WO2017198881A1

WO2017198881A1 - Method for the corrective treatment of aged wines contaminated with lactic and acetic acid bacteria presenting a high volatile acidity

Info

Publication number: WO2017198881A1
Application number: PCT/ES2017/000054
Authority: WO
Inventors: Victor Manuel PALACIOS MACIAS; Ana María Roldan Gomez; Marta Isabel LLORET VIERA
Original assignee: Universidad De Cádiz (Otri)
Priority date: 2016-05-20
Filing date: 2017-05-11
Publication date: 2017-11-23
Also published as: ES2642903A1; ES2642903B2

Abstract

A method for the corrective treatment of aged wines contaminated with lactic and acetic acid bacteria presenting a high volatile acidity. The scientific field to which the invention corresponds is Food Technology, specifically the winemaking industry. The method consists of establishing a submerged culture of flor yeasts in the wine to be treated, with supply and regulation of an air flow by microaeration, and the addition of lysozyme once the exponential growth phase of the yeasts has commenced. The method allows biologically aged wines with a volatile acidity of up to 1.2 g/L to be treated, with growth of the flor yeasts in film-forming culture being inhibited or prevented. The sensory characteristics of the treated wines improve, and said wines can be reincorporated into the industrial system once the alcohol content thereof has been corrected to 15% v/v.

Description

PROCEDURE FOR THE CORRECTIVE TREATMENT OF AGING WINES CONTAMINATED WITH LACTIC AND ACETIC BACTERIA THAT PRESENT A HIGH VOLATILE ACIDITY.

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 are concentrated in the areas from Jerez, Huelva, Montilla-Moriles, Jura (France), etc.

STATE OF THE TECHNIQUE.

The "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 Saccharomyces (Aiexandre, 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 (Martínez de la Ossa, E., Caro, I., 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. (Martínez de la Ossa, E., Caro, I., 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; Martínez de la Ossa, E., Pérez, L., & Caro, I. (1987). Variations 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 bioiogical aging of fino sherry: by products and aroma compounds. American Journal of Enology and Viticulture, 49, 240-250). The Biological aging produces very significant sensory modifications that give rise to the endologies of fine wines and chamomiles over time.

In industrial biological breeding systems, more than 9596 of the microorganisms present in the biofilm are yeasts of the genus S. cerevisiae (Alexandre, H. (2013). Flor yeasts of Saccharomyces cerevisiae— Their ecology, genetics and metabolism. International Journal of Food Microbiology, 167, 269-275). However, under certain conditions, some contaminating microorganisms such as lactic bacteria, acetic bacteria and in some cases non-Saccharomyces yeasts such as Brettanomyces (Alexandre, H. (2013). Flower yeasts of Saccharomyces cerevisiae can coexist and proliferate in the environment. - Their ecology, genetics and metabolism, International Journal of Food Microbiology, 167, 269-275; Moreno-Arribas, MV, & Pola, MC (2008). Occurrence of lactic acid bacteria and biogenic amines in biologically aged wines. Food Microbiology, 25, 875-881; Suarez-Lepe, JA, & Iñigo-Leal, B. (2004). Oenological microbiology. Vinification foundations. Mundi- Prensa Editions, Madrid, Spain.). The consumption of oxygen by yeasts establishes semi-aerobic conditions within the wine that favor, above all, the development of lactic bacteria. The presence of specific substrates such as gluconic acid (derived from the presence of Botrytis cinerea in the grape harvest), can facilitate its proliferation and the development of alterations such as "lactic mincing" or "spreading", which lead in most cases of an increase in volatile acidity and a loss of the characteristic sensory attributes of parenting (Lasanta, C, Roldan, A., Caro, I., Pérez, L., & Palacios, V. (2010). Use of lysozyme for the prevention and treatment of heterolactic fermentation in the biological aging of sherry wines. Food Control, 21, 1442-1447; Pérez, L, Valcárcel, MJ, González, P., & Domecq, B. (1991). Botrytis 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 high levels (> 0.8 g / L) the flower veil becomes inactive, falls and dies, creating ideal conditions for the development of acetic bacteria (dissolved oxygen and presence of acetic acid) (Casas, J . (2008). The vinification of Jerez in the 20th century. Ed. Junta de Andalucía. Sevilla, Spain. 310-311). Acetic bacteria are taxonomically classified as strict aerobic microorganisms and require aerobic conditions for their development and survival (Bartowsky, EJ, & Henschke, PA (2004). Acetic acid bacteria and wine: all is well until oxygen enters the scene. The Australian & New Zealand Grapegrower and Winemaker, 485a, 86-91; Drysdale, GS, & Fleet, GH (1988) Acetic acid bacteria in winemaking: a review, American Journal of Enology and Viticulture, 39, 143-154). The development of acetic bacteria produces a rapid and significant increase in volatile acidity in wines (> lg / L), invalidating the regeneration, presence and growth of the flower veil on the surface of the wine. The boots that present these symptoms are removed from the aging system (you take out and dew on Jerez wines), to avoid contamination of other boots, the wines being destined for vinegar production.

Generally to control the growth of both lactic and acetic bacteria in wines, high doses of sulfur of at least 100 mg / L (total S0 ₂ ) are used (Joyeux, A., Lafon-Lafourcade, S., & Ribereau-Gayon , P. (1984). Evolutfon of acetic acid bacteria during fermentatlon and storage of wine. Applied and Environmental Microbiology, 48, 153-156; Suarez Lepe, JA, & Iñigo-Leal, B. (2004). Oenological microbiology. of vinification, Mundi-Prensa Editions, Madrid, Spain). However, these doses of sulphurous cannot be applied in biological aging for two reasons: one, the flower yeast is very sensitive to this antiseptic (Suarez-Lepe, JA, & Iñigo-Leal, B. (2004). Oenological Microbiology Fundamentals of winemaking, Mundi-Prensa Editions, Madrid, Spain), and two, the presence of high concentrations of Idehido aceta in these wines means that practically all sulfur is in a combined form, which reduces its antiseptic capacity on bacteria ( Casas, J. (2008). The vinification of Jerez in the 20th century. Ed. Junta de Andalucía. Sevilla, Spain. 310-311).

An alternative for the control of lactic bacteria in biological aging has been the use of lysozyme. In studies published by the requesting team, it was found that lysozyme in small doses 5-15 g / hL reduces populations of lactic bacteria by 99% in organic aging wines with an alcohol content between 15-15.5% , so that lysozyme can be a good alternative as a preventive and curative treatment of lactic and possibly preventive pitting and acetic bites in these wines (Lasanta, C, Roldan, A., Caro, I., Pérez, L, & Palacios, V. (2010) Use of lysozyme far the prevention and treatment of heterolactic fermentation 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 occurring at doses lower than 1.5 g / hL (Lasanta, C, Roldan, A., Caro, I., Pérez, L, & Palacios, V. (2010). Use of lysozyme 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, C, Caro, I., & Palacios, V. (2012). Effect of lysozyme 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, severely affecting its hydrophobicity or buoyancy and its state of aggregation (Roldán, A., Lasanta, C, Caro, I., & Palacios, V. (2012). Effect of lysozyme on "flor" velum yeasts in the biological aging of sherry wines Food Microbiology, 30, 245-252) Under these conditions yeasts have no capacity to form stable and covered veils, inhibiting cellular metabolism and the production of the characteristic compounds of biological upbringing. 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 patent granted by prior examination (Palacios Mecías, V. Caro Pina, I .; Roldán Gómez, A .; Lasanta, C. Procedure for the industrial application of lysozymes in the process of preparing organic aging wines Patent Entity rights holder: University of Cádiz. Application number: P201100895. Country of registration: Spain. Registration date: 07/29/2011).

The application of the isozyme together with the inoculation of the flower veil is effective for the treatment of heterolytic fermentations and the reduction of volatile acidity in the wines. On the one hand lysozyme reduces the population of lactic 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 lactic bacteria is rapid, and in about a week the population is reduced to 1 colony / ml. However the decrease in acid? Volatile is much slower, requiring the order of 3 to 6 months to place the initial 0.6-0.8 g / L levels at approximately 0.3 g / L (Palacios Macias, V. Caro Pina, I .; Gómez Roldán, a .; Lasanta, C. Procedure for the industrial application of lysozyme in winemaking process of biological aging Entity patent rights holder.. University of Cádiz N ^{application.?} P201100895 country of registration. : Spain Date of 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 very high levels of volatile acidity (> 0.8 g / L) and with the presence of acetic bacteria. Under these conditions the implantation and development of the flower veil is very difficult, and lysozyme only has an effect on lactic bacteria. The treatment reduced the population of lactic bacteria, but the volatile acidity in the wines continued to increase due to the lack of veil and the growth and metabolism of acetic bacteria.

Some authors in the past showed that flower veil yeasts under submerged culture show an accelerated metabolism both in the production of acetaldehyde, and in the consumption of ethanol and acetic acid (Ough, CS, & Amerine, MA (1958) Studies on alcetaldehyde production under pressure oxygen and agitation. American Journal of Enology and Viticulture, 9, 11-122; Ter-Karapetian, MA (1953). Biochemical reactions in sherry formation. Akad. Nauk SSSR, Biokhim. Vinodeliia, Sbornik, 4, 83-120). The submerged cultivation of flower yeasts accelerates the aging process with respect to the traditional film-forming culture without significantly modifying the sensory profile of the wines, and has been used with yeasts of the Saccharomyces capensis type to reduce gluconic acid levels (Peinado, RA, Mauricio, J. C, Ortega, JM, Medina, M., & Moreno, JJ (2003). Changes in giuconic acid, polyols and major volatile compounds in sherry wine during aging with submerged flower yeast content. 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 g / L) and high gluconic acid content, which is one of the main substrates for the growth of lactic bacteria and whose metabolism derives in the production of acetic acid. In some cases the consumption of acetic acid by the 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 mg / L), 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 iisozyme and phymogenic cultivation may be effective (Palacios Maclas. V. Caro Pina, I .; Roldán Gómez, A; Lasanta, C. Procedure for the industrial application of lysozymes in the winemaking process biological upbringing, patent, rights holder entity: University of Cádiz, application number *: 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 are higher than 24-25 ° C in some wineries. Under these conditions of temperature and with a medium volatile acidity (> 0.5-0.6 g / L) inactivation and the fall of the flower veil can occur, sponsoring the dissolution of oxygen within the wine and growth of the acetic bacteria, which are normally found in the heads (lees) in very small concentration. The growth of acetic bacteria produces a rapid increase in volatile acidity, placing levels above 0.8 g / L in less than a month. In these cases, the treatment methodology of the lysozyme and film-forming culture (Palacios Martas, V. Caro Pina, I .; Roldan Gómez, A; Lasanta, C. Procedure for the industrial application of lysozymes in the process of elaboration of biological aging wines. Patent. Rights holder : University of Cádiz. N ⁹ of application: P20110089 S. Country of registration: Spain. Registration date: 07/29/2011) is not effective, in the first place, because lysozyme only has action on lactic bacteria and not on acetic, and secondly, park at these levels of volatile acidity it is very difficult to implant and grow 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 g / L) and a contamination by both lactic and by acetic bacteria. 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 by means of microairration, so that the levels of dissolved oxygen are below 1 ppm; and second the addition of lysozyme, 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 g / L to 0 , 3-0.1 g / L.

In recent studies carried out by the applicant team, it has been found 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 g / L) 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 inoculum of yeasts (or cuba pie), there is an increase in the iatencia phase and a reduction of the final active population in the exponential phase growth, especially in wines with greater volatile acidity (> 1 g / L). However, when lysozyme is added at the end of the Iatence 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 the population levels of lactic 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 Marías, V. Caro Pina, I .; Roldán Gómez, A .; Lasanta, C. Procedure for application . Industrial lysozyme in winemaking process of biological aging Entity patent rights holder. University of Cádiz N ⁹ application.. P201100895 registration Country: Spain registration date:. 29/07/2011). Stirring conditions favor the diffusion and action of lysozyme on lactic bacteria.

On the other hand, the growth of the flower yeast in submerged culture implies an important consumption of the dissolved oxygen in the wine, so that by means of a regulation of the micraaireation flow, anoxic or semi-aerobic conditions can be established in the medium (0 ₂ <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 foot of cuba 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 10 ⁶ 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 μηι 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 Ig / L (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.

MODE OF EMBODIMENT OF THE INVENTION.

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

10. A variable capacity fermenter dating from microd fusers and a paddle shaker 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 20-22'C and as an auxiliary equipment a pressurized air generator (compressor, gas bottle, etc.) 2 ^{9 will be required} . 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 rearing, and has no indication of contamination or microbial alteration (volatile acidity <0, 3 g / L)

3 ⁹ . With the industrial veil a cuvette or inoculum of flower veil yeasts is prepared in full exponential growth, preferably in the same fermenter where the wine treatment is going to be carried out For the preparation of the cuba pie a healthy wine is used ( with volatile acidity <0.3g / L and an alcoholic strength of 15% v / v) that we will filter through a microfiltration membrane of 0.22 μιη pore size, to ensure its sterility. The wine is inoculated with the flower veil yeasts and by means of microairration in a range of 0.01-0.03 liters of air per minute and liter of wine, stirring between 350 and 450 rpm and temperature controlled in a range of 20 -22 ^fi C, it is expected that a yeast population equal to or greater than 10 cells / ml will be ^reached . The foot of Cuba must represent in all cases 10% of the total volume of the wine to be treated. 4*. The wine to be treated with a volatile acidity of not more than 1.2 g / L is added to the fully growing Cuba foot and in a volume that represents 90% of the total wine in the fermenter.

59. The microaireation flow is regulated by a flowmeter 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 microbubbles of air 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 microairration flows 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.

6 ^°. Once started , the exponential growth phase (2-3 days of starting treatment), and with a population of viable yeast around 10 cel / ml lysozyme was added at a concentration of 10-12 g / hl . Lysozyme is prepared according to the manufacturer's instructions.

7 ". The wine treatment ends when the volatile acidity has been reduced to levels between 0.3 and 0.1 g / L.

8 ". 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 the microairination, stirring and temperature control under established conditions.

99. The treated wine will have an alcohol correction of up to 15% v / v before it is incorporated into the industrial biological aging system.

Claims

1. Procedure for the corrective treatment of aging wines contaminated with lactic and acetic bacteria that have high volatile acidity, comprising:

a) A submerged culture of flower yeasts with contribution and regulation of air flow by microairration, so that the levels of dissolved oxygen are below 1 ppm. b) The wine is put in contact with the submerged culture of flowering yeasts c) 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 high volatile acidity, according to claim 1, characterized by the following phases:

a) Preparation of the cuba pie or inoculum of flower veil yeasts, in which it is used: microfiltered wine with 0.22 μπη membrane with volatile acidity less than or equal to 0.3 g / L and alcoholic strength of 15% v / v, industrial flower veil yeasts extracted from a boot or a biological rearing deposit. b) Submerged cultivation of the cuvette of yeast veil 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 22 ^e C

c) Addition of the wine to be treated when the viable yeast population in the cuba foot is greater than or equal to 10 * cells / ml. The wine must have a volatile acidity not exceeding 1,

2 g / L

d) Submerged culture of veil yeasts in the wine treated under the following conditions: dissolved oxygen less than 1 ppm, stirring between 350 and 450 rpm and a controlled temperature around 20-22 * C.

e) Preparation of lysozyme, according to the manufacturer's instructions.

fj 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 an equal or higher population of viable yeasts of 10 ⁸ cells / ml. g) Completion of treatment when volatile acidity is reduced to levels between 0.3 and 0.1 g / L

*

3. Procedure for the corrective treatment of aging wines contaminated with lactic and acetic bacteria that have a high volatile acidity, according to claims 1 and 2, which comprises the regulation of the air flow by flowmeter in the submerged culture of the treated wine, for keep dissolved oxygen levels below 1 ppm.

4, Procedure for the corrective treatment of aging wines contaminated with lactic and acetic bacteria that have a high volatile acidity, according to claims 1 and 2, characterized in that the cuvette of veil yeast must represent 10% of the total volume in the treatment.