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.
Media Used For Storage, Isolation, Detection, and Enumeration
All cultures were stored at 4°C on MYPG agar (Oxoid) containing: malt extract, 3 g/l; yeast extract, 3 g/l; peptone, 2 g/l; glucose 10 g/l; agar 15 g/l; with the pH adjusted to 5.0 and autoclaved at 121°C for 15 minutes at 15 psi. Initial growth was at 28°C in a dark, aerobic growth chamber.
Media selected for isolated growth of wild yeasts include, Copper Sulfate (CuSO4) agar containing: malt extract agar (Oxiod), 50 g/l; copper sulfate, 0.6 g/l; autoclaved at 115°C for 10 min. MYPG agar (Oxiod) with the addition of a 0.1% solution of cycloheximide(MYPGch), 10 mg/l (10 ppm) added after autoclaving once media was at pouring temperature (45°-50°C). Lysine agar containing: glucose, 10 g/l; L-Lysine, 10 g/l; agar #1, 12 g/l; yeast salts, 1ml/l; with the pH adjusted to 6.0 and autoclaved at 121°C for 15 minutes at 15 psi. Vitamin solution added once media was at pouring temperature, 10 ml.
Further media was selected for isolation and detection of pure cultures being used throughout the experiments. Wallerstein Laboratory Nutrient (WLN) agar (Oxoid) prepared at 75 g/l, with an additional 1% of agar added, 10 g/l; and autoclaved at 121°C for 15 minutes at 15 psi. Additionally WLD agar was made with the addition of a 0.1% solution of cycloheximide, 10 mg/l (10 ppm).
Further media used includes MYPG agar (Oxoid) with the addition of 2% w/v CaCO3 (MYPGcc), 10 g/l and Universal Beer Agar (Sigma-Aldrich) prepared at 61 g/l and solubilized in 750 ml distilled water and 250 ml beer. YPD (Oxoid) containing: yeast extract, 10 g/l; peptone, 20 g/l; glucose 20 g/l; agar 20 g/l; and autoclaved at 121°C for 15 minutes at 15 psi.
Inoculation and Isolation of ‘Drie’ Strain
A 22 oz bottle of Avery 15 (Avery Brewing Co.) was prepared by removing any special foil and scrubbed with hot soapy water to remove any glue around the top neck of the bottle and underside of the cap. It was then rinsed and sterilized with 70% isopropyl alcohol. Using sterile techniques the bottle cap was removed, neck and top lip flamed and the contents poured off, collecting only the visible yeast cells near the bottom of the bottle into three pre-autoclaved, 20 ml glass vials with airtight tops.
For inoculation media MYPG, MYPGcc, Copper Sulfate, and WLN agar plates were prepared as described above. The isolations were performed in triplicate on each media using inoculum from each vial. 0.1ml was pipetted from the vials and plated out using glass bead dispersion method. The plates were then incubated aerobically in a dark growth chamber at 28°C for 7-10 days.
Upon growth of inoculum culture on each media plate, distinct single colonies were loop inoculated and streaked onto the same parent media for confirmation of pure cultures. The media plates were again incubated as described above to allow inoculum growth. From the secondary plates, which showed uniform morphologies, single cells were looped and inoculated into four, 250 ml Erlenmeyer flasks containing a 100 ml solution of MYPG. MYPG solution was prepared with the same methods as MYPG agar excluding the 10 g/l of agar. Prior to inoculation, Erlenmeyer flasks containing 100 ml of MYPG solution, 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 inoculated and the 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 100 rpm and a temperature of 28°C. After 10 days of cell growth, serial dilution of 10-2, 10-4 and 10-6 were made for each of the four flasks using pre-sterilized, distilled water, autoclaved in 20 ml glass vials. 0.1 ml was pipetted from each serial diluted vial and inoculated in duplicate onto MYPG agar and WLN agar using glass beard dispersion.
BSI-Drie and CMY001, two morphologically unique and stable yeast strains isolated as described above were identified according to Kreger-van Rij (1984) for classification purposes and use within the research. The cellular morphology and type of vegetative reproduction was determined microscopically using the wet-mount technique after growing the yeast cells in YPD solution for 48-72 hours at 28°C.
Growth at 30°C on media agar enriched with the antibiotic cycloheximide (actidione) was carried out to test for resistance. MYPG agar supplemented with cycloheximide (MYPGch) along with WLD agar, both prepared as described above, were loop inoculated and streaked from colonies pre-grown on MYPG agar plates and used to test for resistance at 10 mg/l as observed by Gilliland (1961).
To test for the production of acids, MYPG agar supplemented with CaCO3 (MYPGcc) were prepared as described above, loop inoculated and streaked from colonies pre-grown on MYPG agar plates.
Additionally Michael Pearson at the University of California Santa Cruz conducted PCR identification sequencing based on polymorphism in the rRNA internal transcribed spacer region. Pearson identified both strains as B. bruxellensis. Egli and Henick-Kling (2001) previously described the methods used for genus and species identification based on polymorphism in the rRNA internal transcribed spacer region.
Screening of Yeast Culturability on Various Media Agar
The ability of Brettanomyces spp., B. bruxellensis (WLP650), B. lambicus (WLP653), B. claussenii (WLP645), B. bruxellensis (BSI-Drie) and B. bruxellensis (CMY001) to culture on popular brewery related media agar was carried out for the relevance of each media, its use throughout the project, and practicability of use in the brewing industry. The following media was prepared as described above: MYPG, MYPGcc, MYPGch, WLN, WLD, Copper Sulfate, Lysine and UBA. The media plates were loop inoculated and streaked from colonies pre-grown on MYPG agar plates then incubated aerobically in a dark growth chamber at 28°C for 7-10 days. Observations were taken on strength of growth and morphological characteristics.