5.6.3 Analytical means for monitoring spoilage microorganisms

Bactéries acétiques

The most common is the monitoring of volatile acidity or acetic acid which represents 98% of the volatile acidity. Monitoring these parameters is fast, easy and effective. Testing can be carried out using the enzymatic method where solely acetic acid is measured, by FTIR measurement or by acid-base titration (official method) for volatile acidity.
Alternatively, it is also possible to quantify the bacteria via epifluorescence, PCR or on agar media. Counting is a more selective technique to identify the source of contamination in the event of problems.

Brettanomyces bruxellensis

Measurement of phenols (see below)

This technique provides information on phenol concentration in wine and therefore enables intervention if the quantity starts to increase. The technique is called SBSE/GC/MS, SBSE being a phenol extraction technique using magnetic bars (Stir Bar Sorptive Extraction) then gas chromatography (GC) and mass spectrophotometry (MS). It is a fast and precise method but rather expensive. Like the volatile acidity monitoring for acetic bacteria, ethyl-phenols determination for the monitoring of B.bruxellensis is very precise if done regularly. Brettanomyces population levels are not a determining factor in the quantity of phenols quantity produced, it is possible to have a significant population and no phenols and vice versa.

Counting on Petri dishes

This is a selective agar that allows observation of the development of Brettanomyces. This method is inexpensive. However, the delay before obtaining results is rather long (at least 8 days) and the viable but nonculturable population (VNC) is not quantified. To do a plate count, a quantity of wine must be filtered, then 1 ml is applied to the agar. The initial wine quantity is chosen by the winery or according to the laboratory's protocol, it is then possible to detect low population thresholds by filtering, for example, one litre of wine to concentrate the potential quantity of B. bruxellensis.

Flow Cytometry

This method is based on counting cells by means of specific staining with a fluorescent agent. The cells are then detected by a particle counter. This analysis is quite fast and more accurate than using a Petri dish. It enables the quantification of cells from 200 cells/ml.

qPCR (quantitative polymerase chain reaction)

This is a molecular biology technique that enables the amplification of a specific fraction of the B. bruxellensis genome. Once amplified, the number of cells in the medium can be detected and quantified. It enables the quantification of small populations, from 10 cells/ml.
The method is specific, reliable and fast. Note that, depending on the analysis protocol used, it is possible to count dead cells which results in an overestimation of the population.

Setting up monitoring of microorganisms in the envirionment: what is an appropriate testing frequency?

Frequency depends on the risk factors, production stage and microorganism type. If the alcoholic fermentation is stuck or if there is a significant delay between AF and MLF, regular monitoring should be implemented early on to avoid any risks.

**Volatile acidity

For volatile acidity, which can potentially increase due to lactic and/or acetic bacteria, there should be very regular monitoring as soon as AF ends. If there is residual sugar, the lactic bacteria can break down the sugar and produce acetic acid. Vigilance is therefore required.
Similarly, when oxygen is added during the ageing process and/or when the free SO2 is insufficient, the volatile acidity can increase via acetic acid bacteria proliferation.
In all cases, acetic acid should ideally be monitored weekly during malolactic fermentation and monthly during ageing.

**Brettanomyces / Volatile phenols

Traditionally, a preliminary assessment is carried out after MLF to evaluate any risks. Subsequently, frequency is determined by the initial contamination. Monthly or bi-monthly monitoring may be appropriate. For high-risk cases, a follow-up every two weeks is advisable, especially during periods of high risk, such as when cellar temperatures rise in summer. Similarly, when used barrels are employed, initial monitoring should be more frequent.
In a situation where ageing takes place with the bung on the barrel's side, it may be relevant to keep a few barrels with the bung on top to use for monitoring even if the exposure to oxygen will not be quite the same. Another method is to pierce the barrels using a small spike and then close them.
Other cases, such as maturing without SO2, require greater frequency.