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]
• 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
Fe Cu
Color pH-value
60 min at 24 C° (RT) and filtration with a 0.2 µm filter. a.W. – after whirlpool [µg/L] [µg/L]
electron transfer and have catalytic effects on prooxidative 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
Gallotannin 1.78 g/hL 1.32 x 10
Fig. 5: Pictures illustrating the complex formation of Punicalagin with Cu and Fe ions.
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

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

5
simulating different acidities of wort production (Fig. 3). 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 µm 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
• Metal-ion concentration: ICP-OES the well-known gallotannins in terms of oxidative stability enhancement;
Whirlpool (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
[email protected] Beer Model Solutions, Brewing Science Vol. 73; 2020
[2] Mertens, T.; Kunz, T.; Wietstock, P. C.: Complexation of transition metals by
ProTUTec +49 (30) 314-27400 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