METHOD FOR THE DETECTION, QUANTIFICATION AND CHARACTERIZATION OF BRETTANOMYCES SPP. AND OTHER YEASTES CONTAINED IN AN ORGANIC LIQUID SUBSTRATE WITH FERMENTABLE SUGARS

Description

Domaine technique

[0001] The present invention relates to a method enabling, with the same sample and a single passage through a cytomer, the detection, quantification, and discrimination of yeasts. Brettanomyces and other yeasts, in particular Saccharomyces spp contained in an organic liquid substrate containing fermentable sugars. Art antérieur

[0002] Yeasts of the genus Brettanomyces are capable of producing compounds that produce, among other things, stable odors. Brettanomyces It is relatively resistant to alcohol and low pH. In most cases, it is sensitive to SO2, although some strains can be particularly resistant. Yeasts Brettanomyces are smaller in size compared to Saccharomyces cerevisiae However, they remain difficult to identify due to significant polymorphism. Furthermore, in a complex environment such as wine, vinegar, or must, for example, their differentiation is even more complicated due to interactions with the surrounding environment. The most common yeast Brettanomyces East Brettanomyces bruxellensis which includes a sporulating genus called Dekkera bruxellensis. These yeasts produce, among other things, 4-ethylphenol, which is stable in wine and gives it a barnyard odor that is best avoided. The same phenomenon can occur in other fermented and possibly alcoholic beverages such as cider, beer, tequila, kombucha, and kefir, for example. Brettanomyces spp Being present in the surrounding environment, it is likely to contaminate any liquid substrate containing fermentable sugars in which it can grow. Yeasts Brettanomyces Cells can exist in several states: living (therefore active and producing the aforementioned compound), dead (not inactive and not producing the aforementioned compound), or latent. Latent cells are capable of becoming active again and thus producing, among other things, 4-ethylphenol during the aging of wine, for example. Cells in the latent state are not detectable by culturing the substrate, which means that culturing a substrate sample does not guarantee the absence of undesirable fragrances in the future.

[0003] The publication entitled "The application of flow cytometry in microbiological monitoring during winemaking: two case studies" by R. Guzzon et al., published in the journal Ann Microbiol (2015) 65 / 1865-1878, indicates that it is possible to differentiate a living cell from a dead cell in a culture of S . cerevisiae pure or in a wine to which a mixture of sugar and S . cerevisiae. The method applied to the wine before the addition of the aforementioned mixture for a second fermentation yielded no results. A mixture of markers is used in this method: the cFDA mixture combined with propidium iodide.

[0004] The publication entitled "Rapid detection of viable yeasts and bacteria in wine by flow cytometry" by Malacrino et al., published in the Journal of Microbiological Methods (45 (2001) 127-134), indicates that it is possible to determine the number of malolactic yeasts and bacteria in Pinot Noir juice to which pre-cultured yeasts have been added in a specific medium (1% yeast extract + 2% peptone + 2% dextrose) by flow cytometry. The best marker is the cFDA fluorescein mixture. The same method applied to a wine sample does not give results consistent with those obtained by counting cultures. The wine must therefore be washed before measurement. The publication suggests that this method could be used for the detection of undesirable yeasts such as Brettanomyces spp.

[0005] The publication entitled "Survival and metabolism of hydroxycinnamic acids by Dekkera bruxellensis in monovarietal wines" by Nine de Lima, published in the journal Food Microbiology in February 2021 (volume 93), indicates that it is possible to measure the population of Dekkera bruxellensis in wine previously inoculated with a strain of Dekkera bruxellensis by flow cytometry. Two markers were used in combination: propidium iodide and SYTO 9 ®< .

[0006] The publication entitled "Specific Identification and Quantification of the Spoilage Microorganism Brettanomyces in Wine by Flow Cytometry: A Useful Tool for Winemakers," by H. Alexandre et al., published online on February 11, 2010, in the journal Wiley Interscience, describes a method for identifying and quantifying Brettanomyces in wine using flow spectrometry with the fluorescence in situ hybridization (FISH) technique. The fluorescent marker used (Alexa Fluor® < 488) targets specific amino acid sequences that correspond to Brettanomyces RNA. The Brettanomyces cell membrane in wine is initially permeabilized, making it impossible to differentiate between dead, live, or latent cells. The detection threshold is 10² cells / mL.

[0007] The publication entitled "A simple procedure for detecting Dekkera bruxellensis in wine environment by RNA-FISH using a novel probe" by Branco P et al., published in the international Journal of Food Microbiology Elsevier in 2019, describes a method for detecting D . bruxellensis (Brettanomyces dekkera bruxellensis) in wine using a specific fluorescent probe that targets a particular sequence of ribosomal RNA D . bruxellensis. This probe allows for reliable detection if the amount of ribosomal RNA is sufficient to produce a sufficiently strong fluorescent signal. According to this method, samples are incubated for 2 hours at 46°C. A red diode (680 / 30) is used to excite the sample. Cell membranes are permeabilized (for 1 hour), which makes it impossible to distinguish between dead and live cells.

[0008] The publication entitled "Evaluation of damage induced by Kwt and Pikt zymocins against Brettanomyces / Dekkera spoilage yeast, as compared to sulphur dioxide" by Ora L. et al., published in the Journal of Applied Microbiology in 2016, indicates the use of propidium iodide for detection Dekkera bruxellensis. This document discusses the toxicity of two zymocins on Brettanomyces but does not describe a method for detecting Brettanomyces spp.

[0009] The document entitled "Evaluation of yeast viability and concentration during wine fermentation using flow cytometry" by Thornton, p. 209 of the book "Cultivability, mortality and metabolic activity," indicates that it is possible to detect yeast in wine and determine if these yeasts are alive. However, this document does not describe the possibility of discriminating Brettanomyces spp of other yeasts. Problème Technique

[0010] One aim of the present invention is to propose a flow cytometry method that allows for the determination, quantification, and discrimination of yeasts. Brettanomyces spp other yeasts, and in particular yeasts Saccharomyces spp in a liquid substrate containing fermentable sugars and possibly fine particles the size of bacteria or yeasts.

[0011] Another objective of the present invention is to propose a flow cytometry method that allows for the discrimination and quantification of live yeast cells. Brettanomyces spp, dead yeast cells Brettanomyces spp and latent yeast cells Brettanomyces spp.

[0012] Another objective of the present invention is to propose a method that is quick to implement and in particular requires a reduced incubation time.

[0013] Another objective of the present invention is to propose a method that is applicable to a finished product, such as wine, cider, beer, vinegar or fruit juice, possibly put in a vat or bottled and suitable for sale.

[0014] Another aim of the invention is to propose a method which, with an analysis of the same sample, also allows for the counting of bacteria and possibly their state (dead, latent or alive). Brève description de l'invention

[0015] The present invention relates to a method for the detection, quantification and discrimination of yeast cells by flow cytometry. Brettanomyces spp contained in an organic liquid substrate containing fermentable sugars, according to which a sample of said substrate is taken, possibly diluted, at least one first fluorochrome capable of binding to the DNA of dead and / or living cells is added to said possibly diluted substrate, said sample is irradiated so as to obtain fluorescence emission from said first fluorochrome and said sample is also irradiated so as to obtain fluorescence emission from said sample at 670 nm, a biparametric histogram is established giving for each point the intensity of the fluorescence due to the first fluorochrome and the intensity of the fluorescence emitted at 670 nm, thus obtaining at least one first cluster of points corresponding to a fluorescence intensity emitted and detected at 670 nm greater than that detected for the other points, it is deduced that the points of said first cluster correspond to the cells of Brettanomyces spp and we may count the number of cells of Brettanomyces spp by counting the points of said first cloud.

[0016] It is indeed to the Plaintiff's credit that she highlighted the autofluorescence of the cells of Brettanomyces spp at 670 nm after excitation with a laser emitting in the wavelengths corresponding to red (from 620 nm inclusive to 750 nm inclusive), and in particular at 637 nm. The first fluorochrome binds to DNA, thus suppressing background noise due to suspended particles, for example. Only the cells of the microorganisms present, i.e., yeasts, fungi, and bacteria, will be labeled. If the first fluorochrome only penetrates cells with damaged and therefore permeable cell walls (dead cells), the fluorescence signal only allows the detection of dead cells. However, determining the first window, as explained later, and using a second fluorochrome that binds to both dead and living cells allows the detection of all cells. Brettanomyces spp.

[0017] The excitation wavelength is not limited according to the invention. It is advantageously greater than or equal to 620 nm and less than or equal to 750 nm inclusive, and in particular equal to 637 nm.

[0018] Furthermore, the demonstration of autofluorescence of cells Brettanomyces spp allows for the rapid and precise definition of a window on the biparametric histogram, which gives the method of the invention high precision, high reliability and high speed of implementation.

[0019] Advantageously, the first fluorochrome binds to the DNA of both dead and living cells.

[0020] The sample can be diluted to 1 / 10, 1 / 40, 1 / 100, 1 / 300 or 1 / 1000, depending on its yeast and bacteria load. Description détaillée

[0021] According to a specific implementation method, the substrate in question contains primarily yeasts. Brettanomyces spp and yeasts Sacharomyces spp, On the biparametric histogram, we obtain at least two point clouds, one comprising the points corresponding to a fluorescence intensity emitted at 670nm greater than that of the points in the second point cloud; we deduce that the points of the first cloud correspond to the cells of Brettanomyces spp and that the points of said second cloud correspond to the cells of Saccharomyces spp and we may count the number of cells in Brettanomyces spp and / or Saccharomyces spp by counting the points of each of said clouds.

[0022] Advantageously, depending on the complexity of the substrate, before measuring fluorescence, a first discrimination is made between the particles and the cells contained in said sample by measuring the intensity of the reflected and refracted light and the intensity of the diffracted light, a biparametric histogram is established giving for each point corresponding to a detected particle or cell the value of said intensities, a first window is thus determined according to the values ​​of said intensities which contains points attributable to yeast cells and possibly a second window which corresponds to points attributable to bacterial cells, said biparametric histogram is established giving the intensity of the fluorescence due to the first fluorochrome and the intensity of the fluorescence emitted at 670nm for each point located in said first window.Determining the first window (using a "gating" strategy) reduces background noise from particles and bacteria in the substrate. This isolates the points corresponding to yeast cells, and the aforementioned biparametric fluorescence emission histogram is generated for these cells alone. This allows for the detection of points that actually represent either a bacterium or a particle and are still located within the first window.

[0023] Advantageously, the said first fluorochrome being capable of binding to the DNA of living cells and to the DNA of cells whose cell wall is permeable, before any measurement, a second fluorochrome capable of binding only to the DNA of cells whose cell wall is permeable is added to the said sample, possibly diluted; a third window is determined which surrounds the points of the said first cloud; the sample is excited so as to cause fluorescence emission from the first and the second fluorochrome; and for the points located in the said third window, a biparametric histogram is also established giving, for each point, the intensity of the fluorescence of the first and that of the second fluorochrome, or the intensity of the fluorescence per unit area of ​​one of the two fluorochromes and that due to the other fluorochrome; and two groups of points are determined,a first group for which the fluorescence due to the fluorochrome which binds only to the DNA of cells whose cell wall is permeable is greater than that of the second group and possibly we count the number of points of each of the groups, which corresponds to the number of cells of , Brettanomyces sppp living cells for the second group and the number of cells Brettanomyces spp dead for the first group.

[0024] It is also possible to use fluorescence intensity per unit area for either of the fluorochromes.

[0025] The second fluorochrome further suppresses spots corresponding to particles or bacteria; such spots may indeed remain in the first window. The closer the fluorescence emission wavelength of the second fluorochrome is to 670 nm, the greater the fluorescence intensity of the cells. Brettanomyces spp labeling with this second fluorochrome is important. It is thus possible to distinguish cells from Brettanomyces spp dead cells Brettanomyces spp alive.

[0026] It is also possible to distinguish the cells from Brettanomyces spp Dead cells were detected using only autofluorescence at 670 nm. A biparametric histogram was then established in the third window, showing the fluorescence for the first fluorochrome and the fluorescence detected at 670 nm. The fluorescence of dead and / or latent cells at 670 nm is weaker than that emitted by living cells.

[0027] Advantageously, said second window is determined and said sample is excited so as to cause fluorescence emission from the first and second fluorochrome and for the points located in said second window, a biparametric histogram is established giving for each point the intensity of the fluorescence of the first and that of the second fluorochrome or the intensity of the fluorescence per unit area of ​​one of the two fluorochromes and that due to the other fluorochrome and two groups of points are determined, a first group for which the fluorescence due to the fluorochrome which binds only to the DNA of cells whose cell wall is permeable is greater than that of the second group and the number of points in each of the groups is counted, which corresponds to the number of live bacterial cells for the second group and the number of dead bacterial cells for the first group.

[0028] With a single sample and a single pass through the cytometer, it is possible to obtain a simultaneous analysis of yeasts and bacteria.

[0029] A particularly advantageous method is to add, before any measurement, a third fluorochrome, which only emits a fluorescence signal when it reacts with a living cell, to the sample, possibly diluted. The sample is then excited to also elicit fluorescence from the third fluorochrome, and a biparametric histogram is established for the points in the second and / or third window. This histogram shows, for each point, the fluorescence intensity due to the fluorochrome that binds only to the DNA of cells with permeable cell walls and the fluorescence intensity due to the third fluorochrome. For each window, three subgroups of points are then determined: a first subgroup of points corresponding to a fluorescence intensity due to the third fluorochrome higher than that of the other subgroups, this first subgroup of points representing the cells of Brettanomyces spp / live and active bacterial cells, a second subgroup of dots corresponding to a fluorescence intensity due to the third fluorochrome lower than that of the first subgroup and coupled with a fluorescence intensity due to the first / second fluorochrome lower than that of the third subgroup, the dots of this second subgroup correspond to the cells of Brettanomyces spp / bacterial cells in a latent state and a third subgroup of dots corresponding to a fluorescence intensity due to the first / second fluorochrome greater than that of the first and second subgroups, these dots correspond to the cells of Brettanomyces spp / dead bacterial cells.

[0030] Here we see that the presence of the three fluorochromes allows, with a single sample analyzed in one pass through a flow cytometer, not only the simultaneous detection of the presence of yeasts and bacteria, but also the quantification (number of cells) of yeasts and bacteria, and the differentiation of yeasts. Brettanomyces spp other yeasts, in particular Saccharomyces spp but also to determine for bacteria and for yeasts, and more specifically for yeasts Brettanomyces spp their state (dead, alive, or latent). The method of the invention even makes it possible to quantify latent yeasts and bacteria, which are not detectable by culture. It also proves to be much faster than the enumeration method after culturing.

[0031] The shape of the windows is not limited according to the invention. They can advantageously be polygonal.

[0032] According to the invention, fluorochromes are not limited. Thus, said first and said second fluorochrome are different and may be chosen from among fluorochromes capable of binding to cell DNA and having a maximum fluorescence absorption wavelength equal to or greater than 599nm and equal to or less than 657nm, a maximum fluorescence emission wavelength equal to or greater than 619nm and equal to or less than 678nm and a quantum yield equal to or greater than 0.16 and equal to or less than 0.39 and mixtures thereof, in particular fluorochromes capable of binding to cell DNA and having a maximum fluorescence absorption wavelength of 652 nm, a maximum fluorescence emission wavelength of 676 nm and a fluorescence quantum yield on DNA of 0.27 and fluorochromes capable of binding to DNA and having a maximum fluorescence absorption wavelength of 657nm,a maximum fluorescence emission wavelength of 673nm and a fluorescence quantum yield on DNA of 0.17, fluorochromes capable of binding only to the DNA of cells with permeable cell walls and which have a maximum fluorescence absorption wavelength of 547nm, a maximum fluorescence emission wavelength of 570nm and a fluorescence quantum yield on DNA of 0.9 and mixtures thereof.

[0033] When said first fluorochrome is chosen from among fluorochromes capable of binding to DNA and having a maximum fluorescence absorption wavelength of 657nm, a maximum fluorescence emission wavelength of 673nm and a quantum yield of fluorescence on DNA of 0.17 and fluorochromes capable of binding to cell DNA and having a maximum fluorescence absorption wavelength of 652 nm, a maximum fluorescence emission wavelength of 676 nm and a quantum yield of fluorescence on DNA of 0.27, the second fluorochrome is chosen from among fluorochromes capable of binding only to cell DNA whose cell wall is permeable and which have a maximum fluorescence absorption wavelength of 547nm and a maximum fluorescence emission wavelength of 570nm and a quantum yield of fluorescence on DNA of 0.9.Said third fluorochrome is advantageously chosen from 5-carboxyfluorescein diacetate, 6-carboxyfluorescein diacetate, mixtures of 5-carboxyfluorescein diacetate and 6-carboxyfluorescein diacetate and succinimidyl 5,6-fluoroescein diacetate of the following general formula (1): .

[0034] Thus, according to a specific implementation method, the first and second fluorochromes can be chosen from among the fluorochromes marketed by Thermo Fisher under the names SYTOX-orange®, SYTO 62®, and SYTO 63®. When the first fluorochrome is chosen from SYTO 62® and SYTO(63), the second fluorochrome is SYTOX-orange®. Conversely, when the second fluorochrome is SYTOX-orange, the second fluorochrome is chosen from SYTO 62® and SYTO 63® and mixtures thereof.

[0035] The fluorochrome SYTOX Orange ®< penetrates only cells with damaged cell walls, i.e., dead cells. It is preferably used in combination with the third fluorochrome to discriminate between the different states of cells of Brettanomyces spp (alive, latent or dead)

[0036] The fluorochrome SYTOX-orange ®< is marketed by Thermo Fisher Scientific. It is capable of binding to DNA, has a maximum absorption fluorescence wavelength of 547nm and a maximum emission fluorescence wavelength of 570nm and a fluorescence quantum yield on DNA of 0.9.

[0037] A fluorochrome capable of binding to cell DNA and exhibiting a maximum fluorescence absorption wavelength of 652 nm and a maximum fluorescence emission wavelength of 676 nm and a quantum fluorescence yield on DNA of 0.27 is marketed under the name SYTO 62 ®< by the Thermofisher company.

[0038] A fluorochrome capable of binding to DNA and exhibiting a maximum absorption fluorescence wavelength of 657nm and a maximum emission fluorescence wavelength of 673nm and a quantum fluorescence yield on DNA of 0.17 is marketed under the name SYTO 63 ®< by the company Thermofisher.

[0039] The substrate is not limited according to the invention. Thus, the substrate can be chosen from sparkling or still wine, red wine, white wine, rosé wine, cider, beer, sake, fruit juices, in particular grape or apple juice, water kefir, fruit juice kefir, milk kefir, milk, tequila, whisky, vodka, musts in particular grape musts, wines in the process of first or second fermentation, finished wines, sparkling or still, and vinegars.

[0040] The method of the invention can be applied to any type of yeast Brettanomycesspp chosen from the following species B . anomalus, B. bruxellensis, B. custersianus, B. nanus, B. dekkera bruxellensis et B. naardenensis of at least one other species of yeast and in particular

[0041] In particular, it allows us to distinguish between cells of Brettanomyces spp cells of at least one species of Saccharomyces spp chosen from the following species: Saccharomyces bailii Linder, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces delbrueckii, Saccharomyces exiguus, Saccharomyces fermentati, Saccharomyces florentinus, Saccharomyces fragilis, Saccharomyces fructuum, Saccharomyces heterogenicus, Saccharomyces oleaginosus, Saccharomyces rosei, Saccharomyces steineri, Saccharomyces boulardii, Saccharomyces kefir, Saccharomyces kluyveri.

[0042] The method of the invention makes it possible, in particular, to discriminate Brettanomyces dekkera bruxellensis or Brettanomyces bruxellensis from Saccharomyces cerevisiae. The present invention also relates to a fluorochromic mixture containing or consisting of a solvent and a first fluorochrome selected from fluorochromes capable of binding to DNA and having a maximum fluorescence absorption wavelength of 652 nm and a maximum fluorescence emission wavelength of 676 nm and a fluorescence quantum yield on DNA of 0.27, fluorochromes capable of binding to DNA and having a maximum fluorescence absorption wavelength of 657 nm and a maximum fluorescence emission wavelength of 673 nm and a fluorescence quantum yield on DNA of 0.17, and mixtures thereof.a second fluorochrome chosen from among the fluorochromes having a maximum fluorescence absorption wavelength of 547nm and a maximum fluorescence emission wavelength of 570nm and a fluorescence quantum yield on DNA of 0.9 and a third fluorochrome said third fluorochrome is chosen from 5-carboxyfluorescein diacetate, 6-carboxyfluorescein diacetate, mixtures of 5-carboxyfluorescein diacetate and 6-carboxyfluorescein diacetate and succinimidyl 5,6-fluoroescein diacetate of the following general formula (1): ,

[0043] The present invention also relates to the aforementioned fluorochromic mixture for its use in detection in vitro (particularly in a substrate such as the one mentioned above) of Brettamyces spp and its discrimination from other yeasts, in particular Saccharomyces spp.

[0044] The present invention also relates to the use of the aforementioned fluorochromic mixture for the detection and discrimination of Brettamyces spp and its discrimination from other yeasts, in particular Saccharomyces spp, particularly in a substrate such as the one mentioned above.

[0045] In one particular embodiment, the fluorochromic mixture contains SYTOX®-orange, SYTO®-62 or SYTO®-63, and the c-FDA mixture. Similarly, in an advantageous embodiment, the method of the invention uses this aforementioned mixture of three fluorochromes.

[0046] The mixture is added to the substrate, possibly diluted, before its analysis in the flow cytometer. Définitions

[0047] The acronym FSC refers to the signal corresponding to diffracted light (FSC: Forward Scatter); this signal depends on the size and surface area of ​​the cell analyzed; The acronym SSC (SSC: Side Scatter) refers to the signal corresponding to reflected and refracted light; this signal depends on the granularity and cellular complexity of the cell analyzed.

[0048] The acronym SSC-H refers to the signal intensity corresponding to reflected and refracted light; The acronym SSC-A refers to the intensity per unit area of ​​the signal corresponding to reflected and refracted light; The acronyms FSC-H and FSC-A refer, respectively, to the intensity and intensity per unit area of ​​the signal corresponding to diffracted light; The acronym cFDA or c-FDA designates a mixture of 5-carboxy fluorescein diacetate and 6-carboxy fluorescein diacetate.

[0049] Throughout the application, the maximum fluorescence absorption and emission values ​​defining the fluorochromes are determined in the presence of DNA with a ratio of approximately 100 base pairs and specifically 100 base pairs of nucleic acid for one fluorochrome molecule in a Tris medium of pH 7.5 and EDTA concentration equal to 1mM.

[0050] Throughout the application, the fluorescence quantum yield that defines fluorochromes is measured in the presence of DNA and expressed relative to the yield determined for cresyl violet in methanol.

[0051] The term "in the majority" means 50% or more by mass or number. When applied to two entities, it means that the mixture of the two entities is present at 50% or more by mass or number.

[0052] The term "juice" in the context of the present invention refers to any liquid extracted from the pulp or flesh of certain fruits or vegetables.

[0053] The term "must" in the context of this invention refers to a mixture obtained by pressing or cooking plants (seeds, fruits, leaves, etc.) or plant extracts. These plants may be fruits, vegetables such as potatoes, but also cereals such as wheat, barley, malt, corn, or rice, for example.

[0054] The term "wine" in the context of the present invention refers to a juice or must of white and / or black grapes, some or all of whose sugar is transformed into alcohol by fermentation, in particular by alcoholic fermentation due to Saccharomyces cerevisiae. The wine can be white, red, or rosé, according to the invention. The wine can be stored in a wooden barrel.

[0055] The term "finished wine" refers to a wine, red, white, rosé, sparkling or not, whose fermentation is complete (first and second in the case of sparkling wines) and which is stored in vats, wooden barrels or bottles.

[0056] The term "beer" refers to any alcoholic beverage obtained by fermentation of a yeast or a fungus.

[0057] The term "vinegar" in the context of the present invention refers to the result of acetic fermentation produced by microbiological oxidation-reduction of an aqueous solution of ethanol, in particular a wine or a cider, exposed to air. FIGURES

[0058] There Fig. 1a is a biparametric histogram representing the intensity of the SSC signal as a function of the intensity of the FSC signal obtained for the analysis of a finished wine sample; The Fig. 1b is a biparametric histogram representing the fluorescence intensity due to the fluorochrome SYTOX-orange ®< as a function of the fluorescence intensity due to the fluorochrome SYTO 62 ®< for points located in the "Yeast and Bacteria background" window (first window) shown on the Fig. 1a ; There Fig. 1c is a biparametric histogram representing the intensity of fluorescence due to the cFDA fluorochrome as a function of the fluorescence due to the SYTOX-orange ® fluorochrome obtained for the "Brettanomyces" window (second window) visible on the Fig. 1b ; There Fig. 1d is a biparametric histogram showing the intensity of fluorescence detected in the RL1 channel at a wavelength of 670 nm and the intensity of fluorescence detected in the GL1 channel at 575 nm; The Fig. 2 represents a biparametric histogram showing, for each point, the intensity of the SSC signal as a function of the intensity of the FSC signal; the second window, corresponding to bacteria, is visible in this figure; The Fig. 3 represents a biparametric histogram showing the intensity per unit area of ​​the SSC signal as a function of the fluorescence intensity of the SYTO-62 fluorochrome for points located in the "bacteria + background" window (second window) visible on the Fig. 2 ; the window visible on the Fig. 3 Circle the dots corresponding to living bacterial cells; The Fig. 4 represents the intensity of fluorescence emitted by the fluorochrome SYTOX-orange ®< as a function of the fluorescence intensity due to the cFDA mixture for the points located in the second window, it represents the three groups of points which correspond to the three states of bacterial cells (alive, latent and dead). EXEMPLES : Mélange de marquage fluorochromique

[0059] Physiological water (osmosis / ultrapure water + NaCl at 7g / L) is prepared, then autoclaved and filtered before use (filter cutoff threshold 0.22µm).

[0060] A fluorochrome commercially available under the name SYTO 62™ (SYTO 63™ is also usable) (Thermofischer, 5 µM), SYTOX™-Orange (Thermofischer, 5 µM), and a mixture of 5,6-carboxyfluorescein diacetate (c-FDA) are diluted in DMSO (final concentrations of 50 µM, 12.5 µM, and 2 g / L, respectively) and then stored in a freezer. The final concentrations of fluorochromes in the labeling mixture are 0.15 µM of SYTO 62™ or 63™, 0.025 µM of SYTOX™-Orange (SYTOX-Gold), and 2 mg / L of c-FDA in physiological saline.

[0061] The first fluorochrome exhibits a high affinity for DNA and causes biological organisms containing DNA to fluoresce. It allows for the separation of microbiological cells from background noise in wine. The fluorochrome chosen here is SYTO®<-63. Its fluorescence is induced by a red laser (637 nm). Similar results are also obtained under the same conditions with SYTO®<62.

[0062] The second permeable fluorochrome penetrates only cells with compromised cell walls. The aim is to separate cells with permeable membranes (positively labeled), theoretically corresponding to dead cells, from living cells (unlabeled). The fluorochrome chosen here is SYTOX®-Orange, whose fluorescence is induced by the green laser (532 nm).

[0063] The third fluorochrome is inactive in its initial ester form. It becomes active through esterase activity during cellular metabolism. The goal is to separate metabolically active cells from inactive ones. This second category consists of latent cell populations that, in practice, develop little or not at all in petri dish cultures. It corresponds to VNC populations (viable but not cultureable populations). The fluorochrome chosen here is c-FDA. Its fluorescence is induced by a blue laser (488 nm). Préparation des échantillons de substrat Wine and must samples

[0064] Samples of finished or fermenting wines (must) are diluted (1 / 40, 1 / 100, 1 / 300, or 1 / 1000 depending on their biological load) with the fluorochromic labeling mixture. For bottled wines, 50 mL are centrifuged for 8 minutes at 4500 rpm. The supernatant is discarded, and the pellet is resuspended in 10 mL of filtered physiological saline (8.5 g / L NaCl in reverse osmosis water filtered to 0.22 µm). The sample is then diluted 1:2 in the labeling mixture and vortexed for a few seconds. The substrate-fluorochromic mixture is incubated for approximately 30 minutes in the dark before analysis.

[0065] The sample is a finished wine sample from 2020 from the Languedoc region. Equipment used

[0066] Flow cytometer: ATTUNE® NXT acoustic focusing cytometer (thermofischer scientific). This method describes a microbiological analysis protocol using a flow cytometer equipped with three lasers: blue (488 nm), green (532 nm), and red (637 nm). Triple cellular labeling of bacteria and yeasts using the aforementioned fluorochromes is performed.

[0067] The flow cytometer may or may not be equipped with an autosampler for reading 96-well microplates. This equipment features an acoustic flow focusing system allowing the use of flow rates up to 1000 µL / min.

[0068] The flow rate is set at 500 µL / min. This rate is reduced when the microbial load is high. Data is collected on the FSC, SSC, and BL1 (525 / 50) channels for c-FDA, GL1 (575 / 36) for SYTOX-orange, and RL1 (670 / 14) for SYTO 62 or 63. The voltage values ​​for each of these channels are 200V, 300V, 330V, 360V, and 440V, respectively. Different values ​​can be applied. There are no compensation issues to correct in this configuration. Traitement des résultats

[0069] Events that respond to SYTO 6255 ®< or 63 positive (SYTO 62 or 63 +) are considered microorganisms.

[0070] Triple labeling of the samples and a calibration strategy enabled the separation of bacteria and yeasts. Saccharomyces And Brettanomyces in the finished wine. Initially, the majority of bacteria and background noise are separated from the yeasts and some bacteria by the SSC-H / FSC-H plot ( Fig. 1a And 2 ). Then, using the RL1 plot (670 / 14 nm) applied to the yeast-bacteria window of the Fig. 1a , the last bacteria are eliminated and the dichotomy between the Brettanomyces And Saccharomyces appears. This dichotomy is made possible by a manifest property of different relative levels of autofluorescence in the red of the cells of Saccharomyces spp and those of Brettanomyces spp.

[0071] As depicted on the Fig. 1d , even in the absence of SYTO62 / 63 labeling, the difference in fluorescence at 670nm between Saccharomyces And Brettanomyces is observable (see Fig. 1d ). This is therefore a differentiated phenomenon of autofluorescence.

[0072] There Fig. 3 shows the difference in fluorescence between dead bacterial cells and latent or living cells. Fig. 1b shows this difference for bacteria. Then, the different states of Brettanomyces and bacteria are obtained by combining the data obtained with the 3 fluorochromes. Thus, the "dead" state corresponds to SYTOX-Orange + / cFDA -, the "latent live (LLE)" state to SYTOX-Orange - / c-FDA -, and the "active live / vital" state to SYTOX-Orange - / c-FDA + (see Fig. 1c And Fig. 4 ). The windows were able to be validated by comparing cytometry with microscopy and Petri dishes.

[0073] It can be seen from the aforementioned results that the method of the invention makes it possible to achieve at the same time, a separation of the microorganism of interest from the background noise, a separation of living microorganisms from dead microorganisms and within the population of living microorganisms, to separate the physiologically active microorganisms from the dormant microorganisms.

[0074] It provides all the information on vitality and viability simultaneously. It can be implemented quickly, with active labeling in approximately 15 minutes. Furthermore, it is inexpensive because it uses existing reagents. It is suitable for industrial-scale, high-throughput analysis.

[0075] Naturally, the invention described above is by way of example. It is understood that a person skilled in the art is capable of carrying out different embodiments of the invention without departing from its scope.

Claims

1. A method for detecting, quantifying, and discriminating cells of Brettanomyces spp yeasts contained in an organic liquid substrate which contains fermentable sugars, wherein a sample of said substrate is taken, possibly diluted, and at least one first fluorochrome capable of binding to DNA of both dead and / or living cells is added to the possibly diluted substrate, said sample is then irradiated so as to obtain fluorescence emission from the first fluorochrome, and said sample is also irradiated to obtain fluorescence emission from the sample at 670 nm, a dual histogram is established, giving for each point the fluorescence intensity due to the first fluorochrome and the fluorescence intensity emitted at 670 nm, at least one first point cloud corresponding to a fluorescence intensity emitted and detected at 670 nm that is higher than that detected for the other points, it is deduced that the points in said first cloud correspond to the cells of Brettanomyces spp, and the number of Brettanomyces spp cells is optionally counted by counting the points in said first cloud.

2. The method according to claim 1, characterised in that said substrate contains mostly Brettanomyces spp yeasts and Sacharomyces spp yeasts, in that two point clouds are obtained on said dual histogram, a first cloud comprising the points corresponding to a fluorescence intensity emitted at 670nm greater than that of the points of the second point cloud, in that it is deduced therefrom that the points of said first cloud correspond to the Brettanomyces spp cells and that the points of said second cloud correspond to the Saccharomyces spp cells and the number of Brettanomyces spp and / or Saccharomyces spp cells is optionally counted by counting the points of each of said clouds.

3. The method according to claim 1 or 2, characterised in that before measuring fluorescence, a first discrimination is performed between the particles and the cells present in the sample by measuring intensity of the reflected and refracted light as well as intensity of the diffracted light, a dual histogram is then established, giving the values of said intensities for each point corresponding to a detected particle or cell, based on the values of said intensities, a first window that contains points attributable to yeast cells, and optionally a second window corresponding to points attributable to bacterial cells are thus determined, said dual histogram giving fluorescence intensity due to the first fluorochrome and fluorescence intensity emitted at 670 nm for each point located in said first window is established.

4. The method according to any one of the preceding claims, characterised in that the first fluorochrome is capable of binding to DNA of live cells as well as to DNA of cells whose wall is permeable, in that prior to any measurement, a second fluorochrome capable of binding only to DNA of cells whose wall is permeable, is additionally added to the possibly diluted sample, in that a third window is further determined that surrounds the points of said first cloud, in that the sample is excited to cause fluorescence emission from the first and second fluorochromes, and for points located in said third window, a dual histogram is also generated, giving for each point the fluorescence intensity of the first and second fluorochromes, or the fluorescence intensity per unit area of one of the two fluorochromes and that due to the other fluorochrome, and in that two groups of points are further determined, a first group for which fluorescence due to the fluorochrome that binds solely to DNA of cells whose wall is permeable is greater than that of the second group, and the number of points in each group is counted, which corresponds to the number of live Brettanomyces spp cells for the second group and the number of dead Brettanomyces spp cells for the first group.

5. The method according to claims 2 and 3, characterised in that said second window is determined and that said sample is also excited so as to cause fluorescence emission from both the first and second fluorochromes, and for points located in said second window, a dual histogram is established, giving fluorescence intensity for each point of both the first and second fluorochromes, or the fluorescence intensity per unit area of one of both fluorochromes and that due to the other fluorochrome, and in that two groups of points are identified, a first group for which fluorescence due to the fluorochrome that binds only to DNA of cells whose wall is permeable is greater than that of the second group, and the number of points in each group is then counted, which corresponds to the number of live bacterial cells for the second group and the number of dead bacterial cells for the first group.

6. The method according to claims 5 or 6, characterised in that, prior to any measurement, a third fluorochrome which emits a fluorescence signal only when it reacts with a living cell is added to the possibly diluted sample, the sample is then excited so as to obtain fluorescence emission of said third fluorochrome, and for points of the second and / or third window, a dual histogram giving, for each point, fluorescence intensity due to the fluorochrome that binds only to DNA of cells whose wall is permeable, as well as fluorescence intensity due to the third fluorochrome is established, for each window, three subgroups of points are then determined, a first subgroup of points corresponds to a fluorescence intensity due to the third fluorochrome greater than that of the other subgroups, this first subgroup of points representing Brettanomyces spp cells / live and active bacterial cells, a second subgroup corresponding to fluorescence intensity due to the third fluorochrome lower than that of the first subgroup and coupled with a fluorescence intensity due to the first / second fluorochrome lower than that of the third subgroup, the points of this second subgroup corresponding to Brettanomyces spp cells / bacterial cells in a latent state, and a third subgroup of points corresponding to fluorescence intensity due to the first / second fluorochrome that is greater than that of the first and second subgroups, these points corresponding to dead Brettanomyces spp cells / bacterial cells.

7. The method according to any one of the preceding claims, characterised in that said first and said second fluorochromes are different and selected from fluorochromes capable of binding to DNA of cells and having a maximum fluorescence absorption wavelength of 599 nm or greater and 657 nm or less, a maximum fluorescence emission wavelength of 619 nm or greater and 678 nm or less, and a quantum yield of 0.16 or greater and 0.39 or less, and mixtures thereof, in particular, the fluorochromes capable of binding to DNA of cells and having a maximum fluorescence absorption wavelength of 652 nm, a maximum fluorescence emission wavelength of 676 nm, and a fluorescence quantum yield on DNA of 0.27, and fluorochromes capable of binding to DNA and having a maximum fluorescence absorption wavelength of 657 nm, a maximum fluorescence emission wavelength of 673 nm, and a fluorescence quantum yield on DNA of 0.17, the fluorochromes capable of binding only to DNA of cells whose wall is permeable and which have a maximum fluorescence absorption wavelength of 547 nm, a maximum fluorescence emission wavelength of 570 nm, and a quantum fluorescence yield on DNA of 0.9, and mixtures thereof, and in that when the first fluorochrome is selected from fluorophores capable of binding to DNA with a maximum fluorescence absorption wavelength of 657 nm, a maximum fluorescence emission wavelength of 673 nm, and a fluorescence quantum yield on DNA of 0.17, and fluorophores capable of binding to DNA of the cells and having a maximum fluorescence absorption wavelength of 652 nm, a maximum fluorescence emission wavelength of 676 nm, and a fluorescence quantum yield on DNA of 0.27, the second fluorochrome is selected from fluorophores capable of binding only to DNA of cells whose wall is permeable, and which a maximum fluorescence absorption wavelength of 547 nm and a maximum fluorescence emission wavelength of 570 nm, and a quantum fluorescence yield on DNA of 0.9, and in that said third fluorochrome is selected from 5-carboxyfluorescein diacetate, 6-carboxyfluorescein diacetate, mixtures of 5-carboxyfluorescein diacetate and 6-carboxyfluorescein diacetate, and succinimidyl 5,6 carboxylate fluorescein diacetate of the following general formula (1):

8. The method according to any one of the preceding claims, characterised in that said substrate is selected from sparkling or still wine, red wine, white wine, rosé wine, cider, beer, sake, fruit juices, particularly grape or apple juice, water kefir, fruit juice kefir, milk kefir, milk, tequila, whisky, vodka, musts, especially grape musts, wines undergoing primary or secondary fermentation, finished wines, whether sparkling or still, and vinegars.

9. The method according to any one of the preceding claims, characterised in that it enables quantification and discrimination of at least one Brettanomyces spp yeast species selected from the following species: B. anomalus, B. bruxellensis, B. custersianus, B. nanus, B. dekkera bruxellensis and B. naardenensis from at least one other yeast species and especially at least one Saccharomyces spp species selected from the following species: Saccharomyces bailii Linder, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces delbrueckii, Saccharomyces exiguus, Saccharomyces fermentati, Saccharomyces florentinus, Saccharomyces fragilis, Saccharomyces fructuum, Saccharomyces heterogenicus, Saccharomyces oleaginosus, Saccharomyces rosei, Saccharomyces steineri, Saccharomyces boulardii, Saccharomyces kefir, Saccharomyces kluyveri and in particular Saccharomyces cerevisiae.

10. The method according to any one of the preceding claims, characterised in that said sample is excited at a wavelength of greater than or equal to 620 nm and less than or equal to 750 nm inclusive, and in particular equal to 637 nm.