Brettanomyces: Yesterday's Myths and Today's Realities Means of diagnosis and control of the development of Brettanomyces/Dekkera in wines
This saga is divided into 6 articles Read the introduction, Ecology and Origin of Brettanomyces in wines.
What are the factors and practices that influence the development of Brettanomyces in wines?
Means of preventive control against Brettanomyces and Brettanomyces: Means of curative control.
Brettanomyces is a yeast that has been known for a long time but has recently attracted a lot of attention. The latest fashionable subject, controversies, myths, miracle solutions are now legion and contribute to enormously complicating a serious problem but nonetheless not as complex as some would have us believe... The abundance of information of varying quality on this subject has rather detrimental to the management and solution of the Brettanomyces problem in red wines. The main objective of this article is to take stock of the key elements likely to significantly influence the development of microorganisms in wines and to identify practical and effective tools for controlling their development.
Microscopic observation is sometimes used to “identify” the presence of Brettanomyces. It is therefore useful to point out that it is completely illusory to be able to distinguish and even more so identify this type of yeast in a wine because none of the characteristic morphological types is clearly maintained in the physico-chemical conditions of the wine! Those who distinguish Brettanomyces by observation alone are therefore victims most of the time of hallucinations...
The Ethyl-phenol Determination makes it possible to clearly diagnose the current or past presence of Brettanomyces/Dekkera in a wine. Its regular monitoring (monthly in summer) during breeding allows, in the same way as we follow the evolution of acetic bacteria by measuring volatile acidity, to follow the development of yeast populations well. before they reach a critical threshold. This technique that we initiated for the monitoring of barrel aging several years ago (Brett® monitoring from EXCELL), is simple (taking a sample from several representative barrels) and quick (24 hours); it allows for easy reaction in the cellar (drawing, sulfiting, filtration or more energetic action) depending on the intensity of the development detected from the variation in volatile phenol contents. At this stage, microbiological control is of little interest. Microbiological control during breeding is significantly more complicated (sterile sampling), slow to produce (5 to 8 days) and difficult to interpret (see below) if it is carried out by culture. Indeed, due to (i) the variations existing from one strain to another in terms of adjustment of growth kinetics to the production of phenols and (ii) the differences in phenol production capacity from one strain to another. the other27, there is no clear relationship between viable population and volatile phenol content. Consequently, during aging, it ultimately does not matter whether we know the population of Brettanomyces/Dekkera, it is better to know the content and the evolution of the phenol content to predict the practical consequences in terms of wine quality. This technique should be generalized in cellars aging a large number of high-quality wine barrels to prevent any undesirable development thanks to a monthly monitoring frequency, particularly during the summer period from June to October.
Control and detection of populations of microorganisms
However, knowledge of Bretanomyces/Dekkera populations is essential before bottling to estimate the risk of future deviation in the bottle! It is incomprehensible that wines can be packaged today without having been checked beforehand. Population estimation can be carried out using different techniques, but for all the sample collection must be carried out on a homogenized wine because Brettanomyces is not a mobile germ and therefore tends to sediment at the bottom of the wine. containers.
The first method proceeds by counting viable populations obtained after cultivation on a specific medium. This methodology has three main limitations. The first relates to the so-called specificity of the culture media used. It turns out that the vast majority of laboratories and culture media marketed today are not media strictly specific to Brettanomyces/Dekkera. They are (at best) rather specific to non-Saccharomycodes yeasts (use of cycloheximide as a selective agent). Wine (particularly those aged in barrels) can harbor yeasts developing in these environments (Pichia sp., Hansenula sp., Zygosaccharomycodes, Hanseniaspora sp.) but which do not present any risk of producing a “phenolic” character. The use of these environments therefore tends to sometimes greatly overestimate the populations present (wines collected in barrels). Then, the counting of viable cells in the wine requires a culture duration varying between 5 and 8 days, a time long enough to develop spoilage if the wine is significantly contaminated...Finally, the counting by culture does not take into account the " viable but non-culturable germs” (VNC) which correspond to cells in a stressed physiological state, incapable of multiplying in the short term, which leaves them appearing dead when they are in fact capable of multiplying in the medium or long term term. CNVs unfortunately represent the vast majority (70 to 90%) of the microorganisms present in wines that are well aged and correctly prepared for bottling (fining, filtration and active sulfur dioxide levels adjusted to reduce the presence as much as possible). microbiological in general). This always results in an underestimation of risk when traditional microbiology is used. If it is used, it must at least use a high-performance and truly selective medium like the one we have developed and made available (Excell Brett Selective Medium©).
Enumeration using molecular biology techniques provides very specific and rapid access to germs belonging to the Brettanomyces/Dekkera genus. The technique using quantitative real-time PCR (Polymerase Chain Reaction) (RT-PCR) makes it possible to measure precisely and sensitively (10 cells/ml) the number of cells present according to the copy number of a fraction specifically amplified from the genome of these yeasts28. Simultaneous detection with amplification makes it possible to obtain results almost in real time after extracting and purifying the DNA contained in the cells; the laboratory obtains results within a day or half a day, which is extremely useful. Analyzing the DNA contained in healthy cells theoretically allows us to measure viable cells, whether they are in a quiescent state or not. In practice, this technology tends to slightly overestimate actual populations because there is a few weeks between the death, then the loss of integrity of a cell and the total disappearance of amplifiable DNA in the wine! With classic RT-PCR, we therefore instead measure total cells.
A modification of the protocol (EMA-RTPCR) before amplification (intercalation of ethidium bromide monoazide or similar salts) would now make it possible to specifically determine “real viable cells”29,30 when the populations are sufficiently abundant (we speak of QV-RT -PCR). However, in the current state of available reagents and available techniques which induce a certain direct toxicity on cells, the use of this principle of quantification of viable microorganisms remains quite delicate and should be reserved for relatively abundant populations (> 1000 cells). /ml) so as not to induce detrimental underestimation.
The PCR technique is particularly useful in quality control during the preparation of wines for bottling to determine the benefit of microbiological stabilization and specify, if necessary, the ideal filtering medium. It also makes it possible to monitor the effectiveness of the process online before the end of the project and to ship the wines with full knowledge of the facts. With other types of nucleic acid probes, this same technique also makes it possible to simultaneously measure (on the same sample and within the same time frame) total yeasts, other types of contamination yeasts (Zygosaccharomyces bailli for example), the main bacteria lactic acid contamination (Lactobacillus sp. and Pediococcus sp.) or acetic bacteria from wine (Acetobacter and Gluconobacter sp.). The VinoBRETT™ system developed by Invisible Sentinel™ offers a miniaturized RT-PCR system without cyclic thermal amplification with significantly simplified handling which allows semi-quantitative estimation of populations in only 4 h with an advanced detection threshold apparently quite low (10 cells/ml)31.
Flow cytometry is a technique for counting the cells of microorganisms both by their size and structure (particle size and density) and/or by their marking with a specific antibody coupled to a fluorochrome (no staining sensus stricto). The fluorochrome is excited by a laser whose emitted fluorescence is measured. This technique is attractive due to its simplicity and speed. However, the basic technique generally proposed uses marking which is not often specific to Brettanomyces/Dekkera! The promoters of this technique first distinguished Brettanomyces from other yeasts by a size criterion, assuming that the smallest cells (3 µm) correspond to Brettanomyces/Dekkera… However, we know that the shape or size criterion n is not reliable in the physicochemical conditions of the wine. In addition, (quiescent) VNC germs can have very small sizes but also have a weak or at least variable affinity to the antibody-fluorochrome couple. It follows that the rate of detection in wine by flow cytometry of germs supposed to be Brettanomyces turns out to be quite high (>200 cells/ml) and the specificity of the method more than relative.
The recent development of different coloring reagents using in situ fluorescence after hybridization with DNA (FISH), or better the more sensitive and more robust FISH-Peptic Nucleic Acid (PNA-FISH) coupling, allows controls by epifluorescence or cytometry. flow (FISH-FCM) truly specific and more sensitive (<100 cells/ml)32 while remaining less sensitive than PCR.
Alternative techniques using antibodies to specifically label Brettanomyces should use monoclonal antibodies; poly-clonal antibodies (Amarok™)33 which react with elements of the wall of microorganisms are not at all specific and produce cross-reactions with other phylogenetically close microorganisms which are often abundant in wines aged in barrels…
We can also validly distinguish dead cells from living ones by coupling PNA-FISH staining with specific staining of living and dead cells with Baclight™ technology for example34 allowing the combination of biochemical probes sensitive to the redox state of the cells or to the permeability of membranes, for example, capable of reflecting the viable or dead state of microbial cells.
Besides the different methods listed above, there are other solutions which are more of a screening test than a real diagnosis or even a gadget...
The SNIF BRETT® test consists of making a liquid culture of wine to detect the presence of Brettanomyces by the olfactory detection of ethyl phenols after several days of incubation; the relative intensity of the risk would be inversely proportional to the speed of appearance of the odor... However, the detection of the odor of volatile phenols is closely linked to the sensitivity of each person and some people are really not very sensitive to this aroma. Then, as we have already mentioned above, there is no strict relationship between the production of volatile phenols and the population of contamination yeasts. Finally, the response time of the test to be obvious is quite long. In conclusion, only a frankly and quickly positive test allows us to draw a conclusion, but then it is often already too late...A negative test does not allow us to conclude with certainty, but its periodic repetition at short notice nevertheless allows us to ensure a certain follow up.
The detection of Brettanomyces by an immunological reaction using antibodies coupled with a colored enzymatic reaction (ELISA) has been the subject of various attempts. The only commercial formula still apparently available (Z-Brett™ from Z-Enology) uses a strip of polyvinylidene fluoride impregnated with immobilized reagents on which a volume of centrifugate of the wine to be tested is deposited35. The response is reflected after complete reaction by a more or less dark coloring which can be linked qualitatively and approximately to a reference. The test is simple to implement and quite quick. The announced sensitivity is ambitious (10 or 100 cells/ml) but to be achieved it requires a significant concentration of the sample by centrifugation, which is not easy to achieve with honest reproducibility! In addition, the antibodies developed are once again polyclonal and therefore not really specific for Brettanomyces/Dekkera: Zygosaccharomyces sp., Pichia sp., Candida sp. which are common in wines aged in barrels react and disrupt the result by exaggerating the response. This test therefore looks more like a binary positive/negative detection method for populations that are actually quite high (> 1000 cells/ml).
28 CHATONNET P., FLEURY A. 2008 New methods and new applications of microbiological control in near real time using Excell Gen® technology. Revue des Oenologues, 129, 23-27
29 NOVGA HK, DROMTORP SM, NISSEN H, RUDI K. 2003 Ethidium monoazide for DNA based differentiation of viable and dead bacteria by 5-nuclease PCR, Biotechniques, 010, 812-813
30 RUDI K, MOEN B., DROMTORP SM, HOLCK AL. 2005 Use of ethidium monoazide and PCR in combination for quantification of viable and dead cells in complex samples, Applied and Environmental Microbiology, 71, 1018-1024
31 https://invisiblesentinel.com/products/wine-quality-tests/vinobrett-brettanomyces-bruxellensis/
32 SERPAGGI V., REMIZE F., SEGUEIRO-LE-GRAND A. 2010 Specific identification and quantification of the spoilage microorganism Brettanomyces in wine by flow cytometry: A useful tool for winemakers Cytometry Part A, 77A, 497-499
33 http://amarokbiotech.com/pdf/Poster%20AFC%20Bretta%20V3.pdf
34 https://www.alphalabs.co.uk/pnafish
35 O'NEIL M., LEBRUN S. 2007 Rapid, low-cost assay for detecting Brettanomyces and other spoilage yeasts in wine. United States Patent Application Publication, No. US 2007/0196877 A1