While performing the propagation trials, I prepared my research methodology for the primary fermentations, which would make up the bulk of my research. As previously mentioned all mini-fermentations were conducted with wort produced on the 2 hectolitre pilot system. The following details the experiments I put together in researching the characteristics of a Brettanomyces primary fermentation. My intent was to observe what compounds are produced during the fermentation, how those compare to a general ale, and also to observe a few known aroma compounds produced. The main aroma compound I wanted to observe was Ethyl Lactate, an ester produced from ethyl alcohol and Lactic acid. Ethyl lactate is believed to create a pineapple or tropical fruit aroma. By adding Lactic acid to the wort, pre-fermentation in varying amounts, I planned to observe the levels of Ethyl Lactate formed, and correlate it to the aroma found in the specific fermentation conducted.
First Tier: Pitching Rates
• Trial fermentations conducted in duplicate in 2000mL Duran glass bottles. The dimensions were 135mm:200mm (diameter:height) or a ratio of 1:1.5 with a conical headspace of 20% volume.
• 1.8L (1,800mL) of standard (autoclaved) wort used with 10ppm dissolved oxygen diffused into the wort initially. (roughly 1ppm per °Plato)
• Anaerobic fermentations were conducted by inserting rubber bungs air tight into the tops of the glass bottles and connecting rubber air tubing, allowing blow-off of Carbon dioxide gas into sterile water.
• Fermentations conducted at 21-22°C (70-72°F) in temperature-controlled incubators.
• Starting Plato of wort 12° (1.048 gravity)
• Starting pH of 4.95 (±) .05
Primary pitching rate = 1.0×10^6cells/mL×12°P×1800mL=2.16×1010cells
• When counting cells for determining amounts to be measured out to pitch, viability was also determined, recorded, and taken into account to get the actual amount of viable cells for pitching (shown in red). The following shows the calculation used to determine the volume of propagated cells needed within each trial fermentation.
• B. claussenii (WLP645) 2.16×1010cells/6.59×108=32.77mL (vol. pitched into wort)
• B. claussenii (WY5151) 2.16×1010cells/10.80×108=20.00mL (vol. pitched into wort)
• B. bruxellensis (WLP650) 2.16×1010cells/6.64×108=32.53mL (vol. pitched into wort)
• B. bruxellensis (WY5112) 2.16×1010cells/10.66×108=20.26mL (vol. pitched into wort)
• B. lambicus (WLP653) 2.16×1010cells/5.79×108=37.30mL (vol. pitched into wort)
• B. lambicus (WY5526) 2.16×1010cells/13.94×108=15.5mL (vol. pitched into wort)
• B. matted (CMY-001) 2.16×1010cells/5.90×108=36.61mL (vol. pitched into wort)
• B. glossy (CMY-002) 2.16×1010cells/8.18×108=26.40mL (vol. pitched into wort)
For Secondary and Tertiary pitch rates, 4 strains were chosen for observation in the trials.
Secondary pitching rate = .5×10^6cells/mL×12°P×1800mL=1.08×1010cells
• B. claussenii (WY5151) 20.00mL/2=10.00mL (vol. pitched into wort)
• B. lambicus (WY5526) 15.5mL/2=7.75mL (vol. pitched into wort)
• B. matted (CMY-001) 36.61mL/2=18.30mL (vol. pitched into wort)
• B. glossy (CMY-002) 26.40mL/2=13.20mL (vol. pitched into wort)
Tertiary pitching rate = 1.5×10^6cells/mL×12°P×1800mL=3.24×1010cells
• B. claussenii (WY5151) 20.00mL×1.5=30.00mL (vol. pitched into wort)
• B. lambicus (WY5526) 15.5mL×1.5=23.25mL (vol. pitched into wort)
• B. matted (CMY-001) 36.61×1.5=54.91mL (vol. pitched into wort)
• B. glossy (CMY-002) 26.40×1.5=39.60mL (vol. pitched into wort)
Second Tier: Varying Lactic Acid Concentrations
• Trial fermentations conducted in duplicate in 1000mL Duran glass bottles. The dimensions were 100mm:230mm (diameter:height) or a ratio of 1:2.3 with a conical headspace of 20% volume.
• Anaerobic fermentations were conducted by inserting rubber bungs air tight into the tops of the glass bottles and connecting rubber air tubing, allowing blow-off of Carbon dioxide gasses into sterile water.
• .9L (900mL) of standard (autoclaved) wort used in each cylinder with 10ppm dissolved oxygen. (roughly 1ppm per °Plato)
• Fermentations conducted at 21-22°C (70-72°F) in temperature-controlled incubators.
• Pitching rate described previously (1.0×106cells/mL×12°P×900mL=1.08×1010cells)
• When counting cells for determining amounts to be weighed out and pitched the same methods were used as previously described.
• 4 different concentrations of Lactic acid (100ppm, 500ppm, 1000ppm, 3000ppm) added to wort (after autoclaving, before aerating/pitching yeast)
• Lactic Acid (VWR) (89.70% wt/vol, D/L%)
• Calculation used to achieve desired concentrations:
• 100ppm: .1mL/L×.900mL×(100/89.7)=0.1003mL of Lactic acid
• 500ppm: .5mL/L×.900mL×(100/89.7)=.501mL of Lactic acid
• 1000ppm: 1.0mL/L×.900mL×(100/89.7)=1.003mL of Lactic acid
• 3000ppm: 3.0mL/L×.900mL×(100/89.7)=3.01mL of Lactic acid
100 ppm
• B. claussenii (WLP645) Starting pH= 4.55
• B. claussenii (WY5151) Starting pH=4.55
• B. bruxellensis (WLP650) Starting pH=4.55
• B. bruxellensis (WY5112) Starting pH=4.55
• B. lambicus (WLP653) Starting pH=4.55
• B. lambicus (WY5526) Starting pH=4.55
• B. matted (CMY-001) Starting pH=4.55
• B. glossy (CMY-002) Starting pH=4.55
500 ppm
• B. claussenii (WLP645) Starting pH=4.05
• B. claussenii (WY5151) Starting pH=4.05
• B. bruxellensis (WLP650) Starting pH=4.05
• B. bruxellensis (WY5112) Starting pH=4.05
• B. lambicus (WLP653) Starting pH=4.05
• B. lambicus (WY5526) Starting pH=4.05
• B. matted (CMY-001) Starting pH=4.05
• B. glossy (CMY-002) Starting pH=4.05
1000 ppm
• B. claussenii (WLP645) Starting pH=3.75
• B. claussenii (WY5151) Starting pH=3.75
• B. bruxellensis (WLP650) Starting pH=3.75
• B. bruxellensis (WY5112) Starting pH=3.75
• B. lambicus (WLP653) Starting pH=3.75
• B. lambicus (WY5526) Starting pH=3.75
• B. matted (CMY-001) Starting pH=3.75
• B. glossy (CMY-002) Starting pH=3.75
3000 ppm
• B. claussenii (WLP645) Starting pH=3.08
• B. claussenii (WY5151) Starting pH=3.08
• B. bruxellensis (WLP650) Starting pH=3.08
• B. bruxellensis (WY5112) Starting pH=3.08
• B. lambicus (WLP653) Starting pH=3.08
• B. lambicus (WY5526) Starting pH=3.08
• B. matted (CMY-001) Starting pH=3.08
• B. glossy (CMY-002) Starting pH=3.08