Microorganisms
8 strains of Brettanomyces yeasts were used throughout the experiments. The strains were chosen for their availability from commercial yeast companies specializing in brewers yeast. Three strains were attained from Wyeast Laboratories Inc. (Odell, OR. USA) B. bruxellensis (WY5112), B. lambicus (WY5526) and B. claussenii (WY5151). Three strains were attained from White Labs Inc. (San Diego, CA. USA) B. bruxellensis (WLP650), B. lambicus (WLP653) and B. claussenii (WLP645). The final two strains were personally cultured from a bottle of 100% Brettanomyces fermented ale, which used a strain referred to as “Drie” from The Brewing-Science Institute (Divide, CO. USA). After repeated culturing two physiologically distinct morphologies, termed BSI-Drie and CMY001 were established and stored separately.
Wort Preparation
200 liters of wort were produced on the pilot brewery at Heriot-Watt University and used exclusively throughout the entire research. Lager malt (Crisp Maltings, UK) was used to produce standard wort of 12° Plato (1.048 gravity ±0.002), and Hallertauer Magnum (12.7% alpha acid) hops were used to give 22 theoretical IBU. The wort was then stored in 2 litre, Nalgene bottles and frozen at -20°C. Wort to be used for propagations was prepared by de-thawing, homogenizing and filtering through filter cloth into Erlenmeyer flasks to the desired volume. The flasks were then fitted with foam bungs inserted in the tops, loosely covered by aluminum foil and sterilized by autoclaving at 121°C for 15 min. at 15 psi. Wort to be used for fermentations was prepared by de-thawing, homogenizing and filtering through filter cloth into Duran glass bottles. The Duran bottles were then sealed with their lid and autoclaved at 121°C for 15 min. at 15 psi.
Yeast Propagation
Single colonies were looped from MYPG agar and inoculated into 100ml of wort (12°Plato, 1.048 gravity ±0.002) in a 250 ml Erlenmeyer flask prepared as described above. The flasks were then placed in a Gallenkamp – Orbital Incubator with an agitation rate of 80 rpm and a temperature of 28°C. Initial cell growth from single colonies proceeded for 8 days. 100 ml of yeast suspension was then poured aseptically into 500 ml of wort in a 1.5 litre flask. This second propagation step underwent a further 8 days of cell growth, reaching early stationary phase growth, before being used for inoculating into fermentations.
Pure Culture Fermentation, Pitching Rate Impact
The first tier of primary fermentations involved looking at standardizing pitching rates of viable yeast cells. As pitching rate has never before been studied with this genus, three rates were chosen to observe the impact of initial cell concentration on the overall fermentation. Fermentations were conducted in duplicate in 2,000 ml Duran glass bottles. The dimensions of the fermentation vessels were 136 mm diameter by 265 mm height a ratio of 1:1.95 with a conical headspace of 20% volume. 1,800 ml of wort, prepared as described above, was used with 10 ppm dissolved oxygen diffused into the wort before adding yeast cells. Anaerobic fermentations were conducted by inserting rubber bungs air tight into the tops of the glass vessels and connecting rubber air tubing through the center, allowing a one-way exchange of gasses into sterile water. All fermentations were conducted at 21-22°C (70-72°F) in temperature-controlled incubators. Starting wort was 12°Plato (1.048 gravity ±0.002) with a starting pH of 4.95 ±0.05.
Wort was inoculated using early stationary phase cells and a primary pitching rate of 106 cells/ml/°P (12×106 cells/ml). Final cell counts were recorded from each of the single strain propagations with viability factored in to account for the volume of viable cells needing to be added. The yeast suspension was measured and added separately via pipette to each fermentation vessel. Secondary and tertiary pitching rates were chosen to observe the impact of initial cell concentration on fermentation behavior with four strains from the study. The secondary pitching rate of 0.5×106 cells/ml/°P (6×106 cells/ml) was decided as it was half the primary pitching rate while a tertiary pitching rate of 1.5×106 cells/ml/°P (18×106 cells/ml) was chosen being one and a half times the primary pitching rate. The amount of yeast suspension added in each fermentation vessel was calculated as previously mentioned.
Pure Culture Fermentation, Wort Acidification Using Lactic Acid
The second tier of primary fermentations involved dosing wort with different initial concentrations of lactic acid. Fermentations were conducted in duplicate in 1000 ml Duran glass bottles. The dimensions of the fermenting vessels were 101 mm diameter by 230 mm height or a ratio of 1:2.27 with a conical headspace of 20% volume. 900 ml of wort, prepared as described above, was used with 10 ppm dissolved oxygen diffused into the wort before adding yeast cells. Anaerobic fermentations were conducted by inserting rubber bungs air tight into the tops of the glass vessels and connecting rubber air tubing through the center, allowing a one-way exchange of gasses into sterile water. All fermentations were conducted at 21-22°C (70-72°F) in temperature-controlled incubators. Starting wort was 12°Plato (1.048 gravity ±0.002) with a starting pH of 4.95 ±0.05 except when stated otherwise.
Four different concentrations of Lactic acid (89.70% wt/vol, VWR) 100, 500, 1000, and 3000 mg/l, were directly added to wort via pipette after autoclaving and before aerating/pitching yeast suspension. A control group of fermentations were run for each strain with no lactic acid added to the fermentation vessels. Wort was then inoculated with a pitching rate of 12×106 cells/ml as previously described.
Quantitative Analysis
Primary fermentations were given 35 days (5 weeks) to attenuate before being collected for analysis. Prior to fermentation, samples of the wort and wort with lactic acid additions were collected and data recorded for original specific gravity and pH. Samples were then frozen at -20°C and saved for chemical analysis. Specific gravity of the wort and final gravity of the fermented worts were taken using an Anton-Paar DMA 46 with oscillating U-tube, benchtop density meter (Stanton Redcroft, London, UK). The pH of unfermented wort and unfermented wort with varying amounts of lactic acid was measured along with the final pH from each fermentation vessel using a pH 210 Microprocessor pH Meter (Hanna Instruments, USA). Original and apparent extract in degrees Plato was calculated from the gravities measured, using an equation recommended by the American Society of Brewing Chemists (Appendix, 1a). Percent apparent attenuation was further derived from the apparent extract and original extract (Appendix, 1b).
Chemical Analysis
When collecting samples for chemical analysis all volumes collected were doubled to allow duplicate runs, limiting the window of error during a run. Samples were then collected from each fermentation vessel at a volume of 12 ml for analysis of standard beer components and 22 ml for ethyl lactate analysis. Volatile compounds in the fermentations were salted out in a sealed vial and the equilibrium headspace vapor was sampled and analyzed by gas chromatography using a Hewlett Packard 5890 series II GC with split/splitless injector and FID & ECD detectors using a Chrompack CP-Wax-57-CB column. The VDKs were measured by Electron Capture Detector (ECD), all other components are measured by Flame Ionisation Detector (FID). Samples were pre-heated at 60°C for 90min in order to convert acetolactate and acetohydroxy-butyrate to diacetyl (2,3-butanedione) and 2,3 Pentanedione, respectively. Hewlett Packard Chemstation data handling (HP3365) was used for calculation of the detected peak surface.
The reducing sugars glucose, fructose, sucrose, maltose and maltotriose were analyzed in each fermentation in order to better understand sugar utilization of each strain. A 1 ml sample was collected from the fermentations to allow duplicate runs of each sample. Concentrations were determined by High performance anion exchange (HPAE) using a pulsed amperometric detector (Dionex PAD) with gold electrode and a Dionex Carbopac PA-100 column. Hewlett Packard Chemstation data handling (HP3365) was used for calculation of the detected peak surface.
Volatile phenols have been determined to be an important characteristic compound by Chatonnet et al. (1995) and samples were collected at a volume of 1 ml to allow duplicate runs for the analysis of 4-vinyl guaiacol and 4-vinyl phenol. Separation is achieved by gradient elution, high performance liquid chromatography (Gilson), using fluorescence as a means of detection (Waters 420-AC) and run using the Spherisorb S5ODS2 column. Gilson 715 data handling package is used for recording of peak surface.