ALCOHOLIC FERMENTATION

TECHNOLOGICAL FIELD:

The present disclosure relates to a method to reduce the loss of volatile aroma compounds during alcoholic fermentation of a fermentable material.

BACKGROUND:

Fermented beverages, including, for example, wine, champagne, beer, ciders, spirits or liquor are obtained by alcoholic fermentation of fermentable material containing C6 sugar (as for example glucose, fructose, galactose, starch, maltose, etc). The conversion of these fermentable materials into ethanol is induced by yeast such as, for example, Saccharomyces, and the process produce also carbon dioxide plus a wide variety of yeast-derived aroma compounds and waste by-products. These aroma compounds can desirably affect the smell and/or taste of the wine and the enjoyment associated with drinking the fermented wine.

As part of the typical fermentation process, carbon dioxide gas is vented to the atmosphere during fermentation to avoid increasing pressure in the fermentation container. Some exceptions to this involve the production of carbonated beverages where a small supplemental fermentation is conducted inside a close container (tank or bottle). In typical fermentation, the carbon dioxide gas takes with it a non-negligible part of the desirable aroma compounds. As a consequence, the consumable fermented beverage is deprived of a fraction of desirable aroma compounds that would have otherwise enhanced the taste and/or smell of the fermented beverage. In some cases, desirable aroma compounds escape with carbon dioxide by diffusing into carbon dioxide bubbles. In some cases, desirable aroma compounds diffuse directly into the headspace from the surface of a fermenting beverage.

The enjoyment associated with drinking fermented beverages, such as wine or beer, for example, is largely due to aroma compounds present in the beverage. Some of these aroma compounds are representative of the fermentable raw material products, such as the variety of grape used in wine making. Important aromas are also due to yeast metabolism during fermentation. Some of those aroma compounds have a low gas/liquid partition coefficient which makes them less volatile and helps keep them contained in the beverage during fermentation where they remain, for the most part, in the finished beverage. Other aroma compounds have higher gas/liquid partition coefficients. These aroma compounds are relatively more volatile and will diffuse into the headspace above the fermenting beverage, which exposes them to loss during fermentation when carbon dioxide gas, produced during fermentation, is released from the system and takes with it some of the volatile aroma compounds. This decreases the concentration of the aroma compounds in the finished, consumable beverage. This loss of aroma in the escaping carbon dioxide gas is sometimes referred to as "carbon dioxide stripping".

Aroma compounds with higher gas/liquid partition coefficients are important since, in order to detect aroma, the aroma compounds are typically volatile. Notably, more volatile aroma compounds are relatively more susceptible to carbon dioxide stripping. Esters, for example, are a group of aroma compounds important in giving wine or beer their fruity flavor, but they tend to have higher gas/liquid partition coefficients and are relatively susceptible to carbon dioxide stripping.

The issue of aroma loss during fermentation is an important one and a variety of methods have been advanced for preserving some of the aroma in the fermented beverages. However, these methods often require tampering with the beverage or capturing and isolating aroma compounds during fermentation for reintroduction to the beverage after fermentation. Some of these existing methods require the use of complex refrigeration or vacuum equipment for treating or isolating the aroma compounds. Other existing methods to preserve aroma compounds in the fermented beverage, as for example control of the temperature, limitation of the oxidation by addition of glutathion, and maintaining the yeast in good physiological conditions, promote the production of these compounds, but do not prevent their loss due to their volatility or by carbon dioxide stripping. Recent results seem indicate that low fermentation temperature has a higher effect on the decrease of the aroma loss than on the increase of aroma biosynthesis by the yeast (see Morakul and al, J. Agric. Food Chem. 2010 ;58(18):10219-25)

There exists a need for an improved method for preserving the volatile aroma compounds of a fermentable beverage in the fermentation medium during the fermentation. BRIEF SUMMARY

The present disclosure provides a process/method for producing a fermentation product by alcoholic fermentation of a fermentable material in a fermentation medium comprising fermenting the fermentable material in presence of a microorganism to perform alcoholic fermentation into the fermentation product;

Wherein mannoproteins are added to the fermentation medium before and/or during the alcoholic fermentation of the fermentable material.

According to a first aspect, the addition of mannoproteins before and/or during the alcoholic fermentation reduces the loss of at least one volatile aroma compound from the fermentation medium and/or fermentation product.

According to a second aspect, the present disclosure provides a process/method to reduce the loss of at least one volatile aroma compound from a fermentation medium during alcoholic fermentation, the process comprising the addition of mannoproteins to the fermentation medium before and/or during the fermentation.

According to a third aspect, the present disclosure provides a fermentation product made by the process/method of the present disclosure, wherein the fermentation product comprise at least one volatile aroma compound with a concentration at least 1 % higher than the concentration of the same volatile aroma compound in a fermentation product fermented without the addition of mannoproteins before and/or during the alcoholic fermentation.

According to a fourth aspect, the present disclosure provides the use of mannoproteins to reduce the loss of at least one volatile aroma compound from a fermentation medium during alcoholic fermentation.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 illustrates change in the density for the fermentations of the Chardonnay variety.

Figure 2 illustrates change in the density during the fermentations of the Tempranillo variety vinified as rose wine. Figure 3 illustrates change in the density for the fermentations of the Tempranillo variety vinified as red wine.

Figure 4 illustrates the variation in % of the concentration of the fermentative aromas analyzed in the microvinifications treated with addition of mannoproteins with respect to the control microvinifications without addition of mannoproteins of the Chardonnay variety.

Figure 5 illustrates the variation in % of the concentration of the fermentative aromas analyzed in the microvinifications treated with addition of mannoproteins with respect to the control microvinifications without addition of mannoproteins of the of Tempranillo as rose wine.

Figure 6 illustrates the variation in % of the concentration of the fermentative aromas analyzed in the microvinifications treated with addition of mannoproteins with respect to the control microvinifications without addition of mannoproteins of the of Tempranillo as red wine.

Figure 7 illustrates change in the density for the fermentations of the Verdejo assays during alcoholic fermentation at different temperature treated or not treated with mannoproteins.

Figure 8 illustrates change in the density for the fermentations of the Tempranillo assays during alcoholic fermentation at different temperature treated or not treated with mannoproteins.

Figure 9 illustrates the sensory evaluation of wines from Verdejo assays resulting from fermentation at different temperature treated or not treated with mannoproteins.

Figure 10 illustrates the sensory evaluation of wines from Tempranillo assays resulting from fermentation at different temperature treated or not treated with mannoproteins.

DETAILED DESCRIPTION

In accordance with the present disclosure, there is provided a method to reduce the loss of at least one volatile aroma compounds from a fermentation medium during alcoholic fermentation of a fermentable material. The method comprises the addition of mannoproteins to the fermentation medium before and/or during the alcoholic fermentation. Advantageously, the reduction of the loss of volatile aroma compounds from the fermentation medium leads to an improvement of the aromatic content of the fermentation product. As used herein, the reduction of the loss of volatile aroma compounds from a fermentation medium or fermentation product during alcoholic fermentation treated with mannoproteins is evaluated by comparison with the amount (for example in concentration, volume, weight, or other quantity units) of the volatile aroma compounds from a fermentation medium or fermentation product of an alcoholic fermentation not treated with mannoproteins and performed under the same conditions than the fermentation treated with mannoproteins. In one embodiment, the method or process of the present invention reduces the loss of, or increases the concentration of, at least one, two, three, four, five, six, seven, eight, or more volatile aroma compounds from a fermentation medium or fermentation product during alcoholic fermentation.

The present disclosure also provides a method and or process of producing a fermentation product by alcoholic fermentation of a fermentable material in a fermentation medium in presence of a microorganism. The method/process comprises, consist essentially, or consist in addition of mannoproteins to the fermentation medium before and/or during the alcohol fermentation of the fermentable material. Advantageously, the addition of mannoproteins to the fermentation medium before and/or during the alcohol fermentation of the fermentable material reduce the loss of at least one volatile aroma compounds from the fermentation medium during the alcoholic fermentation of the fermentable material.

In one embodiment, the mannoproteins are not generated in-situ by the microorganism that performs the alcoholic fermentation. In one embodiment, the microorganism is a yeast. Suitable yeast can be, for example, from the genus Saccharomyces. Suitable yeast species can include, for example, Saccharomyces cerevisiae (S. cerevisiae) and interspecific hybrids such as for example Saccharomyces uvarum and Saccharomyces pastorianus. In another embodiment, the mannoproteins are not generated by dead or inactivated yeast or by yeast cell walls that performs the alcoholic fermentation.

Apart from the addition of mannoproteins to the fermentation medium before and/or during the alcohol fermentation, the alcoholic fermentation is performed in standard conditions well known in the art for the production of commercial fermented beverages as for example, wine, beer, cider, potable alcohol, sake. Alternatively, the addition of mannoproteins to the fermentation medium before and/or during the alcohol fermentation of the fermentable material provides a method to perform alcoholic fermentation at higher temperature than the standard conditions well-known in the art while reducing the loss of at least one volatile aroma compounds from the fermentation medium during the alcoholic fermentation. As used herein, the term "standard conditions" refers to conditions well known in the art to perform alcoholic fermentation, more particularly to obtain fermented beverages. The skilled person of the art would know which specific conditions are necessary to obtain the desired fermentation product. Such conditions are recommended by regulatory authorities in the field. For examples standard conditions well known in the art for the production of wines is alcoholic fermentation at temperature between 12 to 22 °C for white wines (see Sablayrolles JM, 2013. Fermentation alcoolique in Les vins blancs, de la demarche marketing a la vinification : les cles d'un pilotage reussi. Schneider R. Ed., Editions France Agricole, Paris, p.176-184 and Collectif, 1998. Vinification en blanc in Traite d'cenologie 1. Microbiologie du Vin, Vinification, Ribereau-Gayon P., Dubourdieu D., Doneche B., Lonvaud A. Eds, Dunod, Paris, p. 489-554), 15 to 20 °C for rose wines (see Fauvet J. and Guittard A. , 1998. La vinification en rose, in CEnologie, Fondements scientifiques et technologiques, Flanzy Ed. Lavoirsier tec&Doc, Paris, p. 739-753 and Granes D. and Pic-Blateyron L, 2009. La fermentation alcoolique, in Le vin rose, Flanzy C, Masson G. and Millo F. Eds, Editions Feret, Bordeaux, p. 182-186), and 20 to 35 °C for red wines (see Collectif, 1998. Vinification en rouge in Traite d'cenologie 1. Microbiologie du Vin, Vinification, Ribereau-Gayon P., Dubourdieu D., Doneche B., Lonvaud A. Eds, Dunod, Paris p. 401-484 and Navarre C, 1998. Vinification en rouge, in L'cenologie, Navarre C. Ed, Lavoisier Tec&Doc, Paris, p.149-162). Advantageously, the alcoholic fermentation performed at higher temperature is completed faster than fermentation at lower temperature. In one embodiment, the alcoholic fermentation is performed at a temperature above 16 °C, 16.5 °C, 17 °C, 17.5 °C, 18 °C, 18.5 °C, 19 °C, 19.5 °C, 20 °C, 20.5 °C, 21 °C, 21.5 °C, 22 °C, 22.5 °C, 23 °C, 23.5 °C, 24 °C, 24.5 °C, 25 °C, 25.6 °C, 26 °C, 26.5 °C, 27 °C, 27.5 °C, 28 °C, 28.5 °C, 29 °C, 29.5 °C, 30 °C, 30.5 °C, 31 °C, 31.5 °C, 32 °C, 32.5 °C, 33 °C, 33.5 °C, 34 °C, 34.5 °C, or 35 ° C. In one embodiment, the addition of mannoproteins to the fermentation medium before and/or during the alcoholic fermentation reduces the time of the alcoholic fermentation when compare to the time required to perform the same alcoholic fermentation not treated with mannoproteins.

In one embodiment, the addition of mannoproteins to the fermentation medium before and/or during the alcoholic fermentation allows alcoholic fermentation at higher temperature than the same alcoholic fermentation not treated with mannoproteins.

In one embodiment, the concentration of mannoproteins added in the fermentation medium and/or fermentation product is at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or 200 % higher than in a fermentation medium and/or fermentation product fermented without the addition of mannoproteins. In an alternative embodiment, the concentration of mannoproteins added in the fermentation medium and/or fermentation product is at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or 200 % of the concentration of mannoproteins generated by the fermenting microorganism. In another embodiment, the concentration of mannoproteins in the fermentation medium and/or fermentation product is at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or 200 % higher than in a fermentation medium and/or fermentation product fermented without the addition of mannoproteins.

Surprisingly, the addition of mannoproteins during and/or before the fermentation reduces the loss of at least one volatile aroma compound from the fermentation medium by carbon dioxide stripping during the fermentation. Advantageously, the addition of mannoproteins during or before the fermentation provides a higher concentration of at least one of the volatile aroma compounds in the fermentation medium and/or fermentation product than in a fermented medium and/or fermented product fermented without the addition of mannoproteins. Alternatively, the addition of mannoproteins during and/or before the fermentation maintains the concentration of at least one volatile aroma compound in the fermentation product.

In the context of the present disclosure, the aroma compounds are compounds derived from the fermentable raw material or due to yeast metabolism during fermentation. These aroma compounds can desirably affect the smell and/or taste of the fermented product. For example, the sensory limit for each of the compounds as well as the olfactory sensation for the human nose is shown in Table 1. Those values could be used for calculation of OAV for each of the compounds and will allow one skilled in the art to know how it affects the overall aroma of a wine.

Table 1. Sensory limit for each of the fermentative aromas analyzed and its associated descriptor

In the context of the present disclosure, the volatile aroma compounds are some of the aroma compounds having a high gas/liquid partition coefficient. These aroma compounds are relatively volatile and will diffuse in to the headspace above the fermented product, which expose them to loss during fermentation when carbon dioxide gas, produced during fermentation, is released from the system and takes with some of the volatile aroma compounds. In one embodiment, the addition of mannoproteins during and/or before the fermentation prevents the volatile aroma compounds to escape with carbon dioxide by diffusing into carbon dioxide bubbles. In another embodiment, the addition of mannoproteins during and/or before the fermentation prevents the volatile aroma compounds to diffuse directly in the headspace from the surface of a fermentation product. In a preferred embodiment, the volatile aroma compounds have high gas/liquid partition coefficients. In another embodiment, the concentration of at least one volatile aroma compounds in the fermentation medium and/or the fermentation product is higher than in a fermentation product fermented without the addition of mannoproteins.

In one embodiment, the concentration of at least one volatile aroma compounds in the fermentation medium and/or the fermentation product is at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 % higher than in a fermentation medium and/or fermentation product fermented without the addition of mannoproteins.

In one embodiment, the concentration of at least one volatile aroma compounds in the fermentation product fermented with the addition of mannoproteins during and/or before the fermentation is at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 % higher than in a fermentation medium and/or fermented product fermented without the addition of mannoproteins.

In another embodiment, the addition of mannoproteins during and/or before the fermentation reduces the loss of volatile aroma compounds of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 % from the fermentation medium during the fermentation when compared to the same fermented medium fermented without the addition of mannoproteins. In a preferred embodiment, the loss of volatile aroma compounds is by carbon dioxide stripping.

In another embodiment, the addition of mannoproteins during and/or before the fermentation reduces the loss of volatile aroma compounds of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 % from the fermentation product when compared to the same fermented product fermented without the addition of mannoproteins. In a preferred embodiment, the loss of volatile aroma compounds is by carbon dioxide stripping.

In an embodiment, the reduction of the loss of at least one volatile aroma compounds is between 1-60%, 5-60%, 10-60%, 15-60%, 20-60%, 30-60%, 40-60%, 1-50%, 5-50%, 10-50%, 20-50%, 30-50%, 1-45%, 5-45%, 10-45%, 15-45%, 20-45%, 25-45%, 30-45% or 30-40% when compared to a fermented product fermented or a fermentation medium without the addition of mannoproteins. In another embodiment, the reduction of the loss of at least one volatile aroma compounds is at least 1 %, 5%, 10%, 20%, 30%, 40%, 50%, 60% when compared to a fermented product or a fermentation medium fermented without the addition of mannoproteins.

In the context of the disclosure, the volatile aroma compounds are esters, alcohols, acids, terpenes, aldehydes or ketone compounds, thiol derivatives, or C-13 norisoprenoids. In one embodiment, the volatile aroma compound is an ester, an acetate or a fatty acids. In another embodiment, the volatile aroma compound is an ester, more particularly an ethyl ester.

Alcohol as a volatile aroma compound could be, for example, 1-propanol, isobutyl alcohol, 1-butanol, isoamyl alcohol, 1-pentanol, 4-methyl-1-pentanol, 2-heptanol, 1- hexanyhol, 3-hexen-1-ol, 2-octanol, 1-heptanol, 2-ethyl-1-hexanol, 2-nonanol, 2,3- butanediol, 1-octanol, 3-(methylthio)-2-propanol, 1-decanol, benzyl alcohol, 2- henylethanol, or 1-dodecanol.

Ester as a volatile aroma compound could be, for example, ethyl acetate, isobutyl acetate, Ethyl 3-methylbutanoate, isoamyl acetate, ethyl hexanoate, hexyl acetate, ethyl lactate, heptyl acetate, methyl octanoate, ethyl octanoate, ethyl decanoate, diethyl succinate, phenethyl acetate, or ethyl dodecanoate.

Acid as a volatile aroma compound could be, for example, acetic acid, propanoic acid, isobutyric acid, isovaleric acid, hexanoic acid, octanoic acid decanoic acid.

Aldehydes or ketone compounds as a volatile aroma compound could be, for example, nonanal, benzaldehyde, geranial, /3-ionone, decanal, furfural, vanillin, β-damascenone or acetoin.

Terpenes as a volatile aroma compound could be, for example, geraniol, citronellol, linalool, nerol, hotrienol or limonene.

In one embodiment, ester is ethyl butanoate, ethyl hexanoate, ethyl octanoate, ethyl decanoate, ethyl dodecanoate, diethyl succinate, ethyl acetate, isoamyl acetate, hexyl acetate, or 2-phenyethyl acetate.

In one embodiment, acid is acetic acid, propanoic acid, isobutyric acid, isovaleric acid, hexanoic acid, heptanoic acid, octanoic acid decanoic acid. In one embodiment, alcohol is isoamyl alcohol, isobutanol, benzyl alcohol, hexanol, or 2- phenyl ethyl alcohol.

In one embodiment, terpenes are linalool, nerol, geraniol, or linalool.

In one embodiment, thiol derivatives are 4-methyl-4-mercaptopentan-2-one, 3- mercaptohexanol, or 3-mercaptohexyl acetate.

In one embodiment, norisoprenoids are β-damascenone or β-ionone

In one embodiment, the volatile aroma compound is ethyl octanoate, ethyl hexanoate or isoamyl acetate.

In one embodiment, the volatile aroma compound is ethyl octanoate, linalool, 2- phenylethanol, β-ionone or octanoic acid.

In one embodiment, the volatile aroma compound is isoamyl acetate, hexanol, ethyl hexanoate, or β-ionone.

In one embodiment, the volatile aroma compound is 3-methyl-1-butanol, isoamyl acetate, or geraniol.

In one embodiment, the volatile aroma compound is ethyl butyrate, ethyl hexanoate, ethyl octanoate, ethyl decanoate, ethyl dodecanoate, diethyl succinate, ethyl acetate, isoamyl acetate, hexyl acetate, 2-phenylethyl acetate, isoamyl alcohol, isobutanol, benzyl alcohol, hexanol, 2-phenylethyl alcohol, hexanoic acid, octanoic acid, or decanoic acid.

In another embodiment, the volatile aroma compound is ethyl butyrate, ethyl hexanoate, ethyl octanoate, ethyl decanoate, ethyl dodecanoate, diethyl succinate, ethyl acetate, isoamyl acetate, hexyl acetate, or 2-phenylethyl acetate.

In another embodiment, the volatile aroma compound is ethyl butyrate or diethyl succinate.

In one embodiment, the volatile aroma compounds are compounds which are most contributive to wine aroma. Thus, in one embodiment, the volatile aroma compounds are selected from:

C4 to C10 ethyl esters for the esters, Isoamyl acetate, hexyl acetate and 2-phenyl ethyl acetate for the acetates;

hexanoic acid, octanoic acid or decanoic acid for the fatty acids; and

combinations thereof.

In one embodiment, the quantity of manoproteins added to the fermentation medium is equal or inferior to the quantity of mannoproteins generated by the microorganism performing the alcoholic fermentation.

In one embodiment, the quantity of manoproteins added to the fermentation medium is superior to the quantity of mannoproteins generated by the microorganism performing the alcoholic fermentation.

In the context of the present disclosure, the fermentation product is a fermented beverage. In one embodiment, the fermented beverage is a fermented must or fruit juice, fermented plant juice, fermented sugar material or starchy material. More specifically, the fermented beverage is an alcoholic beverage as for example base for spirit, liquor, wine (including non-sparkling wines as well as sparkling wine, as for example champagne), brandy base wine, beer, champagne, cider, mead or sake. In one embodiment, the wine is red wine, white wine or rose wine.

In the context of the present disclosure, the fermentable material contains at least one fermentable substance. A fermentable substance within the meaning of the present disclosure is a chemical compound which can be used under anaerobic and/or aerobic conditions by microorganisms, such as yeasts and bacteria, as energy source and as carbon source. This includes monosaccharides, disaccharides and polysaccharides, in particular. In a preferred embodiment, the fermentable materials are those which contain C6 sugar as for example fructose, glucose, galactose, sucrose, maltose or starch, or one of their degradation products. In particular, fermentable material is a fruit (apple, grape, pears, plums, cherries, peaches), a plant (sugar cane, agava, cassava, ginger), a sugar material (honey, molasse), a starchy material (rice, rye, corn, Sorghum, millet, barley, wheat, potatoes) or a derived product (grape must, apple mash, malted grain, crushed fruit, fruit puree, fruit juice, fruit must, plant mash, gelatinized and saccharified starch from different plant origins as rice, corn, sorghum, wheat, barley). In the context of the present disclosure, starchy material refers to a material that contains starch that could be converted into alcohol by a microorganism during alcoholic fermentation. Starchy material could be for example, gelatinized and saccharified starch from cereals, grains (wheat, barley, rice, buckwheat) or grain derived-products (malted grain) or vegetable (potatoes, beets). In one embodiment, the fermentable material is grape must.

In the context of the present invention, "mannoproteins" are glycoprotein of proteoglycan macromolecules containing 15 to 90 % mannan by weight and peptides chains, found especially in yeast cell wall of, for example, Saccharomyces cerevisiae and other yeast species belonging to Saccharomyces genus or other genus such as for example Schizosaccharomyces pombe, Schizosaccharomyces japonicus, or Torulaspora sp.. Mannoproteins are different structures depending on their molecular weight, their degree and type of glycosylation and their load size. Mannoproteins can be extracted from Saccharomyces cerevisiae cells and/or yeast cell walls by mean of physico-chemical or enzymatic methods. In one embodiment, the mannoproteins of the present disclosure have a molecular weight between 3 kDa and 100 kDa, between 3 kDa and 50 kDa, between 3 kDa and 40 kDa, between 3 kDa and 30 kDa, between 3 kDa and 20 kDa, or between 3 kDa and 10 kDa.

Mannoproteins can be added to the fermentation medium in the form of granulate, powder or in the form of a solution. In one embodiment, the mannoprotein granulate is a micro-granulate. In an alternative embodiment, the mannoprotein solution according to the present disclosure is preferably an aqueous solution. In order to avoid diluting the fermentation medium too much upon addition of the mannoprotein solution, the concentration of mannoproteins in the solution is preferably between 20 g/l and 400 g/l, between 50 g/l and 300 g/l, between 100 g/l and 400 g/l, between 100 g/l and 300 g/l, between 125 g/l and 275 g/l, between 150 g/l, and 275 g/l, between 150 g/l and 250 g/l, between 175 g/l and 225 g/l, or most preferably about 200 g/l.

In one embodiment, the mannoprotein solution has a turbidity, when measured by nephelometry at a concentration of 200 g/l of mannoprotein and at a pH in the range of pH 4 to pH 8, lower than 70 NTU, preferably lower than 60 NTU, more preferably lower than 50 NTU, most preferably lower than 40 NTU.

This mannoprotein solution is particularly suitable to be used in alcoholic fermentation for reducing the loss of volatile aroma compound. Because the turbidity of a mannoproteins solution is dependent on the concentration and on the pH, the values of turbidity are measured at a concentration of 200 g/l of the solution. However this should not be interpreted as meaning that the solution of the present disclosure has a fixed concentration of mannoproteins of 200 g/l, but that the turbidity thereof is measured at a concentration of 200 g/l. Therefore depending on its initial concentration the mannoproteins solution can be diluted or concentrated up to 200 g/l prior to measurement of turbidity. At a pH of 5.5 the turbidity of the mannoproteins solution, measured by nephelometry at a concentration of 200 g/l mannoproteins, is preferably 100 NTU or lower, more preferably 70 NTU or lower, more preferably 50 NTU or lower, even more preferably 40 NTU or lower, most preferably 30 NTU or lower. The pH of the solution is preferably such that the stability of the mannoprotein is preserved in time and such as to avoid turbidity of the solution. Therefore the pH of the mannoprotein solution is preferably between 3 and 8, more preferably between 4 and 8, more preferably between 4 and 7, or most preferably between 5 and 6.

The mannoproteins solution of the present disclosure finds its preferred use in the reduction of loss of volatile aroma compounds from the fermentation medium during the alcoholic fermentation of a fermented product. For this reason it is important that the solution is devoid of the presence of any harmful microorganism. Furthermore, the presence of any viable microorganism can have an impact on the storage stability of the solution. For these reasons in one embodiment of the invention, the mannoproteins solution of the present disclosure has a total amount of viable microorganisms present in the solution, measured in Colony Forming Unit (CFU) / ml which is lower than 10, preferably lower than 5, more preferably the mannoproteins solution is sterile. In another embodiment, the total amount of viable microorganisms present in the mannoproteins solution, measured in Colony Forming Unit (CFU) / ml of solution is lower than 10 000, 5000, 1000, 100, or 10.

With the wording "total amount of viable microorganisms" is meant the total amount of aerobic and anaerobic psychrophilic microorganisms + aerobic and anaerobic mesophilic microorganisms + aerobic and anaerobic thermophilic microorganisms present in the solution. The total amount of viable microorganisms present in the solution, measured in Colony Forming Unit (CFU) / ml is defined in Materials and Methods and is established by determination of the total plate count (TPC), a method which is well known to those skilled in the art. Most preferably the mannoproteins solution is sterile, for instance the total amount of viable microorganisms present in the solution, measured in Colony Forming Unit (CFU) / ml of solution is 0. The mannoproteins solution is preferably packed in a sterile container which will be hermetically closed.

In one embodiment, the solution of mannoproteins is preferably microbiologically stable in time. Therefore, in a preferred embodiment, at least a stabilizing additive is added to the mannoproteins solution as for example: sulphur dioxide, sodium benzoate, potassium sorbate, sorbitol, or propylene glycol. More preferably the stabilising additive is sulphur dioxide. More preferably sulphur dioxide can be added to the solution in the form of bisulphite salt, more preferably as sodium or potassium bisulphite (K2S2O5). Addition of sulphur dioxide has another advantage next to maintain the mannoprotein solution microbiologically stable. In one embodiment, the amount of sulphur dioxide to be added to the mannoprotein solution is preferably in the range of 1 to 20 g/l (measured as amount of K2S2O5 added).

The mannoproteins solution of the present disclosure may comprise one or more additional components, preferably one or more food grade components. The component is preferably suitable for use in fermented beverage, as defined above. Examples of suitable components are protein substrates, protein hydrolysates, yeast extracts, carboxy methyl cellulose, wine additives or mixtures of two or more of these components. Examples of wine additives are meta-tartrate, or Arabic gum. Another specially preferred component is a protein hydrolysate. Typical protein substrates for the preparation of protein hydrolysates are vegetable proteins such as wheat gluten, corn gluten, soy protein, rape seed protein, pea protein, alfalfa protein, sunflower protein, fabaceous bean protein, cotton or sesame seed protein, maize protein, barley protein, sorghum protein, potato protein, rice protein, or coffee proteins. Other possible protein substrates are animal proteins such as milk protein (for instance casein or whey protein), egg white, fish protein, meat protein including gelatin, collagen, blood protein (for instance haemoglobin), hair, feathers or fish meal. In one embodiment, the protein hydrolysate is preferably selected from a protein hydrolysate of animal, vegetable or microbial origin, more preferably selected from casein hydrolysate, egg albumin hydrolysate, gelatin hydrolysate, bovine serum albumin hydrolysate, lysozyme hydrolysate, wheat protein hydrolysate, soy protein hydrolysate, pea protein hydrolysate or a mixture of two or more of these components The mannoproteins solution according to the present disclosure should be preserved in the time period between the production of the solution by the manufacturer thereof and the use of the solution to reduce the loss of volatile aroma compound in the fermentation medium.

The mannoprotein solution of the present disclosure is preferably characterised by a carbohydrate content in the mannoproteins of at least 50 % w/w, based on the mannoproteins dry matter, of which at least 70 % w/w, based on the total carbohydrate content, consists of mannose residues in the form of mannose oligomers or polymers. Preferably the mannoproteins solution obtainable by the process of the invention is characterised by an amount of phosphorous (measured as P20s) in the mannoproteins which is lower than 2% w/w based on dry matter, more preferably 1 % w/w or lower. The amount of phosphorous in the mannoproteins can be measured according to a well- known AES-ICP method (Atomic Emission Spectroscopy with the aid of Inductive Coupled Plasma).

It will be understood by those skilled in the art that the process of the present invention may encompass a step in which the mannoproteins solution is dried by methods known to those skilled in the art, for instance by lyophilisation, spray, fluidized bed or drum drying, to yield mannoproteins in solid form, for instance in the form of a powder or granulate. Said mannoproteins in solid form are also encompassed by the present invention.

While the invention has been described in connection with specific embodiments thereof, it will be understood that the scope of the claims should not be limited by the preferred embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description as a whole.

EXAMPLES:

Example 1

Microvinifications

The starting grapes were in optimal maturity conditions for the elaboration of white wine from a Chardonnay grape. The grape of the Chardonnay variety was pressed, the resulting must was divided into six 50 L vats, 40 L per unit, and the enzyme Rapidase Clear was added (2 g/hL). Once the grape was processed, divided into the different vats and homogenized, the individual characterization of the different sets was carried out. The following parameters were analyzed: degree Brix, potential alcohol degree (PAD), pH, total tartaric acid (TTA), acetic acid, and yeast assimilable nitrogen (YAN). The average was calculated and results for each of the parameters analyzed are shown in Table 2. All these analyses were carried out according to the protocols established by the Compendium of International Methods of Wine and Must Analysis-OIV [International Organization of Vine and Wine], 2011.

Table 2. Basic characterization of musts of the Chardonnay variety.

The musts were clarified at 12 °C and inoculated with the yeast Anchor Vin 13; they were introduced into a water bath at 15 °C for the performance of the alcoholic fermentation. Of those six vats, three were used as Control microvinifications, while a solution of 20% mannoproteins was added at the recommended dose of 1 mL/L to the remaining three vats. The characteristics of the solution of mannoprotein are shown in Table 3.

Table 3. Characteristics of the mannoproteins solution

The microvinifications were monitored by daily measurement of temperature and density. Figure 1 shows the change in the density of the different microvinifications during the alcoholic fermentation. The total sulfur concentration was kept below 50 mg/L during the processing of the grape and the fermentation.

Once the alcoholic fermentations were completed, the wines elaborated were characterized by analysis of the basic physicochemical parameters well known by the person skilled in the art: alcohol degree, pH, total tartaric acid, acetic acid, malic acid and lactic acid. All the analyses were carried out according to the protocols established by the Compendium of International Methods of Wine and Must Analysis-OIV 201 1. The average was calculated for each parameter analyzed and results are summarized in Table 4.

Table 4. Basic characterization of wines of the Chardonnay variety.

The addition of mannoproteins has no significant impact on the physicochemical parameters of the Chardonnay wine as shown in Table 4.

The analysis of the main fermentative aromas of each of the wines elaborated was performed. These analyses were carried out by solid phase microextraction (SPME), gas chromatography (GC), and mass spectrometry (MS). The average was calculated for each parameter analyzed and results are summarized in Table 5. The percentage of variation between the concentration of the fermentative aromas analyzed in the microvinifications treated with addition of mannoproteins with respect to the control microvinifications without addition of mannoproteins of the Chardonnay variety is shown in figure 4.

Table 5. Concentration of the fermentative aromas analyzed in the Chardonnay microvinifications.

Example 2

Microvinifications

The starting grapes were in optimal maturity conditions for the elaboration of rose wine from a Tempranillo variety. The grape of the Tempranillo variety intended for the elaboration of rose wine was destemmed and divided into six 50 L vats, 40 kg per unit. Once the grape was processed, divided into the different vats and homogenized, the individual characterization of the different sets was carried out. The following parameters were analyzed: degree Brix, potential alcohol degree (PAD), pH, total tartaric acid (TTA), acetic acid, and Yeast assimilable nitrogen (YAN). The results for each of the parameters analyzed are shown in Table 6. All these analyses were carried out according to the protocols established by the Compendium of International Methods of Wine and Must Analysis-OIV [International Organization of Vine and Wine], 201 1.

Table 6. Basic characterization of musts of the Tempranillo variety for the elaboration of rose wine.

Subsequently, the yeast Anchor NT1 16 was inoculated and the vats were introduced into a water bath at 20 °C for the performance of the alcoholic fermentation. Of those six vats, three were used as Control microvinifications, while a solution of 20% mannoproteins was added at the recommended dose of 1 mL/L to the remaining three vats. The characteristics of the solution of mannoprotein are shown in Table 7.

Table 7. Characteristics of the mannoproteins solution

The microvinifications were monitored by daily measurement of temperature and density. Figure 2 shows the change in the density of the different microvinifications during the alcoholic fermentation. The total sulfur concentration was kept below 50 mg/L during the processing of the grape and the fermentation.

Once the alcoholic fermentations were completed, the wines elaborated were characterized by analysis of the basic physicochemical parameters well known by the person skilled in the art: alcohol degree, pH, total tartaric acid, acetic acid, malic acid and lactic acid. All the analyses were carried out according to the protocols established by the Compendium of International Methods of Wine and Must Analysis-OIV 201 1. The average was calculated for each parameter analyzed and results are summarized in Table 8.

Table 8. Basic characterization of the wines of the Tempranillo variety for the elaboration of rose wine.

The addition of mannoproteins has no significant impact on the physicochemical parameters of the rose wine of the Tempranillo variety as shown in Table 8.

The analysis of the main fermentative aromas of each of the wines elaborated was performed. These analyses were carried out by solid phase microextraction (SPME), gas chromatography (GC), and mass spectrometry (MS). The average was calculated for each parameter analyzed and results are summarized in Table 9. The percentage of variation between the concentration of the fermentative aromas analyzed in the microvinifications treated with addition of mannoproteins with respect to the control microvinifications without addition of mannoproteins of the Tempranillo as rose wine is shown in figure 5. Table 9. Concentration of the fermentative aromas analyzed in the microvinifications of Tempranillo as rose wine.

Example 3

Microvinifications

The starting grapes were in optimal maturity conditions for the elaboration of red wine from a Tempranillo variety. The grape of the Tempranillo variety intended for the elaboration of rose wine was destemmed and divided into six 50 L vats, 40 kg per unit. Once the grape was processed, divided into the different vats and homogenized, the individual characterization of the different sets was carried out. The following parameters were analyzed: degree Brix, potential alcohol degree (PAD), pH, total tartaric acid (TTA), acetic acid, and yeast assimilable nitrogen (YAN). The results for each of the parameters analyzed are shown in Table 10. All these analyses were carried out according to the protocols established by the Compendium of International Methods of Wine and Must Analysis-OIV [International Organization of Vine and Wine], 2011.

Table 10. Basic characterization of musts of the Tempranillo variety for the elaboration of red wine.

Subsequently, they were inoculated with the yeast Anchor Exotics and introduced into a thermostatted chamber at 25 °C for the performance of the alcoholic fermentation. Of those six vats, three were used as Control microvinifications, while a solution of 20% mannoproteins was added at the recommended dose of 1 mL/L to the remaining three vats. The characteristics of the solution of mannoprotein are shown in Table 1 1.

Table 1 1. Characteristics of the mannoproteins solution

The microvinifications were monitored by daily measurement of temperature and density. Figure 3 shows the change in the density of the different microvinifications during the alcoholic fermentation. The total sulfur concentration was kept below 50 mg/L during the processing of the grape and the fermentation.

Once the alcoholic fermentations were completed, the wines elaborated were characterized by analysis of the basic physicochemical parameters well known by the person skilled in the art: alcohol degree, pH, total tartaric acid, acetic acid, malic acid and lactic acid. All the analyses were carried out according to the protocols established by the Compendium of International Methods of Wine and Must Analysis-OIV 201 1. The average was calculated for each parameter analyzed and results are summarized in Table 12.

Table 12. Basic characterization of the wines of the Tempranillo variety for the elaboration of red wine.

The addition of mannoproteins has no significant impact on the physicochemical parameters of the red wine of the Tempranillo variety as shown in Table 12.

The analysis of the main fermentative aromas of each of the wines elaborated was performed. These analyses were carried out by solid phase microextraction (SPME), gas chromatography (GC), and mass spectrometry (MS). The average was calculated for each parameter analyzed and results are summarized in Table 13. The percentage of variation between the concentration of the fermentative aromas analyzed in the microvinifications treated with addition of mannoproteins with respect to the control microvinifications without addition of mannoproteins of the Tempranillo as red wine is shown in figure 6.

Table 13. Concentration of the fermentative aromas analyzed in the microvinifications of Tempranillo as red wine.

Example 4

Evaluation of the impact of each aroma in the olfactory perception of the wine

The effect of the aroma compounds considered individually in the olfactory perception of a wine is a complex subject, since sensory perception is a combination of the primary or varietal aromas, secondary or fermentative aromas, and tertiary aromas or aromas resulting from changes over time. Depending on the variety, the aroma of the wine can be formed by impact aromas such as, for example, 3-isobutyl-2-methoxypyrazine (IBMP) in Cabernet Sauvignon, or by a set of aromas the combination of which constitutes the basic aroma of the wine. In any case, one of the most accepted ways of calculating the impact of an aroma in the wine is by means of the OAV (Odor Activity Value), which is the quotient of the concentration of the aroma in the wine and its olfactory perception limit for the human nose (see table 1 ).

The OAV values for the analyzed aromas in the different Chardonnay, Tempranillo rose and Tempranillo red microvinifications are shown in table 14. Table 14. OAV values for the analyzed aromas in the different Chardonnay, Tempranillo rose and Tempranillo red microvinifications

Example 5

Evaluation of the addition of mannoproteins at the beginning of alcoholic fermentation on aromatic profile vs the fermentation temperature

Verdejo (white wine) and Tempranillo (red wine) grapes were used to evaluate the impact of the addition of mannoproteins on the aromatic profiles achieved by fermentation at different temperatures, more particularly higher temperature than the standard conditions well-known in the art. The healthy degree was optimum, and the maturation conditions were corrected to obtain wines with a high alcoholic degree close to 13 %vol. in all the microvinifications. The grape musts were clarified at 12 °C and inoculated with yeast, namely Anchor Vin 13 (for white wine) or Exotics (for red wine). After inoculation, the grape musts were introduced into a water bath at the desired temperature for the performing the alcoholic fermentation. Vats coded DDGF were added with a solution of 20 % mannoproteins at 1 mL/L at just after the yeast inoculation. The characteristics of the solution of mannoproteins are shown in Table 3.

The conditions related to each microvinification were the following:

Condition 1 (Verdejo): Anchor VI N 13 at 16 °C (Assay VE 16)

Condition 2 (Verdejo): Anchor VI N 13 at 18 °C (Assay VE 18)

Condition 3 (Verdejo): Anchor VIN13 at 18 °C + DDGF5 (Assay VE18DDGF)

Condition 4 (Tempranillo): Anchor Exotics at 24 °C (Assay TE24)

Condition 5 (Tempranillo): Anchor Exotics at 26 °C (Assay TE26)

Condition 6 (Tempranillo): Anchor Exotics at 26 °C + DDGF5 (Assay

TE26DDGF) All the microvinifications were conducted in duplicate.

Table 15 and Table 16 show the oenological characteristics of grape juices for Verdejo and Tempranillo that were used in the assay. The sanitary statuses of the grapes were excellent and presented a suitable technology maturity. The different parameters that were analysed before alcoholic fermentation were: Brix degree, Estimated alcoholic strength, Total sulphite, pH, Total Acidity and Yeast Assimilable Nitrogen (YAN).

Table 15. Verdejo grape juices characterization.

Table 16. Tempranillo grape juices characterization.

The fermentations were monitored by daily measurement of density. Figures 7 and 8 show the change in the density of the different iterations during the alcoholic fermentation. These data show that all fermentations have been performed without problems.

Once the alcoholic fermentations were completed, the resulting wines were characterized by analysis of the basic physicochemical parameters well known by the person skilled in the art: Alcoholic strength, pH, Total Acidity, Acetic Acid, and Glucose/Fructose. All the analyses were carried out according to the protocols established by the Compendium of International Methods of Wine and Must Analysis- OIV 201 1. The oenological characteristics obtained for each iteration are summarized in Tables 17 and 18.

The main fermentative aromas of each of the wines produced was analysed. These analyses were carried out by solid phase microextraction (SPME), gas chromatography (GC), and mass spectrometry (MS). The measured levels are summarized for each duplicate in Tables 19, 20, 21 for the Verdejo wines assays and Tables 22, 23, 24 for the Tempranillo wines assays. Table 19. Concentration of ethyl esters compounds analyzed in the Verdejo assays (ppb).

Table 20. Concentration of acetate and alcohol compounds analyzed in the Verdejo assays (ppb).

Table 21. Concentration of fatty acids compounds analyzed in the Verdejo assays (ppb).

Table 22. Concentration of ethyl esters compounds analyzed in the Tempranillo assays (ppb).

Table 23. Concentration of acetate and alcohol compounds analyzed in the Tempranillo assays (ppb).

Table 24. Concentration of fatty acids compounds analyzed in the Tempranillo assays (ppb).

The data related to the compounds that were the most contributive to wine aroma have been summarised for each class of compounds (C4 to C10 ethyl esters for the ethyl ester, isoamyl, hexyl and 2-phenyl ethyl for the acetate, and fatty acids). The percentage variation between the concentrations of the most contributive aroma compounds obtained from the fermentation at higher temperature, with respect to the control fermentation at lower temperature have been calculated. The percentage variation data are summarized in Tables 24 and 25 for the Verdejo wines assays and Tables 26 and 27 for the Tempranillo wines assays. Calculations are presented in ppb as well as in OAV, except for fatty acids result, considered as fruity masks.

Table 25. Variation of fermentation aromas for the Verdejo wines (levels in ppb)

Table 26. Variation of fermentation aromas for the Verdejo wines (levels in OAV)

Table 27. Variation of fermentation aromas for the Tempranillo wine assay (levels in ppb)

Table 28. Variation of fermentation aromas for the Tempranillo wine assay (levels in OAV)

The result for the Verdejo wine assays clearly show a decrease of the aroma contents when fermentation temperature is higher. The addition of mannoproteins induced a lower decrease as compared to fermentation not treated with mannoproteins. Thus, the addition of mannoproteins preserves more aromas compound in the wine. In addition, fatty acids drastically decreased at higher fermentation temperature, thus decreasing their fruity mask impact. The two phenomena observed normally have a synergic effect, leading to a higher aromatic quality of the wines. These results are consistent with the sensory evaluation of the wine obtained after fermentation (see Figure 9), where the wine added with mannoproteins and fermented at higher temperature was of better quality, with significant results for green notes, and hotness.

The results for the Tempranillo wines shows similar behaviour in presence of mannoproteins. The wine with added mannoproteins had higher levels of acetates and esters. Indeed, the addition of mannoproteins during fermentation has been shown to have a greater impact on Tempranillo wines (red wine) fermented at higher temperatures than the results obtained from the Verdejo wines assays (white wines), since we have observed an augmentation of the aroma level in the Tempranillo wines treated with mannoproteins in comparison to the non-treated control fermented at lower temperature.

Sensory evaluation on the Tempranillo wines obtained during the assays is consistent with the variation of fermentative aroma observed above. The evaluation was showing wines of higher quality for fermentation at higher temperature treated with mannoproteins (see Figure 10), with significant results with more fruits notes, green notes, chemical notes, mouthfeel, and astringency.

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(4) Tominaga, T.; Murat, M.L.; Dubourdieu, D. Development of a method for analyzing the volatile thiols involved in the characteristic aroma of wines made from Vitis vinifera L. Cv Sauvignon Blanc. Journal of agricultural and Food Chemistry, 1998, 46, 1044-1048.

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