Yeast Strain and Medium
The experiments were carried out with a production lager yeast strain of S. pastorianus (A-63015) from the VTT Culture Collection, Finland. The yeast was propagated from a freezer stock maintained at −150 °C.
All-malt wort with an extract content of 15.0 °Plato (68 g maltose, 19 g maltotriose, 19 g glucose, 4.7 g fructose and 1.5 g sucrose per litre) and FAN content of 408 mg L−1 (alanine 248.4, arginine 303.4, asparagine 210.5, aspartic acid 128.8, glutamine 54.8, glutamic acid 103.4, glycine 82.5, histidine 98.8, isoleucine 161.2, leucine 380.3, lysine 253.5, methionine 68.1, phenylalanine 283.2, proline 752.5, serine 146.5, threonine 133.2, tryptophan 77.3, tyrosine 273.4 and valine 263.7 mg L−1) was prepared at the VTT Pilot Brewery. Part of the all-malt wort was diluted to a semi-synthetic wort with an extract content of 13.3 °Plato (77.5 g maltose, 8.5 g maltotriose, 19 g glucose, 4.7 g fructose and 1.5 g sucrose L−1) and a FAN content of 204 mg L−1 using a sterile sugar solution made up of maltose (Sigma-Aldrich, Finland), glucose (VWR, USA), fructose (Merck KGaA, Germany) and sucrose (VWR, USA) in deionized water. Amino acids (Sigma-Aldrich, Finland) were supplemented to the worts from sterile-filtered stock solutions prepared in deionized water. The FAN content was determined using method 9.24.2 described in EBC-Analytica (European Brewery Convention 2008).
Three different fermentation trials were performed, the first investigating the effect of supplementing various amounts of valine (100, 200 and 300 mg L−1) to the wort, the second investigating the effect of supplementing valine (300 mg L−1) to worts with standard (408 mg L−1) and reduced FAN content (204 mg L−1), and the third investigating the effects of supplementing various groups of amino acids to the wort (amino acids were supplemented to double their concentration in the wort, with the exception of tyrosine, which concentration was increased by only 10 % because of poor aqueous solubility), on the production of diacetyl and diacetyl precursors and the change of wort valine concentration during fermentation. The amino acids were grouped into the three groups based on their absorption rates or structures (Table 1). The first group, preferred amino acids (PAA), contains the amino acids which had a higher uptake rate than valine during the first 25 h of fermentation. The second group, non-preferred amino acids (NPAA), contains the amino acids which had a lower uptake rate than valine during the first 25 h of fermentation. The third group, branched-chain amino acids (BCAA), contains leucine and isoleucine, which have a similar structure to valine. The pH of the PAA-supplemented wort was adjusted to that of the control wort with 90 % lactic acid (Merck KGaA, Germany).
Yeast propagation was carried out essentially as previously described (Ekberg et al. 2013). Briefly, frozen yeast suspensions in 30 % glycerol were thawed and used to inoculate 500 mL autoclaved YP medium containing 40 g maltose L−1 in 1L Erlenmeyer flasks. Cultures were incubated overnight at 25 °C with shaking (120 rpm) and then transferred to 1.5 L of 15 °P wort to achieve an OD600 of 0.15. These cultures were incubated at 16 °C with shaking for 48 h and then moved to 0 °C. After 16 h, the sedimented yeast was diluted with decanted supernatant to 20 g centrifuged yeast mass/100 g of slurry. Cylindroconical fermentation vessels containing approx. 10 L of oxygenated (10 mg dissolved oxygen L−1) 15 °P wort were pitched with this ‘generation 0’ slurry to a concentration of 5 g fresh centrifuged yeast L−1. This fermentation was allowed to proceed in a room at 15 °C until 80 % apparent attenuation was reached. The partially settled yeast was then cropped from the bottom of the vessels as a slurry mixed with beer (approximately 2 L), transferred to 0 °C and allowed to sediment for 16 h. A slurry containing 200 g centrifuged yeast mass L−1 was prepared as described above and used within 2 h to pitch the ‘generation 1’ experimental yeast fermentations. These ‘repitched’ yeast fermentations were carried out so that the yeast condition would approximate that of yeast used to start industrial fermentations.
The experimental fermentations were carried out in duplicate, in 2-L cylindroconical stainless steel fermenting vessels, containing 1.5 L of wort medium. Yeast was inoculated at a rate of 5 g fresh yeast per litre of wort (corresponding to 20 × 106 viable cells mL−1). The wort was oxygenated to 9 mg L−1 prior to pitching. The fermentations were carried out at 15 °C for 8 days. Wort samples were regularly drawn from the fermentation vessels with a syringe, and placed directly on ice, after which the yeast was separated from the fermenting wort by centrifugation (9,000×g, 10 min, 1 °C). Fermentations were stopped once apparent attenuation of the all-malt wort had reached 80 % or the apparent attenuation of the semi-synthetic wort had reached 95 % (approximate alcohol content of 6.7 %), and the beer was collected in sterile flasks.
The density, specific gravity, ethanol concentration and pH of samples was determined from the centrifuged and degassed fermentation samples using an Anton Paar Density Meter DMA 5000 M (Anton Paar GmbH, Austria) with Alcolyzer Beer ME and pH ME modules (Anton Paar GmbH, Austria). The apparent extract (AE; in degree Plato) of the samples was estimated from the previously measured specific gravities (SG) using the approximations from Kobayashi et al. (2005b). The apparent attenuation (AA; %) of the samples was estimated from the apparent and original extract (i.e. the apparent extract of the wort at the time of pitching) as described in Vidgren et al. (2009). The real extract (in degree Plato) of the samples was estimated from the AE (in degree Plato) and the ethanol content (A
ABW; % (w/w)) using an approximation proposed by Hackbarth (2009).
Fermentable sugars were analysed by high-performance anion exchange chromatography (HPAEC) (Dionex ICS-3000) with pulse amperometric detection using CarboPac PA-1 (4 mm × 250 mm) analytical column and CarboPac PA-1 (4 mm × 50 mm) guard column at 30 °C (Dionex Corp, USA). The system was equilibrated with 100 mM NaOH. After injection of a 100 μL filtered (0.45 μm), diluted sample, 100 mM NaOH was run through the column (5 min). Separation was with a gradient (1 mL min−1) of 100 mM to 300 mM NaOH in 3 min and then 300 mM NaOH to 250 mM NaOH + 75 mM Na-acetate in 15 min and washing was with 100 mM NaOH + 300 mM Na-acetate and 300 mM NaOH. The flow rate was 1 mL min−1. The results were confirmed by MSQ detection (HPAEC-MS) using a CarboPac PA200 (3 mm × 250 mm) with a CarboPac PA200 guard (3 mm × 50 mm) column (Dionex) with a configuration as described by Bruggink et al. (2005) and a gradient as described by Mikkelson et al. (2013).
The yeast dry mass content of the samples was determined by suspending the yeast pellet gained from centrifugation in a total of 6 mL H2O (water was deionized and filtered through active carbon (MilliQ Water System; Millipore Corporation, MA, USA). The suspension was then transferred to a pre-weighed porcelain crucible, and was dried overnight at 105 °C and allowed to cool in a desiccator, before the change of mass was measured.
Vicinal diketone analysis
Total VDKs (free and acetohydroxy acid form) were measured for the centrifuged fermentation samples according to Analytica-EBC method 9.10 (European Brewery Convention 2008). Samples were heated to 60 °C, where they were kept for 90 min, in a headspace auto sampling unit (Headspace Autosampler 7000 HT, Tekmar-Dohrmann, USA). Heating to 60 °C results in the conversion of acetohydroxy acids to VDK. The samples were then analysed by headspace gas chromatography (HP 6890 Series GC System, Hewlett-Packard, USA; HP-5 50 m × 320 μm × 1.05 μm column, Agilent, USA) with 2,3-hexanedione as an internal standard.
Amino acid analysis
Centrifuged fermentation samples were diluted to 1:40. A 10-μL volume of the diluted sample was taken and mixed with 10 μL of norvaline (250 μM, internal standard) and 70 μL of boric acid buffer. The mixture was then vortexed for 30 s. Derivatization was done with AccQ·Fluor reagent kit (Waters Corporation, USA). The AccQ·Fluor reagent was reconstituted with acetonitrile (1 mL), and vortexed for 30 s. The mixture was heated to 55 °C for 8 min, kept in an ultrasound bath for 5 min and finally vortexed for 60 s. The AccQ·Fluor reagent (10 μL) was added to the sample mixture, which was instantly vortexed for 60 s. Samples were kept at 5 °C before and during analysis. Analysis was performed on an Acquity UPLC system (Waters Corporation, USA) with UV detector. Chromatography was performed using an Acquity Mass Trak™ (2.1 × 150 mm, 1.7 μm) column (Waters Corporation, USA), kept at 43 °C. Injection volume was 2.0 μL. Separation was performed using gradient elution with 10 % (v/v) Amino Acid Analysis Concentrate A in water and Amino Acid Analysis Eluent B at a flow rate of 0.4 mL/min. The signal was detected at 260 nm (2.4 nm resolution, 20 points/s).
Aroma compounds analysis
The concentrations of various yeast-derived aroma compounds (acetaldehyde, alcohols, and esters) in the wort samples were determined by headspace-GC/MS. A 10-mL volume of the supernatant was filtered (0.45 μm cellulose acetate filter) before analysis. For analysis, the samples were first thawed and then incubated at 60 °C for 30 min. A 1-mL volume of sample was then injected in the splitless injector (260 °C; flow 14.9 mL min−1) of the gas chromatograph (Agilent 6890 Series; Palo Alto, CA, USA) combined with an MS detector (Agilent 5973 Network MSD, USA) and SPME autosampler (Combipal, Varian Inc., USA). Analytes were separated on a BPX5 capillary column of 60 m × 0.25 mm with phase thickness 1.0 μm (SGE Analytical Science Pty Ltd., Australia). Helium was used as carrier gas on constant flow mode 1.7 mL min−1. The temperature program was started at 50 °C for 3 min, then 10 °C min−1 to 100 °C, followed by 5 °C min−1 to 140 °C and finally 15 °C min−1 to 260 °C, where the temperature was kept for 1 min. MSD was operated in electron-impact mode at 70 eV, in the full scan m/z 40–550. The ion source temperature was 230 °C and the interface was 280 °C. Compounds were identified with retention times of corresponding standards and by comparing the mass spectra on Palisade Complete 600 K Mass Spectral Library (Palisade Mass Spectrometry, USA) and were quantitated with a standard curve. 1-Butanol was used as internal standard.