Treatment method using plasma activated water for treating auxiliary forest materials for wine preservation and use of plasma activated water

Abstract

The present invention relates to a method for treating auxiliary forest materials for wine preservation (such as bottle corks and wooden barrels) using plasma-activated water (PAW). The method disinfects and / or decontaminates the materials by continuously contacting the materials with PAW. The present invention also relates to the use of PAW to disinfect and / or decontaminate auxiliary forest materials used in wine production and preservation.

Description

[Technical Field]

[0001] The present invention relates generally to the field of wine production, and more particularly to a treatment method for disinfecting and / or decontaminating forest materials used in wine preservation, with the aim of maintaining wine quality and food safety. [Background technology]

[0002] The wine industry is one of the most important agri-food sectors in Spain and Southern Europe. According to current statistics (OIV Statistical Report, 2018, www.oiv.int), Spain produces approximately 44 million hectoliters of wine, ranking third after Italy and France. Spain also has the largest vineyard area in the world, accounting for 13% of the total. To meet consumer demand, the wine industry requires constant innovation, resulting in increased competition in the market.

[0003] Furthermore, with the increasing demand for natural foods, the food industry, and therefore the wine industry, is faced with the challenge of providing safe, healthy and minimally processed food.

[0004] The sensory evolution of wine is greatly enhanced when oak barrels are used for wine production and aging. Increasingly demanding consumers seek balanced wines with aromatic complexity. This is achieved through barrel aging, where a series of interactions between the wine and the wood enrich the aroma and mouthfeel, promote micro-oxygenation, and result in a physically and chemically stable wine. Used barrels, while their potential is reduced compared to new barrels, are still viable for cost-effective use in the production of wine and alcoholic products. Therefore, proper maintenance is essential for reusing barrels. Microbiological or chemical contamination is a major problem during wine aging, and the development of undesirable fungi (in the first 8 mm of wood in contact with the wine) can lead to wine deterioration or render it unsuitable for consumption. This problem is exacerbated by the reuse of improperly maintained barrels, which can result in "acetic acid spoilage," phenolic odors or "brettage," or "lactic acid spoilage," "viscosification," or "bitterness." A. Palacios et al., Enologos. 77 (2012) 46-54.

[0005] Furthermore, tartrate precipitates produced during aging adhere to the interior walls of barrels, providing a haven for potentially contaminating microbial strains and even clogging the barrel's porous structure. It has been shown that in a 225-liter barrel, approximately 5 liters of wine can be retained in the first few millimeters of the barrel wood. Furthermore, the wood's microporous structure facilitates deep microbial penetration. All of this complicates the process of barrel cleaning and sanitizing. This problem is particularly severe in the case of contamination with the yeast Brettanomyces ("Brett"). Brett imparts characteristic aromas (descriptors) to wines, such as a plastic-like odor, burnt rubber, stables, or horse sweat, known as phenolic odors. This sensory issue is currently a topic of widespread discussion, particularly affecting aged wines and those stored in barrels for extended periods. A. Palacios et al., Enologos. 77 (2012) 46-54.

[0006] Cork, used to stopper wine bottles, tends to produce TCA (2,4,6-trichloroanisole), a compound known as "cork disease." This problem affects approximately 4% of bottled wines worldwide. TCA is a molecule formed through a chemical process triggered by the presence of chlorophenols. Chlorophenols are present in the environment, with cork oaks and forest soils being some of the main sources. This makes cork a major source of TCA, imparting negative characteristics to wine. However, this process can also occur at various stages of wine production, as chlorophenols are present in the humid environment typical of wineries. This issue is of paramount importance in the wine industry because once wine acquires the sensory characteristics specific to TCA, it is impossible to remove them, resulting in consumer rejection.

[0007] In 2017, a study conducted by Raithwaites Wine observed that 624 million bottles of wine are wasted annually in the UK. The study cited consumer ignorance about wine preservation practices and sensory defects as reasons for this. In this regard, it was observed that unpleasant odors originating from the cork are the main cause of wine's impaired sensory characteristics. While these odors have various causes, the most common is cork contamination by chlorinated compounds, particularly trichloroanisole (TCA), which accounts for approximately 80% of this problem.

[0008] Furthermore, the wine industry is a key customer for the cork industry. The loss of market share for cork stoppers compared to alternative stoppers indicates the need to reevaluate the cork industry, communicating its inherent environmentally friendly and socially responsible benefits through its products. The Iberian Peninsula dominates global cork production, with Spain accounting for 30% of it (506,000 hectares of cork oak forests). One of the challenges in improving the cork industry is reducing the incidence of organochlorine compounds, which cause unpleasant aromas in wine. The interest in eliminating this sensory deviation and its causes is justified by its economic impact on the cork and wine industries. To date, the application of hot water and the use of chemical preservatives have not been effective enough to fundamentally solve this problem.

[0009] To address the above issues, several treatment methods are known in the art for the purpose of preserving and continuously reusing forest products related to wine production and preservation. One of the most widely used methods for sanitizing barrels in wineries is traditional sulfur fumigation, which has been used since ancient Roman times. This method involves burning sulfur pellets inside empty barrels, which produces sulfur dioxide, a compound that has a bactericidal effect on wood. This method helps to keep winery barrels clean and free of microorganisms that can spoil the wine.

[0010] However, the European Commission's Directive 98 / 8 / EC2 prohibits the use of sulfur dioxide to sanitize barrels. This directive creates an urgent need to find new disinfection solutions that can carry out this task in an economically and operationally feasible way. In Spain, a moratorium on the use of sulfur fumigation has been agreed until 2025.

[0011] This situation has led to the emergence of new alternative technologies to sanitize barrels (heating, ozone, cavitation, or sandblasting, among others), but none of these adequately meet the needs of the industry and have the potential for contamination in various aspects.

[0012] Sanitation differs from pasteurization (complete elimination of microorganisms) in that it destroys viable microorganisms by chemical and / or physical action, significantly reducing the number of remaining microorganisms. In this respect, two categories are defined for new methods of sanitizing barrels that replace sulfur fumigation: chemical and physical.

[0013] Chemical methods are carried out using oxidizing agents. For example, sulfur dioxide can be used in liquid form, as a sodium sulfite solution, or in gaseous form, as a solution for wood and wine. This method can be achieved by burning sulfur (as mentioned above) or by using liquefied gas. Other methods include hydrogen peroxide, peracetic acid, potassium permanganate, and ozone (O3). It should be noted that ozone is a highly oxidizing, toxic, and explosive gas. Ozone can only be used if it is dissolved in cold water to produce an activated ozone solution containing 3–5 mg / L of O3. While ozone functions as a disinfectant, it does not possess cleaning properties. Given its mechanism of action, it must be used on perfectly clean surfaces to achieve disinfection. In the case of wooden containers, its effectiveness is severely limited due to the partial reaction with ozone.

[0014] Physical Methods: The most widely used physical method is the thermal method. This involves the use of portable hot water generators capable of generating hot water at 80–90°C and pressures of 80–210 bar. However, using extremely high temperatures is impractical because the flow rate is very low. Pressures of 100–120 bar are sufficient and are within the wood-friendly range. This technology allows cleaning without detergents, but its disinfecting effect is limited to the wood's surface. The heat transmitted by water vapor (105°C) raises the temperature of the wood's inner layers only very slowly. This is due to wood's very low thermal conductivity. Effective use of this technology requires very long treatment times. However, as mentioned above, this is a major limitation, as microorganisms can penetrate more than 8 mm into wood. Considering the various surfaces that may come into contact during wine production, heat treatment appears to be effective on nearly smooth surfaces such as stainless steel and glass. However, it is not effective on the surfaces of forest materials such as wood and cork, which have very high roughness and porosity.

[0015] Another possible method is electromagnetic microwave treatment. This method heats the wood by exciting the water contained within the material. By selecting the wavelength, the wood can be heated from the center of the barrel using minimal energy. Furthermore, the shape and placement of the magnetron and the rotation of the barrel allow for uniform irradiation within a metal Faraday cage, which reflects the waves. Another ultrasonic method involves inserting a sonotron into a barrel filled with recycled water at 60°C. Ultrasound (150 kHz) generates high pressures of over 2000 bar at the surface and within a few millimeters of the wood through a cavitation effect at the microscopic level. This process removes compounds that have penetrated the wood, destroys microorganisms, and simultaneously disinfects the wood. Other less common physical methods include the use of dry ice (efficient and fast, but expensive, and does not guarantee deep disinfection of the barrel) and negative oxygen.

[0016] All of these methods have been scientifically studied and industrially tested, but none have been able to surpass the disinfection results achieved by sulfur fumigation. Furthermore, the implementation costs and processing times of some of the techniques (e.g., ultrasound and microwave) are prohibitively expensive and impractical.

[0017] Another known disinfection method is cold air plasma. While research has been conducted on its direct application to wooden products, such as barrels, its industrial application has not yet been demonstrated. Plasma is the fourth state of matter and is composed of positive ions, negative ions, electrons, excited and neutral atoms, free radicals, ground and excited state molecules, and UV photons. Plasmas are classified as thermal plasma (high temperature) or nonthermal plasma (low temperature) based on the thermodynamic equilibrium of their constituents at their temperatures. The temperature of cold plasma does not exceed 100°C. Nonthermal plasma sources widely used in the food industry include dielectric barrier discharge (DBD), plasma jet, and corona discharge. In recent years, many scientists and researchers have utilized cold plasma in various food applications, including microbial disinfection, enzyme inactivation, improving the cooking quality of rice varieties, starch modification, and seed germination (see, for example, R. Tirumudas et al., "Cold Plasma: A Novel Nonthermal Technology for Food Processing," Food Biophys. 10 (2014) 1-11).

[0018] Most research into the antimicrobial activity of plasma has been conducted by applying plasma directly to food or surfaces containing food. However, in some cases, direct application of a plasma source is technically infeasible or industrially uneconomical. For example, plasma treatment of the interior of a barrel requires disassembly of at least one side of the barrel in order to introduce the plasma device into the barrel. This approach is nearly impossible in wineries.

[0019] Instead of directly applying cold or hot plasma, research has recently begun into the use of plasma-activated water (PAW) as a disinfectant. As described in detail herein, PAW contains reactive nitrogen and oxygen species (primary reactive species) that are generated when plasma contacts water, as well as secondary reactive species that are generated when these primary reactive species interact in subsequent secondary reactions, and these are known to have excellent antibacterial activity.

[0020] For example, documents CN212456771U, RU2746976C1, and ES2314000T3 disclose devices for generating plasma activated water (PAW), but none of these documents teach or suggest using PAW to disinfect the surface of forest materials.

[0021] There have been very few studies using plasma-activated water (PAW) to decontaminate surfaces. Joshi et al. ("Characterization of Microbial Inactivation Using Plasma-Activated Water and Plasma-Activated Acidic Buffer Solution," J. Food Prot. 81 (2018) 1472-1480) investigated the use of PAW in disinfecting glass slides inoculated with Enterobacter aerogenes. Kamgang-Yubi et al. ("Microbial Decontamination of Stainless Steel and Polyethylene Surfaces Using Gliding-Arc Plasma-Activated Water Without Chemical Additives," J. Chem. Technol. Biotechnol. 93 (2018) 2544-2551) confirmed the effectiveness of PAW in decontaminating AISI 304 stainless steel and high-density PET surfaces contaminated with Saccharomyces cerevisiae, Staphylococcus epidermidis, Leuconostoc mesenteroides, and Hafnia alvei. However, there are no known studies that have satisfactorily applied PAW to disinfect and decontaminate rough and porous materials, more specifically forest materials such as wood and cork.

[0022] Furthermore, various methods for treating cork products (e.g., bottle stoppers) to reduce TCA content are disclosed in, for example, ES2423255B1, ES2268459T3, ES2402890T3, ES2019562A6, ES2726598B2, and ES2247180T3. However, all of these methods rely on high temperatures, high pressures, long treatment times, and / or continuous container rotation. However, high pressure and high temperature can have adverse effects, such as irreversible distortion of the stoppers, as demonstrated, for example, in ES2423255B1. On the other hand, long treatment times and container rotation (as well as the use of high pressure and high temperature) increase implementation costs, thereby reducing the profitability of the method.

[0023] It is therefore desirable to provide an alternative method for disinfecting and / or decontaminating auxiliary materials and products for wine production and preservation, particularly forest products such as wood and cork. Specifically, it is desirable to provide an alternative to sulfur fumigation. This method should provide results comparable to or better than sulfur fumigation while overcoming its drawbacks, particularly the harmful problems resulting from the use of sulfur dioxide. It is also desirable that this method not cause any adverse effects, such as irreversible deformation, on the treated materials (cork and wood), and that it reduces the economic costs associated with its implementation compared to the alternatives known in the prior art. Summary of the Invention

[0024] To solve the above-mentioned problems, the present invention proposes a new treatment method for auxiliary forest materials used in wine preservation. The method aims to disinfect and / or decontaminate them, maintaining the quality of the wine and food safety. Specifically, the method is based on contacting the materials that need to be disinfected and / or decontaminated with plasma-activated water (PAW).

[0025] The present invention also proposes the use of plasma activated water (PAW) to disinfect and / or decontaminate auxiliary forest materials used in wine production and preservation.

[0026] The appended dependent claims refer to preferred embodiments of the processing method and use of the PAW of the invention. [Brief explanation of the drawings]

[0027] The present invention will be better understood with reference to the following drawings, which illustrate preferred embodiments of the invention and are not to be construed as limiting the invention in any way. [Figure 1] FIG. 1 shows a schematic diagram of a system for generating plasma activated water for use in a method according to a preferred embodiment of the present invention. [Figure 2] FIG. 2 is a schematic diagram illustrating a procedure for sanitizing a wooden barrel, according to some preferred embodiments of the present invention. [Figure 3] Figure 3 shows chromatograms of PAW samples treated for different times, allowing identification of the phenolic reaction products associated with each reactive species in the PAW samples. [Figure 4] Figure 4 shows an overlay of the phenol reaction product peaks of the hydroxyl radical OH* obtained from the chromatograms of the four PAW samples. [Figure 5] 5 is a bar graph showing the logarithmic growth of viable Brettanomyces cells per gram of wood obtained from contaminated barrel wood treated with distilled water (DW) and various PAW samples in accordance with the present invention. Different letters indicate statistically significant differences (p<0.05). [Figure 6] Figure 6 is a bar graph showing the mean concentrations of TCA (ng / l) in artificially contaminated cork treated with distilled water (DW) and various PAW samples according to the present invention. Different letters indicate statistically significant differences (p<0.05). Detailed Description of the Preferred Embodiments

[0028] As mentioned above, the present invention is based on the use of plasma activated water (PAW) for the disinfection and / or decontamination treatment of auxiliary forest materials used in wine production and preservation.

[0029] PAW technology offers many advantages over current disinfection and / or decontamination methods. First, the technology is low-cost, often requiring only electricity and compressed air to generate the plasma; no chemicals, filters, or other consumables are required. Furthermore, because it is generated at atmospheric pressure and room temperature, no auxiliary equipment is required. It can also be applied in currently used barrel cleaning systems (pressurized water lances), allowing barrels to be cleaned and sterilized simultaneously, saving water, energy, and process time. Finally, the technology is environmentally friendly, producing no toxic chemicals or waste.

[0030] However, PAW technology is an emerging technology that has not yet been extensively studied or applied at an industrial level, and in particular, there is no known literature that mentions the use of PAW for disinfecting surfaces made of wood or other forest materials.

[0031] Plasma-activated liquids are generated by direct or indirect contact between a plasma source and a liquid. This interaction generates or transfers activated chemical species into the liquid. Plasma-activated solutions can be used as disinfectants in the food industry. The most widely studied plasma-activated liquid is plasma-activated water (PAW). Its antibacterial effects have been demonstrated in various publications (see, for example, MJ Traylor et al., "Long-term antibacterial efficacy of air plasma-activated water," J. Phys. D. Appl. Phys. 44 (2011) 472001), and it has attracted considerable attention.

[0032] PAW has different composition and physicochemical properties than water: it typically exhibits an acidic pH, changes in redox potential and conductivity, and contains reactive oxygen species (ROS) and reactive nitrogen species (RNS) (see A. Mai-Prochnow et al., "Microbial decontamination of chicken using atmospheric plasma bubbles," Plasma Process. Polym. (2020)).

[0033] The antibacterial ability of PAW is generated through several processes: [a] Gas phase: Reactive species are formed in the gas phase through the interaction of plasma gases and the surrounding atmosphere with charged particles (electrons, neutrons, etc.) and ultraviolet light. [b] Gas-liquid phase: Reactive species generated in the gas phase and those generated by plasma-liquid interactions, especially relatively long-lived species such as ozone, atomic oxygen, and nitric oxide, dissolve in water and act as precursors for other ROS and RNS, such as hydrogen peroxide, nitrates, and nitrites. [c] Liquid phase: Secondary reactions of long-lived reactive species, for example, caused by the instability of nitrite in acidic media. These cyclic reactions can result in the formation of hydroxyl radicals (OH * ), acidification nitrite (NO * , NO2 * This explains the existence of short-lived, highly cytotoxic reactive species such as peroxynitrite (O=NOOH) for several days.

[0034] Some of the most potent biocidal species in PAW (OH * , NO * , NO2 *, and O=NOOH) are generated after the plasma source is turned off (short-lived reactive species, SLS). SLS can be generated during PAW generation (when the plasma comes into contact with air and water), but its lifetime is extremely short (on the order of milliseconds) and is not thought to be due to dissolution in water. The SLS ultimately found in PAW is generated by reactions from "long-lived" reactive species (hydrogen peroxide, nitrate, and nitrite) generated within the PAW after the plasma source is turned off. The SLS generated by these secondary reactions is a "transient" reactive species that is highly cytotoxic and is responsible for the antibacterial activity of PAW for several days after exposure to plasma discharge.

[0035] The main secondary reaction occurs because nitrite is not stable under acidic conditions (pH < 3.5). Below is a diagram of the main secondary reactions that occur within PAW. Nitrite (HNO2) is in acid-base equilibrium with nitrite [a], and under acidic conditions it decomposes to produce nitric oxide (NO * ) radical and nitrogen dioxide (NO2 * ) radicals [b]. In addition, nitrogen dioxide (NO2 * ) radical undergoes hydrolysis in aqueous media, producing nitrite ions (NO2 - ) [c]. NO and NO2 also react with dissolved oxygen to form nitrite ions (NO2 - ) and nitrate ions (NO3 - ), can undergo general reactions [d] and [e]. The NO and NO2 nitrogen radicals produced in these secondary reactions are highly cytotoxic and are thought to be one of the main causes of the cytotoxic effects of nitrite under acidic conditions. For this reason, they are called "acidified nitrites." Furthermore, under acidic conditions, the nitrite ion (NO2 - ) can react with hydrogen peroxide (H2O2) to produce peroxynitrite (O=NOOH) [f]. Peroxynitrite acts either directly with microorganisms or indirectly by decomposing to produce OH and NO2 [g]. This is due to the hydroxyl radical (OH * ) is one of the pathways that generate

[0036]

number

[0037] Acidified nitrite (NO and NO2) and OH are produced by secondary reactions * Radical formation is key to the long-lasting biocidal properties of PAW.

[0038] Hydroxyl (OH * )radical OH radicals are considered to be the most important reactive species generated by plasma treatment of liquid solutions. They have the ability to nonselectively oxidize most organic compounds that they come into contact with, and radical recombination is the primary source of hydrogen peroxide in plasma systems. In terms of biocidal potential, the part most affected by OH radicals is the outer cell wall of microorganisms, including the cell membrane. Cell membranes are primarily composed of organic compounds such as lipids, proteins, and polysaccharides, and OH radicals are the most important reactive species generated by plasma treatment of liquid solutions. * Cell membranes are susceptible to radical attack. Lipids in particular are the most vulnerable macromolecules to oxidation. The reaction of lipids with OH radicals occurs through the removal of hydrogen (H) from unsaturated carbon bonds in fatty acids, leading to lipid peroxidation in the presence of oxygen. Similarly, OH radicals can damage membrane proteins by abstracting hydrogen from the alpha carbon of the peptide bond -CO-NH- that attaches to the amino acid chain between peptides. This attack causes peroxidation and cleavage of the protein backbone. The combined effect of these damages leads to cell death.

[0039] NO and NO2 radicals ("acidified nitrites") Acidified nitrites have remarkable antibacterial effects against a wide range of pathogenic microorganisms, including viruses (e.g., SARS-CoV-1 and SARS-CoV-2), bacteria, and fungi. Some of the damage they cause to microorganisms includes oxidation of membrane proteins, consumption of available iron through reactions with metalloenzymes, inactivation of metabolic enzymes, DNA damage through oxidative damage, and lipid peroxidation that disrupts cell membranes. These multifaceted damages cause severe dysfunction and ultimately lead to cell death.

[0040] As described above, the present invention relates to a method for treating auxiliary forest materials used in wine preservation using plasma activated water (PAW), with the aim of disinfecting and / or decontaminating these materials. Specifically, the forest materials to be disinfected and / or decontaminated can be selected from the group consisting of bottle corks and wooden containers (such as wooden barrels).

[0041] In the case of cork, the main objective of the method according to the invention is to bring about a reduction in the anisole, in particular TCA (2,4,6-trichloroanisole), present in the cork, preferably by at least 50%, more preferably by at least 75%, of the amount of TCA present in the cork before treatment.

[0042] In the case of wooden vessels, particularly wooden barrels, the primary objective of the method of the present invention is to reduce the amount of yeast, particularly Brettanomyces yeast, present in the vessel prior to treatment, preferably by at least 1 log, more preferably at least 3 logs, relative to the Brettanomyces content in the vessel prior to treatment.

[0043] More specifically, the processing method according to the present invention comprises the following steps: - optionally generating PAW by applying plasma to the initial water; - if necessary, washing the material to be disinfected with pressurized water; - If a cleaning step has been performed, immediately thereafter, continuously contacting the material to be disinfected and / or decontaminated with the PAW for a period of time sufficient to achieve the desired level of disinfection and / or decontamination of the material to be disinfected and / or decontaminated, preferably for a period of at least 3 hours.

[0044] The present invention also relates to the use of plasma activated water (PAW) to disinfect and / or decontaminate auxiliary forest materials used in wine production and preservation.

[0045] As noted above, the reactive species within the PAW that cause the disinfection and / or decontamination effect may exist for a relatively long period of time, e.g., several days. Thus, the process of generating the PAW can be performed as part of the method of the present invention, can be performed as a separate process at the same location, or can be sourced from an external commercial supplier. These options are dependent on the requirements and technical constraints of the facility in which the method is performed.

[0046] According to a preferred embodiment of the present invention, a system for producing cold atmospheric pressure plasma activated water, as shown schematically in FIG. 1, is used to generate PAW suitable for use in the treatment methods disclosed herein.

[0047] The bottom of the figure shows a tank (10) containing distilled water, which is used as the initial water for plasma activation. Distilled water is useful in performing the tests described below because it does not have the biocidal effect of the chlorine contained in tap water. However, according to another preferred embodiment of the present invention, tap water can also be used as the initial water for generating PAW.

[0048] Air (12) is used as the gas for plasma generation. * This is because it is the gas that generates the most radicals and is also the least expensive gas.

[0049] The system further includes a power generator (14) and two electrodes (16, 18). As air (12) passes through the system, a plasma stream (20) is generated and projected into the water tank (10). This causes the plasma to interact with the water (22) to generate reactive species via the chemical reactions described above.

[0050] The parameters used to generate PAW in a preferred embodiment of the present invention are as follows: Plasma gas: Air Plasma gas (air) flow rate: 60 slm Plasma power: 500 W Amount of treated water: 2 liters of distilled water Processing time: between 1.5 and 30 minutes Table 1 below shows the nomenclature of the four PAW samples produced by the tests described herein and the amount of time the initial water was subjected to plasma treatment to produce each sample:

[0051] [Table 1]

[0052] The tests described below confirmed that the physicochemical properties of PAW that significantly reduced Brettanomyces load were as follows: pH: less than 4.5 EC (electrical conductivity): Over 50 μS / cm ORP (oxidation-reduction potential): Over 350 mV No. 3 - (Nitrate ion): Over 3mg / L NO2 - (Nitrite ion): More than 0.5 mg / l.

[0053] After PAW is generated, it is used in the disinfection and / or decontamination process of the present invention. However, prior to the disinfection and / or decontamination process by contact with PAW, the surface of the material to be disinfected and / or decontaminated must be cleaned, especially when applying this process to wooden containers (such as wooden barrels). The purpose of cleaning the interior of the barrel is to remove any adhered material or deposits. This process is common in wineries and is currently performed before sulfur smoking. This cleaning is usually performed with a high-pressure water lance (24) (see FIG. 2a). According to another preferred embodiment of the present invention, the pressurized water used in the cleaning process is also PAW.

[0054] A step of continuously contacting the interior of the barrel with PAW is then carried out. Preferably, this step of contacting with PAW (disinfecting) is carried out immediately after the cleaning step. The PAW is introduced into the interior of the barrel through the same opening as the introduction of the cleaning water. The same cleaning lance (24) can be used for this purpose.

[0055] In order for the PAW to remove Brettanomyces from the barrel (as mentioned above, Brettanomyces present on the barrel walls can reach a depth of up to 8 mm), the PAW must be in contact with the entire interior of the barrel for a sufficient time (preferably at least 3 hours). For this purpose, there are two alternative methods: According to a preferred embodiment, the entire barrel is filled with PAW, as shown in FIG. 2b. Another preferred alternative embodiment is to introduce a minimal amount of PAW into the barrel and rotate the barrel continuously, as shown in FIG. 2c. In this method, the PAW contacts the entire interior of the barrel. In this case, the amount of PAW consumed is significantly reduced compared to the embodiment shown in FIG. 2b. Note that these barrel rotation systems are commonly used in wineries and are also used to drain the contents of barrels.

[0056] When the treatment method of the present invention is applied to reduce TCA in cork, the step of continuously contacting the cork with PAW is carried out by completely immersing the cork in PAW in a suitable container, without the need for vibration or movement of the cork, and the treatment method is entirely static.

[0057] In any case, the process according to the invention is preferably carried out at room temperature and atmospheric pressure. [Example]

[0058] Below are some examples of applications of the processing methods disclosed herein.

[0059] 1. Quantitative analysis of reactive species contained in PAW: First, tests were conducted to quantify the reactive species contained in each PAW sample prepared by the method described above.

[0060] The most biocidal secondary species in PAW (OH * , NO * , NO2, NO2 * The highly reactive radicals (O=NOOH, O=O, and O=NOOH) make direct detection and quantification difficult, especially in the liquid phase. Therefore, these species must be detected and quantified indirectly. The primary detection methods involve the use of chemicals that selectively react with these radicals to produce relatively stable products, which are detected spectroscopically by electron spin resonance (EPR), fluorometry, or high-performance liquid chromatography (HPLC).

[0061] In this study, we used a method by Lukes et al. ("Aqueous-phase chemistry and bactericidal effects from an air discharge plasma in contact with water: evidence for the formation of peroxynitrite through a pseudo-second-order post-discharge reaction of H2O2 and HNO2", Plasma Sources Sci. Technol. 23 (2014) 015019) for the indirect detection and quantification of reactive species contained in PAW. This method utilizes phenol (C6H5-OH) and OH * , NO * , and NO2 * The test procedure is as follows: -2 A phenol solution is prepared in water at a concentration of 1000 M. 5 ml of this phenol solution is taken and mixed with 95 ml of PAW. The mixture is heated at 50 degrees Celsius for 24 hours. The solution is then filtered through a 0.45 μm filter disc. The filtered solution is analyzed by high-performance liquid chromatography (HPLC). Injection volume: 20 μl, column: C18, mobile phase: the following elution gradient at a flow rate of 1.0 ml / min is used:

[0062] [Table 2]

[0063] Total analysis time: 16 minutes, Pressure: 90 / 10: approx. 90 bar, 60 / 40: approx. 60 bar, Detector: DAD (Diode Array Detector), Measurement wavelength: 260 nm, Reference wavelength: 699 nm The reaction product is: benzoquinone (phenol + OH * ), 4-nitrosophenol (phenol + NO * ), 2-nitrophenol (phenol + NO2 * ) The reaction times were as follows: hydroquinone: about 5 minutes, 4-nitrosophenol: about 5.4 minutes, phenol: about 8.2 minutes, 2-nitrophenol: about 11.2 minutes.

[0064] To quantify the reaction products (μg / L), the absorbance values of reaction product solutions with known concentrations were determined, and the corresponding calibration curves were constructed based on these values. Benzoquinone and 2-nitrophenol standards with purity of ≥98% were obtained from Sigma-Aldrich (Madrid, Spain), and 4-nitrosophenol standards with purity of ≥98% were obtained from TCI (Tokyo, Japan). Methanol (purity ≥99.5%) was obtained from Scharlab (Santenate, Barcelona, Spain).

[0065] The stock solution of each standard was prepared in a water-methanol (95:5) mixture at 10 -2 It was prepared to a concentration of M, stored in a glass bottle, and kept at 8 degrees Celsius.

[0066] A calibration curve was prepared by diluting with deionized water, and the following concentrations were used: benzoquinone: 1.35 × 10 -5 M, 2-nitrophenol: 1.60 x 10 -4 M, 4-nitrosophenol: 2.54 × 10 -4 M The calibration curve details are as follows:

[0067] [Table 3]

[0068] Chromatograms were obtained for each PAW sample (PAW-1.5, PAW-5, PAW-15, and PAW-30), which showed signals corresponding to phenolic reaction products, the most important reactive species related to the disinfection and decontamination capabilities of PAW. These reaction products are: * Hydroxyl radical (OH) **: benzoquinone, acidified nitrite; 4-nitrosophenol corresponding to NO and 2-nitrophenol corresponding to NO2, phenol itself. These results are shown in Figure 3.

[0069] Hydroxyl radical (OH * ) is important in both applications: disinfection of Brettanomyces in wooden barrels and reduction of TCA in cork, so the chromatograms related to hydroxyl radicals for all PAW samples are shown in more detail in Figure 4.

[0070] Finally, the reactive species contained in each PAW sample were indirectly quantified from the phenolic reaction products based on the method described above, and the results are shown in Table 4.

[0071] [Table 4]

[0072] 2. Testing on barrel disinfection methods Test materials Oak wooden barrels used in the production of aged wine were used as test materials. These barrels were naturally contaminated with Brettanomyces. The test barrels were disassembled to obtain boards measuring 5 x 5 cm. The average Brettanomyces contamination in a sample consisting of four boards was measured as 4.35 ± 0.26 log viable cells per gram of wood.

[0073] The processing applied Three contaminated pieces (5 x 5 cm) were placed in a 4-liter glass jar. Five grams of sulfur pellets were suspended inside the jar and ignited with a lighter to generate SO2 vapors. The jar was completely sealed with a sealable lid and parafilm and held in this state for 30 minutes. This method is a scaled-down version of the conditions commonly used in sulfur smoking techniques today.

[0074] Immersion treatment with PAW Three naturally contaminated wood pieces were placed in separate containers and immersed in 250 ml of PAW, the same solution used in this study. The sides corresponding to the inside of the barrels were completely immersed, and the wood was submerged to a depth of 3 cm (microorganisms have been shown to penetrate up to 8 mm into wood). Five immersion conditions were considered: distilled water (DW), used as a control, followed by PAW-1.5, PAW-5, PAW-15, and PAW-30.

[0075] Tests conducted Analysis of microbial inactivation by PAW Contaminated boards were marked 10 mm above the toasted surface, the height at which all Brettanomyces bacteria were expected to reside. The boards were individually placed in plastic containers (one board per container) with the toasted side (corresponding to the inside of the barrel and containing Brettanomyces) facing downwards. Then, 250 ml of each PAW (PAW-1.5, PAW-5, PAW-15, PAW-30) and distilled water (as a control) were added to each container. Each sample contained three replicates.

[0076] After the treatment time (3 hours of immersion in all cases), the boards were removed and scraped to a depth of 10 mm, which was collected in pre-weighed sterile plastic bags.

[0077] After collecting the wood chips, each bag containing the wood chips was weighed to confirm the weight of the wood chips collected from each sample. Each bag was then placed, open, in a sterile storage bottle, and 600 ml of warm TSB (tryptic soy broth, recovery medium) was added, ensuring the wood chips were fully submerged. These bottles were then placed on an orbital shaker at 28°C and 80–100 rpm for 24 hours. After the shaking period, the bottles containing the wood chips were removed from the orbital shaker, maintaining the same shaking sequence. The TSB medium for each sample was then extracted in a laminar flow cabinet to maintain sterility. This medium was then filtered through a sterile net curtain and transferred to a sterile centrifuge bottle. The samples were centrifuged at 4°C and 10,000 g for 30 minutes. After centrifugation, the supernatant was immediately removed, and the pellet was resuspended in Ringer's solution (refrigerated to maintain cell viability for 7 days). The suspension was transferred to a sterile plastic tube and adjusted to a volume of 15 ml.

[0078] Finally, to analyze each sample, we first treated it with propidium monoazide (PMA) and extracted DNA. After DNA extraction, quantitative PCR was performed using EvaGreen, and the results were expressed as the number of viable Brettanomyces genomes per gram of wood (see Figure 5).

[0079] Results obtained As shown in Figure 5, treatment with PAW-5 achieved a 3.49 log reduction over the control sample (DW treated with distilled water). In contrast, treatment with PAW-1.5 (PAW obtained after 1.5 minutes of plasma treatment of the initial water) achieved only a 1.46 log reduction, still superior to the sulfur smoke reference treatment. Treatments with PAW-15 and PAW-30 achieved complete reduction.

[0080] According to a preferred embodiment of the present invention, treatment with PAW-5 is considered optimal because it achieved a significant 3.49 log reduction against Brettanomyces. While not achieving complete reduction as with PAW-15 and PAW-30, it offers a significant advantage in that it consumes one-third less energy (one-third less processing time to produce PAW) than PAW-15. However, the choice of which PAW to use is within the scope of protection provided by the appended claims and depends on the preferences and technical constraints of the specific application to which the treatment methods disclosed herein are applied.

[0081] 3) Testing methods to reduce TCA (2,4,6-trichloroanisole) in cork Test materials 100% natural cork stoppers for 750ml wine bottles were used. These corks showed no TCA contamination. Some of the corks used were analyzed to rule out the presence of polyhaloanisoles and polyhalophenols. The results were negative in all cases.

[0082] Four uncontaminated corks were placed in 95-gram glass containers and immersed in 80 ml of a 400 ng / L TCA solution. These containers were placed on a rotary shaker at 50 rpm for 5 hours. After treatment, each cork was analyzed individually. The average TCA concentration of the four artificially contaminated corks was 26.59 ± 1.64 ng / L.

[0083] Application treatments to reduce TCA (2,4,6-trichloroanisole) Five treatments were considered per soaking: distilled water (DW, control), PAW-1.5, PAW-5, PAW-15, and PAW-30. The same PAW was used as that used to disinfect the wooden barrels. For each treatment, three corks artificially contaminated using the method described above were used.

[0084] The corks were each (individually) placed in a 95 gram glass container with 80 ml of each of the various solutions (DW and the four PAWs) and soaked for three hours without any movement, rotation, or shaking. It should be noted that the application method of the different solutions (DW and the four PAWs) was the same as that used to disinfect the wooden barrels, and the three-hour soaking treatment was carried out.

[0085] Tests conducted Analysis of polyhaloanisoles and polyhalophenols: All corks in this study (artificially contaminated corks treated with distilled water and PAW) were analyzed based on the following chemical analytical methods: OIV-MA-AS315-16 "Determination of the potential release of 2,4,6-trichloroanisole from cork stoppers in wine" (International Organization for Vine and Wine OIV / OENO Resolution 296 / 2009); OIV-MA-AS315-17 "Determination of the presence and content of polychlorinated phenols and polychlorinated anisoles in wine, cork stoppers, wood, and air-trapping bentonite" (OIV / OENO Resolution 374 / 2009). These methods are ** International Organisation of Vine and Wine (OIV) ** The test conforms to the "International Methods for the Analysis of Wine and Must" published by the International Wine and Must Association and simulates the migration of 2,4,6-trichloroanisole (TCA), 2,4,6-trichlorophenol, 2,3,4,6-tetrachloroanisole, 2,3,4,6-tetrachlorophenol, pentachloroanisole, and pentachlorophenol that may occur between the cork and the bottled wine. Test procedure: The cork was immersed in a water-alcohol solution and allowed to macerate until equilibrium was achieved. Polyhaloanisoles and polyhalophenols were extracted from the headspace of a portion of the macerate using solid-phase microextraction (SPME) techniques. The extract was ** Gas phase chromatography (GC) and mass spectrometry (MS) ** was analyzed using

[0086] Results obtained The decontamination results are shown in Figure 6. As can be seen, treatment with PAW-5 reduced the TCA content of untreated contaminated cork (control samples) by 75.2%. Treatment with distilled water (DW), on the other hand, was ineffective in both cases. This suggests that in the case of artificially TCA-contaminated cork, the TCA contamination is not superficial (unlike naturally contaminated cork). This is because distilled water did not have the effect of "stripping" the TCA from the surface.

[0087] Assuming that the distilled water and PAW reach the pores inside the cork, all of the reduced TCA is converted to hydroxyls (OH) in the PAW. * ) obtained by decomposition by radicals.

[0088] Treatment with PAW-1.5 (obtained after 1.5 min of processing) reduced TCA in untreated contaminated cork by 18.1%, while treatment with PAW-15 and PAW-30 achieved a similar reduction compared to PAW-5.

[0089] Based on these results, treatment with PAW-5 is considered optimal for reducing TCA in cork, according to a preferred embodiment of the present invention, as it achieved a significant reduction of over 75%. Furthermore, a significant advantage is that it consumes one-third the energy (one-third the processing time for PAW production) compared to PAW-15 (which exhibits a similar TCA reduction effect).

[0090] As can be seen from the above tests, the preferred treatment method is the same for both applications considered here (disinfection of Brettanomyces in wood and reduction of TCA in cork): a 3-hour static immersion treatment (contaminated wood and cork) using PAW-5. PAW (PAW-5) subjected to a 5-minute plasma activation treatment reduced OH *The PAW-1.5 (PAW-1.5) subjected to a 1.5 minute plasma activation treatment has the characteristic of having a concentration of OH at least 23 μg / L (23.1 μg / L as shown in Table 4). However, in other applications, it may be desirable to use PAW-1.5 (protected by the appended claims) depending on the technical constraints and specific requirements of the equipment in which the method of the present invention is applied. * It is characterized by a concentration of 8 μg / l or more (8.2 μg / l as shown in Table 4).

[0091] Similarly, the favorable physicochemical properties of plasma activated water (PAW) for disinfecting and / or decontaminating auxiliary forest materials used in wine production and preservation are considered to be: pH: less than 4.5 Electrical conductivity (EC): Over 50 μS / cm Oxidation-reduction potential (ORP): Over 350mV Nitrate ions (NO3 - ) Concentration: More than 3mg / l Nitrite ion (NO2 - ) Concentration: More than 0.5 mg / l OH * Concentration: greater than 8 μg / l (preferably greater than 23 μg / l) These parameters are already achieved with a plasma activation time of 1.5 minutes (PAW-1.5), although the most preferred plasma activation time is 5 minutes (PAW-5).

[0092] pH value, electrical conductivity (EC) value, oxidation-reduction potential (ORP) value, and nitrate ion (NO3 - ) and nitrite ion (NO2 - ) concentrations were measured using techniques well known in the art.

[0093] Although the present invention has been described based on preferred embodiments, it should be understood that these embodiments are illustrative and not limiting. Those skilled in the art can easily make modifications and variations to the embodiments described herein, without thereby departing from the scope of protection defined by the appended claims.

Claims

1. 1. A method of treating auxiliary forest materials for wine preservation with plasma activated water (PAW) to disinfect and / or decontaminate them, the method comprising contacting the materials to be disinfected and / or decontaminated with PAW.

2. 10. The method of claim 1, wherein the step of contacting the material to be disinfected and / or decontaminated with PAW is carried out for at least three continuous hours.

3. A method as described in claim 1, characterized in that the material to be disinfected and / or decontaminated by contact with PAW is a bottle cork or a wooden container.

4. A method as described in claim 3, characterized in that a reduction in anisole is obtained in bottle corks that have been decontaminated with PAW.

5. A method according to claim 4, characterized in that a reduction in TCA (2,4,6-trichloroanisole) is obtained.

6. A method according to claim 5, characterized in that a reduction in TCA of at least 50% is obtained.

7. A method according to claim 6, characterized in that a reduction in TCA of at least 75% is obtained.

8. A method as described in claim 1, characterized in that it includes a pretreatment step of washing the materials to be disinfected and / or decontaminated with pressurized water immediately before the continuous contact step with PAW.

9. 9. The method of claim 8, wherein the pressurized water used in the washing step is PAW.

10. The method of claim 3, wherein a reduction in Brettanomyces is obtained in a wooden container that is subjected to decontamination with PAW.

11. 11. The method of claim 10, wherein the method provides at least a 1 log reduction of Brettanomyces relative to the untreated wooden container.

12. 12. The method of claim 11, wherein the method provides at least a 3 log reduction of Brettanomyces on a pre-treated wooden container.

13. The method of claim 10, as applied to a wooden barrel.

14. 14. The method of claim 13, wherein the step of continuously contacting the wooden barrel with PAW is carried out by filling the entire volume of the barrel with PAW.

15. 14. The method of claim 13, wherein the step of continuously contacting the wooden barrel with PAW is carried out by introducing a minimum amount of PAW into the barrel and continuously rotating the barrel so that the PAW contacts the entire inner surface of the barrel.

16. A method as described in claim 1, characterized in that it includes a pretreatment step of generating PAW by applying plasma to initial water using air as the plasma gas at a plasma gas flow rate of 60 slm and a plasma power of 500 W.

17. A method as described in claim 1, comprising a pretreatment step of generating PAW by applying plasma to initial water, wherein the time for treating the initial water with plasma is between 1.5 minutes and 30 minutes.

18. 18. The method of claim 16 or 17, wherein the initial water treatment time with plasma is 5 minutes.

19. The method of claim 16 or 17, wherein the initial water is distilled water.

20. A method according to either claim 16 or 17, characterized in that the initial water is tap water.

21. A method as described in claim 1, characterized in that the PAW used has the properties of a pH of less than 4.5, an electrical conductivity (EC) of more than 50 μS / cm, and an oxidation-reduction potential (ORP) of more than 350 mV.

22. The method according to claim 1, wherein the PAW used is nitrate ions (NO ３ － ) Concentration exceeds 3 mg / l, nitrite ions ２ (NO ２ － ) a concentration of greater than 0.5 mg / l.

23. The method according to claim 1, wherein the PAW used is OH ＊ A method characterized by having a concentration of greater than 8 μg / l.

24. 24. The method of claim 23, wherein the PAW used is OH ＊ A method characterized by having a concentration greater than 23 μg / l.

25. Use of plasma activated water (PAW) to disinfect and / or decontaminate auxiliary forest materials used in wine production and preservation.

26. 26. Use of plasma activated water (PAW) according to claim 25, characterized in that the PAW used has the properties of pH less than 4.5, electrical conductivity (EC) greater than 50 μS / cm, and oxidation-reduction potential (ORP) greater than 350 mV.

27. ​​Use of plasma activated water (PAW) according to claim 25, wherein the PAW used contains nitrate ions (NO ３ － ) concentration exceeds 3 mg / l, and nitrite ions (NO ２ － ) Use characterized by having a concentration of more than 0.5 mg / l.

28. Use of plasma activated water (PAW) according to claim 25, wherein the PAW used is OH ＊ Use characterized by the property of having a concentration of more than 8 μg / l.

29. Use of plasma activated water (PAW) as described in claim 28, characterized in that the PAW used has properties exceeding 23 μg / l.

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