J. Agric. Food Chem.

2000, 48, 11291134

1129

Measuring Antioxidant Efficiency of Wort, Malt, and Hops against

the 2,2-Azobis(2-amidinopropane) Dihydrochloride-Induced
Oxidation of an Aqueous Dispersion of Linoleic Acid
Catherine Liegeois, Guillaume Lermusieau, and Sonia Collin*
Universite Catholique de Louvain, Unite de Brasserie et des Industries Alimentaires,
Place Croix du Sud, 2/Bte 7, B-1348 Louvain-la-Neuve, Belgium

This paper presents a simple, convenient method for determining the efficiency of antioxidants in
aqueous systems. Production of conjugated diene hydroperoxide by oxidation of linoleic acid in an
aqueous dispersion is monitored at 234 nm. 2,2-Azobis(2-amidinopropane) dihydrochloride is used
as a free radical initiator. Among 12 antioxidants tested, phenolic compounds proved to be the most
efficient, both kinetically and in terms of the inhibition time (Tinh). Applied to wort, malt, and hops,
the method confirmed a significant antioxidant activity in such products, especially hops. This assay
can be used to follow oxidative changes throughout the brewing process and to understand the
contribution of each raw material.
Keywords: Antioxidant; lipid oxidation; AAPH; beer
INTRODUCTION

As lipid oxidation is the major pathway of alkenal

formation, inhibiting lipid oxidation during the brewing
process should provide brewers with a key to regulate
alkenal production and hence beer staling (Collin et al.,
1999; Lermusieau et al., 1999; Noel et al., 1999a).
Enzymatic degradation of linoleic acid produces trans2-nonenal and many other volatile aldehydes during
mashing, but autoxidation during boiling has emerged
from a recent work as the main source of this cardboardlike off-flavor in aged beer (Noel et al., 1999b). Inhibiting
the oxidative deterioration of wort in the kettle will thus
require protection of the intrinsic antioxidant potential
of malt and hops and/or addition of exogenous antioxidants.
Initiation of lipid autoxidation, enhanced either by
free radicals (O2-, OH) or by singlet oxygen (1O2), can
usually be prevented by singlet oxygen quenchers, free
radical scavengers, or chelatants (Niki, 1987). The
propagation chain reaction can also be broken with
peroxy radical scavengers such as phenolic compounds
(Torel et al., 1986; Gardner, 1989; Porter et al., 1995).
Over the past few years, the antioxidant activity of
wort and beer has been investigated by various methods
including (1) measuring the capacity to reduce the iron(II)-dipyridyl complex (Chapon, 1981), (2) measuring
the ability to scavenge the radical cation of 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) in an aqueous phase (Araki et al., 1999), (3) measuring the 1,1diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging
activity (Kaneda et al., 1995a,b), (4) measuring chemiluminescence, either directly or after reaction with the
radical scavenger 2-methyl-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-3-one (CLA) (Kaneda et al., 1990a,b,
1991, 1994), (5) electron spin resonance (Kaneda et al.,
* Author to whom correspondence should be addressed
(telephone 32 10 47 29 13; fax 32 10 47 21 78; e-mail collin@
inbr.ucl.ac.be).

1988, 1989; Uchida and Ono, 1996), (6) assaying

2-thiobarbituric acid reactive substances (Grigsby and
Palamand, 1976), (7) electrochemical measurement of
the redox potential (van Strien, 1987; Galic et al., 1994;
Buckee et al., 1997), (8) measuring the capacity to delay
methyl linoleate oxidation (in lipidic media and at high
temperature) followed by gas chromatography (Boivin
et al., 1993; Maillard and Berset, 1995), and (9) quantification of linoleic acid hydroperoxide in a Fenton-type
reaction (Bright et al., 1998). Unfortunately, some of
these assays are either time-consuming or specially
designed for lipidic media. Moreover, it appears of great
interest to optimize an antioxidant assay relevant to
wort, which could also be applied to all raw materials.
Azo compounds generating free radicals through
spontaneous thermal decomposition are useful for in
vitro studies of lipid peroxidation (eqs 1-3) (Niki, 1987;
Pryor et al., 1993; Ghiselli et al., 1998). The watersoluble azo compound 2,2-azobis(2-amidinopropane)
dihydrochloride (AAPH) has been extensively used as
a clean and controllable source of thermally produced
alkylperoxyl free radicals (Niki, 1990). To date, however,
only Fantozzi et al. (1998) have applied it in the brewery
field to assess the effect of beer on the oxidation of
human low-density lipoprotein (LDL).
kd

AsNdNsA 98 (1 - e) AsA + 2e A + N2

(1)

A + O2 f AO2

(2)

AO2 + LH f AOOH + L

(3)

with A ) -C(CH3)2C(NH2)dNHHCl and e ) efficiency

of free radical production.
The aim of the present work was to use this hydrophilic azo compound to assess the oxidability of linoleic
acid in an aqueous dispersion (resembling wort) in the
presence of antioxidants from malt or hops. First, the
efficiency of 12 recognized antioxidants, some naturally

10.1021/jf9911242 CCC: $19.00 2000 American Chemical Society

Published on Web 02/29/2000

1130 J. Agric. Food Chem., Vol. 48, No. 4, 2000

Liegeois et al.

Figure 1. Antioxidants used in this study (a, water-soluble; b, oil-soluble). The numbers in brackets give the initial rates of
oxidation in the presence of 2 M antioxidants compared to the controls [Rinh/Ro].

occurring in beers, was investigated to determine the

main contributors to the antioxidant activity of wort.
MATERIALS AND METHODS
Reagents. Twelve antioxidants have been compared (see
Figure 1): catechin hydrate 98%, 1; 4-hydroxy-3-methoxycinnamic acid 99% (ferulic acid), 2; 3,4-dihydroxycinnamic acid
(caffeic acid), 3; reduced glutathione, 4; L-cysteine chlorhydrate, 5; potassium metabisulfite, 6; L-(+)-ascorbic acid (vitamin C), 7; 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic
acid 97% (Trolox), 8; 4-hydroxy-2,5-dimethyl-3(2H)-furanone
95% (Furaneol), 9; (()-R-tocopherol 95% (vitamin E), 10;
quercetin dihydrate, 11; and butylated hydroxytoluene (BHT),
12. Compounds 1, 3, 10, 11, and 12 were from Sigma Chemical
Co. (St. Louis, MO); 2, 4, 7, and 9 were from Acros Organics
(Geel, Belgium); 5 was from Merck (Darmstadt, Germany); 6
was from Vel (Leuven, Belgium); and 8 was from Aldrich
Chemical Co. (Milwaukee, WI).
Linoleic acid (99%) used as substrate was obtained from
Sigma Chemical. The water-soluble radical initiator, AAPH,
was obtained from Aldrich Chemical Co. Tween 20 (polyoxyethylenesorbitan monolaurate), used as a surfactant, and
anhydrous dipotassium hydrogenphosphate p.a., potassium
dihydrogen phosphate p.a., and boric acid p.a., used for buffer
solutions, were purchased from Merck, and dimethylsulfoxide
p.a. (DMSO) was from Fluka Chemie (Buchs, Switzerland).
Methanol 205 super purity from Romil (Cambridge, U.K.) was
used for solvent extraction.
All aqueous solutions and reagents were made using Milli-Q
(Millipore, Bedford, MA) double-distilled water (resistance )
18 m/cm2).
Substrate. An aqueous solution of linoleic acid was prepared weekly according to the method of Surrey (1964), with
minor modifications: under continuous stirring, linoleic acid
(0.25 mL) was added dropwise to 5 mL of 0.05 M borate buffer,
pH 9, containing 0.25 mL of Tween 20. The resulting dispersion was clarified by adding 1 mL of 1 N sodium hydroxide.
The volume was adjusted to 50 mL with additional borate
buffer. This 16 mM linoleic acid solution was stored at 4 C
in the dark under argon until needed. Before use, the substrate
was checked for autoxidation, and solutions exhibiting >3%
autoxidation were discarded.
AAPH Solution. Forty millimolar AAPH was freshly
prepared in 0.05 M phosphate buffer, pH 7.4.
Antioxidants. Stock solutions (0.01 M) of oil-soluble antioxidants were freshly prepared in pure methanol (preparation
at 50 C for antioxidant 11). Stock solutions (0.01 M) of water-

soluble antioxidants were prepared in 0.05 M phosphate buffer,

pH 7.4 (preparation at 60 C for antioxidants 1-3) except for
Trolox 8 solution, which was first prepared in DMSO. Appropriate dilutions were prepared in phosphate buffer (containing 0.05% v/v Tween 20 in the case of lipophilic antioxidants) just before the antioxidant assay.
Extraction of the Antioxidants from Malt and Hop.
Malt was finely ground in a DLFU-mill from Buhler-Miag
(Braunschweig, Germany); hop pellets were crushed in a
mortar. Ground samples (1 g) were extracted four times under
nitrogen with 7 mL of methanol by shaking for 15 min and
sonicating for 5 min. After centrifugation (3500g, 10 min), the
supernatant (25 mL) was collected. For the CO2 hop extracts,
an aliquot (1 g) was dissolved under nitrogen in 25 mL of
methanol by sonicating for 10 min. Appropriate dilutions were
prepared in pure methanol by taking into account a maximum
methanol level of 0.33% (v/v) in the antioxidant assay.
Antioxidant Assay. Thirty microliters of the 16 mM
linoleic acid dispersion described above was added to the UV
cuvette containing 2.81 mL of 0.05 M phosphate buffer, pH
7.4, prethermostated at 40 C. The oxidation reaction was
initiated at 37 C under air by the addition of 150 L of 40
mM AAPH solution. Oxidation was carried out in the presence
of aliquots (10 L) of either wort samples (wort dilution factor
in test ) 700 to 300), methanolic malt extracts (final concentration in test ) 133.3 mg/L), methanolic hop extracts (final
concentration in test ) 1.7-16.7 mg/L), or aqueous solutions
of antioxidants (final concentration in test ) 0-8 M). In the
assay without antioxidant (curve 0), lipid oxidation was
measured in the presence of the same level of methanol
(maximum ) 0.33%). We carried out all of the assays without
chelatants to assess overall antioxidant activity including prooxidant effects of transition metals.
The rate of oxidation at 37 C was monitored by recording
the increase in absorption at 234 nm caused by conjugated
diene hydroperoxides. A molar extinction coefficient of 28000
M-1 cm-1 was used in all calculations (Lulai and Baker, 1976).
A Shimadzu UV-visible 240 spectrophotometer (Antwerp,
Belgium) equipped with an automatic sample positioner allowed analysis of six samples every minute. In all cases, the
measurements were run in duplicate against the buffer and
compared with a separate AAPH-free control to check for any
spontaneous oxidation. AAPH has a relatively high absorbance
below 260 nm, which changes as the compound decomposes:
the A234 of a 2 mM solution was 0.9 at the start of the
incubation, increasing to 1.3 after 4 h at 37 C. Therefore, its
absorbance measured in a separate cuvette in the absence of
linoleic acid was subtracted from each experimental point.

Antioxidant Efficiency of Wort, Malt, and Hops

J. Agric. Food Chem., Vol. 48, No. 4, 2000 1131

Figure 2. Effect of various antioxidants on AAPH-induced

linoleic acid oxidation, as measured by kinetics of conjugated
diene formation. Linoleic acid (0.16 mM) was incubated with
2 mM AAPH in 50 mM potassium phosphate buffer, pH 7.4,
at 37 C under air, in the absence (b) and presence of 2 M
antioxidant. The numbers give the antioxidant shown in
Figure 1.
Table 1. Initial Rates of Oxidation (Ro) of Linoleic Acid
(LH) in an Aqueous Dispersion at 37 C
run

[LH], mM

[AAPH], mM

108 Ro, M
dienes formed/s

1
2
3
4
5
6
7
8

0.1
0.16
0.16
0.16
0.2
0.4
0.5
0.8

2
1
2
4
2
2
2
2

0.95
1.05
1.47
1.62
1.86
3.31
3.88
5.11

The inhibition time (Tinh) was estimated with Microsoft

Excel software as the point of intersection between the
tangents to the inhibitionsand propagationsphase curves,
under precise oxidation conditions. The rate of radical generation from AAPH in an aqueous phase (Ri) was measured with
Trolox, a water-soluble vitamin E analogue, as the radical
scavenger (Niki et al., 1986).
RESULTS AND DISCUSSION

Oxidation of Linoleic Acid in an Aqueous Dispersion and Its Inhibition by Antioxidants. In the
absence of a radical initiator, the rate of spontaneous
oxidation at 37 C can be considered negligible. Addition
of AAPH induces oxidation, which starts at a constant
rate of conjugated diene formation. As shown in Table
1, the concentrations of both linoleic acid and AAPH
strongly influence the initial oxidation rate (Ro) in the
aqueous dispersion. On the basis of this observation, the
concentrations (0.16 and 2 mM for linoleic acid and
AAPH, respectively) and temperature (37 C) were
chosen to obtain appropriate rates of oxidation.
Figure 2 shows the results obtained at the beginning
of the oxidation assay in the presence of several antioxidants (2 M). During this inhibition period, phenolic
compounds (11, 3, 1, and 2) proved to inhibit oxidation most efficiently, thiols (5 and 4) less so. Surprisingly, sulfites 6 did not inhibit AAPH-induced oxidation of linoleic acid. We can assume that the antioxidant activity of sulfites detected by ESR technique
(Uchida and Ono, 1996) is mainly due to changes in
hydroxyl radical concentrations, which are underestimated in the presence of AAPH. The ratio of the
initial oxidation rate in the presence of antioxidants
(Rinh) to the initial oxidation rate in their absence (Ro)
provides a first antioxidant activity index (Iwatsuki et
al., 1995). The Rinh/Ro values for all of the antioxidants tested are shown in Figure 1 (see values in
brackets).

Figure 3. (a) Effect of Trolox concentration on AAPH-induced

linoleic acid oxidation, as measured by kinetics of conjugated
diene formation. Linoleic acid (0.16 mM) was incubated with
2 mM AAPH in 50 mM potassium phosphate buffer, pH 7.4,
at 37 C under air, in the presence of 0, 1, 2, 4, and 8 M Trolox
(curves 0, 1, 2, 3, and 4, respectively). (b) Inhibition time (Tinh)
as a function of the Trolox concentration.

As shown in Figure 3a for Trolox 8, an inhibition time

(Tinh) can also be determined for each antioxidant
concentration. In all cases, once this inhibition time is
over, oxidation proceeds at the same rate as in the
absence of inhibitor. The slope of the curve representing
the Tinh versus the antioxidant concentration, shown in
Figure 3b (S or S), can be used as a second very
interesting antioxidant activity index. Figure 4 shows
the slopes obtained in the same way for the various
tested antioxidants. The measured inhibition time was
directly proportional to the concentration of the additive
in the tested concentration range.
From these data, it is possible to calculate a stoichiometric coefficient n representing the number of peroxyl
radicals trapped by each antioxidant molecule (see
values in Figure 4). Using Trolox as a reference inhibitor
removing two radicals per added molecule (Niki et al.,
1986), it is easy to determine the rate of radical
generation (Ri) under our assay conditions from the
equation Ri ) n[antioxidant]/Tinh.
Using our experimental Ri value [1.2075 10-6
[AAPH] (s-1)], to be compared with that obtained by
Niki et al. (1986) in a similar medium [1.30 10-6
[AAPH] (s-1)], n for the other molecules can be deduced.
Caffeic acid 3, catechin 1, and quercetin 11 produced
a longer inhibition period than vitamin E 10, ferulic acid
2, or BHT 12, indicating that the first three can
scavenge more radicals.
As recently observed by Koga et al. (1998) in emulsified methyl linoleate, Furaneol 9 proved less potent than
vitamin C 7.
Antioxidant Activity of Unboiled Wort. Although
linoleic acid is the major fatty acid in wort (50%,
1-100 mg/L), its concentration is negligible once the
wort is diluted several hundred times. Therefore, addition of wort to the oxidative system elicited a behavior
similar to that reported for pure antioxidants. A preliminary dose-response experiment, performed with

1132 J. Agric. Food Chem., Vol. 48, No. 4, 2000

Liegeois et al.
Table 2. Antioxidant Activity of an Industrial Wort (12
P) and a 1 mg/L Methanolic Solution of Hop Pellets or
Pale Malt Given in Reference Equivalents
mg/L equiv

wort

hop pellets

pale malt

Furaneol
cysteineHCl
glutathione
vitamin C
vitamin E
Trolox
BHT
ferulic acid
catechinH2O
quercetin2H2O
caffeic acid

1550
1312
1080
653
262
245
172
129
93
77
70

0.433
0.367
0.302
0.183
0.073
0.069
0.048
0.036
0.026
0.022
0.019

0.0141
0.0119
0.0098
0.0059
0.0024
0.0022
0.0016
0.0012
0.0008
0.0007
0.0006

Table 3. Efficiency of Four Successive Extractions with

7 mL of Methanol Applied to 1 g of Pale Malt or Hop
Pelletsa
no. of extractions with
7 mL of methanol

Tinh (min)

(A) Pale Malt

1
2
3
4

Figure 4. Comparison of the peroxyl radical scavenging

activity of various antioxidants. n represents the stoichiometric
number of peroxyl radicals trapped per molecule of additive
(*, taken as reference; Niki et al., 1986). Experimental conditions were as in Figure 3.

1
2
3
4

8.6b
8.4b
13.0b
13.0b
14.3b
14.1b
14.6b
16.8b
(B) Hop Pellets
28.6c
32.7c
39.3c
45.3c
45.1c
51.5c
49.1c
51.3c

8.5
13.0
14.2
15.7

30.7
42.3
48.3
50.2

a The final malt concentration in the assay is 133.3 mg/L; the

final hop concentration in the assay is 13.3 mg/L. b Assay duplicates. c Extraction duplicates.

Figure 5. Inhibition time (Tinh) as a function of (a) the

reciprocal of the final wort dilution in test and (b) the final
hop concentration in test. Experimental conditions were as in
Figure 3.

wort at different dilutions, showed a linear relationship

between the inhibition time and the reciprocal of wort
dilution (Figure 5a). On the basis of this first experiment, we chose an appropriate wort dilution: 400-fold
dilution in phosphate buffer for an initial gravity of 12
P. An inhibition time of 33.8 min was found with a
variation coefficient of 2.2% (quadruplicate assays).
The antioxidant efficiency of wort is expressed in
Table 2 in reference antioxidant equivalents according
to the following equation:

antiox activity (12 P wort, antiox equiv in mg/L) )

[Tinh (diluted wort sample) dilution factor
(400 in our case)]/S(ref antiox)

As estimated according to the method of Bishop

(1972), the total polyphenol content of the studied wort
was 107 mg/L. Compared to the antioxidant activity
given in polyphenol equivalents in Table 2, polyphenols
emerge here as the main contributors to the antioxidant
activity of wort.
Antioxidant Activity of Malt and Hops. Methanol
was chosen as a solvent for its capacity to dissolve both
liposoluble and more hydrophilic compounds. As depicted in Table 3A, the malt extraction procedure was
found to be fully satisfactory with four successive 7-mL
methanol aliquots (variation coefficient of 3.2% based
on triplicate extractions). Successive extractions were
also found to be more efficient than one extraction with
28 mL of methanol. Linoleic acid oxidation in the
presence of methanolic malt extracts confirms the
presence of peroxyl radical scavengers in malt. Here
again, the antioxidant activity was inversely proportional to the dilution ratio (data not shown).
The same method was further applied to methanolic
hop extracts. Figure 5b presents results for methanolic
solutions of CO2 hop extracts. On hop pellets, best
results were also obtained with four successive extractions with 7 mL of methanol [variation coefficient of
1.7% based on quadruplicate extractions (Table 3B].
As in the case of wort, the antioxidant activities of
methanolic solutions of hop pellets (1 mg/L) or pale malt
(1 mg/L) can be expressed in equivalents of various well-

Antioxidant Efficiency of Wort, Malt, and Hops

known antioxidants (Table 2) according to the following

equation:

antiox activity (1 mg/L malt or hop,

antiox equiv in mg/L) ) Tinh (133.3 mg/L malt or
13.3 mg/L hop in the test)/[S(ref antiox)
constant (133.3 for malt or 13.3 for hop)]
In this assay hop appeared as an oxidation inhibitor 30
times more efficient than the tested malt. This suggests
a promising means of optimizing the antioxidant activity
in the kettle: selecting hop cultivars with the highest
antioxidant activity. Further research is required, however, to identify the most active hop fractions.
Conclusion. We propose a new reliable, quick, lowcost spectrophotometric method for measuring the antioxidant activity of raw materials, worts, or beers. The
method, based on the inhibition of lipid oxidation,
provides a measure of how efficiently natural antioxidants protect against lipid oxidation in vitro. Oxidation
of exogenous linoleic acid by a thermal free radical
producer (AAPH) is followed by UV spectrophotometry
in a highly diluted sample. The high initial velocity of
the reaction makes it easy to estimate the extent to
which oxidation is delayed in the presence of antioxidants. Our method emerges as very useful for exploring
oxidative changes during the brewing process and
assessing the contribution of all raw materials. Applied
to wort, it confirms the predominant role of polyphenols.
For the first time, hops were found to display much
higher antioxidant activity than pale malt.
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Liegeois et al.
Received for review October 18, 1999. Revised manuscript
received January 14, 2000. Accepted January 14, 2000. Guillaume Lermusieau is grateful to the Interbrew-Baillet Latour
Foundation (Leuven, Belgium) for financial support.
JF9911242