The aim of the study was to determine if Brettanomyces can survive in grape pomace under conditions typically found in vineyards and as a result serve as a source of contamination for future harvests.
Experimental layout of short term incubation studies:
- A Syrah wine was produced and the free run wine separated from the un-pressed pomace.
- The pomace was placed in sterile polypropylene bottles of which half of the pomace containing bottles were autoclaved.
- The bottles were inoculated with one of three different strains of Brettanomyces and kept at 21°C for seven days before incubation at four different temperatures: 21°C, 10°C, 0°C and -18°C.
- At various stages bottles were removed from the given storage temperature and Brett enrichment medium added before further incubation at 21°C for 18, 168 or 720 hours, in order to evaluate the culturability of Brett.
Experimental layout of long term experimental studies:
- In year one pomace from Syrah wine at two different alcohol levels (11.5% and 14.9%) was added to 250 ml receiver flasks that were fitted with 0.22µm PVDF membrane filters.
- Half of the containers were sterilised with gamma rays.
- All containers were inoculated with the same one strain of Brett and incubated for one week at 21°C.
- Containers were then placed in random places in vineyards. Air temperatures of these locations were documented.
- Every ten weeks samples from the different treatments were removed and Brett enrichment medium added and then further incubated at 21°C for 18, 168 or 720 hours to determine Brett culturability.
- In year two the experiment was repeated but with the Syrah pomace having alcohol levels of 11.7% and 15.1%.
- Additional pomace from year two was divided into two lots with the one lot receiving 45 mg/l SO2.
- Both lots were inoculated with Brett and incubated for one week at 21℃.
- Replicate samples were then placed in water baths with different temperatures: 40, 50, 60 or 70℃ for different times.
- After the specific heating times the bottles were removed and Brett enrichment medium added. They were then incubated for up to an additional 60 days at 27℃.
- Brett culturability was determined by plate counts. Brett identity was confirmed by genetic sequencing of random colonies.
- Brett could be recovered from pomace stored at -18℃ after 10 weeks.
- Brett cells could be recovered from pomace stored in vineyards after 130 weeks.
- Total cell recovery was dependent on the vineyard air temperature. Larger recoveries were obtained when the vineyard temperatures were above 10℃.
- Sterilised (before Brett inoculation) pomace always had higher Brett counts than non-sterilised pomace.
- Lower alcohol pomace had higher Brett counts than higher alcohol pomace (inhibition effect of alcohol).
- A minimum temperature of 50℃ for more than 10 minutes is needed to eradicate Brett from pomace.
- Heating time period can be shortened by either increasing the temperature or by adding SO2.
Significance of the study:
The study indicated that Brett is extremely resilient and can survive the extreme conditions in vineyards. As a result untreated infected winery pomace worked back into the vineyard can serve as a source of contamination for future harvests. Large scale composting might be a way to eradicate Brett from pomace destined for vineyards, but must be further investigated.
Survival of Brettanomyces bruxellensis in grape pomace and reduction of populations by application of heat and sulfites.
Z.M. Cartwright B.R. Bondada C.G. Edwards
First published: 11 November 2018 https://doi.org/10.1111/ajgw.12372