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
] 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
], while hop terpenols and polyfunctional thiols bring pleasant citrus and exotic flavors to late- and dry-hopped beers [6
]. 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
] and ribes
off-flavor (a catty smell linked to the presence of 3-sulfanyl-3-methylbutyl formate) [14
]. 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 oxidation and the development of related off-flavors [20
]. 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
]. When Brettanomyces
strains are present in the bottle, production of isovaleric, hexanoic, and octanoic acids is especially promoted [29
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
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
2.3. Short-chain Fatty Acid Analysis
First, 100 µL 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 µL 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 µL 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-µm 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 µL 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 µL 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-µm 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 Na2
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-µm 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 µL 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 1:3n
in diethyl ether. The dilution factor (FD) was calculated as 3n
+ 1 being the number of dilutions (factor 3) required for no odor to be perceived (Log3
FD values in Table 2
equal to 0, 1, 2, …, 10, nd if no odor detected for the undiluted extract). The difference between two Log3
FD becomes significant when above 1.
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.