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Molecules 2024, 29, 2370. https://doi.org/10.3390/molecules29102370 www.mdpi.com/journal/molecules
Article
Use of Botanical Ingredients: Nice Opportunities to Avoid
Premature Oxidation of NABLABs by Increasing Their ORAC
Values Strongly Impacted by Dealcoholization or Pasteurization
Margaux Simon, Hubert Kageruka and Sonia Collin *
Unité de Brasserie et des Industries Alimentaires, Louvain Institute of Biomolecular Science and Technology (LIBST),
Faculté des Bioingénieurs, Université Catholique de Louvain, Croix du Sud, 2 Box L7.05.07,
B-1348 Louvain-la-Neuve, Belgium; margaux.simon@uclouvain.be (M.S.); hubertk2@gmail.com (H.K.)
* Correspondence: sonia.collin@uclouvain.be
Abstract: Even when fresh, non-alcoholic, and low-alcoholic beers (NABLABs) exhibit significant staling
defects due to premature oxidation. In this study, the antioxidant power of eleven fresh commercial
NABLABs was assessed by means of three different assays: the oxygen radical absorbance capacity
(ORAC), the linoleic acid-induced oxidation (TINH), and the indicator time test (ITT). Only the first two
assays, both involving radicalar degradations initiated by AAPH, were found to correlate with each
other. NABLABs displayed lower ORAC values than conventional beers (on average, 6127 µmol eq.
Trolox/L), except for three samples made with special-colored malts or dry-hopped. Dealcoholization
was the step with the greatest impact on the ORAC value (up to a 95% loss) and on flavan-3-ols, sotolon,
and polyfunctional thiols, while pasteurization strongly affected color, TBA, and Strecker aldehydes.
ORAC assays applied to hop, alternative cereals, and various botanical ingredients indicated that mash-
ing with red sorghum, dry hopping/spicing, and wood maturation could bring the antioxidant power of
a NABLAB close to those of conventional beers. With an ORAC value not reached by any other tested
botanical ingredient (5234 µmol eq. Trolox/g), African Vernonia amygdalina leaves (traditionally used for
Rwandan Ikigage beers) emerged here as the best candidate.
Keywords: antioxidant power; ORAC; NABLABs; pasteurization; dark malts; Vernonia amygdalina
1. Introduction
Slowing down aroma staling to extend a beer’s shelf life remains one of the major chal-
lenges for the brewing industry [1,2]. Oxidation is often the primary contributor to flavor in-
stability [3]. Much effort has been devoted to minimizing oxygen uptake during brewing and
packaging [1,4–6]. Increasing antioxidant concentrations can also inhibit the effects of oxygen
by scavenging reactive oxygen species or free radicals, chelating transition metal ions (copper
and iron), decomposing peroxides, etc. [3,7–12]. A large number of assays have been pub-
lished in the literature for measuring antioxidant activity, some of them taking into account
more specific properties [7,11,13,14]. As many oxidative mechanisms can occur in a complex
matrix, it could be advised to combine several assays for beer investigations.
Interest was first concentrated on the oxidoreduction reactions (colorimetric or electro-
chemical methods), which could inform about the reducing power of wort and beer (e.g., 2,6-
dichlorophenolindophenol in the indicator time test (ITT) [15], iron dipyridyl complex [16],
redox potential [17], FRAP (ferric reducing antioxidant parameter) [9], and CUPRAC (cupric
reducing antioxidant capacity) [18]) (Figure 1a). Nowadays, it is accepted that reactive oxygen
species (ROS) such as hydroxyl radical HO° and superoxide radical O2°− are agents causing
beer damage. Therefore, most assays prefer measuring the free radical scavenging activity of
the medium (e.g., DPPH° reducing activity [19], ABTS° decolorization assay [20], superoxide
Citation: Simon, M.; Kageruka, H.;
Collin, S. Use of Botanical
Ingredients: Nice Opportunities to
Avoid Premature Oxidation of
NABLABs by Increasing Their
ORAC Values Strongly Impacted by
Dealcoholization or Pasteurization.
Molecules
2024, 29, 2370. hps://
doi.org/10.3390/molecules29102370
Academic Editor: Encarna
Gómez-Plaza
Received: 21 March 2024
Revised: 17 April 2024
Accepted: 15 May 2024
Published: 17 May 2024
Copyright: © 2024 by the authors. Li-
censee MDPI, Basel, Swierland.
This article is an open access article
distributed under the terms and con-
ditions of the Creative Commons At-
tribution (CC BY) license (hps://cre-
ativecommons.org/licenses/by/4.0/).
Molecules 2024, 29, 2370 2 of 17
scavenging activity in the xanthine/xanthine oxidase system [21], and the scavenging of the
hydroxyl radical in deoxyribose [21] or leucomethylene blue [22] assays) (Figure 1b).
In many cases (including the ORAC and TINH assays), peroxyl radicals are artificially
created by the thermal decomposition of 2,2′-azobis(2-methylpropionamidine) dihydrochlo-
ride (AAPH). A great advantage of the ORAC method (in which the radicalar degradation of
fluorescein is easily monitored by UV fluorescence) is its very high sensitivity [23]. Moreover,
ORAC values have been determined for a wide range of food matrices [24], for example, 5693
µmol eq. Trolox/100 g for red wine, 9645 µmol eq. Trolox/100 g for hazelnut, or 20,823 µmol
eq. Trolox/100 g for black chocolate. Another sensitive method in which the oxidation kinetic
of an aqueous dispersion of linoleic acid is followed was described by Liégeois et al. [25] as
more representative of what happens in a dispersed lipid matrix such as wort or beer. Prod-
ucts resulting from this peroxidation are the conjugated diene hydroperoxides, which absorb
at 234 nm. When antioxidants are present in beer, oxidation is delayed, and the resulting inhi-
bition period (TINH) is determined. In order to assess also the pro-oxidant activity of the me-
dium (iron cations, etc.), radicals in beer can still be monitored by electron spin resonance
(ESR) or luminescence analysis (Figure 1c) [26–28].
(a)
(b)
(c)
Figure 1. Examples of antioxidant activity assays used in the brewing field. * Selected in the present study.
In conventional beers, both endogenous and exogenous antioxidants can play a crucial
role in delaying or preventing oxidative damage [2,29]. Natural antioxidants originate mainly
Molecules 2024, 29, 2370 3 of 17
from barley malt and kettle hopping [11,30]. Both contribute to beer polyphenols [31–33],
while only special malts bring significant amounts of reductones and melanoidins
[3,8,11,34,35]. Therefore, in most cases, the total polyphenol content of a beer correlates directly
(R2 = 0.8) with its antioxidant activity (contribution of 55–88%) [1,8,31,36]. Unfortunately, oxi-
dation products derived from polyphenols can also negatively affect color and colloidal sta-
bility [8,37,38]. During fermentation, yeast also produces antioxidants, mainly sulfites
(through the conversion of sulfates, methionine, or cysteine) and glutathione [3,8,26,39]. More-
over, sulfites and ascorbic acid can be added to the bottle as exogenous antioxidants. In addi-
tion to their antimicrobial activity, sulfites consume bottled oxygen, thus protecting other an-
tioxidant fractions [5,8,35]. Other antioxidants present in beer at very low levels include carot-
enoids and tocopherols [31,32,40], saponarin, and hordatines A-C [8,41]. Some additional an-
tioxidants may come from dry hopping [37], spices/herbs (e.g., hibiscus, juniper, lemon balm,
etc. [42,43]), fruits (e.g., cherry juice and goji berry [44,45]), flavorings and colorings [7], or al-
ternative raw materials (e.g., sorghum and buckwheat [46,47]).
Whatever the process used (dealcoholization, cold contact, special yeast, etc.), non-alco-
holic and low-alcoholic beers (NABLABs, NAB ≤ 0.5% ABV and LAB 0.5–1.2% ABV in most
European countries) are usually brewed at lower original extract levels, leading to lower total
polyphenol contents (75–366 mg GAE/L versus 875 mg GAE/L for bock beer) [30,48,49] and
lower melanoidin levels (0.58 mg/L versus 1.49 mg/L for dark beer) [11,34,35]. Furthermore,
the dealcoholization and stronger pasteurization (at least 50 UP versus 15 UP for conventional
beers [50,51]) usually applied to NABLABs can also degrade the antioxidant capacity of the
medium. Dealcoholized beers have been found to display about a third of the antioxidant
power of bock beers (1525 versus 4663 µmol Fe2+/L as determined by FRAP assay) [31].
Unsurprisingly, fresh NABLABs often suffer from premature oxidation (Figure 2). This
has an impact on both bitterness and astringency, by enhancing isohumulone and flavan-3-ol
oxidation [38]. trans-Isohumulones are known to be the most degraded fraction, given their
propensity to be converted to tricyclohumols. For cis-isohumulones, oxidative degradation to
alloisohumulones is the main concern [52,53]. For flavan-3-ols, it is now recognized that the
oxidation of catechins to dehydrodicatechins increases color, while oligomer oxidation leads
to colloidal instability and astringency [38]. The odorants sotolon (curry), phenylacetaldehyde
(floral, honey), methional (boiled potato), and dimethyltrisulfide (onion) have recently been
detected at higher levels in such beers [54].
Figure 2. Oxidation of flavan-3-ols, isohumulones, and precursors of odorants, impacting color,
haze, astringency, bierness, and flavor.
Molecules 2024, 29, 2370 4 of 17
The aim of the present work was to compare the antioxidant power of eleven commercial
NABLABs with conventional beers. ORAC, TINH, and ITT values were related to levels of
various previously quantitated beer constituents. The impacts of both dealcoholization and
pasteurization on the ORAC value and aromas were further assessed on two pilot samples.
Lastly, to determine the feasibility of increasing NABLAB antioxidant activity, an ORAC assay
was applied to sorghum, spices, wood, and other promising botanical extracts in order to cal-
culate the amount required to reach in NABLABs an ORAC value similar to that of conven-
tional beers.
2. Results and Discussion
2.1. ORAC Values of Fresh NABLABs and Relationship to Color, Phenols, and Bitterness
As depicted in Table 1, almost all fresh NABLABs, whatever the process used, showed
significantly lower antioxidant power (on average 6127 µmol eq. Trolox/L) than a conventional
lager (10,171 µmol eq. Trolox/L), a dry-hopped beer (11,456 µmol eq. Trolox/L), or a Trappist
brown ale (12,332 µmol eq. Trolox/L). The relatively low densities of the worts commonly em-
ployed in NABLAB production (around 5 °P) most probably limit their polyphenol content
(43–150 mg/L, Table 1). Moreover, intrinsic antioxidants can be altered by dealcoholization
and pasteurization, procedures often applied to NABLABs.
Interestingly, beers E and K exhibited the highest values (11,637 and 9193 µmol eq.
Trolox/L, respectively), likely due to the use of special/colored malts known to contain antiox-
idant melanoidins [11]. As shown in Figure 3a, a correlation was observed between the ORAC
value and color (R2 = 0.81 if the red fruit wheat beer G was not included).
Beer B also reached a slightly higher value (7906 µmol eq. Trolox/L) because of its dry
hopping process. Hop is known to show a 30 times greater intrinsic antioxidant capacity than
pale malt [25,55], thanks to its very high level of polyphenols [37,55,56]. The total polyphenol
content, as already shown by other studies [8,41,57], appeared to contribute most to the anti-
oxidant power of each beer (42–100%; Table 1), with a major proportion attributed to flavan-
3-ols (catechin ORAC value = 11.2 µmol eq. Trolox/µmol) [1,31,41] and phenolic amino acids
(2.1 and 1.0 µmol eq. Trolox/µmol for tryptophan and tyrosine, respectively) [41,58]. No rela-
tionship was found here with polyphenols.
Surprisingly, we also observed a correlation between the ORAC value and the isohumu-
lone content (R2 = 0.77 without beer E whose cold contact process provided better protection
against oxidation, Figure 3b). There should be no direct causative link here, as isohumulones
(produced by isomerization in the boiling kettle from hop humulones) showed almost no an-
tioxidant activity (ORAC value = 0.1 µmol eq. Trolox/µmol; Table 1). Yet, the level of bitter
compounds depends on the amount of hop used, as does the level of polyphenols (which in-
directly elucidates this correlation).
Molecules 2024, 29, 2370 5 of 17
Table 1. Ethanol, color, isohumulones, phenols, and antioxidant activity (ORAC, TINH, and ITT values) determined for fresh NABLABs. Values in parentheses
give the contribution (%) of each fraction to the measured ORAC value, determined on the basis of analyses performed on four reference standards (0.1, 11.2, 2.1,
and 1.0 µmol eq. Trolox/µmol for isohumulone, catechin, tryptophan, and tyrosine).
Beer Samples Ethanol (% v/v) Color (°EBC)
Isohumulones (mg/L) Phenols (mg/L) Antioxidant Activity
cis- trans- Total
polyphenols
(+)-Catechin
(−)-Epicatechin
Procyanidin B3
Tryptophan Tyrosine
ORAC value
(µmol eq.
Trolox/L)
TINH (min)
ITT
(min)
NABLABs Special yeasts
A 0.5 6.9 5.5 e (<0.1) 1.7 e,f (<0.1) 43 f (82) 1.0 f (1.9) 0.3 f (0.6) 1.1 c (1.1) 2.7 g (1.3) 4.8 i (1.3) 2014 g 15 f 0.7 d
B 0.3 9.3 16.0 c (<0.1) 4.7 b (<0.1) 124 d (60) 1.5 e (0.7) 1.0 b (0.5) 2.0 b (0.5) 15.9 d (2.0) 25.5 f (1.7) 7906 c 29 c 14 b
C
0.2
4.7
5.8
e
(<0.1)
0.3
g
(<0.1)
135
d
(>100)
3.5
a
(3.0)
1.2
a
(1.0)
1.0
c
(0.4)
15.4
e
(3.5)
26.0
e
(3.2)
4428
e
25
d
14
b
Limited fermentation or cold contact
D 0.8 5.6 4.3 e (<0.1) 0.2 g (<0.1) 56 f (64) 2.1 b (2.3) 0.9 c (1.0) 1.2 c (0.7) nd 12.4 h (2.0) 3382 f 20 e 66 a
E 0.1 19.7 10.9 d (<0.1) 3.6 c (<0.1) 149 d (49) 1.1 f (0.4) 0.8 c,d (0.3) 0.7 c (0.1) 16.0 d (1.4) 35.0 c (1.6) 11,637 a 43 b 0.8 d
Vacuum dealcoholization
F <0.1 8.0 12.0 d (<0.1) 2.6 d,e (<0.1) 84 e (47) 1.4 e (0.8) 0.8 c,d (0.5) 0.9 c (0.3) 17.6 b (2.6) 33.0 d (2.6) 6865 d 21 e 4 c
G <0.1 17.8 9.4 d (<0.1) 0.5 g (<0.1) 171 c (>100) 1.6 d,e (1.1) 0.6 e (0.4) 0.9 c (0.3) 0.7 h (0.1) 3.4 j (0.3) 5420 e 28 c 3 c
H
0.1
7.8
10.7
d
(<0.1)
1.8
e,f
(<0.1)
68
e,f
(52)
1.8
c,d
(1.4)
0.3
f
(0.2)
1.0
c
(0.4)
16.5
c
(3.3)
38.0
a
(4.1)
5047
e
20
e
0.7
d
I <0.1 7.0 15.3 c (<0.1) 1.2 f,g (<0.1) 50 f (42) 0.9 f (0.7) 0.3 f (0.2) 0.7 c (0.3) 8.6 f (1.9) 24.1 g (2.8) 4621 e 20 e 12 b
J
<0.1
10.9
18.9
b
(<0.1)
3.2
c,d
(<0.1)
269
b
(>100)
1.9
b,c
(1.1)
0.7
d
(0.4)
1.1
c
(0.3)
nd
nd
6890
d
24
d
5
c
Filtration dealcoholization
K 0.5 13.9 28.9 a (<0.1) 6.2 a (<0.1) 304 a (>100) 3.6 a (1.5) 1.0 b (0.4) 2.6 a (0.5) 19.0 a (2.1) 35.5 b (2.1) 9193 b 51 a 0.7 d
Conventional beers
Lager 5.2 5.7
10,171 b
Dry-hopped
6.0
18.2
11,456
a
Trappist brown
beer 9.0 60.0 12,332 a
Within a column, values with different letters are significantly different (p < 0.05) according to the Student–Newman–Keuls test; nd: not detected in sample by UPLC.
Molecules 2024, 29, 2370 6 of 17
Figure 3. Correlations for fresh NABLABs between ORAC value and (a) color or (b) total isohumu-
lone concentration (leer: name of sample and cross: sample exclude from correlation).
2.2. Comparison of the ORAC Assay with Two Other Antioxidant Assays Used on NABLABs
In parallel with the ORAC assay, two additional antioxidant power measurements
were applied to the eleven NABLABs: TINH, which also involves a radicalar reaction ini-
tiated by AAPH (linoleic acid used here as substrate instead of fluorescein), and the ITT
test, which involves a simpler redox reaction (Figure 1). Whatever the method used, the
antioxidant power of NABLABs remained poor (Table 1). Not surprisingly, a correlation
was found only between the ORAC and TINH values (R2 = 0.70, Figure 4a). The non-rad-
icalar ITT test showed no correlation with the ORAC value (R2 = 0.13, Figure 4b).
Figure 4. Correlations for fresh NABLABs between ORAC values and (a) TINH or (b) ITT values
(leer: name of sample).
2.3. Impact of NABLAB Dealcoholization and Pasteurization on ORAC Values, Thermal Indica-
tors, Bier Compounds, Phenols, and Aromas
Two pilot blond beers (A and B; initially at 5.6% and 4.7% ethanol (v/v), respectively)
were subjected to vacuum distillation (industrial NABLAB production operating at 35–40
°C and 100 mbar) and tunnel pasteurization (50 UP for A and 90 UP for B). Antioxidant
activity (ORAC), thermal indicators (color and TBA), bier compounds, phenols, stale
odorants, and hoppy polyfunctional thiols were determined before dealcoholization (BD),
after dealcoholization (AD), and after pasteurization (AP) (Table 2).
Molecules 2024, 29, 2370 7 of 17
Table 2. Antioxidant activity, color, thermal load, bier compounds, phenols, and aromas in two
pilot samples before dealcoholization (BD), after dealcoholization (AD), and after pasteurization
(AP).
Sample A Sample B
BD AD AP BD AD AP
Antioxidant Activity
ORAC value (µmol eq. Trolox/L) 8238 a 3372 b 1042 c 7204 a 355 b 291 b
Thermal indicators
Color (°EBC) 9.0 7.0 9.5 6.5 5.5 8.5
TBA
35
c
43
b
60
a
12
c
14
b
36
a
Bitter compounds
Alloisohumulones (mg/L eq. isohumulones) 0.2 a 0.4 a 0.6 a 0.1 b 0.2 a,b 0.3 a
cis-Isohumulones (mg/L) 9.3 a 8.6 a,b 7.6 b 11.4 a 10.0 b 8.8 c
trans-Isohumulones (mg/L) 6.0 a 5.2 a,b 3.5 b 6.0 a 5.1 b 4.8 b
Phenols (mg/L)
Total polyphenols 144 a 134 a 148 a 154 a 89 b 107 a
Catechin
2.0
a
1.2
b
0.5
c
3.2
a
1.5
b
1.3
b
Epicatechin 1.0 a 0.6 a 0.5 a 1.4 a 0.8 a 0.6 a
Procyanidin B3 2.0 a 1.4 a 1.4 a 1.8 a 1.2 a 0.9 a
Stale odorants and pleasant polyfunctional thiols (µg/L)
Sotolon (thr. = 0.8 µg/L) 0.2 c 0.6 b 0.9 a 0.1 c 0.3 b 1.4 a
Methional (thr. = 0.5 µg/L)
0.5
b
0.5
b
1.3
a
0.7
b
0.6
b
2.5
a
Phenylacetaldehyde (thr. = 5.4 µg/L) 7.0 b 8.1 b 28.4 a 7.2 b 5.9 b 10.4 a
3SHol (thr. = 0.055 µg/L)
4.3
a
0.3
b
nd
nd
nd
nd
3SHA (thr. = 0.005 µg/L) nq nd nd 0.3 a 0.1 b 0.1 b
3S4MPol (thr. = 0.07 µg/L) 0.3 a nd 0.3 b 0.7 a 0.4 b 0.3 b
3S4MPA (thr. = 0.16 µg/L) 0.9 a nd 0.5 b 0.7 a nd nd
thr. = perception threshold, nd = not detected in sample, nq = not quantifiable; within a line, values
with different letters are significantly different (p < 0.05) according to the Student–Newman–Keuls test.
The dealcoholization of either sample led to a huge ORAC value decrease (loss of up to
59% for sample A and 95% for sample B). The antioxidant activity decreased further through
pasteurization, leading to only 1042 and 291 µmol eq. Trolox/L (which is even lower than the
values found in the eleven investigated commercial NABLABs, probably due to the lower-
scale experiments). The data of previous chemiluminescence studies confirm an increase in
the level of oxidation in conventional beers (a five times higher OH-radical signal intensity)
after pasteurization [59–62], whereas, surprisingly, Lund et al. found an increased antioxidant
capacity, likely due to formation of Maillard compounds [2,59].
As previously reported by Callemien et al. [63], total polyphenol values are not good in-
dicators of intrinsic oxidative changes in flavan-3-ol chemical structures (loss of only 10 mg/L
after dealcoholization in sample A). On the other hand, catechin and procyanidin B3 dropped
strongly from 3.2 to 1.3 mg/L and from 1.8 to 0.9 mg/L in sample B, respectively, clearly evi-
dencing the occurrence of oxidation through both dealcoholization [64] and pasteurization.
Our two thermal indicators showed that dealcoholization had little impact on heat-re-
lated reactions, compared to pasteurization: Specifically, an increase of 2.5–3 °EBC and 17–22
TBA was observed between AD and AP, whereas color slightly decreased during dealcoholi-
zation [64]. A higher degree of pasteurization (50–90 UP) and, consequently, a greater thermal
load are required for NABLABs. Colored compounds resulting from Maillard reactions are
logically formed at this step.
Oxidation of cis- and trans-isohumulones occurred during both dealcoholization and pas-
teurization (a loss of cis-isohumulones up to 2.6 mg/L, in sample B), in parallel with the syn-
thesis of their oxidative degradation products such as alloisohumulones [53,65], reaching 0.2–
0.4 mg/L isohumulone equivalents (other by-products, including tricyclohumols, were not de-
termined here).
Among the stale odorants often detected in NABLABs even when fresh, sotolon was
found at 0.9–1.4 µg/L after pasteurization (values significantly above its sensory threshold of
0.8 µg/L in both samples). In both cases, dealcoholization already slightly increased the
Molecules 2024, 29, 2370 8 of 17
amount of this oxidative aroma. On the other hand, only pasteurization caused a marked in-
crease in methional and phenylacetaldehyde (oxygen not required for thermal Strecker deg-
radation).
In contrast, most fresh hoppy/citrus polyfunctional thiols were lost upon dealcoholiza-
tion (3SHol dropped from 4.3 µg/L to 0.3 µg/L in sample A). One should note, however, that
some can be added at the time of pasteurization, most probably coming from cysteinyl pre-
cursors.
2.4. Potential to Increase NABLAB ORAC Values by Using Sorghum, Vernonia amygdalina, Spices,
or Wood Chips
In order to assess how to enhance the NABLAB antioxidant capacity, ORAC values of
alternative cereals, spices, other botanical ingredients, and wood chips were determined, and
for each, the quantity needed to achieve the antioxidant power of a conventional beer in
NABLABs was calculated (Table 3). For comparison, ORAC values of ascorbic acid and potas-
sium metabisulfite (KMS) (antioxidants often used in breweries; Table 3) show that extrava-
gant spiking would be required, both with KMS (386 g/hL = 3860 mg/L for a maximum of 20
mg/L allowed) and ascorbic acid (87 g/hL= 870 mg/L; compared to the 30–50 mg/L amount
usually added).
Table 3. ORAC values of brewing antioxidants, alternative cereals, botanical ingredients, spices, and
wood chips, and amounts required to achieve the antioxidant power of a conventional beer.
ORAC Value
(μmol eq. Trolox/g)
Amount Required (g/hL Beer for 100% Recovery) to Bring the
ORAC Value of a NABLAB (on average 6127 μmol eq.
Trolox/L) to the Antioxidant Power of a Conventional Beer
(on average 11,320 μmol eq. Trolox/L)
Common brewing antioxidants
Ascorbic acid 5982 a 87
Potassium metabisulfite
1344
c,d
386
Non-conventional cereals
Unmalted white sorghum 24 k 21,638
Unmalted red sorghum 390 h,i,j,k 1332
Rwandan traditional malted
red sorghum 855 d,e,f,g,h 607
Hops
Citra T-90 pellets 615 e,f,g,h,i,j 844
Saaz T-90 pellets 1101 c,d,e 472
Spices/herbs and other botanical ingredients
Coriander 273 i,j,k 1902
Orange peel 510 f,g,h,i,j,k 1018
Cardamom 56 k 9273
Licorice 212 i,j,k 2450
Cinnamon
907
d,e,f,g,h
573
Ginger 721 e,f,g,h,i 720
Hibiscus 477 g,h,i,j,k 1089
Vernonia amygdalina leaves 5234 b 99
Vernonia amygdalina flowers
1457 c 356
Wood chips
Oak 980 c,d,e,f,g 530
Acacia 1036 c,d,e,f 501
Mulberry 148 j,k 3509
Within a column, values with different leers are significantly different (p < 0.05) according to the
Student–Newman–Keuls test.
Molecules 2024, 29, 2370 9 of 17
As shown here with the Citra hop sample (one of the varieties richest in flavanoids,
along with Saaz [56]), dry hopping above 850 g/hL would effectively boost the ORAC
value of a NABLAB into the target range (this was only partially achieved in the Belgian
dry-hopped commercial beer B, with its 7906 µmol eq. Trolox/L). In the United States, hop
is often used up to 500–1000 g/hL (2000 g/hL even reached for NEIPAs).
Interestingly, Vernonia amygdalina leaves (used in some traditional Rwandan sor-
ghum beers known as Ikigage [66]) exhibited the highest antioxidant power (5234 µmol
eq. Trolox/g). Only 99 g/hL would be needed (if no loss occurs through the process) to
reach the antioxidant capacity of a conventional beer. Malted red sorghum (855 µmol eq.
Trolox/g) should also make it possible to substantially increase the antioxidant activity of
NABLABs (only 5–10% of barley malt should be here replaced by sorghum malt). Red
sorghum is known to contain exceptional amounts of flavan-4-ols, 3-deoxyanthocya-
nidins, flavones, and flavan-3-ols (up to hexamers) [67]. As an additional advantage, this
cereal also contains lile beta-amylase, the enzyme that brewers avoid in NABLAB wort
mashing (lower maltose content). Among the spices/herbs investigated here, the best can-
didates were cinnamon, ginger, and orange peel (ORAC values of 907, 721 and 510 µmol
eq. Trolox/g, respectively), although more than 500 g/hL would be required during boiling
or fermentation/maturation to reach the antioxidant activity of a conventional beer (prob-
ably too much in terms of flavor; generally added from 5 to 225 g/hL, depending on the
type of spice). With their 1036 and 980 µmol eq. Trolox/g, acacia and oak chips, possibly
added during maturation, also appear as reasonable candidates (500 g/hL often used by
brewers for wood-aged beers [68]).
3. Materials and Methods
3.1. Chemicals
Acetic acid, acetone, acetonitrile, ammonia solution 28–30%, anhydrous sodium sul-
fate, citric acid monohydrate, dichloromethane, dipotassium hydrogen phosphate trihy-
drate, ethanol absolute 99%, formic acid, hydrochloric acid 37%, methanol, potassium di-
hydrogen phosphate, potassium hydroxide, sodium chloride, and sodium hydroxide
were purchased from VWR International (Leuven, Belgium). 2-Acetylthiophene, ammo-
nium iron (III) citrate 16%, 2,2′-azobis(2-methylpropionamidine) dihydrochloride
(AAPH), Amberlite XAD-2 resin, boric acid, carboxymethylcellulose sodium salt, >98% L-
cysteine hydrochloride monohydrate, (±)-catechin hydrate, decane, 2,6-dichlorophenolin-
dophenol, 6 mL Discovery Ag-ion SPE tube, (−)-epicatechin, fluorescein sodium salt, lin-
oleic acid 99%, methional, nonadecane, phenylacetaldehyde, Sephadex LH-20 resin, soto-
lon, 3-sulfanylhexan-1-ol (3SHol), 3-sulfanylhexyl acetate (3SHA), 6-sulfanylhexan-1-ol, 2-
thiobarbituric acid, titriplex III, Trolox® ((±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-
carboxylic acid), tryptophan and tyrosine were purchased from Sigma-Aldrich (Overijse,
Belgium). Isohumulone standard was purchased from Labor Veritas Co. (Zürich, Swier-
land). Procyanidin B3 and (+)-taxifolin standards were from Extrasynthèse (Genay,
France). AccQ•Tag Ultra Reagent derivatization (6-aminoquinolyl-N-hydroxysuccin-
imidyl carbamate, AQC), AccQ•Tag Ultra Eluent I, AccQ•Tag Ultra Eluent II, and
AccQ•Tag Ultra borate buffer were purchased from Waters Corporation (Milford, CT,
USA). Milli-Q water was used (Millipore, Bedford, MA, USA).
3.2. Samples
Eleven commercial NABLABs were investigated: Star Light (A; special blond), Ener-
gibajer (B; dry-hopped), Pico Bello (C; dry-hopped), Leopold 7 Road Trip (D; sour beer),
Palm N.A. (E; amber), Maes 0.0% (F; lager), Hoegaarden rosée 0.0% (G; red fruit white
beer), Carlsberg 0.0% (H; lager), Jupiler 0.0% (I; lager), Leffe Blonde 0.0% (J; abbey beer),
and Brugse Sport Zot alcoholvrij (K; special blond). The beers, either received from brew-
ers or bought at Belgian markets (freshly released), were analyzed in duplicate. Pilot sam-
ples of two beers (A and B), taken both before dealcoholization (BD) and after
Molecules 2024, 29, 2370 10 of 17
dealcoholization (AD), as well as after pasteurization (AP; 50 UP for A and 90 UP for B),
were provided by AB-Solutions (Courcelles, Belgium) and brewers. Spices/herbs (corian-
der, orange peel, cardamom, licorice, cinnamon, ginger, and hibiscus) were supplied by
Fagron (Nazareth, Belgium), and wood chips (oak, mulberry, and acacia) were obtained
from Wilhelm Eder GmbH (Bad Dürkheim, Germany). Vernonia amygdalina leaves and
flowers were harvested in Rwanda.
3.3. Standard Analyses on NABLABs and Pilot Samples
Prior to analysis, beers were degassed by shaking and filtered through paper filters
(MN 614 ¼ Macherey-Nagel, Düren, Germany). The alcohol content was determined with
DM4500 apparatus (Anton Paar GmbH, Graz, Austria), and color was analyzed by means
of Analytica-EBC 9.2. and 9.6 [69]. TBA (thiobarbituric Acid Index) was analyzed accord-
ing to the ASBC method Wort 21 [70].
3.4. Antioxidant Assays
The solid matrices (1 g), after grinding, were first extracted with 10 mL of a mixture
of acetone/water/acetic acid (70:29.5:0.5, v/v/v) and centrifuged for 15 min at 3000 rpm.
The extraction and ORAC analysis were conducted in duplicate.
3.4.1. ORAC Values of NABLABs, Chemical Standards, Pilot Samples, and Botanical Ex-
tracts
The ORAC procedure with fluorescein as a “fluorescent probe” (substrate) was car-
ried out at 37 °C on an automated 96 white opaque wells plate reader (Synergy HT, Bio-
Tek, Winooski, VT, USA) working at an excitation wavelength of 485 nm and an emission
wavelength of 520 nm. The reaction was started by the thermal decomposition of AAPH.
Working solutions of fluorescein (55 nM), AAPH (153 µM), and Trolox® (200 µM) were
freshly prepared in phosphate buffer (75 mM, pH 7.4) from stock solutions stored under
refrigeration conditions. In each well, 250 µL of fluorescein and 25 µL of the sample (suit-
able dilution to prepare in advance), blank, or standard (Trolox® at 8, 16, 24, 32, and 40
µM) were added. The plate was then heated to 37 °C for 10 min prior to the addition of 25
µL of AAPH. The fluorescence was measured immediately and every minute for 50 min.
The ORAC values, expressed as µmol Trolox equivalents/g fresh mass (or /L for liquid
extracts), were calculated with the following equation: ORAC value = (AUCsample −
AUCblank)/(AUCTrolox − AUCblank) × Trolox® concentration (µM) × dilution factor with AUC
= area under fluorescence curve.
3.4.2. TINH Values of NABLABs
The antioxidant activity was determined as the inhibition times of linoleic acid oxi-
dation induced in an aqueous solution by the free radical initiator AAPH [25]. Briefly, 30
µL of the 16 mM linoleic acid dispersion (in borate buffer 0.05 M, pH 9) was added to the
UV cuvee containing 2.81 mL of phosphate buffer (0.05 M, pH 7.4), prethermostated at
40 °C. The oxidation reaction was initiated at 37 °C under air by the addition of 150 µL of
40 mM AAPH solution (in phosphate buffer). Oxidation was carried out in the presence
of 10 µL of beer samples. In the assay without antioxidants, lipid oxidation was measured
in the presence of the same level of methanol. The oxidation rate at 37 °C was monitored
by recording the increase in absorption at 234 nm caused by conjugated diene hydroper-
oxides. A Shimadzu UV–visible 240 spectrophotometer (Antwerp, Belgium) equipped
with an automatic sample positioner allowed for the analysis of six samples every minute.
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). Therefore, its
absorbance measured in a separate cuvee in the absence of linoleic acid was subtracted
from each experimental point. The inhibition time (TINH) was estimated with Microsoft
Molecules 2024, 29, 2370 11 of 17
Excel (Microsoft 365 version 2404) and Geogebra Classic software (version 6.0.841.0) as
the point of intersection between the tangents to the inhibition and propagation phase
curves.
3.4.3. ITT Values of NABLABs
The ITT assay measures the discoloration time of an indicator, 2,6-dichlorophenolin-
dophenol (DCPIP, 1450 mg/L), which is blue in its oxidized form and turns colorless when
reduced by antioxidants in beer. First, four samples were prepared: 50 mL water with pH
adjusted to that of beer + 250 µL DCPIP (comparator solution); 10 mL beer + 250 µL DCPIP
(indicator solution); beer; and distilled water. Subsequently, 10 mL of each solution was
placed in a Hellige’s comparator. The comparator solution, with a dilution resembling
80% DCPIP discoloration, was introduced into the left-hand lens of the comparator. This
was positioned in front of the tube containing the beer to simulate the turbidity present in
the indicator solution on the right. The DCPIP indicator, influenced by the antioxidants in
beer, was gradually reduced and discolored. The time required for the indicator solution
to reach the same discoloration as the comparator solution was then measured.
3.5. Analyses of Bier Compounds in NABLABs and Pilot Samples by High-Performance Liquid
Chromatography–Ultraviolet Detection (HPLC-UV)
Beer samples were degassed by shaking and diluted twice in methanol. After 15 min,
the mixture was filtered through a Chromafil polyester filter (0.45 µm, Macherey-Nagel,
Düren, Germany). Separation was performed on two C8 columns in tandem: the Zorbax
Eclipse XDB-C8 150 × 4.6 mm, 5 µm, and the Zorbax Eclipse XDB-C8 150 × 4.6 mm, 3.6 µm
(Agilent Technologies, Santa Clara, CA, USA), using the binary solvent system of Analyt-
ica EBC method 9.47 [69] with A: methanol; B: 1% aqueous citric acid solution (pH 7.0)–
acetonitrile (70:30, v/v). Gradient elution was as follows: 15% A for 5 min, increasing A to
80% over 25 min, and 80% A for 3 min. The column temperature was kept at 35 °C, the
flow rate at 1.0 mL/min, and the injection volume was 50 µL. Chromatograms were rec-
orded throughout elution with the Empower software version 2002 (Build 1154, Waters
Corporation, Milford, CT, USA). The retention time and absorption spectrum of isohumu-
lones were obtained by injection of standards. An absorbance wavelength of 270 nm was
chosen for isohumulone and alloisohumulone quantitation (absorbance spectrum λmax =
228 and 280 nm [65]). Quantitation was performed using a single-point calibration, as sug-
gested by the EBC method 9.47 [69].
3.6. Phenols Quantitation in NABLABs and Pilot Samples
3.6.1. Total Polyphenol Measurement
Total polyphenol content was analyzed according to Analytica EBC method 9.11 [69].
3.6.2. Catechin, Epicatechin, and Procyanidin B3 Determination by HPLC-UV
Beer flavan-3-ols (catechin, epicatechin, and procyanidin B3) were extracted on Se-
phadex LH-20 resin. Briefly, 3 g of resin packed in a 12 mL filtration tube SPE with a pol-
yethylene frit was preconditioned for 4 h with methanol–water (30:70, v/v). The flux was
set at 0.5 mL/min. After loading 50 mL of degassed beer containing 2.8 mg/L of IST ((+)-
taxifolin), the column was washed with 40 mL of methanol–water (30:70, v/v). Flavan-3-
ols were recovered with 70 mL of acetone–water (70:30, v/v). The eluate was concentrated
to dryness by vacuum rotary evaporation (35 °C) and dissolved in 2 mL of acetonitrile–
water (30:70, v/v). The extracts were kept at −80 °C prior to analysis.
An Agilent 1200 Series liquid chromatography system (Agilent Technologies, Santa
Clara, CA, USA) equipped with an autosampler, a quaternary pump, and a UV detector
set at 280 nm was used. A 150 × 2.1 mm, 3 µm C18 Prevail column (HICHROM, Deerfield,
IL, USA) was used at a flow rate of 0.2 mL/min. Chromatographic separation was obtained
using a multilinear gradient of water containing 0.1% formic acid (A) and acetonitrile
Molecules 2024, 29, 2370 12 of 17
containing 0.1% formic acid (B). Gradient elution was 97–91% A, 0–5 min; 91–85% A, 5–30
min; 85–67% A, 30–60 min; 67–0% A, 60–70 min; 0–97% A, 70–75 min; and then return to
the initial conditions for 15 min. Ten microliters of beer extract were injected into the col-
umn kept at 25 °C. Chromatograms were recorded throughout elution using ChemStation
software (version B.04.03). Quantitation was achieved using the calibration curves (rela-
tive to the IST).
3.6.3. Tryptophan and Tyrosine Quantitation by Ultra-Performance Liquid Chromatog-
raphy–UV Detection (UPLC-UV)
Briefly, 10 µL of a degassed beer sample, filtered through a Chromafil polyester filter
(0.22 µm, Macherey-Nagel, Düren, Germany), was mixed with 70 µL of borate buffer and
20 µL of AQC derivatization reagent. The mixture was then heated at 55 °C for 10 min. An
ACQUITY UPLC liquid chromatography system (Waters Corporation, Milford, CT, USA),
equipped with a degasser, an autosampler, an oven, a quaternary pump, and a UV detec-
tor set at 210 nm was used. Separation was carried out on ACQUITY UPLC BEH C18 (100
× 2.1 mm, 1.7 µm column—Waters Corporation) at a flow rate of 0.65 mL/min, with a
mixture of A (Eluent I), B (10% Eluent II in water), C (water), or D (Eluent II). Gradient
elution was as follows: 0.0–0.29 min, 10–9.9% A and 90–90.1% C; 0.29–5.49 min, 9.9–9% A,
0–80% B, and 90.1–11% C; 5.49–7.10 min, 9–8% A, 80–15.6% B, 11–57.9% C, and 0–18.5%
D; 7.10–7.30 min, 8% A, 15.6% B, 57.9% C, and 18.5% D; 7.30–7.69 min, 8–7.8% A, 15.6–0%
B, 57.9–70.9% C, and 18.5–21.3% D; 7.69–7.99 min, 7.8–4% A, 70.9–36.3% C and 21.3–59.7%
D; 7.99–8.59 min, 4% A, 36.3% C, and 59.7% D; 8.59–8.68 min, 4–10% A, 36.3–90% C, and
59.7–0% D; 8.68–10.20 min, 10% A and 90% C. One microliter of mixture was injected into
the column kept at 42 °C. Chromatograms were recorded throughout elution using Em-
power 2 software. Tryptophan and tyrosine identification was performed by the injection
of a commercial mixture of standards. Quantification was achieved using the calibration
curves.
3.7. Pilot Sample Aroma Extraction
3.7.1. XAD-2 Resin Extraction of Sotolon, Methional, and Phenylacetaldehyde, and Quan-
tification by Gas Chromatography-Electron-Impact Mass Spectrometry (GC-MS)
For apolar compounds extraction, 2 g of Amberlite XAD-2 resin was added to a 50
mL degassed beer sample containing 150 µL of 2-acetylthiophene (IST, 8 mg/L, final beer
concentration = 24 µg/L). For sotolon extraction, the pH of the beer was adjusted to 11.5
with sodium hydroxide. The two mixtures were shaken at 200 rpm for 2 h. The content of
the flask was then transferred into a glass column (60 × 1 cm, i.d.). For apolar aromas, the
resin was first rinsed with 4 × 50 mL of Milli-Q water to eliminate sugar and other water-
soluble substances. They were then eluted with 2 × 20 mL of bidistilled dichloromethane.
For sotolon, the eluate from the resin and the first 50 mL of resin washing water were
mixed before bringing the pH to 3.0 with hydrochloric acid. This aqueous phase was ex-
tracted three times with 40 mL of bidistilled dichloromethane (10 min, 2500 rpm). All ex-
tracts were then dried with anhydrous sodium sulfate, and 25 µL of decane or nonadecane
(for sotolon) solution (250 mg/L) was added as EST before concentration reached 500 µL
in a Danish– Kuderna at 45 °C (total concentration factor = 100). The final extracts were
stored at −80 °C until analysis by GC-MS.
One microliter of each aroma extract was analyzed with an Agilent Technologies 7890
NB Gas Chromatograph System equipped with a splitless injector (250 °C). Apolar com-
pounds were separated using a wall-coated open tubular (WCOT) apolar capillary col-
umn (CP-Sil 5 CB, 50 m × 0.32 mm, 1.2 µm). The oven temperature was programmed to
rise from 36 to 85 °C at 20 °C/min, then to 145 °C at 1 °C/min, and to 250 °C at 3 °C/min,
and then held for 30 min. Sotolon was analyzed with a WCOT polar capillary column
(FFAP CB, 25 m × 0.32 mm, 0.3 µm). The oven temperature was programmed to rise from
36 to 85 °C at 20 °C/min, then to 145 °C at 1 °C/min, followed by 160 °C at 3 °C/min, and
Molecules 2024, 29, 2370 13 of 17
230 °C at 3 °C/min, and then held for 30 min. The carrier gas was helium, and the pressure
was set at 100 kPa (50 kPa for sotolon). The column was connected to a quadrupole mass
spectrometer (Agilent 5977 MSD) operating in single-ion monitoring (SIM) mode with
electron ionization at 70 eV. The following m/z values were monitored: 111 and 126 for 2-
acetylthiophene, 71 and 85 for decane and nonadecane, 91 and 120 for phenylacetalde-
hyde, 104 and 76 for methional, and 83 and 128 for sotolon. Chromatograms were rec-
orded throughout elution (Agilent OpenLab software version 2.1 used). Calibration
curves (with areas relative to IST) were constructed for each compound, and the following
equation was used for quantitation of compound A: concentration of A (in µg/L) = IST
concentration (in µg/L) × (A area/IST area) × (IST response coefficient/A response coeffi-
cient). The IST relative recovery factor was set at 1 for all compounds.
3.7.2. Ag Selective Extraction of Polyfunctional Thiols, and Quantification by Gas Chro-
matography–Pulsed-Flame Photometric Detection (GC-PFPD)
Briefly, 2 µg/L 6-sulfanylhexan-1-ol was added as IST to 150 mL beer, which was then
saturated with NaCl and stirred with 50 mL dichloromethane for 15 min. The mixture was
centrifuged at 4500 rpm for 15 min. The recovered organic phase was loaded onto a Dis-
covery Ag-ion SPE cartridge conditioned beforehand with 10 mL dichloromethane. The
cartridge was rinsed with 10 mL dichloromethane, then with 20 mL acetonitrile, and fi-
nally with 10 mL ultrapure water (reversed cartridge in this last case). Free thiols were
released from the Ag cartridge by percolating 20 mL washed cysteine solution (4 × 20 mL
dichloromethane for washing 215 mg cysteine in 20 mL water). The eluent was extracted
twice with bidistilled dichloromethane (5 mL for 5 min and 10 mL for 10 min). The result-
ing organic phase was dried on anhydrous sodium sulfate and concentrated to 250 µL in
a Danish–Kuderna distillation apparatus and to 70 µL on a Dufton column at 45 °C. 2-
Acetylthiophene was added as EST (0.5 mL at 200 µg/L added before concentration).
One microliter of free thiol extract was analyzed with an Agilent 6890N gas chro-
matograph equipped with a splitless injector maintained at 250 °C. Compounds were an-
alyzed with WCOT apolar capillary column (CP-Sil 5 CB, 50 m × 0.32 mm, 1.2 µm). The
helium pressure was set at 90 kPa. The oven temperature was programmed to increase
from 36 to 85 °C at 20 °C/min, then to 145 °C at 1 °C/min, and finally to 220 °C at 3 °C/min,
and was held for 30 min. The column was connected to the OI Analytical PFPD detector
(model 5380, combustor internal diameter: 2 mm). The following parameters were se-
lected for the PFPD detector: temperature, 250 °C; voltage, 600 V; gate width, 18 ms; gate
delay, 6 ms; trigger level, 400 mV; pulse frequency, 3.33 Hz. PFPD chromatograms were
recorded throughout elution. The ChemStation software was used to process the resulting
data. For all thiols, the IST-relative recovery factor was set at 1 (experimental values from
0.8 to 1.2, determined beforehand by standard addition). The following equation was used
for the quantitation of the commercially available standards 3SHol, and 3SHA: thiol con-
centration (in µg/L) = IST concentration (in µg/L) × (thiol area/IST area) × (IST weight re-
sponse coefficient/thiol weight response coefficient). For the commercially unavailable
standards, 3-sulfanyl-4-methylpentanol (3S4MPol), and 3-sulfanyl-4-methylpentyl acetate
(3S4MPA), the good equimolarity of the PFPD detector enabled us to set the IST-relative
molar response coefficients at 1 and to apply only the corrective molar weight ratio: thiol
concentration (in µg/L) = IST concentration (in µg/L) × (thiol area/IST area) × (thiol molar
weight /IST molar weight).
3.8. Statistical Analyses
All analytical measurements were carried out in duplicate. Multiple comparisons of
means were performed with Student–Newman–Keuls tests (JMP Program). Values shar-
ing no common leer are significantly different (p < 0.05).
Molecules 2024, 29, 2370 14 of 17
4. Conclusions
Commercial NABLABs displayed only half the antioxidant capacity of conventional
beers, except for three samples made with special-colored malts or dry-hopped. Surpris-
ingly, a correlation (R2 = 0.77) was observed between the ORAC value and the isohumu-
lone content, even though isohumulones showed almost no antioxidant activity. Phenolic
compounds contributed most to the antioxidant power of NABLABs. Dealcoholization
had a strong impact on the ORAC value, flavan-3-ols, sotolon, and hop polyfunctional
thiols, while pasteurization mainly affected color, TBA, and Strecker aldehydes. Red sor-
ghum mashing, dry hopping/spicing, and wood maturation could reasonably increase the
antioxidant power of a NABLAB to a level approaching those of conventional beers. In-
terestingly, Vernonia amygdalina leaves emerged here as the best candidate, with an ORAC
value (5234 µmol eq. Trolox/g) not reached by any other tested botanical ingredient.
NABLAB production trials should be carried out to confirm these findings.
Author Contributions: Conceptualization, M.S. and S.C.; methodology, M.S. and H.K.; software,
M.S.; validation; S.C.; formal analysis, M.S. and H.K.; investigation, M.S. and H.K.; resources, S.C.;
data curation, M.S. and H.K.; writing—original draft preparation, M.S. and S.C.; writing—review
and editing, S.C. and M.S.; visualization, M.S. and H.K.; supervision, S.C.; project administration,
S.C.; funding acquisition, S.C. All authors have read and agreed to the published version of the
manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data are contained within the article.
Acknowledgments: The authors are indebted to ARES (Académie de Recherche et d’Enseignement
Supérieur) for financial support. We are also indebted to AB-Solutions and Brasserie Licorne for
kindly providing pilot dealcoholized and pasteurized samples.
Conflicts of Interest: The authors declare no conflicts of interest.
Abbreviations
NABLABs: non-alcoholic and low-alcoholic beers, ABV: alcohol by volume, HPLC: high-per-
formance liquid chromatography, UV: ultraviolet detection, GC: gas chromatography, PFPD:
pulsed flame photometric detection, MS: mass spectrometry, IST: internal standard, EST: external
standard, ORAC: oxygen radical absorbance capacity, TINH: inhibition time, ITT: indicator time
test, AAPH: 2,2′-azobis(2-methylpropionamidine) dihydrochloride, TBA: thiobarbituric acid index,
GAE: gallic acid equivalent, UP: pasteurization unit, 3SHol: 3-sulfanylhexan-1-ol, 3SHA: 3-sulfanyl-
hexyl acetate, 3S4MPol: 3-sulfanyl-4-methylpentanol, 3S4MPA: 3-sulfanyl-4-methylpentyl acetate,
BD: before dealcoholization, AD: after dealcoholization, AP: after pasteurization, DCPIP: 2,6-dichlo-
rophenolindophenol, SPE: solid-phase extraction.
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