5-HMF 5-hydroxymethyl furfural
ABTS 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)
ABV alcohol by volume
ANOVA analysis of variance
BAX beverage antioxidant index
BSA bovine serum albumin
Ca calcium
CCD charge coupled device
CGC-MS capillary gas chromatography-mass spectrometry
CL chemiluminescence
CMC carboxymethylcellulose
CO2 carbon dioxide
Cu/CuI/CuII copper/cuprous/cupric
CUPRAC cupric reducing antioxidant capacity
DI deionised
DMS dimethyl sulphide
DO dissolved oxygen
DOE design of experiments
DPPH 2,2-diphenyl-1-picrylhydrazyl
DTPA diethylenetriaminepentaacetic acid
e.g. exempli gratia
EAP endogenous antioxidant potential
EBC European brewery convention
EDTA ethylenediaminetetraacetic acid
EGTA egtazic acid
EJD European joint doctorate
ESR electron spin resonance
et al. et alii
etc. et cetera
FAN free amino nitrogen
Fe/FeII/FeIII iron/ferrous/ferric
FIFO first-in, first-out
FRAP ferric reducing antioxidant power
GC gas chromatography
H2 O 2 hydrogen peroxide
HO•2 perhydroxyl (radical)
HSA hot side aeration
HSO head space oxygen
HS-SPME headspace-solid phase microextraction
i.a. inter alia
i.e. id est
IBU international bitterness unit
ICP-OES inductively coupled plasma optical emission spectrometry
KU(L) katholieke universiteit (Leuven)
List of Abbreviations XIII
LA linoleic acid
LODO low dissolved oxygen
LOQ limit of quantification
LOX lipoxygenase
MBT 3-methyl-2-butene-1-thiol
MEBAK mitteleuropäische brautechnische analysenkommission
Mg magnesium
min. minutes
Mn manganese
MRPs Maillard reaction products
MS mass spectrometry
MW molecular weight
NIR near-infrared
O2 (di)oxygen
O2•- superoxide anion
O22- peroxide
OH• hydroxyl (radical)
ORAC oxygen radical absorbance capacity
PBN N-tert-butyl-α-phenylnitrone
PCT peroxide challenge test
PDMS/DVB polydimethylsiloxane/divinylbenzene
PET polyethylene terephthalate
PFBHA O-(2,3,4,5,6-pentafluorobenzyl)-hydroxylamine-hydrochloride
POBN α(4-pyridyl-1-oxide)-N-tert-butylnitrone
ppb parts per billion
ppm parts per million
PVPP polyvinylpolypyrrolidone
RF radio frequency
ROS reactive oxygen species
RT room temperature
sec. seconds
SI stability index
SMM S-methylmethionine
SO2 sulphur dioxide
T/C trans/cis
TBA thiobarbituric acid
TBI thiobarbituric acid index
TEAC Trolox-equivalent antioxidant capacity
TL;DR too long; didn't read
TPO total package oxygen
TRAP total reactive antioxidant potential
TU(B) technische universität (Berlin)
UV/Vis ultraviolet–visible
v/v volume by volume
w/w weight by weight
Zn zinc
Δ delta (change/variation)
XIV List of Publications
List of Publications
This thesis draws upon a series of original publications, referred to as Paper I-V:
I Mertens, Tuur; Kunz, Thomas; Gibson, Brian R. 2022. Transition metals in brewing and their
role in wort and beer oxidative stability: a review. Journal of the Institute of Brewing.
DOI: 10.1002/jib.699
II Mertens, Tuur; Kunz, Thomas; Methner, Frank-Jürgen. 2020. Assessment of Chelators in
Wort and Beer Model Solutions. BrewingScience.
DOI: 10.23763/BrSc20-01mertens
III Mertens, Tuur; Kunz, Thomas; Wietstock, Philip C.; Methner, Frank-Jürgen. 2021.
Complexation of transition metals by chelators added during mashing and impact on beer
stability. Journal of the Institute of Brewing.
DOI: 10.1002/jib.673
IV Mertens, Tuur; Kunz, Thomas; De Rouck, Gert; Gibson, Brian R.; Aerts, Guido; De Cooman,
Luc†. 2023. Effects of Mash Chelator Addition on Transition Metal Content and Oxidative
Stability of Brewer’s Wort. BrewingScience.
V Mertens, Tuur; Kunz, Thomas. 2021. Application of punicalagin/ellagic acid to improve
oxidative and colloidal stability of beverages (esp. beer). Technische Universität Berlin.
Patent: WO2021170827A1
Author contributions
I Mertens, Tuur: Writing - Original draft.

Kunz, Thomas: Review & Editing (co-supervisor).
Gibson, Brian R.: Review & Editing (supervisor).
II Mertens, Tuur: Conceptualisation & Design, Project administration, Experimentation, Formal
analysis, Data interpretation & Visualisation, Writing - Original draft.
Kunz, Thomas: Conceptualisation, Data interpretation, Review & Editing (co-supervisor).
Methner, Frank-Jürgen: Funding acquisition (supervisor).
III Mertens, Tuur: Conceptualisation & Design, Project administration, Experimentation, Model
creation, Formal analysis, Data interpretation & Visualisation, Writing - Original draft.
Kunz, Thomas: Conceptualisation, Data interpretation, Review & Editing (co-supervisor).
Wietstock, Philip C: Conceptualisation, Data interpretation, Review & Editing.
Methner, Frank-Jürgen: Funding acquisition (supervisor).
IV Mertens, Tuur: Conceptualisation & Design, Project administration, Experimentation, Formal
analysis, Data interpretation & Visualisation, Writing - Original draft.
Kunz, Thomas: Verification, Data interpretation, Review & Editing (co-supervisor).
De Rouck, Gert: Verification, Data interpretation, Review & Editing (co-supervisor).
Gibson, Brian R.: Data interpretation, Review & Editing (supervisor).
Aerts, Guido: Data interpretation, Review & Editing (supervisor).
List of Publications XV
De Cooman, Luc†: Data interpretation, Funding acquisition (supervisor).

V Mertens, Tuur: Scientific discovery (Conceptualisation & Design), Research work
(Experimentation, Model creation, Formal analysis, Data interpretation & Visualisation),
Contribution to patent writing and development.
Kunz, Thomas: Patent arrangement, Significant contribution to patent writing and
development (co-supervisor).
Thesis introduction 1
1. Thesis introduction
Throughout production, packaging and storage—and even as early as malting—beer is subject to a

multifaceted interplay of diverse phenomena that affect its stability. Staling, often referred to as
ageing, is the process by which off-flavours and unsavoury aroma compounds manifest over time,
resulting in an overall decline of freshness and vibrancy. Beer typically develops notes of cardboard or
paper, ribes or black currant, nuttiness and sherry-like flavours [1].
Staling is a complex and intricate culmination of numerous chemical reactions. Nevertheless, it is

recognised that a prominent part of its mechanism is O2-driven oxidation [2, 3], making radical-
mediated processes, by extension, very relevant. Reactive oxygen species (ROS) are the primary radical
entities generated during brewing and storage and play a central role in the oxidation of wort and beer
constituents [4, 5]. Radicals can accelerate the formation of off-flavours through the degradation of
alcohols, fatty acids, hop components, polyphenols, lipids, amino acids, sulphite, etc. [6–8].
Catalytic transition metal ions, including iron, copper and manganese, are key factors influencing ROS
formation. They initiate and propagate (Fenton and Haber-Weiss) reactions that generate highly
reactive hydroxyl radicals (OH•) and other ROS [9, 10]. A deeper understanding of the significance of
these metals in the oxidative degradation of wort and beer is imperative in the challenge to enhance
beer flavour stability. Reducing their negative effects could prove vital in extending shelf life.
This dissertation aims to test the hypothesis that depletion of the transition metal ion pool during the
brewing process can contribute to improved oxidative stability of the wort and, hence, improved
quality and flavour stability of the resulting beer. To achieve this goal, the study proposes an innovative
approach involving the addition of natural chelators during brewing. Chelators, known for their ability
to bind and sequester metal ions, offer a promising way to reduce the availability of catalytic metals
and limit the production of ROS throughout the brewing process.
By incorporating chelating agents during mashing, it is hypothesised that the complexation and
removal of Fe, Cu and Mn will prevent their involvement in radical reactions, leading to reduced
oxidative degradation. This research will investigate the efficacy of nineteen chelators, including tannic
acid, pomegranate and green tea extract, and their impact on staling. Factors such as chelator
concentration, addition time, grain bill and pH were examined on a laboratory scale to determine
optimal conditions for minimising transition metal-mediated oxidation. Further brewing conditions
and potential interactions with wort components (e.g. protein, FAN) were explored at pilot scale.
Addressing our incomplete knowledge of transition metals and chelators in brewing is critical to
advancing our understanding of staling and developing novel strategies to preserve beer flavour
quality over extended periods of time. The outcomes of this research may have significant implications
for the brewing industry, as it has the potential to guide the development of innovative brewing
practices and processing aids to improve the longevity and sensory attributes of beer.
In conclusion, this dissertation aims to explore the role of iron, copper and manganese in oxidative
beer staling and investigate the effectiveness of chelators in depleting these metals during mashing.
By bridging this knowledge gap, we aspire to contribute valuable insights into beer staling and propose
practical solutions to enhance the quality and shelf life of beer.
2 Literature Review - Preamble
2. Literature Review (Paper I)
To improve oxidative wort and beer stability, it is important to understand the dynamics of transition
metals and their effects on staling throughout the brewing process. A comprehensive review article
[11] has been published on this topic and forms an integral component of this thesis (Paper I). The
review discusses the broad facets of oxidative and flavour stability, including the mechanisms of beer
staling, techniques for determining staleness and staling potential, and current methods for preventing
beer ageing. Particular emphasis is placed on the critical role of iron, copper and manganese as
oxidation catalysts during brewing and storage, highlighting the importance of removing or inhibiting
prooxidative transition metal ions to achieve prolonged shelf life.
Building upon the foundation laid by Paper I, this Literature Review section delves deeper into specific
aspects that are pertinent to the thesis but were not fully explored or extensively discussed in the
review article.
2.1. Preamble
The brewing sector is an important industry worldwide, with beer production and distribution
contributing to employment opportunities and overall economic growth [12]. Unfortunately, the
inherent instability of beer as a commodity means that it inevitably deteriorates during storage. As
beer ages, undesirable changes occur, such as colour darkening, haze formation, loss of crispness and
the development of off-flavours [8]. The time it takes for these changes to become noticeable under
standard storage conditions typically determines a beer's shelf life and can be as little as three months
[13].
This presents a challenge to the maintenance of internal quality standards, particularly when it comes
to cost-effective methods of exporting beer internationally. Flavour instability of beer can lead to
consumer dissatisfaction and reduced demand, which is why breweries worldwide invest significant
resources in modern equipment, quality raw materials, novel processing aids, industry production
standards, innovative packaging, and controlled storage and distribution to ensure adequate beer shelf
life. By maintaining consistent quality and flavour profiles over time, brewers can build brand loyalty,
increase market share and maintain profitability [14, 15].
Beyond financial considerations, there is the matter of sustainability. More than ever, there is a need
to minimise the environmental footprint associated with food production. By extending the longevity
of food and drink, including beer, the unnecessary disposal of expired products can be mitigated.
Furthermore, as craftsmen, brewers want consumers to enjoy their beer the way it was intended and
to provide fellow beer enthusiasts with a pleasurable drinking experience.
To ensure optimum beer freshness and shelf life, several factors must be controlled, including oxygen
exposure, heat stress, transition metal content, sulphite levels, pH, wort/beer composition, packaging,
storage time and temperature, etc. [8, 16, 17]. Most of these parameters can be addressed by the
strategies briefly mentioned above, of which some specific examples are given below:
Literature Review - Preamble 3
- Implementation of modern equipment:

o Replacing traditional copper vessels with contemporary stainless steel ones to
reduce copper-mediated oxidation [18].
o Employing efficient mash filters, whirlpools and wort coolers for rapid filtration,
clarification and chilling of hot wort to minimise accumulated heat stress and oxygen
exposure time [19].
o Minimising shear forces, e.g. during wort transfer, to reduce oxygen pickup [20].
o Using well-tuned packaging machines to achieve ultra-low levels of total package
oxygen (TPO) [21].
- Use of quality raw materials:
o Refining malt choice to reduce the amount of aldehydes, radicals, metals and staling
precursors it releases by selecting malts with minimal levels of these, taking into
account factors such as low malt colour, lipoxygenase (LOX) activity, free amino acids
(FAN) and heat load [22, 23].
o Optimising hop cultivar selection (e.g. low transition metal content) and hopping
technology/regime (e.g. use of continuous hop dosing) [24–28].
o Implementing water treatment procedures, such as sand filtration and reverse
osmosis, to ensure minimal concentrations of dissolved substances, including
transition metal ions [29].
o Prioritising yeast health and optimal fermentation performance to ensure minimum
levels of unreduced aldehydes, transition metal ions, dissolved oxygen and
appropriate amounts of yeast-produced sulphite antioxidant [30–32].
- Utilisation of novel processing aids:
o Adding antioxidants and chelating agents to quench free radicals and reduce radical
formation.
o Applying clarifying agents to shorten the maturation period, thereby reducing the
contact time with any oxygen that may be present in the bright beer tank headspace
[33].
o Incorporating sulphite additives as effective scavengers of free radicals and active
oxygen species [34].
- Adherence to industry production standards:
o Avoiding hot side aeration or upstream oxidation, e.g. by gentle transfer and no
splashing of hot wort [35].
o Minimising long periods of high temperature, e.g. during whirlpool rest or beer
pasteurisation.
o Using deaerated water for brewing and packaging purposes to minimise oxygen
loading.
o Flushing hoses and tanks with CO2 or N2 to minimise oxidation. Avoiding excessive
head space in lagering or bright beer tanks [36].
o Employing wet milling to reduce LOX activity in the mash [37].
o Clarifying beer by centrifugation, membrane filtration or using alternative filtration
aids other than kieselguhr/diatomaceous earth to prevent the extraction of soluble
iron into the beer [38–40].
4 Literature Review - Brewing, oxidative stability and beer staling
- Adoption of innovative packaging:

o Choosing packaging options that minimise oxygen ingress, such as cans or oxygen
scavenging bottle caps [41].
o Opting for non-clear or non-green bottles when using glass, to provide better light
protection and prevent light-induced degradation [42].
- Ensuring controlled storage and distribution:
o Implementing temperature control during transport and storage, also known as cold
chain management (arguably the greatest benefactor of beer shelf life, but costly)
[43].
o Avoiding direct light exposure and minimising vibration during storage and
distribution [44].
o Introducing a stock rotation system to ensure first in, first out (FIFO) distribution
[45].
Each of these aspects could be extensively explored in detail, and it should be emphasised that this list
is by no means exhaustive. However, the primary focus of this thesis is the investigation and
identification of novel chelating agents—as a type of processing aid—with the specific aim of
improving beer flavour stability by applying them during brewing.
The following subsection gives a general and broad introduction to brewing, oxidative stability and
beer staling. In Subsection 2.3, chelation and chelators are discussed in more detail. Although these
topics have already been touched upon in the review article (Paper I), they are core aspects of the
thesis and will be further elaborated upon here.
2.2. Brewing, oxidative stability and beer staling
The many steps involved in the brewing process are illustrated in Figure 1. Oxygen will be present to
some extent during each of these stages. When oxygen is introduced during high-temperature steps
(from mashing up to whirlpooling), it is referred to as hot side aeration (HSA) or upstream oxidation.
Conversely, when oxygen is introduced during low-temperature steps (from cooling up to packaging),
it is known as cold side aeration or downstream oxidation.
Literature Review - Brewing, oxidative stability and beer staling 5
Figure 1: The brewing process and potential zones for oxidative reactions.
To ensure the production of a high-quality beer with a suitable shelf life, it is essential to minimise
oxygen ingress as much as possible, starting at the packaging stage and continuing to optimise the
process from the bottom up. The methods mentioned above are commonly used for this reason. In
addition to the production process, the shelf life of beer will be determined by its raw materials and
storage conditions [16, 46, 47]. As a consequence, each beer (style) undergoes a unique ageing
process, and different storage temperature ranges lead to different pathways of deterioration,
resulting in varying staling compounds [48, 49].
Nevertheless, as beer ages, there is an inevitable natural decline in colloidal, foam, colour and flavour
stability, usually resulting from oxidation [16]. The resilience of wort and beer to oxidation is commonly
known as 'oxidative stability', whereas 'flavour stability' refers to a beer's capacity to delay flavour
deterioration and off-flavours. Whilst, as mentioned above, each beer type will have its unique ageing
characteristics, a general representation of sensory changes in beer flavour during storage was
provided by Dalgliesh in 1977 [50], an updated version of which is shown in Figure 2. It is important to
note that such figures represent a simplified view of a complex reality and should be interpreted with
caution.
Figure 2: Graphical representation of the typical changes in flavour characteristics observed during
accelerated ageing of a conventional beer. Adapted with permission from reference [51].
As beer is stored over time, certain flavours will diminish, while others may develop/intensify and
exceed their flavour thresholds. Unlike spirits such as whisky, which benefit from prolonged
maturation [52], these changes in beer flavour are often undesirable. Sulphur notes, initially present
and characteristic of young/fresh beer (especially in lagers), diminish relatively quickly and can be
considered off-flavours if too pronounced [53, 54]. As the ageing process continues, typically desired
beer characteristics (e.g. vibrant hop aroma, floral and fruity/estery notes) and bitterness quality tend
to decrease, often in synergy with an increase in sweetness [16]. The beer becomes dull and might
even turn astringent and harsh [55]. Sherry-like, caramel, nutty and bready flavours can become more
prominent [56, 57]. Additionally, notes reminiscent of cardboard or paper—strongly associated with
staleness—may appear, although these can also dissipate with further ageing [48, 56, 57].
This cardboard/papery flavour in aged beer has been shown to be intricately linked to the development
of trans-2-nonenal (an aldehyde derived from lipid oxidation with a very low flavour threshold of 0.1
µg/L) [58]. Since its identification around 1970 [59, 60], the molecule has been a focal point in the
study of beer flavour stability. However, while trans-2-nonenal undoubtedly has significance as a
component involved in staling, its presence during beer ageing is not always consistent, nor does it
singularly account for the full complexity that is beer staleness. Subsequent research has
demonstrated the contribution of numerous other components, leading to the realisation that stale
flavour is the result of a multitude of beer flavour compounds emerging and dissipating [16]. As a
result, scientists presently use a number of molecules and staling indicators to comprehensively
monitor beer oxidation. In addition to trans-2-nonenal, other relevant markers include Strecker
aldehydes (e.g. phenylacetaldehyde, methional, 3-methylbutanal, 2-methylbutanal and 2-
methylpropanal; which tend to increase with higher oxygen levels) [56, 61, 62], furfural (whose
concentration varies logarithmically with storage temperature) [56, 61, 63], hexanal (from lipid
oxidation) [61], polyphenols [64, 65] and trans/cis isomers of isohumulones [61, 62, 66] (which are
both readily oxidised beer constituents), and many others [61, 67].
Although oxygen is a significant contributor to and a major catalyst for beer (quality) degradation, it is
important to note that oxygen in its typical molecular configuration (3O2) does not readily induce
oxidation of wort and beer components. This is due to the high energy input (e.g. heat) that would be
required for direct oxidation to occur as such, since oxygen adopts a triplet ground state (characterised
by two unpaired electrons) under standard conditions. This inherent electronic arrangement prevents
direct reactivity with molecules that have no unpaired electrons (singlet state), which is the prevalent
state for most molecules in the environment, including those found in food and drink [68].
Consequently, for 3O2 to participate in a conventional chemical reaction, it must first transition to a
state of higher reactivity. This can be achieved by the ground state oxygen accepting an electron from
a suitable electron donor, thereby transforming into the much more reactive superoxide anion (O−2). In
the context of brewing, this electron transfer process occurs via coupled reduction systems in which
polyphenols [69–72] and sugars [9, 73, 74], among others [9, 70], donate electrons to transition metal
ions (FeIII, CuII or MnIII), which then donate electrons to oxygen, and so revert to their reduced state
(FeII, CuI or MnII). The resulting superoxide anion radical can then generate further ROS with increased
reactivities. These include the formation of perhydroxyl radicals (HO●2) by protonation, or the
continuation of coupled reduction pathways leading to peroxide anions (O22− ), hydrogen peroxide
(H2O2) and hydroxyl radicals (OH•). Figure 3 provides a graphical overview of the mechanics.
Figure 3: Schematic depiction of the reactive oxygen species formation mechanism in beer, adapted
from [9, 75], wherein 'M' represents iron, copper or manganese.
When considering this aspect in the overall picture of oxygen-induced staling, it becomes clear that
minimising transition metal ions during brewing and packaging is essential to maximise beer shelf life,
alongside the vital task of reducing oxygen exposure. Elimination of the catalysts crucial to the
initiation of ground state oxygen could significantly impede the overall staling process. Further
exploration of this fundamental concept, which serves as the cornerstone of the dissertation, will be
presented in the following subsections and throughout this thesis.
Table 1 provides an overview of the compounds that can be formed during beer ageing. These may
arise via several reaction pathways, including unsaturated fatty acid oxidation, higher alcohol
oxidation, Strecker degradation of amino acids, hop bitter acid degradation, aldol condensation,
glycoside degradation, acetal formation, Maillard reactions and ester formation [16]. However,
assessing the relevance and extent of each reaction mechanism under typical storage conditions
remains a complex undertaking.
Table 1: Compounds of interest formed during beer ageing, according to Vanderhaegen et al. [16].
Class Compounds
Aldehydes Acetaldehyde 2-Methylbutanal
Trans-2-nonenal 3-Methylbutanal
Trans-2-octenal Benzaldehyde
(E,E)-2,4-decadienal Phenylacetaldehyde
(E,E)-2,6-nonadienal Methional
2-Methylpropanal Hexanal
Ketones ß-Damascenone 4-Methyl-2-butanone
Diacetyl 4-Methylpentan-2-one
3-Methyl-2-butanone 2,3-Pentanedione
Cyclic acetals 2-Isobutyryl-4,5-dimethyl-1,3-
2,4,5-Trimethyl-1,3-dioxolane
dioxolane
2-Isopropyl-4,5-dimethyl-1,3- 2-Sec-butyl-4,5-dimethyl-1,3-
dioxolane dioxolane
Heterocyclic Furfural 2-Ethyoxy-2,5-dihydrofuran
compounds 5-Hydroxymethyl furfural Maltol
5-Methylfurfural Dihydro-5,5-dimethyl-2(3H)-furanone
2-Acetylfuran 5,5-Dimethyl-2(5H)-furanone
2-Acetyl-5-methylfuran 2-Acetylpyrazine
2-Propionylfuran 2-Methoxypyrazine
Furan 2,6-Dimethylpyrazine
Furfuryl alcohol Trimethylpyrazine
Furfuryl ethyl ether Tetramethylpyrazine
2-Ethoxymethyl-5-furfural
Ethyl esters Ethyl-3-methylbutyrate Ethyl lactate
Ethyl-2-methylbutyrate Ethyl phenylacetate
Ethyl-2-methylpropionate Ethyl formate
Ethyl nicotinate Ethyl cinnamate
Diethyl succinate
Lactones γ-Nonalactone γ-Hexalactone
Sulphur (S)
Dimethyl trisulphide 3-Methyl-3-mercaptobutylformate
compounds
Historically, the predominant focus regarding the development of stale beer flavour has primarily
revolved around carbonyl compounds (aldehydes and ketones) and their plausible precursors—and
with good reason. Much like other foodstuffs such as milk [76], chips [77], bread [78] and oils [79],
carbonyls have been considered to be the main contributors to flavour alterations during beer ageing
[80]. This perspective has gained solid support over time. The accumulation of aldehydes beyond their
combined flavour thresholds during storage is generally accepted as the key determinant of beer
flavour instability, particularly in pale lager beers (the most widely consumed beer style globally) [81–
85]. In the context of aged beer, their presence is thought to largely result from three distinct
pathways: lipid oxidation, Strecker degradation and Maillard reactions [81, 86–88].
Oxidation of unsaturated fatty acids was originally thought to be the main mechanism of carbonyl
formation during beer storage [60, 89]. However, this proposition has been contested due to the
apparent limited occurrence of direct fatty acid oxidation in the final beer product [90, 91]. More
recent evidence suggests that the oxidation of unsaturated fatty acids primarily takes place during
malting and wort production [92–94]. This process can be initiated by enzymatic oxidation catalysed
by lipoxygenase (LOX) or by non-enzymatic oxidation involving ROS, with Bamforth postulating that
the latter theoretically transpires at substantially higher rates [95, 96].
Another important pathway leading to the formation of carbonyl compounds is Strecker degradation.
This process involves the oxidative deamination and decarboxylation of α-amino acids (in which the
amino group is attached directly to the carbon atom next to the carboxyl group) in the presence of
dicarbonyl compounds (molecules containing two carbonyl groups, such as certain Maillard reaction
intermediates or polyphenol oxidation products) [97]. Strecker degradation gives rise to so-called
Strecker aldehydes and includes Strecker degradation of amino acids in the strict sense, Strecker-like
reactions, direct formation of Strecker aldehydes from Amadori compounds and direct oxidation of
free amino acids (by ROS-induced radical attack) [23, 85, 98].
Strecker aldehyde formation has been found to increase significantly with elevated oxygen levels, not
only during beer storage but also during sweet wort production [99]. Moreover, similar to lipid
oxidation, a significant amount of Strecker aldehyde formation appears to occur prior to beer storage,
specifically during malting and brewing [23, 85, 100–103]. An estimate by Suda et al., using isotopically
labelled 13C-amino acids added before wort boiling, suggests that 85 % of Strecker aldehydes originate
from the brewing process, with only a small fraction being formed in the package [104].
Aldehydes formed during processing can either be physically removed from the process (e.g. by
adsorption to insoluble compounds or evaporation during wort boiling) [100, 105, 106], be reduced
(e.g. by yeast during fermentation and/or bottle conditioning) [43, 97, 107–109] or be bound to matrix
compounds (e.g. bisulphites, imines, polyphenols, or amino acids like cysteine) [85, 105, 110, 111]. It
is postulated that the primary mechanism for the emergence of staling aldehydes during beer storage
is the release of bound state aldehydes, at least during the first four months of natural ageing—
although both bound state aldehyde release and de novo formation occur concurrently during beer
storage. De novo aldehyde formation appears to take on a more prominent role only after about three
to five months from packaging [112].
Maillard reactions are a considerable third source of carbonyls related to staling. These reactions, also
referred to as non-enzymatic browning reactions, encompass a wide spectrum of chemical interactions
that occur subsequent to an initial reaction between a reducing sugar (e.g. maltose, glucose,
maltotriose) and a free amino group (present in compounds such as amines, amino acids, peptides or
proteins). Maillard reactions primarily operate at elevated temperatures (≥ 50 °C) and are more
favourable within slightly acidic conditions (pH 4-7) [113]. Nevertheless, it should be recognised that
Maillard reaction products (MRPs) can also form at lower temperatures—albeit at slower rates—
including temperatures encountered during beer storage [85, 114–116], and across a broader pH range
[117]. However, given that higher temperatures typically occur during malt and wort production, as
opposed to beer storage, Maillard reactions are again more closely associated with these stages.
Furthermore, MRP formation is more pronounced at the higher end of the pH range of 4-7 [117],
meaning that wort and malt (pH 5.2-6.0) provide a more pH-appropriate environment than beer (pH
3.5-4.5).
The array of different Maillard products present in wort and beer is extensive, giving rise to a wide
range of chemical properties [85, 118]. Among these, the most prominent MRPs in beer, from a
quantitative standpoint, are furfural (derived from pentose sugars) and 5-hydroxymethyl furfural (5-
HMF; derived from hexose sugars) [61, 119]. The concentration of these compounds increases
approximately linearly with time, with the rate being logarithmically influenced by the storage
temperature, and strong correlations exist between their levels and the sensory perception of beer
staling, making them reliable indicators of beer ageing [63, 119].
It should be noted that neither furfural nor 5-HMF is generally considered to be responsible for stale
beer flavour. Their high flavour thresholds (150 mg/L and 1000 mg/L, respectively [120]) are not
exceeded even during prolonged beer ageing [103, 121, 122]. For this reason, Maillard reactions have
historically received much less attention in the field of beer flavour stability research than, for example,
lipid oxidation [16]. Nevertheless, De Clippeleer and colleagues showed that spiking fresh pale lager
beer with 400 µg/L of furfural can directly lead to perceptible negative sensory impressions (changes
in taste and mouthfeel) characterised by increased astringency and reduced bitterness intensity and
quality [103].
More relevant, in terms of their more immediate impact on beer staling, is the ability of certain
Maillard intermediates to not only serve as a potential source of dicarbonyls for Strecker degradation
but also to interact with common beer constituents to form staling compounds. An illustrative example
is the formation of furfuryl ethyl ether, which imparts a solvent-like off-flavour and is formed in beer
via an acid-catalysed condensation reaction involving ethanol and furfuryl alcohol (which is
predominantly formed by the Maillard reaction during malt kilning and wort boiling) [65, 67].
There is also evidence that reductones and melanoidins, both products of the Maillard reaction,
accelerate beer staling through melanoidin-mediated oxidation of alcohols [123, 124] and facilitate
free radical formation via reductone-accelerated metal-catalysed Fenton and Haber-Weiss reactions
[10, 125, 126]. However, similar to polyphenols (discussed below), there are conflicting opinions and
evidence in the literature. This discrepancy arises from the fact that melanoidins can also act as
antioxidants through metal chelation and ROS scavenging, thereby inhibiting processes such as
autoxidation of fatty acids and oxidative degradation of isohumulones [123, 127, 128].
It is important to recognise that all three pathways (lipid oxidation, Strecker degradation and Maillard
reactions) are greatly intensified in the presence of oxygen and transition metal ions [82, 129–133].
This reiterates the primary point and again emphasises the importance of ensuring minimal levels of
oxygen and transition metals during both brewing and packaging to improve the overall oxidative
stability and prolong the shelf life of beer.
Literature Review - Chelation in the beverage industry 11
2.3. Chelation in the beverage industry
Metals are common trace elements in various beverages, originating from their natural presence in
the source water or introduced through raw materials, processing equipment or packaging [134–137].
Many of these metal ions can be considered essential, either because they have important nutritional
value (e.g. calcium and magnesium), or because they play a key role in the production of the beverage,
such as magnesium and zinc in brewing [138], or in the taste of the product, such as copper in tequila
[139]. However, as discussed, transition metal ions like iron, copper and manganese can be detrimental
to the oxidative and colloidal stability of beer and other beverages and are therefore undesirable in
most cases.
Metals are seldom found in their free state in nature [140]. They are usually associated with other
species, called ligands, which surround the metal—acting as a central ion—with varying degrees of
binding strength, depending on several factors [141–143]. This process is known as complexation and
can occur with any ligand bearing a functional group capable of binding to the metal ion. Examples of
some of the coordination complexes that may be found in foods and beverages are illustrated in Figure
4.
Figure 4 (reproduced from Paper II, Figure 2): Chemical structures and possible chelation mechanisms
of chelators that may be found in foods and beverages.
12 Literature Review - Chelation in the beverage industry
While certain complexes can exhibit high reactivity and easily transform into other chemical species,
most metal complexes remain stable during chemical or physical processes and are relatively
unreactive, often allowing them to be isolated (e.g. as solids) [144]. It is important to note that while
complexation is possible with all metals, the electronic structure of the metal ion will largely determine
the nature of the complex and the extent to which it is formed. Transition metals, with their unique
electronic configurations and variable oxidation states, readily engage in complex formation [145]. This
property may be conveniently exploited when the goal is to remove iron, copper and manganese from
liquid solutions, such as in the case of wort and beer in this research.
The effectiveness of metal-chelator binding can also be significantly influenced by the acidity of the
environment. The pH of the solution directly governs the competition between metal cations and
protons (i.e. hydrogen ions, H+) for the active binding sites on chelating agents. As per the principles of
the Henderson-Hasselbach equation, if the pH of the milieu equals or exceeds the pKa of a ligand, more
than 50 % of the ligand concentration will be in a deprotonated state. In this state, the ligand's
functional groups are unoccupied by protons, which is a key requirement for successful metal binding
[146, 147]. A higher matrix pH consequently promotes enhanced interactions with metal ions, thereby
increasing the overall chelation efficacy. This phenomenon has been effectively demonstrated by
Wietstock et al., who observed an increased capacity of hop acids to chelate iron in buffered model
systems with increasing pH [148].
Given the intricate composition of most beverages, a certain degree of complexation will take place
with the wide variety of organic ligands that are naturally present. In the case of tea, for example, its
polyphenols can even bind to iron to such an extent that its bioavailability is reduced [149]. Other
common ligands, besides polyphenols, encompass organic acids (e.g. tartaric acid and malic acid in
wine [150], phytic acid in beer [151]), pectic polysaccharides (e.g. in wine [152, 153]), as well as various
amino acids, peptides, proteins (e.g. in wine, wort and beer [152–155]) and Maillard reaction products
(e.g. melanoidins in beer, sweet wine and coffee [156–159]).
Polyphenols, however, stand out for their remarkable ability to form sizable complexes due to their
generally larger molecular size and the concomitant greater number of reactive functional groups. This
enables polyphenols, like tannic acid, to more effectively establish cross-links, i.e. the bonding of
functional groups within or between molecules [160, 161]. Figure 5 provides a non-exhaustive
illustration of the various versatile pathways by which polyphenols, like tannic acid, can interact with
wort and beer matrix compounds, including metal ions, amino acids, peptides and proteins, to form
cross-linked structures. As an example, polyphenols have been observed to exhibit a preference for
binding to protein proline residues [162]—which are abundant in wort [163]—and to bind to multiple
sites on the peptides [164]. In addition to physical/supramolecular cross-linking, as depicted in Figure
5, chemical cross-linking, such as covalent bonding between oxidised polyphenol and amino groups,
can also occur [160, 165, 166].
Figure 5: Illustration depicting potential physical intermolecular cross-linking interactions involving the
polyphenol tannic acid, the metal iron and two common amino acids found in wort and spent grains
(proline and glutamine).
The molecular structures of epigallocatechin gallate (EGCG) and ellagic acid/punicalagin, which are
important in this work, are shown in Figure 6. Looking at the functional groups present in these
structures, it becomes evident that they share similar binding mechanisms as shown for tannic acid in
Figure 5.
Figure 6: Molecular structures of epigallocatechin gallate (left) and ellagic acid/punicalagin (right).
14 Literature Review - Chelation in the beverage industry
The resulting aggregates have good thermal and mechanical stability due to the durability of the
covalent bonds [167, 168], and can be very large and even precipitate [169], making them easier to
remove along with the metal ions they contain—another exploitable property when it comes to
transition metal removal. In general, the more cross-links present, the stronger and more resistant to
dissolution the aggregate will be. Figure 7 illustrates the potential construction of such an aggregate.
Figure 7: Schematic representation of the formation and precipitation of protein-polyphenol-metal

aggregates, where the symbol ℗ represents a polyphenol (e.g. tannic acid) and the symbol Ⓜ
represents a metal ion (e.g. iron). The diagram also includes molecular details to clarify the aggregation
process.
2.3.1. Antioxidant and chelating properties of polyphenols
Polyphenols are a diverse group of naturally occurring aromatic organic compounds that are abundant
in a wide variety of plants [170]. From an evolutionary perspective, they emerged as a defence
mechanism against the oxidative stress to which all aerobic life on earth is inevitably exposed. Through
i.a. metal binding, radical scavenging and the quenching of molecules in excited states, polyphenols
enable plants to maintain their vitality in the presence of reactive oxygen species and other free
radicals. Because of these properties, the consumption of polyphenols has been associated with
numerous health benefits [171].
Due to their prevalence in plants, polyphenols are naturally present in plant-based beverages like tea,
wine and beer. In the case of beer, a significant proportion of its polyphenols, approximately 70-80 %,
originate from the malt, with the remaining 20-30 % coming from the hops [172]. They are among the
most readily oxidised components of beer, apart from iso-α-acids [16], thus effectively acting as
sacrificial antioxidants, protecting other constituents from being oxidised—although oxidised
polyphenols can themselves impart an aged flavour to beer [173]. The presence of polyphenols in beer
has been associated with both positive and negative effects on beer flavour stability. On the one hand,
polyphenols can act as antioxidants by binding to transition metal ions that catalyse radical reactions
[69, 174], or by acting as radical scavengers or singlet oxygen quenchers [16]. On the other hand,
polyphenols can also act as prooxidants by reducing oxidised metal ions to their lower oxidation states
(e.g. FeIII → FeII), effectively recycling them [69, 174]; or because some oxidised polyphenols are potent
oxidants themselves, capable of oxidising other compounds to form off-flavours [173].
These seemingly contradictory effects of polyphenols can be partly attributed to the diverse nature of
this broad class of compounds. Different subclasses of polyphenols can (bio)chemically behave in
different ways. In addition, the complexity of food matrices like wort and beer further complicates
their analysis and their net effect on flavour stability. For instance, whether a particular polyphenol
behaves antioxidatively or prooxidatively may vary based on its abundance, the pH and the
concentrations of the surrounding metal ions [174–176]. Furthermore, the great complexity of the
antioxidant compound class makes it difficult to establish a universally accepted assay to reliably
determine the antioxidant capacity of foods and beverages [177]. This lack of consensus—and the
resultant diversity of antioxidant assays—renders comparison of results very challenging.
Nevertheless, as with synthetic chelators, the addition of botanicals and their extracts to processed
foods and beverages has been shown to provide multifunctional protection in several cases. Examples
include:
- The prevention of lipid oxidation and oxidative rancidity in fat emulsions like salad dressings
and mayonnaise by adding potent synthetic chelating agents such as EDTA [178, 179].
- The incorporation of gallic acid, ascorbic acid, rosemary extract, grape seed extract, phytic
acid, etc. into mayonnaise [179, 180], as natural substitutes for synthetic antioxidants such as
the aforementioned EDTA.
- The use of tannins, such as catechins, gallo- and ellagitannins, as antioxidants in the
production of wine [181, 182] and beer [11].
- The preservation of various foods, such as caviar, oils and meat, by phytic acid via inhibition
of iron-catalysed hydroxyl radical formation and lipid peroxidation [183, 184].
- The promising capabilities of citric acid as a copper-chelating food additive [185].
- The retardation of lipid oxidation by hops and its extracts, not only in beer but also in other
foods such as German sausages [186].
- The use of curcumin as a bioactive food protector in various foods [187].
- The reduction of lipid oxidation and protein carbonyl formation in Bologna-type sausage by
the addition of green tea extract or rosemary extract [188].
These examples highlight the potential benefits of incorporating external chelators into food systems
to improve their stability. However, this practice has been little explored in the field of brewing.
16 Research Questions & Thesis Overview
3. Research Questions & Thesis Overview
The central theme of the work revolves around the oxidative stability of wort and beer. The thesis
consists of a review article (Paper I) and three research articles (Papers II-IV), which collectively form
the foundation of the dissertation. Primary emphasis was placed on the detrimental role of transition
metal ions (iron, copper and manganese). The main objective of the research was to effectively
reduce the presence of transition metals in the brewing process through chelator-mediated
sequestration, thereby enhancing the oxidative stability of the resulting wort. This approach was
ultimately aimed at achieving the overall goal of extending the shelf life of beer.
Figure 8 provides a schematic overview that contextualises the general research problem, objective
and problem-solving approach (through the research questions and studies listed below) and
illustrates the interrelatedness of the work. Paper I serves as a comprehensive review article that
delves into the current research and existing knowledge regarding oxidative degradation in the
context of brewing. As a review article, it constitutes a significant portion of Section 2 (Literature
Review) and provides a thorough exploration of the subject matter. Paper II explores the selective
complexing ability of nine chelators with seven distinct brewing-relevant metal ions, both individually
and in a mix, in wort and beer model solutions of varying pH and ethanol content. The study also
examines the filtration potential of the complexes formed using a 0.2 µm membrane filter. Paper III
focuses on the evaluation of five notable chelators identified in Paper II under actual wort conditions
during laboratory scale mashing and aims to determine, through experimental design, the optimal
conditions to enhance transition metal depletion after lautering. In addition, the study investigates
the efficacy of fourteen other chelating agents, including ten novel natural chelators derived from
food extracts. Paper IV validates the findings of Paper III at pilot scale and further investigates if and
how chelator addition affects the brewing process, transition metal concentrations, radical
generation and aldehyde formation in two distinct brewery settings. The patent (Paper V) represents
a valorisation of the overall findings of the thesis.
The basic structure of the dissertation is built on the systematic investigation of the overarching
thesis hypothesis, i.e. that the addition of external chelators during brewing can lower the pool of
transition metal ions, thereby increasing the oxidative stability of the wort, leading to improved
overall quality and flavour stability of the resulting beer. This led to the sequential investigation of
the following research questions, the answers to which are deliberated upon in Sections 5-6 (Results
and Summarising Discussion):
1. Primary research questions (Paper II):

- Which chelators, out of the nine selected, are able to form targeted complexes with the
undesirable iron, copper and manganese ions, while avoiding binding to the favourable
calcium, zinc and magnesium ions?
- Does the formation of complexes occur rapidly, or does it require a certain amount of
reaction time for them to establish?
- Are the formed chelator-metal complexes large enough to be retained on a 0.2 µm cellulose
membrane filter?
- Is the chelation process more efficient in (model solutions of) wort or beer, considering
factors such as pH and ethanol content?
Research Questions & Thesis Overview 17
- How does chelation behaviour, in terms of binding affinity and efficiency, alter when exposed
to a mixture of all seven metal ions rather than the individual metal ions?
- Among the nine investigated chelators, which are the most effective for the intended
purpose (Fe, Cu and Mn depletion)?
2. Secondary research questions (Paper III):
- Do the five primary chelators, selected from the previous study, exhibit similar chelation
behaviour in actual wort as in the wort model solution?
- Can the metal-chelator complexes formed with iron, copper, manganese (or zinc) during the
mashing process be removed by crude laboratory scale lautering?
- Are there any alternative chelating agents that outperform tannic acid in its exceptional
ability to sequester iron during mashing?
- How do grain bill, mash pH, mashing out temperature and sparging affect the content of
wort transition metals?
- Can the efficacy of chelators in removing transition metals be optimised by variations in
chelator concentration, addition time, mash pH and mashing out temperature?
- How is the radical formation in lautered wort affected by variations in chelator
concentration, addition time, mash pH and mashing out temperature?
- Does iron removal during mashing by chelation have a positive effect on the oxidative
stability of the wort?
3. Tertiary research questions (Paper IV):
- Can the laboratory scale findings of the previous study be successfully replicated at pilot
scale?
- What are the comparative performances of the top three chelators identified in the previous
study (tannic acid, green tea and pomegranate extract) when applied in a real brewing
setup?
- How do the chelators affect various brewing parameters, including extract yield, pH,
filtration time, thiobarbituric acid index (TBI), total polyphenols, soluble protein content and
FAN?
- What are the effects of chelators on staling-related parameters in wort, such as transition
metal content, radical formation and aldehyde levels?
- Does the timing of chelator addition during mashing have an impact on the outcomes?
- Are there any other variables, such as heat load, that significantly affect the staling-related
parameters?
18 Research Questions & Thesis Overview
Figure 8: Schematic overview of the research problem, objective and investigative approach of the thesis.
Materials & Methods 19
4. Materials & Methods
This section provides an overview of the materials and methods used in the thesis research. Detailed
technical and procedural information pertaining to the equipment and procedures can be found in the
original publications (Papers II-IV).
It should be noted that the patent (Paper V) contains certain specific beer-related results for which the
methodologies are not explained within the publication. Therefore, in order to provide full clarity, the
beer sample preparations and associated beer analysis methods are described in detail in this section.
20 Materials & Methods - Chemicals, consumables, equipment and software
4.1. Chemicals, consumables, equipment and software
Table 2: Summary of chemicals used in the thesis.

Chemical Supplier Purity (%) Paper
Calcium(II) acetate hydrate ≥ 99.0 II
Ferulic acid ≥ 99.0 II-III
Iron(II) sulphate heptahydrate ≥ 99.0 II, IV
Sigma-Aldrich Chemie GmbH (Steinheim, Germany)
Phytic acid sodium salt hydrate ≥ 90.0 II-III
Quercetin dihydrate ≥ 98.0 II-III
Tannic acid ≥ 99.0 II
2-methylbutanal ≥ 95.0 IV
2-methylpropanal ≥ 99.0 IV
2-thiobarbituric acid ≥ 99.0 IV
3-methylbutanal ≥ 97.0 IV
4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL) ≥ 97.0 III-IV
Absolute ethanol ≥ 99.2 IV
Ammonia solution ≥ 25.0 IV
Ammonium iron(III) citrate (FeIII) ≥ 16.5 IV
Anhydrous gallic acid ≥ 98.0 II-III
Bovine serum albumin (BSA, albumin fraction V) ≥ 98.0 IV
Calcium chloride dihydrate ≥ 99.0 IV
Merck KGaA (Darmstadt, Germany)
Carboxymethylcellulose (CMC; low viscosity) - IV
Citric acid monohydrate ≥ 99.5 II-III
Deuterated benzaldehyde (benzaldehyde-d6) 98 atom %D IV
D-fructose ≥ 99.0 IV
Disodium hydrogen phosphate dihydrate ≥ 99.5 IV
Ethylenedinitrilo-tetraacetic acid disodium salt dihydrate
≥ 99.0 II-IV
(EDTA disodium salt or Titriplex® III)
Furfural ≥ 99.0 IV
Glacial acetic acid ≥ 99.0 IV
Glycine ≥ 99.7 IV
Hexanal ≥ 98.0 IV
Materials & Methods - Chemicals, consumables, equipment and software 21

Iron(III) chloride hexahydrate ≥ 99.0 II
Isooctane ≥ 99.8 IV
Manganese(II) sulphate monohydrate ≥ 99.0 II
N-butanol ≥ 99.9 IV
Ninhydrin ≥ 99.0 IV
O-(2,3,4,5,6-pentafluorobenzyl)-hydroxylamine-hydrochloride (PFBHA) ≥ 99.0 IV
Phenylacetaldehyde ≥ 90.0 IV
Potassium dihydrogen phosphate ≥ 99.5 IV
Potassium iodate ≥ 99.7 IV
Tartaric acid ≥ 99.5 II-III
Trans-2-nonenal ≥ 97.0 IV
Chlorogenic acid ≥ 98.0 II-III
Acros Organics (New Jersey, USA)
Methional ≥ 95.0 IV
Magnesium(II) acetate tetrahydrate Honeywell Riedel-de Haën AG (Seelze, Germany) ≥ 99.5 II
Zinc(II) acetate dihydrate AppliChem GmbH (Darmstadt, Germany) ≥ 99.5 II
Copper(II) acetate monohydrate ≥ 99.0 II
Dimethyl sulfoxide ≥ 99.5 II
Glacial acetic acid Carl Roth GmbH (Karlsruhe, Germany) 100 II
Glycerine ≥ 98.0 IV
Hydrogen peroxide (ROTIPURAN®) ≥ 30.0 IV
Absolute ethanol 100 II-III
Hydrochloric acid VWR International S.A.S. (Fontenay-sous-Bois, France) 32.0-37.0 II, IV
Sodium hydroxide ≥ 98.5 II-IV
Concentrated nitric acid 65.0-70.0 III-IV
Th. Geyer GmbH & Co. KG (ChemSolute®, Renningen,
Hydrochloric acid 34.0-37.0 III
Germany)
Sodium acetate ≥ 99.0 II
N-tert-Butyl-α-(4-pyridyl-1-oxide)-nitrone (POBN) TCI Deutschland GmbH (Eschborn, Germany) ≥ 98.0 III-IV
MN-polyamide CC 6 Macherey-Nagel™ GmbH & Co. KG (Düren, Germany) - IV
Reference standards for iron, copper, manganese and zinc PerkinElmer LAS GmbH (Rodgau, Germany) 1000 μg/mL III-IV
Argon (Alphagaz™ 1) ≥ 99.999 III-IV
Carbon dioxide (Aligal™ 2) Air Liquide GmbH (Düsseldorf, Germany) ≥ 99.9 V
Helium (Alphagaz™ 2) ≥ 99.9999 IV

Methane ≥ 99.995 IV
Nitrogen ≥ 99.999 IV
Tannic acid (BrewTan B®) S.A. Ajinomoto OmniChem N.V. (Wetteren, Belgium) ≥ 98.0 III-IV
Green tea extract (epigallocatechin gallate) Fairvital B.V. (Landgraaf, The Netherlands) ≥ 50.0 III-IV
≥ 40.0 &
Healthtonics (Thornaby, UK) III
Pomegranate extract (ellagic acid) ≥ 90.0
PureBulk Inc. (Oregon, USA) ≥ 90.0 IV
Grape seed extract (oligomeric proanthocyanidin) PumpEffect B.V. (Maastricht, The Netherlands) ≥ 95.0 III
Reishi extract (polysaccharides) Kurkraft GmbH (Berlin, Germany) ≥ 35.0 III
Cinnamon extract (polyphenols) ≥ 30.0 III
Extrakt Manufaktur GmbH (Hamburg, Germany)
Curcuma extract (curcuminoid) ≥ 95.0 III
Milk thistle extract (silymarin) Alpha Zwo B.V. (Eindhoven, The Netherlands) ≥ 80.0 III
Ginkgo biloba extract (flavonol glycosides) Nuvi Health B.V. (Heerlen, The Netherlands) ≥ 24.0 III
Grapefruit seed extract (flavonoids) Bafoxx UG (Münster, Germany) ≥ 45.0 III
Raspberry extract (raspberry ketone) GEN Nutrition UG (Aachen, Germany) 100 III
Deuterated 2-methylbutanal (2-methylbutanal-d10) MercaChem (Nijmegen, The Netherlands) 100 atom %D IV
Bradford’s protein assay dye reagent concentrate (Coomassie®
Bio-Rad Laboratories GmbH (Munich, Germany) 0.01 IV
Brilliant blue G-250)
All aqueous solutions were made with ultrapure water, either through Milli-Q® purification or via Sartopore® 2 MidiCap 0.2 µm filtration.
Materials & Methods - Chemicals, consumables, equipment and software 23
Table 3: Summary of consumables used in the thesis.

Consumable Supplier Paper
Munich malt Type I (TU Berlin) III
Munich malt Type II (TU Berlin) Weyermann GmbH & Co. KG (Bamberg, Germany) IV
Pilsner malt (TU Berlin) III
Pilsner malt (KU Leuven) Holland Malt (Lieshout, The Netherlands) IV
Munich malt 15 MD (KU Leuven) Mouterij Dingemans (Stabroek, Belgium) IV
IsoHop® hop extract (20 % iso-α-acids, KU Leuven) Barth-Haas (Paddock Wood, England) IV
SafLager™ S-189 lager yeast (Saccharomyces pastorianus, KU Leuven) Fermentis (Marquette, France) V
Hopsteiner® hop extract (30 % iso-α-acids, TU Berlin) Hopsteiner (Mainburg, Germany) IV
BrewMasters German Classic W34/70 3G lager yeast (Saccharomyces
Erbslöh (Geisenheim, Germany) V
pastorianus, TU Berlin)
Table 4: Summary of equipment used in the thesis.

Equipment Supplier Paper
Sartopore® 2 MidiCap filter system Sartorius AG (Goettingen, Germany II-IV
Milli-Q® filter system Merck Millipore, Darmstadt, Germany IV
Lambda 25 UV/Vis spectrophotometer II-IV
PerkinElmer (Rodgau, Germany)
Avio 200 ICP-OES spectrometer II-IV
0.2 & 0.45 μm cellulose acetate membrane syringe filters II-III
VWR International (Radnor, USA)
Metal-free plastic lab tubes II-IV
Metal-free syringes Henke-Sass Wolf (Tuttlingen, Germany) II-IV
10 mm high UV/Vis precision cell (out of special optical glass) Hellma Analytics (Jena, Germany) II
Mashing apparatus with stainless steel cups Bender & Hobein (Bruchsal, Germany) III
Cytiva filter papers Whatman GmbH (Freiburg, Germany) III-V
DMA 5000 alcolyzer beer analysing system Anton Paar GmbH (Graz, Austria) III-V
X-band ESR spectrometer Bruker BioSpin GmbH (e-scan, Rheinstetten, Germany) III-IV
Varian Cary 100 UV/Vis spectrophotometer Agilent Technologies (California, USA) IV-V
Hydromill® wet disc mill
Meura SA (Péruwelz, Belgium) IV
Membrane-assisted thin-bed filter
Two-roll mill Künzel Maschinenbau GmbH (Mainleus, Germany) IV
65 µm StableFlex PDMS/DVB fibre Supelco (Bellefonte, USA) IV
Equipment Supplier Paper

Thermo Scientific™ FOCUS GC gas chromatograph
Thermo Fisher Scientific Inc. (Waltham, USA) IV
Thermo Scientific™ ISQ™ EC Single Quadrupole mass spectrometer
VOS ROTA 90/25 turbidity meter Haffmans (Venlo, The Netherlands) V
Becopad type 350 cellulose filter sheets Eaton (Nettersheim, Germany) V
America monobloc six-headed rotating counter pressure filler CIMEC (Nizza Monferrato, Italy) V
SvelOx 8500 oxygen scavenger crown corks Actega DS (Bremen, Germany) V
Three-stage membrane candle filter Donaldson Company Inc. (Minneapolis, USA) V
H4 bottle filler JS Maschinen GmbH (Nandlstadt, Germany) V
Table 5: Summary of software used in the thesis.

Software Supplier Paper
ChemBioDraw Ultra (version 12.0) PerkinElmer Inc. (Waltham, MA, USA) II-IV
DesignExpert (version 11) Stat-Ease Inc. (Minneapolis, MN, USA) III
Microsoft Excel (version 211 16.0.12527.22286) Office 365 (Redmond, WA, USA) II-V
OriginPro (version 9.6.5.169) OriginLab Corporation (Northampton, MA, USA) III-IV
Visual MINTEQ (version 3.1) KTH Royal Institute of Technology (Stockholm, Sweden) II
Materials & Methods - Sample preparation 25
4.2. Sample preparation
Table 6 provides an overview of all the sample types investigated in the thesis, along with their
respective characteristics and the corresponding publications in which they were examined.
Summaries of their preparation are given in the following subsections; however, for a more in-depth
understanding, readers are encouraged to refer to the respective original publications. In the case of
Paper V, the preparation of the beer samples was not addressed and therefore a full account is given
in Subsection 4.2.3.
Table 6: Summary of samples investigated in the thesis.

Sample Characteristics Paper
Wort model buffer solution Acetate buffer: pH 5.60, 0.0 ABV II
Beer model buffer solution Acetate buffer: pH 4.30, 5.0 ABV II
Wort pH 5.2-5.6, 12.1-12.7 °P III-IV
Beer pH 4.6-4.1, 4.9-5.8 ABV V
4.2.1. Preparation of metal-chelator mixtures in wort and beer model solutions
Both the 'wort' buffer, with a pH of 5.60 and no ethanol (0.0 vol%), and the 'beer' buffer, with a pH of
4.30 and 5.0 vol% ethanol, were 0.1 M sodium acetate buffers. Individual solutions were prepared for
each metal ion (FeII, FeIII, CuII, MnII, CaII, ZnII, MgII) and each chelator (EDTA, citric acid, tartaric acid,
quercetin, chlorogenic acid, ferulic acid, gallic acid, phytic acid, tannic acid) in both buffer systems at
concentrations of 500 µmol/L. After uniform mixing, a final concentration of 250 µmol/L was achieved
for both the metal ion and the chelator.
The 'metal ion mixture' consisted of six metal ions (FeII, CuII, MnII, CaII, ZnII, MgII), each at a concentration
of 250 µmol/L (half that of the single metal ion solutions). By mixing equal volumes of the metal ion
mixture and the chelating agent solution, a final concentration of 125 µmol/L was obtained for each
metal ion and 250 µmol/L for the chelating agent.
4.2.2. Preparation of laboratory and pilot scale wort samples
For the laboratory scale samples, sweet worts (400 mL) were prepared by mashing a mixture of 50 %
milled Pilsner and 50 % milled Munich malt, unless otherwise stated. Ultrapure water was used at a
weight/weight ratio of 1:4 in conjunction with a congress mashing apparatus with stainless steel mash
cups. Adjustments to the naturally occurring mash pH (approximately 5.6) were made at the start of
the mashing to meet the specific objectives. This involved the addition of either hydrochloric acid for
acidification or sodium hydroxide for alkalisation. The lautering process was simulated by passing the
mash through folded filter papers and then recirculating the first 100 mL of lautered wort over the
spent grains resting on the filter paper.
Pilot scale wort samples were produced at both the KU Leuven research brewery in Ghent (Belgium)
and the TU Berlin research brewery in Berlin (Germany). Table 7 provides a comparison of the two
wort preparation processes, including both similarities and differences in terms of raw materials,
equipment and process protocols for the two distinct brewery setups.
26 Materials & Methods - Sample preparation
Table 7: Comparison of wort preparation at the KU Leuven and TU Berlin research breweries.
KU Leuven TU Berlin
Atmosphere Ambient air
Amount of wort (L) 500 200
Malt:liquor ratio (w/w) 1:2.2 1:3.3
Pilsner:Munich malt ratio (w/w) 1:1
Mill Fine wet disc mill Two-roll mill
Mashing schedule (steps in °C) 64-72-78 62-66-72-78
Water Pre-heated reverse osmosis water
CaII (mg/L) 81 68
Wort separation Membrane-assisted thin-bed filter Lauter tun
First runnings (°P) 25 16.5
Final wort (°P) 12-13
Targeted bitterness (IBU) 29 30
ZnII (µL/L) 10 -
Clarification Whirlpool
4.2.3. Preparation of beer samples
Following the production of the KUL and TUB worts, as briefly outlined above and detailed in Paper IV,
twelve corresponding batches of KUL and TUB beer were subsequently prepared in the pilot scale
breweries of KU Leuven (Ghent, Belgium) and TU Berlin (Berlin, Germany), as described in Subsections
4.2.3.1 and 4.2.3.2 respectively. The sample names and characteristics are repeated in Table 8.
Table 8: Chelator mash addition protocol.

Concentration
Brew Chelator Addition time
(mgchel./kgmalt)
Ref₁ (△)
- - 0
Ref₂ (◻)
PGₑ (▲) Pomegranate extract
GTₑ (▲) Green tea extract Onset of mashing (“early”)
KU Leuven TAₑ (▲) Tannic acid
0.17
PGι (◼) Pomegranate extract
GTι (◼) Green tea extract End of mashing (“late”)
TAι (◼) Tannic acid
Refₔ (◇) - - 0
PGₔ (◆) Pomegranate extract
TU Berlin
GTₔ (◆) Green tea extract Onset of mashing (“early”) 0.20
TAₔ (◆) Tannic acid
4.2.3.1. Pilot scale beer production at the KU Leuven, Technology Campus Ghent
After cooling the clarified wort to fermentation temperature (20 °C), 1 hL of it was transferred to a 1.2-
hL cylindroconical tank for fermentation, after aeration with sterile air and inline pitching of 80 g dry
lager yeast/hL (> 5 x 106 viable cells/mL). After 8 days of fermentation, the beer was matured for 14
days at 0 °C, filtered through cellulose sheets (nominal retention range of 1-2 µm), carbonated (5.6 g
CO2/L) and packaged in 250-mL brown glass bottles. For bottling, a six-headed rotating counter
pressure filler was used, applying double pre-evacuation and intermediate CO2 flushing, along with
Materials & Methods - Analytical methods 27
over-foaming (by hot water injection) before capping with oxygen scavenger crown corks. Beer
samples included freshly bottled beers and their force-aged counterparts (2 weeks, 1 month, 2 months
and 3 months at 30 °C) and were stored at 0 °C until analysis.
4.2.3.2. Pilot scale beer production at the TU Berlin
After cooling the clarified wort to fermentation temperature (12 °C), 1 hL of it was transferred to a
cylindroconical tank for fermentation, after aeration with sterile air and pitching of 100 g dry lager
yeast/hL (> 5 x 106 viable cells/mL). After 7 days of fermentation (apparent extract, < 3.5 °P), the beer
was matured for > 14 days at 1 °C, filtered through a three-stage membrane candle filter (nominal
retention ranges of 5, 1 and 0.45 µm), carbonated and packaged in 500-mL brown glass bottles. For
bottling, an H4 filler was used, applying CO2 flushing and over-foaming before capping. Beer samples
included freshly bottled beers and their aged counterparts (2 months at room temperature) and were
stored at 0 °C until analysis.
4.3. Analytical methods
Table 9 presents an overview of the methodologies employed in the thesis, including the experimental
objectives and corresponding publications. Reference is made to the original publications for further
details of the methodologies applied. For the beer samples discussed in Paper V, relevant information
is provided in the following subsections.
Table 9: Summary of analytical methods used in the thesis.

Determination or
Method Paper Reference
quantification of
ESR spectroscopy Radicals I, III-IV MEBAK, method 2.15.3 [189]
ICP-OES analysis Metal ions II-IV Custom method
CGC-MS analysis Free aldehydes IV Baert et al. [190, 191]
Complex formation II Custom method
TBI IV MEBAK, method 2.4 [189]
FAN IV MEBAK, method 2.6.4.1 [189]
UV/Vis spectrophotometry Beer colour V MEBAK, method 2.12.2 [189]
Total polyphenols IV MEBAK, method 2.16.1 [189]
Soluble protein IV Bradford [192]
Anthocyanogens IV MEBAK, method 2.16.2 [189]
Continuous flow analysis Bitterness IV MEBAK, method 2.17.1 [189]
Oscillating U-tube densimetry Density III-V MEBAK, method 2.9.6.3 [189]
NIR spectroscopy Alcohol V MEBAK, method 2.9.6.3 [189]
Turbidimeter Haze V MEBAK, method 2.14.1.2 [189]
+
pH meter Acidity (H ) II-V MEBAK, method 2.13 [189]
Thermometer Temperature II-V -
Precision balance Weight II-V -
28 Materials & Methods - Standard beer analyses
4.4. Standard beer analyses
Density, extract, alcohol content and pH were evaluated using an Alcolyzer beer analysing system on
clear, undiluted and degassed samples, according to MEBAK (method 2.9.6.3 and 2.13, respectively)
[189]. Beer colour was determined using ultraviolet-visible spectrophotometry, according to MEBAK
(method 2.12.2) [189]. Beer haze was measured at scatter angles of 90° (H90, side scatter) and 25°
(H25, forward scatter) with a turbidity meter at 20 °C (permanent haze) and 0 °C (cold haze), according
to MEBAK (method 2.14.1.2) [189]. Forced hazes were determined similarly, by either keeping the beer
for six days at 60 °C prior to measurement or by subjecting the beer to multiple alternating cycles of
cold and warm days (24 hours at 0 °C and 40 °C, respectively).
4.5. Data analysis
Mean values and standard deviations (SDs; graphically represented through error bars, where
applicable) of the beer data are determined via two technical replicates unless noted otherwise.
Scientific graphing and data analysis were conducted using Microsoft Excel.
Results - Assessment of chelators in wort and beer model solutions 29
5. Results
This section of the thesis offers a comprehensive overview of the most significant findings from the
published papers and formulates answers to the Research Questions from Section 3. The primary
objective is to provide a concise and focused summary of the key results, emphasising the main
outcomes and contributions of each study. Readers are encouraged to refer to the original publications
for more in-depth descriptions of the methods and results. A comprehensive discussion of these
principal findings is reserved for the subsequent Section 6 (Summarising Discussion), where the
implications and potential impacts of the results are further explored.
5.1. Assessment of chelators in wort and beer model solutions (Paper II)
Chemical equilibrium modelling software, like Visual MINTEQ, allows for theoretical calculations of
metal ions binding to specific ligands at various pH levels. Through complex stability constants from
the literature, predictions can be made about the percentage of metal ions that are ligand-bound.
When applied to the wort model solution, considering the seven metal ions (FeII, FeIII, CuII, MnII, CaII,
ZnII, MgII) and nine chelators (EDTA, citric acid, tartaric acid, quercetin, chlorogenic acid, ferulic acid,
gallic acid, phytic acid, tannic acid) investigated in this study, the calculations suggest promising
potential in terms of iron and copper binding for EDTA, citric acid, quercetin, gallic acid and tannic acid
(Paper II, Table 4). Of the selected chelators, only EDTA and phytic acid are anticipated to form
complexes with manganese. However, it should be noted that EDTA can also bind to calcium, zinc and
magnesium—and phytic acid to zinc—demonstrating the low selectivity of these agents.
Changes in UV/Vis absorbance, referring to the difference in absorbance within the buffer solutions
between the metal-chelator combination and the individual metal ion or chelator, serve as an
indication of complex formation. While the observations generally aligned with the aforementioned
predictions, drawing a clear line between (un)anticipated complex formation and spectral changes
proved difficult. It was evident that absorbance changes occurred rapidly and persisted throughout
the 60-minute reaction period, indicating that complex formation continued over time. The changes
in the UV/Vis spectra were consistent across both media, although there were some exceptions (e.g.
tannic acid, which showed different spectra in wort and beer model solutions; Paper II, Figure 3).
The majority of the complexes were found to be too small to be effectively retained by the
microfilter, resulting in little to no reduction in metal content after filtration, as shown in Table 10.
Nevertheless, certain metal-chelator combinations demonstrated high filter retention rates
(decreases of > 90 % compared to the 'no chelator' controls), namely FeII-III-tannic acid, CuII-tannic
acid, FeIII-quercetin and FeIII-chlorogenic acid. Notable reductions (70-73 %) were also observed for
ZnII-phytic acid and FeIII-gallic acid. It is important to note that these decreases all occurred at a wort
pH of 5.60. Based on this finding, it was decided to carry out the chelator additions during mashing,
rather than during or after fermentation, for the two subsequent studies.
30 Results - Complexation of transition metals by chelators added during mashing and impact on beer stability
Table 10 (adapted from Paper II, Table 5): Residual metal content ± standard deviation (μmol/L) of
metal-chelator mixtures after 60 minutes reaction time at room temperature and filtration with a 0.2
μm filter. Standard deviations were calculated from two biological replicates, except for the 'no
chelator' controls which were replicated in triplicate. The heat map shows the extent of metal
depletion relative to the highest value within each row.
Metal Chelator added (250 µmol/L)
ion
added pH Chloro-
No Citric Tartaric Querce- Ferulic Gallic Phytic Tannic
(250 EDTA genic
chelator acid acid tin acid acid acid acid
µmol/L) acid
267.5 259.5 253.9 249.4 239.5 164.6 264.4 236.8 223.1 220.8
4.30
± 6.5 ± 1.1 ± 1.8 ± 2.2 ± 5.4 ± 3.7 ± 0.1 ± 0.2 ± 1.0 ± 5.8
Fe(II)
175.6 243.2 224.6 245.5 114.9 238.6 159.1 251.4 245.6 10.6
5.60
± 30.6 ± 1.2 ± 23.4 ± 0.8 ± 10.4 ± 4.3 ± 27.4 ± 17.9 ± 1.4 ± 2.6
241.1 232.6 236.5 224.6 186.6 213.0 229.9 213.6 215.2 182.2
4.30
± 6.4 ± 19.5 ± 11.6 ± 12.2 ± 10.1 ± 9.4 ± 6.3 ± 11.3 ± 7.8 ± 27.5
Fe(III)
149.5 168.7 207.1 194.7 5.7 14.6 132.1 40.9 211.0 4.8
5.60
± 9.3 ± 8.4 ± 1.2 ± 6.4 ± 0.1 ± 0.5 ± 1.8 ± 6.2 ± 1.6 ± 1.1
244.2 218.0 231.4 231.8 247.7 262.2 264.5 216.2 308.5 218.5
4.30
± 14.1 ± 27.5 ± 11.8 ± 12.6 ± 0.3 ± 2.9 ± 7.8 ± 27.1 ± 3.2 ± 26.0
Ca(II)
237.4 200.6 204.5 208.7 237.8 232.9 246.7 197.9 262.9 195.2
5.60
± 17.5 ± 20.7 ± 14.9 ± 13.3 ± 4.6 ± 1.6 ± 1.1 ± 19.2 ± 6.0 ± 21.9
270.3 267.5 276.2 276.2 235.3 237.8 234.5 264.2 131.9 269.4
4.30
± 14.7 ± 24.9 ± 13.1 ± 15.3 ± 3.6 ± 4.8 ± 0.2 ± 27.5 ± 5.0 ± 25.6
Zn(II)
226.9 212.9 228.2 228.4 229.2 211.9 224.4 222.5 67.3 215.3
5.60
± 1.7 ± 8.2 ± 3.8 ± 3.4 ± 0.6 ± 4.9 ± 1.6 ± 9.0 ± 12.5 ± 14.2
233.4 214.1 221.3 219.7 223.9 231.6 217.6 212.6 288.4 216.8
4.30
± 16.3 ± 15.4 ± 10.0 ± 9.3 ± 4.5 ± 4.4 ± 4.3 ± 15.3 ± 4.6 ± 15.6
Mg(II)
213.2 240.4 202.0 203.5 211.6 204.7 204.8 197.7 241.6 206.1
5.60
± 2.8 ± 43.2 ± 11.0 ± 7.5 ± 1.3 ± 4.7 ± 1.8 ± 14.0 ± 0.2 ± 7.4
268.5 238.7 251.6 251.0 244.8 238.3 277.7 238.2 329.9 240.0
4.30
± 14.6 ± 18.9 ± 6.0 ± 7.5 ± 6.1 ± 0.1 ± 1.4 ± 17.6 ± 16.7 ± 18.1
Mn(II)
241.3 207.5 196.8 201.4 233.0 221.8 232.5 186.2 232.2 227.1
5.60
± 18.9 ± 13.3 ± 17.4 ± 9.4 ± 1.3 ± 0.0 ± 0.4 ± 9.7 ± 2.4 ± 27.3
251.8 229.8 239.7 236.6 225.0 225.8 215.9 229.1 214.4 219.2
4.30
± 19.0 ± 10.5 ± 3.1 ± 1.0 ± 0.6 ± 1.1 ± 5.6 ± 6.9 ± 21.1 ± 3.8
Cu(II)
239.1 198.7 190.6 225.3 233.3 238.1 245.4 143.7 219.8 4.1
5.60
± 11.9 ± 5.5 ± 9.2 ± 34.8 ± 1.2 ± 2.6 ± 1.5 ± 15.3 ± 26.5 ± 0.7
The large discrepancy in chelator efficacy with respect to pH is also apparent in the mixed trials, as
shown in Table 11, where chelators are combined with a blend of six distinct metal ions. In general,
the behaviour of the chelators in the metal ion mixture differed minimally from their performance in
single metal solutions. Tannic acid, in addition to its established ability to deplete iron and copper,
slightly depleted ZnII in the metal mixture. However, markedly different interactions as compared to
the single metal trials were observed with phytic acid, as it led to a depletion of all metal ions except
MgII. It is also evident that chelators can prevent metal ions from binding to endogenous ligands (in
this case acetate and hydroxide), thereby potentially increasing metal permeability.
Results - Assessment of chelators in wort and beer model solutions 31
Table 11 (adapted from Paper II, Table 6): Residual metal content ± standard deviation (in µmol/L) of
metals-chelator mixtures after 60 minutes reaction time at room temperature and filtration with a
0.2 µm filter. Standard deviations were calculated from two biological replicates, except for the 'no
chelator' controls which were replicated in triplicate. The heat map shows the extent of metal
depletion relative to the highest value within each row.
Metal ion Chelator added (250 µmol/L)
added Chloro-
pH No Citric Tartaric Querce- Ferulic Gallic Phytic Tannic
(125 EDTA genic
µmol/L) chelator acid acid tin acid acid acid acid
acid
116.6 118.1 119.0 115.0 95.5 104.2 93.2 114.3 42.6 99.8
Fe(II)
± 2.3 ± 0.0 ± 0.8 ± 1.0 ± 3.3 ± 0.9 ± 7.4 ± 9.1 ± 14.9 ± 9.3
121.6 128.9 129.0 126.1 106.8 158.8 105.4 126.4 121.7 133.6
Ca(II)
± 3.4 ± 4.4 ± 4.0 ± 4.6 ± 5.7 ± 34.9 ± 5.4 ± 1.6 ± 5.1 ± 1.4
114.8 116.0 114.6 115.3 107.5 121.8 103.6 112.2 80.0 120.0
Zn(II)
± 2.1 ± 1.7 ± 0.9 ± 1.2 ± 1.2 ± 15.4 ± 1.0 ± 3.2 ± 4.5 ± 5.8
Mix 4.30
121.2 119.2 122.8 121.4 109.7 141.2 106.6 124.9 125.8 125.2
Mg(II)
± 3.2 ± 6.3 ± 4.0 ± 5.5 ± 2.8 ± 31.6 ± 6.9 ± 1.2 ± 1.6 ± 1.4
116.3 117.5 116.9 114.8 104.5 141.9 102.1 116.8 100.8 122.5
Mn(II)
± 2.7 ± 1.4 ± 1.9 ± 1.7 ± 3.0 ± 11.4 ± 2.1 ± 3.3 ± 0.0 ± 5.5
117.3 117.8 117.7 108.9 109.5 119.8 122.6 105.6 93.5 127.6
Cu(II)
± 1.5 ± 1.8 ± 0.1 ± 3.1 ± 1.9 ± 11.0 ± 18.7 ± 0.9 ± 1.0 ± 10.6
89.0 122.5 138.9 137.8 45.8 84.7 71.1 110.6 15.1 15.9
Fe(II)
± 8.9 ± 1.8 ± 1.5 ± 0.8 ± 1.7 ± 24.1 ± 6.3 ± 30.3 ± 0.5 ± 1.1
91.7 95.7 110.7 97.1 82.9 83.0 90.2 100.9 61.2 97.2
Ca(II)
± 40.4 ± 32.4 ± 25.3 ± 40.9 ± 42.9 ± 44.4 ± 56.5 ± 69.1 ± 55.2 ± 72.9
126.0 122.1 122.9 124.3 122.5 119.9 124.2 125.7 17.2 92.6
Zn(II)
± 8.2 ± 0.8 ± 0.9 ± 0.0 ± 2.0 ± 0.5 ± 3.9 ± 7.2 ± 1.3 ± 7.7
Mix 5.60
127.6 125.3 123.5 128.1 118.9 122.7 135.0 143.3 136.3 155.4
Mg(II)
± 11.4 ± 1.6 ± 1.6 ± 0.4 ± 1.3 ± 0.9 ± 14.6 ± 21.3 ± 20.7 ± 30.6
129.3 124.9 124.3 127.9 124.5 123.7 129.7 135.3 68.0 133.8
Mn(II)
± 5.7 ± 4.0 ± 2.7 ± 4.1 ± 5.8 ± 4.0 ± 0.2 ± 7.2 ± 2.1 ± 11.7
127.6 125.5 123.5 125.4 116.3 114.8 127.1 117.0 63.5 13.4
Cu(II)
± 5.7 ± 2.6 ± 1.8 ± 1.7 ± 4.2 ± 3.2 ± 0.6 ± 11.1 ± 1.8 ± 2.0
In conclusion—and in response to the research questions of Paper II—tannic acid stands out as the
most effective chelator for the selective depletion of catalytic transition metals, outperforming all
other chelators assessed. A common observation among all chelators is their limited ability to remove
manganese. Except for FeII-chlorogenic acid, all chelators demonstrate greater metal-depletion at wort
pH (5.60) compared to beer pH (4.30), prompting the decision to incorporate chelators during mashing
in subsequent studies. This approach offers the added advantage of binding deleterious metal ions
early on, thereby mitigating their detrimental effects sooner. In the follow-up study (Paper III), four
additional chelators (gallic acid, EDTA, citric acid and phytic acid) were selected for further
investigation, as the beneficial effects of tannic acid are already known and utilised within the brewing
industry.
5.2. Complexation of transition metals by chelators added during mashing and

impact on beer stability (Paper III)
The significant influence of pH is further substantiated throughout this laboratory scale mashing study,
corroborating the findings of the model solution study (Paper II). In conjunction with the increased
efficacy of external chelators at elevated mash pH—resulting in diminished (free) metal levels—there
is a decreased release of iron, manganese and zinc into the lautered wort, with the exception of
copper, as illustrated in Figure 9.
Figure 9 (reproduced from Paper III, Figure S6): Levels of manganese, zinc, iron and copper (µg/L) in
lautered wort at increasing mash pH, with mashing conducted with 5.9 µM gallic acid added at the
onset of mashing (0 min) and a mashout of 78 °C. Error bars indicate the least significant difference
observed between the two biological replicates.
This 'pH effect', whereby higher mash pH leads to lower transition metal concentrations in the wort,
persists throughout sparging. While the differences in metal ion concentration decrease between the
pH 5.0 and pH 6.0 wort sparges may appear small (except for zinc; Paper III, Figure 2), a substantial
disparity becomes apparent when absolute concentrations are considered. In other words, sparge
waters derived from acidic mashes have significantly higher transition metal contents, as shown in
Table 12. It is also noteworthy that, in relation to extract, sparge waters hold considerably higher
concentrations of metal ions than the first wort. Therefore, it may be beneficial for brewers to treat
the sparge water with effective chelating agents—and recirculate it— before reintroducing it to the
first wort to remove excess transition metals.
Results - Complexation of transition metals by chelators added during mashing and impact on beer stability 33
Table 12 (adapted from Paper III, Table S1): Averaged metal concentrations ± standard deviation (µg/L)
for the first worts, the first and the second sparges, for both mash pHs, for every chelator. Values and
standard deviations were calculated from sixteen biological samples, encompassing variations in
addition time, mash out temperature and chelator concentration.
First wort metal content (µg/L)
Iron Copper Manganese Zinc
Chelator
pH 5.0 pH 6.0 pH 5.0 pH 6.0 pH 5.0 pH 6.0 pH 5.0 pH 6.0
190.2 101.8 325.7 358.8 660.9 182.4 519.6 270.5
Tannic acid
± 88.5 ± 22.1 ± 23.5 ± 25.1 ± 59.1 ± 11.4 ± 99.5 ± 34.4
346.9 143.2 341.4 367.0 629.6 177.1 414.4 258.3
Gallic acid
± 15.5 ± 5.9 ± 13.8 ± 9.3 ± 39.2 ± 13.1 ± 33.0 ± 18.6
828.4 366.5 355.9 385.8 701.6 304.5 966.5 967.8
EDTA
± 356.8 ± 169.6 ± 23.7 ± 17.7 ± 75.7 ± 105.8 ± 412.5 ± 490.7
384.0 150.3 357.6 398.1 676.3 180.4 461.0 266.4
Citric acid
± 64.1 ± 25.8 ± 86.1 ± 86.7 ± 95.2 ± 23.2 ± 80.2 ± 38.4
387.0 151.6 359.9 397.6 708.6 186.0 451.1 259.2
Phytic acid
± 50.6 ± 11.7 ± 16.0 ± 18.8 ± 86.7 ± 16.5 ± 57.4 ± 18.4
First sparge metal content (µg/L)
Chelator
152.4 72.5 205.3 244.0 621.1 172.8 597.1 228.7
Tannic acid
± 66.2 ± 18.8 ± 14.5 ± 20.8 ± 36.1 ± 8.0 ± 43.4 ± 20.7
256.8 104.7 213.4 244.5 606.7 167.9 521.8 220.7
Gallic acid
± 19.4 ± 15.1 ± 7.0 ± 7.2 ± 44.2 ± 10.3 ± 26.4 ± 11.6
457.1 184.7 216.8 251.1 664.7 227.9 1011.1 782.4
EDTA
± 177.9 ± 66.5 ± 22.1 ± 22.0 ± 103.7 ± 54.1 ± 395.7 ± 393.7
258.0 100.6 194.1 237.1 626.5 165.0 549.5 217.2
Citric acid
± 46.8 ± 16.8 ± 23.9 ± 31.3 ± 68.6 ± 16.5 ± 92.9 ± 33.4
284.9 108.9 223.9 263.2 694.1 184.5 574.2 232.2
Phytic acid
± 44.0 ± 13.4 ± 10.7 ± 9.4 ± 94.7 ± 18.7 ± 75.3 ± 23.3
Second sparge metal content (µg/L)
Chelator
88.2 49.7 114.0 132.9 453.2 147.7 403.6 150.4
Tannic acid
± 34.9 ± 11.6 ± 8.5 ± 7.0 ± 29.3 ± 5.3 ± 44.4 ± 10.1
143.9 70.4 114.0 129.0 423.1 140.2 342.7 142.3
Gallic acid
± 10.5 ± 5.5 ± 5.3 ± 4.0 ± 27.3 ± 11.4 ± 20.5 ± 7.5
231.9 110.1 114.7 133.3 444.7 165.4 582.4 437.9
EDTA
± 78.1 ± 33.6 ± 7.8 ± 8.7 ± 52.2 ± 29.3 ± 194.3 ± 210.0
143.8 70.5 105.7 127.9 455.2 136.2 371.4 140.3
Citric acid
± 27.3 ± 12.4 ± 18.2 ± 20.9 ± 66.2 ± 18.6 ± 69.9 ± 24.3
173.2 81.6 127.5 149.0 552.3 167.0 445.0 165.8
Phytic acid
± 44.9 ± 16.7 ± 11.9 ± 12.3 ± 136.0 ± 32.0 ± 130.3 ± 32.3
The grain bill is another important variable influencing wort metal composition. As illustrated in Figure
10, the values generated by the full central composite design reveal that higher proportions of roasted
malt lead to higher concentrations of iron and manganese, but not copper, especially at low mash pH.
Variations in mashing out temperature and chelator addition time had little impact on metal levels,
although the limited difference in mashing out temperature (ΔT ≤ 8 °C) may have contributed to the
small effects observed; and chelator addition time may still affect wort oxidative stability and
aldehydes formation during brewing. Additionally, for chelators capable of forming metal complexes,
chelator concentration had a direct influence on the final metal levels, up to a certain plateau. Even
for chelating agents that form lauterable chelate complexes, complete removal of metal ions is not
achievable, regardless of their efficacy and concentration.
Figure 10 (reproduced from Paper III, Figure 9): Levels of iron, manganese and zinc (μg/L) at increasing
ratios of Munich malt (left to right) and at two pH levels (pH 5.25, black; pH 5.75, red).
Of the five 'primary' chelators selected from Paper II (tannic acid, gallic acid, EDTA, citric acid and phytic
acid), only tannic acid effectively depleted the iron content of lautered wort when added during
mashing, as shown in Table 13 and Figure 11. Moreover, none of the chelating agents proved effective
in removing copper or manganese—although the latter was expected based on the previous study.
Mash inclusion of phytic acid led to elevated levels of zinc in the wort; and the addition of EDTA even
resulted in major increases in iron (Paper III, Figure 4), manganese and zinc. This phenomenon might
be attributed to the extraction of metal ions from the malt during mashing and the hindrance of metal
ion incorporation into large endogenous complexes, such as with proteins. In the case of EDTA, its
strong chelating properties even masked the pH-related effect normally seen with zinc
(increased/decreased zinc with lower/higher mash pH); and its addition resulted in an increase in
radical formation of the unboiled wort samples, as measured on electron spin resonance (ESR)
spectroscopy—a valuable tool for assessing antioxidant potential and understanding the interplay
between pro- and antioxidants within a matrix.
Table 13 (reproduced from Paper III, Table 5): Pearson correlation coefficients (r) between
experimental factors and metal ion concentrations in lautered wort.
Tannic acid Gallic acid EDTA Citric acid Phytic acid
[Fe] [Cu] [Mn] [Zn] [Fe] [Cu] [Mn] [Zn] [Fe] [Cu] [Mn] [Zn] [Fe] [Cu] [Mn] [Zn] [Fe] [Cu] [Mn] [Zn]
Chelator
0.00 0.74 -0.05 0.04 -0.05 0.32 -0.08 -0.08 -0.01 -0.29 -0.01 -0.28 -0.02 -0.12 -0.10 -0.06 -0.14 0.25 -0.10 -0.11
addition time
Mash pH -0.59 0.49 -0.99 -0.87 -0.99 0.65 -0.99 -0.95 -0.66 0.55 -0.92 -0.05 -0.96 0.47 -0.98 -0.93 -0.96 0.70 -0.98 -0.92
Mash out
-0.07 -0.17 -0.10 -0.35 -0.06 0.08 -0.07 -0.25 0.03 -0.10 -0.18 -0.13 0.08 0.04 -0.05 -0.15 -0.13 0.05 -0.10 -0.22
temperature
Chelator
-0.66 0.08 0.05 0.16 0.01 0.10 0.01 0.04 0.69 0.01 0.27 0.93 -0.01 -0.02 -0.01 0.01 0.02 0.22 0.00 -0.05
concentration
Pearson's r ∈ [-1,1] and coloured dark red at total positive linear correlation (1.00), white at no linear correlation
(0.00) and dark blue at total negative linear correlation (-1.00).
Figure 11 (reproduced from Paper III, Figure 5): Response surface interaction model for the fitted value
levels of iron concentration (μg/L) in lautered wort, after mashing with varying levels of tannic acid
addition (μmol/L) and mash pH, with chelator additions at onset of mashing and mash out at 78 °C.
As shown in Figure 12, of the fourteen ‘alternative’ chelators examined in this study, which included
ferulic acid, tartaric acid, quercetin, chlorogenic acid and ten polyphenolic food extracts (green tea,
pomegranate, grape seed, reishi, cinnamon, curcuma, milk thistle, ginkgo, grapefruit seed and
raspberry), only two demonstrated noteworthy chelating properties during the mashing process in
terms of forming filterable complexes with iron: green tea and pomegranate extract.
Figure 12 (adapted from Paper III, Figures 6-7): Levels of iron (μg/L) with increasing concentrations of
chelators (mg/L), with chlorogenic acid (C) in dark blue, tartaric acid (T) in purple, ferulic acid (F) in red,
quercetin (Q) in green, green tea (G) in light blue, pomegranate extract (P; 40 % ellagic acid) in mauve
and eight polyphenolic food extracts in grey (curcuma, 1; cinnamon, 2; raspberry, 3; grapefruit seed,
4; ginkgo, 5; grape seed, 6; reishi, 7; milk thistle, 8). All values were calculated from four technical
replicates, with an average relative standard deviation below 2 %.
The pomegranate extract with 90 % purity in ellagic acid outperformed its 40 % purity counterpart
(Paper III, Figure 8) and demonstrated superior iron binding performance compared to tannic acid, as
shown in Figure 13. This particular finding is of fundamental importance within the scope of this thesis
and its potential implications within the brewing and beverage industry have been patented (Paper V)
due to its practical applicability in these fields.
Figure 13 (reproduced from Paper III, Figure 10): Response surface interaction model for the fitted
value levels of iron concentration (μg/L) in lautered wort, after mashing with varying levels of
pomegranate extract addition (mg/L) and mash pH, with a 50/50 Pilsner/Munich grain bill, chelator
additions after 27.5 min mashing and mash out at 78 °C.
In conclusion—and in response to the research questions of Paper III—out of the nineteen chelators
investigated in this study, only three demonstrated the ability to bind with iron in a manner that
allowed for its efficient removal by lautering, namely tannic acid, green tea and pomegranate extract.
The metal-depleting properties of the latter exceeded those of the well-established tannic acid.
Acidification of the mash leads to increased levels of iron and manganese in the lautered wort. To
increase the effectiveness of the chelators and minimise the amount of transition metals in the wort
without compromising the brewing process, it is recommended to mash at a natural wort pH of 5.6.
As can be seen in Figure 14, the addition of pomegranate extract successfully improved the oxidative
stability of the wort, as evidenced by reduced radical formation rates assessed by ESR spectroscopy.
38 Results - Effects of mash chelator addition on transition metal content and oxidative stability of brewer’s wort
Figure 14 (reproduced from Paper III, Figure 11): ESR measurements (T450-values) of boiled worts (grain
bill, 50/50 Pilsner/Munich; mash out temperature, 78 °C) with various levels of pomegranate extract
(mg/L), mash pH and chelator addition time (min). Error bars indicate standard deviation based on two
technical replicates.
5.3. Effects of mash chelator addition on transition metal content and

oxidative stability of brewer’s wort (Paper IV)
The laboratory scale findings of Paper III could be successfully replicated at pilot scale in the research
breweries of the KU Leuven (Ghent, Belgium) and the TU Berlin (Berlin, Germany). Figure 15 shows
that all three chelators tested (tannic acid, pomegranate and green tea extract) effectively lowered
iron levels throughout the brewing process when added to the mash, as measured by inductively
coupled plasma optical emission spectrometry (ICP-OES) analysis.
Results - Effects of mash chelator addition on transition metal content and oxidative stability of brewer’s wort 39
Figure 15 (reproduced from Paper IV, Figure 8): Iron concentrations of the KUL and TUB worts during
mashing, measured at the onset of boiling (light grey), end of boiling (grey) and end of cooling (dark
grey). All values were calculated from four technical replicates, with an average relative standard
deviation below 2 %, and normalised to 12 % w/w extract.
Similar to the previous study, these reductions in iron content led to more oxidatively stable wort, as
indicated by decreased radical formation using ESR spectroscopy—the results of which are presented
in Figure 16. Pomegranate extract (90 % ellagic acid) outperformed tannic acid and green tea extract
by a significant margin in both assays.
Figure 16 (reproduced from Paper IV, Figure 9): ESR results of the clarified KUL and TUB worts.
Reference brews (no chelator addition) are in black with hollow symbols (△,◻,◇), pomegranate
extract brews are in red (early addition, ▲ & ◆; late addition, ◼), green tea extract brews are in green
(early addition, ▲ & ◆; late addition, ◼) and tannic acid brews are in blue (early addition, ▲ & ◆;
late addition, ◼). Error bars indicate standard deviation based on two technical replicates.
40 Results - Effects of mash chelator addition on transition metal content and oxidative stability of brewer’s wort
The addition of chelators did not affect the brewing process, as evidenced by the high similarities and
low standard deviations of several brewing-relevant parameters (Paper IV, Table 5), including extract,
pH, filtration time, total polyphenols, soluble protein content and FAN (Paper IV, Figures 2-6).
However, as seen in Figure 17, two of the eight KUL brews exhibited higher TBI values early on,
indicating higher levels of heat stress. This observation is important since heat load can have a
significant impact on staling-related benchmarks such as colour and aldehydes, as confirmed in this
study. It was hypothesised that the presence of Maillard intermediates in the green tea and
pomegranate extracts may have been responsible for the elevated TBI values observed. These
intermediates might not be completely removed during mashing when introduced late in the process,
as appears to have been the case with the early additions.
Figure 17 (reproduced from Paper IV, Figure 7): TBI values of the KUL and TUB worts, measured at
mash filtration (light grey), the onset of boiling (grey) and end of boiling (dark grey). All values were
normalised to 12 % w/w extract. Error bars indicate standard deviation based on two technical
replicates.
Consistent with the previous study and evident from the concentrations listed in Table 14, no chelator-
mediated removal of manganese and copper was observed. Although the content of these two
deleterious transition metals naturally decreases during brewing (Paper IV, Table 5), their presence
can contribute to oxidative damage. However, the successful removal of iron appeared to be of greater
importance, as clear correlations were observed between lower iron levels, reduced radical formation
rates and decreased aldehyde levels in the wort (which was not the case for copper and manganese).
Early addition of chelators generally proved more effective in achieving these results compared to late
mash addition, as can be seen from both Table 14 and Figure 16.
Results - Effects of mash chelator addition on transition metal content and oxidative stability of brewer’s wort 41
Table 14 (reproduced from Paper IV, Table 2): Transition metal concentrations of the clarified KUL and
TUB worts. All values were calculated from four technical replicates, with an average relative standard
deviation below 2 %, and not subjected to normalisation for w/w extract.
Addition [Fe] [Cu] [Mn] Sum
Brew Chelator
time (µg/L) (µg/L) (µg/L) (µg/L)
Ref₁ (△) 339.3 91.3 95.9 526.4
- -
Ref₂ (◻) 298.1 76.9 95.1 470.0
PGₑ (▲) Pomegranate extract Onset of 47.4 89.1 100.1 236.5
KU GTₑ (▲) Green tea extract mashing 274.4 91.7 98.1 464.1
Leuven TAₑ (▲) Tannic acid (“early”) 204.5 91.0 114.6 410.1
PGι (◼) Pomegranate extract End of 76.0 89.7 88.4 254.0
GTι (◼) Green tea extract mashing 312.6 90.7 94.0 497.3
TAι (◼) Tannic acid (“late”) 200.0 94.0 96.5 390.5
Refₔ (◇) - - 180.1 39.1 68.7 287.9
TU PGₔ (◆) Pomegranate extract Onset of 21.1 26.0 71.2 118.3
Berlin GTₔ (◆) Green tea extract mashing 133.3 24.4 68.5 226.2
TAₔ (◆) Tannic acid (“early”) 121.5 21.3 73.6 216.4
In addition to iron content, heat load and appropriate wort boiling with adequate evaporation were
also identified as important factors influencing wort aldehyde content (Paper IV, Table 3). This is
evident from the collective aldehyde levels across all brews, as shown in Figure 18. The brews with the
highest TBI values exhibited the highest levels of pre-boil aldehydes, while the brew with the lowest
evaporation rate during boiling had the highest levels of post-boil aldehydes.
Figure 18 (reproduced from Paper IV, Figure 10): Aldehyde concentrations of the KUL worts, measured
at mash filtration and the end of boiling. All values were normalised to 12 % w/w extract and calculated
from three technical replicates; error bars indicate standard deviation. From bottom to top: 3-
methylbutanal (red), 2-methylpropanal (blue), 2-methylbutanal (yellow), methional (green),
phenylacetaldehyde (purple), furfural (grey), hexanal (pink), trans-2-nonenal (indistinguishable).
42 Results - Application of punicalagin/ellagic acid to improve oxidative and colloidal stability of beverages
In conclusion—and in response to the research questions of Paper IV—the addition of chelating agents
resulted in lower levels of total aldehydes in the boiled wort compared to the ‘no chelator’ reference
brews. Tannic acid, and in particular pomegranate extract, showed great success in improving the
oxidative stability of wort, mainly through iron sequestration and removal during brewing. Since a high-
quality wort—characterised by low levels of (free and bound) Strecker aldehydes, high oxidative
stability and elevated levels of residual reducing substances—is expected to result in a higher-quality
beer with extended shelf life, chelating agents, especially the newly discovered pomegranate extract
presented in this thesis, offer a compelling and innovative addition to the brewer's repertoire to
achieve this goal.
5.4. Application of punicalagin/ellagic acid to improve oxidative and colloidal

stability of beverages (esp. beer) (Paper V)
As Paper V is a patent rather than a research paper, the majority of the results presented in it are
derived from Papers III-IV and will not be readdressed within this context (see above). However, a few
distinctive results relating to beer were highlighted and will be briefly reviewed here.
The invention described in Paper V relates to a technique for producing beverages with improved
oxidative and colloidal stability through the addition of punicalagin/ellagic acid (compounds commonly
found in pomegranates, hence pomegranate extract). The technology aims to slow down the rate of
oxidation and reduce the formation of staling precursors, such as carbonyls, during production and
storage by chelating transition metals and/or quenching free radicals. Pomegranate extract,
particularly punicalagin-rich versions, also has the potential to act as a clarifying agent by binding to
proteins, potentially shortening maturation time—saving production time and costs—and preventing
haze formation. Although the primary focus is on brewing and beer, it is not limited to these areas and
can be used across different production steps (e.g. mashing, boiling, maturation, filtration), setups (e.g.
breweries) and product types (e.g. beer styles).
Pomegranate extract performs a similar function to tannic acid, which is widely used in large-scale
commercial brewing for its colloidal stabilising properties. However, tannic acid, which is commonly
derived from gallnuts, has the disadvantages of being costly and having poor consumer acceptance.
Pomegranate extract, which is derived from an edible fruit, has the potential to overcome these issues,
while also excelling in its ability to remove iron, making it a more potent antioxidant.
The eight beer batches produced at the KU Leuven pilot brewery (Technology Campus Ghent) exhibited
similar alcohol contents (Paper V, Figure 6), with a mean value of 5.8 ± 0.1 ABV, consistent with the
comparability of the wort extracts (see Paper IV). Beer pH values also remained stable, averaging 4.6
± 0.0. These characteristics are consistent with normal brewing and fermentation practices, suggesting
that chelator additions have no adverse effects herein. Similar reproducibility was observed in the four
batches of beer produced at the TU Berlin pilot brewery, with alcohol contents of 4.9 ± 0.1 ABV and
pH values of 4.1 ± 0.1. The slightly lower pH observed in the TUB beers was due to the use of a more
heavily roasted Munich malt, which also resulted in higher beer colouration (data not shown).
As illustrated in Figure 19, the colour of the fresh KUL beer (with a mean value of 14.1 ± 2.2 EBC)
remained stable over two weeks (13.9 ± 1.9 EBC) and one month (14.3 ± 1.9 EBC) of ageing. No notable
differences in colour progression rates or initial colour values were observed between the 'no chelator'
Results - Application of punicalagin/ellagic acid to improve oxidative and colloidal stability of beverages 43
reference beers and the chelator beers, except for two outliers: the late-added pomegranate and
green tea extract beers (PGι and GTι, respectively), both of which showed markedly higher beer colour.
This observation was consistent with the higher heat load (and colour) observed in the wort of these
beers, as shown in Figure 17. Clearly, the underlying factor responsible for these effects—presumably
coloured Maillard reaction products—persisted throughout fermentation.
Figure 19 (adapted from Paper V, Figure 7): Colour progression of KUL beer as it ages, from fresh beer
(white) to two weeks old (light grey), one month old (grey), two months old (dark grey) and three
months old (black). Error bars indicate standard deviation based on two technical replicates.
In terms of colloidal stability, beers with late mash addition of pomegranate extract, and especially
tannic acid, showed less haze development over forced ageing than the blanks, as shown in Figure 20.
Conversely, both the early and late green tea extract addition beers showed increased susceptibility
to haze formation over time. More clarity on the effects of chelator additions on colloidal beer stability
may be obtained by incorporating them during wort boiling or beer maturation rather than during
mashing.
44 Results - Application of punicalagin/ellagic acid to improve oxidative and colloidal stability of beverages
Figure 20 (adapted from Paper V, Figure 12): Forced haze formation in fresh and aged KUL beers.
Reference brews (no chelator addition) are in black with hollow symbols (△,◻), pomegranate extract
brews are in red (early addition, ▲; late addition, ◼), green tea extract brews are in green (early
addition, ▲; late addition, ◼) and tannic acid brews are in blue (early addition, ▲; late addition, ◼).
All values were calculated from two technical replicates, with an average relative standard deviation
below 1 %.
Although a full and comprehensive sensory evaluation with a large panel was not possible for the beer
samples, mainly due to the COVID-19 restrictions in place at the time, sensory evaluations were carried
out at a later date by independent evaluators. The results of these evaluations are documented in
Section 7 (Conclusion & Outlook).
Summarising Discussion 45
6. Summarising Discussion
Maintaining the fresh flavour profile of beer remains an ongoing challenge for brewers. The removal
and inhibition of transition metal catalysts (Fe, Cu, Mn) is expected to improve beer quality and shelf
life by minimising the concentration of reactive oxygen species (ROS) during brewing and storage. This
approach is based, at least in part, on the widespread recognition that oxidation is the primary driver
of beer staling [2, 3], of which a significant proportion is initiated by radicals, and formed the basic
premise on which the thesis was built. The concept is effectively captured by the title of the thesis,
'Strategies to Reduce the Iron Intake during the Brewing Process with respect to Flavour Stability',
which was established prior to any of the research work conducted.
Although the objective was clear from the outset, the exact approach to achieving it had yet to be
determined. The focus was placed on the utilisation of external chelating agents as a means of not only
sequestering iron but also attempting a complete removal of it—preferably along with copper and
manganese—through filtration following complex formation. As a first step, a broad screening of nine
chelating agents was carried out under controlled conditions (Paper II). The selection criteria for the
chelators emphasised food grade status, commercial availability in high purity and relevance to the
food and beverage sector, with a preference for components already naturally occurring in beer. A
further requirement was that the compounds should be selective for iron and, ideally, copper and
manganese while abstaining from removing essential brewing metals such as calcium, zinc and
magnesium. The compounds chosen, along with additional factors contributing to their selection, are
presented in Table 15.
Table 15: Initial chelators selection and rationale.

Chelator Factors that influenced their selection
Renowned powerful synthetic food grade chelator. Known to form highly stable
EDTA
complexes with various metal ions in a wide range of foods and beverages [193, 194].
Naturally found in beer [195, 196] and wine [197].
Citric acid
Promising copper scavenger [185].
Tartaric Naturally found in beer [196] and wine [197, 198].
acid Known to form complexes with iron i.a. in wine [199].
Naturally found in beer [198, 200] and red wines [201].
Quercetin
Capable of binding iron and copper in slightly acidic to neutral pH conditions [202].
Chlorogenic Naturally found in beer [198, 200] and wine [197].
acid Shown to chelate iron in buffer solutions [203].
Naturally found in beer [198, 200] and wine [197].
Ferulic acid
Shown to chelate copper in buffer solutions [204].
A hydrolysis product of tannic acid. Naturally found in beer [198, 200] and wine [201].
Gallic acid
Shown to chelate iron in buffer solutions [203].
Naturally found in malt and its degradation product in beer [205].
Phytic acid
Known to bind to iron in wine [206]. Strong oxidation inhibitor in various foods [184].
Commonly used as a clarifying agent in the beer and wine industries [207].
Tannic acid Renowned for its ability to bind iron and copper, for which it is used commercially i.a.
in brewing [70].
To ensure controlled conditions, acetate buffer model solutions were employed to simulate wort and
beer environments in terms of acidity (pH 5.60 and 4.30) and ethanol content (0.0 and 5.0 vol%).
While complex formation undoubtedly occurred with most of the chelators, as indicated by UV/Vis
46 Summarising Discussion
spectral changes and the appearance of colour within the initially colourless mixtures (Paper II,
Tables 3-4), the study revealed that achieving filterable metal complexes proved challenging, even
with 0.2 µm filtration, as seen in Tables 10-11. However, it is worth mentioning that the inability of a
chelator to form large, filterable complexes with transition metal ions does not necessarily preclude
it from exhibiting antioxidant behaviour. Some metal complexes (e.g. Cu–quercetin) show enhanced
radical scavenging activity relative to the ligand in its free state and reduced reactivity relative to the
metal in its unbound form [208–210].
Of the nine chelators tested in Table 15, only tannic acid met the desired expectations with regard to
the simultaneous removal of iron and copper. Although it should be noted that tannic acid did not
form complexes with manganese, this was a characteristic observed with all the chelators. Tannic acid
was, however, chosen as a positive control in this study because of its established reputation as a
processing aid with chelating properties in the beverage industry [70]. This result confirms existing
knowledge rather than representing a discovery.
Nevertheless, a number of other compounds showed promise in different ways. Although not nearly
as effective as tannic acid, the chelators gallic acid, quercetin, phytic acid, chlorogenic acid and ferulic
acid all caused reductions in iron levels compared to the ‘no chelator’ control samples. Furthermore,
gallic acid slightly scavenged copper, while phytic acid had a high affinity for zinc. The study also
highlighted the significant influence of pH on complex formation, with most chelators forming larger
complexes at wort pH (5.60) as opposed to beer pH (4.30). As a result of this observation, subsequent
work was carried out exclusively in wort.
In the follow-up study (Paper III), the chelators were tested under actual brewing conditions, where
they were added during laboratory scale mashing. While these conditions introduce more variability
than controlled model studies, they more closely mimic real brewing situations. Although only five
chelators from the previous study were initially selected for experimental screening, the remaining
four chelators were eventually also evaluated under mashing conditions, albeit without experimental
design.
Apart from tannic acid, the remaining eight chelators failed to successfully remove iron—although
quercetin did exhibit a small beneficial effect, as depicted in Figure 12. Furthermore, the notion that
permeated chelators could potentially confer antioxidant activity to the lautered wort could not be
substantiated for the chelators tested (gallic acid, citric acid, phytic acid, EDTA) by ESR spectroscopy
on unboiled wort. This was either due to the lack of substantial differences in radical formation rates
between 'high level' (35.3 µM) and 'low level' (5.9 µM) chelator mash additions, or even, as in the case
of EDTA, an increase in radical concentration with higher chelator concentration—attributed to
enhanced extraction of transition metals into the lautered wort and a possible prooxidant effect of the
Fe-EDTA complex [211].
Based on the consistent efficacy of tannic acid, and supported by the minimal differences observed
between classical and 0.45 µm wort filtration, it was concluded that the ability of a chelating agent to
effectively retain metals through filtration, particularly in coarse filtration such as lautering, relies
heavily on its capacity to form large complexes. This in turn is highly dependent on its molecular size,
composition and ability to establish cross-links with other matrix compounds (e.g. peptides and
proteins). Consequently, another broad screening of chelating agents was undertaken, this time
focusing on high molecular weight (MW) chelators, like tannic acid. This requirement naturally led to
the exploration of (poly)phenolic compounds as potential candidates.
Several criteria were considered to ensure the suitability of the compounds for the study. Apart from
having a large molecular size, the compounds had to be food grade and have chelating potential, which
was determined by the presence of functional groups capable of binding to metals (e.g. hydroxyl,
carboxyl, catechol and galloyl moieties) [212, 213]. However, obtaining high-purity polyphenolic
compounds can be costly and challenging. To maximise the number of candidates within the available
budget, it was decided to work with dietary supplements. Polyphenol-rich dietary supplements are
affordable, readily available and safe to consume at the intended concentrations.
However, their use has certain drawbacks that need to be considered. Unlike the chelators in the first
model study, the purity of the compound of interest in extracts may be limited or even unknown. Along
with the active ingredient listed on the label, extracts may contain various other phenolics and organic
components. This variability may potentially complicate the interpretation of results and introduce
uncertainty into the findings. Nevertheless, a selection of ten interesting polyphenolic extracts was
made for broad screening in terms of their ability to remove transition metals, details of which are
given in Table 16.
Table 16: Polyphenolic food extracts with the purity and molecular weight of their active compound.
Extract Active compound Purity (%) MW (g/mol)
Green tea Epigallocatechin gallate 50 458
Pomegranate Ellagic acid 40 & 90 302
Grape seed Oligomeric proanthocyanidin 95 10²-103
Reishi Polysaccharides 35 103-106
Cinnamon Polyphenols 30 10²-103
Curcuma Curcuminoids 95 300-400
Milk thistle Silymarin 80 482
Ginkgo biloba Flavonol glycosides 24 10²
Grapefruit seed Flavonoids 45 10²-103
Raspberry Raspberry ketone 100 164
As a point of reference, tannic acid has a molecular weight of 1701 g/mol. Although some of the
compounds listed in Table 16 have comparatively low MWs, it should be borne in mind that, as
mentioned above, the extracts in question may contain other, possibly larger, polyphenols (e.g.
unhydrolysed gallo- or ellagitannins) in addition to the active constituents mentioned. This presence
of large tannins is highly dependent on the extraction methods and the type of raw materials used,
which are unfortunately unknown in this context. For instance, besides ellagic acid, pomegranate
extract may also contain punicalagin (an ellagitannin found in pomegranates, particularly in the peel)
and pentagalloyl glucose (a gallotannin found in pomegranates, particularly in the leaves) [214]. These
compounds have MWs of 1085 and 941 g/mol, respectively, and are known to precipitate proteins
[215–217].
Out of the ten alternative substances evaluated in Table 16, two demonstrated good results. Green
tea and pomegranate extract were shown to have the ability to form complexes with iron in a manner
that allowed for its effective removal by laboratory scale lautering. The pomegranate extract that
contained 90 % ellagic acid even outperformed tannic acid in terms of transition metal removal (Paper
III, Figure 8). By successfully lowering the prooxidative iron content during mashing, the addition of
pomegranate extract resulted in a reduced level of radical formation in the wort and exhibited an even
greater antioxidant activity than the well-known tannic acid, as measured by ESR spectroscopy.
48 Summarising Discussion
Mash acidity was found to be a critical factor in wort metal loading and chelator efficacy, reaffirming
the significance of pH. Acidic mashes at pH 5.0 yielded higher levels of iron, manganese and zinc than
the more alkaline mashes at pH 6.0. However, copper levels remained largely unaffected by variations
in mash pH. Elevated iron levels resulting from acidic mash conditions (e.g. mash souring or use of
melanoidin-rich malt) could be mitigated by the appropriate addition of pomegranate extract or tannic
acid, highlighting the significant influence of both mash pH and chelator concentration—provided the
chelator is capable of sequestering iron. Similarly, the undesirable tendency of roasted malt to release
higher levels of iron and manganese into the wort can also be alleviated by effective chelator use—
something that is particularly useful when using high proportions of dark roasted malts. In contrast,
mashing out temperature and chelator addition time had relatively little effect on wort metal load.
The oxidative stability of the wort was found to be greater in samples with late additions of
pomegranate extract, contrary to the expected performance of early additions, where it was thought
that endogenous wort antioxidants would be better preserved. It was postulated that the improved
stability of the late addition samples was probably due to their slightly lower iron content. This
hypothesis was later confirmed in the subsequent study (Paper IV), which showed that early chelator
additions generally resulted in reduced radical formation rates due to lower levels of iron in the final
wort. However, when iron levels were comparable, no substantial differences in ESR spectroscopy
were observed between early and late chelator administration, as demonstrated by tannic acid in
Figure 16. Similarly, no noticeable differences were observed in terms of free wort aldehyde levels
between the early and late additions, as depicted in Figure 18.
The laboratory results were validated at pilot scale in two distinct breweries (Paper IV). The primary
objective of the study was to evaluate the reproducibility of the findings and to ensure that the
chelators had no adverse effects on the brewing process. It was found that sequestration of iron could
be successfully reproduced on a large scale, regardless of the wort separation method employed (mash
filtration or lauter tun). Importantly, the addition of chelators did not negatively impact the brewing
parameters assessed, including free amino acids, total polyphenols, soluble protein, extract and pH.
However, as can be seen in Figures 17 and 19, effects on TBI and colour were observed in two brews
with late chelator addition. This was attributed to the potential presence of Maillard reaction products
in the green tea and pomegranate extracts. As mentioned above, this was a possible drawback of using
dietary supplements.
Similar to the findings in the laboratory study, both pomegranate extract and tannic acid proved highly
effective in diminishing iron throughout the brewhouse operations. Early mash addition of
pomegranate extract showed the greatest efficacy, lowering iron concentrations by almost 90 % and
radical levels by nearly 80 % in the final wort compared to the ‘no chelator’ controls. Overall, the
incorporation of chelators resulted in a notable decline in total wort (free) aldehydes post-boil. Given
the chemical equilibrium between free and bound state aldehydes and the established strong positive
correlations between individual free Strecker aldehydes and their cysteinylated counterparts [22], it
can be reasonably inferred that chelator inclusion would also result in reduced levels of bound state
aldehydes. This holds significance because, due to their lower volatility, bound aldehydes are not
evaporated during wort boiling [85, 218]; nor are they readily reduced during fermentation [85, 218].
As a result, they can persist into the finished beer, where they can transform into their free and flavour-
active forms, ultimately contributing to the development of a stale beer taste [219].
It is essential to emphasise that both the overall heat load of the brewing process and the amount of
evaporation during wort boiling also play essential roles in determining wort aldehyde levels, as seen
in this and other work [220]. The former leads to the formation of aldehydes through i.a. heat-induced
Maillard reactions, whereas the latter leads to the depletion of aldehydes through vaporisation.
Furthermore, the study revealed strong to moderate correlations between wort iron content, total
polyphenols and free aldehydes.
The results indicate that natural chelators such as tannic acid, ellagic acid and punicalagin (an ellagic
and gallagic acid bound to the glucose moiety) are very likely to improve the freshness and shelf life of
the resulting beer by decreasing the levels of transition metals, radical formation and aldehydes during
brewing. However, the complexity of staling necessitates further study to fully comprehend the
implications of these findings for beer stability.
Addressing the following research questions would undoubtedly push the current knowledge about
beer flavour stability and the effects of chelators even further:
- Does the inclusion of iron-targeting chelators during mashing truly result in a measurable
enhancement of beer quality and freshness?
- Does the addition of chelators contribute to enhanced beer flavour stability, as evidenced by
changes over time in sulphite content, bitterness, oxidative stability, aldehyde concentration
and trans/cis iso-α-acids ratio?
- How do chelator additions influence the formation of haze during the ageing of beer?
- What are the effects of chelator additions on head formation in fresh beer and the stability
of the foam during ageing?
- Can remnants of the chelators, or their breakdown products, be detected in the beer?
- Do the chelating agents impart any discernible taste to the beer?
- What are the sensory differences between fresh and aged beers brewed with and without
chelators, as evaluated by a trained sensory panel?
However, it must be recognised that the feasibility of chelator addition has its limits in terms of
economic viability—as per the principle of diminishing returns. There also exists a practical threshold
as to the extent to which iron can be separated from wort and beer, making complete removal
unachievable. This is an important point to consider, as even at the lowest attainable iron
concentrations, a sufficient amount of iron may persist during brewing and/or in the final product to
sustain radical chain oxidation reactions. This concern is further exacerbated by the ability of iron to
undergo redox cycling (FeIII ⇄ FeII) and the fact that copper and manganese, which also act as reaction
catalysts in the Fenton and Haber-Weiss mechanisms, remain ineffectively removed by the external
chelators studied in this work. Furthermore, the presence of Cu and Mn, together with phenolics,
appears to play an important role in the redox cycling of iron [221–223].
50 Conclusion & Outlook
7. Conclusion & Outlook
Oxidative stability is a critical factor in beer production as it affects shelf life and overall product quality.
Iron, copper and manganese play a catalytic role in radical-associated staling. The hypothesis and
objective of this thesis were to investigate the potential of removing these metals to promote beer
flavour stability. It was found that this could be achieved by the incorporation of external chelating
agents during brewing, specifically high molecular weight polyphenols. The addition of either
pomegranate extract or tannic acid during mashing resulted in wort with enhanced oxidative stability
due to the early sequestration and precipitation of a large proportion of detrimental iron. It was also
demonstrated that the addition of tannic acid, green tea and pomegranate extract all resulted in
reduced aldehyde levels in boiled wort at pilot scale.
The results strongly suggest that polyphenolic compounds with iron scavenging properties—such as
tannic acid, ellagic acid and punicalagin—are valuable agents for improving the shelf life and flavour
characteristics of fresh beer. However, it must be recognised that staling is a complex interplay of
diverse chemical and physical reactions; and while chelators can certainly help combat oxidative
degradation and prolong beer shelf life, they are not a panacea ("cure-all"). Factors like excessive heat
exposure during brewing and storage, as well as elevated levels of TPO, can significantly undermine
the benefits of chelator use. Achieving optimum beer flavour stability therefore always necessitates a
combination of best practices and technology; while, of course, enjoying the beer promptly.
Further research is warranted to fully assess the effects of mash chelator additions on the
characteristics of the resulting (fresh and aged) beers, as outlined in Section 6 (Summarising
Discussion). Building upon the points discussed in that section, several interesting avenues for future
research and potential prospects can be considered in this field, including:
- Identification of suitable food grade chelating agents that are capable of effectively removing
copper and manganese from the brewing process.
- Investigation of the effects of incorporating pomegranate extract at other stages of the
brewing process, such as wort boiling and fermentation, characterised by a pH of 5.4 ± 0.3.
- Examination of the impact of adding pomegranate extract post-fermentation, during lagering
or before filtration, as well as direct addition to beer ready for packaging, characterised by a
lower pH of 4.1 ± 0.7.
- Evaluation of the potential of pomegranate extract as a clarifying agent for beverages.
- Assessment of the possible synergistic benefits of adding a combination of tannic acid and
pomegranate extract during brewing, compared to individual additions.
- Study of the application of chelators to enhance the stability of other beverages, such as
wine, juice and soft drinks, which may undergo similar ageing processes.
- Exploration of alternative extraction methods for the production of pomegranate extract
tailored for brewing and beer shelf life enhancement. This could include increasing the
punicalagin content (to enhance its iron binding ability) and minimising the heat impact
during extraction (to reduce the presence of reductone intermediates capable of metal ion
redox cycling).
As with any idea, its true value is revealed in its execution. It is with this in mind that the final point
deserves further elaboration.
Conclusion & Outlook 51
As of the time of writing, the rights to the patent for the technology of incorporating pomegranate
extract into beverage production (as outlined in Paper V) have been procured by a company
specialising in brewing processing aids. Concurrently, a pomegranate extract, specifically tailored for
brewing purposes, is actively undergoing development. As postulated, the aim is to create a more
refined and punicalagin-rich product to surpass the metal binding and antioxidant capabilities of the
pomegranate extract used throughout this research. While the prototype is still undergoing extensive
testing and refinement, a commercial version is anticipated to be available in the foreseeable future.
Although these advancements are not directly part of the work, they provide a valuable preview of the
practical implications and prospects of the thesis findings. Several unpublished tests conducted by
third parties using the punicalagin-rich (≥ 40 % pure) prototype extract have yielded promising
outcomes. These include:
- In buffer solutions at pH 5.0 and 5.7, punicalagin showed superior iron removal to tannic acid
(≥ 99 % pure) and ellagic acid (≥ 94 % pure) after 0.2 µm membrane filtration. At pH 5.7,
punicalagin also outperformed tannic acid and ellagic acid in the removal of copper [224].
- In mashing trials using 100 % Pilsner malt, punicalagin was more effective than tannic acid
and ellagic acid at removing iron from the wort, both after lautering and after whirlpooling. It
also gave the lowest radical concentration after an 800-minute assay (ESR T800-value). Similar
results were seen in mashing trials with a 10 % Cara-malt inclusion.
- In pilot scale brewing trials, the effects of ellagic acid and punicalagin as stabilisers were
investigated by membrane filtration and compared with established stabilising agents,
namely tannic acid, polyvinylpolypyrrolidone (PVPP) and silica gel. While both PVPP and silica
gel strongly reduced permeability, ellagic acid, punicalagin and tannic acid did not. All agents
improved haze stability except for ellagic acid. Punicalagin outperformed ellagic acid and
tannic acid in terms of decreasing iron content and enhancing beer oxidative stability [225].
- In a small commercial brewery, the addition of punicalagin resulted in significantly lower iron
levels and ESR T-values throughout the brewing process up to beer centrifugation, compared
to no-addition controls. Forcing tests also demonstrated reduced formation of permanent
haze in beers where punicalagin had been added during mashing. Similar outcomes were
observed in a separate brewery following an analogous approach.
- In a large commercial brewery, sensory evaluations of beers brewed with tannic acid, ellagic
acid and punicalagin were conducted by a trained panel of eight. The panellists rated the
control samples higher in terms of oxidation while noting that the punicalagin-treated beers
had better head retention, fruity esters, bitterness and drinkability compared to the fresh
control beers—which were less preferred, as they gave off some malt oxidation notes. The
control beers also had the highest iron concentrations, from cold wort to packaged beer,
although the differences in the beer could be considered negligible.
These findings strengthen the thesis conclusion that chelating agents have the potential to enhance
beer quality and extend shelf life. The results also underscore the effectiveness of polyphenolic
compounds such as tannic acid, ellagic acid and punicalagin in achieving these goals.
In summary, the exploration of various chelators and their effects on the brewing process and metal
removal in this work has contributed to the existing body of knowledge. The results obtained with
pomegranate extract, and particularly with the punicalagin-rich prototype, show great potential and
call for further exploration in the field of brewing and beverage production. In this respect, the thesis
has successfully met its objectives, while also contributing practical insights to the current
understanding of crafting a more flavour-stable beer.
52 References
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[221] Danilewicz, J.C.: Chemistry of manganese and interaction with iron and copper in wine,
American Journal of Enology and Viticulture, 67 (2016), no. 4, pp. 377–384.
[222] Danilewicz, J.C.; Tunbridge, P. and Kilmartin, P.A.: Wine Reduction Potentials: Are These
Measured Values Really Reduction Potentials?, Journal of Agricultural and Food Chemistry, 67
(2019), no. 15, pp. 4145–4153.
[223] Jenkins, D.; James, S.; Dehrmann, F.; Smart, K. and Cook, D.: Impacts of Copper, Iron, and
Manganese Metal Ions on the EPR Assessment of Beer Oxidative Stability, Journal of the
[224] Kunz, T.; Mertens, T.; Hahne, K.; Kuhn, F.; Bergmann, T. and Gibson, B.: Punicalagin - a novel
brewing stabilizing agent for beer shelf-life enhancement, 15th International Trends in Brewing
Symposium, Ghent (Belgium), 2023.
[225] Burrer, F. and Kunz, T.: Alternative stabilization agents for cross-flow membrane filtration, 15th
International Trends in Brewing Symposium, Ghent (Belgium), 2023.
64 Theses
9. Theses
1. Mash pH has a substantial influence on the effectiveness of both endogenous and exogenous
chelators, resulting in significant variations in the metal composition of wort and beer. This
influence is particularly pronounced for iron and manganese, where lower mash pH leads to
increased concentrations, ultimately affecting beer staling potential.
2. Roasted grains exhibit higher propensities for the release of iron and manganese into the wort.
This effect can be corrected by the use of effective chelating agents.
3. External chelating agents do not readily remove manganese from wort and beer model solutions,
nor from actual wort. While copper removal was observed with some chelators in model
solutions, the results did not transfer to actual wort.
4. Copper forms strong complexes with proteins and peptides during the brewing process, making
its impact on wort staling less pronounced compared to that of iron.
5. Lowering iron levels during the brewing process improves the oxidative stability of the wort and
reduces its aldehyde content.
6. The ability of chelators to form large complexes through the process of cross-linking is a critical
attribute for their effectiveness in removing transition metal ions by lautering or mash filtration.
7. Pomegranate extract outperforms the renowned tannic acid in its ability to sequester iron during
brewing, thereby improving wort oxidative stability to a greater extent.
8. Thermal load experienced during wort and beer production plays a pivotal role in the process of
staling. However, achieving appropriate wort boiling and evaporation rates is critical for effective
aldehyde removal.
9. Incorporation of certain chelating agents (e.g. EDTA, phytic acid) during mashing promotes the
extraction of transition metal ions, increasing the wort metal load, resulting in worts with reduced
oxidative stability and higher rates of radical formation.
10. Effective chelators (e.g. tannic acid, ellagic acid, punicalagin) have considerable potential to
improve product quality, enhance flavour stability and extend shelf life, not only in brewing but
also in various other beverage production processes.
Publications 65
Publications
A peer-reviewed review article (Paper I) was written and published and, in conjunction with Section 2
(Literature Review), serves to contextualise the research within the existing academic landscape,
identify research gaps, justify the need for the study, guide the formulation of research objectives and
methods, critically analyse previous research and provide a basis for discussion.
The practical research conducted for this thesis has led to the publication of a total of three research
articles (Papers II-IV) in two international peer-reviewed journals (BrewingScience and Journal of the
Institute of Brewing). Additionally, out of the experimental findings of Papers III-IV, a patent application
(Paper V), focusing on the use of ellagic acid/punicalagin in brewing, was filed and published with the
financial support of the Technische Universität Berlin.
Each research paper is based on independent experiments, although the findings and conclusions of
the publications are closely interrelated, as illustrated in Figure 8. Papers II-IV specifically address the
topic of transition metal chelation in brewing, with Paper II focusing on a broad screening of chelators
in brewing-relevant model solutions and Papers III-IV concentrating on the effects of optimised mash
chelator incorporation during laboratory and pilot scale brewing, with particular emphasis on oxidative
wort stability and staling-related aldehydes.
The experimental work for Papers II-III was carried out at the Technische Universität Berlin (Berlin,
Germany) between 2017 and 2019. The experimental work for Paper IV was conducted at both the TU
Berlin and the KU Leuven (Ghent, Belgium) from 2019 to 2021. It should be noted that the research
and publication of Paper IV was significantly hampered by the COVID-19 pandemic, causing delays in
data collection, experiments and the programme as a whole.
All publications were authored as the first author and received funding from the European Union’s
Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant
agreement No. 722166. Detailed author contributions can be found in the List of Publications. The
articles presented in this work have been reproduced with the permission of the publishers and are
arranged logically, giving priority to coherence rather than adhering to chronological order by date of
publication.
I
Transition metals in brewing and their role in wort and beer

oxidative stability: a review
Tuur Mertens, Thomas Kunz and Brian R. Gibson
Journal of the Institute of Brewing. 2022; 128:77-95.
DOI: 10.1002/jib.699
Review article
Received: 5 December 2021 Revised: 16 April 2022 Accepted: 24 June 2022 Published online in Wiley Online Library: 2 August 2022
(wileyonlinelibrary.com) DOI 10.1002/jib.699
Transition metals in brewing and their role in

wort and beer oxidative stability: a review
Tuur Mertens,* Thomas Kunz and Brian R. Gibson
Beer inevitably changes over time: the colour will darken, haze may form, and stale flavours develop, while others fade. The chal-
lenge of maintaining the fresh flavour quality of beer (over a typical 9-12 month storage period) is generally the determining fac-
tor of a beer’s shelf-life for brewers, as opposed to colloidal or microbiological stability. Fortunately, as early as the brewhouse,
oxidative degradation can - to a certain extent - be controlled, enabling the shelf-life to be increased. This review considers the
general issues of oxidative stability, mechanisms of ageing, ways of quantifying staleness and staling potential, and current prac-
tical approaches to prevent oxidative beer ageing. Emphasis is placed on the catalytic role of iron, copper and manganese on ox-
idation during brewing and storage; and how the removal and/or inhibition of these prooxidative transition metal ions leads
to prolonged beer (flavour) stability.
Keywords: Beer ageing; flavour stability; staling; oxidation; transition metal catalysts; chelation
Introduction rough and general estimate. Actual dates will vary depending on
multiple factors, including beer style, storage conditions, total
Beer is the most widely consumed alcoholic drink and - together package oxygen (TPO), packaging, and agitation (8). Oxidative pro-
with coffee and tea - one of the most popular beverages. The cesses are widely recognised to be the main force behind product
beer market continues to expand (1), as does consumer knowl- degradation (9–12) and the resulting flavours are described as
edge and demand for high(er) quality. Beer is expected to be ‘oxidised’, ‘aged’ or ‘stale’. These are umbrella terms for describing
fresh and free of any contamination or inappropriate haze. Fortu- a combination of oxidation notes found in beer, such as card-
nately, the latter are no longer serious problems for the brewer board/papery, sherry/Madeira, honey, ribes/blackcurrant/catty,
to control, as microbial and colloidal stability are, respectively, leathery, etc.
taken care of by removal of bacteria and wild yeast (e.g. by hy- With high oxygen ingress (e.g. during the brewing process, filtra-
gienic working practices, pasteurisation, 0.45 μm membrane fil- tion (13), poor filling practices, or usage of PET bottles (14) or inad-
tration) and application of stabilisation agents (e.g. PVPP, silica equate bottle caps (15)), these flavour transitions take place more
gel, tannic acid, etc.) (2). Accordingly, flavour stability - the ability rapidly. Off-flavour formation occurs, but also degradation of ini-
of a product to retain freshness and resist physicochemical tial, fresh flavours, such as hop aromas and pleasant bitterness.
changes – is the key factor in determining beer shelf-life. The organoleptic outcome of the shifts will depend on the concen-
Beer flavour stability has been the subject of more than 500 tration of the formed and degraded substances, and their respec-
publications (SciFinder). This is because, for any business to attract tive flavour activities.
and keep customers, the production and delivery of a consistent Besides flavour deterioration, aged beers typically appear darker
quality product is key. Creating a well known identity is vital to than their fresh counterparts, due to the oxidised polyphenols be-
maintain customer selection preference (3). Delaying in-pack fla- ing colourants (16). These oxidised polyphenols can also cause col-
vour changes as long as possible is of great commercial impor- loidal and foam stability issues, due to the polymerisation of pro-
tance (4). tein-polyphenol-metal complexes (forming haze), which
The issue of beer flavour (in)stability is more relevant than ever, concurrently diminishes the foaming ability through precipitation
given the international market where packaged beer can be of foam positive polypeptides (17,18).
shipped around the globe, often under harsh conditions (e.g. hot
containers, vibrating trucks) and for extended periods of time (5).
Even though it has been intensively studied since the 1960s, the
science behind beer staling is still not fully understood and contro-
versies remain.
The chemical non-equilibrium of beer, emanating from its intri-
cate matrix composition, gives rise to a complex set of ageing phe- * Corresponding author: Tuur Mertens, Technische Universität Berlin, Institute
nomena; suggesting that fresh beer, as a natural product, will of Food Technology and Food Chemistry, Chair of Brewing and Beverage
Technology, Berlin, Germany. Email: mertens.tuur@gmail.com
never be a fully stable commodity (6). Nonetheless, considerable
improvements in beer flavour stability have been made over the Institute of Food Technology and Food Chemistry, Chair of Brewing and Bev-
years and further advances can be expected, because of ongoing erage Technology, Technische Universität Berlin, Berlin, Germany
research and the continuous improvement of technology.
This is an open access article under the terms of the Creative Commons At-
Noticeable flavour changes may become apparent three tribution License, which permits use, distribution and reproduction in any
months from packaging (at room temperature) (7), but this is a medium, provided the original work is properly cited.
77
J. Inst. Brew. 2022; 128: 77–95 © 2022 The Authors. Journal of the Institute of Brewing published by John Wiley
& Sons Ltd on behalf of The Institute of Brewing & Distilling.
20500416, 2022, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/jib.699 by Technische Universitaet Berlin, Wiley Online Library on [30/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
T. Mertens et al.
Beer ageing mechanisms alcohol-free beer is unknown, but presumably, would also be car-
bon centred, since, in the absence of ethanol, the nonselective hy-
Oxidative beer ageing droxyl and alkoxyl radicals will react in a non-specific manner with
any nearby carbohydrate, protein, polyphenol, or other organic
Oxygen exposure is detrimental to beer stability and causes accel- molecules present in the matrix (36).
erated aldehyde formation (19). Fresh beer typically only contains Although the mechanisms in Figure 1 are well understood, and
low levels of aldehydes which are below the flavour threshold. In their significance is not in question, it is still uncertain exactly how
the past, the aldehyde trans-2-nonenal was subject to a lot of at- big a part they play in the overall staling picture. Oxidation, for ex-
tention since increasing concentrations of this potent, volatile car- ample, also occurs enzymically in malting and mashing through
bonyl were often seen in conjunction with beer the action of lipoxygenase (LOX) (37). There are however some ca-
staling, contributing to the ‘aged beer’ flavour characterised by veats, as iron besides promoting free radical and ROS formation
stale, oxidised, and cardboard/papery notes. These days, an array through Fenton and Haber-Weiss reactions, also plays a role in
of marker aldehydes is typically used to determine flavour instabil- lipoxygenase-induced oxidation (38,39). To further illustrate the
ity (20,21): including Strecker degradation aldehydes (2- and 3- complexity, other enzymes such as catalase conversely protect
methylbutanal, 2-methylpropanal, methional, benzaldehyde, and against oxidative damage (36,40).
phenylacetaldehyde), lipid oxidation aldehydes (hexanal and In addition, staling compounds (such as aldehydes) that are
trans-2-nonenal), and aldehydes formed during Maillard reactions formed during malting and mashing, may bind to other com-
(furfural). With the exception of furfural, these aldehydes can be pounds, resulting in adduct formation (41,42). While the influence
formed through oxidative reactions (6). of oxygen (and other parameters) on the formation of bound state
Oxidative degradation is the main cause of rapid beer staling aldehydes has yet to be fully investigated (43), their gradual release
(22) and, in most cases, the principal oxidising agent will be oxy- may be considered an indirect form of oxidative staling, as they
gen. However, as is later discussed in ‘oxygen-free beer ageing’, progressively become ‘unmasked’ during storage (44–47).
other reagents can act as an oxidant, such as mineral ions (e.g. Protecting the malt and wort from oxidation could prove benefi-
Fe3+ or Cu2+), oxidised organic compounds (e.g. melanoidins, phe- cial to shelf-life, since it would limit the endogenous ‘ageing poten-
nols), and halogens (e.g. from cleaners/sanitisers). Regardless, the tial’ carried over to the fresh beer. However, it is still unknown to
presence of oxygen will invariably promote oxidation. which degree oxidised substances in the free or bound state are
Remarkably, molecular oxygen (3O2) - partially dissolved in beer, formed during malting and brewing, or how much of the intrinsic
present in the headspace and diffusing through the crown cap antioxidative power of malt and wort is lost during these stages,
liner during storage - is relatively unreactive, as the reaction of ox- and how substantial this is in the ageing of packaged beer. In sum-
ygen with organic compounds is hindered kinetically (23). How- mary, beer staling is an immensely complex interplay of reactions,
ever, ground state oxygen can be converted to highly reactive with its kinetics still mostly unclear (e.g. enzymatic versus non-en-
forms, either by chemical reduction or collision with other organic zymatic) (48).
radicals, which can be formed during high energy stages, includ- As briefly mentioned, and displayed in Figure 1, transition
ing kilning of malt, wort boiling, beer pasteurisation, exposure to metals, such as ferrous (Fe2+) and cuprous (Cu+) ions, play a vital
light (24), and even milling (25). These highly reactive oxygen role in ROS formation (49). The participation of manganese ions
forms are collectively termed ‘reactive oxygen species’ (ROS) and (Mn2+) in generating radicals has been investigated but to a lesser
are potent oxidisers. Each is more reactive than oxygen itself extent. Although, like iron and copper, manganese is a d-block el-
and, in increasing order of reduction state and reactivity, involve ement (found from the third group to the twelfth group of the
superoxide anion (O●- ●
2 ) < perhydroxyl radical (HO2 ) < hydrogen modern periodic table), making it a viable metal catalyst, as it
●
peroxide (H2O2) < hydroxyl radical (OH ) (6). shares the tendency to exhibit two or more oxidation states. Since
Bamforth and Parsons (26) first noted the importance of ROS in the initial report by Zufall and Tyrell in 2008 (50), few recent studies
beer regarding flavour stability. At the time (1985), little was known have confirmed the augmenting effects of manganese ions on
about the mechanisms at play; but, involvement of the Fenton and beer staling (51,52).
Haber-Weiss mechanism was likely, as transition metal ions in beer Fe2+, Cu+ and Mn2+ serve as electron donors to reduce oxygen
are known to catalyse these radical-creating reactions. In 1988, species and are oxidised to Fe3+, Cu2+ and Mn3+. Oxygen (3O2) cap-
Kaneda and colleagues (27) first monitored free radicals in beer turing an electron forms superoxide (O●- 2 ), which can become pro-
by electron spin resonance (ESR) spectroscopy. In further studies tonated to generate the perhydroxyl ion (HO●2 ), or is further re-
(28–33), they uncovered the effects these radicals have on beer fla- duced to peroxide (O2- 2 ) which is then protonated twice, forming
vour deterioration, and the pathways involved in their formation, hydrogen peroxide (H2O2). Both perhydroxyl and hydrogen perox-
by trapping the short-lived radicals with spin-trapping reagents. ide are reduced to hydroxyl (OH●) via the Fenton and Haber-Weiss
The technique to trap short-lived radicals, creating detectable reactions. Because beer is relatively acidic (pH ∼ 4.3), most of the
long-lived spin adducts is still used in beer ageing research today. superoxide will be in the more reactive perhydroxyl radical form
Supporting Bamforth’s claim, Kaneda and colleagues suggested (pKa 4.8).
the free radicals formed in beer, to be hydroxyl radicals which, as Prooxidant molecules present in beer can reduce oxidised metal
the most reactive oxygen species, attack almost every substance ions back to their reduced state, so that they can contribute to the
with poor selectivity. While this is indeed taking place, Andersen activation of ground state oxygen (or a ROS) again. This is aggra-
and Skibsted (34) later showed the 1-hydroxyethyl radical to be vated by a process of ‘free radical atom abstraction’, where the pro-
the most abundant radical in beer, due to hydroxyl radicals oxidant itself can turn radical, furthermore degrading or reacting
reacting with ethanol a good radical scavenger and copious in with other compounds, producing off-flavours. Examples of wort/
beer. The generated alkyl radicals subsequently form acetaldehyde beer species, which have easily abstractable hydrogen and can be-
and hydroperoxyl radicals, after electron loss by metal ions or oxy- come secondary organo-radicals, are alcohols (primarily ethanol),
gen (see Figure 1) (34,35). The dominant radical species in sugars (e.g. glucose), free-thiol group (e.g. cysteine) containing
78
wileyonlinelibrary.com/journal/jib © 2022 The Authors. Journal of the Institute of Brewing published by John Wiley J. Inst. Brew. 2022; 128: 77–95
Review of metals and oxidative stability in brewing
Figure 1. Mechanism of reactive oxygen species formation in beer, adapted from (33,34)
proteins, organic acids (e.g. isohumulones), and phenolic sub- needed for the quenching of free radicals, reduction of H2O2 and
stances (e.g. hydroquinone, catechols) (53–56). adduct formation, excessive HSO-3 is converted to SO●- 3 (a free rad-
Some prooxidants, such as cysteine or other sulfhydryl-group ical detrimental to beer flavour stability).
containing proteins, can act as a direct source of H2O2, when The oxidative mechanisms in wort and beer are an intertwined
heated or illuminated (57). Examples of compounds that can act chain of electron transfer (oxidation-reduction/redox) reactions.
as prooxidative electron donors are reductones (e.g. early and in- Each reaction has a reaction rate (the speed at which a chemical
termediate stage Maillard reaction products) (58), polyphenols, reaction proceeds) and this rate is affected by the type of reaction,
sugars, iso-humulones, oxidised melanoidins, and alcohols (33). the substrate concentration, and the temperature. Thus, that a re-
Antioxidants, on the other hand, inhibit the effects of oxygen, by action is thermodynamically probable, does not guarantee that it
either chelating metal ions and/or capturing ROS and other free happens under storage conditions.
radicals (‘quenching’) (59). Some native beer and wort components As noted - apart from obvious parameters (storage temperature
- when present in appropriate (typically high) amounts - already ex- and in-pack oxygen) - transition metals play a key role in the loss of
hibit these protective properties, including yeast produced sulphite beer freshness (22). They may even be responsible for the majority
and certain malt and hop products (such as tocopherols, flavo- of oxidative degradation reactions in foods, as oxidation in the ab-
noids, tannoids, phenolic acids, reduced/unoxidised melanoidins, sence of catalysts is often negligible (67). Transition metal ions in-
reductones, and specific amino acids and peptides (60)). fluence a myriad of chemical reactions and aid in the creation of
Sulphite tends to get the most attention, as it is arguably the free radicals. This causes oxidative degradation of various organic
best naturally occurring antioxidant found in beer that can, in ad- molecules in wort and beer, including iso-humulones (68), unsatu-
dition, mask aged flavours by forming flavour inactive adducts rated fatty acids (69), amino acids (70), polyphenols (68), sugars
with carbonyl staling compounds (61–63). Nevertheless, some au- (71), alcohols (19), and melanoidins (72) - although melanoidins re-
thors claim that there may be an upper limit regarding the ideal quire high temperatures to oxidise (72).
SO2 content in a beer (22,64). Yeast, for example, cannot readily re- Iron, copper and manganese are directly or indirectly involved in
duce carbonyl-SO2 adducts. Reduced formation of sulphite during the formation of aldehydes (stale flavours) and (di)ketones (27,70).
fermentation will equate to higher levels of free aldehydes, which These contribute to a rapid alteration of the original/intended fla-
the yeast can then reduce to alcohols. This may mitigate the car- vour profile of a beer. Due to their abundance in beer, the degra-
bonyl ‘blooms’ often seen during (warm) beer storage (65). Addi- dation of hop bittering acids (mainly iso-α) is especially noticeable;
tionally, when it comes to anti/prooxidants, concentration is deci- making the continuous loss in bitterness over time a defining pa-
sive. Even with sulphite, a recent study by Foster and Ranguelova rameter of beer ageing, which can be used as a quantitative mea-
(66) found that when the total beer SO2 is in excess of what is sure for staling (trans/cis-ratio) (73). To produce beers that are
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T. Mertens et al.
consistently bitter, even after prolonged storage, brewers may use whether this non-oxidative reaction causes off-flavours in a signif-
tetrahydro-iso-α-acids, which are resistant to oxidative deteriora- icant way (90). Similarly, Strecker degradation of amino acids in
tion, more bitter and enhance foam stability (17). beer can happen in the absence of oxygen, but is of questionable
The concentration of metals is important. Even more significant, relevance at low total package oxygen (TPO) (70,91).
however, is the chemical state (speciation) in which these metallic A further example of oxygen free off-flavour formation is beer
ions are present (74,75), which depends heavily on the physico- becoming ‘sun-struck’, ‘light-struck’ or ‘skunked’. This involves the
chemical conditions of the system: pH, composition, temperature, occurrence of a sulphury, skunky note when beer is exposed to vis-
and oxidation-reduction potential. These same variables also influ- ible and ultra-violet (UV) light. Light-struck flavour is strongly asso-
ence precipitation, dissolution, redox and complexation reactions. ciated with isohumulones, which decompose - under the influence
The ligand speciation can also drastically affect the nature and of light and the photosensitiser riboflavin - to 3-methyl-2-butene-
characteristics of the formed metallic complexes (76). The chelator 1-thiol (MBT), the main chemical related to the odour and flavour
size and the strength of the complex, for example, determine of light struck beer (92). Interestingly, Lusk et al (93) found that
whether the electron is transferred by an inner- or outer-sphere MBT can also form in beer in the absence of light, through thermal
mechanism, which influences the reactivity of the complexed ageing, although slowly.
metal (77). An inner-sphere mechanism involves electron transfer
through a bridging ligand, with bonds being broken and new ones
Measuring beer staling and stability
formed. An outer-sphere mechanism involves electron transfer be-
tween complexes that do not undergo substitution, with no bonds Over the years, multiple techniques have been developed to mon-
broken or formed. itor beer staling. Sensory analysis is a well-established approach
While determining the speciation of metals in beer is important, (94), but suffers from poor reproducibility and requires a lot of re-
only limited experimental research has been reported with the pi- sources (time, people). Chemical analyses are usually more sensi-
oneering work of Svendsen and Lund (78), and subsequent studies tive but unfortunately, there is no absolute test or
of Pohl and colleagues (79–82) on metal species in beer. Both iron all-encompassing assay for quantifying beer staling or flavour sta-
and copper seem to be mostly complexed (78,80,83). The state of bility, as flavour changes are not due to one single reaction (95).
the bound iron is reported to be negatively charged (although The most used methods are listed in Table 1.
later findings also imply positive and neutral species), while copper Of these methods, two address the direct and indirect detection
was found as neutrally, negatively (∼ 70%) and positively (< 30%) of free radicals and ROS in real-time. Electron spin resonance spec-
charged species (78,80,84). Manganese (which has a much weaker troscopy (ESR) measures electrical signals whilst chemilumines-
ability to form complexes with beer components), remains mostly cence uses photochemical detection. In both methods, samples
unbound (> 90% at pH 4.0), as simple Mn2+ cations (78,80). The are forcibly oxidised (typically by applying heat) and the resulting
small bound fraction presumably exists as polyphenolic free radicals measured. The time before detection and formation
complexes. rate of the radicals offers information about the oxidative stability
Even from the limited data available, it is obvious that a wide and staling potential. Both methods, however, require expensive
range of ligands are at play. More research into the different forms equipment with high operating costs and the need for skilled
of Cu, Fe and Mn in beer is needed to learn more about which re- operators.
actions drive flavour instability. The ESR assay is a widely used holistic and established tech-
nique for predicting beer flavour stability and the technology is in-
Oxygen-free beer ageing. Although oxygen is the main staling creasingly available commercially. The main advantage of ESR is its
agent, it would be incorrect to regard (O2-driven) oxidation as the capability to unambiguously detect unpaired electrons in complex
sole force behind beer ageing. Glycoside and ester hydrolysis, ester biological samples (quantification), and its capacity to shed light
and etherification and Maillard reactions are all flavour deteriorat- on the molecular structure of the free radicals (identification), while
ing reactions that can operate non-oxidatively (6). Even trans-2- requiring only small amounts of sample (96–98).
nonenal which is a major indicator of beer staling and an impor- Uchida and Ono (99,100) first applied ESR to predict beer flavour
tant oxidative off-flavour is released during beer storage, indepen- stability in 1996. They observed, when force ageing beer, that long
dently of oxygen content (46). lived spin adducts were not detected immediately after the start of
Reactive oxygen species are not the only (re)active molecules in heating but were produced after a period of incubation. This
beer. Melanoidins, for example, can oxidise higher alcohols to alde- inhibited oxidation phase (‘lag time’) is the result of naturally pres-
hydes in the absence of oxygen although O2 does accelerate the ent antioxidants in the beer quenching the generated radicals or
reaction (19) and metals can catalyse some (e.g. fatty acid) radical ROS. Once the antioxidants are sufficiently depleted, the radical
formation independent of oxygen (85). β-Damascenone - an products react covalently with the spin-trapping agent to form
aroma compound that behaves similarly to trans-2-nonenal during (more) stable adducts, which can then be determined. Figure 2 dis-
extended storage of beer - has been reported to develop indepen- plays two typical ESR spectra of wort and beer, with successive
dently of the total oxygen and SO2 content in bottled beer (86,87). measurements taken during forced ageing.
It has also been reported that, in the presence of suitable electron The endogenous antioxidant activity of beer - its natural ability
acceptors, flavour active beer constituents (e.g. five-membered- to quench radicals - can be estimated through its lag time deter-
ring hop derivatives, including trans-isohumulones, mined (using N-tert-butyl-α-phenylnitron/PBN as spin-trap) (27)
dihydroisohumulones, tetrahydroisohumulones, and or the endogenous antioxidant potential (EAP) value (using α(4-
humulinones) can be oxidatively degraded, even without the in- pyridyl-1-oxide)-N-tert-butylnitrone/POBN as spin-trap) (101). Both
volvement of any oxygen containing entities (88). metrics correlate strongly with the sulphite content of the beer
Aldol condensation such as the formation of trans-2-nonenal, by and give an indication of its inherent antioxidant capacity and po-
condensation of acetaldehyde and heptanal (89) is another oxygen tential flavour stability. Only the EAP value shows a linear correla-
independent process linked to staling. It is unclear, however, tion with the SO2 content, while the lag time portrays an
80
Table 1. Methods for determining staleness, staling potential and flavour stability
Type Method
I Measuring fluctuations in marker compounds
Increase of staling components, such as Decrease of ‘fresh’ components, such as
• 5-Hydroxymethyl furfural (5-HMF) (256) • Iso-α-acids (bitterness); trans/cis-ratio (73)
• Acetaldehyde (257) • Pro-anthocyanidins (20)
• Ethylene (258) • Sulphur dioxide (SO2) (260)
• Furfural (259) and other marker aldehydes (2- and 3-methylbutanal, hexanal, 2- • Total flavanoids (261)
methyl-propanal, etc.) (20)
• β-Damascenone (86)
II Antioxidant capacity assays

• 2,2’-Azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical scavenging or Trolox-equivalent antioxidant capacity
(TEAC) (262)
• 2,2-Diphenyl-1-picrylhydrazyl (DPPH) reducing activity (263,264)
• Cupric reducing antioxidant capacity (CUPRAC) (265)
• Ferric reducing antioxidant power (FRAP) (266)
• Hydrogen peroxide scavenging (267)
• Linoleic acid (LA) assay (268)
• Metal-chelating activity (of Fe2+ with ferrozine) (269,270)
• Oxygen radical absorbance capacity (ORAC) (271)
• Peroxyl radical scavenging (β-carotene bleaching) (272)
• Superoxide and hydroxyl radical scavenging (273)
• Thiobarbituric acid (TBA) index (274,275)
• Total reactive antioxidant potential (TRAP) (276)
III Others
• Chemiluminescence (CL) (277–279)
• Electron spin resonance (ESR) spectroscopy (99,100)
• Nonenal potential concept (69)
• Peroxide challenge test (PCT) (96)
exponential relationship (102). Because of the linearity between

sulphite and EAP value, the time to consume one mg of SO2 per
litre of beer can be determined. Expressed in min*L/mg, this ratio
is termed the Beverage Antioxidant IndeX or BAX value and pro-
vides information about the interplay of anti- and prooxidative
beer components independent of the sulphite content and the
rate of consumption of the existing antioxidative potential during
storage (102).
Beers with high endogenous antioxidant activity show retarded
formation of stale flavours (103). A supplementary ESR metric is the
T-value: an indicator for the quantitative radical generation after a
certain time, typically around minute 450 (although T150 and T600
are also used). It is mainly influenced by pH and substances that
suppress or promote radical formation, such as complexing
agents, transition metals, and intermediates of the Maillard reac-
tion (99,102,104). A high T-value has been shown to correlate with
the rapid development of Strecker aldehydes (65). Thus, an ESR
graph provides information about the anti/prooxidative balance
in a wort or beer, where a longer lag phase = higher EAP value =
greater antioxidant potential = higher flavour stability; and a
steeper slope = higher radical generation = higher T-value =
Figure 2. ESR analysis of wort and beer during an oxidative forcing test at 60°C. An
increase in free radical formation is observed in beer after the lag phase. Wort typically greater prooxidant potential = faster staling.
does not have a lag phase. Note: while the lag time and EAP-value might seem inter- However, the ESR technique is not free from criticism. Studies
changeable, consider that lag time is determined by using PBN as a spin-trap reagent, (77,105–107) have demonstrated certain chelators to form strong
while EAP-value (and T-value) is determined by using POBN. oxidative metal complexes, capable of oxidising biomolecules, that
81
T. Mertens et al.
are not ESR-detectable (by being spin-trap inaccessible). Accord- of ∼2; Figure 3) (115,116). Accordingly, a beer held at 0-4°C will
ingly, ESR does not detect every oxidative species; contrary to keep four times longer than one held at room temperature
chemiluminescence, which does not rely on spin-trapping. (Figure 3) (95). Refrigerated transport and storage is used by some
It is important that a spin-trap reagent does not affect the pH, as brewing companies but is a logistically complex and expensive af-
a small change in acidity will dramatically affect the autoxidation fair. So, despite being one of the most powerful tools to prevent
rate of a metal (77). However, several studies make use of PBN as staling, it is not always an economically feasible option (117).
the ESR spin-trap, which can increase the sample pH. This causes It is not only during storage that (high) temperatures drive stal-
lag time measurements to be significantly distorted (up to 500% ing. Throughout the brewing process, and especially during the
in high EAP beers) through accelerated radical generation (102). high heat stages, a variety of staling relevant compounds are
For this reason, using a spin-trap agent without any effect on pH formed. These include the Maillard reaction products (reductones
(such as POBN) is recommended (101,102,108). and melanoidins), formed via the reaction of reducing sugars with
A further concern involves the typical ESR measurement being proteins or amino acids (present in malt, wort, and beer). They add
conducted in vials that are open to the atmosphere, leading colour and flavour to malt and beer and are responsible for a myr-
to oxygen ingress and volatilisation of ethanol. However, a recent iad of (pro- and anti) oxidative effects throughout the production
development rectifies this by encapsulating the beer sample in a chain and in the final product (102,118).
sealed capillary tube (66). This substantially reduces sample oxida- The TBA method (Table 1) is used to gauge heat load in the
tion during analysis and better represents packaged beer in trade. brewhouse. Thiobarbituric acid forms complexes with many
Lastly, the calculated lag time is not always an ideal metric in Maillard intermediates but is particularly sensitive towards furfu-
predicting sensory flavour stability, as it can be imprecise and inac- rals. One complex (5-hydroxymethylfurfural-TBA) acts as a yellow
curate due to high variability of the fitted sigmoidal curve (22). This indicator, with a maximum absorption at 448 nm that can be used
is especially the case with beer styles that have low or no lag time as a quantitative measure for thermal load (119). A downside of the
(stale lagers, red beers, dark beers) (22). The ESR area under the TBA method is its limited specificity, since thiobarbituric acid can
curve has been suggested as a better metric, since it correlates also react with other substances, including proteins and sugars,
more strongly with sensory data and consumer acceptance (22). to form coloured species that can interfere with the assay.
Others, because of the complexity of beer ageing, propose an even The heat/thermal load received by wort, beer, and malt, gives an
broader approach to measuring flavour stability (109), such as the indication of the expected flavour stability of beer. A high (er) heat
‘stability index’ (SI) method, which combines the results of four dif- load equates to high (er) rates of unwanted staling reactions (free
ferent antiradical analyses (110,111). radical generation, autoxidation of unsaturated fatty acids, Maillard
reactions, Strecker degradation), which equates to high (er) levels
of aldehydes, ageing precursors and prooxidative compounds.
Preventing beer ageing
Reduction of heat load typically involves reducing the time and/
Keep it dark, cool, and still. A principle for slowing down any or temperature of the high heat process steps (kilning, mashing,
chemical reaction, including those involved in beer staling, is to wort boiling, pasteurisation). Mashing-in above 63°C can, for in-
keep the system energy low. For beer, this typically involves no stance, limit unnecessary thermal stress. Additionally, it will inhibit
light irradiation (keep dark) and low temperatures (keep cold). Less enzymatic (lipid) oxidation of unsaturated fatty acids to trans-2-
significant, but not inconsequential, is to limit vibrational energy nonenal by deactivating the lipoxygenase (LOX) enzyme (120).
(keep still), particularly during transport (112). Agitation enhances
the diffusion of any headspace oxygen into the beer, such that ox-
ygen involved reactions proceed at a faster rate.
In terms of minimising the penetration of light, cans and kegs in-
evitably outperform glass bottles. With glass, brown/amber bottles
are best at protecting the product against the damaging effects of
ultraviolet light, with green bottles being a poor second, followed
by blue/cobalt and uncoloured/flint glass (113). Besides preventing
beer from being light-struck, it is important to shield beer from ir-
radiation to prevent it undergoing other photo-oxidative reactions.
Examples include the photochemical oxidation of unsaturated
fatty acids through the formation of singlet oxygen via
photosensitisers (e.g. riboflavin) (45), and the photo-oxidation of
sulphur containing amino acids/polypeptides/proteins by triplet-
excited riboflavin (and other flavins), delivering S-centred radicals
(114).
Formation of 3-methyl-2-butene-1-thiol (MBT) can also be
avoided by using modified/advanced hop products, such as rho,
tetra and hexa hop extracts (containing rho-, tetra-, and
hexahydro-iso-α-acids). Because these hop products do not
photodecompose into MBT, there is the misconception that these Figure 3. Calculated time before noticeable beer staling in relation to storage tem-
compounds are light stable. However, they do break down, but perature, based on a 90 day shelf-life assumption at room temperature (20°C), with
into light-struck flavours with higher aroma thresholds (114). the following Q10 values: 1.5 (▼-line), 2 (bars) and 3 (▲-line). Note: The Q10 describes
the ratio by which reaction rates change when the temperature is increased by 10°C
It is a general rule of thumb for most food systems that a 10°C and is used to predict the expected shelf-life of a food product. Typically, a Q10 value
decrease in temperature roughly halves the rate of all chemically of 2 is used as an initial shelf-life estimate, but it can range anywhere from 1.1-3 for
based deterioration reactions (i.e. a Q10 temperature coefficient beer (depending on the temperature and the system/product).
82
Another possibility to lower heat stress is to brew with raw Table 2. Industry standard oxygen levels across the brewery
(unmalted, unkilned) barley (121) or with green (germinated, (131)
unkilned) malt (122,123); to cool the wort before the whirlpool rest
with the added benefit of limiting SMM (DMS precursor) cleavage Stage Oxygen content (μg/L)
(124), or to centrifuge the wort instead of using a whirlpool. With
boiling, many alternative systems that reduce heat load and save Aerated/oxygenated wort 8000 – 17000+
energy are available, including internal boiling/heating system Fermentation < 10 – 30
(9), thin-film evaporation (125,126), dynamic low-pressure boiling Filtration 5 – 50
(127), vacuum boiling (128) and innovative wort production (with Bright beer after filtration 10 – 50
mashing-off at 95°C) (129). Beer at the filler 10 – 30
Package dissolved O2 (bottle) 20 – 50
Avoid oxygen. To battle oxidative ageing, exposure to oxygen Package dissolved O2 (can) 30 – 60
must be avoided as best as possible. This means oxygen ingress Total package O2 (dissolved + 40 – 150
throughout the brewing process is as low as is reasonably achiev- headspace)
able, and headspace oxygen in packaging is as low as is practically
attainable, with avoidance of any oxygen ingress during storage.
Bamforth (64) estimated that 0.1 μg/L of oxygen would incite oxi- the pre-seal fobbing of beer). Regardless of the container type,
dative mechanisms to a damaging degree. With modern filling beer should be stored upright and vibration/transportation
machines achieving in-pack dissolved oxygen levels of 20-50 μg/ minimised. This way, the beer has less surface area to interact with
L in beer (130,131), it is debatable whether we should continue trace amounts of oxygen in the headspace, slowing down
to strive for even lower oxygen concentrations. oxidation.
Good manufacturing practices will improve the shelf-life of beer A practical but niche option to lower in-pack oxygen is to add
by minimising the formation of ROS. Kuchel et al (132) suggest that yeast to beer in bottle or can, which scavenges some of the re-
lowering the in-pack oxygen of beer to 1 μg/L should, at a mini- maining free amino acids and removes oxygen (144,145). In addi-
mum, extend its shelf-life beyond the typical 120 days. It can also tion, the yeast also removes aged flavour notes (such as aldehydes)
be reasonably assumed that ‘downstream’ oxygen ingress is a big- from the beer, prolonging its overall freshness (146,147). However,
ger concern than ‘upstream’ oxidation (95); notwithstanding that refermentation or secondary conditioning is a complex biochemi-
both should be prevented. Upstream entails the materials needed cal process that involves more than oxygen removal. Suspended
for production and any part of the production involving the extrac- yeast can cause haze and the additional carbon dioxide may lead
tion (making of wort); downstream includes processing after fer- to excess carbonation and gushing. Furthermore, there is the risk
mentation, the finished product (packaged beer), distribution of yeast autolysis and the release of intracellular enzymes, lipids,
and retail. amino acids, and metal ions, which can increase beer pH and cause
For this reason, any unnecessary transfer of beer should be off-flavours (148–151).
prevented, whether in the brewery or in retail. When required, it
should be done gently, to avoid splashing and turbulence, which
leads to aeration (133). Where possible, wort and beer should be Add antioxidants. Antioxidants are substances that prevent,
‘pushed’ with CO2 or another inert gas. It is best to purge any con- delay or remove oxidative damage, either by eliminating superox-
tainer (bottles, kegs, cans, carboys, tanks) with CO2 or N2 before fill- ide, hydroxyl radicals or other reactive species (like peroxides,
ing and to fill from the bottom up. Hoses and pumps can also be which sulphite will quench), or by inactivating trace amounts of
purged, or better still, prefilled with deaerated water to expel any Fe, Cu and Mn (through complexation/chelation) (61). They are al-
air. When bottling or canning, always ‘cap on foam’ by agitating/ ready present in beer and play a vital role as endogenous staling
fobbing the beer slightly so that headspace oxygen is minimised inhibitors, even at low concentrations. However, there are ways
(134). to naturally enrich the antioxidant content, such as ageing the
The caps on glass bottles are another unavoidable contributor beer in wooden barrels (152), by using ingredients or brewing pro-
to flavour instability, as air can permeate into the headspace. There cesses that favour a high polyphenol content in wort and/or beer
are, however, differences among cap/crown types when it comes or, as noted above, the use of re-fermentation, which will produce
to oxygen ingress (135). Pry-off bottle caps are better at keeping in-pack SO2 (146–148). Other options to enhance the sulphite con-
oxygen out than twist-offs (14,136,137). The crown liner material tent are to use a high-sulphite producing yeast strain (153,154),
is also instrumental, as polymers vary in the extent of oxygen per- zinc addition (155), reduced fermentation temperature (155,156),
meation (135,138,139). To minimise the problem, innovative oxy- lower dissolved oxygen in pitching wort (154,157), clear/bright
gen-scavenging caps can be used. The scavengers within the wort (154,158,159), and a higher wort pH (160).
liner react with gaseous oxygen, reducing the overall oxygen con- Depending on the regional legislation, antioxidants may be
tent in the bottle (140–142). added to the brewing process or the final product to potentially
With keg beer, gases can permeate through some grades of dis- prolong shelf-life. However, better packaging technologies with
pense tubing with CO2 leaving and air entering the system (143). lower oxygen pick-up have made the practice of adding exoge-
This is of greater concern with keg beers that are ‘slow moving‘. nous antioxidants less common (161). Additionally, regulations
The use of CO2 as top pressure gas for beer dispense may contrib- have become stricter in recent years and consumers increasingly
ute oxygen, as commercially available food-grade CO2 contains prefer ‘clean label’ products with no artificial ingredients or syn-
trace amounts of O2 (131). Conversely, canned beer has zero oxy- thetic chemicals. In Europe and the United States, beers with
gen ingress after sealing, but bears the risk of having a higher sulphite contents > 10 mg/L must be labelled as such, as high
TPO than bottled beer (Table 2), as cans cannot be vacuum evacu- sulphite residues can trigger allergenic effects in susceptible
ated without collapsing and have wide mouths (which impedes individuals (63).
83
T. Mertens et al.
Common antioxidants used in brewing are ascorbic acid (vita- The metal content of a beer varies depending on the quality of
min C; E300) and sulphur dioxide (90). The latter can be adminis- the materials and processing aids used, though it is unavoidable
tered by dissolving (potassium or sodium) metabisulphite at vari- that a portion of the metal ions are present in the final beer. A
ous stages in the brewing process. Apart from the quenching of study by Wietstock et al demonstrated transfer rates, from raw ma-
ROS (61), sulphites will form adducts with unwanted aldehydes terials to the finished beer, of 0.1% Fe, 0.4% Zn, 3.1% Cu, 6.3% Ca
(acetaldehyde and other carbonyl compounds (162,163)), making and 15.1% Mg (181), with the biggest metal source being (pale
them less flavour active, although this masking effect may only lager) malt (ca. 96%). With darker malt types, such as crystal or
be temporary (43,65). Ascorbic acid, like most antioxidants, also roasted malts, different transfer ratios would be anticipated;
has prooxidative capabilities (164,165). This is due to ascorbate, be- namely, higher for Fe and Mn and lower for Cu, due to changes
ing a typical reductone, having a strong affinity to reduce oxidised in the binding capacity of malt solids (166,173,182).
metal ions back to their catalytic state (e.g. Fe3+ to Fe2+) so that they Beer has a much lower transition metal content than wort (183),
are again available to activate oxygen by electron transfer (58). which make the ransfer rates appear low. The explanation for this
Whether a chemical entity behaves as an anti- or prooxidant de- is that a large fraction of the metals in wort are bound to nitroge-
pends greatly on the type and the concentration of the com- nous and polyphenolic compounds and are removed with the hot
pound, oxidation state, pH, the type and concentration of transi- break and trub during mashing, wort boiling, and in the whirlpool
tion metals present and the matrix. It can be difficult to make (181,184–186). The spent grains, left after lautering or mash filtra-
clear cut predictions, as what works in one medium might not tion, are also a great sink for metals, particularly transition metal
work in another. This may explain why, for some substances, con- ions (187,188). Moreover, yeast in addition to removing oxygen
flicting results have been reported in the literature. Indeed, in this (145,189), scavenges metals during fermentation (especially Cu,
review, melanoidins have had both pro- and antioxidative effects Fe and Zn), lowering the final metal content in beer (190–193). In
attributed to them (catalysing oxidation of higher alcohols into contrast, manganese is not significantly lost during the brewing
their equivalent aldehydes, but also chelating transition metals) process, making it a potent beer prooxidant (50,52).
(58,166–169). The same is true of certain phenolic compounds However, it is important to note that the metal concentration in
(chelating transition metals, but also reducing them back to their finished beer does not need to be high to cause noticeable de-
prooxidative form) (170–172). fects. The damaging properties of copper occur at < 50 μg/L (50)
A novel antioxidative compound that can be employed during and a transition metal addition of 10 μg/L results in a measurable
brewing is punicalagin (173) a water-soluble ellagitannin, found decrease in oxidative stability (52,102,194). Some metal ions may
in pomegranates, which can be hydrolysed to smaller phenolic be introduced after the brewhouse and fermentation stages,
compounds (such as ellagic acid). Both punicalagin and ellagic acid bypassing the protective effects of spent grains, hot/cold break/
are capable of forming chelates with iron and copper ions at beer trub formation, and yeast. The increasingly popular practice of
and wort pH (174,175), inhibiting the production of reactive oxy- dry hopping is one such example. Considering that hops are rich
gen species and scavenging them (176). in metal ions (181), dry-hopping is presumably detrimental to beer
flavour stability, although this has yet to be adequately investi-
gated (51,179,195,196). Furthermore, such additions to finished
Remove transition metals. Metals in beer are mainly derived beer, can inadvertently result in oxygen pick up.
from the raw materials (malt, hops, water, yeast), but also from It is likely that a high amount of transition metals, present during
the brewing equipment and additives (filter media (102), pipes, brewing, will negatively impact the final quality and stability of the
tanks, vessels, packaging (177,178), adjuncts, stabilisers, finished beer; even though a beer made from an iron-rich wort
pesticides (179)). Table 3 summarises the range of might finish with a similar iron content as a beer from an iron-poor
concentrations reported for iron, copper and manganese from wort. This notion is often overlooked, despite the known capacity
different raw materials and stages of the brewing process. of (metal ion) oxidation catalysts to facilitate oxidative degradation
Whether good or bad, their presence plays a substantial and of wort (and its antioxidative compounds) especially during high
overlooked part in the palatability, conservation and overall heat stages and regardless of the catalysts being in a bound state
stability of beer. Positive effects include supporting yeast and or not.
fermentation and contributing to the nutritional value. Negative To reduce any negative flavour effects, the content of transition
effects involve spoilage - due to haze formation, oxidative metals (Fe, Cu and Mn) needs to be lowered in some way, ideally as
processes, gushing - and other sensory defects (31,180). early as possible. One approach is by complexing metals during
Table 3. Transition metals throughout the brewing process
Fe Cu Mn
Malt (mg/kg dm) 25-32a 2-3a 8-12a
Hops (mg/kg dm) 300-800b 5-10b 40-60b
Filtered wort (μg/L) 200-500a 50-100a 70-150a
Pitching wort (μg/L) ≥ 200a ≥ 70a ≥ 70a
Beer (μg/L) 20-80a 20-60a 70-130a
a
Range according to Zufall and Tyrell (50).
b
Range according to Lie et al. (280).
84
the brewing process so that they are no longer chemically in- ‘Simple’ chelation might not suffice in protecting wort and beer
volved in activating oxygen (Reaction 1). Certain chelators can do from oxidative damage. A chelator that forms an insoluble metal
this by one or more of the following: interruption of the metal re- complex has a better chance of diminishing oxidation, by decreas-
dox cycle; occupation of all coordination sites; formation of insolu- ing the mobility, and the reactivity of the metal. Ideally, a chelator
ble metal complexes; steric hindrance between metals and oxida- should also possess a high enough binding affinity for transition
tion intermediates (e.g. peroxides) (197). However, perfect ‘catch- metals, so that it can strip them away from other molecules,
all’ chelators may not exist (49). protecting possibly flavour relevant molecules from site-specific
degradation by the metal-catalysed radicals (213). Good examples
of site-specific degradation reactions in beer are when
isohumulones or polyphenols are directly attacked by ●OH radi-
(Reaction 1) cals, generated by iron ions that they captured, destroying them;
Some native wort/beer components seem to portray such char- respectively resulting in cardboard off-flavour and haze formation
acteristics naturally, through donor N-, O- and S-atoms, and in- (31). In addition, chelators must be food safe and flavour neutral;
clude polyphenols (198,199), amino acids (192), phytic acid not remove metals required for brewing (yeast requires trace
(200,201), melanoidins (202–204), and hop acids (205,206). These amounts of Zn2+, Ca2+ and Mg2+) (84); they must perform well
mostly derive from malt, but also from hops. So, even without at high temperature and low pH; destroy O●- 2 and H2O2 without
the addition of complexing agents, a natural and complex equilib- reducing agents; and, preferably, be low in cost and practical
rium already exists between free and bound metal ions in wort and in use.
beer with an inclination to the bound, organometallic state (78). In 1999, Bamforth et al (133) called for a deeper exploration of
The benefit of scavenging metal ions is clear, but the reality the aspects of chelation, but little has been subsequently been
proves challenging. To prevent Fenton chemistry, a chelator must published. A few studies have examined the metal ion scavenging
stabilise the metal ion in a state inert to either oxidation by H2O2 or capabilities in beer of added, exogenous compounds. Effective
reduction by reducing agents (207). But the bound-state metal ones include diethylenetriaminepentaacetic acid (DTPA) (26), eth-
ions can even - depending on the concentration and type of che- ylenediaminetetraacetic acid (EDTA) (31,99,165), egtazic acid
lator - end up promoting oxygen radical formation (Reaction 2 and (EGTA) (214), bipyridine (99), phenanthroline (99), Divergan® HM
3, forming a cycle), instead of quenching it (90,99,133). (102,215), gallotannins/tannic acid (Brewtan®) (13,216,217).
All these chemicals are foreign to beer, but a few chelation stud-
ies in brewing have shown promising results with native beer and
wort compounds. As noted, phytic acid is a strong antioxidant be-
cause of its metal scavenging ability (Cu and Fe, but also Zn and
(Reaction 2) Ca) (184,218,219). Proteins of all fractions especially bind Cu and
Fe ions, with their amino acids acting as ligands (214,220), which
explains why trub contains a high amount of metals. Despite this,
the nature and extent of the binding do not prevent copper from
(Reaction 3) participating in oxidative reactions (221). Hop α-acids
(isohumulones) and hop β-acids can firmly bind Fe ions by com-
The degradation of ascorbic acid serves as an illustration of this. plex formation (195,205,222). The organic compound citric acid
The oxidation of ascorbate is catalysed by both copper and iron, forms complexes with Fe and Cu (80,214). Melanoidins are known
with free Cu+2 being roughly 80 times more potent than unbound to strongly capture Cu, Fe and Zn strongly. Beer flavonols (e.g. from
Fe3+. The presence of EDTA diminishes the ability of copper to ca- hops) can bind Cu at beer pH; myricetin and quercetin can chelate
talyse ascorbate oxidation. However, this is not the case for Fe3+, as Fe (183,223). Oxalic acid (oxalate) is reported to modulate the activ-
EDTA bound iron is four times more potent in degrading ascorbate ity of iron (224).
than its free form (208). It is important to note that most of these studies were con-
The extent of metal ion binding does not necessarily correlate ducted by examining chelating agents in finished beer. However,
with the extent of protection against oxidation from that metal. chelators are often unstable at low pH (211,225) and typically per-
This may also explain why there are still disagreements on whether form better in less acidic environments (35,67,212). Accordingly, it
some compounds are anti- or prooxidative (e.g. melanoidins and might prove more effective to add chelators during mashing, as
polyphenols), especially in complex systems such as wort and wort has a higher pH. Additionally, the high level of amino acids
beer. Model studies with lipid peroxidation show that when the ra- in wort may help complex formation (amino acid + organic acid
tio of chelator (EDTA or DTPA) to transition metal (Fe) is high (> 1), + metal ion). Such mixed complexes are often stronger than those
oxidation is inhibited. When the ratio is low (< 1), oxidation is stim- composed solely of organic and amino acids (226).
ulated (209,210). This suggests that chelator/metal-ratio also plays A recent study explored the binding capacities of 19 chelators
an important role and that chelators must be present at sufficiently added during mashing (173). The findings highlight the advanta-
high concentrations. geous effect of some chelating agents, of which the most effective
Overall, chelation properties are influenced greatly by the pH of were green tea extract, tannic acid and pomegranate extract. The
the solution, which in turn will also influence the reactivity of the latter two were especially successful in reducing the iron content
metal species present (211). The complexity of metal and chela- of wort after lautering. The addition of pomegranate extract (60
tion/coordination chemistry is immense. In addition to the factors mg/L, 90% ellagic acid) resulted in an 80% decrease in radical gen-
already noted, many others will affect the outcome, including che- eration. The study also showed that excessive release of iron and
lator size, nature of the ligand (and whether it is capable of forming manganese during mashing can be avoided by not acidifying
a multiligand complex), buffer system, competing ions, and the the mash, but instead mashing at a ’natural’ pH of 5.6. This is in
matrix (77,212). accordance with the findings of Narziß et al (227), who observed
85
T. Mertens et al.
an increase of ageing related compounds with the reduction of the stress that these malts undergo during kilning, resulting in de-
mash pH (from 5.8 to 5.5 and 5.2). Not acidifying the mash might creased beer flavour stability (166,168,241).
also result in a less acidic beer, which according to Grigsby et al Brewing with green malt or unmalted barley (or a combination
and Kaneda et al (32,228), will lead to a more flavour stable beer of both, making use of the high diastatic power of green malt to
that tastes less oxidised when aged. At lower pH, more of the su- compensate for the enzyme deficiency in raw barley) would re-
peroxide radical will be in its protonated state (the more damaging duce the staling potential considerably, since these starch sources
perhydroxyl radical). do not have the above defects - although they might contribute
The free radical and ROS scavenging properties that some che- other shortcomings. Green malt, for example, must be produced
lators also possess should not be overlooked. Caffeic acid shows and used on-site, as it is microbiologically unstable raw material
low iron binding ability but has outstanding antioxidant proper- with a limited shelf-life (123). Contrary to expectation, the height-
ties, as it can scavenge several reactive species (DPPH, peroxyl ened LOX content of green malt does not result in beer with signif-
and hydroxyl radicals) (229,230). There is still much room for re- icant taints or obvious defects, even when using it at a 100% (122).
search in the field of ‘chelation therapy’ with respect to improve-
Milling. Aggressive milling of malt results in excessive fracture
ment of beer oxidative stability. It is well known that phenolic
of the husks and embryo, which increases wort polyphenol levels,
acids, containing the catechol or galloyl moiety (an
but also leads to increased LOX, lipase, and lipid release, leading to
ortho-dihydroxy functional group), are effective in chelating transi-
elevated nonenal potential (60,242). Finely milled grist (from ham-
tion metals (231,232); and that beer contains a number of these
mer milling) would, typically be utilised by modern mash filters,
acids, including caffeic acid, chlorogenic acid, protocatechuic acid,
which provide reduced filtration time, less heat load, lower oxygen
and gallic acid. Accordingly, enhancing these phenolic acids dur-
pick-up, and shorter mashing times (due to the larger surface-to-
ing the brewing process could potentially improve the shelf-life
volume ratio of the fine grist, leading to higher enzyme and extract
of beer (233). However, at certain concentrations - depending on
yields).
the polyphenol type and beer composition - they may lead to
Alternatively, or in addition, the malt acrospire can be kept in-
the formation of protein-polyphenol hazes over time (234).
tact by employing steep conditioned or wet milling. Wet milling
systems can use deaerated water to minimise oxidation during
Control of flavour stability in practice milling and mashing. To achieve this with steep conditioned mill-
ing, the milling and mashing chambers should be flooded with ni-
Table 4 provides a summary of the strategies to prevent beer trogen or carbon dioxide.
ageing.
Mashing, wort separation, boiling, and clarification. Al-
Raw materials, equipment and additives. The excellence of though chelating agents, such as tannic acid and ellagic acid, can
the raw materials (malt, water, hops, yeast), processing aids, and be used successfully across the brewing process, addition during
equipment is paramount to ensure good beer quality. They will in- the mashing stage is recommended. Firstly, the early elimination
fluence the overall metal load throughout the entire process. of transition metals will more promptly negate their catalytic ef-
Brewing water can be used ‘as is’, if the local raw water quality is fects on oxidation. Secondly, wort has a higher, more suitable pH
adequate, but will commonly have to be deionised to remove min- for chelation, compared to beer (206,212). Hop acids also possess
erals and unwanted metal ions. Water treatment, such as reverse chelating capacity, of which the non-isomerised α-acids have the
osmosis, may come with high costs and process complexity, as highest binding efficiency (206). Application of optimised hopping
salts must be added back to the brewing liquor (235,236). The regimes, where hops are added incrementally - rather than a single
metal content of the other inputs is not always as transparent or dosage at the beginning of wort boiling - can achieve lower levels
easy to control. The use of whole hops, rather than pellets or ex- of iron and copper in the pitching wort (195).
tracts, raises the antioxidative polyphenol content of the wort At mashing-in temperatures of ≥ 63°C, LOX is inactivated and
but also tends to be higher in heavy metals (237). With malt, the fewer staling components are retained in the beer (241,243). A fur-
wort metal content can vary by variety, cultivation, and roast inten- ther advantage of mashing-in above the gelatinisation tempera-
sity (182,185). Similar considerations also apply to spices, herbs, ture of barley malt starch is shortened vessel occupation time,
and other additives (238). Brewing equipment should not leach which in turn limits the total heat damage (244). Wort kettles are
metal ions into the wort or beer and accordingly, passivated stain- usually fitted with vents that aid the removal of unwanted volatiles
less steel ‘coppers’ should be used for wort boiling, rather than (such as staling aldehydes and DMS), and condensate traps that
copper. Similarly, membrane filtration of beer is recommended prevent their re-entrance (129). The addition of oxygen to warm
rather than filtration through kieselguhr/diatomaceous earth, wort (upstream oxidation or ‘hot side aeration’) should be avoided
which is rich in iron (216,239). The design of process equipment as oxidation of wort compounds (proteins, fatty acids, melanoidins,
(pumps, stirrers, pipes, vessels) should be to reduce high mechan- polyphenols) will happen rapidly at high temperatures (72). Wort
ical stress and shear, to minimise oxygen pick-up, to impede β-glu- should be aerated/oxygenated on the cold side of the heat ex-
can extraction during mashing, and to avoid disruption of any ag- changer. The hot wort should be treated gently, by filling the mash
gregates, such as coagulated protein/polyphenols. The primary tun and kettle from the bottom up and avoiding turbulence during
purpose is to prevent reduced filterability by averting the forma- transfers. Where possible, mashing and sparging should use
tion of large hydrogel complexes and, thus, poorer flavour stability. deaerated liquor. Better still perform mashing, wort filtration, and
With dark, roasted malts, more metal ions are transferred to the boiling anaerobically (under an inert atmosphere). Another ’inno-
wort during mashing (173). This explains why dark beers, such as vative’ concept to reduce hot side aeration, heat load, and energy
stouts, typically have a higher Fe content (52,240). Roasted malts costs is to brew without wort boiling, which can either be done
also contribute higher levels of organic radicals, deterioration-rele- through the use of near-boiling temperatures and stripping (129)
vant carbonyl compounds (Strecker aldehydes), and reductone or by omitting wort boiling entirely. Traditional no-boil beers (or
compounds (Maillard reaction products), due to the elevated heat raw ales) have been brewed for centuries including Finnish sahti,
86
J. Inst. Brew. 2022; 128: 77–95

Table 4. Strategies for preventing wort and beer staling (adapted from (281))
Raw materials, Downstream

© 2022 The Authors. Journal of the Institute of Brewing published by John Wiley
equipment, Mashing and Boiling and Fermentation processing Distribution

Process stage and additives Milling wort separation clarification and conditioning and packaging and storage
High Add antioxidants: Avoid excess iron Limit heat load: Ensure vigorous Minimise in-pack Keep it dark
relevance, low sulphur dioxide, and manganese: avoid prolonged fermentation: use oxygen: cap on
cost, effort whole hops mash pH ∼ 5.6 heating, cool healthy yeast, foam, purge
or risk Add effective efficiently and adequate containers, limit
chelators: tannic timely pitching rates, transfers and
acid, punicalagin/ Avoid excess of recommended filtration
ellagic acid iron and copper: temperature Avoid pick-up of
optimised iron, copper and
hopping regime manganese
High Avoid pick-up of Limit heat load: alternative boiling Minimise in-pack Keep it cold
relevance,high iron, copper and systems oxygen: oxygen Limit storage
(er) cost, effort manganese scavenging time: stock
or risk Limit heat load: bottle caps, cans rotation and
pale malt, green logistics
malt and/or
unmalted barley
Low (er) Non-aggressive Limit hot side aeration Bright/clear worts Store it upright
relevance, low milling Inhibit LOX: Remove trub and Keep it still
cost, effort mash-in ≥ 63 °C break
or risk
Low (er) Use Flush grist with Bottle
relevance, lipoxygenase-null inert gas conditioning with
wileyonlinelibrary.com/journal/jib
high (er) cost, barley Mill anaerobically yeast

effort or risk
87
T. Mertens et al.
Danish gammeltøl, Norwegian kornøl, and German Berliner Weisse economically viable, brewers and retailers can agree on positive
(245). release systems, where the beers released for consumption are
There has been much debate about the impact of trub rich (still) true to their brand specification. Additionally, brand owners
(cloudy or turbid) worts on beer quality and flavour stability, with can enforce ’pull dates’ - deadlines where unsold beer should
most research focusing on the potential negative effects of the return to the brewery, usually ranging from 60 to 180 days.
higher lipid content (246,247). However, little consideration is
given to trub being high in iron and copper (181,248). In terms of
transition metal content, trub removal for a bright/clear wort is Concluding remarks
recommended. Fresh beer is not in chemical equilibrium and flavour shifts inevita-
bly occur over time. This inherent flavour instability of beer re-
Fermentation and conditioning. Healthy yeast and vigorous
mains a major challenge facing brewers. Each of the reactions in-
fermentation are important for flavour stability (95,249). Yeast re-
volved is subject to numerous determinants, including
duces aldehydes to their corresponding alcohols and produces
temperature, oxygen, time, transition metal content and specia-
low levels of sulphur dioxide. The use of yeast of high viability
tion, pH, and beer composition. The multitude of variables make
and good physiological state enhances flavour stability and the or-
beer ageing an immensely complex chemical process that is not
ganoleptic properties of the final beer (250). Appropriate levels of
fully understood. Although multiple methods for measuring beer
zinc (and magnesium) are required in the pitching wort to facilitate
staling and stability are available, none are absolute. ESR spectros-
yeast performance (251,252).
copy has been among the most adopted analytical techniques in
Anaerobic, repitched yeast requires trace amounts of dissolved
recent years and gives valuable information about the endoge-
oxygen (5-20 mg/L) in the wort to synthesise sterols and unsatu-
nous antioxidant potential and the interplay between the pro-
rated fatty acids, which are needed for membrane formation and
and antioxidants in wort or beer.
cell multiplication. Accordingly, pitching wort is aerated or oxygen-
Although oxygen and oxidation are not the sole reasons for stal-
ated, post heat exchanger, on route to the fermenter. Although a
ing, they play a central role in beer ageing, together with transition
necessary process, the addition of oxygen to wort is counter intu-
metals. Iron, copper and manganese are major drivers of oxidation,
itive in managing flavour stability. To limit the oxidative damage,
as they catalyse the production of reactive oxygen species. As
oxygen is added on the cold side and yeast is pitched without
brewing and packaging technology may be approaching the prac-
delay.
tical limit for in-pack oxygen, it is wise to explore other pathways in
Alternatives to wort aeration have been explored, including the
restricting oxidation, such as the depletion and inhibition of transi-
direct addition of oxygen to yeast slurries. In one study (253),
tion metal catalysts. Their chemical or physical removal from the
pitching yeast was exposed to olive oil prior to fermentation, so
brewing process is desirable and can be achieved by chelation,
as to supply unsaturated fatty acids (such as oleic acid). This ap-
an uncharted area in brewing science. Because of the complexity,
proach does not satisfy the nutritional need for sterols (anabolic
contradictory results are found in the literature about the anti- or
or exogenous) but was said to produce beers that were less
prooxidative effects of chelating compounds, such as polyphenols,
oxidised.
melanoidins, and ascorbic acid. Nevertheless, chelation and flavour
Downstream processing and packaging. In-pack oxygen as stability warrant further investigation.
low as is reasonably achievable is critical for the prolonged shelf-
life of beer. Due to improved beer processing and packaging tech-
niques, the TPO can be as low as 40-150 μg/L. But even at these
Author contributions
levels, oxidative staling of beer is still taking place. While it is true Tuur Mertens: writing (original draft).
that the oxygen initially present is already enough for oxidation Thomas Kunz: writing (review and editing).
to occur (as it will be recycled through the Fenton and Brian R. Gibson: writing (review and editing).
Haber-Weiss reaction), a large factor is ‘new’ oxygen finding its
way into the beer package by penetration through the closure Acknowledgements
and/or packaging material. As such, a continuous dynamic situa-
This project was funded by the European Union’s Horizon 2020 re-
tion exists, where in-pack oxygen lost through reaction with beer
search and innovation programme under the Marie Skłodowska-
constituents, can be supplemented by atmospheric oxygen. This Curie grant agreement No. 722166.
is why aged bottled beer can still have dissolved oxygen levels of Open Access funding enabled and organized by Projekt DEAL.
30 μg/L (254), rather than near-zero as with aged canned beer
(255). In the absence of oxygen scavenging caps, ingress rates of
1-5 μg/L O2 per day can be anticipated (131,135). Conflicts of interest
Distribution and storage. Storage temperature may be the sin- The authors declare no conflicts of interest.
gle most important quality factor in beer stability. However, there
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95
II
Assessment of Chelators in Wort and Beer Model Solutions
Tuur Mertens, Thomas Kunz and Frank-Jürgen Methner
BrewingScience. 2020; 73:58-67.
BrewingScience May / June 2020 (Vol. 73) 58
Monatsschrift für Brauwissenschaft
T. Mertens, T. Kunz and F.-J. Methner
Assessment of Chelators in Wort and Beer

The scientific organ
of the Weihenstephan Scientific Centre of the TU Munich
Yearbook 2006
of the Versuchs- und Lehranstalt für Brauerei in Berlin (VLB)
of the Scientific Station for Breweries in Munich
Model Solutions
of the Veritas laboratory in Zurich
of Doemens wba – Technikum GmbH in Graefelfing/Munich www.brauwissenschaft.de
Long-term storage of beer often results in flavour deterioration and quality reduction. Prolonging beer
freshness would benefit both brewers and consumers worldwide. Oxygen has always been a major focus
in regard to oxidation. Less naturally is the key involvement of catalytic transition metals (Fe, Cu, Mn) in
oxidative beer ageing. Physicochemically removing these entities through chelation – rendering them
incapable of forming reactive oxygen species – could greatly benefit flavour stability. This study aims to
explore nine chelating compounds (EDTA, citric acid, tartaric acid, quercetin, chlorogenic acid, ferulic acid,
gallic acid, phytic acid, and tannic acid) for their capacity to form complexes with seven metal ions (FeII, FeIII,
CuII, MnII, CaII, ZnII, MgII) and to examine whether the complexes can effectively be removed by filtration.
Chelators and metal ions were mixed and incubated in two distinct acetate buffer solutions, one with the pH
and ethanol content of wort (5.60; 0.0 vol%) and one with that of finished beer (4.30; 5.0 vol%). Measurements
were conducted by UV-Vis and ICP-OES spectroscopy, to respectively assess complex formation (through
absorbance changes) and filterability (through metal level reductions). Ideally, chelators deplete iron, copper,
and manganese, without affecting any metals vital for brewing (Ca, Zn, Mg). The findings suggest tannic acid
to be the most promising chelator in this aspect, followed by quercetin, gallic acid, chlorogenic acid, and
ferulic acid. EDTA, citric acid and tartaric acid did not form filterable complexes with any of the metal ions.
Phytic acid chelated out zinc; among others when introduced to a mix of metal ions. Compared to beer pH,
wort pH proved far superior overall in terms of transition metal removal by complex formation.
Descriptors: chelation, transition metals, flavour stability, oxidation, UV/Vis, ICP-OES
1 Introduction all very off-putting for the consumer. Given that buyers are becom-
ing more aware and increasingly demanding of beer quality and
Beer is one of the most all-time beloved beverages around the flavour [2] – along with an ever-expanding global beer distribution
globe [1 chap. 1]; and in several aspects, it is also an exceedingly chain and a continuous rise in craft beer popularity – it’s safe to
complex drink. Beer’s intricate nature brings forth a broad range say that the issue of beer flavour stability has never been more
of different styles and an almost inexhaustible range of possibili- relevant than today.
ties. This complexity, however, comes with the downside of (fresh)
beer being unstable, as it is not in a state of chemical equilibrium. A big culprit in beer staling is oxygen, as oxidation is the main
cause of beer turning ‘aged’ [3, 4]. That is why breweries go to great
The poor (flavour) stability causes beer to change and turn unpal- lengths avoiding any unnecessary oxygen pickup during brewing
atable rather fast; especially considering the detrimental effects and why good bottling practices are so important. Big improvements
of transport and unrefrigerated storage. And even though beer with total package oxygen (TPO) were already seen over recent
will technically not expire and become unsafe to drink, its lack- years – bottle fillers now allow for a TPO of 50 ppb or less [5]. But
ing stability is a modern brewer’s biggest headache. The flavour even though it continues to improve, complete avoidance of oxygen
changes that occur over time are almost always unpleasant and has its practical limits and is downright impossible.
unintended. Adverse storage conditions with time can also cause
haze defects and lack of foam in the beer. These phenomena are But there is more to oxidation than oxygen alone. Due to spin
restrictions, molecular (ground state) oxygen is rather inert [6].
Unfortunately, transition metals (iron, copper, and manganese)
are – through electron exchange – able to undermine this energy
barrier and cause oxygen to go out of its latent state. Resulting
derivatives are called ‘reactive oxygen species’ (ROS), of which
https://doi.org/10.23763/BrSc20-01mertens O2-, OOH•, H2O2, and OH• – generated by the Fenton and Haber-
Weiss reaction mechanism [7] – are chief examples. These ROS
Authors are highly reactive entities and cause substantial oxidative damage
by attacking compounds directly, whilst also inducing a cascade
Tuur Mertens, Thomas Kunz, Frank-Jürgen Methner, Technische Uni- of additional oxidizing reactions, resulting in the formation of
versität Berlin, Department of Food Technology and Food Chemistry,
Chair of Brewing Science, Berlin, Germany; corresponding author: tuur. flavour-active, volatile substances (such as aldehydes), which
mertens@tu-berlin.de are generally – depending on their concentrations – responsible
59 May / June 2020 (Vol. 73) BrewingScience
for atypical tastes and odours. Thus, when it comes to preventing ion will often get degraded through site-specific oxidation [23].
oxidation, one school of thought is to shift the focus more towards This is problematic, as these ligands are usually biomolecules
debilitating or removing these catalytic transition metals, instead which we wish to keep intact. On the other hand, by introducing a
of solely restricting oxygen. strong chelator, transition metal ions can be detachedYearbook
from these
2006
vulnerable bio-compounds, thus enhancing flavour stability. For

1.1 Chelation chelation strength, the rule of thumb is: the more donor atoms a
given chelator has, the stronger and more stable it will bind with
The detrimental effects of transition metals should not be made metal ions [24].
light of; without them, the vast majority of the “auto-oxidative” reac-
tions in beer would cease to happen [8]. Despite this, content of Because of the obscure nature that complexes have on oxidative
transition metals is seldom monitored and/or controlled. stability, we tried to focus more in this study on actual removal of
catalytic transition metals. In practice, this meant screening for
The bulk of metals, found in wort and beer, unavoidably enter chelators that would form complexes with them, large enough so
through the grain at mashing [9]. Despite being added at significantly that they could effectively be filtered out. However, an important
lower quantities, also hops are a relatively substantial source of aspect of the study was to also monitor the beneficial metal ions
manganese in beer (and especially so with dry hopping) [10]. Apart (Ca, Mg, Zn). As mentioned earlier, we would prefer that these
from the unwanted catalytic metals, there is also a leaching-out of metals remain available for the workings of the brewing process.
desirable metal ions for brewing – such as calcium, magnesium,
and zinc. The most feasible way of strictly removing the undesir-
able metal ions whilst brewing would have to be physicochemical, 2 Materials and method
through means of chelation (the act of binding metal ions to other
molecules or chelators). Only limited research has been conducted 2.1 Chemicals
on the captivation of stale inducing metal ions in brewing, albeit
with some very positive outcomes. Wietstock, Kunz and Methner Ferulic acid (99.0 %), phytic acid sodium salt hydrate (90 %), tan-
showed that addition of a strong chelator – ethylenediaminetet- nic acid (99.0 %), quercetin dihydrate (98.0 %), iron(II) sulphate
raacetic acid (EDTA) – to beer actively reduces the amount of heptahydrate (99.0 %) and calcium(II) acetate hydrate (99.0 %)
Strecker aldehydes formed during its storage [4] (note that fresh were purchased from Sigma-Aldrich Chemie GmbH (Steinheim,
beer is low in aldehydes). Analogous anti-oxidative effects through Germany). Ethylenedinitrilo-tetraacetic acid disodium salt dihydrate
iron complexation are seen with hop acids, albeit weaker [11–15]. (Titriplex® III; 99.0 %), citric acid monohydrate (99.5 %), tartaric
Amino acids and melanoidins have been known to chelate copper acid (99.5 %), anhydrous gallic acid (98.0 %), iron(III) chloride
ions [16], which is also the reason why the ‘hot break’ (which largely hexahydrate (99.0 %), and manganese(II) sulphate monohydrate
consists out of coagulated protein) is rich in metals [17]. Certain (99.0 %) were obtained from Merck KGaA (Darmstadt, Germany).
polyphenols, like prenylflavonoids and pro-anthocyanidins, should Chlorogenic acid (98.0 %) was acquired from Acros Organics (New
also act as anti-oxidants by similar metal-confining mechanisms, Jersey, USA). Magnesium(II) acetate tetrahydrate (99.5 %) was
yet some debate still exists [18, 19]. bought from Honeywell Riedel-de Haën AG (Seelze, Germany).
Zinc(II) acetate dihydrate (99.5 %) was acquired from AppliChem
The existing amount of controversy can largely be attributed to GmbH (Darmstadt, Germany). Copper(II) acetate monohydrate
the fact that complexes may act as double-edged swords; behav- (99.0 %), dimethyl sulfoxide (99.5 %), and glacial acetic acid
ing oxidation retardant under certain conditions, but accelerative (100 %) was attained from Carl Roth GmbH (Karlsruhe, Germany).
under others. A certain crossline exists on which every chelator Hydrochloric acid (37 %), ethanol absolute (100 %) and sodium
balances: the agent being reducing enough for it to serve protec- hydroxide came from VWR International S.A.S. (Fontenay-sous-
tively, by reducing ROS; and it being overly reducing, causing the Bois, France). Sodium acetate (99.0 %) was obtained from Chem-
metal ions to be converted back to their reduced states [20]. The solute (Renningen, Germany). All aqueous solutions were made
cross-over phenomenon – whether a ligand
acts pro- or anti-oxidative – is influenced by Table 1 Instrumental parameters for the ICP-OES analysis
factors like chelator concentration and metal-
to-chelator ratio [21, 22]. In this aspect, high Baffled Cyclonic Spray Chamber, Meinhard K1 Nebuliz-
Parameter er, 2.0 mm Alumina Injector, Two-Dimensional Charge
chelator concentrations are usually preferred. Coupled Device (CCD) Detector
Radio Frequency (RF) Power 1500 Watts
To add to the complexity of it all, the pH and
the way the complex is chemically structured Nebulizer Flow 0.35 L/min
will play a role too [13]. If the chelating agent Auxiliary Flow 0.2 L/min
succeeds in shielding off the central metal ion Plasma Flow 11 L/min
from its environment, it prevents the elec- Sample Flow Rate 1.00 mL/min
trons from taking part in the whole electron
Equilibration Time 20 sec
exchanging interplay, which greatly reduces
Torch Position – 3.00 mm
the system’s redox potential. However, if the
complexed transition metal is still oxygen ac- Carrier and purge gas Gaseous argon
cessible, the ligands surrounding the metal Shear gas Air
Table 2 Emission lines used for the quantification of Fe, Cu, Mn, trophotometry: a shift in absorbance (spectrum change) should
Zn, Ca, and Mg by ICP-OES indicate that chelation occurred.
Element Emission line used [nm]
Yearbook 2006 Spectra of metal ion complexes, as well as each individual metal
Fe
238.204
ion and chelator, were examined with a Lambda 25 UV/Vis spec-
Cu 327.398 trophotometer (PerkinElmer, Waltham, USA) and a 10 mm high

Mn 257.612 precision cell made of special optical glass (Hellma Analytics, Jena,
Zn 213.857 Germany), in a wavelength range of 200 – 800 nm (full spectrum).
Ca 317.933
2.4 Determination of complex filterability and metal
Mg 279.553
concentration
with ultrapure water (by Sartopore® 2 MidiCap 0.2 µm filtration; The filterability of metal complexes was tested by pushing each
Sartorius AG, Goettingen, Germany). metal-chelator mixture through a microfilter (0.2 µm) and analys-
ing the filtrate for residual metal with inductively coupled plasma-
2.2 Preparation of reagent optical emission spectroscopy (ICP-OES). For the latter, an Avio
200 spectrometer (PerkinElmer, Rodgau, Germany) was employed
Buffer 1 (‘wort’ buffer; pH 5.60, 0.0 vol% ethanol) and buffer 2 (‘beer’ with following instrumental parameters (Table 1).
buffer; pH 4.30, 5.0 vol% ethanol) were both 0.1 M sodium acetate
buffers. Where necessary, minor pH adjustments were made with The analytical wavelengths used for the determination of each metal
either hydrochloric acid (HCl) or sodium hydroxide (NaOH). ion are stated in table 2. A seven-point matrix-matched calibration
curve, ranging from 0 to 300 µmol/L for each individual metal ion,
All metal ion solutions (FeII, FeIII, CuII, MnII, CaII, ZnII, MgII) and was used to quantify the sample concentrations (r² > 0.99).
chelator solutions (EDTA, citric acid, tartaric acid, quercetin, chlo-
rogenic acid, ferulic acid, gallic acid, phytic acid, and tannic acid) 2.5 Main methodology
had concentrations of 500 µmol/L – allowing for a final concentra-
tion of 250 µmol/L, of both metal ion and chelator, when mixed In order to expand upon the current knowledge of metal chelation
in equal volumes. All solutions were aqueous, with exception of in wort and beer, we tested nine food-grade chelators (EDTA, citric
quercetin (dissolved in dimethyl sulfoxide) and ferulic acid (80:20 acid, tartaric acid, quercetin, chlorogenic acid, ferulic acid, gallic
water:ethanol).
The ‘metal ion mix’ contained six

metal ions (FeII, CuII, MnII, MgII,
ZnII, and CaII) with concentrations
of 250 µmol/L (half that of the sole
metal ion solutions). Blending
equal volumes of metal ion mix
and chelator solution yield final
concentrations of 125 µmol/L for
each metal ion and 250 µmol/L
for the chelating agent.
2.3 Spectrophotometric
determination of com-
plex formation
The stabilization of any charge-

transfer complex accompanies
the transferring of electrical
charges between electron-donor
and -acceptor (e.g. metal ions and
ligands). This electron distribution
change gives rise to the creation
of charge-transfer bands – which
are optical absorption bands – that
are detectable through ultravio-
let–visible (UV/Vis) spectroscopy.
So technically, complex formation
can be monitored through spec- Fig. 1 Experimental setup for the measurement of complex formation and the filterability
was always 1:1. The only exception being the trials with
metal ion mix, which had final molar concentrations of
125 µmol/L for each of the six different metal ions and
250 µmol/L for the chelator. All samples wereYearbook
carried2006
out
in duplicate; except for the blanks (no chelator added),

which were conducted in triplicate.
The chelator-metal mixtures were analysed by spectro-

photometer: once at the beginning (within 1 minute from
mixing) and once at the ‘end’ (60 minutes from mixing).
Separate absorbance spectra of the (blank) buffer solu-
tions, chelators, and metal ions were also collected.
After that, the chelator-metal mixtures were filtered

through a syringe filter with 0.2 µm cellulose acetate
membrane (VWR International, Radnor, USA) and the
filtrates analysed for residual metal ions by ICP-OES. All
syringes (Henke-Sass Wolf, Tuttlingen, Germany) and
tubes (VWR International, Radnor, USA) were single-use
and metal-free.
3 Results & Discussion
In order to find compounds with the potential of slowing

down radical formation in beer and brewing, a multi-
tude of chelators was screened on two key features:
Whether the chelator forms complexes with the metal
ion(s) in question and, if so, whether these complexes
are large enough to be effectively filtered out. Figure 2
displays the chemical structures and possible chelation
mechanisms of all tested chelators. A quick and easy
experimental setup allowed for the numerous combina-
tions to be explored.
Fig. 2 Chemical structures and possible chelation mechanisms of all tested
chelators Due to the high variability and extreme complexity of beer
and wort matrices, acetate buffer solutions were used.
acid, phytic acid, and tannic acid) on their capacity to form filterable Using buffers negate any unwanted interferences coming from
complexes with seven metal ions (FeII, FeIII, CuII, MnII, CaII, MgII, and endogenous beer/wort components, such as naturally present
ZnII) in both ‘wort’ and ‘beer’ buffer solutions (buffer 1 and buffer chelators, metal ions, colour, etc.
2, respectively). Likewise, each chelator was also subjected to a
mix of these metals to check the binding affinities – chelators bind Some metal-chelator combinations produced distinct colour forma-
metals in order of decreasing complex-stability constants – and tions, visible to the naked eye (Table 3); meaning that complex
performance consistency – whether similar efficiencies to sole formation indisputably occurred.
metal ions are seen.
UV/Vis spectrophotometry was used to more accurately detect
As depicted in figure 1, both metal and chelator solution were whether complexes were formed between metal and chelator.
transferred simultaneously into a beaker and mixed. Molar concen- The findings can be found in table 4 (see page 62). The table only
trations were 250 µmol/L for each, entailing that chelator:metal-ratio addresses buffer 1 (‘wort’), but the spectrum changes generally
Table 3 Combinations leading to visibly coloured complex formations
Quercetin Chlorogenic acid Gallic acid Tannic acid

Fe(II) – Green – Dark Green – Dark purple Purple Dark purple
Fe(III) Green Dark green Green Dark green Dark purple Purple Dark purple Purple
White White
Cu(II) – – – – – –
(cloudy) (cloudy)
Table 4 Complex formation – study findings and percentage of bound metal ion (as calculated by Visual MINTEQ software) in ‘wort’
buffer 1 (pH 5.60)
Cation Chelator
Yearbook 2006
No Citric Tartaric Chloro- Ferulic Gallic Phytic Tannic
EDTA Quercetin
chelator* acid acid genic acid acid acid acid acid

99.8 56.5 3.3 56.6 0.0 28.8 5.1 99.8

Fe(II) (49.2) –
(0.1) (21.4) (47.5) (21.2) (49.1) (35.0) (45.6) (0.1)
100 90.4 0.5 100 83.7 8.6 100 99.4
Fe(III) (100)a –
(0.0) (9.6)b (99.5)c (0.0) (16.3)d (91.4)e (0.0) (0.6)f
91.6 21.9 2.1 0.0 63.3 0.0
Ca(II) (37.3) – – –
(3.2) (29.1) (36.5) (37.0) (13.7) (36.2)
100 58.1 5.1 0.0 0.0 81.3 97.5 0.1
Zn(II) (62.1) –
(0.0) (26.0) (58.9) (61.8) (62.1) (11.6) (1.5) (62.1)
47.3 21.2 0.7 0.0 28.5 0.0
Mg(II) (41.7) – – –
(22.0) (32.8) (41.3) (41.4) (29.8) (40.6)
99.7 27.0 5.8 0.0 98.8 0.1
Mn(II) (49.3) – – –
(0.1) (35.9) (46.3) (49.2) (0.6) (49.2)
100 79.8 3.3 0.0 10.3 95.8 0.0
Cu(II) (93.1)g – –
(0.0) (18.8)h (90.0)i (93.1)j (83.5)k (3.9)l (92.7)m
Grey cells imply substantial UV/Vis absorbance changes were seen (indicating complex formation occurred).
The primary value indicates the percentage of chelator-bound metal. The secondary value (in brackets) indicates the percentage of metal bound to
other species present (acetate, hydroxide). Even though these latter complexes are weak (log k < 2; specified if otherwise), the metal ions are techni-
cally not considered free.
Vacant cells (–) are due to lacking component information.
* In the ‘no chelator’ column, the buffer’s acetate operates as the active chelator.
[Fe(acetate)2]+ (log K 7.6), [Fe(acetate)3]0 (log K 9.6) and [Fe(OH)2]+ (log K -5.8): a 11.9 %, 68.2 %, 19.9 %; b 1.1 %, 6.6 %, 1.9 %; c 11.9 %, 0.6 %,
19.8 %; d 1.9 %, 11.1 %, 3.2 %; e 10.9 %, 62.3 %, 18.2 %; f 0.1 %, 0.4 %, 0.1 %
[Cu(acetate)3]- (log K 3.9), [Cu(acetate)]+ (log K 2.2) and [Cu(acetate)2]0 (log K 3.4): g 11.0 %, 43.9 %, 38.2 %; h 2.2 %, 8.9 %, 7.7 %; i 10.6 %,
42.5 %, 36.9 %; j 11.0 %, 43.9 %, 38.2 %; k 9.8 %, 39.4 %, 34.3 %; l 0.5 %, 1.8 %, 1.6 %; m 10.8 %, 44.3 %, 37.6 %
Table 5 Residual metal content (μmol/L) of metal-chelator mixtures after 60 minutes reaction time at room temperature and filtration with
a 0.2 μm filter
Chelator added (250 µmol/L)
Metal ion
added pH Chlorogenic
(250 µmol/L) No chelator EDTA Citric acid Tartaric acid Quercetin Ferulic acid Gallic acid Phytic acid Tannic acid
acid
4.30 267.5 259.5 253.9 249.4 239.5 164.6 264.4 236.8 223.1 220.8
Fe(II)
5.60 175.6 243.2 224.6 245.5 114.9 238.6 159.1 251.4 245.6 10.6
4.30 241.1 232.6 236.5 224.6 186.6 213.0 229.9 213.6 215.2 182.2
Fe(III)
5.60 149.5 168.7 207.1 194.7 5.7 14.6 132.1 40.9 211.0 4.8
4.30 244.2 218.0 231.4 231.8 247.7 262.2 264.5 216.2 308.5 218.5
Ca(II)
5.60 237.4 200.6 204.5 208.7 237.8 232.9 246.7 197.9 262.9 195.2
4.30 270.3 267.5 276.2 276.2 235.3 237.8 234.5 264.2 131.9 269.4
Zn(II)
5.60 226.9 212.9 228.2 228.4 229.2 211.9 224.4 222.5 67.3 215.3
4.30 233.4 214.1 221.3 219.7 223.9 231.6 217.6 212.6 288.4 216.8
Mg(II)
5.60 213.2 240.4 202.0 203.5 211.6 204.7 204.8 197.7 241.6 206.1
4.30 268.5 238.7 251.6 251.0 244.8 238.3 277.7 238.2 329.9 240.0
Mn(II)
5.60 241.3 207.5 196.8 201.4 233.0 221.8 232.5 186.2 232.2 227.1
4.30 251.8 229.8 239.7 236.6 225.0 225.8 215.9 229.1 214.4 219.2
Cu(II)
5.60 239.1 198.7 190.6 225.3 233.3 238.1 245.4 143.7 219.8 4.1
The larger the decrease in metal ion concentration after filtration, the more intensely shaded the cell.
did not differ greatly between the two buffers. The represented According to the results depicted in table 4, all investigated iron
values were calculated by using chemical equilibrium modelling species easily form complexes with all tested chelators (cells
software (Visual MINTEQ version 3.1). The data depict the (pre- highlighted in grey). Even though the calculated values for tartaric
dicted) percentages of metal ion bound to any given chelator in acid with iron predict only very low percentages of tartrate-bound
buffer 1 (pH 5.60). Predictive calculations could not be made for all Fe, we do observe a clear spectrum change. The opposite effect
metal-chelator combinations, because of lacking stability constants is also seen: high calculated ratios of bound metal, yet no noted
published in literature for some. absorbance changes. Thus, at least for this setup, it seems dif-

Yearbook 2006
Fig. 3 Absorbance spectra of tannic acid with Fe(II) in ‘wort’ buffer 1 (a) & ‘beer’ buffer 2 (b); and Fe(III) in ‘wort’ buffer 1 (c) & ‘beer’
buffer 2 (d)
ficult to draw a hard line between complex formation and a clear some of these chelators in wort during mashing; and electron spin
change in absorbance. resonance (ESR) spectroscopy will be employed to look deeper
into their anti/pro-oxidative effects.
Additionally, and unfortunately, it appeared that in many cases
the chelator-metal complexes were small enough to allow for filter We could deduct, however, that decreases in residual metal after
penetration, given that often no substantial metal decrease could filtration (Table 5: shaded cells) are always accompanied with big
be noticed after filtration. Nevertheless, as said, even unfilterable/ changes in UV/Vis absorbance (e.g. Fig. 3a and 3c). This only
small complexes could have anti-oxidative properties, as the tran- goes one way, because – like already explained – some formed
sition metals could be bound in such a way that they no longer complexes will be small enough to permeate the microfilter. Thus,
behave pro-oxidatively. This potential should not be dismissed. For an obvious absorbance change is no guarantee that a decrease
this study, it was decided not to focus on this aspect. However, of the particular metal after filtration will take place (e.g. Fig. 3b
follow-up studies will further investigate the chelation behaviour of and 3d).

Yearbook 2006
Fig. 4 Absorbance spectra of tannic acid with Cu(II) in ‘wort’ buffer 1 (e) & ‘beer’ buffer 2 (f)
Table 6 Residual metal content (in µmol/L) of metals-chelator mixtures after 60 minutes reaction time at room temperature and filtration
with a 0.2 µm filter
Chelator added (250 µmol/L)
Metal ion
added pH Tartaric Chlorogenic
(125 µmol/L) No chelator EDTA Citric acid Quercetin Ferulic acid Gallic acid Phytic acid Tannic acid
acid acid
Fe(II) 116.6 118.1 119.0 115.0 95.5 104.2 93.2 114.3 42.6 99.8
Ca(II) 121.6 128.9 129.0 126.1 106.8 158.8 105.4 126.4 121.7 133.6
Zn(II) 114.8 116.0 114.6 115.3 107.5 121.8 103.6 112.2 80.0 120.0
Mix 4.30
Mg(II) 121.2 119.2 122.8 121.4 109.7 141.2 106.6 124.9 125.8 125.2
Mn(II) 116.3 117.5 116.9 114.8 104.5 141.9 102.1 116.8 100.8 122.5
Cu(II) 117.3 117.8 117.7 108.9 109.5 119.8 122.6 105.6 93.5 127.6
Fe(II) 89.0 122.5 138.9 137.8 45.8 84.7 71.1 110.6 15.1 15.9
Ca(II) 91.7 95.7 110.7 97.1 82.9 83.0 90.2 100.9 61.2 97.2
Zn(II) 126.0 122.1 122.9 124.3 122.5 119.9 124.2 125.7 17.2 92.6
Mix 5.60
Mg(II) 127.6 125.3 123.5 128.1 118.9 122.7 135.0 143.3 136.3 155.4
Mn(II) 129.3 124.9 124.3 127.9 124.5 123.7 129.7 135.3 68.0 133.8
Cu(II) 127.6 125.5 123.5 125.4 116.3 114.8 127.1 117.0 63.5 13.4
The larger the decrease in metal ion concentration after filtration, the more intensely shaded the cell.
Copper behaves very similarly to iron with tannic acid, in terms The first column shows the quantified metal levels without any ad-
of diminishment after filtration (see Table 5: big reduction at pH dition of chelator. Ideally, these values should all deviate around
5.60 and significantly less at pH 4.30). However, when comparing 250 µmol/l, which seems to generally be the case (considering
the absorbances, copper behaves differently than both iron ions. also the varying purities of the used chemicals). However, for
Whereas a clear signal change is seen at pH 5.60 (Fig. 4e), one both Fe-species, values appeared noticeably lower at pH 5.60.
does not take place at pH 4.30 (Fig. 4f). This can be explained due to formation iron-hydroxides [13, 25,
26]. With Visual MINTEQ, it was calculated that – in absence of
Concerning the question on whether the formed complexes are chelators and under buffer 1 (pH 5.60) conditions – 19.9 % of
large enough to be filtered out: table 5 depicts all the sole metal ion filterable [Fe(OH)2]+ is formed, but only for FeIII (0.0 % for FeII).
ICP-OES results, featuring the mean residual metal ion concentra- It thus seems that FeII is capable of oxidizing to FeIII under these
tions (in µmol/) found in the filtrate, after running the metal-chelator conditions – albeit slowly [27, 28].
mixtures through a 0.2 µm filter. Table 6 presents the results of
the metal ion mix. This precipitation of iron-hydroxides does not happen as consid-
erably in acidic media or when a chelator is present [13, 29, 30].
This may be the reason why FeII is the most commonly found form stable bridge structures with phosphates [52–54], makes
iron-species in beer [31, 32], as it is low in pH and contains calcium the likely cause of the decreased dialysability seen with
endogenous chelators. Although wort is less acidic than beer, the metal ion mix and phytic acid.
iron-hydroxide formation is unlikely, because of many chelators Yearbook 2006
naturally being present.

4 Conclusions
EDTA makes strong complexes with Fe, Ca, Zn, Mg, Mn, Cu [33,
34], which is also seen from the calculations in table 4. However, Nine compounds (EDTA, citric acid, tartaric acid, quercetin, chlo-
the EDTA-complexes are small enough to penetrate the filter, rogenic acid, ferulic acid, gallic acid, phytic acid, and tannic acid)
which is the reason why we see only small drops in metal levels were screened for their complexing capacities with seven metal ions
after filtration. (FeII, FeIII, CuII, MnII, CaII, ZnII, MgII; both separately and mixed) in
both a ‘wort’ and ‘beer’ buffer solution (pH 5.60, 0.0 vol% ethanol
There are no noteworthy metal decreases with citric and tartaric and pH 4.30, 5.0 vol% ethanol, respectively). The aim was to find
acid. Good iron-catching compounds are quercetin (especially FeIII) compounds that can aid brewers in producing a more flavour stable
[35], chlorogenic acid (ditto) [36, 37], ferulic acid [38], gallic acid beer, by lowering the stale-inducing transition metals.
(solely FeIII) [39, 40], and tannic acid [41, 42]. The latter performs
extraordinarily well for both iron species and copper [41–43]. Gallic Amongst the tested chelators, there is a definite pH effect where
acid also chelates copper, but not as effectively [44]. Judging from they seem to make larger complexes (as appraised by filterability of
these results, tannic acid seems the most promising compound in the complexes) with metal ions at pH 5.60 (the approximate acidity
terms of large complex-making capability. This is consistent with of wort) than at 4.30 (the approximate acidity of beer).
tannic acid possessing multiple phenolic hydroxyl groups (Fig. 2),
which allows for copious metal-chelator cross-links. Phytic acid is Most chelators do not cause a decrease in the beneficial metal
the only chelator binding with zinc in a way that it becomes filter- ions (Ca, Zn, Mg), with phytic acid being the exception by chelat-
able [45, 46]. As zinc is important for yeast health [47], this would ing i.a. zinc. Tannic acid is by far the best performing chelator in
be unfavourable in brewing practice. Edney and team made worts terms of making large complexes with both FeII, FeIII and CuII. And
from different low-phytate barleys and saw significantly higher lev- although in this study it primarily shows this behaviour at the wort
els of zinc and magnesium present [48]. Because of this mineral pH of 5.60, filtration trials have shown that tannic acid is able to
absorption tendency, phytic acid is sometimes referred to as an form iron-complexes in finished beer as well [42].
anti-nutrient.
Iron reducing effects were also witnessed with chelators like
In table 5, we also behold a clear connection between a more quercetin, chlorogenic acid, and gallic acid. The last one, however,
alkaline pH and the forming of better/larger complexes (with chlo- did not bind FeII. Apart from tannic acid, gallic acid was also the
rogenic acid as the sole exception, because of its low pKa, and only compound capable of chelating out copper (albeit to a lesser
only for FeII). pH has a big impact on chelation behaviour, as it extent). This is in line with tannic acid being composed out of eight
determines the ionic species of the chelating agent (a chelator must to nine gallic acid molecules, bound to a central glucose [55].
be ionized to be active) as well as the speciation and hydrolysis
products of the metal ions. Complexes are less stable in acidic As mentioned, almost any concentration decrease was witnessed
systems, due to protonation of ligand functional groups; and the at pH 5.60. Noticeable exceptions were chlorogenic acid, which
protons will compete with the metal ions for binding. Thus, in terms succeeds in binding FeII at the more acidic pH of 4.30, and phytic
of pH, wort is generally a better environment for complex formation acid with zinc. Manganese is, unfortunately, never significantly
than beer [13, 17, 49]. removed by any of the tested chelators. The best effect is seen with
gallic acid. This stability of manganese is also seen in the brewing
Unfortunately, manganese did not form filterable complexes with process, where it is found in the final beer at higher concentrations,
any of the chelators. In contrast to the other transition metals, MnII relative to the other heavy metals (such as iron and copper), which
is known for its rather weak ability to form organic complexes [50, are more readily removed during wort boiling and fermentation [56].
51]. However, in the presence of other metal ions (mix), a reduction
in Mn by phytic acid did take place (Table 6). In the metal ion mix trial, gallic acid does not chelate copper any-
more, chlorogenic acid chelates FeII slightly better, and tannic acid
Phytic acid is also the only chelator that shows highly erratic takes away some zinc; all at pH 5.60. Apart from that, phytic acid
behaviour compared to what is seen with the single metal ions is the only chelator that is acting remarkably differently compared
(Table 5). Whereas most chelators retain (e.g. tannic acid) or even to its chelating behaviour seen with the single metal ions, suddenly
lose (e.g. gallic acid) some of their catching potential in the metal chelating out every metal ion except for MgII. Calcium is thought to
ion mix, phytic acid suddenly exceeds in capturing Fe, Ca, Zn, lie at the basis of this effect. These results are in accordance with
Mn, and Cu (especially at pH 5.60). On suspicion that ZnII played how phytic acid behaves in more complex food systems [57, 58].
an important role in this, a follow-up chelation test was conducted
with zinc being left out of the ion mix (data not shown). The results, The high tendency of tannic acid to form large, precipitating com-
however, were not significantly different from what was seen with plexes with both iron and copper ions could be used to remove
the mix including zinc; except for the CaII being noticeably higher. these pro-oxidative metals from wort (and beer). It is highly likely
That observation, together with the fact that calcium is known to for the resulting beer to achieve an improved oxidative stability,
which would benefit brewers and consumers alike. Hence, follow- 415-425.
up studies will be conducted to check whether the complexing 15. Wietstock, P.C.; Kunz, T. and Methner, F.-J.: Influence of Hopping
tendencies of the most successful chelators can be replicated in Technology on Oxidative Stability and Staling-Related Carbonyls in
wort during mashing,
Yearbook 2006 and how we can potentially optimize their
Pale Lager Beer, BrewingScience, 69 (2016), no. 11/12, pp. 73-84.
chelation towards catalytic transition metals. 16. Plavšić, M.; Ćosović, B. and Lee, C.: Copper complexing properties of
melanoidins and marine humic material, Science of The Total Environ-

Acknowledgements ment, 366 (2006), no. 1, pp. 310-319.
17. Jacobsen, T. and Lie, S.: Metal binding in wort – an evaluation of prac-
This project has received funding from the European Union’s Ho- tical stability constants, EBC Proceedings of the European Brewery
rizon 2020 research and innovation programme under the Marie Convention Congress, Nürnberg, Germany, 1979, pp. 117-129.
Skłodowska-Curie grant agreement No. 722166. 18. Aron, P.M.: The Effect of Hopping Technology on Lager Beer Flavor and
Flavor Stability and the Impact of Polyphenols on Lager Beer Flavor
and Physical Stability, 2011.
5 References 19. Wannenmacher, J.; Gastl, M. and Becker, T.: Phenolic Substances
in Beer: Structural Diversity, Reactive Potential and Relevance for
1. Preedy, V.R.: Beer in Health and Disease Prevention, Elsevier, 2008. Brewing Process and Beer Quality, Comprehensive Reviews in Food
2. Aquilani, B.; Laureti, T.; Poponi, S. and Secondi, L.: Beer choice and Science and Food Safety, 17 (2018), no. 4, pp. 953-988.
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III
Complexation of transition metals by chelators

added during mashing and impact on beer stability
Tuur Mertens, Thomas Kunz, Philip C. Wietstock and Frank-Jürgen Methner
Journal of the Institute of Brewing. 2021; 127:345-357.
DOI: 10.1002/jib.673
Research article
Received: 6 January 2021 Revised: 7 August 2021 Accepted: 18 September 2021 Published online in Wiley Online Library: 25 October 2021
(wileyonlinelibrary.com) DOI 10.1002/jib.673
Complexation of transition metals by chelators

added during mashing and impact on beer
stability
Tuur Mertens,* Thomas Kunz, Philip C. Wietstock
and Frank-Jürgen Methner
Beer inevitably changes due to an array of staling reactions. A major factor in beer ageing is the involvement of transition metals
(iron, copper, manganese) in oxidative reactions. To tackle the flavour stability issue, metal chelation was investigated. Based on
previous research, five primary chelators (tannic acid, gallic acid, EDTA, citric acid and phytic acid) were screened using experi-
mental design for their capacity to reduce the content of wort transition metals. The chelating agents were added under varying
conditions (mash out temperature, mash pH, grain bill, chelator concentration, addition time) during laboratory scale mashing to
assess how they altered complexation and metal load. Fourteen alternative chelators (ferulic acid, tartaric acid, quercetin,
chlorogenic acid and ten polyphenolic food extracts: green tea, pomegranate, grape seed, reishi, cinnamon, curcuma, milk thistle,
ginkgo, grapefruit seed and raspberry) were also explored. Metal ions were analysed using inductively coupled plasma optical
emission spectrometry and wort oxidative stability by electron spin resonance spectroscopy. Mash pH was the most decisive
of all tested process variables: acidified mashing (pH 6 to 5) produced worts with more iron, manganese and zinc (230, 320
and 150%, respectively). Addition of effective chelators counteracted this undesirable effect for iron. Green tea extract, tannic
acid and, particularly, pomegranate extract all resulted in lower wort iron. Conversely, addition of EDTA, caused iron, manganese
and zinc to increase. Pomegranate extract (90% ellagic acid) was the best performing chelator and reduced radical generation in
wort (80% reduction by 60 mg/L addition), making it a promising novel compound in the improvement of beer shelf life. © 2021
The Authors. Journal of the Institute of Brewing published by John Wiley & Sons Ltd on behalf of The Institute of Brewing &
Distilling.
Additional supporting information may be found online in the Supporting Information section at the end of the article.
Keywords: Oxidative beer stability; transition metals; chelate complexes; chelator efficacy; inductively coupled plasma optical emission
spectrometry (ICP-OES); electron spin resonance (ESR) spectroscopy
Introduction Avoidance or removal of transition metal catalysts should slow

the reaction rates, lowering the production of free radicals and
Problems with beer flavour stability can result in brand rejection other reactive species, and reducing the oxidative damage
and a decrease in sales (1). Accordingly, it is paramount that beer inflicted on wort and beer. This was already observed in work by
retains its freshness until at least the best-before date. Unfortu- Wietstock and colleagues, where mash hopping resulted in a more
nately for brewers and consumers alike, this is not always the case: oxidative stable beer, with diminished aldehyde formation during
the issue of beer flavour (in)stability remains the unsolved Achilles’ ageing, through iron chelation by hop α-acids (14–16).
heel of the brewing industry. The problem is particularly apparent, Research (19)—conducted in beer and model wort solutions—
now that beer is produced and retailed both nationally and inter- showed several promising food-grade chelators for the removal
nationally. Noticeable deterioration in flavour may start three of catalytic transition metals from the brewing process, such as
months from packaging when stored at room temperature. Typical tannic acid, gallic acid, and phytic acid. However, wort and beer
signs of beer ageing involve - but are not limited to - the character- matrices are more complex in their chemical make-up, requiring
istic decline in pleasant, crisp or ‘fresh’ bitterness (2–4) and the a fuller investigation of their chelation behaviour in those systems.
formation of undesirable stale flavours or off-flavours (often associ- The previous investigation (19) also showed that mash pH (5.4 ±
ated with aldehydes) (5–7). 0.2) was superior to that of beer (4.3 ± 0.3) in terms of complex
The complex nature of beer degradation (8–10) involves staling
catalysts, of which transition metal ions (iron, copper, manganese)
are the main concern (11,12). Their presence originates primarily * Correspondence to: Tuur Mertens, Technische Universität Berlin, Institute of
(96%) from malt (13). These transition metal ions accelerate oxy- Food Technology and Food Chemistry, Chair of Brewing and Beverage
gen activation per electron transfer by the Fenton and Technology, Berlin, Germany. E-mail: tuur.mertens@tu-berlin.de
Haber-Weiss reaction (Figure 1). The formed reactive oxygen spe- Institute of Food Technology and Food Chemistry, Chair of Brewing and
cies (ROS) - generated from ground state in-pack oxygen - trigger Beverage Technology, Technische Universität Berlin, Berlin, Germany
oxidative stress. ROS are aggressive pro-oxidative entities that
chemically ‘attack’ any molecule in proximity (including desired This is an open access article under the terms of the Creative Commons At-
tribution License, which permits use, distribution and reproduction in any
flavour compounds). medium, provided the original work is properly cited.
345
J. Inst. Brew. 2021; 127: 345–357 © 2021 The Authors. Journal of the Institute of Brewing published by John Wiley
20500416, 2021, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/jib.673 by Tuur Mertens - Technische Universitaet Berlin , Wiley Online Library on [30/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
T. Mertens et al.
Figure 1. Fenton and Haber-Weiss reaction mechanism and the formation of reactive oxygen species, with M = transition metal (Fe, Cu or Mn); adapted with permission from
(17,18)
formation. An additional advantage that chelation in mashing has Materials and methods
over beer - apart from the higher pH - is the added protection pro-
vided by early catalyst removal, as opposed to late removal (e.g. Chemicals
during filtration). This is because transition metals actively promote
Ferulic acid (≥ 99%), phytic acid sodium salt hydrate (≥ 90%) and
staling throughout the whole brewing chain, not only during stor-
quercetin dihydrate (≥ 98%) were purchased from Sigma-Aldrich
age, and are especially reactive during high energy stages
Chemie GmbH (Steinheim, Germany). Ethylenedinitrilo-tetraacetic
(mashing and boiling) (20).
acid disodium salt dihydrate (EDTA disodium salt or Titriplex® III; ≥
The production of wort is a complex and dynamic process, and
99.0%), citric acid monohydrate (≥ 99.5%), tartaric acid (≥ 99.5%)
it is uncertain if chelators have the same impact in a wort/
and anhydrous gallic acid (≥ 98%) were obtained from Merck KGaA
mashing regime, as such a system is (in many ways) more
(Darmstadt, Germany). Chlorogenic acid (98%) was from Acros
complex than model solutions. Chelation and mineral load can
Organics (New Jersey, USA). N-tert-Butyl-α-(4-pyridyl-1-oxide)
be influenced by a myriad of unknown matrix effects (owing to
nitrone (POBN; > 98%) was from TCI Deutschland GmbH
the intricate makeup of wort as the active medium) and by the
(Eschborn, Germany). Hydrochloric acid (34-37%) and
various process parameters (time of addition of the chelator, mash
concentrated nitric acid (67-70%) were from Th. Geyer GmbH &
pH (19,21), mashing temperature (21,22), chelator concentration,
Co. KG (ChemSolute®, Renningen, Germany). Absolute ethanol
grain composition (21,23–25), mechanical stirring etc). All these
(100%) and sodium hydroxide (≥ 98.5) were from VWR
factors will ultimately determine the final metal content of wort
International S.A.S. (Fontenay-sous-Bois, France). Reference stan-
and beer.
dards (1000 μg/mL) for iron, copper, manganese and zinc were
In this study, five previously investigated ‘primary chelators’
from PerkinElmer LAS GmbH (Rodgau, Germany). Argon
(tannic acid, gallic acid, citric acid, EDTA and phytic acid) were
(99.999%) was from Air Liquide GmbH (Düsseldorf, Germany).
assessed for their ability to diminish transition metals during the
Tannic acid (BrewTan B®; ≥ 98%) was from S.A. Ajinomoto
mashing stage of brewing (wort), as opposed to fermentation or
OmniChem N.V. (Wetteren, Belgium). Other polyphenolic (food)
post-fermentation (beer). To reduce the number of trials per
extracts (Table 1) were from various sources: green tea (Fairvital
chelator for the screening of their best working conditions, ‘design
B.V., Landgraaf, Netherlands), pomegranate (Healthtonics,
of experiments’ (DOE) software was used. Additionally, rapid
Thornaby, United Kingdom), grape seed (PumpEffect BV,
screening was also conducted on fourteen (‘alternative’) chelators
Maastricht, Netherlands), reishi (Kurkraft GmbH, Berlin, Germany),
and experimental design was employed for the final optimisation
cinnamon & curcuma (Extrakt Manufaktur Hamburg GmbH,
of the overall best working chelating agent (out of the 19
Hamburg, Germany), milk thistle (Alpha Zwo B.V., Eindhoven,
chelators).
Netherlands), Ginkgo biloba (Nuvi Health B.V., Heerlen,
Chelation efficacy and metal load were assessed by inductively
Netherlands), grapefruit seed (Bafoxx UG, Münster, Germany) and
coupled plasma optical emission spectrometry (ICP-OES) (26),
raspberry (GEN Nutrition UG, Aachen, Germany). All aqueous
and wort oxidative stability was quantified by electron spin
solutions were made with ultrapure water (by Sartopore® 2
resonance (ESR) spectroscopy (27). Ideally, this would replicate
MidiCap 0.2 μm filtration; Sartorius AG, Goettingen, Germany).
the results with the model wort solution (19). The ambition of this
work was to identify food grade chelators - and their optimal work-
ing conditions during mashing - capable of forming filterable
Screening by two-level full factorial design
complexes with the transition metals iron, copper and manganese
(but not with zinc), so that their effective removal (through filtra- A two-level full factorial screening design of experiments (DOE)
tion or lautering) resulted in heightened oxidative stability and in- was conducted for each of the five primary chelators: tannic acid,
creased shelf-life. gallic acid, EDTA, citric acid and phytic acid. A screening DOE
346
Complexation of transition metals by chelators during mashing
Table 1. Polyphenolic (food) extracts
Extract Active compound Purity (%)

Green tea Epigallocatechin gallate 50 (total polyphenol content of 98%)
Pomegranate Ellagic acid 40, 90
Grape seed Oligomeric proanthocyanidin 95
Reishi Polysaccharides 35
Cinnamon Polyphenols 30
Curcuma Curcuminoid 95
Milk thistle Silymarin 80
Ginkgo biloba Flavonol glycosides 24
Grapefruit seed Flavonoids 45
Raspberry Raspberry ketone 100
determines whether the factors of choice (process parameters)

have significant linear effect(s) on the responses of interest (level Table 2. Experimental parameters (factors and levels used) for
of metal ions) and if there are factor interactions. Accordingly, by the two-level full factorial design for testing the effect of chela-
varying two factor levels per experimental unit, the key determi- tor addition time, mash pH, mash out temperature and chela-
nants and their impacts can be identified. tor concentration on the wort metal content (Fe, Cu, Mn, Zn)
Here, the factors were four independent variables: addition time
Factor level
of chelator, mash pH, mash out temperature and chelator concen-
tration. The two factor levels (low and high) used for each variable Experimental factor Low (-1) High (+1)
are reported in Table 2. The reasoning behind the ‘chelator
concentration’ levels (through preliminary trials) can be found in Addition time of chelator [min] 0 55
Supplementary Figure S1. The responses of interest were the four Mash pH [-] 5.0 6.0
dependent (outcome) variables: concentration of iron, copper, Temperature (mash out) [°C] 72 80
manganese and zinc in lautered wort. Chelator concentration [μmol/L] 5.9 35.3
To estimate the pure error for the Lack-of-Fit test, all screening
runs were conducted in duplicate, resulting in 32 runs (2 x 24)
per design. The sequence of the experimental units was deter- In this case, the selected factors were four independent vari-
mined randomly for all DOEs. Randomisation protects against ables: addition time of pomegranate extract, grain bill (Pilsner/
confounding (the distortion of associations, between the Munich), mash pH and pomegranate extract concentration. The
experimental factors and the outcomes, by extraneous factors), responses of interest were - as with the screening DOE - the four
an important source of bias. dependent (outcome) variables: concentration of iron, copper,
manganese and zinc in lautered wort.
Each experimental unit was varied over five factor levels
Rapid screening
(Table 3): eight axial points (±α), sixteen factorial points (±1) and
For rapid screening, chelators were added to the mash at five or six six centre points (0), resulting in 30 runs. The design is rotatable,
concentrations, from 5 mg/L up to 100 or 200 mg/L for quercetin, with α = ±2. The centre point run was performed in sextuplicate
ferulic, tartaric and chlorogenic acid and from 25 or 62.5 mg/L up and was used to estimate pure error for the Lack-of-Fit test. Analo-
to 1000 mg/L for the ten polyphenolic food extracts (green tea, gous to the screening DOE, the run sequence of the experimental
pomegranate, grape seed, reishi, cinnamon, curcuma, milk thistle, units was determined randomly to prevent confounding bias.
ginkgo, grapefruit seed and raspberry).
The lautered worts were analysed for metal content and the
Wort production
data were plotted to check if fluctuations in metal load occurred.
Mashing was performed with unadjusted pH (≈ 5.6), constant grain Laboratory scale sweet worts (400 mL) were prepared from
bill (50/50 Pilsner/Munich), mash out temperature of 78°C and che- mashing milled Pilsner and Munich malt Type I from Weyermann
lator addition time of 0 minutes (onset of mashing). GmbH & Co. KG (Bamberg, Germany) and ultrapure water (1:4 w/
w), using a congress Bender & Hobein mashing apparatus
(Bruchsal, Germany) with stainless steel mashing cups. The water
Optimisation by rotatable full central composite design
lost during mashing due to evaporation was replaced (by weight)
A rotatable full central composite optimisation DOE was con- after mashing, to ensure a grain/liquor-ratio of 1:4 w/w.
ducted with the most promising chelator, pomegranate extract Adjustments of the natural mash pH (≈ 5.6) were made at the
(90% ellagic acid content). Like the screening DOE, the response beginning of mashing either by addition of 2 M hydrochloric acid
surface (optimisation) DOE determines factor importance and in- (acidification) or 1 M sodium hydroxide (basification). Unless other-
teractions. It is, however, not limited to linearity, also allowing to wise stated, there was no wort boiling and mashing was with 50%
check for quadratic or higher order trends. This helps determine Pilsner and 50% Munich malt with the following temperature pro-
the factor configuration leading to minimal metal load and gram: mashing in at 63°C; rest for 30 min; heating up (1°C/min) to
maximal chelation of detrimental transition metal ions (through 72°C; rest for 15 min; heating up (1°C/min) to 78°C and rest for 1
statistical modelling). min (mash out). Boiling was by simmering wort (150 mL) for 60
347
T. Mertens et al.
Table 3. Experimental parameters (factors and levels) for the rotatable full central composite design for testing the effect of pome-
granate extract addition time, grain bill, mash pH and pomegranate extract concentration on wort metal content (Fe, Cu, Mn, Zn)
Factor level
Experimental factor Axial point (-α) Low (-1) Centre point (0) High (+1) Axial point(+α)
Time of addition of pomegranate [min] 0 13.75 27.5 41.25 55
Grain bill (Pilsner/Munich) [%] 0 25 50 75 100
Mash pH 5 5.25 5.5 5.75 6
Pomegranate extract concentration [mg/L] 10 22.5 35 47.5 60
minutes in a 250 mL flask under reflux, followed by trub removal

Table 4. Instrumental parameters for the ICP-OES analysis
through simulated whirlpooling (swirling) and decantation (50
mL).
Baffled Cyclonic Spray Chamber, GemCone Low-Flow
Lautering was simulated by filtering the mash through folded
Nebuliser, 2.0 mm Alumina Injector, Two-Dimensional Charge
Whatman filter papers (Cytiva, Freiburg, Germany) and
Coupled Device (CCD) Detector
recirculating the first 100 mL of lautered wort through the filter
paper (containing spent grains) for enhanced wort clarification. Parameter Level
Sparged wort #1 and #2 were collected separately by consecu-
tively pouring 100 mL of warm (78°C) ultrapure water (without Radio Frequency (RF) power 1500 Watts
pH adjustment) over the ‘filter dry’ spent grains. Nebuliser flow 0.4 L/min
Wort analyses (extract and pH measurement) were performed Auxiliary flow 0.3 L/min
on samples at room temperature. For the determination of metal Plasma flow 8 L/min
content and radical levels, the lautered wort samples were held Sample flow rate 1.00 mL/min
frozen (-18°C) in metal free tubes (VWR International, Radnor, Equilibration time 15 sec
USA) and thawed in a 40°C water bath for 20 minutes before Torch position 0.00 mm
usage. Carrier and purge gas Gaseous argon
Shear gas Air
Wort analysis
Extract and pH determination were on clear, undiluted wort sam-
settings: centre field, 3475 G; attenuation, 3.0 dB; power, 8.920
ples according to MEBAK (27) (method 2.9.3 and 2.13, respectively)
mW; sweep width, 14 G; receiver gain, 3.99 × 103; resolution, 512;
with an Anton Paar GmbH Alcolyzer Beer Analyzing System (Graz,
modulation amplitude, 1.47 G; modulation frequency, 86.00 kHz;
Austria).
conversion time, 10 ms; time constant, 40 ms; number of scans, 25.
Using the method described in MEBAK (27) by Kunz et al. (28), 10
Determination of the metal content ml of sample was mixed with 700 μL absolute ethanol and 100 μL
0.82 M POBN in a glass vial (final sample concentrations: 6.5 v/v
Metal content in lautered wort was determined by inductively
ethanol and 7.6 mM POBN). On adding the spin trap agent (POBN)
coupled plasma optical emission spectroscopy (ICP-OES) using
to the sample, the vial was placed in the heating block at 60°C until
an Avio 200 spectrometer (PerkinElmer, Rodgau, Germany) with
the end of the analysis (ca. 10 hours). All vials were in duplicate and
the instrumental parameters in Table 4. The 20 mL wort samples
sampled by the autosampler, which periodically (every 30-70 mi-
were diluted to 90.9% with 1 mL ultrapure water and 1 mL nitric
nutes) measured the concentration of POBN spin adducts, formed
acid (end concentration: 3-3.2% HNO3) and vortexed before each
by the emerging hydroxyl- and hydroxyethyl radicals.
analysis.
The time dependent radical concentration data were graphically
For the determination of each metal ion, the following analytical
plotted using the statistical software package OriginPro (version
emission lines were used: Fe, 238.204 nm; Cu, 327.398 nm; Mn,
8.0891, OriginLab Corporation, Northampton, MA, USA). Final
257.612 nm; Zn, 206.200 nm. For quantification, four-point
radical concentrations were reported as the radical intensities at
calibration curves (R2 > 0.99) were constructed for each analyte
minute 450 (T450-value).
in wort by standard addition. This was by spiking a ‘blank’
reference wort of equal extract with increasing levels of metal stan-
dards, achieving respective end concentrations, for iron, copper,
manganese and zinc, of (in μg/L): 0, 0, 0, 0 (addition 0); 50, 50,
Data analysis
100, 150 (addition 1); 100, 100, 200, 300 (addition 2); 200, 200,
400, 600 (addition 3). Two-level factorial experimental design (screening), response sur-
face experimental design (optimisation) and multivariate data
analysis were performed with Design-Expert® software (version
Determination of radical levels
11, Stat-Ease, Inc., Minneapolis, MN, USA). The statistical signifi-
Radical formation was measured in wort by electron spin reso- cance (confidence level of 95% or p 0.05) of the numerous
nance (ESR) spectroscopy using an X-band spectrometer (e-scan, factors and their interaction was determined with analysis of vari-
Bruker BioSpin GmbH, Rheinstetten, Germany) with the following ance (ANOVA).
348
The removal of insignificant model terms was done with back- mashing, such as amino nitrogen compounds and organic acids,
ward elimination (with α > 0.1), by applying all blocks and forced which have buffering capability. The first and second wort sparges
terms to the model. This stepwise regression is used to algorithmi- had average gravities of 10.5 ± 0.7°P and 5.7 ± 0.4°P. But here, the
cally reduce the design to the minimum number of needed terms opposite effect could be seen with mash pH and extract: the first
while giving all model terms a chance to be included. and second sparges of the spent grain mashed at pH 5.0 had lower
Non-significant terms were kept in the model when they were extracts on average (10.3°P and 5.4°P) than the ones mashed at pH
involved in interaction(s) with significant terms (‘principle of 6.0 (10.7°P and 5.9°P). This effect was particularly noticeable in the
hierarchy’). tannic acid sparges (Δ°P of 0.7 between different pHs).
For metal ion content (concentration in the sparged worts com-
pared to the first worts), a progressive reduction could be seen.
Results and discussion Zinc and manganese showed substantial displacement from the
The lautered first worts had an average specific gravity of 16.5 ± spent grains to the sparged worts. For zinc, the extent was mainly
0.2°P and were made with all-grain pale malts (Pilsner and dependent on the mash pH. On average, over all the chelator trials,
Munich), because of the predominance of beers utilising this malt the first and second sparges of the pH 5 mashes had 121% and
type (29). Mixing Munich with Pilsner malt was because of its 82% of the Zn content of the first wort, respectively. In comparison,
slightly higher roasting, making it extrude more iron, manganese grains mashed at pH 6 contained only 86% and 56%.
and zinc during mashing. In our preliminary trials, the all-Munich The trials with phytic acid are reported in Figure 2. Diagrams for
wort contained 160-210% more iron than three diverse brands the other four chelators can be found in the supporting informa-
of all-Pilsner worts. This is a known effect, albeit not tion (Figures S2-5), together with an overview of the average abso-
well-documented (14,30). lute metal values for every chelator (Table S1). As previously
Depending on the malts used, a standard 12°P gravity wort has described, one screening DOE was conducted per chelator,
levels of around 100-270 μg/L iron, 20-400 μg/L copper and 80- resulting in a total of 20 models with one model for every metal
150 μg/L manganese with 100-5000 μg/L of the beneficial zinc ion (n = 4) for every chelator (n = 5). A summary of the data with
(31,32). Calcium and magnesium - two other beneficial brewing correlation matrices is given in Table 5. It features the correlation
metals found in wort - were not screened in our trials. Neither ap- coefficients between every experimental factor and metal ion.
pear to substantially chelate out of solution (19) and they are also Strong linear dependencies, between variable and response, are
present in wort at concentrations two orders of magnitude higher coloured red for positive and blue for negative correlations. Some
than the detrimental iron, copper and manganese ions (namely, caution is needed when interpreting this data as some non-linear
50-90 mg/L for Mg and 15-35 mg/L for Ca) (31). associations may be underestimated or overlooked (monotonic
Mashing at pH 5.0 gave a slightly higher extract (16.6°P) com- and non-monotonic relationships, respectively). The ANOVA
pared to mashing at pH 6.0 (16.3°P), which is in accordance with results and fit statistics for every model can be found in the supple-
the report of Taylor and MacWilliam (33,34). This is likely to be mentary info (Table S2).
due to the more efficient availability or activation of limit dextrinase From Table 5 it can be observed that - for the five chelators - the
(35,36). This higher extract does not always indicate higher time of addition does not have a high impact on the levels of iron,
fermentability. The mash pH for maximised fermentability is manganese and zinc found in the lautered wort. However, copper
around 5.5-5.6 (37–39), since this is the pH optima at mashing behaved differently as the time of addition did a to influence the
temperatures (60-70°C) of α- and β-amylase, measured in wort at concentration in wort slightly. But even with tannic acid, where
room temperature. this effect was particularly strong, late addition only led to a minor
During the mashing process, the pH of the adjusted mash increase in copper of 20-30 μg/L (depending on the concentration
drifted towards a more natural wort pH: the pH 5.0 mash had an of the added tannic acid). Further, the mash pH has a major effect
average pH of 5.07, while the pH 6.0 mashes had an average pH on the levels of metal ions that leach into the lautered wort. For
of 5.92. This is due to the release of various wort substances during iron, manganese and zinc, a strong negative correlation exists with
Figure 2. Decrease in metal ion concentration between the first wort and two consecutive sparges (first sparge: dense pattern; second sparge: sparse pattern), average at pH 5.0
349
(left, n = 16) and pH 6.0 (right, n = 16) of phytic acid ± standard deviation
T. Mertens et al.
Table 5. Pearson correlation coefficients (r) between experimental factors and metal ion concentrations in lautered wort
Pearson’s r ∈ [-1,1] and coloured dark red at total positive linear correlation (1.00), white at no linear correlation (0.00) and dark blue at total
negative linear correlation (-1.00)
a lower mash pH leading to a higher amount of Fe, Mn and Zn likely however, although the mash out temperature was found to
leaching out into the wort (see Supplementary Figure S6). A similar be a significant factor in most models, that the difference between
observation was reported by Holzmann and Piendl in 1977 (21). the low (72°C) and high ( 80°C) mash out temperature was too
This is likely to be due to most metal ions having an increased sol- small to cause any tangible effect. There were weak correlations
ubility in more acidic environments, as a higher pH promotes the in concordance with the findings of Holzmann and Piendl (21).
hydroxide precipitation of metals. Additionally, the higher level Higher mashing temperature reduced the level of zinc and manga-
of protons in an acidic mash compete with the metal cations for nese, but for iron this was unnoticeable across 72-80°C.
any binding sites within the wort matrix (such as endogenous Copper behaved atypically, especially with Cu-Citric acid and
polyphenols), thus increasing the number of free metal ions. Fur- Cu-EDTA. As can be seen from the surface plots (Figure 3), a
thermore, the activity of antioxidants (e.g. chelators) is also often non-linear and non-monotonic relationship was found between
linked to their pH dependent solubility, as was seen in previous the response factor (copper) and the two interacting variables
studies (19, 40). Polyphenols, among other chelators, are more sol- (chelator addition time and mash out temperature), leading to
uble and active at alkaline pH (41). Again, copper is the exception. the misleadingly low linear correlation coefficients (Table 5).
A lower pH leads to a slightly - but statistically significant - lower The correlation coefficients for chelator concentration (Table 5)
level of Cu (20-30 μg/L) which is independent of other factors show which chelator agents influence the metal ion composition
(see Supplementary Figure S6). This could be attributed to the en- of the lautered wort. Gallic, citric and phytic acids have a negligible
hanced protein coagulation during mashing at pH 5 (as opposed impact, leastways at the concentration employed (5.9-35.3 μmol/
to pH 6), as the isoelectric point of most wort proteins is around L). EDTA enhanced the solubility of iron (Figure 4), copper,
pH 5.2 or below (37,42–44). Copper binds with the coagulating pro- manganese and zinc, presumably by competing with endogenous
teins, precipitates and is removed with the hot break during chelators (47,48) and possibly stripping them away from other
lautering (45,46). With EDTA, there is the absence of the usual complexes consisting of ‘soft’ ligands (such as thiol groups). In
strong negative correlation of zinc level with the mash pH. It a that agreement with Formanek and Bonte (49), of the five screened
the chelation effect of EDTA outweighs the pH effect. Even at low chelators, tannic acid was the only chelator that successfully re-
concentrations of EDTA (5.9 μM), only a slight increase in zinc is ob- duces the level of iron in wort after lautering (Figure 5). This iron
served when mashing at pH 5 compared to pH 6. chelating capability was also observed in model solutions (19).
From the low correlation coefficients, the mash out temperature The copper binding potential seen in the wort model solutions
did not impact on the final metal content across the board. It is could, however, not be reproduced in the mash. Copper may
Figure 3. Response surface interaction model for the fitted value levels of copper concentration (μg/L) in lautered wort, after mashing at pH 5.6 with (A, left) 35.3 μM citric acid
and (B, right) 35.3 μM EDTA, at varying levels of mash out temperature (°C) and chelator addition time (min) [Colour figure can be viewed at wileyonlinelibrary.com]
350
of removal by tannic acid at the higher pH is likely due to a more

deprotonated state, which facilitates coordination interactions
(19,51).
The successful chelating capacity of some of the other chelators
(gallic acid, phytic acid) in wort buffer solutions could not be
reproduced during mashing. Indeed, even tannic acid was not ca-
pable of chelating out copper. This was not unexpected, as wort is
a complex matrix and lautering is a crude form of filtration com-
pared with microfiltration used in the previous study (19). Even
so, when comparing lautered wort with 0.45 μm microfiltered wort
(data not shown), the difference (on average) was less than 3% for
all metal ions and all chelators with the exception of phytic acid. In
this case, a difference of 8-12% were seen for all metals and was
similar to how phytic acid behaved at wort pH in the trials with
the metal ion mix (19).
What sets tannic acid apart from the other tested chelating
agents is its capability (due to its large size and many hydroxyl
Figure 4. Response surface interaction model for the fitted value levels of iron con- groups) to form multiple crosslinks through coordination with
centration (μg/L) in lautered wort, after mashing with varying levels of EDTA (μmol/L) metal ions (19,52). Tannic acid contains a central glucose spine to
and mash pH, with chelator additions at the onset of mashing and mash out at 78°C which ten galloyl moieties are bound (deca-galloyl glucose), and
[Colour figure can be viewed at wileyonlinelibrary.com]
each galloyl group has two adjacent hydroxyl groups that serve
as binding sites for metal ions. At wort pH, bis- or tris-polyphenol
complexes can be formed with iron (two or three tannic acid
ligands octahedrally coordinated around one metal ion) (53–55).
Accordingly, tannic acid can form large (supramolecular)
organic-metal aggregates, as there are more sites involved in
metal-binding than just the galloyl groups (56). Among other
polyphenols, tannic acid is also known to strongly interact with
proteins (particularly amino and hydroxyl groups) by hydrogen
bonding, covalent bonding, ionic interactions and hydrophobic
interactions (57,58). These polyphenol-protein complexes also
form strong chelates with metal ions (particularly iron and copper)
(11,59,60). Transition metal ions play an important role in
polyphenol-protein association and precipitation (61,62).
Other factors that will influence the size and grade of
polyphenol-protein complexation are the presence of oxidising
agents (e.g. hydrogen peroxide), heightened temperatures and ox-
ygen exposure, as these factors facilitate the formation of covalent
bonds that irreversibly link polyphenols to proteins (63,64).
For every chelator, a selection of unboiled wort samples (addi-
tion time, 0 min; mash out, 80°C; mash pH, 5.0 and 6.0; chelator
Figure 5. Response surface interaction model for the fitted value levels of iron con- concentration, 5.9 and 35.3 μM) were analysed by electron spin
centration (μg/L) in lautered wort, after mashing with varying levels of tannic acid ad- resonance (ESR) spectroscopy, which measures the radical concen-
dition (μmol/L) and mash pH, with chelator additions at onset of mashing and mash
out at 78°C [Colour figure can be viewed at wileyonlinelibrary.com]
tration. Lower free radical intensity is a sign of lower oxidative
stress and potentially reduced product degradation. Among other
things, radical generation is influenced by transition metal content
and the form in which the metal ions are present (free or bound)
(65). Among the ESR samples (data not shown), the only notable
already be too tightly bound to other matrix components, such as difference between ‘high’ and ‘low’ chelator addition was with
amino acids, peptides or proteins (50). EDTA. At both pH values, the area under the curve (radical inten-
In Figure 5, the large effect of mash pH on the iron content (as sity) was larger with the 35.3 μM addition. This can be explained
previously described) is clearly noticeable. Without the addition by the observation that the addition of EDTA (a strong chelating
of tannic acid, reducing the mash pH from 6 to 5 causes a more agent) caused more (transition) metals to leach out into the
than two-fold increase in iron content (from 140 to 310 μg/L, or lautered wort. Further, EDTA metal complexes are known to
221%). On adding a low dosage of tannic acid (5.9 μM or 10.0 behave pro-oxidatively at molar ratios of EDTA and iron below 1,
mg/L), a comparable (high) iron level was found in the wort at spurring free radical generation (66,67).
pH 5. However, this pH effect can largely be diminished (for iron) In summary, from the primary five chelators screened by DOE,
by applying a high dosage of tannic acid (35.3 μM or 60.0 mg/L) only tannic acid was of interest in enhancing beer flavour stability.
to the acidic mash. Unsurprisingly, the lowest Fe level (66 μg/L) This is not a new finding (68). Thus, further examination was re-
was found with 35.3 μM of tannic acid and a mash pH of 6.0. quired, and additional (rapid) screening was performed for other
With the same (high) concentration of tannic acid and a mash chelators that - like tannic acid - perform well during mashing.
pH of 5.0, the iron content was 110 μg/L. The greater efficiency Four chelators (ferulic acid, tartaric acid, quercetin and chlorogenic
351
T. Mertens et al.
acid) - also employed in the previous study - were screened, to- and depletion of iron (Figure 7). What distinguishes the green
gether with ten polyphenolic food extracts (curcuma, cinnamon, tea and pomegranate extracts from the other food extracts is that
raspberry, grapefruit seed, ginkgo, grape seed, green tea, reishi, they are rich in phenolics that, like tannic acid, possess multiple
pomegranate and milk thistle). Many of these fourteen chelators catechol or galloyl groups. The green tea extract contains 50% epi-
are similar to tannic acid in that they possess multiple hydroxyl gallocatechin gallate and the pomegranate extract, 40% ellagic
and/or carboxylate groups, capable of chelation (69). acid. Both molecular structures- and their possible binding sites-
In the range of 0-200 mg/L, ferulic, tartaric and chlorogenic are shown in the supporting information (Figures S7-8).
acids did not reduce the concentration of iron, copper or manga- As the pomegranate extract performed exceptionally well as a
nese. Indeed, at high concentration they led to slightly higher chelating agent, the evaluation was performed with an extract
levels of Fe and Mn, which is likely to reflect the slight containing 90% ellagic acid (rather than 40%). The results (Figure 8)
decrease in pH. Quercetin, however, did cause a reduction in iron are compared with a 60 mg/L addition of tannic acid. It can be
with 3 μg/L at 50.0 mg/L (147.8 μM) and 39 μg/L at 200 mg/L concluded that the pomegranate extract with the 90% ellagic acid
(591.2 μM) (Figure 6). However, this was far from the efficacy is the best chelator of iron. No significant effects were seen with
found with tannic acid. other metal ions, albeit the wort with the addition of tannic acid
The ten polyphenolic food extracts were tested at a broader added was slightly higher in copper. An advantage of using ellagic
range (0-1000 mg/L). Most (curcuma, cinnamon, raspberry, grape- acid is that it is naturally found in some beers (0-1.5 mg/L) (70,71).
fruit seed, ginkgo, grape seed, reishi and milk thistle) had no im- Accordingly, an optimisation experimental design was per-
pact on the transition metal content (Figure 6). Green tea and formed with pomegranate extract (90% ellagic acid). An optimisa-
pomegranate extract however, were successful in the chelation tion DOE requires the most runs per factor, but it also gives the
Figure 6. Levels of iron (μg/L) with increasing concentrations of chelators (mg/L), with ferulic acid (F) in red, tartaric acid (T) in purple, quercetin (Q) in green, chlorogenic acid (C)
in blue and eight polyphenolic food extracts in grey (curcuma, 1; cinnamon, 2; raspberry, 3; grapefruit seed, 4; ginkgo, 5; grape seed, 6; reishi, 7; milk thistle, 8) [Colour figure can be
viewed at wileyonlinelibrary.com]
Figure 7. Levels of iron, copper, manganese and zinc (μg/L) with increasing concentrations of green tea (left) and pomegranate extract (right) (mg/L: 0, 25, 62.5, 125, 250, 500 and
352
1000), with iron in black (■), copper in green (●), manganese in red (▲) and zinc in blue (▼) [Colour figure can be viewed at wileyonlinelibrary.com]
Table 7. Pearson correlation coefficients (r) between experi-

mental factors and metal ion concentrations in lautered wort
Figure 8. Levels of iron, copper, manganese and zinc (μg/L) with increasing concen-
trations of pomegranate extract (mg/L: 0, 5, 10, 20, 60 and 120), with iron in black (■), Pearson’s r ∈ [-1,1] and coloured dark red at total positive linear
copper in green (●), manganese in red (▲) and zinc in blue (▼). The dotted line is of correlation (1.00), white at no linear correlation (0.00) and dark
pomegranate extract (40% ellagic acid) and the solid line with 90% ellagic acid. Tannic
acid (TA) addition (60 mg/L) is included for comparison. [Colour figure can be viewed
blue at total negative linear correlation (-1.00)
at wileyonlinelibrary.com]
Table 6. ANOVA and fit statistics for the response surface

models (optimisation)
Pomegranate extract (90% ellagic acid)

Model Reduced Reduced Reduced Reduced
Cubic Cubic Quartic Cubic
Transformation None None None None
R2 0.99 0.71 0.99 0.94
Adjusted R2 0.99 0.55 0.98 0.91
Predicted R2 0.97 - 0.58 NA 0.84
Adequate 50.3 8.1 38.5 21.6
precision
Model F-value 171 2.6 85.9 29.1
Lack of fit F- 1.32 0.76 0.12 0.54
value
most information of any design. As with the screening DOE, this

approach determines the important factors, fits a quadratic poly-
nomial model to the response to model second-order effects (cur-
vature) and can be used to find the factor settings that lead to a
maximised/optimised response, which (in this case) is the point Figure 9. Levels of iron, manganese and zinc (μg/L) at increasing ratio of Munich
of minimal transition metal content. The ANOVA results and statis- malt (left to right) and two pH levels (pH 5.25, black; pH 5.75, red) [Colour figure can
be viewed at wileyonlinelibrary.com]
tics of the resulting models can be found in Table 6. A summary of
the pomegranate trials by correlation matrix is in Table 7.
In agreement with the chelators tested in the screening
trials (Table 5), the time of addition had little impact. The grain
bill- which was kept at a constant 50/50 ratio in the screening 0.20, this is only part of the explanation. It is likely that an addi-
design- had a low negative correlation with iron, manganese tional mechanism is at play, such as the loss of metal binding
and zinc, with higher ratios of Munich malt aligned with higher capacity of malt solids by roasting (24,30)- with copper, again,
levels of Fe, Mn and Zn in the wort (see Figure 9). It is assumed being the exception.
that this results from the more acidic mash from using In Figure 9 it is also notable that, in terms of mash pH, similar
more highly kilned malts containing higher levels of acidic effects were seen as with the other chelators for iron, manganese
melanoidins, facilitating metal release. However, since the pH dif- and zinc. With respect to the chelator concentration, the correla-
ference between an all-Pilsner wort and an all-Munich wort is tions were almost identical to those of tannic acid, suggesting that
353
T. Mertens et al.
concentrations and different mash pH, is presented in Figure 10.

The iron level with additions of tannic acid are included for refer-
ence. In all, pomegranate extract is more effective at diminishing
iron than tannic acid at the given concentrations.
As can be seen from the correlation matrices (Tables 5 and 7),
mash pH and (if the chelators are effective) chelator concentration
are the two main contributing factors to the metal content of wort.
This derivation is seen in the ANOVA results of all models (namely,
the F- and p-values of the experimental factors). Table 8 and 9, re-
spectively, feature a summary of these outcomes for the
iron-tannic acid model and the iron-pomegranate extract model.
In both models, the chelator concentration term leads the total
model contribution, with mash pH second. Together with iron-
EDTA, and presumably with iron-green tea extract (which was
not investigated by DOE), these are the only instances where this
occurs. In all other models, mash pH is the main contributor to
the model (with Cu-tannic acid, Cu-EDTA and Cu-citric acid being
exceptions).
Electron spin resonance (ESR) spectroscopy was employed to
Figure 10. Response surface interaction model for the fitted value levels of iron
concentration (μg/L) in lautered wort, after mashing with varying levels of
further investigate the effects of pomegranate extract on mashing.
pomegranate extract addition (mg/L) and mash pH, with a 50/50 Pilsner/Munich A selection of the pomegranate worts were boiled and subse-
grain bill, chelator additions after 27.5 min mashing and mash out at 78°C. The quently measured on ESR (Figure 11). The data are from a single
responses with tannic acid are included for reference. [Colour figure can be viewed experiment with technical replicates per sample. The ESR assay al-
at wileyonlinelibrary.com]
lows for a deeper understanding of the antioxidative mechanism
of pomegranate as ICP-OES does not differentiate between free
- like tannic acid - the pomegranate extract effectively reduces the and bound (transition) metal ions within the lautered wort, and
amount of iron during mashing. The three dimensional plot of iron there are important oxidative differences between free and bound
fluctuation, with pomegranate extract added at varying metals. Even among (bound) metal-complexes, there can be great
Table 8. ANOVA results of the Fe-tannic acid model terms
Factor Contribution (%) F-value p-value

[Tannic acid] 44.2 1423 < 0.0001
Mash pH 34.0 1095 < 0.0001
Mash pH * [Tannic acid] 18.9 606 < 0.0001
Mash pH * Addition time 1.3 42.5 < 0.0001
Temperature mash out 0.5 14.5 0.0009
[Tannic acid] * Addition time 0.3 7.9 0.0098
Mash pH * Temperature mash out 0.2 5.0 0.0358
Addition time 0.0 0.1 0.8128
Table 9. ANOVA results of the Fe-pomegranate extract model terms
Factor Contribution (%) F-value p-value

[Pomegr. extr.] 38.4 867 < 0.0001
Mash pH 30.7 694 < 0.0001
Mash pH * [Pomegr. extr.] 10.8 245 < 0.0001
Mash pH2 6.8 154 < 0.0001
Grain bill 3.7 84.4 < 0.0001
Mash pH * Grain bill 3.3 73.9 < 0.0001
[Pomegr. extr.]2 2.2 50.4 < 0.0001
[Pomegr. extr.] * Grain bill 1.4 31.8 < 0.0001
Mash pH * [Pomegr. extr.] * Grain bill 0.8 18.4 0.0006
Mash pH * Addition time 0.5 10.3 0.0055
Addition time 0.4 8.9 0.0086
[Pomegr. extr.] * Addition time 0.2 3.9 0.0667
Addition time2 0.1 3.1 0.0952
354
Conclusions
The results show that pomegranate extract (high in ellagic acid) is
an excellent chelator in lowering the prooxidative iron content of
wort during mashing. Its antioxidative capabilities were reaffirmed
in boiled wort, where it reduced radical generation during
prolonged heating (ESR analysis). Tannic acid and, to a lesser de-
gree, green tea extract share iron chelating properties.
With regard to mashing conditions, it is noteworthy that acidic
(5.2 ± 0.2) mashes will result in significantly higher levels of iron,
manganese and zinc in the wort (and possibly the beer), as com-
pared to mashing at more alkaline pH (5.6 ± 0.2). This effect can,
to a great extent, be nullified for the detrimental iron by sufficient
(early) addition of pomegranate extract or tannic acid. Manganese
content, however, was not readily diminished and its final concen-
tration remained pH dependent. Copper largely remained unaf-
Figure 11. ESR measurement (T450-values) of boiled worts (grain bill, 50/50 Pilnser/
fected. Apart from less metal leaching out with non-acidified
Munich; mash out temperature, 78°C) with various levels of pomegranate extract (mg/
L), mash pH and chelator addition time (min) [Colour figure can be viewed at mashing, a more natural mash pH (≈ 5.6) provides an environment
wileyonlinelibrary.com] that is better suited to chelation, since the chelating agents are less
protonated. Further, mashing at a pH of 5.5-5.6 produces a more
fermentable wort, since it is the optimum working range for both
α- and β-amylase.
disparity, with some complexes being antioxidative and others Mash out temperature and chelator addition time were not as
prooxidative. ESR spectroscopy can help resolve this. significant a parameter as chelator concentration or mash pH in
The reference wort (no pomegranate extract added) shows the terms of influencing metal content. This does not, however, imply
highest radical intensity (T450-value) of all samples, indicating the that these factors have no impact on oxidative stability. As seen
antioxidative capabilities of pomegranate. The wort with the with the chelator addition time, samples with late mash addition
highest amount of pomegranate extract added (60 mg/L) shows of pomegranate extract appeared to show lower radical genera-
the lowest radical intensity or the highest oxidative stability. Apart tion, compared to earlier additions (although this may be due to
from the established iron chelation - which is the main antioxida- minor differences in iron content). It is also likely that a higher
tive effect - further antioxidative properties, such as radical mash out temperature (although it causes slightly more transition
quenching (72–74), may contribute to the effectiveness of pome- metals to drop out of solution) will result in a more oxidised wort
granate extract in lowering the radical generation. For the worts due to higher thermal load.
that had the same concentration of pomegranate extract added, Pomegranate extract containing ellagic acid has exciting poten-
pH was a significant factor, with a lower mash pH leading to a tial as a flavour (and colloidal) stability enhancer, not only by
higher generation of radicals. This effect cannot be solely ex- adding it during mashing but also during wort boiling, beer matu-
plained by the greater iron content of the pH 5 mash since it is only ration and filtration. Further investigation is needed and a
12 μg/L higher than that of the pH 6 mash. An additional follow-up study will explore whether beer shelf-life can be in-
determinant is the pH dependent speciation of some metal ions creased by applying these findings in (pilot scale) brewing trials.
(particularly Fe) and the pH dependent coordination number of Similarly, further work is required on the influence of chelators
some organic ligands (e.g. ellagic acid), all of which will change on de novo aldehyde formation and whether or not, by decreasing
the coordination chemistry of the metal complexes that will the generation of reactive oxygen species, their formation can be
ultimately affect the reactivity of the Fenton and Haber-Weiss slowed down substantially.
mechanism (75,76).
Later addition times of the extract appear to be more beneficial
than an early addition time. From an oxidative standpoint this is Author contributions
surprising, since with the early addition of the chelating agent,
the endogenous wort antioxidants would be better preserved (as Tuur Mertens – conceptualisation of the research plan, project
they are already protected at the onset of mashing from oxidising administration, conceived and performed experiments,
early), resulting in a lower radical concentration. This is may be due created the models and conducted formal analysis, writing (origi-
to the earlier additions having a slightly higher (5-17 μg/L) iron nal draft).
content than the late addition, and ESR measurements are very Thomas Kunz - conceptualisation of the research plan and inter-
sensitive to iron. Jenkins et al. (12) reported that as little as 10 pretation of the data, writing (review & editing).
μg/L transition metal ion can make a detectable difference to the Philip Wietstock - conceptualisation of the research plan and inter-
oxidative stability. This effect was also seen by Maxminer (32), pretation of the data, writing (review & editing).
where a control beer (with no tannic acid added) had a lower Frank-Jürgen Methner - acquisition of funding, supervisor.
ESR area under the curve than two other beers (that did have
tannic acid additions) since the control beer was ca. 20 μg/L lower
in Fe than the two other samples. Other authors (16,77) have also Acknowledgements
reported the accelerative ability of iron to activate oxygen by elec- The authors are grateful for the assistance given by Markus
tron transfer with an excellent correlation (R2 = 0.99) between ESR Heisinger, for his help in the investigation by mashing numerous
area and Fe content. concoctions and gathering samples.
355
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Title
Complexation of transition metals by supplemental chelators during mashing
and their impact on beer stability
Authors
Tuur Mertensa#, Thomas Kunza, Philip C. Wietstocka, Frank-Jürgen Methnera
a
Technische Universität Berlin, Institute of Food Technology and Food Chemistry, Chair of
Brewing and Beverage Technology, Berlin, Germany
#
Corresponding author:
E-mail: tuur.mertens@tu-berlin.de
ORCID iD: https://orcid.org/0000-0002-3143-6099
Supplementary information
Figure S1: Levels of iron, copper, manganese and zinc (µg/L) over increasing concentrations of tannic
acid added (in mg/L: 0, 5, 10, 20, 60 and 120), with iron in black (■), copper in green (●), manganese
in red (▲) and zinc in blue (▼). A ‘chelation plateau’ for iron is reached at circa 60 mg/L of tannic
acid. Vertical lines indicate the low and high factor levels of the two-level full factorial screening
design for chelator concentration: 5.9 µM (10 mg/L tannic acid) and 35.3 µM (60 mg/L tannic acid),
respectively
Figure S2: Metal concentration decrease between the first wort and the two consecutive sparges
(first sparge: dense pattern; second sparge: sparse pattern), averaged over the pH 5.0 runs (left, n =
16) and pH 6.0 runs (right, n = 16) of tannic acid ± standard deviation
16) and pH 6.0 runs (right, n = 16) of gallic acid ± standard deviation
16) and pH 6.0 runs (right, n = 16) of EDTA ± standard deviation
16) and pH 6.0 runs (right, n = 16) of citric acid ± standard deviation
Table S1: Averaged metal concentrations for the first worts, the first and the second sparges, for
both mash pHs, for every chelator
First wort metal content (µg/L)

Chelator Iron Copper Manganese Zinc
Tannic acid 190.2 101.8 325.7 358.8 660.9 182.4 519.6 270.5
Gallic acid 346.9 143.2 341.4 367.0 629.6 177.1 414.4 258.3
EDTA 828.4 366.5 355.9 385.8 701.6 304.5 966.5 967.8
Citric acid 384.0 150.3 357.6 398.1 676.3 180.4 461.0 266.4
Phytic acid 387.0 151.6 359.9 397.6 708.6 186.0 451.1 259.2
First sparge metal content (µg/L)
Tannic acid 152.4 72.5 205.3 244.0 621.1 172.8 597.1 228.7
Gallic acid 256.8 104.7 213.4 244.5 606.7 167.9 521.8 220.7
EDTA 457.1 184.7 216.8 251.1 664.7 227.9 1011 782.4
Citric acid 258.0 100.6 194.1 237.1 626.5 165.0 549.5 217.2
Phytic acid 284.9 108.9 223.9 263.2 694.1 184.5 574.2 232.2
Second sparge metal content (µg/L)
Tannic acid 88.2 49.7 114.0 132.9 453.2 147.7 403.6 150.4
Gallic acid 143.9 70.4 114.0 129.0 423.1 140.2 342.7 142.3
EDTA 231.9 110.1 114.7 133.3 444.7 165.4 582.4 437.9
Citric acid 143.8 70.5 105.7 127.9 455.2 136.2 371.4 140.3
Phytic acid 173.2 81.6 127.5 149.0 552.3 167.0 445.0 165.8
Table S2: ANOVA and fit statistics for the selected factorial models (screening)
Iron
Transformation None Inverse square root Natural log Inverse square root Inverse square root
R² 0.99 1.00 1.00 0.99 0.99
Adjusted R² 0.99 1.00 1.00 0.98 0.99
Predicted R² 0.99 1.00 0.99 0.98 0.99
Adequate precision 56.2 70.0 67.8 52.0 62.9
Model F-value 403 1086 554 460 821
Lack of fit F-value 0.85 1.10 NA 1.77 0.22
Copper
Transformation None None None None None
R² 0.90 0.64 0.85 0.99 0.81
Adjusted R² 0.87 0.55 0.80 0.98 0.71
Predicted R² 0.83 0.41 0.71 0.98 0.52
Model F-value 35.7 7.3 16.3 331 7.9
Lack of fit F-value 0.87 0.63 0.63 0.32 0.63
Manganese
Transformation Natural log Natural log Inverse Inverse Inverse
R² 1.00 1.00 1.00 1.00 1.00
Adjusted R² 1.00 1.00 1.00 1.00 1.00
Predicted R² 1.00 1.00 0.99 1.00 1.00
Model F-value 1365 3603 813 1987 2212
Lack of fit F-value 0.58 1.08 NA 0.85 0.74
Zinc
Transformation Inverse square root Inverse square root Square root Power Inverse square root
R² 0.99 0.99 1.00 0.98 0.95
Adjusted R² 0.99 0.99 0.99 0.98 0.95
Predicted R² 0.98 0.98 0.99 0.97 0.94
Model F-value 260 294 607 196 189
Lack of fit F-value 0.47 0.97 1.10 0.68 0.88
Figure S6: Levels of manganese, zinc, iron and copper (µg/L) in lautered wort at increasing mash pH,
with mashing conducted with 5.9 µM gallic acid added at onset of mashing (0 min) and a mashout of
78°C
Figure S7: Chemical structure and possible chelation mechanism of epigallocatechin gallate
Figure S8: Chemical structure and possible chelation mechanism of ellagic acid
IV
Effects of Mash Chelator Addition on Transition Metal

Content and Oxidative Stability of Brewer’s Wort
Tuur Mertens, Thomas Kunz, Gert De Rouck, Brian R. Gibson, Guido Aerts, Luc
De Cooman†
BrewingScience. 2023; 76:58-72
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BrewingScience Fachverlag Hans Carl GmbH. May / June 2023 (Vol. 76) 58
T. Mertens, T. Kunz, G. De Rouck, B. Gibson, G. Aerts and L. De Cooman (†)
Effects of Mash Chelator Addition on

Yearbook 2006
Transition Metal Content and Oxidative

Stability of Brewer’s Wort

Oxidative stability in brewing refers to the ability of wort and beer to resist degradation by free radicals
and reactive oxygen species. It is a critical quality aspect in beer production, as it affects both the shelf
life and overall excellence of the final product. The catalytic role of iron, copper and manganese in
radical-associated staling is widely acknowledged. In this context, the present study investigates the
effectiveness of polyphenolic chelators (tannic acid, pomegranate and green tea extract) in enhancing
oxidative wort stability by sequestering transition metals during mashing. The results, obtained from 12
comparable brews from two distinct pilot breweries, show that incorporating either pomegranate extract
or tannic acid during mashing effectively lowers the levels of transition metals, specifically iron, in the
brewhouse. Early mash addition of pomegranate extract (90 % ellagic acid) demonstrates the highest
efficacy, with an almost 90 % decrease in iron levels and a nearly 80 % reduction in radical concentration,
as measured in the final wort by ICP-OES and ESR spectroscopy, respectively. While the investigated
chelators do not facilitate the removal of copper or manganese, their levels naturally decline during the
brewing process. Chelator addition yields an average reduction of 40 – 60 % in total post-boil aldehydes.
Furthermore, strong correlations are identified between iron levels, polyphenols and wort aldehydes after
boiling, whereas only weak to moderate correlations with copper and manganese. Aldehydes levels, however,
are greatly influenced by thermal stress throughout the brewing process. The findings suggest that natural
chelators have the potential to enhance beer flavour stability by diminishing radical formation during brewing
and lowering the amount of transition metals and aldehydes in the final product. However, further research is
needed to fully understand the implications of these findings on beer stability, given the intricacy of staling.
Descriptors: oxidative stability; transition metals; chelators; pilot scale brewing; radical generation; staling aldehydes
1 Introduction To maintain the flavour of a beer over time – or, in other words,
increase its flavour stability – it is crucial to prevent the beer from
Beer is the oldest alcoholic beverage that is universally consumed. (oxidative) ageing. Common industry practices include the control
In recent times, however, the beer industry has become increas- of dissolved oxygen levels (throughout brewing and particularly
ingly competitive and, for a business to attain or keep a leading packaging), the limit of heat load and the use of healthy yeast with
position in the current market, delivering a product that exhibits high oxygen scavenging activity, antioxidants that quench free
both high quality and an extended shelf life is essential. Consumers radicals and chelators that remove oxidation catalysing transition
expect beer to taste similar to prior batches they have enjoyed and metal ions (iron, copper, manganese) [1].
consistent throughout storage. Accomplishing that often proves
challenging. Despite being primarily made from only four ingre- The deleterious effects of metals – particularly iron – on wort and
dients (water, malt, hop, yeast), beer is incredibly complex; and beer oxidative stability are well documented [1–11] and are also
the mechanisms involved in beer staling present an even greater seen in other beverages, such as wine [12–14], coffee [15] and
level of complexity. spirits [16]. Through the activation of molecular oxygen by reduced
transition metal ions, reactive oxygen species (ROS) can be gener-
ated, such as superoxide anions (O2●-), perhydroxyl radicals (HO2●),
https://doi.org/10.23763/BrSc23-06mertens
hydrogen peroxide (H2O2) and hydroxyl radicals (OH●). These highly
reactive entities play a crucial role in the formation of many stale
Authors flavour characteristics by oxidising various components present
in wort and beer (fatty acids, hop compounds, amino acids, thiols,
Tuur Mertens, Thomas Kunz, Brian Gibson, Technische Universität Berlin,
Institute of Food Technology and Food Chemistry, Chair of Brewing and polyphenols, etc.).
Beverage Technology, Berlin, Germany; Gert De Rouck, Guido Aerts, Luc
De Cooman (†), Katholieke Universiteit Leuven, Faculty of Engineering In addition to reducing the number of metal ions potentially ending
Technology, Department of Microbial and Molecular Systems, Laboratory
of Enzyme, Fermentation and Brewing Technology, Ghent, Belgium; cor- up in the beer, effectively removing Fe, Cu and Mn during brew-
responding author: mertens.tuur@gmail.com ing should minimise the formation of ROS and mitigate oxidative
59 May / June 2023 (Vol. 76) Fachverlag Hans Carl GmbH. BrewingScience
damage throughout the process. By decreasing radical forma- of the effects of external chelators in brewing and the influence
tion – especially during the high-temperature stages of mashing of transition metal concentrations on wort aldehyde content. The
and boiling – de novo formation of wort aldehydes is anticipated authors hope that this work may serve as a cornerstone for future
to be partially impeded since Strecker aldehydes can be directly research on the topic of metal chelation in brewing –Yearbook
an area in
2006
generated through radical attack of their parent amino acid [17, the field of beer flavour stability still underexplored – and to put
18]. A reduction in bound-state aldehydes would also be expected, another tool in the brewer’s arsenal to combat oxidative ageing.
consequently [19].
The focus of this work is to achieve wort transition metal removal 2 Materials and methods
through the employment of polyphenolic chelators (tannic acid,
pomegranate and green tea extract) during mashing. By seques- 2.1 Chemicals and consumables
tering transition metals early, we aim to positively impact wort
oxidative stability, and thus, beer flavour stability. This assertion Calcium chloride dihydrate (≥ 99.0 %), glacial acetic acid (≥ 99.0
is substantiated by studies demonstrating a positive relationship %), ethanol absolute (≥ 99.2 %), 2-thiobarbituric acid (≥ 99.0 %),
between heightened oxidative stability in wort and improved disodium hydrogen phosphate dihydrate (≥ 99.5 %), potassium
quality, as well as extended shelf life, of the resultant beers under dihydrogen phosphate (≥ 99.5 %), D-fructose (≥ 99.0 %), ninhydrin
comparable conditions [20–32]. (≥ 99.0 %), potassium iodate (≥ 99.7 %), glycine (≥ 99.7 %), carboxy-
methylcellulose (CMC; low viscosity), ethylenedinitrilotetraacetic
Some researchers, however, have expressed scepticism regard- acid disodium salt dihydrate (EDTA, Titriplex® III; ≥ 99.0 %), n-butanol
ing this claim. Arguments on the insignificance of wort oxidation (≥ 99.9), isooctane (≥ 99.8 %), iron(II) sulphate heptahydrate (≥ 99.0
include the inherent capacity of the brewing process to strongly %), ammonium iron(III) citrate (≥ 16.5 % Fe), ammonia solution (≥
negate the content of transition metals (without the help of pro- 25.0 %), bovine serum albumin (BSA, albumin fraction V; ≥ 98.0 %),
cessing aids) during wort separation [33, 34] and fermentation [2, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL; ≥ 97.0
35, 36], the oxygen ingress during brewing being low (especially %), O-(2,3,4,5,6-pentafluorobenzyl)-hydroxylamine-hydrochloride
for large vessels) [18, 21, 37], the aldehydes evaporating during (PFBHA; ≥ 99.0 %), trans-2-nonenal (≥ 97.0 %), phenylacetaldehyde
wort boiling [4, 38] and the ability of yeast to scavenge any of the (≥ 90.0 %), furfural (≥ 99.0 %), hexanal (≥ 98.0 %), 3-methylbuta-
undesirable compounds that may reside after boiling [38–40]. It nal (≥ 97.0 %), 2-methylbutanal (≥ 95.0 %), 2-methylpropanal (≥
is important to note though that a wort with high oxidative stabil- 99.0 %) and deuterated benzaldehyde (benzaldehyde-d6; 98 atom
ity does not inherently ensure good flavour stability, as a beer’s %D) were purchased from Merck KGaA (Darmstadt, Germany).
shelf life is ultimately determined by its weakest component. In Deuterated 2-methylbutanal (2-methylbutanal-d10; 100 atom %D)
the absence of adequate control over downstream factors, such was synthesised upon ordering from MercaChem (Nijmegen, the
as total package oxygen, optimisation of upstream processes will Netherlands). MN-polyamide CC 6 was from Macherey-Nagel™
have little impact. GmbH & Co. KG (Düren, Germany). Bradford’s protein assay dye
reagent concentrate (0.01 % Coomassie® Brilliant blue G-250) was
This study seeks to address a specific facet within the complex made by Bio-Rad Laboratories GmbH (Munich, Germany). Concen-
mechanism of staling by investigating the impact of chelator ad- trated nitric acid (≥ 65.0 %) was obtained from Th. Geyer GmbH &
dition (at different mashing stages) on metal ion concentration, Co. KG (ChemSolute®, Renningen, Germany). Hydrochloric acid (≥
prooxidative radical generation and aldehyde levels in wort. The 32 %) and sodium hydroxide (≥ 98.5 %) were acquired from VWR
results of which augment the current, albeit limited, understanding International (Radnor, USA). Glycerine (≥ 98.0 %) and hydrogen
Fig. 1 Process outline of the 5-hL pilot brewery of KU Leuven, Technology Campus Ghent
peroxide (ROTIPURAN®; ≥ 30 %) were procured from Carl Roth collected in metal-free tubes (VWR International) at the onset of
GmbH (Karlsruhe, Germany). Methional (≥ 95.0 %) was attained mashing, end of mashing, mash filtration, onset of boiling, end
from Acros Organics (New Jersey, USA). N-tert-Butyl-α-(4-pyridyl-1- of boiling and end of cooling (before wort aeration) and stored at
oxide)-nitrone 2006 ≥ 98.0 %) was bought from TCI Deutschland
(POBN;
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– 20 °C until analysis.
GmbH (Eschborn, Germany). Reference standards (1000 µg/mL)
for iron, copper, manganese and zinc were acquired from Perkin 2.2.2 Pilot scale production at the Technische Universität
Elmer LAS GmbH (Rodgau, Germany). Argon (Alphagaz™ 1; ≥ Berlin
99.999 %), nitrogen gas (≥ 99,999 %), helium (Alphagaz™ 2; ≥
99.9999 %) and methane (≥ 99,995 %) were from Air Liquide GmbH Likewise, four batches of wort were produced at the pilot brewery
(Düsseldorf, Germany). Tannic acid (BrewTan B®; ≥ 98.0 %) was of TU Berlin, Department of Brewing and Beverage Technology. All
supplied by S.A. Ajinomoto OmniChem N.V. (Wetteren, Belgium), batches were brewed on a 2-hL scale by mixing 24 kg of kilned malt
pomegranate extract (ellagic acid, ≥ 90.0 %) by PureBulk Inc. (50:50 Pilsner:Munich) with 80 L of mash water (malt/liquor-ratio of
(Oregon, United States) and green tea extract (epigallocatechin 1:3.3 w/w) after milling with a two-roll mill (grinding gap, 1.2 mm;
gallate, ≥ 50.0 %) by Fairvital B.V. (Landgraaf, The Netherlands). All Künzel Maschinenbau GmbH, Mainleus, Germany). Mashing-in
aqueous solutions were made with ultrapure water, either through occurred at 62 °C for 5 min, followed by a temperature rest at 66
Milli-Q® purification (Merck Millipore, Darmstadt, Germany) or via °C for 30 min, one at 72 °C for 20 min (ΔTrise = 0.5 °C/min) and
Sartopore® 2 MidiCap 0.2 µm filtration (Sartorius AG, Goettingen, mashing-off at 78 °C for 10 min. The mashing liquor was pre-heated
Germany). For the KU Leuven brewing trials, pilsner malt was reverse osmosis water, enriched with 68 mg/L of Ca2+ ions, but
sourced from Holland Malt (Lieshout, The Netherlands) and Munich without acid addition (unadjusted mash pH of 5.4 at the onset of
(15 MD) malt from Mouterij Dingemans (Stabroek, Belgium). Hop boiling, measured at 20 °C). For the non-reference brews, chelator
extract (IsoHop®; 20 % iso-α-acids) was provided by Barth-Haas addition occurred solely at the onset of mashing (mashing-in), as
(Paddock Wood, England). For the TU Berlin brewing trials, both summarised in table 1. Mash filtration was performed by lauter tun.
pilsner and Munich (type II) malt were sourced from Weyermann The spent grain was sparged with 40 L of water (first running, 16.5
Malting Company (Bamberg, Germany). Hop extract (30 % iso-α- °P; collected wort, 11.3 °P). The sweet wort was adjusted to 12.0
acids) was provided by Hopsteiner (Mainburg, Germany). °P during boiling and isomerised hop extract was added, aiming
for a final bitterness of 30 mg/L iso-α-acids (presumed yield, 85
2.2 Wort samples %). After the 60 min boil, the hot wort was pumped into an open
whirlpool (filling, 10 min; rest, 20 min; emptying, 10 min), where
2.2.1 Pilot scale production at the Katholieke Universiteit trub was removed. The clarified wort was subsequently cooled
Leuven to 12 °C. Wort samples were collected in metal-free tubes at the
onset of mashing, end of mashing, onset of boiling, end of boiling
Eight batches of wort were produced at the pilot brewery of KU and end of cooling (before wort aeration) and stored at – 18 °C
Leuven, Technology Campus Ghent (Fig. 1), under analogous, until analysis.
atmospheric conditions and with identical materials and equipment.
All batches were brewed on a 5-hL scale by mixing 88 kg of kilned 2.3 Standard wort analyses
malt (50:50 Pilsner:Munich) with 1.94 hL of mash water at a rate
of 2.2 L/kg (malt/liquor ratio of 1:2.2 w/w) during fine milling with Density, extract and pH were evaluated using a DMA 5000 Alcolyzer
a wet disc mill (disc gap, #19; Hydromill®, Meura SA, Péruwelz, beer analysing system (Anton Paar GmbH, Graz, Austria) on clear,
Belgium). Mashing-in occurred at 64 °C for 30 min, followed by undiluted samples, according to MEBAK (method 2.9.6.3 and 2.13,
an intermediate temperature rest at 72 °C for 15 min (ΔTrise
= 1.5 °C/min) and mashing-off at 78 °C for 1 min.
Table 1 Chelator mash addition protocol
The mashing liquor was pre-heated reverse osmosis water,
Addition Concentration
enriched with 81 mg/L of Ca2+ ions, but without acid addition Brew Chelator
time (mgchel./kgmalt)
(unadjusted mash pH of 5.6 at the onset of boiling, measured Ref1 ( ▲ )
at 20 °C). For the non-reference brews, chelator addition – – 0
Ref2 ( ■ )
occurred either at the onset of mashing (mashing-in) or at
the end of mashing (mashing-off), as summarised in table 1. PGₑ (▲) Pomegranate extract
Onset of
Mash filtration was performed with a membrane-assisted KU GTₑ (▲) Green tea extract mashing
thin-bed filter (Meura 2001, Meura SA). The resulting filter Leuven (“early”)
TAₑ (▲) Tannic acid
cake was sparged with 2 hL of water (sparging rate, 2.3 L/kg; 0.17
PGι (■) Pomegranate extract
first running, 25 °P; last running, 3 °P; collected wort, 16-17 End of
GTι (■) Green tea extract mashing
°P). The sweet wort was adjusted to 13.4 °P during boiling. (“late”)
Additionally, isomerised hop extract was added, aiming for TAι (■) Tannic acid
a final bitterness of 29 mg/L iso-α-acids (presumed yield, 50 Refₔ (◆) – – 0
%), and 10 µL/L of zinc. After the 60 min boil, the hot wort was PGₔ (◆) Pomegranate extract
TU Onset of
pumped into an open whirlpool (filling, 6 min; rest, 20 min; Berlin GTₔ (◆) Green tea extract mashing 0.20
emptying, 20 min), where trub was removed. The clarified (“early”)
TAₔ (◆) Tannic acid
wort was subsequently cooled to 20 °C. Wort samples were

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Fig. 2 Original extracts of the KUL and TUB worts. Reference brews (no chelator addition) are in black with hollow symbols ( , , ),
pomegranate extract brews are in red (early addition, ▲ & ◆; late addition, ■), green tea extract brews are in green (early addition,
▲ & ◆; late addition, ■) and tannic acid brews are in blue (early addition, ▲ & ◆; late addition, ■)
respectively) [41]. Thiobarbituric acid index (TBI) and soluble protein propanal) were quantified by capillary gas chromatography-mass
content were determined using ultraviolet-visible spectrophotometry spectrometry (CGC-MS), based on the method of Baert et al. [43,
(Varian Cary 100 UV-Vis spectrophotometer, Agilent Technologies, 44]. The setup employs headspace-solid phase microextraction
California, USA), according to MEBAK (method 2.4) [41] and the (HS-SPME), using a 65 µm polydimethylsiloxane/divinylbenzene
Bradford protein assay [42]. Anthocyanogens and bitterness were (PDMS/DVB) fibre (StableFlex; Supelco, Bellefonte, USA) and
quantified by continuous flow analysis (continuous flow analyser, on-fibre PFBHA derivatisation, coupled with a Thermo Scien-
type San+; Skalar Analytical B.V., Breda, The Netherlands), ac- tific™ FOCUS GC gas chromatograph (Thermo Fisher Scientific
cording to MEBAK (method 2.16.2 and 2.17.1, respectively) [41]. Inc., Waltham, USA) and a Thermo Scientific™ ISQ™ EC Single
Free amino nitrogen (FAN) and total polyphenols were analysed Quadrupole mass spectrometer. Standards for external calibration
according to MEBAK (method 2.6.4.1 and 2.16.1, respectively) were prepared from stock solutions of eight ethanol-dissolved
[41], using UV-Vis spectrophotometry for the KU Leuven samples aldehyde markers.
and continuous flow analysis for the TU Berlin samples.
2.4 Determination of the metal content
Quantification of metal ions (iron, copper, manganese and zinc) in

wort was performed by inductively coupled plasma-optical emis-
sion spectroscopy (ICP-OES), for which an Avio 200 spectrometer
(PerkinElmer, Rodgau, Germany) was employed, in accordance
with a preceding study [6]. Preliminary sample preparation consisted
of centrifugation of the wort.
2.5 Determination of radical levels
Radical formation was measured in the wort samples under force-

ageing conditions (60 °C) through electron spin resonance (ESR)
spectroscopy by using an X-band spectrometer (e-scan, Bruker
BioSpin GmbH, Rheinstetten, Germany), in accordance with a
preceding study [6] and MEBAK (method 2.15.3) [41]. Final radical
concentrations were reported as the radical intensities at minute
450 (T450-value).
Fig. 3 Mash filtration flow rates of the KUL worts. Reference brews
2.6 Determination of (free) aldehydes (no chelator addition) are in black with hollow symbols ( ,
), pomegranate extract brews are in red (early addition,
▲; late addition, ■), green tea extract brews are in green
Aldehydes (methional, trans-2-nonenal, phenylacetaldehyde, (early addition, ▲; late addition, ■) and tannic acid brews
furfural, hexanal, 3-methylbutanal, 2-methylbutanal and 2-methyl- are in blue (early addition, ▲; late addition, ■)
2.7 Data analysis
Mean values and standard deviations (SDs; graphically represented

through error bars,
Yearbook where applicable) are determined via two tech-
2006
nical replicates unless noted otherwise. Scientific graphing and

data analysis were conducted using OriginPro (version 9.6.5.169,

OriginLab Corporation, Northampton, MA, USA) and Microsoft Excel
for Office 365 (version 16.0.12527.22286, Redmond, WA, USA).
3 Results and discussion
3.1 Chelator effects on brewing-relevant parameters
The eight batches of wort produced at the KU Leuven pilot brew-

ery, henceforth referred to as the ‘KUL worts’, were very similar
in terms of extract (SDs of 0.1 °P for every mashing stage). It
verifies suitable batch reproducibility and the inconsequentiality
of chelator addition on starch conversion. The same applied to
the four batches of wort produced at the TU Berlin pilot brewery, Fig. 4 Soluble protein contents of the clarified KUL worts. Cal-
culated from 2 x 2 biological/technical replicates
henceforth referred to as the ‘TUB worts’.
The graphed data are shown in figure 2 and are also summarised viations were found for the KUL wort mash filtration times (Fig. 3).
in table 5 (see page 68). The latter serves as a comprehensive It is not unthinkable that an effect would have been observed if a
compilation of all the analyses and results of the study, allowing more oxidised wort or a lauter tun was used, since tannic acid – at
a comparison of averages between both breweries and providing the concentrations employed – has been shown to positively influ-
insight into parameter fluctuations during brewing. In addition, the ence lautering performance when added to the brewing liquor [45,
inclusion of standard deviations alongside the means provides 46]. It does this i.a. by preventing top-dough or ‘teig’ formation (a
information on the impact of chelator additions on each variable. layer of oxidised cross-linked proteins that hinders wort separation)
A large standard deviation indicates a noticeable effect, whereas through coagulation and flocculation of thiol-containing proteins [47].
a small SD suggests a negligible impact.
The ability to precipitate sensitive proteins makes tannic acid a
Extract yields are important, as considerable variations in the valuable physicochemical stabilisation agent during wort boiling
extract can lead to different concentrations of staling-related wort [48]. Given that pomegranate and green tea extract also contain
compounds that are extracted from the malt (such as polyphenols, polyphenols with protein-binding capabilities [49–51], it is reason-
transition metals, aldehydes, etc.). Similarly, no consequential de- able to assume that these chelators may have similar potential for
Fig. 5 pH of the KUL and TUB worts. Reference brews (no chelator addition) are in black with hollow symbols ( , , ), pomegranate
extract brews are in red (early addition, ▲ & ◆; late addition, ■), green tea extract brews are in green (early addition, ▲ & ◆; late
addition, ■) and tannic acid brews are in blue (early addition, ▲ & ◆; late addition, ■)

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Fig. 6 FAN contents of the KUL and TUB worts, measured at the onset of boiling (light grey) and end of cooling (grey)
beer stabilisation. However, as yet, no studies have explored their during boiling by i.a. addition of hop bitter acids and production of
effectiveness in this regard. Although this study found no measur- Maillard reaction products (MRPs) [53].
able differences in soluble protein content among the clarified KUL
worts (Fig. 4), pronounced differences in protein concentration Negligible inter-batch pH variations were detected for both brewer-
would likely have been observed if the chelators were added to ies, with SDs ∈ [0.01−0.05] and ΔpHmaxima ∈ [0.03−0.14], depending
the boiling kettle, due to the lower protein content of filtered wort. on the mashing stage. The larger inter-brewery variation (ΔpHmeans
∈ [0.16−0.29], depending on the mashing stage) seen is likely
At the concentrations employed (75 and 60 mgextract/Lmash liquor for due to the utilisation of more strongly roasted Munich malt in the
the KUL and TUB worts, respectively), the chelators did not affect TU Berlin trials (25 EBC, as opposed to the KU Leuven’s 15 EBC
mash pH. The acidities of the KUL and TUB worts (measured at Munich malt), leading to more acidic melanoidins being released
20 °C) did, however, show natural pH fluctuations throughout the in the wort.
brewing process. As seen in figure 5, wort pH gradually declines
during mashing mainly due to the liberation of (organic and inor- Only minor deviations were seen in the free amino nitrogen contents
ganic) malt phosphates [52]. Further pH decrease takes place of the KUL and TUB worts (Fig. 6), with both breweries achieving
Fig. 7 TBI values of the KUL and TUB worts, measured at mash filtration (light grey), the onset of boiling (grey) and end of boiling (dark
grey). All values were normalised to 12 % w/w extract
FAN levels of ca. 180 ± 13 mg/L after clarification. This is relevant to originate prior to wort boiling. One potential explanation is the
because significant differences in amino acid content can lead to possible presence of Maillard intermediates in the pomegranate
a high disparity in aldehydes, due to amino acids being important and green tea extracts, carrying over into the filtered/boiled wort
ageing precursors [54–56].
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when added late to the mash but not with early mash addition, due
to cross-linking and co-precipitation of the MRPs with wort proteins
The levels of total polyphenols in the clarified KUL and TUB worts during mashing [59, 60]. The elevated TBI values observed in the
were all very similar (Table 5), with an average of 241 ± 15 mg/L TUB worts, e.g. when comparing the 'end of boiling' data points from
for both breweries. As with the FAN content, the significance is both breweries, can be attributed to the use of the more intensely
that polyphenols can also influence staling by i.a. increasing the roasted Munich malt, as previously mentioned.
reducing activity and decreasing the formation of carbonyls [57].
The utilisation of isomerised (CO2) hop extract in this study was The worts with higher TBI values, PGι and GTι, were visibly darker
motivated by the aim to exclude the potential influence of hop- than the others. This observation is in line with the evident correlation
derived polyphenols as a confounding factor. that exists between the thermal loads of malt, wort and beer and
the corresponding increase in their colour, due to greater produc-
These results suggest that the additions of the polyphenolic extracts tion of coloured Maillard reaction products [61–63]. It is important
(pomegranate, green tea, tannic acid) during mashing did not lead to note that similar associations have been established between
to a measurable increase in the polyphenol content of the clarified these factors and prooxidative radical generation [8, 62, 64–66].
worts. The excess polyphenols seem to remain in the brewhouse This is attributed to specific MRPs, such as reductone intermedi-
(during mash filtration and clarification, adsorbed to wort proteins ates, which can rapidly reduce oxidised metal ions, promoting e.g.
and trub) and, thus, do not carry over into fermentation. To substanti- catalytic iron redox cycling (Fe3+  Fe2+) [67, 68].
ate this claim, however, a more elaborate examination is needed.
Tannic acid, for example, has been known to impart (di)gallic acid 3.2 Chelator effects on metal ion, free radical and
residues to finished beer when added in the brewhouse [46, 58]. aldehyde content in wort
A significant discrepancy was observed in the thiobarbituric acid Transition metal (Fe, Cu, Mn, Zn) concentrations naturally fluctuate
index – a value used for gauging cumulative thermal stress in malt, throughout the brewing process (Table 5). Copper concentrations
wort and beer, brought about by heat exposure during kilning, decrease every subsequent step, whereas manganese is less
mashing, boiling, etc. – for two of the KUL brews (Fig. 7). Both readily retained in the brewhouse but ultimately declines during
brews PGι and GTι (late additions of pomegranate and green tea whirlpooling. Overall, metal levels decline during mashing, wort
extract, respectively) had higher TBIs compared to the other KUL boiling and/or at the whirlpool step as a result of metal retention
brews, before and after boiling. Worts and beers with higher TBIs with spent grain, hot break and/or trub [1, 8, 35].
generally have heightened staling-related benchmarks, such as
increased aldehyde levels, colour and radical concentrations, as In the present work, however, the primary objective was to coer-
will be discussed in greater detail later on. cively reduce wort transition metal levels further. This was suc-
cessfully achieved for iron in both pilot breweries by the addition
As stated, the elevated heat stress in brews PGι and GTι appeared of the external chelators, as shown in figure 8, which replicates
Fig. 8 Iron concentrations of the KUL and TUB worts during mashing, measured at the onset of boiling (light grey), end of boiling (grey)
and end of cooling (dark grey). All values were calculated from four technical replicates, with an average relative standard devia-
tion below 2 %, and normalised to 12 % w/w extract
Table 2 Transition metal concentrations of the clarified KUL and TUB worts. All values were calculated from four technical replicates,
with an average relative standard deviation below 2 %, and not subjected to normalisation for w/w extract
[Fe] [Cu] [Mn] Sum

Brew Chelator Addition time
(µg/L) (µg/L) (µg/L)
(µg/L) 2006
Yearbook
Ref1 ( ▲ ) 339.3 91.3 95.9 526.4

– –
Ref2 ( ■ ) 298.1 76.9 95.1 470.0
PGₑ (▲) Pomegranate extract 47.4 89.1 100.1 236.5
Onset of
KU GTₑ (▲) Green tea extract mashing 274.4 91.7 98.1 464.1
Leuven (“early”)
TAₑ (▲) Tannic acid 204.5 91.0 114.6 410.1
PGι (■) Pomegranate extract 76.0 89.7 88.4 254.0
End of
GTι (■) Green tea extract mashing 312.6 90.7 94.0 497.3
(“late”)
TAι (■) Tannic acid 200.0 94.0 96.5 390.5
Refₔ (◆) – – 180.1 39.1 68.7 287.9
TU PGₔ (◆) Pomegranate extract 21.1 26.0 71.2 118.3

Onset of
Berlin GTₔ (◆) Green tea extract mashing 133.3 24.4 68.5 226.2
(“early”)
TAₔ (◆) Tannic acid 121.5 21.3 73.6 216.4
the findings of a previous lab-scale study [6]. Similarly, ellagic acid separated from their binding partners [75–78]. This behaviour is
and tannic acid – despite their ability to chelate copper in wort-pH confirmed by studies that have shown minimal losses of Mn during
buffer solutions [69, 70] – appeared unable to influence the final the brewing process [2, 7, 9].
wort concentrations of copper and manganese. Their low standard
deviations, observed in table 5, indicate little variation among the A slight discrepancy with the preceding lab-scale study [6] was
brews and provide further confirmation of the chelators' inefficacy the better performance of the early (pomegranate) extract addition
in removing Cu and Mn ions. towards iron removal at pilot scale. This effect explains the lower
ESR signal intensities observed after the 450-minute assay (repre-
Copper ions likely have stronger binding affinities for amino acids sented by T450-values) with the early additions in this study (Fig. 9).
and proteins, which are abundant in wort, than for endo- or exo-
genous polyphenols. According to Jacobsen et al., certain amino The KUL reference brews exhibited an average, unnormalised iron
acids can form complexes with copper at wort pH that may com- concentration of 320 ± 30 µg/L in the clarified worts, which was
pete with potent chelating agents such as EDTA and EGTA [71]. higher than any of the 'chelator brews'. In terms of iron removal,
This assertion is supported by brewing studies that have found compared to the reference brews, both the early and late additions
heightened concentrations of copper bound to proteinaceous mat- of pomegranate extract were the most effective (– 85 % and – 76 %,
ter [2, 72–74]. In contrast, divalent manganese ions tend to form respectively), followed by tannic acid (– 36 % and – 38 %) and
unstable complexes with organic ligands and are therefore easily green tea extract (– 14 % and – 2 %). These results were reaffirmed
Fig. 9 ESR results of the clarified KUL and TUB worts. Reference brews (no chelator addition) are in black with hollow symbols ( , ,
), pomegranate extract brews are in red (early addition, ▲ & ◆; late addition, ■), green tea extract brews are in green (early ad-
dition, ▲ & ◆; late addition, ■) and tannic acid brews are in blue (early addition, ▲ & ◆; late addition, ■)
Table 3 Concentrations of eight distinct aldehydes, as measured in the respective KUL worts. (lautering vs. mash filtration) – although
All values were normalised to 12 % w/w extract, are means ± standard deviation of these factors are often interrelated.
eight brews and include both reference and chelator brews

Yearbook 2006 Aldehyde content (µg/L) Due to the variations in the total transi-
tion metal content among the different
Change over boiling
Mash filtration End of boiling brews, different radical formation rates
(%)
Furfural 73.1 ± 6.1 92.9 ± 7.5 +28 ± 10 are anticipated in the worts. Figure 9
depicts the results of the electron spin
2-Methylpropanal 567.6 ± 137.4 109.0 ± 102.4 -80 ± 20
resonance (ESR) assay for the clari-
2-Methylbutanal 440.8 ± 85.2 66.4 ± 66.5 -84 ± 18
fied worts. Compared to the reference
3-Methylbutanal 683.2 ± 158.2 108.7 ± 109.3 -83 ± 19 brews, the early additions of pome-
Methional 397.1 ± 53.2 207.5 ± 22.8 -47 ± 5 granate extract resulted in a ca. 80 %
Phenylacetaldehyde 87.8 ± 11.5 78.4 ± 17.1 -11 ± 10 reduction in T450-values, indicating a
significant decline in radical generation.
Hexanal 37.7 ± 4.6 3.0 ± 3.9 -91 ± 12
Incorporations of tannic acid also led to
Trans-2-nonenal 0.1 ± 0.0 0.0 ± 0.0 -79 ± 20
reduced T450-values, seemingly inde-
pendent of addition time. The radical
in the TUB trials (– 88 %; – 33 %; – 26 %), meaning that the find- reduction efficacies of green tea extract were observed to be mini-
ings are brewery independent and irrespective of mash filtration or mal, as they also did not demonstrate very effective iron removal.
lautering. It should be noted that the TUB worts had smaller iron
contents at mashing-in (Table 5), resulting in their lower final iron While the low metal content of pomegranate brews certainly con-
concentrations. Comparatively, however, larger quantities of iron tributes to their low T450-values, it is plausible that an additional
were removed from the KUL brews. antioxidative effect is at play. Notably, the additions of tannic acid
and green tea in the TUB trials show a lower amount of total tran-
Concentrations of iron, copper and manganese in the clarified sition metals (216 and 226 µg/l, respectively), compared to the
worts can be found in table 2. Apart from the declarations already pomegranate additions in the KUL trials (237 and 254 µg/L, early
made, it is noticeable that the metal levels were lower in the TU and late); yet, the latter two worts have lower T450-values.
Berlin trials, even though a more roasted Munich malt was used,
which typically bestows excess iron and manganese to the wort Ellagic acid, for example, is also likely to engage in free radical
[6, 33]. While there was, indeed, a slightly higher concentration scavenging [81, 82]. It is questionable, however, whether the low
of the chelators used, this would not account for the differences residual concentrations would account for the impact observed in
seen in the reference brews. The observed effect is likely caused figure 9. In reality, there is a strong correlation between the T450-
by differences in barley cultivar [79], malt roasting conditions values and the wort Fe content (r of 0.95; R² of 0.90). The coefficients
[80], as well as the use of high extract brewing with fine milling of correlation (r) and determination (R²) are significantly lower with
at KU Leuven, as opposed to e.g. the method of wort separation Cu (0.39; 0.15), Mn (0.33; 0.11) and even the sum of transition
Fig. 10 Aldehyde concentrations of the KUL worts, measured at mash filtration and the end of boiling. All values were normalised to
12 % w/w extract and calculated from three technical replicates. From bottom to top: 3-methylbutanal (red), 2-methylpropanal
(blue), 2-methylbutanal (yellow), methional (green), phenylacetaldehyde (purple), furfural (grey), hexanal (pink), trans-2-nonenal
(indistinguishable)
Table 4 Notable coefficients of correlation (r) and determination and weakens post-boiling. Furfural, however, does not exhibit this
(R²) between seven selected variables and total wort alde- behaviour and seems to be the more reliable predictor for post-boil
hyde concentrations at two brewing stages (pre- and
post-boil), with inclusion of brew PGₑ. Correlations ex- wort aldehydes in this study. Nevertheless, it should be emphasised
cluding the brew PGₑ (as an outlier in terms of post-boil that the thiobarbituric acid index has been proposed by De Schutter
Yearbook 2006
aldehydes) are provided and discussed in the text
et al. as the superior method for determining various types of heat

load on wort [94, 95]. This is because, in addition to the volatile

Mash filtration End of boiling
5-hydroxymethylfurfural, the TBI also accounts for non-volatile
r R² r R²
precursors of volatile ageing compounds (such as Amadori rear-
TBI 0.86 0.74 – 0.53 0.28 rangement products and α-dicarbonyl compounds). It is therefore
Furfural 0.82 0.67 0.76 0.57 less susceptible to the shortcomings of volatile heat load indicators
FAN* 0.64 0.41 – 0.57 0.32 such as furfural, where information about the applied thermal load
Iron 0.39 0.15 – 0.07 0.00
on wort is lost through evaporation.
Copper – 0.21 0.04 – 0.26 0.07

As illustrated in figure 10, the brew (PGₑ) with the least amount of
Manganese 0.09 0.01 0.02 0.00 transition metals and the lowest T450-value remarkably demonstrated
Total polyphenols* 0.00 0.00 – 0.48 0.23 the highest aldehyde levels after boiling, exceeding even the boiled
*Clarified wort data reference worts. This unexpected outcome can be solely attributed
to an aberration during the boiling process, which impeded the
metals (0.88; 0.78). Accordingly, the three worts with the lowest evaporation of aldehydes. The elevated presence in brew PGₑ of
amount of iron (Table 2, bolded) display the lowest T450-values. aldehydes in the higher volatility range (2-methylpropanal, 2- and
3-methylbutanal) bolsters this hypothesis. It is plausible that the
It is reasonable to anticipate distinctive behaviour in beer, consid- brewing process for PGₑ involved a relatively subdued boiling
ering how radical formation, and hence ESR values, are highly state (simmering, rather than a rolling boil), leading to inefficient
dependent on various matrix factors [1], including extract, pH, levels volatilisation [96, 97]. A comparison of the brewing log boiling times
of MRPs and reductones, presence of transition metals, complexing revealed that brew PGₑ spent a considerably shorter duration at
agents (e.g. hop acids), radical-quenchers (e.g. sulphite, ethanol), temperatures of ≥ 100 °C (22 min), in stark contrast to the other
and the mutual concentration ratios of all pertinent compounds. brews (58 ± 7 min).
Indeed, copper and manganese have been shown to affect beer
lag times (i.e. the duration required for antioxidants to be depleted Consequently, including outlier brew PGₑ in the calculations of
during ESR analysis) [5, 7]. the coefficients presented in 4 introduces potential inaccuracies.
The exclusion of brew PGₑ causes minimal changes in the ‘mash
Table 3 depicts the normalised aldehydes contents, before and after filtration’ coefficients (Δabs ≤ 0.1) but considerably alters the ‘end of
wort boiling. The data show that despite the added heat stress – and boiling’ coefficients for certain variables. Omitting the outlier reveals
consequent furfural increase – over the course of boiling, there is positive correlations between the levels of free aldehydes detected
a notable decrease in the overall aldehyde levels. This trend has in the boiled wort and the quantities of iron (r of 0.83; R² of 0.69)
been observed in several other studies [53, 83–86], and is attributed and manganese (0.45; 0.20), while a strong negative correlation
to volatilisation, particularly for the aldehydes with boiling points with total polyphenols (– 0.81; 0.66) emerges. The recalculated
below 100 °C (2-methylpropanal, 2- and 3-methylbutanal). It is coefficients align with the expected prooxidative influence of iron
apparent that the amount of aldehydes lost through evaporation and manganese as transition metal catalysts and the antioxida-
during boiling surpasses the amount that is generated through tive effects of polyphenols, as discussed previously. It is worth
release or de novo formation (e.g. via Strecker degradation of noting that a weak negative correlation between wort aldehydes
amino acids, the Maillard reaction or oxidative processes) [87]. and copper (– 0.32; 0.10) persists, which contradicts the prevail-
ing understanding but is in line with recent observations made by
The total aldehyde contents of each brew are visually represented other researchers [5, 33, 74].
in figure 10 through stacked column graphs for both brewing
stages. The highest aldehyde levels were observed at mash filtra- Each chelator addition led to a lower level of total aldehydes in the
tion; particularly in brews PGι and GTι, which correlate with the boiled worts when compared to the reference brews. On average,
elevated TBIs and colour intensities of these two worts (Fig. 7). there was a reduction of – 39 % for green tea extract, – 55 % for
The latter observation strengthens the existing understanding tannic acid and – 58 % for pomegranate extract (excluding brew
that aldehydes are closely linked to heat load, as recognised by PGₑ). Although these results are promising, further investigation
numerous researchers [8, 20, 88–93] and evident from table 4. is needed to determine the impact of the depleted iron levels dur-
In addition to demonstrating the high predictive capability of TBI ing the brewing process, and the subsequent lower T450-values
and furfural (both heat load indicators) for pre-boil total aldehyde and aldehyde levels of the final worts, on the final beer quality
levels, the coefficients in 4 also reveal a moderate association and flavour stability. A follow-up study will analyse the fresh and
with iron in this regard. force-aged beer samples resulting from these worts to address
these queries. The potential benefits of these specified conditions
It is worth noting that the correlation between TBI and furfural prior have been speculated and are supported by the widespread use
to wort boiling (r of 0.69) attenuates after boiling (r of – 0.09). Like- of tannic acid-based products (such as gallotannins) in the brew-
wise, the correlation between TBI and total aldehydes also inverts ing industry.
Table 5 Summary of results obtained from the analyses of worts produced at the KU Leuven and TU Berlin pilot breweries. The reported
values are means ± standard deviation of either eight (KUL) or four (TUB) brews and include both reference and chelator brews
KU Leuven worts (n = 8) TU Berlin worts (n = 4)

Yearbook 2006
Onset End Mash Onset End End Onset End Onset End End
Stage
mashing mashing filtration boiling boiling cooling mashing mashing boiling boiling cooling
Analysis
25.6 ± 12.9 ± 12.7 ± 12.7 ± 16.3 ± 11.6 ± 12.8 ± 12.1 ±
Extract (°P)
0.1 0.1 0.1 0.1 0.8 0.1 0.4 0.1
pH 5.6 ± 0.0 5.6 ± 0.0 5.5 ± 0.0 5.6 ± 0.0 5.5 ± 0.1 5.5 ± 0.0 5.4 ± 0.0 5.3 ± 0.0 5.2 ± 0.0
Flow rate of initial
3.1 ± 0.2
100 min (L/min)
59.9 ± 81.0 ± 97.0 ± 114.7 ±
TBI*
10.2 13.1 3.0 4.3
184.3 ± 168.5 ± 179.1 ±
FAN (mg/L)
16.3 6.2 4.6
507.8 ±
Soluble protein (mg/L)
14.8
Total polyphenols 236.6 ± 255.9 ±
(mg/L) 10.8 15.1
49.4 ±
Anthocyanogens (mg/L)
11.3
486.3 ± 290.4 ± 380.0 ± 193.5 ± 200.7 ± 202.9 ± 236.8 ± 180.3 ± 179.2 ± 126.2 ± 112.7 ±
Iron* (µg/L)
95.0 60.9 130.6 70.8 88.8 101.0 173.5 114.1 119.3 79.3 66.0
326.1 ± 288.2 ± 277.7 ± 190.3 ± 134.5 ± 82.7 ± 234.7 ± 191.6 ± 106.4 ± 28.8 ± 27.3 ±
Copper* (µg/L)
38.2 11.3 5.6 10.6 28.3 4.8 9.8 32.5 15.3 4.6 7.6
631.4 ± 242.4 ± 133.2 ± 145.7 ± 168.8 ± 90.6 ± 854.4 ± 376.8 ± 220.9 ± 135.0 ± 69.6 ±
Manganese* (µg/L)
85.2 10.4 9.1 6.5 25.0 6.7 117.2 36.7 10.3 34.3 2.7
1093.6 ± 430.3 ± 152.4 ± 183.7 ± 395.2 ± 251.5 ± 970.1 ± 504.3 ± 202.5 ± 93.2 ± 19.8 ±
Zinc* (µg/L)
162.7 38.8 17.8 10.3 103.3 58.0 143.9 50.3 11.8 30.0 3.2
T450-value (x 106) 6.7 ± 2.4 4.8 ± 2.6
23.7 ±
Bitterness (IBU)
1.0
*Data normalised to 12 % w/w extract
The application of gallotannins (either pre- and post-wort boil, dur- an approximate 90 % reduction in the iron content of the final wort
ing maturation or before filtering/centrifugation) has been shown and a nearly 80 % reduction in prooxidative radical generation.
to enhance beer stability [45, 47, 98, 99]. Nevertheless, recent None of the examined chelators significantly affect concentrations
brewing research, including the present study, has shown that el- of copper and manganese. However, the levels of these metals
lagic acid [6], and in particular punicalagin (a glycoside of ellagic naturally decline during brewing through wort separation (filtration
acid) [69, 100], outperform gallotannins in terms of iron chelation and clarification).
and overall antioxidative capabilities. Punicalagin, like tannic acid,
is a high molecular weight polyphenol with a large number (16) of On average, the chelator additions result in a 40 – 60 % reduc-
phenolic hydroxyl groups per molecule and has the potential to be tion in total post-boil aldehydes compared to the reference brews,
an effective stabilising and antioxidative agent. Further scientific depending on the chelator used. However, only the pomegranate
exploration, however, is required to determine its full potential in extract brews exhibit significantly higher oxidative wort stabilities,
brewing. as evidenced by the diminished radical generation in ESR spec-
troscopy analysis (indicated by low T450-values), which strongly
correlate with wort iron content. Our investigation also identifies
4 Conclusion strong correlations between iron levels, polyphenols and total wort
aldehydes after boiling, while only weak to moderate correlations are
Incorporation of chelators during mashing influences the transition seen with copper and manganese. These associations, however,
metal content of wort and does not adversely affect any of the per- are not evident before boiling. It must furthermore be noted that
tinent brewing parameters (extract, pH, filterability, soluble protein, aldehydes display a strong dependence on heat load throughout
free amino acid content, total polyphenols, heat load). Among the the brewing process.
polyphenolic substances investigated, pomegranate extract (90 %
ellagic acid) and tannic acid are particularly effective in lowering Wort oxidative stability is important, as it can affect the quality
iron levels in the wort throughout the brewing process. The addition and shelf life of the final beer product. In this regard, the use of
of pomegranate extract during the early stage of mashing leads to pomegranate extract in brewing demonstrates great potential,
particularly for high-iron beers such as stouts and other dark ales. Chemists, 74 (2016), no. 2, pp. 87-90.
However, staling is complex; and the relationship between wort 10. Ferreira, I.M.; Carvalho, D.O. and Guido, L.F.: Impact of storage
oxidative stability and beer flavour stability is not necessarily di- conditions on the oxidative stability of beer, European Food Research
rect. Further investigation is warranted to determine the impact of and Technology, 249 (2023), no. 1, pp. 149-156. Yearbook 2006
these findings on oxidative beer stability and beer flavour stability. 11. Irwin, A.J.; Barker, R.L. and Pipasts, P.: The Role of Copper, Oxygen,
Subsequent studies will evaluate the fresh and aged characteristics and Polyphenols in Beer Flavor Instability, Journal of the American
of the resulting beers. Society of Brewing Chemists, 49 (1991), no. 3, pp. 140-149.
12. Comuzzo, P. and Zironi, R.: Biotechnological Strategies for Control-
Acknowledgements ling Wine Oxidation, Food Engineering Reviews, 5 (2013), no. 4, pp.
217-229.
This article is dedicated to the loving memory of prof. Luc De 13. Kreitman, G.Y.; Danilewicz, J.C.; Jeffery, D.W. and Elias, R.J.: Re-
Cooman. The author would also like to express gratitude to col- action Mechanisms of Metals with Hydrogen Sulfide and Thiols in
leagues from the Laboratory of Enzyme, Fermentation and Brew- Model Wine. Part 2: Iron- and Copper-Catalyzed Oxidation, Journal
ing Technology (Katholieke Universiteit Leuven) and the Chair of of Agricultural and Food Chemistry, 64 (2016), no. 20, pp. 4105-4113.
Brewing and Beverage Technology (Technische Universität Berlin) 14. Danilewicz, J.C.: Review of Reaction Mechanisms of Oxygen and
for their valuable contributions to several of the experiments per- Proposed Intermediate Reduction Products in Wine: Central Role
formed in this study. of Iron and Copper, American Journal of Enology and Viticulture, 54
(2003), no. 2, pp. 73-85.
This project has received funding from the European Union’s Ho- 15. Yeretzian, C.; Pascual, E.C. and Goodman, B.A.: Probing Free
rizon 2020 research and innovation programme under the Marie Radical Processes during Storage of Extracts from Whole Roasted
Skłodowska-Curie grant agreement No. 722166. Coffee Beans: Impact of O2 Exposure during Extraction and Storage,
Journal of Agricultural and Food Chemistry, 61 (2013), no. 13, pp.
The author(s) would like to declare that a patent application was 3301-3305.
filed on the application of punicalagin/ellagic acid to improve the 16. Wang, L.; Chen, S. and Xu, Y.: Distilled beverage aging: A review on
oxidative and colloidal stability of beverages (esp. beer). aroma characteristics, maturation mechanisms, and artificial aging
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agents, Food Chemistry, 125 (2011), no. 3, pp. 850-859. Spectroscopy, 173 (2017), pp. 910-917.
66. Kunz, T.; Woest, H.; Lee, E.J.; Müller, C. and Methner, F.-J.: Improve- 83. Ditrych, M.; Filipowska, W.; De Rouck, G.; Jaskula-Goiris, B.; Aerts,
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68. Kunz, T.; Müller, C. and Methner, F.-J.: EAP Determination and Bever- International, 140 (2021), no. 4, p. 110049.
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ence – Monatsschrift für Brauwissenschaft, 65 (2012), no. 1/2, pp. of different lauter fractions, Monatsschrift für Brauwissenschaft, 59
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69. Kunz, T.; Mertens, T.; Hahne, K.; Kuhn, F.; Bergmann, T. and Gibson, 86. Kretschmer, H.; Miedaner, H.; Narziss, L. and Back, W.: Der Einfluss
B.: Punicalagin – a novel brewing stabilizing agent for beer shelf-life des Sauerstoffgehaltes beim Keimen, Schwelken und Darren auf die
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(Belgium), 2023. the European Brewery Convention, Brussels (Belgium), 1995, pp.
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Convention Congress, Nürnberg, Germany, 1979, pp. 117-129. development of Strecker aldehydes during beer aging, 31st Con-
74. Pagenstecher, M.; Bolat, I.; Bjerrum, M.J. and Andersen, M.L.: Cop- gress of the European Brewery Convention, Fachverlag Hans Carl
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Agricultural and Food Chemistry, 69 (2021), no. 23, pp. 6613-6622. 90. Jaskula-Goiris, B.; De Causmaecker, B.; De Rouck, G.; Aerts, G.;
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91. Gastl, M.; Spieleder, E.; Hermann, M.; Thiele, F.; Burberg, F.; Kogin, 96. Hudson, J.R. and Birtwistle, S.E.: Wort-boiling In Relation To Beer
A.; Ikeda, H.; Back, W. and Narziss, L.: The influence of malt quality Quality, Journal of the Institute of Brewing, 72 (1966), no. 1, pp. 46-
and malting technology on the flavour stability of beer, Monatsschrift 50.
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Yearbook 2006 59 (2006), no. 9/10, pp. 163-175. 97. Bamforth, C.W.: Dimethyl Sulfide – Significance, Origins, and Control,
92. Malfliet, S.; Opstaele, F.; Clippeleer, J.; Syryn, E.; Goiris, K.; Cooman, Journal of the American Society of Brewing Chemists, 72 (2014), no.
L. and Aerts, G.: Flavour Instability of Pale Lager Beers: Determina- 3, pp. 165-168.
tion of Analytical Markers in Relation to Sensory Ageing, Journal of 98. Formanek, J.A. and Bonte, P.: Use of Tannic Acid in the Brewing
the Institute of Brewing, 114 (2008), no. 2, pp. 180-192. Industry for Colloidal and Organoleptic Stability, Master Brewers
93. Baert, J.J.; De Clippeleer, J.; Hughes, P.S.; De Cooman, L. and Association of the Americas Technical Quarterly, 54 (2017), no. 1,
Aerts, G.: On the Origin of Free and Bound Staling Aldehydes in pp. 11-16.
Beer, Journal of Agricultural and Food Chemistry, 60 (2012), no. 46, 99. Reinhardt, C.; Kunz, T. and Methner, F.-J.: Gallotannins - A useful
pp. 11449-11472. tool to improve colloidal and oxidative Beer Stability, Master Brewers
94. De Schutter, D.P.; Saison, D.; Delvaux, F.; Derdelinckx, G.; Rock, Association of the Americas Annual Conference, Austin, Texas (USA),
J.-M.; Neven, H. and Delvaux, F.R.: Release and Evaporation of 2013.
Volatiles during Boiling of Unhopped Wort, Journal of Agricultural 100. Burrer, F. and Kunz, T.: Alternative stabilization agents for cross-flow
and Food Chemistry, 56 (2008), no. 13, pp. 5172-5180. membrane filtration, 15th International Trends in Brewing Symposium,
95. Miedaner, H.; Narziß, L. and Schneider, F.P.: Influence of Evapora- Ghent (Belgium), 2023.
tion on Wort and Beer Quality, EBC Symposium Wort Boiling and
Clarification, Strasbourg (France), 1991, pp. 37-44. Received 23 May 2023, accepted 7 June 2023
V
Application of punicalagin/ellagic acid to improve oxidative

and colloidal stability of beverages (esp. beer)
Tuur Mertens and Thomas Kunz
Technische Universität Berlin. 2021.
Patent: WO2021170827A1
(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
9
(l ) Worl~~;:::~~:! Property 1111111111111111 IIIIII IIIII 111111111111111 II Ill lllll lllll 111111111111111 IIII 1111111111111111111
International Bureau lntoern2atoio2nall /Plu b7h0"ca8ti2on7NAumlber

(tow)
(43)International Publication Date ;;;;;..,,-""
02 September 2021 (02.09.2021) WI PO I PCT
(51) International Patent Classification: DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN,
C12C 5/02 (2006.01) A23L 2/62 (2006.01) HR, HU, ID, IL, IN, IR, IS, IT, JO, JP, KE, KG, KH, KN,
C12C 7104 (2006.01) C12C 7106 (2006.01) KP, KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD,
ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO,
(21) International Application Number:
NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW,
PCT/EP202 l/054885
SA, SC, SD, SE, SG, SK, SL, ST, SV, SY, TH, TJ, TM, TN,
(22) International Filing Date: TR, TT, TZ, UA, UG, US, UZ, VC, VN, WS, ZA, ZM, ZW.
26 February 2021 (26.02.2021)
(84) Designated States (unless otherwise indicated, for every
(25) Filing Language: English kind of regional protection available): ARIPO (BW, GH,
GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ,
(26) Publication Language: English
UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ,
(30) Priority Data: TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK,
20160167.l 28 February 2020 (28.02.2020) EP EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV,
MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM,
(71) Applicant: TECHNISCHE UNIVERSITAT BERLIN TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW,
[DE/DE]; Strafie des 17. Juni 135, 10623 Berlin (DE). KM, ML, MR, NE, SN, TD, TG).
(72) Inventors: MERTENS, Tuur; Gleimstr. 44, 10437 Berlin
(DE). KUNZ, Thomas; Rathausstr. 17, lO 178 Berlin (DE). Published:
- with international search report (Art. 21 (3))
(74) Agent: ENGELHARD, Markus; Boehmert & Boehmert
Anwaltspartnerschaft mbB Pettenkoferstrafie 22, 80336
=
=
Munich (DE).
(81) Designated States (unless otherwise indicated, for every
kind of national protection available): AE, AG, AL, AM,
iiiiiiiiiiiiiii AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ,
CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO,
(54) Title: APPLICATION OF PUNICALAGIN/ELLAGIC ACID TO IMPROVE OXIDATIVE AND COLLOIDAL STABILITY
-
OF BEVERAGES (ESP. BEER)
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(57) Abstract: The present invention relates to a method for producing a beverage, preferably a fermented beverage with improved
oxidative and colloidal stability. The present invention further relates to a beverage produced by a method for producing a beverage.
The present invention also relates to a beverage having increased stability, preferably increased oxidative flavor stability and colloidal
0 stability. The present invention further relates to a use of a stabilizing agent for preparing a beverage having increased stability.
WO 2021/170827 PCT/EP2021/054885
Application ofpunicalagin/ellagic acid to improve oxidative and colloidal

stability of beverages (esp. beer)
FIELD OF THE INVENTION
The present invention relates to a method for producing a beverage, preferably a fermented
beverage with improved oxidative and colloidal stability. The present invention further
relates to a beverage produced by a method for producing a beverage. The present invention
also relates to a beverage having increased stability, preferably increased oxidative flavor
stability and colloidal stability. The present invention further relates to a use of a stabilizing
agent for preparing a beverage having increased stability.
BACKGROUND OF THE INVENTION
A beer's freshness is the key to good drinkability. Compared with other beverages, beer is
subject to complex reactions in the final package that can harm the product's character. Stale
flavours result from the formation of unsaturated, volatile carbonyl compounds, e.g. 2-
methylbutanal, 3-methylbutanal, phenylacetaldehyde, benzaldehyde, 2-furfural,
hydroxymethylfurfural, and trans-2-nonenal. Among the pathways thought to be involved in
the formation of such carbonyls and other aging components are the Strecker degradation of
amino acids, the melanoidin-mediated oxidation of higher alcohols, the autoxidation of
unsaturated fatty acids, and the aldol condensation of short-chain aldehydes. A beer
deterioration mechanism was proposed in which free radicals are formed from Fenton-type
reactions during aging of beer which, in turn, can initiate a series of radical reactions that are
responsible for the generation of aging components by oxidative processes. Atmospheric
oxygen and transition metal ions in the wort or beer matrices such as iron ions are proposed
be detrimental substances for beer flavour stability, since the presence of oxygen and
transition metal ions leads to the production of highly reactive oxygen species.
Beer deterioration reactions are dependent on the reactants, catalysts, pH, and temperature.
Beer should therefore be stored cool and the level of oxygen should be kept as low as
practically possible during production and packaging. However, even though oxygen uptake
is mitigated, atmospheric oxygen may ingress through the crown cork's compound. Beer
deterioration reactions can be suppressed by antioxidants which are present in the raw
materials, added artificially, or generated during fermentation. Pro-oxidative substances may
promote staling reactions by, for example, recycling Fe3+ to its lower valance state Fe2 +,
thereby making it available for an activation of oxygen and for a catalysis of radical
1
WO 2021/170827 PCT/EP2021/054885
2
generation by the Fenton reaction system [8]. Many attempts have been made to find a
solution for inhibiting or delaying the process of beer deterioration during storage.
The fundamental approaches to solve the problem of flavor deterioration is masking stale
flavors by other substances, reducing the precursors for beer staling, and minimizing the rate
of oxidation. It is important to consider that all raw materials which are used for brewing, i.e.
malt, hops, water, yeast, and adjuncts, may influence flavor stability and the staling potential
of a resultant beer.
It has been shown that adding hops during beer production can markedly enhance the flavor
stability of beer and decrease oxidation indicators during beer storage. Hops contain several
antioxidative substances, whereby hop a- and ~-acids contribute to the antioxidative
potential to a high extend, as measurements using ESR spectroscopy showed [1, 2]. Hop a-
acids and ~-acids were demonstrated to have a highly positive effect on the reduction of the
formation of pro-oxidative radicals, whereas iso-a-acids had a smaller effect. Hence, the
isomerization of hop-a-acids to iso-a-acids was described to yield a lower antioxidant
capacity. However, newer studies using a 2-deoxyribose oxidative degradation assay revealed
that both hop a-and iso-a-acids are capable of suppressing radical formation by complexing
iron; but are incapable of scavenging hydroxyl radicals [3]. It has been proposed that the
main mechanism of hop acids acting as antioxidants is by chelating iron and by scavenging
radicals [1, 2, 4-7]. However, the problem of beer staling may still occur.
A positive effect on the reduced formation of pro-oxidative acting radicals by chelating iron
may be observed upon an application of gallotannins in the brewing process. Gallotannins
(tannic acid) are applied in a lot of breweries worldwide as a stabilization agent, particularly
to get an increased colloidal beer stability by removing haze active protein fractions [9, 10].
However, with regard to the German purity law, an addition of gallotannins during the
brewing process is not allowed, and customer acceptance in Germany is low. Furthermore,
gallotannins are costly and the problem of beer staling may still occur upon addition of
gallotannins.
In consideration of the extra costs caused by gallotannin addition during the brewing
process, the lack of consumer acceptance, and the possibility of beer staling still occurring
after application of gallotannin, it is a challenge to find an efficient substance to improve the
stability of a beer, such as oxidative beer stability, preferably a substance that is cost-efficient
and that has high costumer acceptance. Beside the haze active polyphenol and protein
fractions, the colloidal beer stability is also influenced by oxidative beer stability and specific
transition ions, such as iron.
WO 2021/170827 PCT/EP2021/054885
3
Therefore, it is an aim of the present invention to find an efficient means for increasing the
stability, particularly the oxidative stability, of a beverage, such as beer. It is also an aim of
the present invention to provide a means for increasing colloidal stability of a beverage,
preferably a fermented beverage. The invention further aims at providing a means for
chelating transition metals in a beverage, preferably in a beer, wherein said means is safe for
foodstuff. Furthermore, it is an aim of the present invention to provide a beverage that can be
stored for at least several months without deterioration of the taste and/ or flavor. It is also an
aim of the present invention to provide a method for producing a beverage having increased
oxidative and/ or colloidal stability.
SUMMARY OF THE INVENTION
In the following, the elements of the invention will be described. These elements are listed
with specific embodiments, however, it should be understood that they may be combined in
any manner and in any number to create additional embodiments. The variously described
examples and preferred embodiments should not be construed to limit the present invention
to only the explicitly described embodiments. This description should be understood to
support and encompass embodiments which combine two or more of the explicitly described
embodiments or which combine the one or more of the explicitly described embodiments
with any number of the disclosed and/ or preferred elements. Furthermore, any permutations
and combinations of all described elements in this application should be considered disclosed
by the description of the present application unless the context indicates otherwise.
In a first aspect, the present invention relates to a method for producing a beverage,
preferably a fermented beverage with improved oxidative and colloidal stability, comprising
the following steps:
(i) providing a sugar-containing raw material,
(ii) mashing the sugar-containing raw material to obtain a wort,
(iii) lautering and boiling of the wort,
(iv) optionally, whirlpool rest of the wort,
(v) fermenting the wort by using a yeast,
(vi) obtaining a beverage,
(vii) optionally, maturation of the beverage,
(viii) optionally, filtration of the beverage,
wherein said method further comprises adding a stabilizing agent before, during, or after
any of steps (ii), (iii), (iv), (v), (vi), (vii), and/ or (viii), wherein said stabilizing agent
comprises and/ or consists of ellagic acid and/ or punicalagin.
WO 2021/170827 PCT/EP2021/054885
4
In one embodiment, the step of adding a stabilizing agent is performed before or during
any of the steps (ii), (iii), (iv), (v), (vi), (vii) and/ or (viii).
In one embodiment, the step of adding a stabilizing agent is performed before or during
any of the steps (ii), (iii), (iv), and/ or (viii)
In one embodiment, said sugar-containing raw material is any of malt, barley, maize, rice,
wheat, oat, rye, sorghum, triticale, fonio, and millet, preferably is malt.
In one embodiment, said stabilizing agent is any of a natural extract comprising ellagitannin
and/ or ellagic acid, such as pomegranate extract, an ellagitannin, such as punicalagin, a
hydrolysis product thereof, ellagic acid, and/ or combinations thereof.
In one embodiment, said stabilizing agent is added in step (ii) before, during, or after
mashing, preferably is added before or during onset of mashing.
In one embodiment, said stabilizing agent is added in step (iii) before, during, or after said
boiling of the wort, preferably is added before or during said boiling of the wort, and/ or
said stabilizing agent is added in step (iv) before, during, or after said whirlpool rest,
preferably is added before or during said whirlpool rest, and/ or
said stabilizing agent is added in step (v) before or during fermentation, preferably is
added before fermentation, and/ or
said stabilizing agent is added in step (vii) before, during, or after maturation, preferably
is added before maturation, and/ or
said stabilizing agent is added in step (viii) before or during filtration.
In one embodiment, said stabilizing agent is added more than once in any of steps (ii)-(viii)
and/or is added in more than one of steps (ii)-(viii).
In one embodiment, said stabilizing agent is added in the form of a solid, such as in the form
of a powder, pressed powder, capsule, pill, granules, or tablet, and/ or is in the form of a
liquid, such as in the form of a suspension, emulsion, or solution.
WO 2021/170827 PCT/EP2021/054885
5
In one embodiment, said stabilizing agent is added at a concentration in the range of from
0.01 mg/L to 1500 mg/L, preferably in the range of from 0.01 mg/L to 600 mg/L, more
preferably in the range of from 0.01 mg/L to 500 mg/L, more preferably in the range of from
0.01 mg/L to 250 mg/L, even more preferably in the range of from 0.01 mg/L to 200 mg/L,
and even more preferably in the range of from 0.01 mg/L to 100 mg/L
In one embodiment, wherein said stabilizing agent is ellagic acid, said ellagic acid is
preferably added at a concentration in the range of from 200 mg/L to 500 mg/L, preferably
in the range of from 200 mg/L to 400 mg/L, more preferably in the range of 200 mg/L to
300 mg/L, or in any subrange of concentrations between 200 mg/L and 500 mg/L.
In one embodiment, wherein said stabilizing agent is punicalagin, said punicalagin is

preferably added at a concentration in the range of from 600 mg/L to 1500 mg/L, preferably
in the range of from 600 mg/L to 1200 mg/L, more preferably in the range of from 600 mg/L
to 900 mg/L, or in any subrange of concentrations between 600 mg/L and 1500 mg/L.
In one embodiment, said natural extract comprises punicalagin, and/ or wherein said
ellagitannin is punicalagin.
In one embodiment, said stabilizing agent has an ellagic acid content of at least 10 wt%,
preferably at least 40 wt%, more preferably at least 90 wt%.
In one embodiment, said stabilizing agent is a natural extract having a purity of ellagitannin
and/or ellagic acid of at least 40 %, preferably at least 90 %, and/or
said stabilizing agent is ellagitannin having a purity of at least 40 %, preferably at least

90 %, and/or
said stabilizing agent is one or several hydrolysis products obtained from ellagitannin
having a purity of at least 40 %, preferably at least 90 %, and/ or
said stabilizing agent is ellagic acid having a purity of at least 40 %, preferably at least
90%.
In one embodiment, said malt comprises Pilsner malt, Munich malt, and/ or an other special
malt type, such as a color malt, a flavor malt, a caramel malt, a roast malt, and a melanoidine
malt.
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In one embodiment, said stabilizing agent binds to, preferably chelates, a pro-oxidative
acting transition metal in said wort and/ or beverage.
In one embodiment, said pro-oxidative acting transition metal is any of iron, manganese, and
copper, preferably is iron.
In one embodiment, said stabilizing agent reduces a radical intensity in said wort, as
measured by electron spin resonance spectroscopy and/ or chemiluminescence.
In a further aspect, the present invention relates to a beverage produced, obtained or

obtainable by a method as defined in any of the embodiments above or below, wherein said
beverage is preferably a fermented beverage, such as a beer, a beer-based beverage, and/or a
wme.
In a further aspect, the present invention relates to a beverage having increased stability,
preferably increased oxidative flavor stability and colloidal stability, wherein said beverage
comprises any of a natural extract comprising ellagitannin and/ or ellagic acid, such as
pomegranate extract, an ellagitannin, such as punicalagin, a hydrolysis product thereof,
ellagic acid, and/ or combinations thereof, wherein said beverage is preferably a beer.
Said natural extract, said ellagitannin, said ellagic acid, said pomegranate extract, said
punicalagin, and said beer are as defined above.
In a further aspect, the present invention relates to a beverage having increased stability,
preferably increased oxidative flavor stability and colloidal stability, wherein said beverage
comprises a stabilizing agent comprising and/ or consisting of ellagic acid.
Said stabilizing agent and said ellagic acid are as defined above.
In a further aspect, the present invention relates to a use of a stabilizing agent for preparing a
beverage having increased stability, preferably increased oxidative flavor stability and
colloidal stability, wherein said stabilizing agent is any of a natural extract comprising
ellagitannin and/ or ellagic acid, such as pomegranate extract, an ellagitannin, such as
punicalagin, a hydrolysis product thereof, ellagic acid, and/ or combinations thereof, wherein
said beverage is preferably a beer.
Said natural extract, said ellagitannin, said ellagic acid, said pomegranate extract, said
punicalagin, and said beer are as defined above.
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In a further aspect, the present invention relates to a use of a stabilizing agent for preparing a
beverage having increased stability, preferably increased oxidative flavor stability and
colloidal stability, wherein said stabilizing agent comprises and/ or consists of ellagic acid.
DETAILED DESCRIPTION
The present invention aims at providing an efficient brewing process for obtaining beverages
with increased stability, namely by chelating pro-oxidative transition metals, preferably iron,
in the wort and/ or beverage, preferably beer. Particularly, a stabilizing agent, such as a
punicalagin is added to the brewing process to prolong beer freshness. Punicalagin is a water-
soluble polyphenol, particularly an ellagitannin, that is hydrolysable into smaller phenolic
compounds, such as ellagic acid. In one embodiment, an ellagitannin, such as a punicalagin,
and/ or an hydrolysis product thereof, such as ellagic acid, binds to a pro-oxidative acting
transition metal, such as iron, which is a key player in the beer staling mechanism. In one
embodiment, by chelating the pro-oxidative transition metals, preferably iron, in the wort
and/ or beverage using a stabilizing agent comprising ellagic acid, beer staling is effectively
inhibited and the stability of the wart/beverage is increased. In one embodiment, a natural
extract, preferably pomegranate extract, comprises ellagitannin and/ or ellagic acid, and
effectively chelates iron in a wort and/ or beverage. In one embodiment, ellagitannins from
pomegranate, such as punicalagins, effectively chelate pro-oxidative transition metals,
preferably iron, in a mashing liquor, for example at a concentration of about 0,01 mg/L to
1500 mg/L, preferably about 0.01 mg/L to 1200 mg/L, more preferably 0.01 mg/L to 900
mg/L, more preferably 0.01 mg/L to 600 mg/L, even more preferably 0.01 mg/L to 500
mg/L, even more preferably 0.01 mg/L to 250 mg/L, even more preferably 0.01 mg/L to 200
mg/L, and even more preferably in the range of from 0.01 mg/L to 100 mg/L, and in any
subrange of any of the foregoing .. In one embodiment, ellagitannins, such as punicalagins,
have an effect on the stability of a wort and/ or beverage by chelating transition metal ions
and/ or by quenching radicals during brewing and storage. In one embodiment, ellagitannins
and/ or ellagic acid have an efficient antioxidative effect in a wort and/ or beverage. In one
embodiment, the antioxidative effect of an ellagitannin is evoked by the ellagic acid
comprised by said ellagitannin.
In accordance with embodiments of the present invention, the addition of a stabilizing agent,
as defined herein, has the additional positive advantage in that it allows a shortening of any
maturation time that might be necessary before a possible filtration step. This is because in
accordance with embodiments of the present invention, a stabilizing agent, as defined herein,
when added, may also act as a clarifying agent. This makes the maturation process more
efficient and reduces production costs.
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In one embodiment, a stabilizing agent used in the present invention derives from a natural
origin, i.e. is a natural extract and/ or is a composition comprising or consisting of
ellagitannin and/or ellagic acid obtained from a natural product, and thus said stabilizing
agent has a high consumer acceptance. In one embodiment, the stabilizing agent has a high
costumer acceptance since it is derived from pomegranate fruit extract and/ or pomegranate
juice. In one embodiment, the stabilizing agent used according to the present invention has a
higher consumer acceptance than currently available products, such as tannic acid which is
found in oak bark, leaves, and oak galls, since the stabilizing agent used in the present
invention is derived from an edible fruit.
The term "beverage", as used herein, relates to a liquid intended for human consumption. In
one embodiment, the beverage is a fermented beverage which refers to a beverage produced
by a method comprising a step of fermentation, such as a beer, a beer-based beverage, a
kombucha, a kwass, and/ or a wine. A beer-based beverage is a beverage that comprises beer
and/ or is derived from beer, such as a beer mix beverage, e.g. shandy. In one embodiment, a
beverage produced by a method of the present invention is preferably a beer.
The term "stability", as used herein, relates to a stability of a beverage, such as beer, including
microbiological stability, resistance against haze formation, flavor stability, and foam
stability. In one embodiment, stability of a beverage such as beer preferably relates to
oxidative flavor stability and/ or colloidal stability. Oxidative flavor stability refers to the
stability against beer oxidation and/ or beer staling. Beer oxidation may result in beer staling.
Colloidal stability relates to the resistance against haze formation.
The term "sugar-containing raw material", as used herein, relates to a raw material used in
mashing for obtaining a wort, wherein said raw material comprises sugars. The fermentation
in a subsequent step of producing a beverage is based on the sugars comprised in the raw
material. In one embodiment, said sugar-containing raw material comprises any of malt,
barley, maize, rice, wheat, oat, rye, sorghum, triticale, fonio, millet, grapes, bread, sugar-
containing tea, and combinations thereof, preferably comprises any of malt, barley, maize,
rice, wheat, oat, rye, sorghum, triticale, fonio, millet, and combinations thereof, more
preferably comprises at least malt. In one embodiment, the sugar-containing raw material is
in the form of malt and/ or unmalted grains such as barley, maize, and wheat. In one
embodiment, said malt is a special malt such as colour malt, flavor malt, Munich malt,
caramel malt, roast malt, melanoidine malt, Pilsner malt, or a combination thereof. In one
embodiment, when the beverage to be produced is wine, the sugar-containing raw material
comprises grapes. In one embodiment, when the beverage to be produced is kwass, the sugar-
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containing raw material comprises bread. In one embodiment, when the beverage to be
produced is kombucha, the sugar-containing raw material comprises sugar-containing tea. In
one embodiment, when the beverage to be produced is beer, the sugar-containing raw
material comprises any of malt, barley, maize, rice, wheat, oat, rye, sorghum, triticale, fonio,
millet, and combinations thereof, preferably comprises at least malt.
The term "mashing the sugar-containing raw material", as used herein, relates to a process of
combining a sugar-containing raw material, such as a mix of grains, e.g. malted barley and
optionally supplementary grains such as corn, sorghum, rye, or wheat, or other sugar-
containing raw materials, such as grapes, bread, sugar-containing tea, with a liquid such as
water and then heating the mixture to obtain a wort. In one embodiment, mashing comprises
processing a sugar-containing raw material, such as malt, in a mashing liquor to obtain a
wort. In one embodiment, a mashing liquor is obtained using any sugar-containing raw
material, such as any of malt, barley, maize, rice, wheat, oat, rye, sorghum, triticale, fonio,
millet, grapes, bread, sugar-containing tea, and combinations thereof. In one embodiment,
mashing is performed for about 30 min to 2 h, preferably for about 1 hour. In one
embodiment, a stabilizing agent is added to a mashing liquor prior to heating of said mashing
liquor in a mashing process, or is added at the beginning of mashing, preferably when the
mash reaches a first temperature step of about 63 °C. In one embodiment, mashing
comprises a first temperature step at about 63 °C for about 30 min, a second temperature
step at about 72 °C for about 15 min, a third temperature step at about 78 °C for about 1 min,
wherein, preferably, there is a temperature rise of about 1 °C/min between the temperature
steps. In one embodiment, said mashing liquor comprises water, and optionally further
components such as a salt or a chelator. In one embodiment, a mashing liquor is a liquid
processed in step (ii), such as before, during, or after step (ii). In one embodiment, when the
beverage to be produced is wine, the mashing liquor is obtained from grapes. In one
embodiment, when the beverage to be produced is kwass, the mashing liquor is obtained
from bread. In one embodiment, when the beverage to be produced is kombucha, the
mashing liquor is obtained from sugar-containing tea. In one embodiment, when the
beverage to be produced is beer, the mashing liquor is obtained from any of malt, barley,
maize, rice, wheat, oat, rye, sorghum, triticale, fonio, millet, and combinations thereof,
preferably comprising at least malt.
In one embodiment,
in step (ii), said mashing is performed at a temperature of from about 63 °C to about
78 °C for a period of from 30 min to 2 hours, preferably comprising a first temperature
step at about 63 °C, a second temperature step at about 72 °C, and a third temperature
step at about 78 °C, and/ or
WO 2021/170827 PCT/EP2021/054885
in step (iii), said boiling is performed at a temperature of about 100 °C for a period of
about 1 hour, and/or
in step (iv), said fermenting is performed at a temperature of from 6 to 30 °C for a period

of from 1 day to 20 days.
The term "lautering and boiling", as used herein, relates to a process step in which the mash
is separated into the clear liquid wort and the residual grain (mash filtration), and to a
process of boiling the mash and/ or wort, respectively. In one embodiment, lautering is
performed prior to boiling. In one embodiment, lautering a wort results in "lautered wort". In
an alternative embodiment, boiling is performed prior to lautering. In one embodiment,
lautering is performed for about 10 min to about 4 hours, preferably for about 2 hours. In one
embodiment, boiling is performed for about 1 min to about 2 hours, preferably for about 1
hour. In one embodiment, boiling is performed at at least 100 °C. In one embodiment, a wort
is boiled to obtain a "boiled wort". In one embodiment, a temperature during lautering is
between the mashing-out temperature, such as about 78 °C, and the start of boiling, such as
about 100 °C.
For the fermentation, different yeast strains can be applied to achieve various flavors of the
beverage. Fermentation is performed for at least one day, preferably several days, and
depends on the fermentation temperature and the yeast strain. In one embodiment,
fermentation is a cold fermentation, for example at about 10-12 °C, or a fermentation at room
temperature, or a fermentation at a temperature above or below room temperature. In one
embodiment, the term "young beer" relates to pitching wort which has been brought into
initial contact with yeast for fermentation, for example at the beginning of step (v). In one
embodiment, when alcohol is being produced during fermentation, for example in step (v),
young beer is converted to beer.
The term "whirlpool rest", as used herein, relates to a step in which hot trub, for example
proteins and/ or hops, is separated from the wort. In one embodiment, a whirlpool rest is a
process step after wort boiling to separate the formed hot break from wort matrix. In one
embodiment, a whirlpool rest is performed after boiling of the wort. In one embodiment,
whirlpool resting comprises clarification. In one embodiment, a whirlpool rest allows to get a
good fermentation performance. In one embodiment, trub can be removed by alternative
steps such as decanting and/ or filtration. In one embodiment, whirl pooling/ clarification is
performed to obtain "whirlpooled wort"/"clarified wort".
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The term "maturation of the beverage", as used herein, relates to any transformation of a
wort and/ or beverage between the end of fermentation and the preparation of a beverage for
packaging, such as removing unwanted flavors. In one embodiment, maturation comprises
adding a stabilizing agent to said beverage. In one embodiment, maturation comprises
removing unwanted flavor compounds such as diacetyls while other wanted flavors are being
formed. In one embodiment, maturation may comprise sedimentation, i.e. an extra beverage
clarification.
The term "stabilizing agent", as used herein, relates to any agent that increases the stability of
a beverage, such as beer, preferably an agent that increases oxidative flavor stability and/or
colloidal stability. In one embodiment, a stabilizing agent relates to any of a natural extract
comprising ellagitannin and/ or ellagic acid, such as pomegranate extract, an ellagitannin,
such as punicalagin, a hydrolysis product thereof, ellagic acid, and/or combinations thereof.
In one embodiment, said natural extract comprises punicalagin. In one embodiment, said
ellagitannin is punicalagin. In one embodiment, said stabilizing agent is hydrolyzed when
present in said wort and/ or beverage, and thus releases ellagic acid. In one embodiment, said
stabilizing agent has an ellagic acid content of at least 10 wt%, preferably at least 40 wt%,
more preferably at least 90 wt%. In one embodiment, said stabilizing agent is a natural
extract having a purity of ellagitannin and/ or ellagic acid of at least 40 %, preferably at least
90 %, and/ or said stabilizing agent is ellagitannin having a purity of at least 40 %, preferably
at least 90 %, and/ or said stabilizing agent is one or several hydrolysis products obtained
from ellagitannin having a purity of at least 40 %, preferably at least 90 %, and/ or said
stabilizing agent is ellagic acid having a purity of at least 40 %, preferably at least 90 %. In
one embodiment, when referring to percentages with regard to a stabilizing agent, the terms
"content" and "purity" are used interchangeably. In one embodiment, when referring to the
"purity" of an extract, such as a pomegranate extract having an ellagic acid "purity" of 90%,
the purity refers to the ellagic acid content within the extract. In one embodiment, said
stabilizing agent binds to, preferably chelates, a pro-oxidative acting transition metal,
preferably iron, in said wort and/ or beverage, and thereby increases the stability of said wort
and/ or beverage, particularly the oxidative flavor stability and/ or colloidal stability. In one
embodiment, said stabilizing agent increases the stability of a wort and/ or beverage by
reducing radical intensity and/ or by chelating a pro-oxidative transition metal, preferably
iron. In one embodiment, said stabilizing agent is added at a concentration to provide an
ellagic acid concentration, directly or indirectly via hydrolysis, in the range of from 0.01 to
1500 mg/L, preferably in the range of from 0.01 to 600 mg/L, more preferably in the range of
from 0.01 mg/L to 500 mg/L, more preferably in the range of from 2oomg/ml to 5oomg/ml,
more preferably in the range of from 0.01 mg/L to 250 mg/L, even more preferably in the
range of from 0.01 mg/L to 200 mg/L and even more preferably in the range of from 0.01
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mg/L to 100 mg/L, in a mashing liquor, in a wort, and/ or in a beverage. In one embodiment,
when referring to "ellagic acid" being added as a stabilizing agent, the ellagic acid may be in
the form of pomegranate extract, such as pomegranate extract having a purity of at least
40 %, preferably of at least 90 %. In one embodiment, ellagic acid is the active compound
within pomegranate extract, i.e. a compound having a stabilizing effect. In one embodiment,
a stabilizing agent used in (a method of) the present invention is any of a natural extract
such as punicalagin, a hydrolysis product thereof, and ellagic acid, wherein each of said
natural extract, said ellagitannin, said pomegranate extract, said punicalagin, said hydrolysis
product, said ellagic acid comprises and/ or consists of ellagic acid.
In one embodiment, said radical intensity is measured by electron spm resonance

spectroscopy and/ or chemiluminescence, preferably by electron spin resonance. In one
embodiment, a T6ooDvalue is defined as an ESR signal intensity measured after 600 min of
reaction time at a forced aging temperature of 6o°C and estimates the amount of radicals that
are generated [11].
The term "ellagic acid", as used herein, relates to a natural phenol antioxidant found in
numerous fruits and vegetables. Ellagic acid is the dilactone of hexahydroxydiphenic acid.
Plants produce ellagic acid from hydrolysis of tannins such as ellagitannin. Ellagic acid may
be extracted from, for example, walnuts, pecans, cranberries, raspberries, strawberries,
grapes, peaches, and pomegranates. The term "ellagitannin", as used herein, refers to a
diverse class of hydrolyzable tannins, a type of polyphenol formed primarily from the
oxidative linkage of galloyl groups in 1,2,3,4,6-pentagalloyl glucose. Ellagitannins differ from
gallotannins, in at least that their galloyl groups are linked through C-C bonds, whereas the
galloyl groups in gallotannins are linked by depside bonds. Ellagitannins comprise
hexahydroxydiphenoyl units. Hexahydroxydiphenic acid, which is created upon hydrolysis an
ellagitannin, spontaneously lactonizes to ellagic acid.
In one embodiment, ellagic acid chelates pro-oxidative transition metals, such as iron. In one
embodiment, a concentration of a stabilizing agent in the range of from 0.01 to 250 mg/L, for
example of about 60 mg/L, is highly efficient with regard to the ratio of the amount of
stabilizing agent used to the chelating effect elicited. In one embodiment, in addition to
chelating iron, ellagic acid has a positive impact on haze, and thus increases colloidal
stability. A stabilizing agent used in a method of the present invention effectively chelates
iron both in worts having a higher transition metal content and in worts having a lower
transition metal content, for example in a 100 % Munich wort having an iron concentration
of around 300 µg/L and a 100% Pilsner wort h~vin2" an iron concentration of 180 µg/L. In
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one embodiment, a stabilizing agent decreases the transition metal content, preferably iron
content, by at least 250 %.
The term "adding a stabilizing agent", as used herein, relates to an addition of a stabilizing
agent in a method of producing a beverage. In one embodiment, a stabilizing agent is added
to any step of the method of producing a beverage, optionally more than once in said method,
and/ or more than once in any step of said method. In one embodiment, said stabilizing agent
is added at least once in at least one step of said method in any form that is suitable, such as
in the form of a solid, e.g. as a powder, pressed powder, capsule, pill, granules, or tablet,
and/ or in the form of a liquid, e.g. as a juice, a suspension, emulsion, or solution. In one
embodiment, said liquid is obtained from dissolving and/ or suspending a stabilizing agent.
In one embodiment, said stabilizing agent is added at a concentration in the range of from
0.01 to 250 mg/L, preferably in the range of from 0.01 to 100 mg/L. In one embodiment, a
method for producing a beverage, preferably a fermented beverage with improved oxidative
and colloidal stability, comprises the following steps:
wherein said method further comprises adding a stabilizing agent before, during, or after
any of steps (ii), (iii), (v), and/ or (vi), optionally any of steps (ii), (iii), (iv), (v), (vi), (vii),
and/or (viii), wherein said stabilizing agent comprises and/or consists of ellagic acid and
/ or punicalagin. Preferably in one embodiment, the step of adding a stabilizing agent is
performed before or during any of steps (ii), (ii), (iv), (v), (vi), (vii) and/ or (viii), more
preferably before or during any of steps (ii), (iii), (iv) and/ or (viii).
The term "malt", as used herein, relates to germinated cereal grain, such as barley, that has
been dried in a malting process. In one embodiment, malt relates to malted grains of any
cereal. In one embodiment, malt comprises a mixture of several types of malt, for example
Pilsner malt, Munich malt, and other special malt types, such as a color malt, a flavor malt,
caramel malt, roast malt, and a melanoidine malt. In one embodiment, when adding 35 mg/L
of ellagic acid to a mash, such as a 100% Pilsner mash or 100% Munich mash, the iron
concentration decreases by at least 250%. In one embodiment, the concentration of the
stabilizing agent used depends on the amount of malt used in a mash and/ or the type of malt.
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For example, the higher the amount of malt used in the mash, the higher the stabilizing
concentration. Roasted malts which are, for example, used for dark beers, release high
amounts of transition metal ions, and thus a higher concentration of stabilizing agent is
needed than for other malts. In one embodiment, higher amounts of stabilizing agent need to
be added to a method using special malt types such as roast malt and caramel malt, since
special malt types such as roast malt and caramel malt have a significantly higher iron entry
caused by the roasting process, such as up to 700 % higher iron entry than Pilsner malt types.
In one embodiment, the term "iron entry" may relate to "iron release" into the mashing
liquor, wort, and/ or beverage.
In one embodiment, the terms "ppm" and "mg/L" are used interchangeably. In one
embodiment, the term "comprising" may relate to the term "consisting of'. In one
embodiment, when referring to steps of a method of the present invention, the steps may be
performed in any order. In one embodiment, steps (i)-(vi), optionally steps (i)-(viii) of a
method of the present invention may be performed in any order. In another embodiment,
steps (i) to (vi) and, optionally, steps (i) - (viii), are performed in the order according to their
respective numbering.
BRIEF DESCRIPTION OF THE FIGURES

The present invention is now further described by reference to the following figures.
All methods mentioned in the figure descriptions below were carried out as described in
detail in the examples.
Figure 1 shows a proposed mechanism for the accelerated oxygen activation and generation
of reactive hydroxyl radicals by Fenton reaction system based on the strong reducing
potential of reductones toward oxidized metal ions and consideration of well-known basic
mechanisms [8]. In one embodiment, a stabilizing agent used in a method of the present
invention chelates transition metals such as iron, and thereby increases oxidative stability of
a beverage.
Figure 2 shows the results of an electron spin resonance (ESR) spectroscopy for assessing
the antioxidant capacity of wort.
ESR results including the T6oo-value [11] for boiled worts without an addition of ellagic acid
(o mg/L, blank, black line) and with an addition of ellagic acid (10 mg/L, low concentration,
brown line; 60 mg/L, high concentration, red line) are shown. The T6oo-value indicates the
radical intensity reached after 600 min. The lower the ESR-signal-intensity (T6oo-value), the
lower the quantitative amount of prooxidative actin2: radicals are generated during heating
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(boiling), which indicates that the respective wort/beer has a greater oxidative flavor stability.
As can be seen in Figure 2, the addition of punicalagin, which is 90% pure in ellagic acid,
reduces the overall intensity. The effect achieved by an addition of a high concentration of
ellagic acid (60 mg/L) is stronger than the effect achieved by an addition of a low
concentration of ellagic acid (10 mg/L). The effect achieved upon addition of a low
concentration of ellagic acid was stronger than the blank in which no ellagic acid was added.
Figure 3 shows the effect of ellagic acid on the metal content of unboiled wort.
The results show that ellagic acid, having a concentration in the range of from o mg/L to
120 mg/L, influences the metal content of unboiled wort during mashing, as measured by
inductively coupled plasma optical emission spectrometry (ICP-OES). ICP-OES results of
unboiled worts are depicted which indicate the metal contents (Fe, Cu, and Mn) observed for
increasing ellagic acid concentration (full line: ellagic acid; dotted line: tannic acid).
Manganese and copper are neither significantly influenced by increasing concentrations of
ellagic acid, nor by increasing concentrations of tannic acid/ gallotannins (as indicated by the
dotted lines). Iron, however, is significantly bound, i.e. ellagic acid effectively chelates iron.
Furthermore, ellagic acid chelates iron more efficiently than tannic acid/gallotannins (as
indicated by the dotted line). The comparison between ellagic acid (solid lines) and tannic
acid (dotted lines) with regard to metal depletion shows that the content of iron in the wort,
such as a wort comprising 50% Pilsner malt (pale malt) and 50% Munich malt (slightly more
roasted), effectively decreases upon addition of 90 % pure ellagic acid, and that the iron
decreasing effect of ellagic acid is higher than of tannic acid.
Figure 4 shows the effect of adding food extracts during mashing on the iron content of
wort.
The figure shows ICP-OES results of the iron content of unboiled worts upon addition of
increasing chelating compound concentrations (grapefruit in green, green tea in purple,
pomegranate - ellagic acid (40%) in blue, raspberry in red). A comparison between four
different chelating compounds is made. Most of the investigated extracts did not have a
significant effect on the iron levels, similar to what is seen with grapefruit and raspberry
extract. However, pomegranate extract, which has an ellagic acid purity of 40 % (i.e. content
of ellagic acid of 40 wt%), effectively decreases the iron content in wort. Green tea extract
also decreases the iron content in wort, but shows a lower effectiveness in decreasing the iron
content compared to pomegranate.
Figure 5 shows a comparison between two ellagic acid purities and between different
concentrations of ellagic acid.
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Iron concentrations (blue dotted line) in the wort are decreased after addition of various
concentrations of ellagic acid (of two purities: 40 % and 90%, for example a natural extract
comprising a content of 40 wt% and 90 wt% ellagic acid, respectively). The levels of copper
and manganese (red unbroken line and green dashed line, respectively) do not significantly
change after addition of ellagic acid.
Ellagic acid having a purity of 90 % has a stronger effect than ellagic acid having a purity of
40 %. At about 60 mg/L of ellagic acid, a plateau is reached with regard to the effect of
decreasing the iron level.
Figure 6 shows AntonPaar data of KU Leuven beers.
Beers were analyzed at different time points, i.e. fresh, after 2 weeks, and after 1 month of
ageing. Parameters such as the density, specific gravity, sugar content, carbohydrate content,
and alcohol content of the different beers were analyzed. All beers show normal
characteristics of the measured parameters when compared to blank measurements.
Therefore, a beer having normal density, specific gravity, sugar content, carbohydrate
content, and alcohol content, can be brewed upon addition of a stabilizing agent such as
ellagic acid.
Figure 7 shows beer colors of various beers (blue (="1"): fresh; orange (="2"): two weeks
aged: grey (="3"): one month aged). A higher EBC (European Brewery Convention)
corresponds to a higher beer color.
The following beers are depicted:
•CLl & CL5: blanks (no additions)
•CL2: 75 mg/L ellagic acid (added at the begin of mashing)
•CL3: 75 mg/L green tea extract (added at the begin of mashing)
•CL4: 75 mg/L tannic acid (added at the begin of mashing)
•CL6: 75 mg/L ellagic acid (added at the end of mashing)
•CL7: 75 mg/L green tea extract (added at the end of mashing)
•CL8: 75 mg/L tannic acid (added at the end of mashing)
All beers showed comparable beer color properties. The color of the beer remained relatively
constant upon addition of a stabilizing agent even after one month of aging. Particularly,
when added at the beginning of mashing, the addition of ellagic acid provided a beer with
high color stability over at least four weeks.
Figure 8 shows the influence of various added components on radical intensity observed
during ESR measurement.
WO 2021/170827 PCT/EP2021/054885
17
Lower radical intensity values indicate a more oxidation-resistant wort. Pomegranate extract,
i.e. a natural extract used in a method of the present invention comprising and/ or consisting
of ellagic acid, achieves the lowest radical intensity of the tested compounds. Thus, ellagic
acid effectively decreases radical intensity and increases oxidation-resistance of a wort.
Figure 9 shows the total polyphenol levels of the different worts.

Two sampling stages of worts were analyzed; at the end of mashing and after
whirlpooling/clarification. Similar total polyphenol levels were observed for the various
brews.
•CLl & CL5: blanks (no additions)
•CL2: 75 mg/L ellagic acid (added at the begin of mashing)
•CL3: 75 mg/L green tea extract (added at the begin of mashing)
•CL4: 75 mg/L tannic acid (added at the begin of mashing)
•CL6: 75 mg/L ellagic acid (added at the end of mashing)
•CL7: 75 mg/L green tea extract (added at the end of mashing)
•CL8: 75 mg/L tannic acid (added at the end of mashing).
Figure 10 shows Fe-levels (black, diagonal lines) in wort and their variation in relation to
the added ellagic acid concentration (y-axis) and the grain bill (x-axis).
The left part of the diagram are worts with a high amount of Munich malt (up to 75%), the
right side of the diagram are worts with a high amount of Pilsner malt (also up to 75%).
Munich malt is slightly roasted and thus releases higher amounts of transition metals such as
iron into the wort than Pilsner malt. When the concentration of added ellagic acid is "low",
e.g. 22.5 mg/L of ellagic acid (bottom part of the diagram), the iron concentration is 21%
higher when using high amounts of Munich malt, e.g. 75 % Munich malt, than when using
high amounts of Pilsner malt, e.g. 75 % Pilsner malt. However, when the added ellagic acid
concentration is "high", e.g. 47.5 mg/L of ellagic acid, the difference in iron concentration
when using 75% Munich malt and 75% Pilsner malt, respectively, is only 16%.
Figure 11 shows iron concentrations in eight different brews.

Wort results are shown, i.e. from the beginning of mashing until the finished pitching wort.
Samples of every brew were taken at six stages during mashing; 1. onset of mashing, 2. end of
mashing, 3. at mash filtration, 4. at the onset of boiling, 5. at the end of boiling, and 6. at
clarification. It is observed that ellagic acid is the most effective agent in decreasing the iron
content during the wort production process. Tannic acid is second best in iron content
decreasing effectiveness. Green tea has only a small effect. The first brew and the fifth brew
are blank brews (no chelator addition). In the first four brews, the chelators were added at
WO 2021/170827 PCT/EP2021/054885
18
the onset of mashing. In the last four brews, the chelators were added at the end of mashing.
Chelators were added at a concentration of 75 mg/L.
Figure 12 shows that a stabilizing agent provides colloidal stability of a wort and/ or beer.
Haze formation over time was determined. For example, haze formation was measured at
room temperature and/ or at about o °C after forced and/ or unforced aging. Samples CL1-
CL8 were measured freshly, after two weeks, after one month, and after two months. An
increased colloidal stability was observed upon addition of a stabilizing agent according to
the present invention. The representative figure shows cold haze formation at o °C in samples
CL1-CL8 at four time points after production, particularly at time points "fresh" (1), two
weeks (2), one month (3), and two months (4).
In the following, reference is made to the examples, which are given to illustrate, not to limit
the present invention.
EXAMPLES
Example 1: Assessment of the antioxidant capacity of wart and/or beer.
The antioxidant capacity of wort and/ or beer was assessed using electron spin resonance
(ESR) spectroscopy. The samples were heated to a temperature of about 60 °C causing the
samples to generate radicals, and the amount of radicals generated over time was measured.
Optionally, a spin-trap agent was used to stabilize radicals during the measurements. A
higher oxidative flavor stability is indicated by a lower amount of radicals generated during
heating which is indicated by a lower intensity in ESR spectroscopy. Punicalagin, having a
content of 90% ellagic acid, was added to a wort, such as at the beginning of mashing, at a
lower concentration of 10 mg/L and at a higher concentration of 60 mg/L. The addition of
punicalagin reduced the intensity measured in ESR spectroscopy, and thus increased
oxidative flavor stability of the wort. The higher concentration of punicalagin was more
efficient in lowering the intensity, and thus in increasing oxidative flavor stability, than the
lower concentration of punicalagin (Figure 2).
Example 2: Ellagic acid effectively chelates the pro-oxidative transition metal iron.
Different concentrations of ellagic acid were added to unboiled wort during the mashing
process, namely o mg/L, 10 mg/L, 20 mg/L, 60 mg/L, and 120 mg/L ellagic acid.
Furthermore, tannic acid was used as a control. The metal contents of Fe, Cu, and Mn were
analyzed using inductively coupled plasma ootical emission spectroscopy (ICP-OES). The
WO 2021/170827 PCT/EP2021/054885
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contents of manganese and copper were not significantly changed by increasing

concentrations of ellagic acid. However, increasing concentrations of ellagic acid showed
effective chelation of iron in the wort. The chelation of transition metals using ellagic acid
was more efficient than the chelation of transition metals using tannic acid (Figure 3).
Example 3: Pomegranate extract effectively chelates the pro-oxidative transition metal

iron.
Several natural extracts were added during mashing and the content of iron in unboiled wort
upon addition of different concentrations of these extracts was analyzed. Four different
natural extracts were compared, namely grapefruit extract, green tea extract, raspberry
extract, and pomegranate extract. Grapefruit extract and raspberry extract did not show a
significant reduction of the iron content. Green tea extract showed a reduction of the iron
content, however the reduction was smaller than the reduction of iron using pomegranate
extract. Pomegranate extract, having an ellagic acid purity of 40%, effectively decreased the
iron content in the wort (Figure 4). Pomegranate extract showed the highest efficiency with
regard to chelating iron compared to the other tested natural extracts.
Example 4: Ellagic acid effectively chelates transition metals in various concentrations

and purities.
Various concentrations of ellagic acid, and two purities of ellagic acid, namely having a purity
of 40 % or 90 %, were added to wort, such as at the beginning of mashing. The contents of
transition metals Fe, Cu, and Mn in the wort were analyzed using ICP-OES. The levels of
copper and manganese were not significantly changed by any of the concentrations and/ or
purities of ellagic acid. In contrast thereto, iron was effectively chelated by ellagic acid in
various concentrations and in both purities (Figure 5). A concentration of about 60 mg/L is
highly efficient with regard to the amount of compound needed to evoke the iron reducing
effect and the respective effect evoked.
Example 5: Analysis of aging of different beers.
Eight KU Leuven beers (samples CL1-CL8) were analyzed at three different time points,
namely directly after brewing ("fresh"), after two weeks, and after one month of aging, using
AntonPaar analysis. Two beers were blank beers which did not contain a stabilizing agent of
the present invention (CL1 and CL5); CL2 contained 75 mg/L ellagic acid which was added at
the begin of mashing; CL3 contained 75 mg/L green tea extract which was added at the begin
of mashing; CL4 contained 75 mg/L tannic acid which was added at the begin of mashing;
WO 2021/170827 PCT/EP2021/054885
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CL6 contained 75 mg/L ellagic acid which was added at the end of mashing; CL7 contained
75 mg/L green tea extract which was added at the end of mashing; and CL8 contained
75 mg/L tannic acid which was added at the end of mashing.
Furthermore, the color of these beers at the three time points (fresh, two weeks aged, one
month aged) was analyzed. The color of the beer remained relatively constant upon addition
of a stabilizing agent even after one month of aging (Figure 7).
Example 6: Ellagic acid effectively decreases radical intensity when added at different
time points during the brewing process.
Green tea extract, tannic acid, and pomegranate extract comprising ellagic acid were added to
a wort at the beginning of mashing or at the end of mashing. Green tea extract and tannic
acid did not significantly decrease the radical intensity measured in ESR spectroscopy. In
contrast thereto, ellagic acid effectively decreased radical intensity both when added at the
beginning of mashing or when added at the end of mashing. Ellagic acid can thus be added at
different steps during the brewing process to obtain a wort and/ or beverage with higher
oxidation resistance.
Example 7: Ellagic acid increases colloidal stability.
A stabilizing agent of the present invention reduced haze formation over time (Figure 12) and
thus showed a colloidal stability enhancing effect.
Example 8: Ellagic acid effectively chelates transition metals in warts comprising malt
with high transition metal contents.
Worts with different types of malts, such as Munich malt and Pilsner malt, were analyzed,
particularly a 100% Munich wort and a 100% Pilsner wort, using ICP-OES analysis. Due to
the grain composition, Munich worts have much higher transition metal contents,
particularly iron contents, than Pilsner worts. For example, a Munich wort may have an iron
concentration of about 300 µg/L and a Pilsner wort may have an iron concentration of about
180 µg/L. It was observed that, both in worts having a higher iron content and in worts
having a lower iron content, it was possible to effectively chelate iron. Particularly, higher
amounts of stabilizing agent allowed to counterbalance higher amounts of transition metal
present in the wort (Figure 10 ). Therefore, when using worts comprising sugar-containing
raw materials with higher transition metal content, higher amounts of stabilizing agent
should be used, such as a concentration of at least 4.0 mg/L.
WO 2021/170827 PCT/EP2021/054885
21
Example 9: Effect of the time point of the addition of the stabilizing agent during the
brewing process.
It was assessed whether the time point of the addition of the stabilizing agent has an
influence of the chelating efficiency of the stabilizing agent. Eight different brews were
analyzed. In four brews, 75 mg/L of a stabilizing agent were added at the onset of mashing
("early addition"), and in another four brews, 75 mg/L of a stabilizing agent were added at
the end of mashing ("late addition"). There was one control brew without addition of a
stabilizing agent, one brew with an addition of ellagic acid, one brew with an addition of
green tea extract, and one brew with an addition of tannic acid, in each of the "early addition"
group and the "late addition" group. In each brew, a sample for analyzing the transition metal
content was taken at the onset of mashing, at the end of mashing, at mash filtration, at the
onset of boiling, at the end of boiling, and at clarification. It was observed that ellagic acid is
effective in reducing the iron content during the brewing process, and is effective both when
added at the onset of mashing or at the end of mashing. Ellagic acid showed a superior
reducing iron effect compared to green tea extract and tannic acid. The iron reducing effect
was slightly higher when ellagic acid was added at the onset of mashing compared to when
ellagic acid was added at the end of mashing. In contrast to green tea extract and tannic acid,
ellagic acid chelates iron even during the boiling step (Figure 11).
The project leading to this application has received funding from the European Union's
Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant
agreement No 722166.
REFERENCES
[1] Ting, P., Lusk, L., Refling, J., Kay, S., and Ryder, D. (2008) Identification of antiradical
hop compounds. J. Am. Soc. Brew. Chem. 66(2), 116-126.
[2] Wietstock, P. Kunz, T., Shellhammer, T., Schi:5n T., and Methner, F.-J. (2010) Behavior of
antioxidants derived from hops during wort boiling. J. Inst. Brew. 116(2), 157-166.
[3] Wietstock, P. and Shellhammer, T. (2011) Chelating properties and hydroxyl-scavenging

activities of hop-a and iso-a-acids, J. Am. Soc. Brew. Chem. 69(3), 133-138.
[4] Lermusieau, G., Liegeois, C., and Collin, S. (2001) Reducing power of hop cultivars and
beer ageing. Food Chem. 72(4), 413-418.
WO 2021/170827 PCT/EP2021/054885
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[5] Liu, Y., Gu, X. ,Tang, J., and Liu, K. (2007) Antioxidant activities of hops (Humulus
lupulus) and their products. J. Am. Soc. Brew. Chem. 65(2), 116- 121.
[6] Mikyska, A., Hrabak, M., Haskova, D., and Srogi, J. (2002) The role of malt and hop
polyphenols in beer quality, flavour and haze stability. J. Inst. Brew. 108(1), 78-85.
[7] Wietstock, P., Kunz, T. Pereira, F. Methner F.-J. (2016) Metal chelation behaviour of hop
acid in buffered model systems. Vol. 69, 56-63.
[8] Kunz,T.; Strahmel, A.; Cortes, N.; Kroh, L.W.; Methner, F.J.; Influence of Intermediate
Maillard Reaction Products with Endiol Structure on the Oxidative Stability of Beverages.
American Society of Brewing Chemists, 71, 2013, p.114-123.
[9] Aerts, G.; De Cooman, L.; DeRouck, G. Penzes, Z.; De Buck, A.; Musche, R.; Van
Wasberghe, J; (2004) evaluation of the Additiion of Gallotannnins to the Brewing Liquor for
the improvement of the Flavor Stability of Beer. Technical quarterly - Master Brewers
Association of the Americas 41 (3), 298-304.
[10] Reinhardt, C.; Kunz, T.; Methner, F.-J. (2013) Gallotannins - A usefull tool to improve
colloidal and oxidative beer stability, in Master Brewers Association of Amerika, annual
meeting, Austin, Texas, Poster-presentation.
[11] Kunz, T.; Miiller,C.; Methner, F.J.; EAP Determination and Beverage Antioxidative
IndeX(BAX)-Advantageous tools for Evaluation of the oxidative flavor stability of beer and
beverages. In: Brewing Science,65, Jan/Feb 2012, p.12-22.
The features of the present invention disclosed in the specification, the claims, and/ or in the
accompanying figures may, both separately and in any combination thereof, be material for
realizing the invention in various forms thereof.
WO 2021/170827 PCT/EP2021/054885
23
CLAIMS
1. A method for producing a beverage, preferably a fermented beverage with improved

oxidative and colloidal stability, comprising the following steps:
wherein said method further comprises adding a stabilizing agent before, during, or after any
of steps (ii), (iii), (iv), (v), (vi), (vii), and/or (viii),
wherein said stabilizing agent comprises and/ or consists of ellagic acid and/ or punicalagin.
2. The method of claim 1, wherein said sugar-containing raw material is any of malt, barley,
maize, rice, wheat, oat, rye, sorghum, triticale, fonio, and millet, preferably is malt.
3. The method of claim 1 or 2, wherein said stabilizing agent is any of a natural extract
such as punicalagin, a hydrolysis product thereof, ellagic acid, and/or combinations
thereof.
4. The method of any of the foregoing claims, wherein said stabilizing agent is added in step
(ii) before, during, or after mashing, preferably is added before or during onset of
mashing.
5. The method of any of the foregoing claims, wherein said stabilizing agent is added in step
(iii) before, during, or after said boiling of the wort, preferably is added before or during
said boiling of the wort, and/ or
wherein said stabilizing agent is added in step (iv) before, during, or after said whirlpool
rest, preferably is added before or during said whirlpool rest, and/ or
WO 2021/170827 PCT/EP2021/054885
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wherein said stabilizing agent is added in step (v) before or during fermentation,
preferably is added before fermentation, and/ or
wherein said stabilizing agent is added in step (vii) before, during, or after maturation,
preferably is added before maturation, and/ or
wherein said stabilizing agent is added in step (viii) before or during filtration.
6. The method of any of the foregoing claims, wherein said stabilizing agent is added more
than once in any of steps (ii)-(viii) and/ or is added in more than one of steps (ii)-(viii).
7. The method of any of the foregoing claims, wherein said stabilizing agent is added in the
form of a solid, such as in the form of a powder, pressed powder, capsule, pill, granules,
or tablet, and/ or is in the form of a liquid, such as in the form of a suspension, emulsion,
or solution.
8. The method of any of the foregoing claims, wherein said stabilizing agent is added at a
concentration in the range of from 0.01 mg/L to 1500 mg/L, preferably in the range of
from 0.01 mg/L to 600 mg/L, more preferably in the range of from 0.01 mg/L to 500
mg/L, more preferably in the range of from 0.01 to 250 mg/L, even more preferably in
the range of from 0.01 to 200 mg/L, and even more preferably in the range of from 0.01
mg/L to 100 mg/L.
9. The method of any of the foregoing claims, wherein said natural extract comprises
punicalagin, and/ or wherein said ellagitannin is punicalagin.
10. The method of any of the foregoing claims, wherein said stabilizing agent has an ellagic
acid content of at least 10 wt%, preferably at least 40 wt%, more preferably at least
9owt%.
11. The method of any of the foregoing claims, wherein said stabilizing agent is a natural
extract having a purity of ellagitannin and/ or ellagic acid of at least 40 %, preferably at
least 90 %, and/ or
wherein said stabilizing agent is ellagitannin having a purity of at least 40 %, preferably at

least 90 %, and/ or
wherein said stabilizing agent is one or several hydrolysis products obtained from
ellagitannin having a purity of at least 40 %. oreferably at least 90 %, and/ or
WO 2021/170827 PCT/EP2021/054885
25
wherein said stabilizing agent is ellagic acid having a purity of at least 40 %, preferably at
least 90 %.
12. The method of any of the foregoing claims, wherein said malt comprises Pilsner malt,
Munich malt, and/ or an other special malt type, such as a col or malt, a flavor malt, a
caramel malt, a roast malt, and a melanoidine malt.
13. The method of any of the foregoing claims, wherein said stabilizing agent binds to,
preferably chelates, a pro-oxidative acting transition metal in said wort and/ or beverage.
14. The method of any of the foregoing claims, wherein said pro-oxidative acting transition
metal is any of iron, manganese, and copper, preferably is iron.
15. The method of any of the foregoing claims, wherein said stabilizing agent reduces a
radical intensity in said wort, as measured by electron spin resonance spectroscopy
and/ or chemiluminescence.
16. A beverage produced by a method of any of claims 1-15, wherein said beverage is
preferably a fermented beverage, such as a beer, a beer-based beverage, and/or a wine.
17. A beverage having increased stability, preferably increased oxidative flavor stability and
colloidal stability, wherein said beverage comprises any of a natural extract comprising
ellagitannin and/ or ellagic acid, such as pomegranate extract, an ellagitannin, such as
punicalagin, a hydrolysis product thereof, ellagic acid, and/ or combinations thereof,
wherein said beverage is preferably a beer.
18. Use of a stabilizing agent for preparing a beverage having increased stability, preferably
increased oxidative flavor stability and colloidal stability, wherein said stabilizing agent is
any of a natural extract comprising ellagitannin and/ or ellagic acid, such as pomegranate
extract, an ellagitannin, such as punicalagin, a hydrolysis product thereof, ellagic acid,
and/or combinations thereof, wherein said beverage is preferably a beer.
19. The use according to claim 18, wherein said natural extract comprises punicalagin,
and/ or wherein said ellagitannin is punicalagin.
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1 2 3 4
INTERNATIONAL SEARCH REPORT
International application No
PCT/EP2021/054885
A. CLASSIFICATION OF SUBJECT MATTER
INV. C12C5/02 C12C7/04 A23L2/62 C12C7/06
ADD.
According to International Patent Classification (IPC) or to both national classification and IPC
B. FIELDS SEARCHED
Minimum documentation searched (classification system followed by classification symbols)
C12C A23L
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X CN 109 971 573 A (UNIV NORTHEAST FORESTRY) 1-19

5 July 2019 (2019-07-05)
paragraph [0063] ; claim 1.• examples
-----
X basicbrewing: Pomegranate Pale Ale -
11
1-19
Basic Brewing Video - October 31, 2014 11
,
YouTube
•
31 October 2014 (2014-10-31), XP054980732,
Retrieved from the Internet:
URL:https://www.youtube.com/watch?v=Zn0Paw
HgihQ
[retrieved on 2020-07-24]
minute 2:50-3:58 of the video;
the whole document
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IT] Further documents are listed in the continuation of Box C. [K] See patent family annex.
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A EP 2 499 226 Al (MILLER BREWING 1-19

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19 September 2012 (2012-09-19)
the whole document
A M. T. WALTERS ET AL: "Comparison of 1-19
(+)-Catechin and Ferulic Acid as Natural
Antioxidants and Their Impact on Beer
Flavor Stability. Part 2: Extended Storage
Trials",
JOURNAL OF THE AMERICAN SOCIETY OF BREWING
CHEMISTS.,
vol. 55, no. 3, 6 June 1997 (1997-06-06),
pages 91-98, XP055717884,
us
ISSN: 0361-0470, DOI:
10.1094/ASBCJ-55-0091
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Appendix - Other contributions 187
Appendix
I. Other contributions
a. Conference proceedings at international malting and brewing symposia
Oral:
I Assessment of Metal Chelators in Wort- and Beer-Simulating Buffer Solutions.

Tuur Mertens, Thomas Kunz, Frank-Jürgen Methner. XIX Szkoła Technologii Fermentacji (19th
School of Fermentation Technology) Conference, 2018. Kocierz, Poland.
II Metal Ions in Brewing - Their Role in Beer Staling and Current Remedies.
Tuur Mertens, Thomas Kunz, Frank-Jürgen Methner. 6th International Symposium for Young
Scientists and Technologists in Malting, Brewing and Distilling, 2018. Trier, Germany.
IIIa Improving Beer Flavour Stability by Removal of Transition Metals during Mashing.
Tuur Mertens, Thomas Kunz, Philip C. Wietstock, Frank-Jürgen Methner. VLB-Jahrestagung
Online, 2021. Berlin, Germany.
IIIb Improving Beer Flavour Stability by Removal of Transition Metals during Mashing.
Tuur Mertens, Thomas Kunz, Philip C. Wietstock, Frank-Jürgen Methner. 2nd VLB
International Brewing Web Conference, 2021. Berlin, Germany.
Poster:
Ia Assessment of Metal Chelators in Wort- and Beer-Simulating Buffer Solutions.

Tuur Mertens, Thomas Kunz, Frank-Jürgen Methner. 13th International Trends in Brewing
Conference, 2018. Ghent, Belgium.
Ib Evaluation of Metal Chelators in Wort- and Beer-Simulating Buffer Solutions.

Tuur Mertens, Thomas Kunz, Frank-Jürgen Methner. Brewing Summit Conference, 2018. San
Diego, USA.
II Direct analysis of trace elements in beer by ICP-OES.

Tuur Mertens, Daniela Grothusheitkamp, Thomas Kunz, Olaf Paulsen, Michael Petrich, Erica
Cahoon. 18th European Winter Conference on Plasma Spectrochemistry, 2019. Pau, France.
III Assessment of Metal Chelation during the Mashing Stage of Brewing.

Tuur Mertens, Thomas Kunz, Philip C. Wietstock, Frank-Jürgen Methner. 37th Congress of the
European Brewery Convention, 2019. Antwerp, Belgium.
IV Punicalagin - a novel brewing stabilizing agent for beer shelf-life enhancement.

Thomas Kunz, Tuur Mertens, Kristin Hahne, Florian Kuhn, Tim Bergmann, Brian R. Gibson.
15th International Trends in Brewing Conference, 2023. Ghent, Belgium.
The four posters are featured on the next pages in the aforementioned order.
Assessment of Metal Chelators in Wort-
and Beer-Simulating Buffer Solutions
Tuur Mertens1, Thomas Kunz1, Frank-Jürgen Methner1
1Technische Universität Berlin, Institute of Food Technology and Food Chemistry, Department of Brewing Science, Germany
INTRODUCTION: Restricting the beer staling mechanism RESULTS & DISCUSSION

Flavor stability remains one of the most challenging quality aspects in By individually mixing each metal ion with every complexing compound, we can check whether a
brewing. Transition metals (M: iron, copper, and manganese) constitute in complexation occurs, expectedly indicated by changes in UV-VIS absorbance. It is an easy method
beer staling and oxidation, as they catalyze and accelerate the Fenton and that allows for rapid screening. A few examples are presented in Figure 2: Panels 1-5 show clear
Haber-Weiss reactions in the formation of reactive oxygen species (ROS) [1]: evidence of (complexation) reactions taking place. However, it is not always so clear-cut, as can be
noticed in the 6th-7th panel. Panels 8-9 demonstrate no evidence of any reaction occurring.
1.0 1.0 1.0
0.9 0.9 0.9

Buffer 1 (wort) Buffer 1 (wort)
Buffer 1 (wort)
Buffer 1 + Gallotannin (GT) Buffer 1 + Tannic Acid (TA)
0.8 Buffer 1 + EDTA 0.8 0.8
Buffer 1 + Fe(III) Buffer 1 + Cu(II)

Buffer 1 + Fe(III)
0.7 0.7 0.7 Buffer 1 + Cu(II) + TA (t0)
Buffer 1 + Fe(III) + EDTA (t0) Buffer 1 + Fe(III) + GT (t0)
Buffer 1 + Fe(III) + GT (t60) Buffer 1 + Cu(II) + TA (t60)

0.6 Buffer 1 + Fe(III) + EDTA (t60) 0.6 0.6
Absorbance (A)
Absorbance (A)
Absorbance (A)
0.5 0.5 0.5
0.4 0.4 0.4
0.3 0.3 0.3
0.2 0.2 0.2
0.1 0.1 0.1
0.0 0.0 0.0

250 300 350 400 450 500 550 600 650 700 750 800 250 300 350 400 450 500 550 600 650 700 750 800 250 300 350 400 450 500 550 600 650 700 750 800
1 Wavelength (nm) 2 Wavelength (nm) 3 Wavelength (nm)
1.0 1.0 1.0
0.9 0.9 0.9

Buffer 2 (beer)
Buffer 1 (wort)
Figure 1: Mechanism of reactive oxygen species formation in beer (adapted from [1] and [2]) 0.8 Buffer 1 + Gallic Acid (GA) 0.8
Buffer 2 (beer)
0.8
Buffer 2 + Tannic Acid (TA)
Buffer 2 + Cu(II)
Buffer 1 + Fe(III)
Buffer 2 + Gallotannin (GT)
0.7 0.7 0.7 Buffer 2 + Cu(II) + TA (t0)
Chelating these metals whilst brewing could potentially reduce deterioration of 0.6
Buffer 1 + Fe(III) + GA (t0)
Buffer 1 + Fe(III) + GA (t60) 0.6

Buffer 2 + Fe(III)
0.6
Buffer 2 + Cu(II) + TA (t60)
Buffer 2 + Fe(III) + GT (t0)
beer freshness during storage and ultimately prolong shelf-life.
Absorbance (A)
Absorbance (A)
Absorbance (A)
Buffer 2 + Fe(III) + GT (t60)
0.5 0.5 0.5
In this study, 10 compounds were selected to be screened for their 0.4 0.4 0.4
complexing capacities with 8 metal ions (and an all-including mix) in both a 0.3 0.3 0.3
wort- and beer-simulating solution, using acetate buffer. Measurements were 0.2 0.2 0.2
done by UV/VIS and ICP-OES spectroscopy, respectively, to assess changes 0.1 0.1 0.1
in absorption; and monitor reductions in metal concentration after incubation 0.0

250 300 350 400 450 500 550 600 650 700 750 800
0.0
250 300 350 400 450 500 550 600 650 700 750 800
0.0
250 300 350 400 450 500 550 600 650 700 750 800
and filtration. Ideal chelators should bind iron, copper, and manganese in a 4
1.0
Wavelength (nm) 5
1.0
Wavelength (nm) 6
1.0
Wavelength (nm)
way that they can no longer participate in the generation of ROS, but without 0.9 0.9 0.9
removing metals that are vital for the yeast and the brewing process (e.g. 0.8
Buffer 1 (wort)
Buffer 1 + Gallic Acid (GA) 0.8

Buffer 2 (beer)
Buffer 2 + Citric Acid (CA) 0.8

Buffer 1 (wort)
Buffer 1 + Citric Acid (CA)
zinc). At the time of writing, the results (partial and ongoing) suggest 0.7
Buffer 1 + Mn(II)
0.7
Buffer 2 + Mn(II)
0.7
Buffer 1 + Zn(II)
gallotannin, gallic acid, and tannic acid to be selective chelators for iron and 0.6
Buffer 1 + Mn(II) + GA (t0)
Buffer 1 + Mn(II) + GA (t60) 0.6

Buffer 2 + Mn(II) + CA (t0)
Buffer 2 + Mn(II) + CA (t60) 0.6

Buffer 1 + Zn(II) + CA (t0)
Buffer 1 + Zn(II) + CA (t60)
copper. Manganese, unfortunately, is not readily complexed.

Absorbance (A)
Absorbance (A)
Absorbance (A)
0.5 0.5 0.5
Keywords: Metal chelation; Reactive oxygen species; Flavor stability 0.4 0.4 0.4
0.3 0.3 0.3
SAMPLE PREPARATION / ANALYTICS 0.2 0.2 0.2
0.1 0.1 0.1
0.0 0.0 0.0

250 300 350 400 450 500 550 600 650 700 750 800 250 300 350 400 450 500 550 600 650 700 750 800 250 300 350 400 450 500 550 600 650 700 750 800
7 Wavelength (nm) 8 Wavelength (nm) 9 Wavelength (nm)
Figure 2: Examples of trial mixes with their respective UV-VIS spectra (metal ion and chelator are always mixed at a
1:1 ratio, each at 250 M)
After 60 minutes reaction time, the samples were filtered and measured for their respective metal ion
content. The metal ion concentration for all mixtures done so far (in duplicate) are shown in Table 1:
Table 1: Metal ion concentration in M after 60 min reaction time (room temperature) and filtration (0.2 m)
No complexing
Metal ion pH EDTA Citric acid Tartaric acid Gallotannin Gallic acid Tannic acid
compound
4.30 241 ± 8 233 ± 20 237 ± 12 225 ± 12 193 ± 23 214 ± 11 182 ± 27
Fe(III)
5.60 232 ± 92 169 ± 8 207 ± 1 195 ± 6 8±0 41 ± 6 5±1
4.30 235 ± 5 218 ± 27 231 ± 12 232 ± 13 233 ± 11 216 ± 27 219 ± 26
Ca(II)
5.60 234 ± 21 201 ± 21 204 ± 15 209 ± 13 206 ± 13 198 ± 19 195 ± 22
4.30 280 ± 8 268 ± 25 276 ± 13 276 ± 15 279 ± 11 264 ± 28 269 ± 26
Zn(II)
5.60 282 ± 57 213 ± 8 228 ± 4 228 ± 3 228 ± 1 223 ± 9 215 ± 14
4.30 222 ± 4 214 ± 15 221 ± 10 220 ± 9 221 ± 11 213 ± 15 217 ± 16
Mg(II)
5.60 246 ± 36 240 ± 43 202 ± 11 204 ± 7 201 ± 9 198 ± 14 206 ± 7
4.30 258 ± 1 239 ± 19 252 ± 6 251 ± 7 253 ± 6 238 ± 18 240 ± 18
Mn(II)
5.60 230 ± 12 208 ± 13 197 ± 17 201 ± 9 207 ± 6 186 ± 10 227 ± 27
4.30 259 ± 20 230 ± 11 240 ± 3 237 ± 1 223 ± 1 229 ± 7 219 ± 4
Cu(II)
5.60 199 ± 52 199 ± 6 191 ± 9 225 ± 35 5±0 144 ± 15 4±1
Very high reductions in metal ion concentration are marked in dark green; lesser decreases are
iCAP 6200 Inductively indicated in light green to yellow. Mixes that constitute to a high metal ion decrease clearly form
Coupled Plasma-Optical complexes which can be filtered out of solution. However, a small decrease in metal ion concentration
Emission Spectroscopy
system, fitted with a CID 86
does not necessarily mean that no complexes are formed, as some complexes could be small enough
detector and argon as the to penetrate through the filter. In these cases, the UV-VIS absorbances should provide concluding
carrier gas information.
• Buffers: Both acetate buffers. CONCLUSION / SUMMARY
• Metal ions: Fe3+, Fe2+, Ca2+, Zn2+, Mg2+, Mn2+, Cu2+, Cu+ (and a mix). The results show that—out of the chelators tested so far—gallotannin, gallic acid and tannic acid are
• Chelating compounds: EDTA (Merck), citric acid (Merck), tartaric acid able to chelate out Fe(III) very efficiently; but particularly at pH 5.60. There is still an effect at pH 4.30,
(Merck), gallotannin (Ajinomoto OmniChem), quercetin, chlorogenic but it is considerably lower. At pH 5.60, gallotannin and tannic acid are able to greatly chelate Cu(II);
acid, ferulic acid, gallic acid (Merck), phytic acid, tannic acid (Sigma- gallic acid does it to a lesser extent. None of the tested chelating compounds significantly decrease the
Aldrich) and a zero addition. metals that are beneficial for the brewing process, which is positive. Unfortunately, this trend also
applies for the unwanted Mn(II) ion. The highest manganese removal is seen with gallic acid at pH
Contact: Tuur Mertens, M.Sc. 5.60, and is even more effective than EDTA with a concentration decrease of ± 10 %.
E-mail: tuur.mertens@tu-berlin.de For now, gallotannin, gallic acid and tannic acid seem to be promising, which is in accordance with the
literature. This study will be further elaborated upon in the near future, i.a. studying the remaining metal
Phone: +49 (30) 314-27403
ions and chelators.
Acknowledgement
This project has received funding BIBLIOGRAPHY
from the European Union’s Horizon [1]: H. Kaneda, N. Kobayashi, M. Takashio, T. Tamaki, and K. Shinotsuka, “Beer Staling Mechanism,” Master Brew. Assoc. Am. Tech. Q., vol. 36, no.
2020 research and innovation 1, pp. 41–47, 1999.
program under the Marie Skáodowska- [2]: M. L. Andersen and L. H. Skibsted, “Electron Spin Resonance Spin Trapping Identification of Radicals Formed during Aerobic Forced Aging of
Curie grant agreement No. 722166. Beer,” J. Agric. Food Chem., vol. 46, no. 97, pp. 1272–1275, 1998.
Direct analysis of trace elements in beer by ICP-OES
Tuur Mertens1, Daniela Grothusheitkamp1, Thomas Kunz1, Olaf Paulsen2, Michael Petrich2, Erica
Cahoon3
1 TU Berlin, Institute of Food Technology and Food Chemistry, Chair of Brewing Science, Berlin,
Germany
2 PerkinElmer LAS GmbH, Rodgau, Germany
3 PerkinElmer Inc., Shelton, CT, USA
1 Analysis of Beer - challenges 4 Matrix Effects to be evaluated 6 Spectral Interference

Trace metal determination in Beer has become Sugar, alcohol, carbon dioxide and other organic Carbon from CO2 and organic matrix components
more and more important. In the past, beer compounds produce matrix effects as they influence (i.e. sugar, malt and other organic compounds)
analysis required a 1:10 dilution to reduce surface tension, volatility and the plasma generate strong spectral interferences in the low UV
interferences that may occur: temperature. For these reasons, the matrix effects region that may only be overcome by digestion or by
Different amounts of alcohol (typically 5%) have to be evaluated, how strong they are and what using a hydride system that can separate elements
Alcohol may destabilize the plasma actions have to be taken to overcome these like As or Se from their sample matrix by forming
CO2 may affect precision and plasma stability by transportation- and nebulization interferences. their hydrides. Otherwise misleading false-positive
foaming These effects can be overcome with: results could be generated. This can easily be shown
C from CO2 may generate spectral interferences Matrix matching of external calibration standards by comparing to a blank.
for the analysis of As (right percentage of Ethanol)
Internal standard(s)
Method of addition (calibrate)
In this work the method of addition and the addition
of internal standards have been used to understand
the extent of the matrix effects.
5 Matrix Effects comparison

Figure 7. 5% (v/v) HCl without (bottom) and with 5% (v/v) Ethanol (top)
The three Internal Standards Sc, Y and Yb show a
similar behavior. All samples differ from the acidified
Figure 1 Beer samples prepared for the analysis
HCl-blank solution, the sensitivity is elevated 7 Analyte Concentration
because of the higher volatility of the alcohol.
It is not a good idea to correct against the intensity
2 Instrument Setup and Conditions of an Internal Standard in a blank that had not been
matrix-matched with alcohol.
Table 3. Concentration of Elements in different beer samples

The results for As are false-positive (Carbon).
8 Results and Discussion

Figure 2. Plasma operating conditions utilizing reduced argon plasma flow rate Figure 5. comparison of Internal Standards (IS) in Method of Additions analyzed Beer can easily be analyzed with ICP-OES. The
Method of addition calibrate should be used for
A Meinhard K1 nebulizer with a baffled cyclonic
calibration, CO2 should be removed for better
chamber and a 2mm alumina injector shipped with
precision and 5% ethanol should be added to
the instrument was used for this work. For unfiltered
alcohol-free beers to overcome sensitivity increases
beers, a Burgener MiraMist or a Glass Expansion Sea
(50%) due to alcohol. Even when using method of
Spray nebulizer may be a better choice to reduce
Additions Calibrate we recommend the use of an
the chances of clogging the nebulizer. The plasma
Internal standard to overcome differences in the
conditions can be kept the same. For plasma
content of alcohol between different types of beer.
stability (Ethanol) the torch position was changed
Results for As or Se (here not analyzed) should not
from -3mm to 0mm.
be considered as the UV lines are spectrally
disturbed by carbon lines. A Hydride Kit would give
LOQs of about 0.5g/l or even better.
It is also shown in this application that plasma argon
consumption can be reduced from the normal 15
Figure 6. Method of Addition calibration curve using 100 and 200 ppb spikes L/min to 10 L/min with no degradation of analytical
For all calibrations, the method of standard additions performance by employing a newly designed RF
was used to compensate for the varying suppression generation system.
of the analytes. Samples were diluted with
Figure 3/4. Sample introduction system / torch position deionizedwater and HCl and spiked with increasing
concentrations to generate an appropriate
calibration curve.
3 Sample Preparation The results of the method of additions have been
compared with those acquired with the method of
Different types of Beer were analyzed:
additions calibrate to identify how much the results
malt beer (alcohol-free)
differ from each other. A method of additions
wheat beer
calibrate can be used when the samples behave very
Pilsner beer
similar. Only one sample is being used for the
Export beer
additions, all other samples are being measured like
Standard-type light beer
with an external calibration against that calibration.
All samples were thoroughly shaken and degassed in
Typically, the results should not differ more than
an ultrasonic bath for 30min. To 8.5ml beer, 0.5ml
15%.
HCl (30% v/v), 1ml DI water and 0.1ml Internal
Standard from each solution (100mg/l Sc,Y,Yb)
were added and then well mixed.
Table 1. Sample preparation Table 2. Comparison of Method of Additions and method of Additions Calibrate Figure 11. Avio 200 ICP-OES system
PerkinElmer, Inc., 940 Winter Street, Waltham, MA USA (800) 762-4000 or (+1)
203 925-4602 www.perkinelmer.com
Assessment of Metal Chelation during the
Mashing Stage of Brewing
Tuur Mertens, Thomas Kunz, Philip Wietstock, Frank-Jürgen Methner
Technische Universität Berlin, Institute of Food Technology & Food Chemistry, Department of Brewing Science, Germany
INTRODUCTION RESULTS & DISCUSSION

Beer aging Example: tannic acid (chelator) and iron (metal ion)
Beer tends to inevitable change over time, due to an array of different staling With help of statistical software (Design-Expert® 11), response surface analyses can be made and
reactions. This limited flavour stability remains one of the most challenging the significances of every factor and their interactions estimated (ANOVA). One of these models
quality aspect in brewing to date, as there is still a lot unknown. can be seen in Figure 2.
It is evident, however, that transition metals
(iron, copper, and manganese) play a key role in
staling, as they drive the formation of ‘reactive
oxygen species’ (ROS) through the Fenton and Time
Haber-Weiss reaction mechanism (Figure 1).
So, oxidative deterioration of beer freshness Oxidation
could potentially be reduced by chelating these
catalytic transition metals whilst brewing. Fresh Old/Stale
Model Fit Statistics
R² 0.99
R²adjusted 0.99
R²predicted 0.99
F-value 402.86
p-value < 0.001 Significant
Figure 1: Mechanism of ROS formation, adapted from [1] and [2]
Complexation
Mashing seems to be the most logical stage for chelation: Previous research
showed enhanced complexation at mash pH (opposed to that of beer) [3]; and
Figure 2: Response surface of the 4-factor interaction model for tannic acid and iron
moreover, the earlier that transition metals are removed from the process, the
less oxidative damage they can inflict. This study therefore plans to assess five
This particular example portrays the effects that the mash pH and the tannic acid concentration
specific chelators on their chelating abilities during mashing:
have on the content of iron found in the lautered wort. The other two factors (addition time of
EDTA, citric acid, tannic acid, gallic acid, and phytic acid.
chelator and temperature of mashing out) did not have a big impact on the iron level. This is also
It is unsure how the high temperatures and intricate (wort) matrix will influence prominent when looking at the different factor contributions on the model (Table 2).
the metal-complex formation. To see whether transition metals are effectively Table 2: The factors and their significance on the predictive model for tannic acid and iron
being removed after lautering, the wort is to be examined for metal content by
inductively coupled plasma optical emission spectrometry (ICP-OES), and for Factor Contribution (%) F-value p-value
oxidative stability by electron spin resonance (ESR) spectroscopy. The wort D: Chelator concentration 44.2 1422.70 < 0.0001
samples are made on lab scale in a congress mash bath, with lautering B: Mash pH 34.0 1094.69 < 0.0001
simulated by spent grain on
filter paper. The grain bill is BD 18.9 606.39 < 0.0001
kept constant for all samples. AB 1.3 42.52 < 0.0001
C: Temperature (mashing out) 0.5 14.53 0.0009
Goal AD 0.3 0.0098
7.94
The aim is to find the most suitable chelating agent, and its optimal working
conditions in mashing. Screening is done, for each chelator, by statistical BC 0.2 4.97 0.0358
experimental design, and investigates how varying four selected parameters A: Addition time of chelator 0.0 0.06 0.8128 Not significant
affect the chelation and the resulting amounts of permeated transition metal. A
future study will explore whether beer flavour stability can be enhanced by It can be concluded that the mash pH has a big effect on the amount of Fe that is being leached
applying the findings in (pilot scale) brewing trials. out of the malt during mashing (meaning: a more acidic mash = higher iron entry). Fortunately, this
effect can be diminished by upping the tannic acid concentration. The best/lowest c(Fe) point (68
DESIGN OF EXPERIMENTS g/L) is achieved with a high tannic acid addition and a “high” mash pH; and the worst/highest
c(Fe) point features 288 g/L (low tannic acid addition and mash pH of 5.0).
For the screening, a two-level full factorial design was constructed. This
implies—with four factors and duplicates—a number of 32 runs/samples (24x2) One important nuance to make is that ICP-OES analysis does not differentiate between bound and
to analyze, per chelator. Each of the four experimental factors has a ‘low’ and free metal ions. While it is certain that removing the catalytic transition metals from the wort
‘high’ level, and every mutual combination is tested within the design. Through positively influences the flavour stability of beer, the effect that metal-complexes have is not always
this, the most influential factors can be revealed and a predictive model made. clear-cut; as they can act pro- and anti-oxidative, depending on the circumstances (pH,
concentration, etc.). In other words: even if the chelator does not remove transition metals by
More specifically: relationships between the independent factors in Table 1 and lautering, an anti-oxidative effect is still possible. The permeated transition metals could be in such
the dependent variables (concentrations of Fe, Cu, Mn and Zn in the lautered a (complexed) state, that they become unable to partake in ROS formation. This can (and will) be
wort; in g/L) can be identified. tested by ESR spectroscopy, later in this study.
Table 1: Factors and levels used in the two level-full factorial design
Level SUMMARY
Code Experimental factor Unit
Low (-) High (+) In order to minimize the content of iron (and copper and manganese, for that matter), it is worth
A Addition time of chelator 0 60 min avoiding the mash pH from getting too acidic, as this causes a significant leaching-out effect. Keep
B Mash pH 5.0 6.0 in mind that a lot of other processes in brewing are also pH dependent (e.g. enzymatic activity).
C Temperature (mashing out) 72 82 °C The addition of chelators may lower the overall transition metal concentration. As this is certainly
D Chelator concentration 5.9 35.3 mol/L proven to be the case for tannic acid—a compound that exceeds in depleting iron, and increasingly
well at higher concentrations.
Contact: Tuur Mertens, M.Sc. During this study, other chelators will be screened in similar fashion. Apart from their ability to
remove transition metals through lautering, the anti-oxidative potential of the chelators will be
E-mail: tuur.mertens@tu-berlin.de
examined, as some chelator-bound metal ions might be rendered incapable of activating oxygen.
Phone: +49 (30) 314-27403
BIBLIOGRAPHY
Acknowledgement
[1] H. Kaneda, N. Kobayashi, M. Takashio, T. Tamaki, and K. Shinotsuka, “Beer Staling Mechanism,” Master Brew. Assoc. Am. Tech. Q., vol. 36, no. 1, pp. 41–
This project has received funding
47, 1999.
from the European Union’s Horizon
[2] M. L. Andersen and L. H. Skibsted, “Electron Spin Resonance Spin Trapping Identification of Radicals Formed during Aerobic Forced Aging of Beer,” J. Agric.
2020 research and innovation
Food Chem., vol. 46, no. 97, pp. 1272–1275, 1998.
p
program under the Marie Skáodowska- [3] T. Mertens, T. Kunz, and F.-J. Methner, “Assessment of Metal Chelators in Wort- and Beer-Simulating Buffer Solutions,” in 13th International Trends in
Curie grant agreement No. 722166. Brewing Symposium, 2018.
Punicalagin - a novel brewing stabilizing agent
for beer shelf-life enhancement
Thomas Kunz, Tuur Mertens, Kristin Hahne, Florian Kuhn, Tim Bergmann, Brian Gibson
Technische Universität Berlin, Institute of Food Technology and Food Chemistry, Chair of Brewing and Beverage Technology, Germany
INTRODUCTION RESULTS
Table 3: Results wort analyses – Brewing trial with 100 % Pilsner malt
• Oxidative and colloidal stability are the main issues The lab-tests demonstrated that pH-values have a major
regarding beer shelf-life. influence on complexing properties of the applied chelators Analyses Metal chelating effects Effect on wort
calculated to 14,2 [°P] Fe Cu
• Prooxidative acting ions like iron can activate oxygen by Table 1: Residual metal contents (ppb) of the single metal-chelator solutions after a.L. – after lautering Color pH-value
60 min at 24 C° (RT) and filtration with a 0.2 m filter. [g/L] [g/L]
electron transfer and have catalytic effects on prooxidative a.W. – after whirlpool
red marked - active ingredient mass [EBC] [-]
chelator added
radical generation by the Fenton reaction system. black marked - dosage mass a. L. a. W. a. L. a. W.
concentration
• Two novel natural stabilization agents extracted from metal ion
metal-buffer- Reference 156 80 420 131 7.8 5.59
solution pH Gallotannins Ellagic Acid Punicalagin
pomegranate were evaluated on their capacity to chelate added
calculated
Gallotannin = 0.89 g/hL 108 58 347 111 7.8 5.58
pro-oxidative transition metal ions during mashing and (ppb) Gallotannin = 1.78 g/hL 105 71 349 120 7.9 5.60
wort boiling: the high molecular weight polyphenol 5.0 2017 2808 1738 Gallotannin = 3.56 g/hL 69 58 337 107 7.8 5.60
Fe (II) 14703
punicalagin and ellagic acid. 5.7 838 1917 444
Gallotannin = 7.12 g/hL 72 51 335 72 8.2 5.53
5.0 9125 9036 9149
• Research idea: Increasing oxidative stability with Ca (II) 9567 Gallotannin = 14.24 g/hL 64 39 342 82 8.0 5.52
5.7 9642 9609 10015
alternative natural stabilization agents without altering 5.0 16161 16523 16234 Ellagic acid 94% = 1.87 g/hL / 1.99 g/hL 104 55 430 114 8.5 5.69
beer quality. Zn (II) 16083
5.7 15484 11843 15862 Punicalagin 40% = 0.45 g/hL / 1.125g/hL -- 52 -- 97 8.0 5.58
5.0 13798 13647 13823
Mn (II) 13337 Punicalagin 40% = 1.78 g/hL / 4.45 g/hL -- 33 -- 115 7.8 5.57
5.7 13001 13426 13361
5.0 12444 6723 9033 Punicalagin 40% = 3.56 g/hL / 8.9 g/hL 67 36 335 128 7.8 5.56
Cu (II) 16233
5.7 8467 6087 2158 Punicalagin 40% = 7.12 g/hL / 17.8 g/hL 47 33 308 126 8.1 5.60
• Prooxidative acting metal ions are reduced after lautering

and wort boiling in correlation to applied chelator dosage;
• Punicalagin shows the most efficient iron reduction;
100 % Pilsner Malt
5 min Fe 60 min 6 Reference
T800
ESR Signal Intensity

1,4x10
Punicalagin 0.45 g/hL 6
1.32 x 10
Fig. 5: Pictures illustrating the complex formation of Punicalagin with Cu and Fe ions. Gallotannin 1.78 g/hL
6 Gallotannin 7.12 g/hL
1,2x10 1.15 x 10
6
Fig. 1: Punicalagin Fig. 2. Ellagic acid • In general, the prooxidative metal ions and Fe2+ Cu2+ are Ellagic acid 1.87 g/hL
Punicalagin 1.78 g/hL 1.12 x 10
6
strongly bound and precipitated by all chelators; 1,0x10

6 Gallotannin 14.24 g/hL
Punicalagin 3.56 g/hL
METHODS • Zn2+, Ca2+, Mn2+ are not significant precipitated by all

8,0x10
5
Punicalagin 7.12 g/hL
0.84 x 10
6
chelators with exeption of Zn2+ by ellagic acid at pH 5.7; 0.68 x 10

6
The complexing properties of punicalagin, ellagic acid and 0.67 x 10

6
• A lower pH-value (pH 5.0) leads to a reduction and 6,0x10

5
0.67 x 10
6
tannic acid were pre-evaluated in the lab. Chelating power

approximation of the complexation effects;
was analyzed through a mixture of various metal ions (Fe2+, 4,0x10
5
0.40 x 10
6
Cu2+, Zn2+, Ca2+, Mn2+) in two distinct buffer solutions, • Punicalagin shows the best complexation properties; 0.30 x 10
6
simulating different acidities of wort production (Fig. 3).

5
2,0x10
Table 2: Residual metal contents (ppb) of both metal-mixture-chelator solutions
after 60 min at 24 C° (RT) and filtration with a 0.2 m filter.
0,0
chelator added 0 100 200 300 400 500 600 700 800
time [min]
concentration
metal ion metal-buffer-solution Fig. 6: ESR results (T800 -values / Wort): Brewing trials 100 % Pilsner malt
pH Gallotannins Ellagic Acid Punicalagin
added calculated
1,4
(ppb)
Radical generation T800-Value [10 ]
Gallotannin
6
1,3
Pomegranate extract (40% Punica.)
Fe (II) 3077 59 114 47 1,2
Punicalagin - active ingredient
Ca (II) 2242 2980 2930 2971 1,1
Mix Zn (II) 3576 5.0 3285 2708 3361 1,0
Mn (II) 2837 2566 2414 2616 0,9
Cu (II) 3469 2617 620 670 0,8
Fe (II) 3077 50 80 91 0,7
Ca (II) 2242 3180 3060 3248
0,6
Mix Zn (II) 3576 5.7 3234 2848 2867
0,5
Mn (II) 2857 2663 2394 2533
0,4
Cu (II) 3469 1544 194 201
0,3
Metal-mixture chelator solutions show comparable effects 0,2
Fig. 3: Experimental setup during the preliminary test according to Mertens et al. [1, 2] as detected with the single metal chelator solutions before. 0,1
0 2 4 6 8 10 12 14 16 18 20
In brewing trials, where the polyphenolic compounds were • Cu2+ and Fe2+ are strongly bound and precipitated with 3.56 g/hL 8,9 g/hL Dosage [g/hL]
applied during “mashing in”. The wort samples were reduced effects of gallotannins on Cu2+ precipitation; Fig. 7: Reduced prooxidative radical generation in correlation to the dosage
collected and analyzed as shown in Fig. 4. • A lower pH-value (pH 5.0) led to a slight reduction and of stabilization agents [g/hL] during “mashing in”
approximation of the complexation effects; • The ESR-results (T800-value) show that prooxidative
Mashing Brewing trials • Zn2+, Ca2+, Mn2+ are not significantly precipitated with radical generation can be reduced by all stabilization
100% Pilsner Malt the exeption of slight effects on Zn2+ by ellagic acid; agents in correlation to the applied dosage;
• In comparison to galloannins, the punicalagin contributes
Stabilization agent –„mashing in“
to an eightfold reduction in prooxidative radical generation,
Lautering
Gallotannin: 0.89 / 1.78 / 3.56 / 7.12 / 14.24 g/hL CONCLUSIONS promising an improved oxidative wort and beer stability;
Ellagic acid: 1.99 g/hL (in 10 mL 0,05M NaOH)
Punicalagin: 1.125 / 4.45 / 8.9 / 17.8 g/hL
• Stabilization agents with chelating capacities against prooxidative acting metal ions can be
Reference: no stabilization agent
great aids in the prolongment of beer flavor and colloidal stability;
Wort boiling Analyses: • Ellagic acid requires intricate handling because of its solubility properties and smaller flock
• Extract: MEBAK[4] 2.9.6.3 formation, making it complicated to remove from wort/beer matrices by lautering/filtration;
• Color: MEBAK[4] 2.12.2
• pH-value: MEBAK[4] 2.9.6.3 • Punicalagin in particular proved to be a highly beneficial natural compound, exceeding even
Whirlpool
• Metal-ion concentration: ICP-OES the well-known gallotannins in terms of oxidative stability enhancement;
(Fe2+, Cu2+, Zn2+, Ca2+, Mn2+)
• ESR analyses: Tx-min-value [3]; MEBAK[4] 2.15.3 • Based on the results a dosage of 4 g/hL calculated to percentage of the active component
Fig. 4: Experimental design of the brewing trials including analyses list. punicalagin from pomegranate extract can be recommended for typical pilsner beer types;
ACKNOWLEDGMENTS Dipl.-Ing. Thomas Kunz REFERENCES

This research project was supported by [1] Mertens, T.; Kunz, T.; Methner F.-J.: Assessment of Chelators in Wort and
thomas-kunz@tu-berlin.de Beer Model Solutions, Brewing Science Vol. 73; 2020
ProTUTec +49 (30) 314-27400
[2] Mertens, T.; Kunz, T.; Wietstock, P. C.: Complexation of transition metals by
chelators added during mashing and impact on beer stability, J.Inst Brew., 2021
Internal research funding [3] Kunz, T.; Müller, C.; Methner, F. J. : EAP determination and Beverage
Antioxidative IndeX (BAX); Advantages tools… , Brewing Science 66, 2013
Technische Universität Berlin Follow us: @tuberlin_brew @tuberlin_brew [4] MEBAK, Wort, Beer, Beer-based Beverages, Freising-Weihenstephan 2013
192 Appendix - Other contributions
b. Popular science
Oral:
I The why and how of reducing (transition) metal content during brewing.
Tuur Mertens. 51st PubhD ‘Brewing Science Special’, 2018. Nottingham, UK.
II What is Happening to my Beer!? The Headache called ‘Beer Flavour Stability’.

Tuur Mertens. Berlin Science Talk – Explain Your Research, 2018. Berlin, Germany.
III Improving beer flavour stability by removal of transition metals during mashing.
Tuur Mertens. EJD Food Science – Final Symposium, 2020. Krakow, Poland.
IV Interview on Beer Flavour Stability.

Tuur Mertens. EJDFoodSci Youtube Channel, 2020. Online.
Article:
I Flavour Stability in Home Brewing.

Tuur Mertens. The Modern Brewhouse; EJD Food Sci; MoreBeer; among others. 2020.
The popular science article is featured on the following pages.

Author: Tuur Mertens
Date: 05/2020
Flavour stability in home brewing

Introduction
Famous painter Edgar Degas once declared the frame to be ‘the pimp of the painting’. If he were a
brewer, he might have made a similar statement about beer freshness; as it is exactly that what
makes a good beer pop.
Sadly enough, for both brewers and consumers alike, beer is unresistant to the tooth of time.
Noticeable staling can already occur 2-3 months from packaging when stored at room temperature.
As beer starts to fade, it loses precious aromas and pleasant (hop) bitterness, all while developing
unpalatable or harsh off-flavours. The question is: does this phenomenon really concern a home
brewer?
Well actually, yes. A home brew will suffer the same fate as any commercially brewed beer,
probably even quicker. Not much oxidation is needed to turn a price-winning home brew into a just
“okay” beer. Luckily, when keeping certain codes of conduct in mind, it is more than manageable to
keep your beers from tasting like wet cardboard, paper, cherry, honey, leather, horse stable, cat
urine, ...
The following article will delve into the ‘what’ and ‘how’ of brewing long(er)-lasting beers, with
information roughly ordered from ‘easily implementable’ to ‘advanced tinkering’.
(TL;DR: Check the summary table at the end)
High relevance and easy to do
Limiting storage time (drink it fresh)

There is always the option of not giving beer the time to turn stale. Although this is more a
bypassing-the-problem than a solution, it works. So always label your beers properly with a
‘packaged on’ date and drink the older beers first (FIFO method: First-In, First-Out). Make the fact
that home brewers deal with small batch sizes work in your favour and keep in mind that some
beer styles are more susceptible to ageing than others (e.g. hoppy beers, pilsners).
Storing dark and cold
From all the things a brewer can do to maintain freshness, this is top of the list. Firstly: Protect your
beer from any damaging (ultraviolet AND visible) light; mainly the sun or fluorescent lamps.
Storing beer in the dark will prevent it from getting “skunked” or “light-struck”, terms used for
describing beer that tastes like skunk spray—as literally the same molecule (3-methyl-2-butene-1-
thiol, a sulphur compound) is being formed by photodecomposition of hop alpha-acids. Cans and
kegs will keep light out perfectly; brown bottles less so—albeit better than green or clear
containers—so best to preventively keep them covered during storage.
Secondly: Keep your beer cold! This is, by far, the biggest component. Those familiar with the
Arrhenius equation will tell you that a temperature rise (or drop) of 10 °C roughly equates to a
doubling (or halving) of the reaction rate. Here is what this means for your beer:
Storage Staling rate compared Estimated time before Examples

temperature (°C) to room temp. storage staling occurs
0 ¼x > 1 year Fridge, bucket with ice
10 ½x 4-6 months Fridge, cellar
20 (room temp.) - 2-3 months Living room, indoors
30 2x 1-1.5 months Warm room
40 4x 2-3 weeks Garage, attic, car trunk (sunny)
60 16 x << 3 days Garage, attic, trunk (hot day)
Due to the difference in production size, cooled storage/transport is an area where home brewers
have an edge on the ‘big guys’. However, not every home brewer possesses the luxury of having lots
of refrigerated space.
Limiting oxygen downstream

Although it is hard to tell for sure, it can be reasonably assumed that limiting oxygen downstream
is more relevant/significant than limiting O2 upstream. Which is why oxygen entry on the cold side
(after fermentation) is likely the second biggest detractor of flavour stability that home brewers
face after warm storage.
Prevent any unnecessary transferring of your finished beer, and when required, do it gently (avoid
splashing/turbulence/aeration) and push with CO2 where possible. It’s best to purge any container
(such as bottles, carboys, kegs, etc.) multiple times with CO2 or N2 before filling and to fill from the
bottom up. Hoses and pumps can also be purged, or better, prefilled with deaerated water to expel
oxygen. When bottling, always ‘cap on foam’ (by agitating the bottle slightly), so that headspace
oxygen is minimized. That said, bottling by hand unfortunately often results in very high oxygen
pickup, which is the reason why beers at home brew contests frequently suffer from staling.
Bottle caps are another unavoidable detractor from beer flavour stability when bottling, but not all
caps are created equal. Pry-offs are better than twist-offs, which is why, for example, Sierra Nevada
changed their twist-off caps to pry-offs in 2007. The liner material of the cap also matters, as some
allow for more oxygen ingress than others. Then there are also specialized oxygen-scavenging caps,
specially designed to combat this issue. If you want to go all-in, you can even dip the capped end of
the bottle in melted wax or paraffin (perfect for e.g. barley wines); and buying a ‘dissolved oxygen’
meter can be a very valuable tool in the brewhouse (albeit expensive).
Kegged beer is less prone to oxidative ageing, because the headspace-to-beer volume ratio
(initially) is much lower compared to a bottle. Canned beer has the advantage of having absolutely
no oxygen ingress after sealing. Regardless of the container type, always store a beer upright and
keep vibration/transportation to a minimum. This way, the beer has less chance/surface to
interact with the oxygen-containing headspace, thus slowing down oxidation.
Side note: Be aware that commercially available carbon dioxide (CO2) is never pure. While even the
purest commercially available grade (99.9 %) may seem “practically pure”, it is not. Force
carbonation of the beer will result in a certain amount of O2 getting dissolved; enough to elicit
staling. To avoid this problem, brewers can choose to carbonate their beer naturally—via e.g.
spunding/bunging—with 100 % oxygen-free yeast-produced CO2.
Using healthy, vigorous yeast

Never forget that it is the yeast that makes beer, the brewer “just” makes wort. In other words: pick
the proper yeast and treat it like a queen. It can make the difference between an amazing beer and
unpalatable swill.
More than just creating beer, the yeast also does a great job in cleaning up aldehydes (off-flavour
contributors) and scavenging detrimental transition metals (see later). Researchers were even
able to chiefly remove the stale flavour from aged beer by adding fresh yeast to it. Aside from these
rectifications, it will also help the beer to stay fresh over time by producing sulphur dioxide (SO2, a
potent antioxidant and carbonyl binder) during fermentation and lowering the level of free amino
acids and dissolved oxygen.
So, a healthy yeast and vigorous fermentation are important for flavour stability. To improve
yeast performance, you may supply extra zinc and oxygenate the pitching wort (but only when
cold!), and that should be the only point during the brewing process where oxygen is deliberately
introduced. Keep in mind that different yeast strains have different oxygen demands. When
fermentation is prematurely halting or “stuck”, it’s even worth risking oxidation by aerating the
semi-fermented beer to help restart the yeast. Of course, it is better to prevent it altogether by
fermenting at the recommended temperature—preferably on the lower side—and pitching
enough fresh yeast (make a yeast starter or, if using dry yeast, rehydrate before pitching).
Limiting heat load during brewing

As touched upon earlier, temperature is a huge driving force behind any chemical reaction,
including those involved in the formation of off-flavours (Maillard and Strecker degradation
reactions). Some of these created off-flavours are non-volatile and will migrate into the final beer,
where they can lead to flavour deterioration. Additionally, more heat load equates to more free
radicals and other reactive entities, which equates to more oxidation and further off-flavour
formation (e.g. aldehydes).
It is important not to mash below 62 °C. More than limiting unnecessary heat load, starting the
mash at 62 °C will make sure lipoxygenase (LOX) is largely inhibited, which benefits flavour
stability greatly (LOX enzymatically oxidizes unsaturated fatty acids to E-2-nonenal, causing
cardboard flavour). With the well-modified malt of today, there is no need to do any enzymatic
steps under 62 °C—such as acid rest, ferulic acid rest and protein rest—and there can even be
argued that multiple temperature steps, in general, are obsolete with the current quality of malt (as
done in ‘single infusion’ mashing). Another way of limiting lipoxygenase activity is to use LOX-less
barley malt.
Also avoid excessively long boiling. Do this by utilizing an energetic “rolling” boil, which will
ensure all necessary boiling requirements are well met within the hour. A rolling boil also helps
achieve higher clarity (clearer wort), greater hop utilization, lower levels of dimethyl sulphide
(DMS), and a better hot break. It is best not to cover the boil; even though it might help speed up the
heating process, it will hinder DMS and aldehyde removal.
Another possibility (albeit more challenging) is to utilize a soft “simmering” boil—or even just
below the boiling point—and bubble an inert gas into the mash, to still ensure sufficient volatile
removal. Heating by direct steam diffusion is yet another way to lower heat load and mash
oxidation (lower shear forces). Always cool boiled wort down to pitching temperature as quickly
as possible (to reduce excess heat load) and remove the trub (to avoid fatty acid excess).
High relevance but more challenging
Avoiding transition metals

Iron, copper and manganese promote oxidative (Fenton and Haber-Weiss) reactions in wort and
beer; and thus, the formation of aged flavour compounds (such as cardboard, papery and cherry)
and the decrease of wanted compounds associated with freshness (such as hop aromas and
bitterness). They can also contribute to haze formation and gushing.
Prevent your mash/beer from picking up unnecessary traces of iron, copper and manganese. They
can emanate from various sources: untreated water, Fe- or Cu-containing brewing equipment
(certain kettles, pipes, pumps, buckets, tanks, filters, kegs, cans, crown caps), raw materials (malt,
adjuncts, hops, yeast), process agents (kettle finings, stabilizers, filtration aids). While some sources
will be hard to control or get rid of—such as the raw materials—others can be dealt with—e.g. the
usage of iron-rich kieselguhr for beer filtration.
Luckily, a lot of the said transition metals (Fe, Cu, Mn) will drop out with the trub and hot break
during mashing and boiling. Unfortunately, some of their damaging effects (e.g. formation of
reactive oxygen species) will already have occurred by then, so “less is definitely more”. Also, metal
picked up after boiling will not drop out anymore—although some metal ions will be scavenged by
the yeast during fermentation and drop out during conditioning. Be aware that dry hopping can
introduce “high” amounts of manganese (and oxygen!).
It is possible to lower the ‘active’ transition metal content during brewing by adding chelating
agents/chelators. Two examples of chelators that are successfully being employed in brewing are
tannic acid (a gallotannin found in e.g. oak) and ellagic acid (a polyphenol found in e.g.
pomegranate). They do this in two ways: Firstly, by forming large complexes with the transition
metals present (i.a. iron). This inhibits the metal ion to partake in oxidative mechanisms.
Secondly—because the formed metal-complexes are so large—by effectively lautering and/or
filtering the metal ions out of the wort/beer, thus removing them completely out of the process.
This tendency to form large complexes also makes tannic and ellagic acid suitable for beer
clarification, behaving as clarifying/fining agents.
Another way to lower the amount of metals overall is to not acidify the mash. A mash pH of 5.2 will
cause a much higher leaching-out of iron, manganese and copper (from the malt) into the wort as
compared to a mash pH of 5.4-5.6. A higher mash pH will also promote enzymatic activity and allow
for faster DMS removal.
Of low(er) relevance or controversial
Limiting oxygen upstream

There is zero disagreement on whether oxygen will damage your brew—it will; be it upstream
(‘hot side’) or downstream (‘cold side’). It is the relevance of limiting oxygen upstream that still has
some brewers—and scientists—debating. The “does hot side aeration matter?” forum discussions
can get quite heated at times, which just shows the ambiguity of the topic.
Whether you believe it’s relevant or not, there is no excuse for splashing around hot wort or mash
liquor. Apart from it being hazardous, mash oxidation will occur very rapidly at these high
temperatures, ultimately compromising the final quality of the beer. Hot side aeration (HSA) may
also cause a lot of other things:
x Darken the colour of the wort and final beer, because of polyphenol oxidation (which, in
turn, will lower the antioxidative potential and, ultimately, the flavour stability)
x Promote enzymatic oxidation (by e.g. lipoxygenase)
x Make wort more cloudy
x Cause immediate or indirect flavour changes
Especially for beginning home brewers, the whole HSA-topic can get quite daunting. In the end, it all
boils down to what the brewer wants to achieve, is technically able to realise and what she/he feels
comfortable with. There is probably not much point in meticulously minimizing HSA if the
downstream oxygen pickup is not yet optimized. But for a seasoned or advanced home brewer, ‘low
dissolved oxygen’ (LODO) brewing can be an exciting way to give the beers an extra dimension.
Here are a few examples of how diminished hot side aeration can benefit the brewing process and
the resulting beer:
x Less crosslinking of proteins through disulphide bridges (formed by mash oxidation of
sulfhydryl containing proteins), which results in:
o A faster and more complete lautering, because of increased beta-glucan breakdown
o A higher attenuation limit, because of the more complete lautering, and because of
less starch and malt endosperm being coated with protein
o Less oxidation, because of less formation of hydrogen peroxide (H2O2, a potent
oxidizer)
o Improved proteolysis and amylolysis (breakdown of proteins and conversion of
starch into sugar, respectively)
x Less polyphenol oxidation, so a paler wort/beer (oxidized polyphenols are colourants) and
a higher carry-over of hop’s and malt’s inherent antioxidative power to the final beer
x Less unsaturated fatty acid oxidation—so less aldehydes, like E-2-nonenal (cardboard
flavour)—both enzymatically (through lipoxygenase) and non-enzymatically (through
radical-induced autoxidation)
x Less astringency (coarse bitterness) in the final beer—because of less proanthocyanidin
oxidation—and a more refined beer flavour in general
x Better beer colloidal stability
x Less stale/oxidized wort and improved flavour stability overall (a fresher/crispier tasting
wort)
Unfortunately, when it comes to hot side aeration, home brewers are at a disadvantage compared to
(bigger) commercial breweries, because home brewing elicits a much higher oxygen ingress due to
a larger surface-area-to-volume ratio. The good news is that home brew setups are extremely
flexible and certain oxygen-introducing production steps, that a large brewery will have to deal
with, can therefore be avoided more easily (e.g. big tank transfers, force carbonation, addition of
residual beer).
Apart from not splashing around hot liquids, other examples of limiting upstream oxygen are ‘wet
milling’ and/or blanketing the malt mill with inert gas (nitrogen or carbon dioxide), deaerating the
brewing water (e.g. by boiling it, which will also help remove the chlorine), bottom filling of the
mash tun and boiling kettle, usage of a ‘mash cap’ during mashing, avoiding turbulence during
transfer or stirring, etc. While these actions may not make a huge impact by themselves, many
(renowned) brewers feel that these precautions do make a difference in flavour and shelf-life when
combined.
Adding antioxidants
‘Antioxidant’ is a term widely used, but surprisingly difficult to clearly define. A basic description
would be that it is any substance that delays, prevents or removes oxidative damage. Antioxidants
can do this in a number of ways, e.g. by quenching free radicals or other reactive species, chelating
transition metals, becoming oxidized themselves in place of other biomolecules, etc.
Beer is naturally rich in antioxidants, which help block oxidation:

x Polyphenols: scavenge free radicals & reactive oxygen species, inhibit lipoxygenase, and
chelate transition metals
x Melanoidins: scavenge reactive oxygen species
x Sulphur dioxide and other sulphites: scavenge free radicals and bind carbonyl compounds
x Chelators (phytic acid, amino acids, melanoidins, …): chelate transition metals
If it only were this simple… Unfortunately, it is not. Oxidative mechanisms are immensely
complex, which is the reason why ‘adding antioxidants’ is listed under ‘controversial’. Whether a
chemical entity behaves anti- or pro-oxidant depends highly on the type and the concentration of
the compound, their oxidation state, the pH, the type and concentration of transition metals
present, the matrix, etc. It can be very difficult to make clear-cut predictions and what works in one
medium might not work in another.
Here are some brewing examples to illustrate the intricacy: Melanoidins have also been known to
exert pro-oxidant activity and catalyse oxidation of higher alcohols into their equivalent aldehydes.
Polyphenols can reduce transition metals back to their pro-oxidative form and small/simple
polyphenols (such as gallic acid) may exhibit pro-oxidant activity once oxidized. There is also the
matter of adding vitamin C (ascorbic acid)—a well-known antioxidant found in foods—to the mash
or beer, which some home brewing books will even recommend doing. However, ascorbic acid’s
beneficial effects on beer flavour stability are dubious at best, as it tends to behave pro-oxidatively
at the (low) concentrations you would employ in beer.
This being said, adding sulphite (e.g. potassium metabisulfite)—another well-known antioxidant—
to beer will bind carbonyl compounds, eliminating many of the “stale” notes. That their use is
restricted or prohibited in some countries is nothing to worry about as a home brewer, but be
aware that too much sulphite is toxic to yeast, causes beer to taste “sulphury” (like rotten eggs or
burned matches) and can invoke allergic reactions in some people.
Bottle conditioning
Although the general consensus is that bottle refermentation aids flavour stability, there is still
some room for debate, as it does not come without its risks.
Bottle conditioning benefits shelf-life because active yeast will absorb small amounts of dissolved
oxygen remaining in the beer. Contrary to popular belief, it will only marginally remove any
headspace and/or ingressing oxygen. Yeast will, however, reduce several aldehydes to alcohols,
making them less flavour-active (which is a good thing); and it will assimilate residual free amino
acids (another good thing, as these can be converted into off-flavour aldehydes with time). Flavour-
wise, bottle conditioning can also add an extra layer of “complexity” and uniqueness to the beer. To
that extent that certain styles, of which many are Belgian strong ales, cannot be accurately
reproduced without bottle conditioning.
The downside of bottle conditioning is that, after this active refermentation phase, the beer will
remain in contact with the yeast during the whole storage period. This sometimes results in the
yeast dying, especially if stored inappropriately, resulting in the cell contents spilling out into the
beer—also known as ‘yeast autolysis’. This causes an immediate flavour change: a sharp, bitter
taste, often called “yeast bite”, accompanied with a meaty, sulphury edge, caused by the re-release
of amino acids and yeast nucleotides. Indirect flavour changes also occur, because of altered acidity,
lipid release (rancidity) and enzymatic digestion—mainly by yeast proteases—of beer proteins
(resulting in haze and reduced head retention). All the yeast-accumulated metal ions will also be
released back into the beer, where it shall wreak oxidative havoc.
Using the same yeast from primary fermentation, as done by most amateur home brewers, will
increase the chance of complications; as this yeast is in a depleted and stressed state by then,
because of being in a low pH and high ethanol environment. Even when using fresh, healthy yeast,
bottle refermentation can get tricky at times: Miscalculations can result in under-carbonation (flat
beer) or over-carbonation (gushing, or worse, exploding bottles). Floating/suspended yeast will
cause haze. And there is a risk of introducing unwanted spoilage organisms (such as lactic acid
bacteria or wild yeast).
Innovation in home brewing (experimental)

As a finishing note, here are a few concepts that home brewers can experiment with that might
benefit flavour stability:
x Brewing without boiling

A brewing process that completely skips the wort boiling step is nothing ground-breakingly new.
Quite the contrary; it is likely that a lot of prehistoric beers were “no-boil” or “raw” ales. While
making beer this way will definitely cut down on the heat load and hot side oxidation (depending
on the mash length), it comes with its own set of technical challenges (such as more remaining
protein). Nevertheless, some very interesting and successful beers have already been made this
way.
x Brewing with green malt

Traditionally, brewing is done with kilned or roasted malt. However, it is not impossible to brew a
good beer even with 100 % green/unkilned malt (it has been done). Since this is going against the
grain, the brewer will have to deal with a fair share of technical challenges; but green malt brewing
comes with the advantage of introducing significantly less heat load to the wort, less reactive
compounds (like free radicals, formed in the husk during kilning/roasting), likely less transition
metals (roasting facilitates their release), more intact polyphenols and substantially more diastase
enzymes (which could mean faster and more efficient mashing).
x Brewing with unmalted grain
Unmalted/raw grain is grain that, not only did not undergo kilning (like with green malt), but also
skipped the whole process of malting altogether (steeping and germination). This causes the grain
to not have its enzymes readily available for converting starch into sugar during mashing. So, when
brewing with e.g. 100 % unmalted barley, addition of technical enzymes is a must. While this is not
a new concept—the first commercial all-barley beer being brewed and sold in 1963—it is not a
very common practice among home brewers. Nonetheless, like with the green malt, brewing with
raw grain results in a paler, less heat-stressed beer with enhanced flavour stability. Since green
malt has way more diastatic power than kilned malt, it might be interesting to experiment with
combining green malt and unmalted barley to omit the need of having to add technical enzymes.
x Brewing without malt and without hops

This last suggestion is just to have a bit of a laugh, although there is a point to be made: While this
method would definitely make for an extremely flavour stable “beer”, no one would drink it. We
should never lose our aim in trying to achieve perfection. At the end of the day, beer is there to be
drunk, enjoyed and to have fun with. Cheers to that!
Summary
Stage Raw Milling Mashing & Boiling & Fermentation Bottling, Storage
materials, lautering clarification & canning,
equipment conditioning kegging
& additives
High relevance Add sulphite Use healthy Minimize in-pack Keep it dark
and easy to do yeast oxygen: oxygen
Add effective scavenging caps Keep it cold
chelators Ensure
(tannic acid, vigorous Avoid pick-up of Limit storage
ellagic acid) fermentation iron, copper and time
manganese
Low(er) relevance Use low-LOX Inhibit LOX Bottle- Store it

or controversial barley (mash-in ≥ conditioning upright
but easy to do 62°C) (residual yeast)
Keep it still
High relevance Avoid pick- Limit heat load Minimize in-pack
but more up of iron, oxygen: cap on
challenging copper and foam, purge
manganese containers, limit
transfers, …
Low(er) relevance Add Flush grist Limit hot side aeration Clear wort
or controversial antioxidants with CO2
and more
challenging Mill
anaerobically
Adjust pH Remove trub

(if necessary)
Experimental Usage of No boiling,

green malt “raw ale”
Usage of
unmalted
grain
Further reading
I am part of the European Joint Doctorate Food Science (EJD) project, funded by the European
Union’s Horizon 2020 research and innovation programme. For those who wish to know more
about this project, about us (seven international early stage researchers) or about the work we do,
please check out the official website: https://ejdfoodsci.eu/
In a nutshell, my research focusses mainly on the removal of transition metals during brewing in
order to improve flavour stability. My EJD colleagues are working on topics such as: brewing with
innovative raw materials, the effect of malt bill on metal composition in wort, the influence of malt
quality and malting process on beer staling compounds, brewing with green malt, the evolution of
aldehydes during brewing, fermentation and storage, and the monitoring of yeast performance and
fermentation through proteomics.
Author: Tuur Mertens

PŚƚŚĞƐŝƐͲϮϬϮϯ
Tuur Mertens
Strategies to Reduce the Iron Intake during the Brewing Process with respect to Flavour Stability
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Strategies To Reduce The Iron Intake During The Brewing Process With Respect To Flavour Stability

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Strategies to Reduce the Iron Intake during the Brewing

Process with respect to Flavour Stability

/ŶƐƟƚƵƚĞŽĨ&ŽŽĚdĞĐŚŶŽůŽŐǇĂŶĚ&ŽŽĚŚĞŵŝƐƚƌǇ Faculty of Engineering Technology

an der Fakultät III – Prozesswissenschaften

Dr. der Ingenieurwissenschaften

Vorsitzender: Prof. Dr. Hajo Haase, Technische Universität Berlin

Tag der wissenschaftlichen Aussprache: Berlin, 6 November 2023

Members of the Examination Committee:

Belgium, 26 September 2023

I Mertens, Tuur: Writing - Original draft.

De Cooman, Luc†: Data interpretation, Funding acquisition (supervisor).

Throughout production, packaging and storage—and even as early as malting—beer is subject to a

Staling is a complex and intricate culmination of numerous chemical reactions. Nevertheless, it is

2. Literature Review (Paper I)

- Implementation of modern equipment:

- Adoption of innovative packaging:

2.2. Brewing, oxidative stability and beer staling

2.3. Chelation in the beverage industry

Figure 7: Schematic representation of the formation and precipitation of protein-polyphenol-metal

2.3.1. Antioxidant and chelating properties of polyphenols

3. Research Questions & Thesis Overview

1. Primary research questions (Paper II):

4. Materials & Methods

4.1. Chemicals, consumables, equipment and software

Table 2: Summary of chemicals used in the thesis.

Chemical Supplier Purity (%) Paper

Chemical Supplier Purity (%) Paper

Table 3: Summary of consumables used in the thesis.

Table 4: Summary of equipment used in the thesis.

Equipment Supplier Paper

Table 5: Summary of software used in the thesis.

4.2. Sample preparation

Table 6: Summary of samples investigated in the thesis.

4.2.1. Preparation of metal-chelator mixtures in wort and beer model solutions

4.2.2. Preparation of laboratory and pilot scale wort samples

4.2.3. Preparation of beer samples

Table 8: Chelator mash addition protocol.

4.2.3.2. Pilot scale beer production at the TU Berlin

4.3. Analytical methods

Table 9: Summary of analytical methods used in the thesis.

4.4. Standard beer analyses

4.5. Data analysis

5.2. Complexation of transition metals by chelators added during mashing and

5.3. Effects of mash chelator addition on transition metal content and

5.4. Application of punicalagin/ellagic acid to improve oxidative and colloidal

Table 15: Initial chelators selection and rationale.

7. Conclusion & Outlook

Cardboard Flavor in Beer, Proceedings. Annual meeting - American Society of Brewing

on brewing and distilling fermentations, Proceedings of the Convention of the Institute of

effective replacement of EDTA in mayonnaise, Applied Food Research, 3 (2023), no. 1, p.

(2023), no. 5, p. 2295.

Transition metals in brewing and their role in wort and beer

Tuur Mertens, Thomas Kunz and Brian R. Gibson

Journal of the Institute of Brewing. 2022; 128:77-95.

(wileyonlinelibrary.com) DOI 10.1002/jib.699

Transition metals in brewing and their role in

II Antioxidant capacity assays

exponential relationship (102). Because of the linearity between

Table 3. Transition metals throughout the brewing process

Review of metals and oxidative stability in brewing

Raw materials, Downstream

equipment, Mashing and Boiling and Fermentation processing Distribution

high (er) cost, barley Mill anaerobically yeast

/ŶƐƟƚƵƚĞŽĨ&ŽŽĚdĞĐŚŶŽůŽŐǇĂŶĚ&ŽŽĚŚĞŵŝƐƚƌǇ Faculty of Engineering Technology