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beverages
Article
Why Craft Brewers Should Be Advised to Use Bottle
Refermentation to Improve Late-Hopped
Beer Stability
Carlos Silva Ferreira , Etienne Bodart and Sonia Collin *
Earth and Life Institute ELIM, Universitécatholique de Louvain, Croix du Sud, 2 box L7.05.07,
B-1348 Louvain-la-Neuve, Belgium; carlos.silva@uclouvain.be (C.S.F.); etienne.bodart@uclouvain.be (E.B.)
*Correspondence: sonia.collin@uclouvain.be; Tel.: +32-10472913
Received: 28 February 2019; Accepted: 13 May 2019; Published: 4 June 2019
Abstract:
The aromatic complexity of craft beers, together with some particular practices (use of
small vessels, dry hopping, etc.), can cause more oxidation associated with pre-maturated colloidal
instability, Madeira off-flavors, bitterness decrease, and aroma loss. As bottle refermentation is widely
used in Belgian craft beers, the aim of the present work is to assess how this practice might impact their
flavor. In fresh beers, key flavors were evidenced by four complementary techniques: short-chain
fatty acids determination, esters analysis, XAD-2 extract olfactometry, and overall sensory analysis.
In almost all of the fresh beers, isovaleric acid was the sole fatty acid found above its sensory threshold.
Selected samples were further analyzed through natural aging at 20
◦
C. The presence of yeast in the
bottle minimized the trans-2-nonenal released from Schiffbases and proved less deleterious than
suggested by previous studies with regard to fatty acid release and ester decrease through aging.
Furthermore, according to the yeast species selected, some interesting terpenols and phenols were
produced from glucosides during storage.
Keywords: craft beer; bottle refermentation; AEDA; short-chain fatty acids; beer aging
1. Introduction
Worldwide, over the last few decades, the production of craft beers has grown significantly,
with new commercial products launched onto the market every day. Craftsmen can bring distinctive
flavors to their beers by working with special malts, dual-purpose hop varieties (with or without
dry hopping), spices, and/or specialty yeasts. These are known to impart fruity esters [
1
,
2
] and, in
some cases, typical phenolic flavors [
3
] (e.g., 4-vinylguaiacol brought by phenolic off-flavor (POF(+))
yeasts). In addition, odorous heterocyclic compounds can be issued from colored malts [
4
,
5
], while hop
terpenols and polyfunctional thiols bring pleasant citrus and exotic flavors to late- and dry-hopped
beers [
6
–
8
]. Unfortunately, the use of small vessels and craftsmanship, by definition, lead to a higher
risk of oxidation and shelf-life decrease.
Beer aging has been the focus of much interest for decades, with the development of worldwide
beverage exchanges. The Dalgliesh plot [
9
] describes aromatic changes occurring in lager beers
during storage. A linear decrease in bitterness (degradation of isohumulones and/or humulinones)
coincides with an increase in sweet aroma and toffee flavor, together with the well-known cardboard
taint (caused by trans-2-nonenal) [
10
–
13
] and ribes off-flavor (a catty smell linked to the presence of
3-sulfanyl-3-methylbutyl formate) [
14
,
15
]. Aging of specialty beers is even more complex, with defects
such as Madeira off-flavor [
16
], phenolic perception [
17
], a change in hoppy aromas [
18
], and a detected
ether taint [19].
Bottle refermentation has been widely used by Belgian craft brewers for its carbonation effect,
giving beer the desired effervescence, and also for the associated oxygen consumption, which limits
Beverages 2019,5, 39; doi:10.3390/beverages5020039 www.mdpi.com/journal/beverages
Beverages 2019,5, 39 2 of 11
oxidation and the development of related off-flavors [
20
–
22
]. About half a million yeast cells per mL
are pitched into the beer before bottling, in the presence of added fermentable sugars. The beer is then
kept in a warm room (20–28 ◦C) from two to four weeks.
According to Saison et al. [
23
], however, refermentation can be damageable, causing loss of
flowery, fruity, and ester notes that are highly appreciated by consumers. Long storage can lead to
yeast autolysis with release of esterases (deleterious to fruity esters) and to excretion of amino acids,
peptides, and short-chain fatty acids [
24
–
28
]. When Brettanomyces strains are present in the bottle,
production of isovaleric, hexanoic, and octanoic acids is especially promoted [29–31].
The aim of the present paper was to assess how bottle refermentation impacts the flavor
properties of Belgian craft beers. As bottle refermentation was already known to significantly
improve the release of free-hop thiols from cysteine and glutathione conjugates [
22
], we decided to
investigate only non-dry-hopped commercial samples. First, short-chain fatty acids were investigated
in 16 bottle-refermented and two unrefermented Belgian craft beers to determine whether they were
present above their sensory threshold. In a few selected samples, more flavors were then analyzed
through natural aging at 20
◦
C in the dark. Esters (isoamyl acetate, ethyl hexanoate, and ethyl octanoate)
were quantitated by headspace-GC-FID, and most trace aromas were monitored by GC-olfactometry
after XAD-2 aroma extraction. Lastly, some cardboard defects (trans-2-nonenal) and a few other
changes in aroma were evidenced by overall sensory analysis.
2. Materials and Methods
2.1. Materials
Isoamyl acetate (99%), ethyl hexanoate (99%), ethyl octanoate (99%), 2-pentanol (98%), isovaleric
acid (99%), hexanoic acid (
≥
98%), octanoic acid (
≥
98%), nonanoic acid (99%), and decanoic acid
were purchased from Sigma Aldrich GmbH (Bornem, Belgium); n-hexanol from Acros Organics
(Geel, Belgium); ethanol (99.8%) from Merck (Darmstadt, Germany); XAD-2 resin from Supelco Inc.
(Bellefonte, United States of America); and sodium chloride, copper sulfate (II), and diethyl ether from
VWR International (Leuven, Belgium). Authentic standard flavor compounds for olfactometry were of
pure grade (purity >98%) from Sigma-Aldrich. Milli-Q water was used (Millipore, Bedford, MA, USA).
2.2. Beer Samples and Aging Procedure
A total of 18 commercial, top-fermented, late-hopped beers (here listed as A–R for reasons of
confidentiality) were kindly supplied by Belgian craft brewers. All were bottle-refermented, except
beers A and B. Six representative samples (A–F), same lot as above, were further selected for more
in-depth investigations through natural aging (20
◦
C in the dark). The main characteristics of these
beers are depicted in Table 1.
Table 1. Main characteristics of the six selected craft beers.
Beer Alcohol
(% vol)
Real Extract
(◦P) pH Bitterness
(BU)
Color
(EBC) Sensorial Characteristics
A 6.5 4.6 4.2 15 12.5 Butter, apple, hop, green
B 12.3 6.6 4.4 20 25 Alcohol, banana, cheese, phenols
C * 7.9 5.1 4.5 21 16.5 Butter, sulfur, hop
D * 8.8 5.8 4.4 14 66 Malt, sulfur, green
E * 8.1 4.2 4.4 29 14.5
Lemon, banana, apple, spicy, phenols
F * 7.5 3.7 4.5 24 15.5 Orange, pineapple, spicy, phenols
*: with bottle refermentation.
Beverages 2019,5, 39 3 of 11
2.3. Short-chain Fatty Acid Analysis
First, 100
µ
L of internal standard (IST—1000 mg/L nonanoic acid) was added to 10 mL of beer
in a 20-mL vial flask, which was immediately closed and shaken for 10 s. Then, 300
µ
L of n-hexanol
was added before shaking again for 5 min. Compounds were recovered in assembled n-hexanol
fractions after 2 successive centrifugations (14,000 rpm) [
32
]. Next, 1
µ
L of extract was analyzed on an
Agilent 6890N gas chromatograph equipped with a split injector maintained at 200
◦
C (
split ratio =73.6
).
The FID (flame ionization detector) was set at 220
◦
C. Compounds were injected into a CP-Wax 58 column
(Agilent, 60 m
×
0.32 mm i.d., 0.5-
µ
m film thickness). The carrier gas was nitrogen, and the pressure was
set at 60 kPa. The oven temperature was programmed to rise from 125 to 140
◦
C at 8
◦
C/min and then to
180
◦
C at 15
◦
C/min. Quantitation was done by determining the relative response of each compound to
IST (done by standard addition to beer B). Results are expressed as the average of duplicates.
2.4. Static Headspace Analysis of Esters
Prior to analysis, the beers were stored for 2 h at 4
◦
C to avoid excessive foaming. The whole
procedure was carried out in a cold room (4
◦
C). Then, 40
µ
L of internal standard (IST, 2500 mg/L
2-pentanol) and 1.9 g of sodium chloride were added to 5 mL of beer in a 20-mL screw vial flask, which
was closed immediately and kept closed until analysis. A total of 500
µ
L of extract were analyzed on a
Thermo Finnigan Trace gas chromatograph, equipped with a splitless injector maintained at 250
◦
C;
the split vent was opened 1 min post-injection. The FID detector was set at 260
◦
C. Compounds were
injected into a VF-Wax MS column (Agilent, 60 m
×
0.32 mm i.d., 0.5-
µ
m film thickness). The carrier
gas was nitrogen, and the pressure was set at 100 kPa. The oven temperature was programmed to
rise from 40 to 140
◦
C at 8
◦
C/min and then to 180
◦
C at 15
◦
C/min. Quantitation was performed by
standard addition (relative response of each compound to IST). Results are expressed as the average
of duplicates.
2.5. Flavor XAD-2 Extraction and Gas Chromatography—Olfactometry Analytical Conditions
An extraction procedure based on that of Lermusieau et al. [
33
], was used to recover aroma
compounds from beer. First, 4 g of XAD-2 resin were added to 50 mL of beer in a 250-mL flask.
The flask was sealed with a Teflon-lined cap and shaken in the dark for 2 h at 200 rpm. After extraction,
the contents were poured into a glass column with a coarse frit and Teflon stopcock, and the liquid
was drained off, leaving a small bed of resin, which was further rinsed with 100 mL of distilled
water (
4×25 mL
). Aroma compounds were then eluted with 40 mL of diethyl ether (2
×
20 mL).
The extract was dried with Na
2
SO
4
and concentrated to 0.5 mL in a Kuderna-Danish evaporator at
39
◦
C. A Chrompack CP9001 gas chromatograph equipped with a splitless injector maintained at 250
◦
C
was used for the olfactometry analyses, and the split vent was opened after 0.5 min. Compounds were
separated using a wall-coated open-tubular (WCOT) apolar CP SIL5 CB capillary column (Agilent,
50 m
×
0.32 mm, 1.2-
µ
m film thickness) connected to a GC-odor port at 250
◦
C. The eluent was diluted
with a large volume of air (20 mL/min) previously humidified using aqueous copper (II) sulfate solution.
The oven temperature was programmed from 36 to 85
◦
C at 20
◦
C/min, to 145
◦
C at 1
◦
C/min, to 250
◦
C
at 3
◦
C/min, and then to remain constant at 250
◦
C for 30 min. A volume of 1
µ
L of extract was injected.
Sniffing was performed by two experienced panelists. Serial dilutions were prepared from the initial
XAD-2 extract at a ratio of 1:3
n
in diethyl ether. The dilution factor (FD) was calculated as 3
n
,n+1
being the number of dilutions (factor 3) required for no odor to be perceived (Log
3
FD values in Table 2
equal to 0, 1, 2,
. . .
, 10, nd if no odor detected for the undiluted extract). The difference between two
Log3FD becomes significant when above 1.
Beverages 2019,5, 39 4 of 11
Table 2.
GC-olfactometric analysis of XAD-2 extracts issued from beers A, C, E, and F (fresh and after 6
months of storage at 20 ◦C). RI: retention index; nd: not detected.
Log3FD
A C E F
Compound RI Odor Fresh Aged Fresh Aged Fresh Aged Fresh Aged
Ethyl butyrate 778 Red fruits 1 1 4 6 nd 3 3 4
3-Methyl-2-
buten-1-thiol 809 Garlic, hoppy 7 8 7 8 10 10 10 10
Isovaleric acid 811 Sweat, rancid 2 2 2 2 4 5 5 5
2-Methylbutanoic
acid 828 Sweat 2 2 2 2 4 5 3 6
2-Methyl-3-
furanthiol 850 Broth 5 7 5 2 10 10 10 10
Isoamyl acetate 854 Fruity, banana 1 1 2 2 nd nd nd nd
Ethyl hexanoate 979 Fruity, candy 2 2 4 4 3 4 4 4
Furaneol 1037 Cotton candy 4 5 6 7 5 5 4 7
Linalool 1089 Flowery,
coriander 7 6 6 7 nd nd 5 2
trans-2-Nonenal 1127 Cardboard nd 6 nd 5 3 4 4 5
Citronellol 1216 Fruity, flowery nd nd 1 4 3 2 nd 8
4-Vinylguaiacol 1294 Clove 3 4 4 4 5 6 4 6
γ-Nonalactone 1327 Coconut nd 3 0 4 1 2 3 5
Vanillin 1365 Vanilla 0 nd 6 7 nd 4 1 5
β
-Damascenone
1374 Fruity, apricot 2 3 4 5 4 4 3 6
4-Vinylsyringol 1543 Clove 3 3 0 3 2 3 5 5
2.6. Sensory Analyses
A group of 10 panelists (all trained scientists, non-smokers, and regular consumers of craft beers,
including three women and seven men aged 23–55 years) scored four aging attributes on a scale of
0–4: cardboard, bread, cooked fruit, and dried fruit. A score of 0 meant the panelist did not detect the
aroma, whereas a score of 4 meant the aroma was strongly perceived.
3. Results and Discussion
3.1. Short-Chain Fatty Acids
GC analyses revealed considerable variability of short-chain fatty acid profiles among fresh
Belgian craft beers (Figure 1). Most samples contained isovaleric acid (Figure 1a) at a concentration
above its threshold (1 mg/L) [
34
]. In all cases, on the other hand, hexanoic (Figure 1b), octanoic
(Figure 1c), and decanoic acids (Figure 1d) were below their sensory thresholds (10, 10, and 5 mg/L,
respectively [
35
]). A good correlation (R
2
=0.72) was found, as expected, between the concentrations
of hexanoic acid and octanoic acid (Figure 2).
Beverages 2019,5, 39 5 of 11
Beverages 2019, 4, x FOR PEER REVIEW 5 of 11
Figure 1. Concentrations of isovaleric (a), hexanoic (b), octanoic (c), and decanoic (d) acids (mg/L) in
18 fresh Belgian craft beers. The dotted lines in each graph indicate the compound sensory threshold
(Thr.).
Figure 2. Correlation between the concentrations (mg/L) of octanoic acid and hexanoic acid (in
eighteen fresh beers: A–R).
In a representative set of beers (A–F), short-chain fatty acids were determined after 3, 6, and 12
months of natural aging (Figure 3). No increase in isovaleric, hexanoic, octanoic, or decanoic acid
concentration was observed over the aging period. Worth mentioning, however, is the significant
decrease in octanoic acid in beer C (Figure 3C). This was the only beer tested here in which the
concentration of this compound reached 9 mg/L before aging. In conclusion, bottle refermentation
does not cause significant release of fatty acids through natural aging and thus does not negatively
impact flavor.
R² = 0.7281
0
2
4
6
8
10
12
0246
Octanoic a cid (mg/L)
Hexanoic acid (mg/L)
Figure 1.
Concentrations of isovaleric (
a
), hexanoic (
b
), octanoic (
c
), and decanoic (
d
) acids (mg/L) in
18 fresh Belgiancraft beers. The dotted lines in each graph indicate the compoundsensory threshold (Thr.).
Beverages 2019, 4, x FOR PEER REVIEW 5 of 11
Figure 1. Concentrations of isovaleric (a), hexanoic (b), octanoic (c), and decanoic (d) acids (mg/L) in
18 fresh Belgian craft beers. The dotted lines in each graph indicate the compound sensory threshold
(Thr.).
Figure 2. Correlation between the concentrations (mg/L) of octanoic acid and hexanoic acid (in
eighteen fresh beers: A–R).
In a representative set of beers (A–F), short-chain fatty acids were determined after 3, 6, and 12
months of natural aging (Figure 3). No increase in isovaleric, hexanoic, octanoic, or decanoic acid
concentration was observed over the aging period. Worth mentioning, however, is the significant
decrease in octanoic acid in beer C (Figure 3C). This was the only beer tested here in which the
concentration of this compound reached 9 mg/L before aging. In conclusion, bottle refermentation
does not cause significant release of fatty acids through natural aging and thus does not negatively
impact flavor.
R² = 0.7281
0
2
4
6
8
10
12
0246
Octanoic a cid (mg/L)
Hexanoic acid (mg/L)
Figure 2.
Correlation between the concentrations (mg/L) of octanoic acid and hexanoic acid (in eighteen
fresh beers: A–R).
In a representative set of beers (A–F), short-chain fatty acids were determined after 3, 6, and
12 months of natural aging (Figure 3). No increase in isovaleric, hexanoic, octanoic, or decanoic acid
concentration was observed over the aging period. Worth mentioning, however, is the significant
decrease in octanoic acid in beer C (Figure 3C). This was the only beer tested here in which the
concentration of this compound reached 9 mg/L before aging. In conclusion, bottle refermentation
does not cause significant release of fatty acids through natural aging and thus does not negatively
impact flavor.
Beverages 2019,5, 39 6 of 11
Beverages 2019, 4, x FOR PEER REVIEW 6 of 11
Unrefermented
Bottle-refermented
Figure 3. Concentrations (mg/L) of isovaleric acid (▲), hexanoic acid (■), octanoic acid (●), and
decanoic acid (♦) in fresh beers (A–F) and their evolution during natural aging (3, 6, and 12 months).
Variation coefficients under 5%.
3.2. Esters
Esters were determined on the same sampling of craft beers (A–F) in the course of one year of
natural aging (Figure 4). For fresh beers, a strong correlation (R2 = 0.86) was again observed between
the concentrations of ethyl hexanoate and ethyl octanoate (Figure 5a), although these ester levels did
not correlate well with the hexanoic and octanoic acids concentrations (Figure 5b,c). Ethyl hexanoate
and ethyl octanoate, were found very close to their sensory thresholds (0.2 and 0.9 mg/L, respectively
[36]) in all fresh samples and remained relatively stable during aging, with no significant difference
between the unrefermented (A and B) and bottle-refermented (C–F) samples. On the other hand, the
fruity banana-like isoamyl acetate was found to be partially degraded throughout the year of storage
in all beers except B (characterized by a much higher level of ethanol, Table 1). The similar trend
observed in A and C–F confirms that esters can be broken down even without the release of esterases
upon yeast autolysis. In all of the beers, the isoamyl acetate concentration remained above the sensory
threshold (0.5 mg/L [37]) after one year.
0
2
4
6
8
10
Fresh 3 months 6 months 12 months
Conc entr at ion (mg/L)
AIsovaleric acid
Hexanoic acid
Octanoic acid
Decanoic acid
0
2
4
6
8
10
Fresh 3 months 6 months 12 months
Concent ration (mg/L)
B
0
2
4
6
8
10
Fresh 3 months 6 months 12 months
Concentration (mg/L)
C
0
2
4
6
8
10
Fresh 3 months 6 months 12 months
Concentration (mg/L)
D
0
2
4
6
8
10
Fresh 3 months 6 months 12 months
Concen tration (mg/L)
E
0
2
4
6
8
10
Fresh 3 months 6 months 12 months
Concentration (mg/L)
F
Figure 3.
Concentrations (mg/L) of isovaleric acid (
N
), hexanoic acid (
), octanoic acid (
), and decanoic
acid (
) in fresh beers (
A
–
F
) and their evolution during natural aging (3, 6, and 12 months). Variation
coefficients under 5%.
3.2. Esters
Esters were determined on the same sampling of craft beers (A–F) in the course of one year of
natural aging (Figure 4). For fresh beers, a strong correlation (R
2
=0.86) was again observed between
the concentrations of ethyl hexanoate and ethyl octanoate (Figure 5a), although these ester levels did not
correlate well with the hexanoic and octanoic acids concentrations (Figure 5b,c). Ethyl hexanoate and
ethyl octanoate, were found very close to their sensory thresholds (0.2 and 0.9 mg/L, respectively [
36
])
in all fresh samples and remained relatively stable during aging, with no significant difference between
the unrefermented (A and B) and bottle-refermented (C–F) samples. On the other hand, the fruity
banana-like isoamyl acetate was found to be partially degraded throughout the year of storage in all
beers except B (characterized by a much higher level of ethanol, Table 1). The similar trend observed in
A and C–F confirms that esters can be broken down even without the release of esterases upon yeast
autolysis. In all of the beers, the isoamyl acetate concentration remained above the sensory threshold
(0.5 mg/L [37]) after one year.
Beverages 2019,5, 39 7 of 11
Beverages 2019, 4, x FOR PEER REVIEW 7 of 11
Unrefermented
Bottle-refermented
Figure 4. Concentrations (mg/L) of isoamyl acetate (▲), ethyl hexanoate (■), and ethyl octanoate (●)
in fresh beers (A–F) and their evolution during natural aging (3, 6, and 12 months). Variation
coefficients under 5%.
0.0
1.5
3.0
4.5
6.0
Fresh 3 months 6 months 12 months
Conc entr at ion (mg/L)
AIsoam yl acetate
Ethyl hexanoate
Ethyl octanoate
0.0
1.5
3.0
4.5
6.0
Fresh 3 months 6 months 12 months
Concentration (m g/L)
B
0.0
1.5
3.0
4.5
6.0
Fresh 3 months 6 months 12 months
Conc entr at ion (m g/L)
C
0.0
1.5
3.0
4.5
6.0
Fresh 3 months 6 months 12 months
Concentration (mg/L)
D
0.0
1.5
3.0
4.5
6.0
Fresh 3 months 6 months 12 months
Concen tr at ion (mg/L)
E
0.0
1.5
3.0
4.5
6.0
Fresh 3 months 6 months 12 months
Conc entr at ion (m g/L)
F
Figure 4.
Concentrations (mg/L) of isoamyl acetate (
N
), ethyl hexanoate (
), and ethyl octanoate (
) in
fresh beers (
A
–
F
) and their evolution during natural aging (3, 6, and 12 months). Variation coefficients
under 5%.
Beverages 2019,5, 39 8 of 11
Beverages 2019, 4, x FOR PEER REVIEW 8 of 11
Figure 5. Correlations between the concentrations (mg/L) (a) ethyl octanoate and ethyl hexanoate, (b)
ethyl hexanoate and hexanoic acid, and (c) ethyl octanoate and octanoic acid (in six fresh beers: A–F).
3.3. Olfactive Analysis of XAD-2 Extracted Flavors
Samples A, C, E, and F (all blond beers to avoid the complexity of special malt-derived molecules
largely investigated elsewhere [5,17]) were subjected to XAD-2 resin extraction followed by GC-
olfactometric (GC-O) analysis. Beer odor intensities were determined by the aroma extract dilution
analysis (AEDA) [38]. To focus on beer flavor-active compounds, we list in Table 2 only those
compounds whose FD was as high as that of ethyl hexanoate, an ester known to be present in the
samples at concentrations close to its sensory threshold.
3-Methyl-2-buten-1-thiol (Log3FD = 7–10, a pleasant hoppy flavor here but also known as skunky
at a much higher level [22]), 2-methyl-3-furanthiol (Log3FD = 2–10, broth), furaneol (Log3FD = 4–7,
cotton candy), and linalool (Log3FD = 2–7, flowery/coriander) emerged as the most potent odorants
in all four beers. The persistent detection of 4-vinylguaiacol (Log3FD = 3–6) in all samples indicates
that POF(+) strains had been used by the brewers.
As already mentioned above, even in bottle-refermented beers (C, E, and F), isoamyl acetate
(Log3FD = 1–2, undetectable in E and F due to the strong previous odor) and ethyl hexanoate (Log3FD
= 2–4) showed good stability through aging, with no significant changes in FD. On the other hand,
the red-fruit ethyl butyrate was produced during storage in the bottle-refermented beers (from not
detected to Log3FD = 3 in beer E and from 4 to 6 in beer C).
Although trans-2-nonenal showed an increase in all four beers during aging, the highest FD jump
was observed for the unrefermented A (from not detected to Log3FD = 6).
An interesting result was the strong increase, especially in aged beer F, of compounds suspected
to be released during storage through glucoside hydrolysis: citronellol, 4-vinylguaiacol, vanillin, and
β-damascenone [39,40]. In this case, the selected yeast clearly brought new flavors to F, explaining
why some consumers may prefer the six-month-aged beer. The efficiency of the yeast β-1,3-glucanase
or β-glucosidase should be taken into account to predict the amounts in which aglycons can be
released through aging. Linalool (detected in fresh beers A, C, and F) was the sole hop terpenol found
R² = 0.8665
0.0
0.2
0.4
0.6
0.8
1.0
1.2
0.00.20.40.60.8
Ethyl octanoate (mg/L)
Ethyl hexanoate (mg/L)
(a)
R² = 0.2476
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0246
Ethyl hexnaoate (mg/L)
Hexanoic acid (mg/L)
(b)
R² = 0.0985
0.0
0.2
0.4
0.6
0.8
1.0
1.2
0246810
Ethyl octanoate (mg/L)
Octanoic acid (mg/L)
(c)
Figure 5.
Correlations between the concentrations (mg/L) (
a
) ethyl octanoate and ethyl hexanoate,
(
b
) ethyl hexanoate and hexanoic acid, and (
c
) ethyl octanoate and octanoic acid (in six fresh beers: A–F).
3.3. Olfactive Analysis of XAD-2 Extracted Flavors
Samples A, C, E, and F (all blond beers to avoid the complexity of special malt-derived
molecules largely investigated elsewhere [
5
,
17
]) were subjected to XAD-2 resin extraction followed
by GC-olfactometric (GC-O) analysis. Beer odor intensities were determined by the aroma extract
dilution analysis (AEDA) [
38
]. To focus on beer flavor-active compounds, we list in Table 2only those
compounds whose FD was as high as that of ethyl hexanoate, an ester known to be present in the
samples at concentrations close to its sensory threshold.
3-Methyl-2-buten-1-thiol (Log
3
FD =7–10, a pleasant hoppy flavor here but also known as skunky
at a much higher level [
22
]), 2-methyl-3-furanthiol (Log
3
FD =2–10, broth), furaneol (Log
3
FD =4–7,
cotton candy), and linalool (Log
3
FD =2–7, flowery/coriander) emerged as the most potent odorants in
all four beers. The persistent detection of 4-vinylguaiacol (Log
3
FD =3–6) in all samples indicates that
POF(+) strains had been used by the brewers.
As already mentioned above, even in bottle-refermented beers (C, E, and F), isoamyl acetate
(Log
3
FD =1–2, undetectable in E and F due to the strong previous odor) and ethyl hexanoate
(
Log3FD =2–4
) showed good stability through aging, with no significant changes in FD. On the other
hand, the red-fruit ethyl butyrate was produced during storage in the bottle-refermented beers (from
not detected to Log3FD =3 in beer E and from 4 to 6 in beer C).
Although trans-2-nonenal showed an increase in all four beers during aging, the highest FD jump
was observed for the unrefermented A (from not detected to Log3FD =6).
An interesting result was the strong increase, especially in aged beer F, of compounds suspected
to be released during storage through glucoside hydrolysis: citronellol, 4-vinylguaiacol, vanillin, and
β
-damascenone [
39
,
40
]. In this case, the selected yeast clearly brought new flavors to F, explaining why
some consumers may prefer the six-month-aged beer. The efficiency of the yeast
β
-1,3-glucanase or
β
-glucosidase should be taken into account to predict the amounts in which aglycons can be released
Beverages 2019,5, 39 9 of 11
through aging. Linalool (detected in fresh beers A, C, and F) was the sole hop terpenol found to
decrease in beer F (Log
3
FD from 5 to 2), suggesting yeast terpenol biotransformations in the bottle [
41
].
3.4. Sensory Analyses
Beers A–F were investigated by a trained panel while fresh and then after 3 and 6 months of
storage. As suspected from the GC-olfactometry results, unrefermented beers A and B developed
a relatively intense cardboard flavor (trans-2-nonenal), already strongly perceived after 3 months
(Figure 6). Despite the increase in FD for furaneol and
β
-damascenone after 6 months of storage, the
attributes bread, dried fruit, and cooked fruit remained absent or relatively weak.
Beverages 2019, 4, x FOR PEER REVIEW 9 of 11
to decrease in beer F (Log3FD from 5 to 2), suggesting yeast terpenol biotransformations in the bottle
[41].
3.4. Sensory Analyses
Beers A–F were investigated by a trained panel while fresh and then after 3 and 6 months of
storage. As suspected from the GC-olfactometry results, unrefermented beers A and B developed a
relatively intense cardboard flavor (trans-2-nonenal), already strongly perceived after 3 months
(Figure 6). Despite the increase in FD for furaneol and β-damascenone after 6 months of storage, the
attributes bread, dried fruit, and cooked fruit remained absent or relatively weak.
Figure 6. Spider diagram of cardboard flavor intensity in fresh (●), and naturally aged (3 and 6
months) beers (A–F).
4. Conclusions
Bottle refermentation of craft beers can be promoted for its ability both to protect beer against
oxidation, (this protection is required to avoid colloidal instability, bitterness decrease, and aroma
loss) and to avoid the accumulation of trans-2-nonenal through enzymatic reduction to nonenol and
nonanol. Moreover, the presence of yeast in the bottle proved not so deleterious as it regards fatty
acid excretion and ester hydrolysis during the first year of storage, while leading to the release of
interesting terpenols and phenols from aglycons.
Author Contributions: Conceptualization, S.C.; Data curation, C.S.F., E.B., and S.C.; Formal analysis, C.S.F., E.B.,
and S.C.; Writing—original draft, C.S.F. and S.C.
Funding: This research received no dedicated funding.
Conflicts of Interest: The authors declare no conflicts of interest.
References
1. Pires, E.J.; Teixeira, J.A.; Brányik, T.; Vicente, A.A. Yeast: The soul of beer’s aroma—A review of flavour-
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0
1
2
3
A
B
C
D
E
F
Fresh 3 months 6 months
Figure 6.
Spider diagram of cardboard flavor intensity in fresh (
), and naturally aged (3 and 6 months)
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Bottle refermentation of craft beers can be promoted for its ability both to protect beer against
oxidation, (this protection is required to avoid colloidal instability, bitterness decrease, and aroma
loss) and to avoid the accumulation of trans-2-nonenal through enzymatic reduction to nonenol and
nonanol. Moreover, the presence of yeast in the bottle proved not so deleterious as it regards fatty acid
excretion and ester hydrolysis during the first year of storage, while leading to the release of interesting
terpenols and phenols from aglycons.
Author Contributions:
Conceptualization, S.C.; Data curation, C.S.F., E.B., and S.C.; Formal analysis, C.S.F., E.B.,
and S.C.; Writing—original draft, C.S.F. and S.C.
Funding: This research received no dedicated funding.
Conflicts of Interest: The authors declare no conflicts of interest.
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