ES2642903B2 - Procedure for the corrective treatment of aging wines contaminated with lactic and acetic bacteria that have high volatile acidity - Google Patents

Abstract

Procedure for the corrective treatment of aging wines contaminated with lactic and acetic bacteria that have a high volatile acidity. # The scientific area to which the invention corresponds is food technology, and the industrial wine sector. # Consists of establishing on the wine a treat: a submerged culture of flower yeasts with contribution and regulation of air flow by means of micro-aeration and the addition of lysozyme, once the exponential phase of yeast growth has begun. # The procedure allows to treat biological aging wines with volatile acidity of up to 1.2 g / L, where the development of veil yeasts in film-forming culture is inhibited or invalidated. Treated wines improve their sensory characteristics and can be reincorporated into the industrial system once their alcohol content is corrected up to 15% v / v.

Description

PROCEDURE FOR THE CORRECTIVE TREATMENT OF AGRICULTURAL WINES CONTAMINATED WITH LACTIC BACTERIA AND ACtTlCAS THAT PRESENT A ELEVATED VOLATILE ACIDITY.

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SECTOR OF THE TECHNIQUE.

The scientific area to which the invention corresponds is the area of Food Technology, and the industrial sector in which it can be applied is the wine industry, and more specifically in the traditional wineries for the production of bio-aging wines that concentrate on areas of Jerez, Huelva, Montilla-Moriles, Jura (France), etc.

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STATE OF THE TECHNIQUE.

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"biological aging" or "breeding under a veil of flower" is a phenomenon constituted by the development of a film-forming crop on the surface of the wine of some species of yeasts of the genus Soccharomyces (Alexandre, H. (2013). Flor yeasts of Saccharomyces cerevisiae-Their ecology, genetics and metabolism. International Journal of Food Microbiology, 167, 269-275). The flower veil develops spontaneously on the surface of the wine, once the alcoholic fermentation is completed and the young wine is headed with wine alcohol up to 15% v / v (Martinez de la Ossa, E., Caro, L, Bonat, M., Pérez, L., & Domecq, B. (1987). Dry extract in sherry and its evolution in the aging of sherry. American Journal of Enology and Viticulture, 38, 321-325). During biological aging, veil yeast develops an aerobic metabolism, in which it consumes ethanol, glycerin, acetic acid, etc., to give rise to other compounds such as acetaldehyde, acetoin, diacetal, etc. (Martinez de la Ossa, E., Caro, l., Bonat, M., Pérez, L., & Domecq, 8. (1987). Dry extract in sherry and its evolution in the aging of sherry. American Journal of Enology and Viticulture, 38, 321-325; Martinez de la Ossa, E., Pérez, L., & Caro, l. (1987). Variatíons of the major volatiles through ageing of sherry. American Journal of Enology and Viticulture, 38, 293-297; Martínez, P., Valcarcel, MJ, Pérez, L., & Benítez, T. (1998) Metabolism of Saccharomyces cerevisiae flor yeasts during fermentation and biological aging of fino sherry: by products and aroma compounds. American Journal of Enology and Viticulture, 49, 240-250). the

biological upbringing produces very significant sensory modifications that give

place after time to the typologies of fine wines and chamomiles.

In industrial biological breeding systems, more than 95% of microorganisms

present in the biofilm are yeasts of the genus S. cerevisiae (Ale) (andre, H. (2013). Flor

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yeasts of Saccharomyces cerevisiae-Their ecology, genetics and metabolism.

International Joumal of Food Microbiology, 167, 269-275). However, under

certain conditions may coexist and proliferate in the environment some

contaminating microorganisms such as lactic bacteria, acetic and in some

yeast cases of the non-Saccharomyces genus such as the Brettanamyces (Alexandre, H.

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(2013). Flor yeasts of Saccharomyces cerevisíae-Their ecology, genetics and

metabolism International Journal of Food Microbiology, 167, 269-275; Moreno-Arribas,

M. V., & Polo, M. C. (2008). Occurrence of lactic acid bacteria and biogenic amines in

biologically aged wines. Food Microbiology, 25, 875-881; Sua rez-Lepe, J. A., & lñigo-Leal,

B. (2004). Oenological Microbiology Fundamentals of winemaking Mundi editions

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Press, Madrid, Spain.). The consumption of oxygen by yeasts establishes

semi-aerobic conditions within the wine that favor development above all

of lactic bacteria. The presence of specific substrates such as gluconic acid

(derived from the presence of Botrytis cinerea in the vintage), they can facilitate their

proliferation and the development of alterations such as the "lactic chop" or "the spun", which

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they lead in most cases to an increase in volatile acidity and a loss of

the characteristic sensory attributes of parenting (Lasanta, C., Roldan, A., Caro, l.,

Pérez, L., & Palacios, V. (2010). Use of Iysozyme for the prevention and treatment of

heterolactic fermentatlon in the biological aging of sherry wines. Food Control, 21, 1442

1447; Pérez, L., Valcárcel, M.J., González, P., & Domecq, B. (1991). Influence of Botrytis

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infection of the grapes on the biological aging process of fino sherry. American Journal

of Enology and Viticulture, 42 (1), 58-62). When volatile acidity reaches levels

high (:> 0.8 giL) the flower veil becomes inactive, falls and dies, generating conditions

Ideal for the development of acetic bacteria (dissolved oxygen and presence of acid

acetic) (Casas, J. (2008). The vinification of Jerez in the 20th century. Ed. Junta de Andalucía.

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Seville, Spain 310-311). Acetic bacteria are taxonomically classified as

strict aerobic microorganisms and require aerobic conditions for their

development and survival (Bartowsky, E. J., & Henschke, P. A. (2004). Acetic acid

bacteria and wine: all is well untit Q) (ygen enters the scene. The Australian & New

Zealand Grapegrower and Winemaker, 485a, 8 &-91; Drysdale, G. S., & Fleet, G. H.

(1988). Acetic acid bacteria in winemaking: a review. American Journal of Enology and

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Viticulture, 39, 143-154). The development of acetic bacteria produces an increase

rapid and significant volatile acidity in wines (> 19J1), invalidating the

regeneration, presence and growth of the flower veil on the surface of the wine. the

boots that show these symptoms are removed from the aging system (bags and dew

in Jerez wines), to avoid contamination of other boots, the wines being destined to

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vinegar production.

Generally to control the growth of both lactic bacteria in wines

high doses of sulphurous of at least 100 mg / L (total S02) are used as acetics

{Joyeux, A., lafon-lafourcade, S., & Ribereau-Gayon, P. (1984). Evolution of acetic acid

bacteria during fermentation and storage of wine. Applied and Environmental

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Microbiology, 48, 153-156; Suarez-lepe, J. A., & lñigo-leal, B. (2004). Microbiology

winemaking Fundamentals of winemaking Mundi-Prensa editions, Madrid, Spain}. Without

However, these doses of sulphurous cannot be applied in biological upbringing by two

Reasons: One, the flower yeast is very sensitive to this antiseptic (Suarez-lepe, J. A., &

lñigo-Leal, B. (2004). Oenological Microbiology Fundamentals of winemaking Editions

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Mundi-Prensa, Madrid, 5pain), and two, the presence of high concentrations of

Acetaldehyde in these wines means that practically all sulfur is in the form

combined, which reduces its antiseptic ability on bacteria (Casas, 1. (2008).

the vinification of Jerez in the twentieth century. Ed. Andalusian Board Seville, Spain 310--311).

An alternative for the control of lactic bacteria in biological rearing has been the

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use of lysozyme. In studies published by the requesting team, it was found

that lysozyme in small doses 5-15 g / hl reduces lactic bacteria populations

99% in organic aging wines with an alcoholic strength between 15

15.5%, so lysozyme can be a good alternative as a treatment

preventive and curative of lactic and possibly preventive pitting bites and

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acetic chopped in these wines (lasanta, C., Roldán, A., Caro, l., Pérez, L., & Palacios, V.

(2010). Use of Iysozyme for the prevention and treatment of heterolactic fermentatlon

in the biological aging of sherry wines. Food Control, 21, 1442-1447). When the applied dose is less than 5 g / hL the effectiveness is significantly reduced, with no effect taking place at doses below 1.5 g / hL (lasanta, e, Roldán, A., Caro, l., Pérez, L. , & Palacios, V. (2010). Use of Iysozyme for the prevention and treatment of heterolactic fermentation in the biological aging of sherry wines. Food Control, 21, 1442

1447).

Lysozyme, when applied under certain conditions, can have an inhibitory effect on the development of flower veil yeasts on the surface of the wine, which can affect both the normal biological aging process and the quality of the wines produced ( Roldan, A., lasanta, e, Caro, l., & Palacios, V. (2012). Effect of Iysozyme on "flor" velum yeasts in the biological aging of sherry wines. Food Microbiology, 30, 245-252). When lysozyme is added in doses greater than 1 g / hl on organic aged wines (15-15.5% voL) that lack a flower veil, yeasts present difficulties for their ascension and development on the surface of the wine, affecting severely to its hydrophobicity or buoyancy and to its state of aggregation (Roldán, A., lasanta, e, Caro, l., & Palacios, V. (2012). Effect of Iysozyme on "flor" velum yeasts in the biological aging of sherry wines, Food Microbiology, 30, 245-252). Under these conditions, yeasts do not have the capacity to form stable and covered veils, inhibiting cellular metabolism and the production of the characteristic compounds of biological aging. However, when lysozyme is applied, in the prescribed doses (5-15 g / hl), on a biological aging wine, which already has a flower veil with the minimum state of aggregation (isolated small points or islets) , or it is grafted or cultivated on the surface afterwards (after application), by means of a sowing handle or spatula, the yeasts do not show any alteration in the growth, as well as in the evolution in the different states of aggregation (fine covered, covered rough), and buoyancy in wine. Under these conditions, the yeast metabolism does not undergo any type of modification, and the wine presents the sensory evolution foreseen in this type of process. The results of this work gave rise to a pattern granted by prior examination (Palacios Macías, V. Caro Pina, l .; Roldán Gómez, A .; lasanta, and Procedure for the industrial application of lysozymes in the elaboration process of wines of biological aging Patent.

rights holder: University of Cádiz. Application NV: P201100895. Country of

Registration: Spain. Registration date: 07/29/2011).

the application of lysozyme together with the inoculation of the flower veil is effective for

Heterolytic fermentation treatment and the reduction of volatile acidity in the

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wines. On the one hand lysozyme reduces the population of tactical bacteria up to 99%, and

on the other, the metabolism of flower veil yeasts decreases the concentration of

acetic acid (volatile acidity). On an industrial scale (boots), the elimination of bacteria

tactics is fast, V approximately in a week the population is reduced to 1

colony / mi. However, the decrease in volatile acidity is much slower,

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needing the order of 3 to 6 months to place the initial 0.6-0.8 giL levels at

approximately 0.3 giL (Palacios Macias, V. Caro Pina, lo; Roldán Gómez, A .; Lasanta,

C. Procedure for the industrial application of lysozymes in the process of

elaboration of wines of biological aging. Patent. Entity holder of rights:

Cadiz University. Neither! of application: P201100895. Country of registration: Spain. Date

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registration: 07/29/2011).

Despite this, it has been possible to corroborate both laboratory and industrial scale that

This treatment is not effective when applied in wines with acidity levels

Very high volatile (> 0.8 giL) And with the presence of acetic bacteria. In these

conditions the implantation and development of the flower veil is very difficult, and lysozyme alone

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It has an effect on lactic bacteria. The treatment reduced the population of bacteria

lactic, but the volatile acidity in the wines continued to increase due to the lack of veil and

to the growth and metabolism of acetic bacteria.

Some authors in the past showed that flower veil yeasts under cultivation

submerged show an accelerated metabolism in both acetaldehyde production,

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as in the consumption of ethanol and acetic acid (Ough, C. S., & Amerine, M. A. (1958)

Studies on alcetaldehyde production under pressure oxygen and agitation. American

Joumal of Enology and Viticulture ,. 9, 11122; Ter-Karapetian, M. A. (1953). Biochemical

reactions in sherry formation. Akad Nauk S.S.S. R., Biokhim. Vinodeliia, Sbornik, 4, 83

120). The submerged cultivation of flower yeasts accelerates the aging process with

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compared to traditional film-forming culture without significantly modifying the profile

sensory of wines, and has been used with yeasts of the Saccharamyces capensis type

to reduce 105 levels of gluconic acid (Peinado, RA, Mauricio, J. C "Ortega, J. M" Medina, M "& Moreno, JJ (2003). Changes in gluconic acid, polyols and major volatile compounds in sherry wine during aging with submerged flower yeast happy Biotechnology letters, 25, 1887-1891).

At present there is no effective technique or procedure to treat acetic bites derived from lactic bites in wines subjected to the biological aging process. As a preventive measure to minimize the development of acetic bites in organic aging wines, some wineries perform veil and inoculation presence controls, especially in those boots that have a moderate volatile acidity (0.50-0.8 gil) And high content of gluconic acid, which is one of the main substrates for the growth of lactic bacteria V whose metabolism derives in the production of acetic acid. In some cases the consumption of acetic acid by flower veil yeasts may be sufficient to avoid an excessive increase in volatile acidity. But when starting from a high concentration of gluconic acid (> 500 mgJl), yeasts cannot maintain volatile acidity levels in wines, resulting in a gradual increase in the concentration of acetic acid. Under these circumstances the application of lysozyme and film-forming culture can be effective (Palacios Macias, V. Caro Pina, l .; Roldán Gómez, A .; lasanta, C. Procedure for the industrial application of lysozymes in the winemaking process of biological upbringing, patent, right-holder entity: University of Cádiz, application N2: P201100895, Country of registration: Spain, Registration date: 07/29/2011).

However, it has been found on an industrial scale that sometimes the phenomenon of acetic acid production is very fast, especially in boots with a lack of veil or very little active veil. Generally this occurs in the summer season, when temperatures in some wineries are higher than 24-25 Oc. Under these conditions of temperature and with an average volatile acidity (> 0.5-0.6 gil) inactivation and falling of the flower veil can occur, sponsoring the dissolution of oxygen within the wine and the growth of the acetic bacteria, which are normally found in the heads (aunts) in very small concentration. The growth of acetic bacteria produces a rapid increase in volatile acidity, placing levels above 0.8 gil in less than a month. In these cases, the treatment methodology of the

Lysozyme Film culture (Palacios Macias, V. Caro Pina, L; Roldán Gómez, A; Lasanta,

C. Procedure for the industrial application of lysozymes in the process of elaboration of wines of biological aging. Patent. Entity holder of rights: University of Cádiz. Or application: P201100895. Country of registration: Spain. Registration date: 07/29/2011) It is not effective, first, because lysozyme only has an effect on tactical bacteria V, not acetic, V second, because at these levels of volatile acidity it is very difficult to implantation and growth of veil yeasts on the surface of the wine, given its high sensitivity to acetic acid.

DESCRIPTION OF THE INVENTION.

A new methodology for applying lysozyme in submerged culture is proposed as patentable for the curative and corrective treatment of organic aging wines that have a very high volatile acidity (> 0.8 gil) and a contamination by both lactic and bacterial bacteria. acetic The methodology consists in establishing on the wine to be treated: first a submerged culture of flower yeasts with contribution and regulation of air flow through microairration, so that it allows dissolved oxygen levels to be below 1 ppm and second the Hsozyme addition, once the exponential phase of yeast growth has begun. Under these conditions, on the one hand, there is a reduction in the populations of acetic and lactic bacteria in contaminated wines by 99%, and on the other, a reduction in volatile acidity from 0.8-1.2 gil to 0, 3-0.1 g / l.

In recent studies carried out by the applicant team, it has been proven that veil yeasts under submerged cultivation have the capacity to develop with an active aerobic metabolism in wines with high volatile acidity (0.8-1.2 gil) and in the presence of lysozyme (10-12 g / hl). In these studies it has been corroborated that if lysozyme is added at the beginning, together with the yeast inoculum (or cuba pie), there is an increase in the latency phase and a reduction in the final active population in the exponential phase growth, especially in wines with greater volatile acidity (> 1 giL). However, when lysozyme is added at the end of the latency phase or beginning of the exponential growth phase (t = 3 days approximate in all cases), there is no effect on the growth kinetics of yeasts, reaching viable population levels even higher than in control wines without lysozyme. Lysozyme under submerged culture conditions does not lose any activity, and reduces population levels of tactical bacteria up to 99% in approximately 30 hours. This time is much shorter than what is needed when operating in film-forming culture (of the order of 120 hours) {Palacios Macias, V. Caro Pina, l .; Roldán Gómez, A .; lasanta, C. Procedure for the industrial application of lysozymes in the process of elaboration of wines of biological aging. Patent. Entity holder of rights: University of Cádiz. Application N2: P201100895, Country of registration: Spain. Registration date: 07/29/2011). Stirring conditions favor the diffusion and action of lysozyme on lactic bacteria.

On the other hand, the growth of the flower yeast in submerged cultivation implies an important consumption of the dissolved oxygen in the wine, so that by means of a regulation of the microaireation flow, anoxic or semi-aerobic conditions can be established in the medium (02 < 1 ppm), which are lethal to acetic bacteria (strict aerobic) without affecting the development of yeasts. In order for these anoxic conditions to be achieved, an air flow must be established in the range of 0.01-0.03 L of air per minute and liter of wine, and stirring by means of blades with a speed between 350 and 450 rpm. At oxygen concentrations below 1 ppm there is a drastic reduction in the population of acetic bacteria of the order of 99% on the second or third day of the process, almost always coinciding with the beginning of the exponential phase of growth of the yeasts In order for the oxygen consumption to be significant from the beginning of the process, it is necessary to inoculate a cuvette or yeast inoculum in a volume percentage of 10% v / v, so that the

Final concentration of yeasts in the wine to be treated is around viable cells / ml. The initial cuba foot should be made with fortified young wine at a graduation of 15% v / v and sterilized by 0.22 ~ m membrane microfiltration. The addition of the foot of Cuba only has to be done in the first treatment. For later you can leave a volume of wine already treated in the fermenter (of the order of 10%) as a foot of Cuba, and the procedure is equally effective.

Finally, the development of flower yeasts in submerged cultivation accelerates the metabolism and consumption of acetic acid and volatile acidity in wines. The process

deacidification is very fast, in less than 7 days a wine with a volatile acidity of 19l1 (high acetification) reduces its levels below 0.1 g / l. When lysozyme is added to the submerged crop, the reduction is more significant, mainly because the growth of yeasts is enhanced.

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MODE OF CARRYING OUT THE INVENTION.

Procedure for the corrective treatment of aging wines contaminated with lactic and acetic bacteria that have high volatile acidity

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12, A variable capacity fermenter equipped with air micro-diffusers and a vane stirrer is required, which have the mission respectively of providing and homogenizing the oxygen necessary for the growth of flower veil yeasts in submerged cultivation. The fermenter must be provided with a system to control the temperature around 20-222 (and as an auxiliary equipment a pressurized air generator (compressor, gas bottle, etc.) will be required.

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22 • Industrial flower veil yeasts are required, which can be obtained by means of a handle, spatula, venence or other similar tool in a boot or deposit that is carrying out biological aging, and has no indication of contamination or microbial alteration (volatile acidity < 0.3 gil)

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32 (On the industrial veil a cuba pie or inoculum of flower veil yeasts is prepared in full exponential growth, preferably in the same fermenter where the wine treatment is to be carried out. For the preparation of the cuba pie a wine is used healthy (with volatile acidity <O, 3g / l and an alcoholic strength of 15% vlv) that we will filter through a microfiltration membrane of 0.22 Ilm pore size, to ensure its sterility. The wine is inoculated with the yeasts of flower veil and by means of microairration in a range of 0.01-0.03 liters of air per minute and liter of wine, agitation between 350 and 450 rpm and temperature controlled in a range of 20-222 (, it is expected that reach a population of yeasts equal to more than 108 cells / ml, and in all cases the foot of Cuba must represent 10% of the total volume of the wine to be treated.

42, The wine to be treated with a volatile acidity not exceeding 1.2 gil is added on the foot of Cuba in full growth and in a volume that represents 90% of the total wine in the fermenter.

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51 !, The microaireation flow is regulated by a flow meter to maintain a dissolved oxygen concentration of less than 1 ppm throughout the process. The oxygen will be measured by a digital oximeter, equipped with an electrode that will be arranged horizontally in the fermenter to avoid the accumulation of micro air bubbles in the measuring membrane. Normally a steady state is achieved with stable concentrations of dissolved oxygen below 1 ppm (between 0.6 and 0.8 ppm) with microairflows of between 0.01-0.03 liters of air per minute and liter of wine , without affecting the growth of yeasts and without producing any oxidation of the wines.

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62 • Once the exponential phase of growth has begun (2-3 days after the start of treatment), and with a viable yeast population around 108 cells / ml, lysozyme is added at a concentration between 10-12 g / hl. Lysozyme is prepared according to the manufacturer's instructions.

72 • Wine treatment ends when volatile acidity levels between 0.3 and 0.1 g / l

be I have reduced this

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82 • Once the treatment is finished, the wine is transferred, leaving approximately 10% of the volume in the fermenter as a cuvette, if a new treatment is to be continued. In these cases it is recommended during this process to maintain microairration, agitation and temperature control under the established conditions.

9 !! The treated wine will have an alcohol correction of up to 15% v / v before its incorporation into the industrial biological aging system.

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Claims (3)

1. Procedure for the corrective treatment of aging wines contaminated with 1-lactic V acetic bacteria that have high volatile acidity, comprising: a) A submerged cultivation of flower yeasts with contribution and regulation of air flow by microairination, so that allows dissolved oxygen levels to be below 1 ppm. b) The wine is put in contact with the submerged culture of flowering yeasts e) the addition of lysozyme, once the exponential phase of yeast growth has begun. 2. Procedure for the corrective treatment of aging wines contaminated with lactic and acetic bacteria that have a high volatile acidity, according to claim 1, characterized by the following phases: to the Preparation of the cuba foot or inoculum of flower veil yeasts, in the one that is used: microfiltered wine with 0.22 Ilm membrane with volatile acidity less than or equal to 0.3 gil and alcoholic strength of 15% v / v, industrial flower veil yeasts extracted from a boot or a breeding deposit biological bJ Submerged cultivation of the cuvette of veil yeast according to the following conditions: 0.01-0.03 liters of air per minute and liter of wine, agitation 350-450 rpm and controlled temperature around 20-222C cl Addition of came to treat when the population of viable yeasts in the foot of Cuba is greater than or equal to 10 'cells / ml. The wine must have a volatile acidity not exceeding 1.2 g d) Submerged culture of veil yeasts in the treated wine under the following conditions: dissolved oxygen less than 1 ppm, stirring between 350 and 450 rpm and a controlled temperature around at 20-222C. Preparation of lysozyme, according to the manufacturer's instructions. f) Addition of lysozyme in a dose between 10-12 g / hl a when the culture has begun the exponential phase of growth, which coincides with a similarly higher population of viable yeasts of 10 'cells / ml.

g)

End of treatment when volatile acidity is reduced to levels

between 0.3 and 0.1 g / l.

3.

Procedure for the corrective treatment of contaminated aging wines

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with tactical and acetic bacteria that have high volatile acidity, according to

claims 1 and 2, comprising the regulation of air flow by

caudatimetro in the submerged cultivation of treated wine, to maintain levels

of dissolved oxygen below 1 ppm.

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Four. Procedure for the corrective treatment of contaminated aging wines

with tactical and acetic bacteria that have high volatile acidity, according to

claims 1 and 2, characterized in that the yeast bowl foot of

Veil should represent eliDo / .. of the total volume in the treatment.