ES2311425A1 - Method for the detection and quantification of yeast of the genera brettanomyces and/or dekkera in red wine and other fermented or carbonated drinks - Google Patents

Abstract

The invention relates to the use of selective-differential media for stimulating the growth of yeast of the genera Brettanomyces/Dekkera, associated with the monitoring of the degradation of p-cumaric acid and the subsequent formation of 4-ethylphenol (smell defect) by means of instrumental analysis HPLC/PDA/ESI-MS, enabling the estimation and/or quantification of the existing microbial population and the potential vinylphenol-reductase enzymatic activity thereof by means of a statistic regression model. This technique is applicable to any stage of an oenology industry and other types of drinks industries.

Description

New method of detection and quantification of yeasts of the Brettanomyces and / or Dekkera genera in red wines and other fermented or carbonated beverages

Technical sector

Oenology: Winemaking in Red.

Prior art

The yeasts of the Brettanomyces / Dekkera genera deteriorate the quality of red wines mainly during aging in the barrel. Having analytical techniques of easy application, which indicate the presence of these microorganisms in a sensitive way, allows the winery to detect and isolate the affected barrels and apply corrective and prophylactic measures that help to avoid and / or minimize the problem. .

Brettanomyces and Dekkera have a characteristic morphology with ogival and small cells. However, neither the morphological characteristics nor the classic identification methods are suitable for routine enological microbiology work, in addition to not allowing the detection of viable but non-cultivable populations, so in recent years different techniques have been developed aimed at a quick and safe identification of these altering yeast species of other different species present in the wine (Suárez et al . Food Chem. 2007, 102, 10-21).

In the detection of Brettanomyces / Dekkera colonies, fluorescence microscopy of the cells previously labeled with fluorescent probes (RNA FISH hybridization ) that target the 26S fragment of the ribosomal RNA (Stender) has been used with acceptable results. et al . J. Microbiological Methods, 2002, 48, 1-17; Stender et al . Appl. Env. Microbiol., 2001, 67, 938-941). This method has a high specific sensitivity for Dekkera bruxellensis .

Another technique used to identify Brettanomyces / Dekkera type altering yeasts in wines consists in growing the isolated strains, extracting the fatty acids from the cell wall and quantifying the type and quantity of each one by gas chromatography after derivatizing them as methyl esters ( Sancho, et al . Food Microbiol. 2000, 17, 613-624).

However, techniques based on molecular biology methods such as PCR ( Polymerase Chain Reaction ) have now been imposed as the fastest sensitive and specific. Generally, these methods have focused on the amplification of certain specific fragments of DNA and ribosomal RNA.

Techniques such as electrophoretic determination of karyotypes, and random amplification of polymorphic DNA (RAPD-PCR) have been used to adapt them to Brettanomyces / Dekkera strains isolated from wines (Mitrakul et al . Food Microbiol., 1999, 16, 3-14) . Also, specific sequences of ITS ( internal transcribed spacer regions ) regions located between ribosomal RNA genes that are divergent enough (especially two of them ITS 1 and ITS2) have been detected to be used as identification targets specifically and reliably for Brettanomyces / Dekkera species (Egli and Henick-Kling. Am. J. Enol. Vitic., 2001, 52, 241-247). There are specific protocols to distinguish Brettanomyces bruxellensis and Brettanomyces anomalus using the DB90F and DB394R primers that use the D1-D2 loop of the 26S rRNA gene as a target that do not lead to the formation of amplification products when other species of Brettanomyces spp. nor with other yeasts (Cocolin et al . Appl. Env. Microbiol., 2004, 70, 1347-1355).

Nested-PCR amplification techniques have also been used that using four primers, two external and two internal, allow the detection of Brettanomyces spp. starting directly from wine, without the need to isolate or cultivate the cells and with great reproducibility and specificity (Navascués and Rasines. Enologos, 2003, 23, 24-26). This new technique allows great versatility and rapid detection of the development of Brettanomyces from early stages. The main problem of molecular techniques lies in the sensitivity and difficulty in differentiating genetic material from non-viable or dead cells and living cells.

Finally, another possible form of action is the detection in wines of a metabolic product of the activity of Brettanomyces / Dekkera , the ethylphenols. However, this method presents the problem that unless the monitoring is continuous and very exhaustive, it may be that these compounds are detected when it is too late to act. Despite the analysis and quantification of ethylphenols in wines using couplings of separative techniques such as gas chromatography, and analytical such as mass spectrometry (GC / MS) allows the identification and quantification of 4-ethylphenol and 4-ethylguaiacol with high sensitivity.

This instrumental analytical technique is often associated with serial sensory analysis, thus becoming a powerful tool for quality control in wines (GC / MS / olfactometry ). With this procedure, once the chromatographic separation has occurred and the different volatile compounds are isolated at the end of the capillary column, a flow division is made and part is diverted to a device that allows a trained taster to indicate the intensity and olfactory quality of the separated volatile compound, and another fraction thereof is directed to the ionization source of a mass spectrometer to accurately identify the molecule that has produced said sensation.

Most of the work done to detect this problem has been approached from almost strictly microbiological points of view (presence of Dekkera / Brettanomyces yeasts) or chemicals (detection of ethylphenols) and not from a purely oenological perspective. For this we use the combination of a liquid selective-differential medium of our own formulation and instrumental analysis by means of HPLC technique in order to evaluate the "Potential hydroxycinnamate decarboxylase and vinylphenol reductase activity of a wine".

There are several modern methods based on molecular biology techniques that can be applied to the identification of these yeasts, within the most important ones should consider: analysis of partial DNA sequences 26S ribosomal; Random enlargement of polymorphic DNA (RAPD) -PCR; PCR using sequence probes specific; hybridization with specific DNA probes and detection by fluorescence microscopy. Generally these methods do not allow quantifying small populations of few cells per liter that go unnoticed and then proliferate in the barrel producing ethylphenols. Nor can they differentiate between cells viable and dead (Suárez-Lepe and Iñigo. Oenological Microbiology Fundamentals of Vinification. Mundi-Press. 2004, 422-424), being able to induce confusion in wines already stabilized. The technique proposal allows to detect small populations (up to 1 cfu / ml) and detects only viable cells that are the ones that can cause problems, so it improves techniques in these aspects molecular.

Another option is instrumental analysis techniques for the detection of ethylphenols such as gas chromatography associated with mass spectrometry (GC / MS). The detection of ethylphenols has been performed by gas chromatography of extracts obtained by liquid-liquid extraction with organic solvents (Chatonnet and Boidron. Sciences des Aliments, 1988, 8, 479-488.), Solid phase microextraction by head space (HSPME) (Monje et al . (2002). Anal. Chimica Acta, 2002, 458, 111-117 .; Martorell et al . J. Chromatography, 2002, 975, 349-354.) And twister absorption by extraction (SBSE) ( Díez et al . J. Chromatography A, 2004, 1025, 263-267.). But the problem of these analyzes lies in the required equipment and in that the detection of the problem occurs once it has been developed, which compromises the application of corrective measures in the wine within a reasonable period of time. High pressure liquid chromatography (HPLC) is not used due to the lack of sensitivity in the analysis of small concentrations (10-100 ppb) of these compounds, which makes their direct application in wines difficult, but has been successfully applied in the detection of enzymatic activity hydroxycinnamate decarboxylase and vinylphenol reductase in synthetic media (Benito et al . Wine Technology, 2006, 32, 27-31). In the face of these techniques, the proposed new technique allows high sensitivity without requiring complex sample preparation such as GC techniques.

Another option is the isolation by solid selective-differential means in Petri dish (Rodrigues et al . J. Appl. Microbiol. 2001, 90, 588-599), used for yeast counting and contamination evaluation by Dekkera / Brettanomyces , whose main drawbacks are the possible false positives (Benito et al . Wine Technology, 2006, 32, 27-31), waiting times and contamination by opportunistic fungi. While the positive ones in liquid media (Couto et al . Lett. Appl. Microbiol. 2005, 41, 505-510) have to be determined generally by human smell, which gives rise to subjectivity, discrepancies between different evaluators and lack of rigor when approving a result. If "Potential hydroxycinnamate decarboxylase and vinylphenol reductase activity of a wine" is evaluated in a liquid medium, as proposed in the present invention, the drawbacks of the isolates in solid media are solved and by determination by HPLC instrumental techniques It acts with a scientific rigor that can allow homologations to official or specialized laboratories.

Explanation of the new technique 1) Problem that exists

Yeasts of the Brettanomyces / Dekkera genera have specialized in colonizing certain substrates using ethanol as the sole source of carbon. Although they have very specific physiological requirements, they are perfectly adapted to grow in the conditions that occur in the aging in red wine barrels. These yeasts have two enzymatic activities (hydroxycinnamate decarboxylase and vinylphenol reductase) that allow them to transform the hydroxycinnamic acids from the grape into ethylphenols (4-ethylphenol, 4-ethylguaicol, 4 ethylcatechol) with very unpleasant aroma and low sensory threshold which deteriorates the olfactory quality of the wines. To this it also contributes that modern winemaking tends to reduce technological practices such as the dose of addition of SO2 or the use of clarifications and filtrations, so as not to modify the organoleptic quality of the wines, but which were positive since they allowed an improvement of its microbiological stability. By reducing these techniques, yeasts of the Brettanomyces / Dekkera genera find less barriers when it comes to proliferating and altering wines.

Normally the red wines that are destined for aging in the barrel are the ones with the highest added value, in addition the aging involves periods of storage of the product that are usually superior to the year which obviously makes it more expensive. If at this stage the yeasts of the Brettanomyces / Dekkera genera sensoryly alter the wine, significant economic losses occur.

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2) Advantages of the invention

one.

Speed with result detection positive in periods between 3 and 5 days.

2.

Higher sensitivity than molecular biology or microscopy techniques of fluorescence allowing to detect up to 1 cfu in volume analyzed (150 ml).

3.

It allows quantifying the population of Brett in a standard volume (1 bottle 750 ml).

Four.

Be They detect only viable forms (living cells). PCR gives false positive with remains of dead cell DNA.

5.

The Sample preparation is simple with no need for extraction or concentration

6.

It allows to reduce false problems positives caused by other yeasts without activity vinylphenolreductase.

7.

I dont know the determination by opportunistic fungi that prevent plate counts in contamination from contamination selective-differential solids conventional.

8.

He Analysis cost is low.

9.

I dont know They use mutagenic agents as in PCR.

10.

Allow Brett detection before Let the formation of ethylphenols begin. It can be used in warehouse for prophylactic-preventive monitoring of the barrels.

eleven.

He Instrumental analysis (HPLC) eliminates the uncertainty caused by human smell and that exists in the selective media differentials

3) Description of the invention

The proposed technique uses a specific and rapid sensitive means for the detection of yeasts of the Brettanomyces / Dekkera genera in red wines but which is applicable to other fermented or carbonated beverages such as beer or soft drinks. It consists of the addition of a liquid selective-differential medium with a high content of p- fumaric acid to the volume of wine or other type of fermented or carbonated beverage (beer, soft drinks, etc.) that is considered contaminated. The medium must also be poor in nitrogen and glucose as a carbon source to be selective against other fermentative yeasts. The growth of other unwanted microorganisms that may give rise to false positives is reduced by the use of antibiotics and antifungals (chloramphenicol and actidione).

The medium is subsequently incubated at optimum growth temperature of Brettanomyces . The detection of the population development of Brettanomyces / Dekkera is carried out by monitoring the degradation of p- fumaric acid and the corresponding formation of 4-ethylphenol using instrumental analysis HPLC / PDA / ESI-MS, which allows quantifying the contents of these molecules and intermediate 4-vinylphenol. These metabolic transformations are produced by the series effect of two enzymes: hydroxycinnamate decarboxylase and vinylphenol reductase, of which the second is specific to very few genera of yeasts, including Brettanomyces / Dekkera . The intensity of degradation of p- fumaric acid and its transformation into 4-ethylphenol is proportional to the polluting population.

The method allows estimating the initial polluting population in red wine (or another beverage) using a regression model that correlates the time necessary for the complete degradation of p- fumaric acid with the population necessary to produce such degradation by a set of strains of collection of the previously characterized Brettanomyces / Dekkera genera.

Exhibition of an embodiment

one.

Taking of the sample in the desired volume 100-1000 ml.

2.

Adding the medium selective-differential liquid with the following composition:

to.

Base nitrogen without added amino acids and without ammonium sulfate 14 g l -1.

b.

Glucose 20 g l -1.

C.

P- fumaric acid 100 mg l -1.

d.

Actidione 20 mg l -1.

and.

Chloramphenicol 400 mg l -1.

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3.

Mix with the wine in conditions of asepsis and in a previously sterilized container.

Four.

Incubation of the mixture to a temperature between 25 and 30 ° C.

5.

Activity Tracking hydroxycinnamate decarboxylase and vinylphenolreductase by HPLC / PDA / MS.

6.

Chromatographic conditions:

to.

Be uses an HPLC device equipped with a matrix detector photodiodes (DAD) and optionally to increase sensitivity with an MS detector.

b.

Column chromatographic separation of reverse phase C18 of 300 mm x 3.9 mm.

C.

Gradient of two eluents A (water MQ / formic acid, 90:10, v / v) and B (methanol / formic acid, 90:10, v / v), 10-50% linear B (0.8 ml min -1) from the minute 0 to 25; 50-10% B from minute 25 to 30. A rebalancing of the 30 minute column is performed at 33.

d.

Detection by monitoring the absorption spectrum between 200 and 400 nm.

and.

Quantification using external standards of p- fumaric acid and 4-ethylphenol.

F.

Quantification of initial population through regression model.

Industrial application

In the elaboration of red wines especially in those aged in barrels for:

-

To know the presence and evolution of altered yeast populations of the Brettanomyces / Dekkera genera in barrels,

-

Pollution control in bottling,

-

Quality control in the material of Winery: Valvulerfa, deposits, pipes, bottling machines,

-

Method of possible approval for the performance of quality control in  Oenological stations and accredited laboratories.

Claims (4)

1. Procedure for the detection and quantification of the yeast population of the Brettanomyces / Dekkera genera of wines and other beverages characterized in that it comprises the following stages:

-

Taking of a sample of wine, or other fermented or carbonated beverage of a  volume between 100 and 1000 ml,

-

preparation of a medium selective-differential,

-

mixture of the wine sample and the medium selective-differential under conditions of asepsis,

-

incubation of the mixture at temperature between 25 and 30 ° C,

-

quantification of p- fumaric acid by means of HPLC / PDA / ESI-MS instrumental analysis over time and estimation of the time necessary for its degradation.

-

Detection and quantification of the yeast population of the Brettanomyces / Dekkera genera by means of a regression model that correlates the time necessary for the complete degradation of p- fumaric acid with the initial yeast population.

2. Method for detecting and quantifying the yeast population of the Brettanomyces / Dekkera genera according to claim 1, characterized in that instead of p- fumaric acid, the contents of 4-vinylphenol and 4-ethylphenol formed from p- acid are quantified Cumaric degraded by Brettanomyces / Dekkera . 3. Selective-differential medium for use in the method of detection and quantification of the yeast population of the Brettanomyces / Dekkera wines genus , described in claims 1 to 2 characterized by having the following qualitative composition:

to.

Base nitrogen without added amino acids or ammonium sulfate,

b.

Glucose,

C.

P- fumaric acid,

d.

Act and

and.

Chloramphenicol

4. Selective-differential medium for use in the Brettanomyces / Dekkera wine detection and quantification method according to claim 3 characterized in that in an experimental embodiment, the optimal results are obtained with the following composition:

to.

Base nitrogen without added amino acids or ammonium sulfate 14 g l -1,

b.

Glucose 20 g l -1,

C.

P- fumaric acid 100 mg l -1,

d.

Actidione 20 mg l -1 and

and.

Chloramphenicol 400 mg l -1.