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.
Results:
- 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.
Reference:
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
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