Brettanomyces
Study of red wine contamination by Brettanomyces yeast
Equipment and Methods
Experimental wine storehouse
Eight experimental (test) stainless steel 100L vats were used in this experiment. Each vat was equipped with :
- 3 taps (to draw wine from the bottom, the middle and the surface of the vat).
- A micro-oxygenation diffuser (ceramic)
- A nitrogen protection (inertage) on the surface
- The vats were in an artificial wine storehouse, insulated and with air conditioning (photo 1).
Photo 1 : Experimental wine storehouse and vats
Yeast colony, culture medium and cellular concentration count.
The Brettanomyces colony came from an isolation on a sample of contaminated Madiran wine. This colony can form a veil on the surface of the wine after 20 days culture. It is called B3.
The Brettanomyces leaven used to inoculate the vats was obtained after 10 days culture on the wine, to which 5g/l of glucose and 0.2g/l of chloramphenicol were added.
The wine used was a Madiran wine (Tannat - Cabernet Franc) and had the following characteristics :
- free SO2 = 24mg/L
- pH = 3.7
- sterilised by flash pasturisation
- residual sugar < 2g/L
Brettanomyces populations were calculated by counting the colonies after spreading out 1ml on a Petri dish containing a selective nutritive medium. The results are shown in CFU/mL
(CFU - Colony Forming Unit), and represent the average of the samples taken at the top, middle and bottom of the vat.
Cellular concentration conditions permitting (>100000 CFU/mL), the CFU were counted directly on Thoma's cell.
Physico-chemical measurements
Dissolved oxygen measurements were carried out between the centre and the bottom of the vats using an external oxygen meter (Orbisphère) and a pump which enabled the wine to circulate.
Free SO2 concentration was measured using iodine titration N/50.
The explosion of the AZF factory in Toulouse prevented us from carrying out further measurements.
Experiment conditions
Five vats were inoculated with 3000 CFU/mL of Brettanomyces. Three uninoculated vats were used as dissolved oxygen reference vats; one inoculated vat was not oxygenated.
Vat n° 1 was oxygenated once on the 17th day, using an "accentuated running", so as to supply a dose of oxygen equivalent to that of a moderate racking or to that of cliqueur usage (about 2mg/L).
These supply techniques are similar, and cause a sudden increase in dissolved oxygen in the wine, just the opposite of micro-oxygenation. The doses studied are 5, 10 mL/L/month and an excessive dose of 20mL/L/month.
The doses Oenodev recommend for a wine at the post-malolactic fermentation stage are lower than 10mL/L/month, and about 2mL/L/month for a wine nearing the end of maturation (as is the case with the wine used during this study).
Table 1 shows experiment conditions.
| Vats | Oxygen (mL/L/month) | Brettanomyces (UFC/mL) | Free SO2 (mg/L) |
|---|---|---|---|
| 1 | 1.5 | 3000 | 24 |
| 2 | 5 | 0 | 24 |
| 3 | 5 | 3000 | 24 |
| 4 | 10 | 0 | 24 |
| 5 | 10 | 3000 | 24 |
| 6 | 20 | 0 | 24 |
| 7 | 20 | 3000 | 24 |
| 8 | 0 | 3000 | 24 |
Gilis J-F., Cabri C. et Ducournau P.
A joint study carried out by the Oenodev Research & Development Team, along with Professor Stréhiano's Fermentation and Bioreactor Team at the ENSIACET* at Toulouse.
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