Use of at least one amphidinol in an alcoholic beverage, particularly as a substitute for sulfites

Abstract

The present invention relates to the use in an alcoholic beverage of a product comprising at least one amphidinol, a product intended for use in alcoholic beverages, a particular alcoholic beverage, and its manufacturing process. The present invention aims to provide a new means of controlling undesirable microorganisms in alcoholic beverages without impacting the organoleptic qualities of said beverages, in order to obtain an alcoholic beverage without added sulfites and without alteration of aromas. The abbreviation does not designate a figure.

Description

Title of the invention: Use of at least one amphidinol in an alcoholic beverage, in particular as a substitute for sulfites Technical field

[0001] The invention relates to the technical field of alcoholic beverages. In particular, the subject of the invention is the use in an alcoholic beverage of a product comprising at least one amphidinol, a product intended to be used in alcoholic beverages comprising amphidinols, a particular alcoholic beverage and its manufacturing process. State of the art

[0002] Sulfites are chemical compounds containing sulfur and oxygen. Due to their ability to inhibit the growth of unwanted microorganisms and their antioxidant properties, they are commonly used in the food industry.

[0003] In alcoholic beverages, sulfites can form naturally during fermentation or can be added during the manufacturing process.

[0004] Natural sulfites are found in varying amounts in alcoholic beverages, and their content depends on various parameters such as the type of beverage, fermentation time and storage conditions.

[0005] In contrast, added sulfites are additional sulfites added to the alcoholic beverage during or after fermentation to increase stability and shelf life. Added sulfites may be in the form of sulfur dioxide or sulfites, including potassium sulfite.

[0006] For example, added sulfites, such as sulfur dioxide (SO2), can be used to control yeasts and other undesirable microorganisms in alcoholic beverages.

[0007] In particular, the use of SO2 is currently the most widely used means of control to prevent the development of Brettanomyces bruxellensis, which is considered a particularly undesirable microorganism in alcoholic beverages and in particular wine. Indeed, the uncontrolled presence of the Brettanomyces species in the manufacturing processes of alcoholic beverages is problematic insofar as this yeast is responsible for the irreversible degradation of the organoleptic qualities of said beverages, such as wine or beer. The Brettanomyces bruxellensis species in particular is usually associated with odors of gouache, cloves, leather, horse sweat and stable due to the production of volatile phenols, essentially 4-ethylphenol and 4-ethylguaiacol.

[0008] The use of sulfites, such as SO2, therefore helps to slow down the development of Brettanomyces bruxellensis in alcoholic beverages while also controlling other microorganisms such as Saccharomyces cerevisiae and / or bacteria and exhibiting an antioxidant effect.

[0009] However, chemicals such as added sulfites are difficult to degrade: they are found in processed alcoholic beverages and are ingested by consumers. However, sulfites have a negative impact on human health, due in particular to their allergenic power.

[0010] In addition, the use of added sulfites is becoming increasingly controversial due to the emergence of resistance. Recent studies have indeed demonstrated that the Brettanomyces species exhibits great genetic diversity allowing a number of strains of the Brettanomyces species to resist and survive sulfites.

[0011] Thus, the use of sulfites to control undesirable microorganisms such as the Brettanomyces species in alcoholic beverages shows its limits and more natural alternative solutions are therefore necessary.

[0012] To replace sulfites, it is possible to use physical treatments as a means of combating undesirable microorganisms in alcoholic beverages, but these treatments are used curatively when contamination is detected and cannot be used preventively. In addition, the implementation of a physical treatment, such as microfiltration, within the manufacturing processes of alcoholic beverages can be expensive and can affect the organoleptic qualities of said beverages. However, in the field of alcoholic beverages, it is particularly important not to affect the aromas and texture of the beverage.

[0013] Alternatives are also appearing with the use of natural molecules to control unwanted bacteria and / or yeasts.

[0014] Chitosan is a natural yeast-killing molecule that has a biocidal action against the yeast Brettanomyces bruxellensis but, however, has no action against S ac-charomyces cerevisiae. In addition, certain strains of Brettanomyces bruxellensis are insensitive to the action of chitosan. This yeast-killing molecule is therefore not suitable for the treatment of alcoholic beverages, particularly as a substitute for sulfites.

[0015] There is therefore a need to have new means of combating undesirable microorganisms in alcoholic beverages, adapted to the conditions of alcoholic beverages, having no impact on their organoleptic qualities, in order to obtain an alcoholic beverage without added sulfites, without alteration of the aromas. Summary of the invention

[0016] While searching for substitutes for sulfites, the inventors discovered that adding at least one amphidinol to an alcoholic beverage made it possible to prevent and / or fight against unwanted microorganisms present in the said drink, while presenting an anti-oxidant effect and preserving its organoleptic qualities.

[0017] Thus, the subject of the invention is the use in an alcoholic beverage of a product comprising at least one amphidinol.

[0018] Amphidinols are organic compounds produced by certain species of di-noflagellates and marine microorganisms.

[0019] As an example, the structures of amphidinol 18 and amphidinol 19 are given below:

[0020] [Chem.l]

[0021] Amphidinols are polyhydroxy-polyenes (long-chain polyketides) known for various biological activities, including anticancer, antifungal and antiviral properties.

[0022] Surprisingly, the inventors have demonstrated: - the effect of amphidinols on controlling the growth of undesirable microorganisms in alcoholic beverages, in particular on yeasts belonging to the genus brettanomyces and Saccharomyces and / or on bacteria of the genus Acetobacter, oenococcus and Pediococcus, - the resistance of amphidinols to the conditions of alcoholic beverages, and its slow degradation during the manufacturing process of the beverage, until it disappears completely in the beverage intended for consumption, - the antioxidant effect of amphidinols in alcoholic beverages, - the preservation of the organoleptic properties of the alcoholic beverage.

[0023] According to a particularly suitable embodiment, the product comprising at least one amphidinol used in an alcoholic beverage comprises at least one amphidinol chosen from amphidinol 1 to 19, amphidinol 22, amphidinol A and mixtures thereof.

[0024] In other words, according to a particularly suitable embodiment, the product comprising at least one amphidinol used in an alcoholic beverage comprises at least one amphidinol chosen from amphidinol 1, amphidinol 2, amphidinol 3, amphidinol 4, amphidinol 5, amphidinol 6, amphidinol 7, amphidinol 8, amphidinol 9, amphidinol 10, amphidinol 11, amphidinol 12, amphidinol 13, amphidinol 14, amphidinol 15, amphidinol 16, amphidinol 17, amphidinol 18, amphidinol 19, amphidinol 22, amphidinol A and mixtures thereof.

[0025] Preferably, the product comprising at least one amphidinol used in an alcoholic beverage comprises amphidinol 18.

[0026] Preferably, the product comprising at least one amphidinol is used in an alcoholic beverage as a bactericide, yeasticide and antioxidant.

[0027] According to another suitable embodiment, the product comprising at least one amphidinol is a cellular extract of one or more micro-algae of the genus Am-phidinium.

[0028] Advantageously, according to this embodiment, the product comprising at least one amphidinol used in an alcoholic beverage is of natural origin.

[0029] The invention also relates to a product intended to be used in alcoholic beverages, comprising at least one amphidinol. According to one embodiment, the product according to the invention comprises amphidinol 18, amphidinol 19 and amphidinol 22.

[0030] Such a product is particularly useful in the context of the invention, thus providing an alternative to the use of sulfites in alcoholic beverages.

[0031] According to another object, the invention relates to an alcoholic beverage comprising at least one amphidinol.

[0032] Finally, according to a last object, the invention relates to a method for manufacturing an alcoholic beverage comprising a step of adding at least one amphidinol to the beverage.

[0033] Other characteristics and advantages will emerge from the detailed description of the invention and from the illustrative but non-limiting examples of the scope of the invention. Brief description of the figures

[0034] [Fig. 1 A] [Fig. 1 A] is a photograph taken 3 days after incubation of three strains of Brettanomyces bruxellensis.

[0035] [Fig. IB] [Fig. IB] is a graphic representation of the experimental protocol of example 2 with the objective of highlighting the effect of the product according to the invention on three strains of Brettanomyces bruxellensis.

[0036] [Fig. IC] [Fig. IC] is a graphical representation of the measurement of optical density over time after incubation of three strains of Brettanomyces bruxellensis in the presence of YPD medium alone (negative controls), ethanol or in the presence of a concentration of 0.06 g / L to 2 g / L of product according to the invention. Two replicates (repi and rep2) were carried out for each condition.

[0037] [Fig.2] [Fig.2] is a photograph of an observation made using a mi optical microscope at X600 magnification of the viability of Brettanomyces bruxellensis previously incubated with ethanol, the product according to the invention or a high temperature.

[0038] [Fig.3A] [Fig.3A] is a graphical representation illustrating the degradability of amphidinol 18 in light and at room temperature for 120 days.

[0039] [Fig.3B] [Fig.3B] is a graphical representation of the monitoring of the growth of Brettanomyces bruxellensis by measuring the optical density at 550 nm at 0, 1, 2, 3 and 7 days (T0, T1, T2, T3 and T7) after incubation of the yeasts at 28°C in the presence of YPD medium alone (negative control), ethanol or in the presence of a concentration of 1 g / L of product according to the invention or of inactivated product according to the invention. Two replicates (repi and rep2) were carried out.

[0040] [Fig.4] [Fig.4] is a graphical representation of the measurement of the minimum inhibition concentration (MIC) of the product according to the invention for different concentrations of Brettanomyces bruxellensis by monitoring the optical density.

[0041] [Fig.5A] [Fig.5A] is a graphical representation illustrating the chromatographic data of a product according to the invention after injection into an HPLC column.

[0042] [Fig.5B] [Fig.5B] is a graphical representation of a tandem mass spectrometry (MS / MS) analysis of peak 0 in [Fig.5A].

[0043] [Fig.5C] [Fig.5C] is a comparative photograph of the purified fraction of peak 0 of [Fig.5A] and of a product according to the invention.

[0044] [Fig.5D] [Fig.5D] is a graphical representation of the growth monitoring of Brettanomyces bruxellensis by measuring the optical density at L550 nm at 0, 3 and 6 days (T0, T3 and T6) after incubation of the yeasts at 28°C in the presence of YPD medium alone (-), methanol or in the presence of peak 0 at a dilution of 1 / 32 to 1 / 256 corresponding to a concentration of 40 to 5 ppm of amphidinol 18 (AM18). As a positive control, the substance ICC001 at 0.25 g / L was tested. Two replicates (repi and rep2) were carried out.

[0045] [Fig.6] [Fig.6] is a graphical representation of the measurement of the minimum inhibition concentration (MIC) of the purified fraction of picO on Saccharomyces ce-revisiae.

[0046] [Fig.7] [Fig.7] is a graphical representation of the measurement by optical density monitoring of the minimum inhibition concentration (MIC) of the purified fraction of picO on Acetobacter aceti.

[0047] [Fig.8A] [Fig.8A] is a graphical representation of the experimental design used during example 9.

[0048] [Fig.8B] [Fig.8B] is a graphical representation of the logarithmic measurement of the number of colony forming units (log cfu / mL) of Brettanomyces bruxellensis NL6293 present in wine at 1, 2, 4 and 11 days (Tl, T2, T4 and Tl 1) after inoculation wine with Brettanomyces bruxellensis NL6293 for different conditions.

[0049] [Fig.8C] [Fig.8C] is a graphical representation of the logarithmic measurement of the number of colony forming units (log cfu / mL) of Brettanomyces bruxellensis NL6300 present in wine at 1, 2, 4 and 11 days (Tl, T2, T4 and Tl 1) after inoculation of the wine with Brettanomyces bruxellensis NL6293 for the different conditions.

[0050] [Fig.8D] [Fig.8D] is a graphical representation of the logarithmic measurement of the number of colony forming units (log cfu / mL) of Saccharomyces cerevisiae present in wine at 1, 2, 4 and 11 days (Tl, T2, T4 and Tl 1) after inoculation of the wine with Brettanomyces bruxellensis NL6293 for the different conditions.

[0051] [Fig.9A] [Fig.9A] is a graphical representation of the measurement of the degradability of amphidinol 18 in wine. For each modality, the amphidinol 18 content was determined in points per million (ppm) by HPLC at times T6, T16 and T44 (6 days, 16 days and 44 days after addition of a product according to the invention).

[0052] [Fig.9B] [Fig.9B] is a graphical representation of the measurement of the degradability of amphidinol 18 in wine. For each modality, the AM 18 content is expressed as a percentage, considering that at time T0, 100% of AM18 is present in the samples.

[0053] [Fig. 10] [Fig. 10] is a graphic representation of an experimental scheme allowing the production of a product according to the invention in the form of a cell extract of Amphidinium carterae.

[0054] [Fig. 11] [Fig. 11] is a graphical representation of a comparative study of chitosan and a product according to the invention comprising amphidinol 18 through their bactericidal and yeasticidal activities. Detailed description of the invention

[0055] Definitions

[0056] For the purposes of the invention, the term “alcoholic beverage” means a beverage comprising at least 1% ethyl alcohol.

[0057] For the purposes of the invention, the term "cell extract" means an extract obtained by lysing cells so as to release intracellular components such as proteins, carbohydrates and metabolites. Preferably, the metabolites present in the cell extract are amphidinols.

[0058] For the purposes of the invention, the term “undesirable microorganisms” means a bacterium, a yeast or a fungus which, with respect to one or more alcoholic beverages, has more negative effects than positive effects. In certain cases, the same microorganism may, within the same beverage, be considered an “undesirable microorganism” at certain concentrations and a “non-undesirable microorganism” at other concentrations or at other stages of the preparation of the beverage. alcoholic beverage.

[0059] For the purposes of the invention, the term “organoleptic qualities” means the sensory properties of the alcoholic beverage which are perceived by the sense organs, namely the appearance, the smell, the taste, and the texture.

[0060] Uses in an alcoholic beverage of a product comprising at least one amphidinol

[0061] The subject of the invention is the use of a product comprising at least one amphidinol in at least one alcoholic beverage.

[0062] Advantageously, the product comprising at least one amphidinol makes it possible to combat undesirable microorganisms without altering the organoleptic qualities of the alcoholic beverage. The product also has an antioxidant effect in the alcoholic beverage.

[0063] The product comprising at least one amphidinol can therefore be used according to the invention in an alcoholic beverage specifically as a substitute for sulfites.

[0064] By product comprising at least one amphidinol is meant any product in any form comprising one or more amphidinol molecule(s) and optionally one or more other elements such as in particular excipients or another active molecule. According to one embodiment, the product comprising at least one amphidinol consists solely of one or more amphidinol molecules.

[0065] The invention therefore also relates to the use of at least one amphidinol for combating at least one undesirable microorganism present in an alcoholic beverage. In particular, an object of the invention is the use of at least one amphidinol in an alcoholic beverage for controlling and / or limiting the growth of at least one undesirable microorganism.

[0066] Preferably, the product comprising at least one amphidinol used in an alcoholic beverage comprises at least one amphidinol chosen from amphidinol 1 to 19, amphidinol 22, amphidinol A and mixtures thereof.

[0067] Preferably, the product comprising at least one amphidinol used in an alcoholic beverage comprises at least one amphidinol chosen from amphidinol 3, amphidinol 18, amphidinol 19, amphidinol 22, amphidinol A and mixtures thereof.

[0068] Preferably, the invention relates to the use of at least one amphidinol chosen from amphidinol 1 to 19, amphidinol 22 and their combinations, to combat at least one undesirable microorganism present in an alcoholic beverage. According to a particularly suitable embodiment, the invention relates to the use of amphidinol 18 to combat at least one undesirable microorganism present in an alcoholic beverage.

[0069] According to one embodiment, the invention relates to the use of a product comprising at least amphidinol 18 in an alcoholic beverage.

[0070] Preferably, the product comprising at least one amphidinol used in an alcoholic beverage comprises at least amphidinol 18 and amphidinol 3.

[0071] According to one embodiment, the product comprising at least one amphidinol used in an alcoholic beverage comprises at least amphidinol 18 and amphidinol 22.

[0072] According to a particular embodiment of the invention, the product comprising at least one amphidinol used in an alcoholic beverage comprises amphidinol 18, amphidinol 19 and amphidinol 22.

[0073] Preferably, the product comprising at least one amphidinol used in an alcoholic beverage comprises at least amphidinol 18 and at least one amphidinol chosen from amphidinol 1 to 17, amphidinol 19 to 22, amphidinol A and mixtures thereof.

[0074] The product comprising at least one amphidinol used in an alcoholic beverage may specifically be a product according to the invention as described in the present application.

[0075] Preferably, the product comprising at least one amphidinol used according to the invention in an alcoholic beverage is of natural origin. Preferably, the product comprising at least one amphidinol used in an alcoholic beverage is obtained from a cellular extract of one or more micro-algae of the genus Amphidinium.

[0076] Preferably, the product comprising at least one amphidinol used in an alcoholic beverage is a cellular extract of one or more micro-algae of the genus Amphidinium.

[0077] In one embodiment, said micro-alga of the genus Amphidinium is chosen from the following species: Amphidinium carterae or Amphidinium klebsii.

[0078] In a particularly preferred embodiment, the product comprising at least one amphidinol used in an alcoholic beverage is or comprises a cell extract of &Amphidinium carterae.

[0079] In one embodiment, the product comprising at least one amphidinol used in an alcoholic beverage is or comprises a cell extract of a strain of Amphidinium carterae selected from the group consisting of CCMP 124, CCMP 1314, CCMP 3177, AC 208, AC 792, and BEA 01198. In the context of the invention, CCMP means "Culture Collection of Marine Phytoplankton", AC means "Algobank Cean" and BEA means "Banco Espanol de Algas".

[0080] Preferably, the product comprising at least one amphidinol used in an alcoholic beverage is or comprises a cell extract of a strain of Amphidinium carterae chosen from the group consisting of CCMP 1314, AC 208 and AC 792.

[0081] When the product comprising at least one amphidinol used in an alcoholic beverage is or comprises a cellular extract of one or more microalgae of the genus Amphidinium said extract can be prepared by any method of cellular extraction known to those skilled in the art, solid-liquid, liquid-liquid or gas-liquid, for example an extraction in inorganic or organic solvent, which can be chosen from the group consisting of water, aqueous solutions, ketones, esters, acids, ethers, alcohols and mixtures in all miscible proportions of these solvents.

[0082] According to a variant, when the product comprising at least one amphidinol used in an alcoholic beverage is or comprises a cellular extract of one or more micro-algae of the genus Amphidinium, said extract can be prepared by supercritical CO2 / H2O extraction.

[0083] According to one embodiment, the product comprising at least one amphidinol used in an alcoholic beverage is or comprises a cellular extract of one or more microalgae of the genus Amphidinium, said extract being obtained by extraction in an alcohol, preferably in an alcohol comprising between 1 and 4 carbon atoms, even more preferably methanol or ethanol.

[0084] Preferably, when the product comprising at least one amphidinol used in an alcoholic beverage is or comprises a cellular extract of one or more microalgae of the genus Amphidinium, said extract is a water-soluble fraction.

[0085] In one embodiment, the invention relates to the use of a product comprising at least one amphidinol, preferably at least amphidinol 18, in an alcoholic beverage, at a concentration greater than 1% by weight relative to the total weight of the product, preferably between 1% and 100% by weight relative to the total weight of the product, in particular between 50% and 90% by weight relative to the total weight of the product, even more preferably between 75% and 90% by weight relative to the total weight of the product.

[0086] In one embodiment, the invention relates to the use of a product comprising at least one amphidinol, preferably at least amphidinol 18, in an alcoholic beverage, at a concentration of between 1 and 50 g / l, preferably between 5 and 40 g / L, preferably between 20 and 50 g / L, preferably 33 g / L.

[0087] In one embodiment, the invention relates to the use of a product comprising at least one amphidinol, preferably at least amphidinol 18, in an alcoholic beverage, at a concentration of between 0.06 g / L and 10 g / L relative to the total volume of said beverage, preferably between 0.06 and 5 g / L, between 0.06 and 2 g / L, between 0.06 and 1 g / L, between 0.06 and 0.5 g / L, between 0.06 and 0.25 g / L relative to the total volume of said beverage. According to one embodiment, the product comprising at least one amphidinol used in an alcoholic beverage is present in said beverage at a concentration of approximately 0.125 g / L, relative to the total volume of said beverage.

[0088] Preferably, the product comprising at least one amphidinol used in an alcoholic beverage according to the invention does not comprise at least one of the molecules chosen from lingshuiols, karatungiols, luteophanols, and colopsinols.

[0089] According to a suitable variant, the product comprising at least one amphidinol used in an alcoholic beverage according to the invention does not comprise at least one molecule chosen from carteraol E, amphezonol A and their mixture.

[0090] Preferably, the product comprising at least one amphidinol used in an alcoholic beverage according to the invention does not comprise carteraol.

[0091] The use of the product comprising at least one amphidinol can be carried out in any type of alcoholic beverage, preferably beverages obtained by alcoholic fermentation.

[0092] According to a preferred embodiment, the alcoholic beverage is chosen from wine, cider, beer.

[0093] Advantageously, the use of a product comprising at least one amphidinol in such an alcoholic beverage allows: - to control and / or limit the growth of unwanted microorganisms - to have an anti-oxidant effect and to fight against free radicals - to preserve the organoleptic qualities of the drink.

[0094] Preferably, the product comprising at least one amphidinol is used according to the invention in an alcoholic beverage having a pH of less than 7, preferably less than 4.

[0095] Preferably, the product comprising at least one amphidinol is used according to the invention in an alcoholic beverage containing at least 1% ethyl alcohol, in particular at least 4%, even more preferably at least 12%.

[0096] According to a variant of the invention, the product comprising at least one amphidinol can be used in a non-alcoholic drink.

[0097] Preferably, the product comprising at least one amphidinol is used in an alcoholic beverage such as: - yeasticide, in particular for controlling and / or limiting the growth of yeasts belonging to at least one genus Brettanomyces, Hansaenula, Saccharomyces, Hanse-niaspora, Pichia, Candida, Kloeckera, Torulopsis, Schizosaccharomyces and combinations thereof, even more preferably for controlling and / or limiting the growth of yeasts belonging to the genus Brettanomyces, in particular for controlling and / or limiting the growth of yeasts belonging to the species Brettanomyces bruxellensis, and / or - bactericidal, even more preferably to control and / or limit the growth of bacteria belonging to the genus chosen from Gluconobacter, Acetobacter, Oe-nococcus, Lactobacillus, Pediococcus, Pectinatus, Megasphaera, Citrobacter, Ente-robacter, Obesumbacterium, Klebsiella, Proteus, Serratia, Zymomonas in particular to control and / or limit the growth of bacteria belonging to the species Ace-tobacter aceti, and / or - antioxidant.

[0098] Preferably, the product comprising at least one amphidinol is used in an alcoholic beverage, preferably in wine, as a bactericide, yeasticide and antioxidant.

[0099] According to one embodiment, the invention relates to the use of at least one amphidinol to combat at least one undesirable microorganism in an alcoholic beverage, preferably chosen from alcoholic beverages obtained by alcoholic fermentation, in particular wine, cider, and beer.

[0100] According to one embodiment, the product comprising at least one amphidinol is used in an alcoholic beverage as a yeasticide for controlling and / or limiting the growth of at least one yeast of a genus chosen from Brettanomyces, Hansaenula, Saccharomyces, Hanseniaspora, Pichia, Candida, Kloeckera, Torulopsis, Schizosac-charomyces and combinations thereof. Advantageously, the product comprising at least one amphidinol used in an alcoholic beverage makes it possible to prevent a yeast of the aforementioned genus from becoming the majority in an alcoholic beverage, and consequently, prevents the production of undesirable metabolites, the formation of which in large numbers can induce undesirable tastes in an alcoholic beverage.Preferably, the product comprising at least one amphidinol is used in an alcoholic beverage as a yeasticide for controlling and / or limiting the growth of at least one yeast of a species selected from Kloeckera apiculata, Brettanomyces bruxellensis, Saccharomyces cerevisiae, Pichia membranefaciens, Hansenula anomala and combinations thereof.

[0101] According to another embodiment, the product comprising at least one amphidinol is used in an alcoholic beverage as a bactericide to control and / or limit the growth of at least one bacterium of a genus chosen from Gluconobacter, Acetobacter, Oenococcus, Lactobacillus, Pediococcus, Pectinatus, Megasphaera, Citrobacter, Ente-robacter, Obesumbacterium, Klebsiella, Proteus, Serratia, Zymomonas. Advantageously, the product comprising at least one amphidinol used in an alcoholic beverage makes it possible to prevent a bacterium of the aforementioned genus from becoming the majority in an alcoholic beverage, and consequently, prevents the production of undesirable metabolites, the formation of which in large numbers can induce undesirable tastes in an alcoholic beverage.Preferably, the product comprising at least one amphidinol is used in an alcoholic beverage as a bactericide to control and / or limit the growth of at least one species of bacteria chosen from Oenococcus oenii, Lactobacillus brevis, Lactobacillus plantarum, Pediococcus damnosus, Pediococcus in-opinatus, Pediococcus pentosaceus, Enterobacter cloaceae, Enterobacter ag- . glomerans, Citrobacter freundii, Obesumbacterium proteus, Zymomonas mobilis and their combinations.

[0102] According to one embodiment, the product comprising at least one amphidinol is used in wine at least as a yeasticide.

[0103] Preferably, the product comprising at least one amphidinol is used in wine to control and / or limit the growth of at least one yeast of a genus chosen from Brettanomyces, Hansaenula, Saccharomyces, Hanseniaspora, Pichia, Candida, Kloeckera and combinations thereof.

[0104] Preferably, the product comprising at least one amphidinol is used in wine to control and / or limit the growth of at least one yeast of species chosen from Kloeckera apiculata, Brettanomyces bruxellensis, Saccharomyces cerevisiae and combinations thereof.

[0105] In a particularly preferred manner, the product comprising at least one amphidinol is used in wine to control and / or limit the growth of Brettanomyces bruxellensis.

[0106] In a particular embodiment of the invention, the product comprising at least one amphidinol is used in wine to control and / or limit the growth of at least one undesirable yeast of the species Brettanomyces bruxellensis and of strain(s) NL6295, NL6300 and / or NL6293 (these strains being strains having been isolated from contaminated wine).

[0107] Thus, according to a variant, the invention relates to the use of at least one amphidinol in a wine to combat at least one undesirable yeast chosen from yeasts of the genus Brettanomyces (in particular Brettanomyces bruxellensis) Candida (in particular Candida stellata, Candida pulcherima or Candida vini), Hansenula (in particular Hansaenula anomala), Hanseniaspora, Kloeckera (in particular Kloeckera apiculata), Pichia (in particular Pichia membranefaciens), Zygosac-charomyces and Saccharomyces (within this latter genus, only certain wild strains of Saccharomyces cerevisiae: var. diastaticus, pastorianus, el-lipsoideus and willianus, can be considered as being undesirable yeasts). Preferably, the invention relates to the use of at least one amphidinol in a wine to combat a yeast Brettanomyces bruxellensis, preferably against a Brettanomyces bruxellensis yeast of strain NL6295, NL6300 or NL6293.

[0108] In one embodiment, the invention relates to the use of at least one amphidinol in a wine to combat at least one bacterium chosen from undesirable acetic bacteria and undesirable lactic bacteria.

[0109] According to one embodiment, the invention also aims at the use of a product comprising at least one amphidinol in wine as a bactericide.

[0110] Preferably, the product comprising at least one amphidinol is used according to the invention in wine to control and / or limit the growth of at least one bacterium chosen from bacteria of the genus Gluconobacter, Acetobacter, Oenococcus, Lactobacillus, Pediococcus and combinations thereof.

[0111] Preferably, the product comprising at least one amphidinol is used in wine to control and / or limit the growth of Oenococcus oenii.

[0112] In one embodiment, the alcoholic beverage is a wine, and the at least one undesirable microorganism is at least: - an acetic acid bacterium chosen from bacteria of the genus Acetobacter (notably Acetobacter aceti) and Gluconobacter and / or - a lactic acid bacterium chosen from bacteria of the genus Oenococcus (notably Oenococcus oenii) Lactobacillus and Pediococcus.

[0113] According to one embodiment, the product comprising at least one amphidinol is used in beer as a yeasticide.

[0114] Preferably, the product comprising at least one amphidinol is used in beer to control and / or limit the growth of at least one genus of yeast chosen from Torulopsis, Schizosaccharomyces, Brettanomyces, Hansaenula, Rhodotorula, Saccharomyces, Pichia, Candida, Kloeckera and combinations thereof.

[0115] Preferably, the product comprising at least one amphidinol is used in beer to control and / or limit the growth of at least one yeast species chosen from Pichia membranefaciens, Hansenula anomala, Saccharomyces cerevisiae and combinations thereof.

[0116] Particularly preferably, the product comprising at least one amphidinol is used in beer to control and / or limit the growth of Brettanomyces bruxellensis.

[0117] In one embodiment, the invention relates to the use of at least one amphidinol in a beer to combat at least one undesirable yeast chosen from yeasts of the genus Brettanomyces, Candida (in particular Candida Rhodotorula), Hansenula (in particular Hansenula anomala), Kloeckera, Pichia (in particular Pichia membranefaciens) and Saccharomyces (within the latter genus, only certain wild strains of Saccharomyces cerevisiae: var. diastaticus, pastorianus, ellipsoideus and willianus, can be considered as being undesirable yeasts).

[0118] According to one embodiment, the product comprising at least one amphidinol is used in beer as a bactericide.

[0119] Preferably, the product comprising at least one amphidinol is used in beer to control and / or limit the growth of at least one genus of bacteria chosen from Gluconobacter, Pectinatus, Megasphaera, Acetobacter, Lactobacillus, Pediococcus, Citrobacter, Enterobacter, Obesumbacterium, Klebsiella, Proteus, Serratia, Zymomonas and their combinations.

[0120] Preferably, the product comprising at least one amphidinol is used in beer to control and / or limit the growth of at least one species of bacteria chosen from Lactobacillus brevis, Lactobacillus plantarum, Pediococcus damnosus, Pediococcus inopinatus, Pediococcus pentosaceus, Enterobacter cloaceae, Enterobacter agglomérons, Citrobacter freundii, Obesumbacterium proteus, Zymomonas mobilis.

[0121] In one embodiment, the invention relates to the use of at least one amphidinol in a beer to combat at least one undesirable bacterium chosen from bacteria of the genus Acetobacter, Citrobacter (in particular Citrobacter freundii), Enterobacter (in particular Enterobacter cloaceae or Enterobacter agglomerons), Gluco-nobacter, Lactobacillus (in particular Lactobacillus brevis or Lactobacillus plantarum), Megasphaera, Obesumbacterium (in particular Obesumbacterium proteus), Pectinatus, Pediococcus (in particular Pediococcus inopinatus or Pediococcus pentosaceus), Zymomonas (in particular Zymomonas mobilis), Klebsiella, Proteus, Serratia, Hafnia, Selenomonas (in particular Selenomonas lacticifex) and Zymophilus.

[0122] According to one embodiment, the product comprising at least one amphidinol used in an alcoholic beverage is in powder form or in liquid form.

[0123] Unlike the solutions proposed by the prior art, and other bactericides and / or yeasticides, the use of a product comprising at least one amphidinol is suitable for beverages intended to be consumed and can be carried out in an alcoholic beverage having a pH of less than 7, preferably less than 4, without being degraded rapidly.

[0124] Advantageously, the product comprising at least one amphidinol used in an alcoholic beverage retains its properties at acidic pH, in particular its capacity to control and / or limit the growth of yeasts belonging to the genus Brettanomyces.

[0125] Preferably, the product comprising at least one amphidinol used in an alcoholic beverage, in particular in wine, is degraded by at least 40% by weight of the total weight of the product in 144 hours within said beverage.

[0126] Advantageously, amphidinols, more particularly amphidinols 18, 19 and 22, degrade naturally over time within the alcoholic beverage. Thus, unlike sulfites, it is possible to introduce a high concentration of amphidinol into the alcoholic beverage to combat undesirable microorganisms without it being ingested by the consumer.

[0127] A person skilled in the art is capable of calculating an initial concentration necessary to combat undesirable microorganisms while measuring the degradation of amphidinols over time to limit their ingestion by the consumer.

[0128] In wine, with a concentration of 60 to 480 ppm, it is degraded 90% in 44 days.

[0129] Advantageously, the product comprising at least one amphidinol used in a alcoholic beverage mixes to form a homogeneous alcoholic beverage.

[0130] Product for use in alcoholic beverages, comprising at least one amphidinol

[0131] The invention also relates to a solid product intended to be used in alcoholic beverages, comprising at least one amphidinol.

[0132] Such a product is particularly useful in the context of the invention, thus making it possible to combat undesirable microorganisms such as yeasts and / or bacteria present in alcoholic beverages while having no impact on the organoleptic characteristics of said beverages.

[0133] According to a preferred embodiment, the product according to the invention comprises at least one amphidinol chosen from amphidinol 1 to 19, amphidinol 22, amphidinol A and their mixtures.

[0134] Preferably, the product according to the invention comprises at least one amphidinol chosen from amphidinol 18, amphidinol 19, amphidinol 22 and their mixtures.

[0135] Preferably, the product according to the invention comprises at least two amphidinols chosen from amphidinol 1 to 19, amphidinol 22, amphidinol A and their mixtures.

[0136] Preferably, the product according to the invention comprises at least two amphidinols chosen from amphidinol 18, amphidinol 19, amphidinol 22 and their mixtures.

[0137] According to one embodiment, the product according to the invention comprises amphidinol 18 and at least one amphidinol chosen from amphidinol 1 to 17, amphidinol 19 to 22, amphidinol A and their mixtures.

[0138] Preferably, the product according to the invention comprises at least one amphidinol chosen from amphidinol 18 and / or amphidinol 22.

[0139] Preferably, the product according to the invention comprises at least one amphidinol chosen from amphidinol 18 and / or amphidinol 3.

[0140] According to another preferred embodiment, the product according to the invention comprises at least amphidinol 18, amphidinol 19 and amphidinol 22.

[0141] Advantageously, the yeasticidal and / or bactericidal and / or antioxidant activity, more particularly the control of the growth of yeasts belonging to the genus bret-tanomyces, is solely due to the amphidinols present in the product according to the invention.

[0142] According to a particular embodiment, the product according to the invention comprises at least 50% of amphidinol 18 and at least one amphidinol chosen from amphidinol 1 to 17, amphidinol 19 to 22, amphidinol A and their mixtures.

[0143] Preferably, the product according to the invention comprises by weight of the total weight of the product: - between 40 and 60% of amphidinol 18, - between 35 and 45% of amphidinol 19, and / or - between 35 and 45% of amphidinol 22.

[0144] The product according to the invention is particularly suitable for use in alcoholic beverages.

[0145] According to one embodiment, the product according to the invention is in the form of a powder or in liquid form.

[0146] Preferably, the product according to the invention is obtained from a cellular extract of one or more micro-algae of the genus Amphidinium.

[0147] The product according to the invention is preferably a cellular extract of one or more micro-algae of the genus Amphidinium.

[0148] Said microalgae of the genus Amphidinium may in particular belong to the species Amphidinium carterae.

[0149] When the product according to the invention is a cellular extract of one or more micro-algae of the genus Amphidinium, said extract can be prepared by any cellular extraction method known to those skilled in the art, solid-liquid or liquid-liquid, for example an extraction in inorganic or organic solvent, which can be chosen from the group consisting of water, aqueous solutions, ketones, esters, acids, ethers, alcohols and mixtures in all miscible proportions of these solvents.

[0150] According to one embodiment, the product comprising at least one amphidinol is a cellular extract of one or more micro-algae of the genus Amphidinium, said extract being obtained by extraction in an alcohol, preferably in an alcohol comprising between 1 and 4 carbon atoms, even more preferably methanol or ethanol.

[0151] Preferably, when the product comprising at least one amphidinol is a cellular extract of one or more microalgae of the genus Amphidinium, said extract is a water-soluble fraction.

[0152] When the product according to the invention is a cellular extract of one or more microalgae of the genus Amphidinium, its preparation process is characterized by the following steps: a) harvesting fresh cells of one or more microalgae of the genus Amphidinium; a') optionally freezing and / or freeze-drying of said cells; b) suspending said fresh or frozen cells or said lyophilisate in an inorganic or organic solvent in a lyophilisate or biomass / solvent weight ratio of between 1 / 200 and 1 / 2; b') possibly freeze-drying of the extract obtained.

[0153] Preferably, the inorganic solvent may be chosen from water or aqueous solutions.

[0154] Preferably, the organic solvent of step b) can be chosen from: - hydrocarbon solvents such as aliphatics or aromatics; - oxygenated solvents such as alcohols, ketones, acids, esters and ethers; and - mixtures in all miscible proportions of these solvents.

[0155] According to a preferred embodiment, the organic solvent of step b) is an alcohol having from 1 to 4 carbon atoms, such as methanol or ethanol.

[0156] When the solvent of step b) is an organic solvent, the suspension can be carried out at a temperature between 4 and 60°C, preferably between 18 and 60°C, particularly preferably at room temperature.

[0157] Preferably, the suspension of step b) of said fresh or frozen cells or said lyophilisate in an inorganic solvent is carried out at room temperature. Preferably, the suspension of step b) of said fresh or frozen cells or said lyophilisate in an inorganic solvent lasts less than 5 minutes, preferably less than 3 minutes, preferably less than 1 minute.

[0158] The suspension of step b) is carried out either by adding to said fresh or frozen cells or to said lyophilisate the solvent previously brought to the desired temperature, or by adding the solvent, re-suspending the mixture and adjusting to the desired temperature.

[0159] Preferably, the suspension of step b) of said fresh or frozen cells or said lyophilisate in an inorganic or organic solvent is carried out in a lyophilisate or biomass / solvent weight ratio of between 1 / 100 and 1 / 50.

[0160] Advantageously, the fresh cells harvested and extracted from step a) come from a cell culture under temperature, photoperiod and salinity conditions adapted to the strain concerned up to a cell concentration of between 5.104 cells / mL and 5.107 cells / mL, preferably a cell concentration of between 5.105 cells / mL and 2.107 cells / mL. The cells are cultured in batch for 5 to 20 days, then continuously or semi-continuously for several months. The light intensity is between 40 pE and 2000 pE, preferably between 70 pE and 500 pE. The culture temperature is generally between 17°C and 30°C, preferably between 17°C and 26°C. The day / night photoperiod is preferably between 8h / 16h and 16h / 8h. The minimum salinity is 15 ppt (parts per thousand).

[0161] According to a particular embodiment, when the fresh cells harvested and extracted from step a) come from the strain Amphidinium carterae, they have been cultured according to the following parameters: the cells are incubated in natural or artificial seawater medium at a temperature between 17 and 25°C with a day / night cycle between 8h / 16h and 16h / 8h, preferably 16h / 8h.

[0162] Alcoholic beverage comprising at least one amphidinol

[0163] The invention also aims at a key developmental intermediate for obtaining an alcoholic beverage without added sulfites, without undesirable microorganisms and without alteration of the aromas.

[0164] For this purpose, the invention relates to an alcoholic beverage comprising at least one amphidinol. This beverage comprising at least one amphidinol is a beverage during aging, a phase during which the amphidinol(s) introduced according to the invention are still present.

[0165] The presence of at least one amphidinol is temporary within the alcoholic beverage due to the degradability of amphidinols.

[0166] According to one embodiment, the alcoholic beverage according to the invention comprises at least one amphidinol chosen from amphidinol 1 to 19, amphidinol 22, amphidinol A and their mixtures.

[0167] According to a preferred embodiment, the alcoholic beverage according to the invention comprises at least one amphidinol chosen from amphidinol 18, amphidinol 19, amphidinol 22 and their mixtures.

[0168] According to a particular embodiment, the alcoholic beverage according to the invention comprises at least two amphidinols chosen from amphidinol 1 to 19, amphidinol 22, amphidinol A and their mixtures.

[0169] Preferably, the alcoholic beverage according to the invention comprises amphidinol 18 and at least one amphidinol chosen from amphidinol 1 to 17, amphidinol 19 to 22, amphidinol A and their mixtures.

[0170] Preferably, the alcoholic beverage according to the invention comprises at least one amphidinol chosen from amphidinol 18, amphidinol 22 and their mixture.

[0171] Preferably, the alcoholic beverage according to the invention comprises at least one amphidinol chosen from amphidinol 18, amphidinol 3 and their mixture.

[0172] According to a preferred embodiment, the alcoholic beverage according to the invention comprises a product described according to any of the embodiments previously described.

[0173] The alcoholic beverage according to the invention does not include added sulfites.

[0174] Process for manufacturing an alcoholic beverage

[0175] According to a final object, the invention relates to a method for manufacturing an alcoholic beverage comprising the implementation of a step of adding at least one amphidinol to the beverage.

[0176] According to a particular embodiment of the invention, the addition of at least one amphidinol to the alcoholic beverage can be carried out at any time during the process of preparing an alcoholic beverage.

[0177] According to a preferred embodiment, the method of manufacturing a beverage al cooled according to the invention comprises at least one step of adding at least one product described according to any of the embodiments previously described.

[0178] Preferably, the alcoholic beverage according to the invention may be any beverage obtained by alcoholic fermentation. Preferably, the alcoholic beverage according to the invention may be chosen from wine, whisky, cider, beer and kombucha. Examples

[0179] Example 1: Obtaining a product according to the invention in the form of an extract of the microalgae Amphidinium carterae.

[0180] Laboratory cultivation of the microalgae Amphidinium carterae, strain ICC001, is carried out in a ventilated T25, T50 flask or in a 5 L flask in an L1 medium (see medium composition below) in a culture chamber at 18°C ​​with a day / night cycle of 12h / 12h at a light intensity of 50-100 pE.

[0181] Scaling up the culture volume allows for moving from less than one liter of culture to several m3 and requires the use of a photobioreactor (PBR) containing culture bags. This is done by seeding 100 L bags of PBR culture medium with 5 L cultures of the microalgae. Culture in PBR is done at a temperature of 22-23°C with pH control by adding CO2 and setting up bubbling (aeration flow rate of IL / min). Lighting at 6,500 K is between 150 and 500 pE in continuous light over 24 hours. When the culture reaches the desired optical density, the microalgae are harvested continuously by daily sampling of 10 to 20% of the culture by adding an equivalent volume of culture medium.These microalgae samples are taken using a tangential filtration system that concentrates the microalgae, which are then centrifuged to obtain a microalgae paste with a dry matter content of 25-35%. This paste is then resuspended in 100% ethanol (EtOH) to obtain a final EtOH concentration of 60-70%. A content of 100 g of dry matter per IL of solution (100 g / L) is thus obtained. This extract is centrifuged at 5,000 rpm to obtain a clarified supernatant that corresponds to the product according to the invention and which theoretically contains 100 g / L of dry matter. The product according to the invention has an amphidinol 18 concentration of 33 g / L. This product according to the invention will be diluted in 100% EtOH at different concentrations from 0.006 g / L to 2 g / L and used for Figures 1 to 4 and for [Fig.9A] and 9B.

[0182] The product according to the invention was subsequently purified by reverse-phase exclusion chromatography on a C18 column in order to obtain an elution peak containing amphidinol 18 (AM18), called peak 0. This peak 0 was used for figures 5 to 8. To do this, the paste was resuspended in methanol (MetOH) at 3 mg of dry matter per 1 mL of MetOH. After filtration of this paste at 0.2 pm, 10 pL of the sample were injected into the C18 column. The elution of the different components was carried out using a mixture of solvent A (0.1% formic acid) and solvent B (MetOH) which differed according to the elution time: T0 min (A / B: 50 / 50); T25 min (A / B: 0 / 100); T40 min (A / B: 0 / 100); T43 min (A / B: 50 / 50) and T48 min (A / B: 50 / 50). Peak 0 corresponds to the first elution peak and for which the presence of AM18 (molecular weight: 1381.8 Da and molar mass: 1358.83 g / mol) was confirmed by mass spectrometry.

[0183] Peak 0 was used in Figures 5, 6, 7 and 8. It will sometimes be referred to as the "purified fraction of picO".

[0184] The general diagram of the process is given in [Fig. 10].

[0185] Composition of IL of the L1 medium 1 L of SSW medium 2 mL of NP solution 1 mL of metal solution 0.5 mL of F / 2 vitamins.

[0186] SSW Solution: NaCl: 24.5 g Na2SO4: 4.09 g KC1: 0.7 g NaHCO3: 0.2 g KBr: 0.1 g H3BO3: 0.03 g NaF: 0.003 g MgC12 6H2O: 11.1 g CaCl2 2H2O: 1.54 g H20 qsp 1 L

[0187] NP Solution (50X): NaNO3: 37.5 g NaH2PO4-H2O: 2.5 g H20: qsp 100 mL.

[0188] Metal solution (1000X): FeC13 • 6H2O: 3.15 g Na2EDTA • 2H2O: 4.36 g CuSO4 • 5H2O: 0.00245 g Na2MoO4 • 2H2O: 0.0189 g ZnSO4 • 7H2O: 0.022 g CoC12 • 6H2O: 0.01 g MnC12 • 4H2O: 0.18 g H2SeO3: 0.0013 g NiSO4 • 6H2O: 0.0027 g Na3VO4: 0.00184 g K2CrO4: 0.00194 g H20: qsp 1 L.

[0189] Solution of F / 2 vitamins: Thiamine (B 1): 400mg Cobalamin (B 12): 2mg Biotin (H): 2mg H20: qsp 1 L

[0190] Example 2: Demonstration of the effect of a product according to the invention on three strains of Brettanomyces bruxellensis

[0191] Three strains of Brettanomyces bruxellensis, and more precisely two triploid strains (NL6293 and NL6293) and one diploid strain (NL6300), were grown independently on YPD agar medium in petri dishes.

[0192] After three days of incubation at 28°C and atmospheric pressure, a photo highlighting the presence of the three colonies was taken, it is given in [Fig.lA].

[0193] Then, dilutions of these three strains are prepared, at 5.104 cfu / mL.

[0194] Furthermore, dilutions in YPD of the product according to the invention are carried out.

[0195] Then, the three strains were incubated in the presence or absence of product according to the invention at different concentrations for seven days at 28°C and atmospheric pressure.

[0196] The different samples are as follows: - YPD alone (negative control; 100 pL yeast dilution + 100 pL YPD); - YPD with the presence of ethanol (100 pL of yeast dilution + 100 pL of YPD containing 0.7 pL of ethanol); - YPD with the presence of product according to the invention (100 pL of yeast dilution + 100 pL of YPD containing 0.06 g / L to 2 g / L of product according to the invention).

[0197] Two replicates were performed for each condition.

[0198] A general diagram of this protocol is given in [Fig.lB].

[0199] Yeast growth is measured by reading the optical density at 550 nanometers at the following times: T = 0, T = 2 days, T = 3 days, T = 5 days, and T = 7 days.

[0200] The summary of the measured optical densities is given in [Fig.lC].

[0201] It should be noted that the populations of the three strains of Brettanomyces bruxellensis are particularly sensitive to ICCOOl, and in particular when the concentration of product according to the invention is 0.250 g / L.

[0202] It has therefore been demonstrated that the product according to the invention has an anti-inflammatory activity. crobian against different strains of Brettanomyces bruxellensis.

[0203] Furthermore, if we compare the activity of the product according to the invention with that of ethanol, it turns out that it is better, whatever the concentration tested.

[0204] Example 3: Demonstration of the effect of the product according to the invention on the viability of Brettanomyces bruxellensis

[0205] A strain NL6293 of Brettanomyces bruxellensis was, depending on the samples: - previously incubated in the presence of 1 / 300th of ethanol for twenty-four hours; - previously incubated in the presence of 1 g / L of product according to the invention for twenty-four hours; or - subjected to a temperature of 95°C for five minutes.

[0206] Then, all samples were incubated for one minute with trypan blue, which is a cell viability marker (it is excluded from viable cells but is present in non-viable cells and marks the nucleus) acting as a DNA intercalator.

[0207] Then, a photo of each of the three samples was taken. It is given in [Fig.2],

[0208] It has therefore been demonstrated that the product according to the invention causes a loss of viability of Brettanomyces bruxellensis.

[0209] Furthermore, if we compare the activity of the product according to the invention with that of ethanol, it turns out that it is better.

[0210] Example 4: Demonstration of the effect of amphidinol 18 on three strains of Bret-tanomyces bruxellensis

[0211] A product according to the invention at one gram per liter (in 70% ethanol) was placed in the light and at room temperature for 120 days.

[0212] Regularly, the concentration of amphidinol 18 within the product according to the invention was measured by high performance liquid chromatography.

[0213] All the data collected were put into the form of a graph, given in [Fig.3A],

[0214] It was demonstrated that the concentration of amphidinol 18 in the product according to the invention decreased; at T = 0 days, it is 24 mg / L, while at T = 120 days, it is close to 0 mg / L.

[0215] In parallel, different samples were prepared using the same three strains of Brettanomyces bruxellensis as in example 2: - YPD alone (negative control; 100 pL yeast dilution + 100 pL YPD); - YPD with the presence of ethanol (100 pL of yeast dilution + 100 pL of YPD containing 0.7 pL of ethanol); - YPD with the presence of the product according to the invention (100 pL of yeast dilution + 100 pL of YPD containing 1 g / L of product according to the invention; T = 0 days); g / L- YPD with the presence of inactivated product according to the invention (100 pL of yeast dilution + 100 pL of YPD containing 1 g / L of product according to the invention having a degree of aging of T = 7 days);

[0216] Two replicates were performed for each condition.

[0217] Yeast growth is measured by reading the optical density at 550 na nomometers.

[0218] The summary of optical densities is given in [Fig.3B].

[0219] It should be noted that the populations of the three strains of Brettanomyces bruxellensis are particularly sensitive to the product according to the invention at all times: no strain of Brettanomyces grows in the presence of the product according to the invention, whatever the time.

[0220] From T = 3 days, the effects in the presence of inactivated product according to the invention are similar to those of ethanol.

[0221] Comparing the two tests presented in this example, it appears that amphidinol 18 is a particularly effective amphidinol for controlling and / or limiting the growth of Brettanomyces bruxellensis.

[0222] Example 5: Determination of the minimum inhibition concentration of the product according to the invention at different concentrations of Brettanomyces bruxellensis

[0223] The minimum inhibition concentration of the product according to the invention was determined for three concentrations of Brettanomyces bruxellensis: 2.5.102 cfu / mL, 2.5.103 cfu / mL and 2.5.104 cfu / mL according to the protocol described in example 2 and [Fig.lB].

[0224] The OD550 nm at 0, 3, 5 and 6 days (T0, T3, T5 and T6) was measured after incubation of the yeasts at 28°C in the presence of YPD medium alone (negative control), ethanol or in the presence of product according to the invention at a final concentration of 0.03 g / L to 0.25 g / L. Two replicates were carried out for each condition.

[0225] The summary of optical densities is given in [Fig.4].

[0226] It should be noted that whatever the concentration of Brettanomyces bruxellensis, a concentration of 0.125 g / L of ICCOOl is very effective in inhibiting Brettanomyces bruxellensis.

[0227] Example 6: Determination of the minimum inhibition concentration of a fraction purified from the product according to the invention on Brettanomyces bruxellensis.

[0228] The chromatographic data of the product according to the invention after injection into the high performance liquid chromatography column are given in [Fig.5A].

[0229] The analysis of peak 0 by tandem mass spectrometry (MS / MS) is given in [Fig.5B]. The peak at 27.759 corresponds to amphidinol 18 (AM18).

[0230] The photo of the fraction of peak 0 and of the product according to the invention is given in [Fig.5C].

[0231] The minimum inhibition concentration of peak 0 (purified fraction) was determined for two strains of Brettanomyces bruxellensis (NL6293 and NL6300) according to the protocol described in example 2 and [Fig.lB].

[0232] The OD550 nm at T = 0 days, T = 3 days and T = 6 days was measured after incubation of the yeasts at 28°C in the presence of YPD medium alone (negative control), methanol or in the presence of peak 0 at dilutions ranging from 1 / 32 to 1 / 256 corresponding to concentrations of 40 to 5 ppm of amphidinol 18. The product according to the invention at 0.25 g / L was also tested (positive control). Two replicates were carried out.

[0233] All the data obtained are given in [Fig.5D].

[0234] It has been demonstrated that all the dilutions as well as the product according to the invention (0.25 g / L) are effective against the NL6293 strain.

[0235] It was also demonstrated that all concentrations except the 5 ppm concentration of AM18 as well as the product according to the invention (0.25 g / L) are effective against the NL6300 strain.

[0236] Example 7: Determination of the minimum inhibition concentration of a purified fraction of the product according to the invention on the yeast Saccharomyces cerevisiae

[0237] The minimum inhibition concentration (MIC) of peak 0 described in [Fig.5D] was determined for Saccharomyces cerevisiae according to the protocol described in example 2 and [Fig.1B],

[0238] The OD550 nm at 0, 3 and 6 days (T0, T3 and T6) was measured after incubation at 28°C of the yeasts at 2.5.104 cfu / mL in the presence of YPD medium alone (negative control), methanol (MEtOH) or in the presence of peak 0 at a dilution of 1 / 32 to 1 / 256 corresponding to concentrations of 40 to 5 ppm of amphidinol 18. The product according to the invention at 0.25 g / L was also tested under the same conditions. Two replicates (repi and rep2) were carried out.

[0239] All the data obtained are given in [Fig.6].

[0240] It has been demonstrated that concentrations of 40 and 20 ppm of AM18 as well as the product according to the invention (0.25 g / L) are effective against Saccharomyces cerevisiae.

[0241] Example 8: Determination of the minimum inhibition concentration of a fraction purified from the product according to the invention on the b&clene Acetobacter aceti

[0242] The minimum inhibition concentration (MIC) of peak 0 described in [Fig.5D] was determined for Acetobacter aceti according to the protocol described in example 2 and [Fig.1B].

[0243] The OD550 nm at 0, 3 and 6 days (T0, T3 and T6) was measured after incubation at 28°C of the yeasts at 105 cfu / mL in the presence of the MRS medium alone (negative control), methanol (MEtOH) or in the presence of peak 0 at a dilution of 1 / 32 to 1 / 256 corresponding to concentrations of 40 to 5 ppm of amphidinol 18. The product according to the invention at 0.25 g / L was also tested under the same conditions. Two replicates (repi and rep2) were carried out.

[0244] All the data obtained are given in [Fig.7].

[0245] It has been demonstrated that the concentrations 40, 20 and 100: of AM18 as well as the product according to the invention (0.25 g / L) are effective against Acetobacter aceti.

[0246] Example 9: Study of the antimicrobial action of the purified fraction of the product according to the invention on Brettanomyces bruxellensis and Saccharomyces cerevisiae in wine.

[0247] At time T0, unsulfured wine previously heated to 100°C for 5 min then supplemented with 20 g / L of glucose was inoculated with Brettanomyces bruxellensis, strain NL6293 or NL6300, or with Saccharomyces cerevisiae at a final concentration of 5.104 cfu / mL.

[0248] After 24 hours of incubation at room temperature and in the dark (Tl), 500 pL of these samples were inoculated with 1 / 32nd of YPD medium (negative control) or 1 / 32nd of methanol or 1 / 32nd or 1 / 64th of the purified peak 0 fraction of the product according to the invention.

[0249] These samples are incubated at room temperature and in the dark with gentle shaking.

[0250] At the indicated times (T = 1 day, T = 2 days, T = 4 days and T = 11 days), 10 pL of each of the samples are taken in order to determine the quantity of colony-forming yeast units (CFU) by counting on Petri dishes.

[0251] The collected data were arranged in Figures 8A, 8B and 8C.

[0252] The logarithmic measurement of the number of colony forming units (log cfu / mL) of Brettanomyces bruxellensis NL6293 present in the wine at T = 1 day, T = 2 days, T = 4 days and T = 11 days after inoculation of the wine with Brettanomyces bruxellensis NL6293 for the different conditions described is given in [Fig.8B].

[0253] In the presence of YPD medium (negative control), CFU growth is in the exponential phase from T = 4 days.

[0254] In the presence of methanol, the growth of CFU is in the exponential phase from T = 4 days.

[0255] In the presence of 1 / 32nd of the purified fraction of the product according to the invention, absence of CFU growth, at T = 2 days no viable CFU is detected.

[0256] In the presence of 1 / 64th of the purified fraction of the product according to the invention, no viable CFU is detected at T = 4 days but the growth of viable CFU becomes detectable at T = 11 days.

[0257] In summary, the purified fraction of the product according to the invention is very effective in combating Brettanomyces bruxellensis NL6293 in wine.

[0258] The logarithmic measurement of the number of colony-forming units (log cfu / mL) of Brettanomyces bruxellensis NL6300 present in wine at T = 1 day, T = 2 days, T = 4 days and T = 11 days after inoculation of wine with Brettanomyces bruxellensis NL6300 for the different conditions described is given in [Fig.8C].

[0259] In the presence of YPD medium (negative control), CFU growth is in the exponential phase from T = 4 days.

[0260] In the presence of methanol, the growth of CFU is in the exponential phase from T = 4 days.

[0261] In the presence of 1 / 32nd of the purified fraction of the product according to the invention, absence of CFU growth, at T = 2 days no viable CFU is detected.

[0262] In the presence of 1 / 64th of the purified fraction of the product according to the invention, no viable CFU is detected at T = 4 days but the growth of viable CFU becomes detectable at T = 11 days.

[0263] In summary, the purified fraction of the product according to the invention is very effective in combating Brettanomyces bruxellensis NL6300 in wine.

[0264] The logarithmic measurement of the number of colony forming units (log cfu / mL) of Saccharomyces cerevisiae present in the wine at T = 1 day, T = 2 days, T = 4 days and T = 11 days after inoculation of the wine with Saccharomyces cerevisiae for the different conditions described is given in [Fig.8D].

[0265] In the presence of YPD medium (negative control), CFU growth is very strong from T = 2 days.

[0266] In the presence of methanol, the growth of CFU is increasing from T = 2 days.

[0267] In the presence of 1 / 32nd of the purified fraction of the product according to the invention, absence of CFU growth, and at T = 4 days no viable CFU is detected.

[0268] In the presence of 1 / 64th of the purified fraction of the product according to the invention, the cfu are stabilized up to T = 4 days.

[0269] In summary, the purified fraction of the product according to the invention is very effective in combating Saccharomyces cerevisiae in wine.

[0270] Example 10: Study of the degradation kinetics of amphidinol 18 in wine.

[0271] Different doses of the product according to the invention, containing from 60 ppm to 480 ppm of amphidinol 18 were inoculated into the wine. The quantity of amphidinol 18 was determined by HPLC assay over time, up to day 44 (T44). The percentage of amphidinol 18 relative to the initial dose was thus evaluated over time.

[0272] These results are described in Figures 9A and 9B.

[0273] This example shows that amphidinol 18 degrades consistently in wine. After 44 days in the wine, similar amounts of amphidinol are found, regardless of the initial concentration.

[0274] Example 11: Study of the yeasticidal activity of a product according to the invention in wine.

[0275] A natural wine containing yeasts was treated either with the product according to the invention comprising 10 ppm of amphidinol 18 (AM18), i.e. with a commercial dose of chitosan. An untreated control (TNT) was also performed.

[0276] The counting of yeasts and bacteria present in each condition was carried out by epifluorescence observation before treatment (D0) and 3 days after treatment (D3).

[0277] The results of this example are illustrated in [Fig.l 1].

[0278] It can be observed that the product according to the invention comprising amphidinol 18 has better bactericidal and yeasticidal efficacy than chitosan after 3 days of treatment.

[0279] Example 12: Study of the activity of the product according to the invention to control and / or limit the growth of yeasts belonging to the genus brettanomyces under acidic pH conditions.

[0280] Brettanomyces strains (NL6293 and NL6200) at a concentration of 2.5 105 cfu / mL were incubated in the presence of a range of concentrations of the product according to the invention in a YPD medium at different pHs. A minimum concentration of product according to the invention inducing 100% inhibition (MIC) of the yeasts could be determined in each condition. [Tables 1] Brettanomyces MIC pH 3.7 pH 4.0 pH 6.5 NL6293 0.03 0.015 0.015 NL6200 0.03 0.03 0.03 These results indicate that the product according to the invention retains its capacity to control and / or limit the growth of yeasts belonging to the genus Brettanomyces at acidic pH.

Claims

Claims

1. Use in an alcoholic beverage of a product comprising at least one amphidinol.

2. Use according to the preceding claim, as a substitute for sulfites.

3. Use according to one of the preceding claims, characterized in that the alcoholic beverage has a pH of less than 7, preferably less than 4.

4. Use according to one of the preceding claims, characterized in that the alcoholic beverage comprises at least 1% ethyl alcohol, preferably at least 4%.

5. Use according to one of the preceding claims, characterized in that the product comprises at least one amphidinol chosen from amphidinol 1 to 19, amphidinol 22, amphidinol A and their mixtures.

6. Use according to one of the preceding claims, characterized in that the alcoholic beverage is a beverage obtained by alcoholic fermentation.

7. Use according to the preceding claim, characterized in that the alcoholic beverage is chosen from wine, cider and beer.

8. Use according to one of the preceding claims, in an alcoholic beverage as a bactericide, yeasticide and / or antioxidant.

9. Use according to one of the preceding claims, in an alcoholic beverage for controlling and / or limiting the growth of at least one yeast of a genus chosen from Brettanomyces, Hansaenula, Sac-charomyces, Hanseniaspora, Pichia, Candida, Kloeckera and combinations thereof.

10. Use according to one of the preceding claims, in an alcoholic beverage, for controlling and / or limiting the growth of yeasts belonging to the genus Brettanomyces, preferably at least Brettanomyces bruxellensis yeasts.

11. Use according to one of the preceding claims, in an alcoholic beverage, for controlling and / or limiting the growth of at least one bacterium of a genus chosen from Gluconobacter, Acetobacter, Oe-nococcus, Lactobacillus, Pediococcus, Pectinatus, Megasphaera, Ci-trobacter, Enterobacter, Obesumbacterium, Klebsiella, Proteus, Serratia, Zymomonas.

12. Use according to one of the preceding claims, characterized in that

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25. whether the product is in powder or liquid form. Product intended for use in alcoholic beverages, comprising at least one amphidinol. Product according to the preceding claim, characterized in that it comprises at least one amphidinol among amphidinol 1 to 19, amphidinol 22, amphidinol A and their mixtures. Product according to one of claims 13 or 14, characterized in that it is a cellular extract of one or more micro-algae of the genus Amphidinium. Product according to the preceding claim, characterized in that said micro-algae of the genus Amphidinium is Amphidinium carterae. Product according to one of claims 13 to 16, characterized in that it does not include carteraol. Method for manufacturing an alcoholic beverage, comprising at least one step of adding at least one amphidinol to the beverage. Method according to the preceding claim, characterized in that the alcoholic beverage is chosen from wine, cider and beer. Method according to one of claims 18 or 19, comprising a step of adding at least one amphidinol chosen from amphidinol 1 to 19, amphidinol 22, amphidinol A and their mixtures. Method according to one of claims 18 to 20, characterized in that it comprises a step of adding at least one product according to one of claims 13 to 17. Alcoholic beverage containing at least one amphidinol. Alcoholic beverage according to the preceding claim, characterized in that it comprises at least one amphidinol chosen from amphidinol 1 to 19, amphidinol 22, amphidinol A and their mixtures. Alcoholic beverage according to the preceding claim, characterized in that it does not contain added sulfites. Use of at least one amphidinol in an alcoholic beverage to control and / or limit the growth of at least one undesirable microorganism.