Brettanomyces testing

Brettanomyces bruxellensis (Brett) is a major wine spoilage yeast responsible for producing volatile phenol compounds. These compounds; 4-ethylphenol (4-EP), 4-ethylguaiacol (4-EG) and 4-ethylcatechol (4-EC), impart characteristics described as “horsy”, “Band-Aid”, “barnyard” and “sweaty saddle”. Collectively these are known as “Brett character”. More information on mitigation and control strategies can be found in the AWRI Fact sheet – Controlling Brettanomyces during winemaking.

In conjunction with control strategies, regular monitoring for Brett is recommended. Regular monitoring allows for the early detection and quantification of Brett in wine before volatile phenols can be detected sensorily. Affinity Labs offers numerous methods for the detection of Brett.

Methods for the detection of Brettanomyces bruxellensis

Selective plating

The plating of wine samples onto nutrient agar is considered the gold standard in Brett detection. Up to 50 mL of wine is filtered through a 0.45 uM membrane filter, which is then placed onto Wallerstein Nutrient agar (WL), supplemented with 100 mg/L cycloheximide, 50 mg/L Chloramphenicol and 500 mg/L biphenyl for the inhibition of Saccharomyces cerevisiae, bacteria and moulds. As some Brett strains are slow growing, plates are incubated for 10 days before results are reported. Any growth is analysed microscopically to identify characteristics that indicate presumptive Brett. Selective plating is a highly sensitive method, with a limit of detection of 1 cell/50 mL of wine. Only viable cells are detected using this method. Though this method is selective for Brett, it is not exclusive to Brett. Confirmation of Brett contamination can be performed using either of the below nucleic acid based methods.

BrettCount (Flow cytometry)

Affinity Labs offers a flow cytometry-based rapid detection method, the Sysmex CyFlow BrettCount system. This method uses a mixture of fluorescent gene probes targeting different regions of the 16s ribosomal RNA of Brett cells to rapidly detect and quantify them in wine. This method has high specificity for Brett and does not cross react with other species that may be present in wine. The limit of detection is 100 cells/mL. Only viable cells are detected using this method, though some cells that are not culturable under lab conditions can also be detected. BrettCount is rapid, and results are obtained within one day.

PCR (Veriflow)

This method uses polymerase chain reaction (PCR) to specifically target Brett DNA. This method has high specificity for Brett and does not cross react with other species that may be present in wine. The limit of detection is 10 cells/mL of wine. Being a PCR based method it is unable to differentiate between live cells, dead cells and DNA present in the wine. Veriflow is rapid, and results are obtained within one day.

Comparative study

Method	Volume required	Turnaround time (days)	Limit of detection	Detects dead cells	Specific to Brettanomyces bruxellensis	Quantitative	Suitable sample types
Selective plating	Up to 50 mL	10	1 cell/50mL	N	N	Y	Wine, beer, other beverages
Flow cytometry	Up to 1 mL	1	100 cells/mL	N	Y	Semi	Wine, beer
PCR	Up to 25 mL	1	10 cells/mL	Y	Y	N	Wine

Table 1. A comparison of the Brettanomyces bruxellensis detection methods offered by Affinity Labs.

Affinity Labs performed a comparative study on 21 Brett positive wines to compare the three different Brett detection methods. Wines were initially determined to be positive by Veriflow analysis and then analysed by both plating and BrettCount. Comparative analysis demonstrated that Veriflow results correlated with plating 38% of the wine. BrettCount results correlated with plating 67% of the time. Differences in results between Veriflow and plating were attributed to the presence of residual DNA and/or dead cells in the wine sample. Differences in results between BrettCount and plating were attributed to cells being below detection limits and/or the presence of non culturable cells. Overall, we found that BrettCount correlates most highly with selective plating for the detection of Brett in wine, which is the current gold standard.

Testing for Brett taint

4-ethylphenol and 4-ethylguaiacol are products of metabolism by Brettanomyces yeast. 4-ethylphenol is responsible for the ‘sweaty saddle/band-aid’ aroma of red wines when present in high concentration, and is generally regarded as detrimental to wine quality. Analysis of wine in barrels will determine whether this compound has been formed in high concentration during maturation and may indicate the need for changes in how the wine is subsequently handled, including blending options, sterile filtration or sulfur management. These compounds can be analysed separately to the oak flavour compounds where the latter is not required.

How does the analysis work?

The analytical method uses headspace-solid phase microextraction (HS-SPME) combined with GCMS analysis to only detect these volatile compounds. Alternatively, a simple liquid-liquid extraction can be performed to extract these same compounds.

How can this assist winemakers?

The analytical results will enable winemakers to determine significant barrel-to-barrel variation in perceived wine quality. If differences are found, it may be due to Brettanomyces activity. Analysis of wines during barrel storage may provide information that will enable winemakers to manage sulfur levels and capture early signs of Brettanomyces activity.

Sample submission

If submitting samples for 4-EP and 4-EG analysis and any associated microbiological testing, such as PCR-based Veriflow, BrettCount or plating for Brettanomyces, please send duplicate samples to avoid potential delays in results. Samples for microbiological testing are unable to be opened prior to these tests being performed, due to the risk of contamination and ‘false positive’ results. This can lead to delays in the 4-EP/4-EG analysis when a single sample is submitted. Duplicate samples will enable each replicate to be handled separately without compromising on turnaround time or results.