Microorganisms
Eight 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.
Cell Growth During Batch Culture Propagation
Single colonies were looped from MYPG agar and inoculated into 100 ml of wort (12°Plato, 1.048 gravity) in a 250 ml Erlenmeyer flask. Prior to inoculation, Erlenmeyer flasks containing 100 ml of wort, with foam bungs inserted in the top were loosely covered by aluminum foil and sterilized by autoclaving at 121°C for 15 min. at 15 psi. After cooling to 25°C the flasks were then inoculated with the cells from a single colony. After the inoculation, foam bungs were re-inserted with the aluminum foil placed over the top. 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 12 days under semi-aerobic conditions. The yeast suspension was then poured aseptically into 500 ml of wort in a 1.5 litre flask prepared as described above. This second propagation step underwent a further 12 days of cell growth before it was collected for chemical analysis.
Further propagations of WY5112 (B. bruxellensis), WY5526 (B. lambicus) and WY5151 (B. claussenii) were carried out by looping single colonies from MYPG agar and inoculating into 100 ml of MYPG solution in a 250 ml Erlenmeyer flask. MYPG solution was prepared as previously described and 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 7 days. The yeast suspension was then poured aseptically into 500 ml of wort in a 1.5 litre flask prepared as described above. This second propagation step underwent a further 7 days of cell growth.
Analytical Techniques
Cell morphology was monitored qualitatively using a Medilux 12 microscope at 400x enlargement. Cell growth was quantitatively recorded along with viability by taking a daily 1 ml sample from each propagation flask using sterile techniques. Numerations were conducted by taking 1 ml of slurry and mixing it with 1 ml of methylene blue solution. From that mixture, 1 ml was pipetted into 9 ml of distilled water, giving a 1:10 ratio and an over all 1:20 dilution. Cells were then counted with a hemocytometer (Thoma cell). The percentage of viable cells was measured using the methylene blue staining method.
The reducing sugars glucose, fructose, sucrose, maltose and maltotriose were analysed in batch culture propagations of B. bruxellensis (WLP650), B. lambicus (WLP653), B. claussenii (WLP645), B. bruxellensis (BSI-Drie) and B. bruxellensis (CMY001). A 1ml sample was collected 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.
Batch culture propagations from B. bruxellensis (WLP650), B. lambicus (WLP653), B. claussenii (WLP645), B. bruxellensis (BSI-Drie) and B. bruxellensis (CMY001) were further collected in duplicate at a volume of 12 ml for analysis of standard beer components and 22 ml for ethyl lactate. Volatile compounds in the propagations were salted out in a sealed vial and the equilibrium headspace vapour was sampled and analysed 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 were measured by Flame Ionisation Detector (FID). Samples were pre-heated at 60°C for 90 min 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.
Volatile Phenols from B. bruxellensis (WLP650), B. lambicus (WLP653), B. claussenii (WLP645), B. bruxellensis (BSI-Drie) and B. bruxellensis (CMY001) were collected at a volume of 1ml to allow duplicate runs for the analysis of 4-vinylguaiacol and 4-vinylphenol. 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.