IT202100025343A1 - IMPROVED OENOLOGICAL PRODUCT FOR WINE MAKING - Google Patents

Description

?IMPROVED OENOLOGICAL PRODUCT FOR WINE MAKING?

FIELD OF INVENTION

The present invention concerns an improved oenological product for winemaking comprising a yeast-based biocatalyst, capable of stabilizing both the wine and the must from a microbiological point of view, in particular by reducing the concentration of malic acid in them, without an unwanted increase in lactic acid and acetic acid. In preferred aspects, the present invention concerns the oenological product comprising a yeast lysate and the use of said oenological product as a yeast activator, to reduce the concentration of malic, lactic and acetic acids both in the must and in the finished wine.

STATE OF THE TECHNIQUE

Acids are an essential component of wine, both during winemaking and in the finished product. Are they already present? in the grapes and influence the chemical, physical and biochemical characteristics of the grapes and then of the must, acting on the ripening of the grapes, as well as on the preparation and evolution of the wine, thus also influencing the final characteristics of the wine, such as colour, balance and taste. Three primary acids in particular are found in wine grapes: citric acid, tartaric acid and malic acid. During winemaking and in the finished wines, further acids are found, such as acetic and lactic acid, deriving from fermentation.

Malic acid (C4H6O5) ? one of the main organic acids present in wine grapes. Malic acid levels in grape berries are at their peak just before veraison, when they can be present in concentrations of up to 20 grams/litre. As the grapes progress through the ripening phase, malic acid is metabolised in the respiration process and, at harvest, its concentration, in the L-form, is ? generally between 1 and 9 g/l (2 - 7 g/l in the must). During the winemaking process, its concentration is reduced both following alcoholic fermentation and, above all, malolactic fermentation, in which microorganisms convert malic acid into lactic acid.

Lactic acid (C3H6O3), generally present in wine in quantities? of 1? 5 g/l, therefore derives mainly from malic acid, following malolactic fermentation and, to a minimal extent, from alcoholic fermentation.

Red wines are more often subjected to malolactic fermentation compared to whites, therefore the latter generally contain higher concentrations of lactic acid.

Furthermore, during alcoholic and malolactic fermentations, acetic acid (C2H4O2) is formed, which determines the so-called acidity. wine volatile.

From an organoleptic point of view, acids give wine freshness, youth and also influence its color. A certain amount of acids? therefore essential to avoid having flat wines without personality, but an excess of them can? lead to making a wine undrinkable. For example, does community legislation provide for a maximum acidity value? volatile (EC Reg. 934/19) equal to 18 meq/l (equal to 1.35 g/l expressed in tartaric acid) for white and rosé wines and 20 meq/l (equal to 1.50 g/l expressed in tartaric acid) for red wines.

The presence of malic acid can? compromise shelf life? of a wine, making it more subject to the development of microorganisms (indigenous malolactic bacteria, Brettanomyces yeasts, acetic acid bacteria, etc.). Furthermore, if a wine is bottled or placed in soft packaging (e.g. bag in box, tetrapack) in the presence of malic acid, bacterial colonies can develop with the development of CO2 following malolactic fermentation. This leads to an organoleptic deviation (wine perceived as sparkling or too aggressive) and the development of unpleasant odors which compromise its shelf life. Furthermore, the swelling of soft packages highlights conservation problems.

Currently, there are various techniques to degrade malic acid, such as adding selected malolactic bacteria (Oenococcus and Lactobacilli), or stimulating natural malolactic fermentation, but these techniques always involve the production of lactic acid, which is undesirable especially for some wines (white and sparkling bases) for their lactic olfactory notes. Furthermore, these techniques also develop acetic acid, compromising the development of fresh aromas with fruity and floral notes. The uncontrolled transformation of malic acid into lactic acid also makes the wine less acidic (it increases the pH of the wine and lowers the titratable acidity), making it difficult to drink. In addition to reducing the presence of malic acid, it? therefore it is preferable that it does not degrade to form lactic acid.

In recent years, to microbiologically stabilize the wine, i.e. to control the activity? metabolism of the microorganisms present, yeasts capable of carrying out a malo-alcoholic fermentation were used, such as Schizosaccharomyces pombe, or others such as Issatchenkia orientalis. The problem with the use of these yeasts? however, the lack of formation of fermentation aromas and the fact that they are unable to close the alcoholic fermentation, forcing a second inoculation with Saccharomyces to be carried out, aggravating production costs and producing significant organoleptic deviations.

To protect malic acid from its degradation into lactic acid, highly impactful techniques are also used from the point of view of both the addition of additives and health. The "P? used? a very important addition of sulfur dioxide, which blocks the activity? microbial. Sulfites are for? allergens that can cause even serious reactions in some individuals. Another technique? the use of lysozyme, but this? effective only against enococci. Lately, ? Chitosan is also widely used, but since it is insoluble it causes precipitation in the bottle which is not welcome, so even if the wine is protected until bottling, there may be undesirable consequences in the bottle. Lastly, filtration can eliminate microorganisms, but there are frequent bottles or other packages in which lactic acid develops from malic acid after packaging, creating the problems described above.

? It has been noted that yeasts of the S. Cerevisiae strain are able to use malic acid as an energy source and metabolize it into oxaloacetic acid through an enzymatic reaction carried out by malate dehydrogenase (MDH).

Malate dehydrogenase? an enzyme involved in the Krebs Cycle, of which it catalyzes the last reaction.

L-malate NAD+ ? oxaloacetate NADH H+

The malate-oxaloacetate reaction? reversible, but in metabolism reversibility? of malate dehydrogenase? controlled by the subsequent reaction of the cycle, catalyzed by citrate synthase: the subtraction of oxaloacetate therefore keeps the reaction equilibrium shifted to the right, allowing the continuous oxidation of the malate.

For the reaction? a cofactor is necessary: this can? be NAD+ or NADP+, depending on the malate dehydrogenase. With reference to enzymes that use the NAD+ cofactor, malate dehydrogenase can be obtained from microbial sources, such as Thermus flavus and various thermophilic bacteria, so? such as from yeasts and also animal sources, from bovine and porcine organs. To these sources are also added the enzymes produced with heterologous expression, mainly in Escherichia coli.

Does malate dehydrogenase have good thermostability? up to approximately 55?C and, in 24 hours at 4-8?C, presents good stability? in the pH range 5.5-11 with an optimal pH range for the activity? of the enzyme between 7.5 and 9.0.

Advantageously, the transformation reaction of malate into oxaloacetate can therefore reduce the concentration of malic acid in the must or wine, with the production of oxaloacetic acid. Oxalacetic acid reacts with polyphenols (in particular with resveratrol), increasing their antioxidant power. Is the wine obtained from fermentation processes that use these yeasts? therefore very resistant to conservation, microbiologically stable, not producing acetic acid and maintaining an aromaticity? complex comparable to that obtained with other strains.

Furthermore, these yeast strains have a rather high fermentative power and a very high alcohol tolerance (18% vol.). Finally, the sensorial analysis of wines obtained from processes using these yeasts did not highlight significant differences, both on an olfactory and gustatory level.

However, since these yeasts are typically produced and marketed in live and active liquid form, packaged in plastic bottles, production of acetic acid may occur within the product during storage in the refrigerator and transport, before use in the production process. winemaking. Although this does not affect the qualities? of the final wine, when the packages are opened, unwanted odors (acetic or lactic) are released, which may cause concern in the user technician about the possibility of that the use of these yeasts leads to an increase in the concentration of acetic or lactic acid in the wine.

Purpose of the present invention? therefore to provide an improved oenological product for the fermentation of wine, which is capable of stabilizing both the wine and the must from a microbiological point of view, in particular by reducing malic acid, without unwanted production of lactic acid and acetic acid, and which does not present an undesirable increase in the aforementioned acids during its storage and transport, before its use.

SUMMARY OF THE INVENTION

Said purpose? was achieved through the production of a yeast-based wine product, according to the attached claims, capable of acting as a biocatalyst for the fermentation of wine, improving the properties of the wine. organoleptic characteristics of the must and wine, thanks to the capacity to degrade malic acid, without increasing the concentration of lactic and/or acetic acid. Said product? It can also be used in the form of yeast lysate as an activator for active yeast, intended for the fermentation of musts and wines to reduce or eliminate malic acid, acetic acid and lactic acid, both on the must and on the finished wine.

Said oenological product includes yeast, or a derivative thereof, and acetylcoA synthase enzyme (ACS; EC 6.2.1.1). Said product? obtainable through a preparation process according to the present invention, comprising incubating a yeast, preferably of the S. Cerevisiae strain, endogenously expressing the acetyl-coA synthase enzyme, in a medium comprising an acetic acid salt (acetate salt).

Therefore, the present invention is also directed to said process of preparing the wine product.

Optionally, the process of preparing the wine product can? further understand the addition of exogenous acetyl-coA synthase enzyme to yeast.

Therefore the acetyl-coA synthase enzyme in said wine product can be the endogenous yeast enzyme, internal to the yeast cells, and/or an exogenously added enzyme.

This invention? furthermore directed to a winemaking process that uses the oenological product of the invention, in the must and/or in the wine.

In preferred aspects of the invention, the final wine product is a yeast lysate including the acetyl-CoA synthase enzyme, obtainable by lysing the yeast after incubation in the medium including the acetate salt.

BRIEF DESCRIPTION OF THE FIGURES

The characteristics and advantages of the present invention will be evident from the following detailed description, from the embodiments provided by way of illustrative and non-limiting examples, and from the attached figures, in which:

- Figure 1 shows the chromatogram of the HPLC analysis of a wine sample, compared with the malic acid and acetic acid standards;

- Figure 2 shows the chromatogram (A) and an enlargement (B) of a yeast starter treated with enzymes (darker line) compared to the untreated yeast starter (lighter line), according to the example 1;

- Figure 3 shows the chromatogram (A) and its enlargement (B) of the wine treated with lysate 2 (darker line) compared to the untreated wine (lighter line), according to Example 3;

- Figure 4 shows the chromatogram of the yeast starter as is (darker line) and after treatment with lysates 1, 2 and 3 (lighter lines), according to Example 3;

- Figure 5 shows the? malic acid in the chromatograms of the yeast starter as is (darker line) and after treatment with lysates 1, 2 and 3 (lighter lines), according to Example 6;

- Figure 6 shows the chromatograms (in detail in the expanded boxes) of the biotransformation of the white (A) and red (B) must, according to example 7;

- Figure 7 shows optical microscope views of samples of the fermentation product of Example 6: a) at the beginning of the lysis phase, in which ? It is possible to observe a greater concentration of whole and viable-looking cells and b) in the advanced lysis phase, in which? It is possible to observe a significant reduction in whole cells and an increase in cellular debris. In c) ? shown a detail at higher magnification of the lysis state at the end of the sonication treatment (the arrow and the dotted arrow show viable cells and lysed cells respectively).

DETAILED DESCRIPTION OF THE INVENTION

The invention therefore concerns an improved oenological product, based on yeast, for the fermentation of wine, capable of reducing malic acid, without increasing acetic acid and/or lactic acid in the wine and/or must.

For ?oenological product? means a product used or usable in winemaking and during all phases of wine refinement.

The winemaking product of the present invention comprises yeast, or a yeast derivative, and acetyl-CoA synthase enzyme (ACS; EC 6.2.1.1).

Acetyl-CoA synthase? an enzyme belonging to the ligase class, which catalyzes the reaction of formation of acetyl-CoA starting from acetate and coenzyme A (CoA), in the presence of ATP.

ATP acetate CoA <=> AMP pyrophosphate acetyl-CoA

The optimal pH range for the reaction? 7.5-9.0; below 7.0, performance and stability? of the enzyme decrease.

Yeast derivatives include yeast lysates and yeast hulls. Preferably the yeast derivative in the wine product? a yeast lysate including the cytoplasmic fraction of the yeast, plus? preferably separated from the peels, obtained by lysing the yeast.

In preferred aspects, said oenological product essentially consists of yeast, or a yeast derivative, and acetyl-CoA synthase enzyme (ACS; EC 6.2.1.1).

Preferably called yeast? a saccharomycete, more? preferably of the S. Cerevisiae strain. In S. cerevisiae, acetic acid is taken up both by passive diffusion of undissociated acid and through the Fps1 channel. Once acetic acid enters the cell, it dissociates into an acetate anion and a proton why? its pKa (4.78) ? much more? low of the almost neutral pH of the cytoplasm. The acetic acid present in must or wine can what? become a substrate for the acetyl-CoA synthase present in the oenological product of the invention.

The oenological product of the invention? preferably obtainable through a process comprising the culture of yeast, said yeast endogenously comprising the acetyl-CoA synthase enzyme, in conditions such as to induce the endogenous enzyme.

By indicating that yeast endogenously includes an enzyme, it is meant that this enzyme is? constitutively expressed in yeast, and/or the yeast? capable of (over)expressing this enzyme. Preferably this enzyme? autologous to the yeast strain, but it cannot be excluded that the yeast is a recombinant yeast expressing a heterologous enzyme.

By ?induction? of an enzyme, here we mean the increase in the expression of that enzyme in the yeast, and/or the increase in the activity? catalytic activity of this enzyme, compared to the basal level of the yeast before induction.

The fermentative process of producing the wine product therefore includes cultivating a yeast in conditions such as to induce the endogenous ACS enzyme. Said conditions include culturing the yeast in a medium comprising an acetate salt. Therefore the oenological product includes yeast, or a derivative thereof, and the endogenously induced ACS enzyme.

This invention? then directed to a preparation process of a yeast-based wine product comprising the phases of:

a) provide a yeast, preferably of the S. Cerevisiae species;

b) cultivating said yeast in a culture medium comprising an acetic acid salt, preferably at a concentration of 5-20 g/l of medium, plus? preferably approximately equal to 10 g/l of soil;

c) recover the yeast at the end of phase b).

Preferably said soil does not include sugars.

Said acetate salt can? be an inorganic acetate salt, such as any alkali metal or alkaline earth metal salt or any other metal acetate salt. Easily dissociable complex salts of acetate or acetic acid esters which release acetate into the medium during culture can also be used.

Preferably, the acetate salt? an acetate salt of an alkali metal, more? preferably? sodium acetate (NaAc). Further usable acetate salts are for example potassium acetate, magnesium acetate, ammonium acetate.

Without wanting to be tied to theory, it is believed that the acetate salt can act both as a carbon source for the growth of yeast biomass and as an inducer of the yeast's endogenous acetyl-CoA synthase enzyme. Yeast growth medium including acetate salt can? therefore it can also be indiscriminately called growth medium or induction medium.

The yeast culture medium further includes elements suitable for yeast growth. Preferably, said soil comprises an organic matrix.

For ?organic matrix? refers to the set of organic sources for the growth of microorganisms. Preferably, such organic sources include at least one of peptone, yeast extract and malt extract.

Table 1 below indicates the ingredients preferably present in the yeast culture medium and their preferred concentrations.

Table 1

The different organic sources, the different carbon sources, so? like the different inorganic salts, they can be present individually, variously combined with each other, or all together in the yeast culture medium.

For example, the ground can include peptone and acetate salt without distinction, or peptone, yeast extract, malt extract and acetate salt, with one or more? of the inorganic salts of table 1.

In preferred aspects, the culture medium includes acetate salt as the carbon source and includes peptone, yeast extract, and malt extract, as the organic source.

Optionally, the yeast culture medium further includes glucose (Glu) as a carbon source, plus? preferably in concentrations between 5 and 20 g/l, more? preferably between 7 and 12 g/l, even more? preferably approximately equal to 10 g/l, volume of soil. Preferably, the culture medium comprises an acetate salt and optionally glucose, each in a concentration between 5 and 20 g/l, plus? preferably between 7 and 12 g/l, even more? preferably approximately equal to 10 g/l, based on the volume of the soil.

In preferred aspects, the culture medium includes glucose in a first culture phase, to allow the growth of biomass, while it includes only acetate salt in a second culture phase, as a carbon source and as an inducer of the ACS enzyme. In this case, the biomass obtained at the end of the first cultivation phase in a first medium with glucose? preferably collected and transferred to a second minimal medium (without sugars) including the acetate salt for the production of the wine product.

Preferably, the production process of the oenological product therefore includes:

a) provide a yeast, preferably of the S. Cerevisiae species;

b0) cultivating said yeast in a first culture medium comprising glucose, preferably at a concentration of 5-20 g/l, more? preferably 7 - 12 g/l, even more? preferably approximately equal to 10 g/l, volume of soil,

and optionally transfer said yeast to a second culture medium at the end of the yeast biomass growth,

b) add an acetic acid salt to the first or second culture medium, preferably at a concentration of 5-20 g/l, more? preferably more? preferably 7 - 12 g/l, even more? preferably approximately equal to 10 g/l, volume of medium and cultivate the yeast in the first or second culture medium comprising the acetic acid salt.

As known in the art, the yeast growth phase ends when that is. the yeast growth curve reaches a plateau.

In preferred aspects of the present invention, the yeast ? of the S. Cerevisiae strain, more? preferably of the cerevisiae physiological breed. Suitable S. Cerevisiae yeast strains according to the purposes of the present invention are: S. Cerevisiae 101S, 228 CU-2, A364A, AWRI1631, AWRI796, BMN1-35, BY2961, BY4741, BY4741-AV16, BY4741-AV8, BY4741-BV19 , BY4741-E18, BY4743, CAT-1, CBS 1585, CBS 2910, CBS 7833, CBS 7834, CBS 7835, CBS 7836, CBS 7837, CBS 7838, CBS 7839, CBS 7840, CBS 7960, CBS 9562, CBS 9563, CBS 9564, CBS 9565, CEN.PK113-7D, CLIB215, CLIB324, CLIB382, EC1118, EC9-8, FL100, Fleischmanns baking yeast, FostersB, FostersO, FY, G600, GSY1135, I14, IL-01, IR-2, JAY291, JWY6147, KRY8, Kyokai no. 7, Lalvin L2056, Lalvin QA23, LAN211, Lindquist 5672, M1-2, M13, M2-8, M22, M32, M34, M3707, M3836, M3837, M3838, M3839, M5, M52bB, M52cW, M8, MMY112, MUCL 28177, N85, NAM34-4C, NC-02, NRRL Y-12632, NY1308, P283, P301, PE-2, PW5, R008, R103, Red Star baking yeast, RM11-1a, S228, S288C, S288C x YJM789, S96 x YJM789, SGU57, Sigma1278b, SK1, T7, T73, UC5, UFMG A-905, var. ellipsoideus, var. diastaticus, Vin13, VL3, VR1, W303, WE372, Y10, Y12, Y2209, Y389, Y9, YB210, YJM1004, YJM1005, YJM1006, YJM1078, YJM1083, YJM1094, YJM1095, YJM1096, YJM1 097, YJM1098, YJM1099, YJM1100, YJM1101, YJM1102, YJM1103, YJM1104, YJM1105, YJM1106, YJM1107, YJM1108, YJM1109, YJM1110, YJM1111, YJM1112, YJM1113, YJM1114, YJM1115, YJM1116, YJ M1117, YJM1118, YJM1119, YJM1120, YJM1121, YJM1122, YJM1124, YJM1125, YJM1129, YJM1133, YJM1135, YJM1138, YJM1139, YJM1140, YJM1141, YJM1142, YJM1143, YJM1144, YJM1145, YJM1146, YJM1178, YJM1190, YJM1199, YJM1202, YJM1208, YJ M1242, YJM1244, YJM1248, YJM1250, YJM1252, YJM1259, YJM1273, YJM1289, YJM1292, YJM1304, YJM1307, YJM1311, YJM1326, YJM1332, YJM1336, YJM1338, YJM1341, YJM1342, YJM1355, YJM1356, YJM1381, YJM1383, YJM1385, YJM1386, YJM1387, YJ M1388, YJM1389, YJM1399, YJM1400, YJM1401, YJM1402, YJM1415, YJM1417, YJM1418, YJM1419, YJM1433, YJM1434, YJM1439, YJM1443, YJM1444, YJM1447, YJM14, YJM1450, YJM1460, YJM1463, YJM1477, YJM1478, YJM1479, YJM1526, YJM1527, YJM1 549, YJM1573, YJM1574, YJM1592, YJM1615, YJM189, YJM193, YJM195, YJM223, YJM244, YJM248, YJM269, YJM270, YJM271, YJM280, YJM308, YJM320, YJM326, YJM332, YJM339, YJM421, YJM428, YJM432, YJM434, YJM435, YJM436, YJM437, YJM 439, YJM440, YJM450, YJM451, YJM453, YJM454, YJM455, YJM456, YJM464, YJM466, YJM467, YJM470, YJM521, YJM525, YJM541, YJM554, YJM555, YJM560, YJM561, YJM627, YJM634, YJM653, YJM669, YJM670, YJM671, YJM 672, YJM674, YJM676, YJM677, YJM678, YJM681, YJM682, YJM683, YJM689, YJM693, YJM789, YJM810, YJM811, YJM813, YJM815, YJM816, YJM936, YJM945, YJM946, YJM947, YJM948, YJM949, YJM950, YJM951, YJM952, YJM 953, YJM954, YJM955, YJM956, YJM957, YJM958, YJM959, YJM960, YJM961, YJM962, YJM963, YJM964, YJM965, YJM966, YJM967, YJM969, YJM972, YJM975, YJM978, YJM981, YJM984, YJM987, YJM990, YJM993, YJM996, YJSH 1, YPH499N, YPS1009, YPS163, ZTW1 (GenBank database), or others .

In preferred aspects, the process of the invention provides for fed-batch cultivation. For ?fed-batch? cultivation we mean a semi-open cultivation system, also called "variable volume", open only at the entrance, but not at the exit, divided into several phases: initially there is a batch biomass growth phase, then, once adequate biomass has been reached, the fed-batch process begins, in which the nutrient is supplied, until other factors cause growth to slow down, such as the scarcity? of oxygen.

Preferably, in the process of the invention the culture conditions are maintained within the parameters indicated in Table 2 below.

Table 2

Preferably, the production process of the oenological product takes place in conditions of high oxygenation, in which the dissolved oxygen (dO2) is greater than or equal to 40% saturation, with a continuous oxygen supply between 10 and 25 mg/L/day, more? preferably between 10 and 15 mg/L/day, measured by oximeter with polarigraphic sensor. This high oxygenation can? be guaranteed for example by growing the yeast in a whisker flask stirred at 100-200 rpm or higher.

Preferably, the production process takes place in conditions of constant pH, included in the range between 5 and 7; more? preferably the pH in the fermentative growth process of the yeast? approximately equal to 6.

Preferably the growth of the yeast in the production process of the wine product lasts 24h-48h.

In particularly preferred aspects, the preparation process of the oenological product takes place in the following conditions: pH between 5 and 7, plus? preferably approximately equal to 6; temperature between 20? and 35? C, more? preferably from 25?C to 30?C, even more? preferably about equal to 28°C; stirring at 100-500 rpm, more? preferably at 400-500 rpm.

Particularly preferred processes according to the present invention are also described in the following examples.

In further aspects of the present invention, the production process of the wine product further includes: inducing the endogenous malate dehydrogenase enzyme of the yeast, incubating the yeast with a solution capable of inducing this enzyme. This solution can include yeast or a yeast derivative, such as yeast hulls or autolyzed yeasts, rich or enriched in fatty acids, sterols, mixtures of long-chain fatty acids (fatty acids with an aliphatic chain of at least 12 carbon atoms), polysorbates, or alcohols; preferably, this solution comprises at least one of oleic acid, polysorbate, and ethanol, plus? preferably at the concentrations indicated in table 3 below.

Table 3

This incubation can take place by placing said solution in contact with the yeast before the cultivation phase in the medium including the acetate salt, or by adding it to said medium substantially simultaneously with the acetate salt, or by adding it to the wine inoculated with the oenological product of the invention.

Preferably, the induction conditions of the malate dehydrogenase enzyme are maintained within the parameters indicated in Table 4 below.

Table 4

In further aspects, the oenological product of the present invention can? also include exogenous enzymes, acetyl-CoA synthase (ACS) and optionally malate dehydrogenase (MDH).

Preferably, said exogenous enzymes are added to the yeast in concentrations between 0.5-50 U/ml, more? preferably between 5-50 U/ml, even more? preferably approximately equal to 10 U/ml on the yeast volume.

In preferred aspects said exogenous enzymes further include lactate dehydrogenase (LDH), more preferably in a concentration between 10-20 U/ml based on the volume of yeast.

Therefore, preferably, the process of the present invention further comprises: adding to the culture medium at least one of: acetyl-CoA synthase enzyme, malate dehydrogenase enzyme, lactate dehydrogenase enzyme. For example, said enzymes can be added, in solution, to the culture medium.

Preferably, said enzymes to be added exogenously are purified from cells of the same yeast strain as the wine product and added at the desired concentrations to the yeast, during the yeast culture, or subsequently to the same in the final wine product.

In particularly preferred aspects, the oenological product essentially comprises or consists of a yeast derivative, in particular a yeast lysate.

Therefore, in preferred aspects, the process of preparing the oenological product further includes the phase of: lysing the yeast recovered in phase c), at the end of the cultivation phase in the medium comprising the acetic acid salt. Preferably the lysis occurs by sonication.

Surprisingly, yeast lysate is so obtained maintains the capacity? to degrade malic acid without causing an increase in the concentration of lactic or acetic acids. Also called lysate? capable of acting as an activator of the yeast itself, to help a (non-lysed) yeast reduce or eliminate both acetic and lactic acid.

Said yeast lysate includes yeast hulls and cytoplasmic fractions of yeasts. Preferably, after lysis, the yeast hulls are separated and the yeast lysate then consists only of the cytoplasmic fraction of the yeast.

Optionally, the oenological product obtained is ? dried, therefore the wine product can? be a dry product.

The wine product comprising yeast, optionally inactivated by sonication or ball milling, and/or a lysate of such yeast, is? advantageously used in a winemaking process, directly as an activator (starter) to add to the must or wine and as a corrector of possible defects in the final product that arise during the maturation, refinement or aging of the wine.

Additionally or alternatively, the yeast lysate can? be added to other yeast, of the same strain or different strains, to obtain a starter comprising a yeast and the yeast lysate comprising induced ACS, to be added to the must or wine to activate alcoholic fermentation and to reduce or eliminate unwanted acids , such as malic acid, lactic acid and acetic acid.

Can the wine product it can therefore be added to the must before vinification, or to the wine before refining and in all phases of vinification, before bottling, optionally together with additional yeast.

Preferably, the oenological product in lysate form? added at a concentration of 10-30 g/l, more? preferably approximately equal to 20 g/l, optionally together with additional yeast, must or wine.

The oenological product of the invention allows to obtain a very important reduction of unwanted acids both in solutions including yeast of the same or other strain, and on must and wine, even in conditions considered prohibitive. This solves many of the problems that arise in the cellar by allowing the degradation of organic acids and their transformation into harmless compounds, improving the general performance of the fermentation and the wine produced.

? it is to be understood that all possible combinations of the preferred aspects of the product and process, as indicated above, are also described, and therefore similarly preferred.

? Furthermore, it is to be understood that all aspects identified as preferred and advantageous for the product are to be considered similarly preferred and advantageous also for the preparation and uses of the same.

Below are examples of implementation of the present invention provided for illustrative and non-limiting purposes.

EXAMPLES

Analytical procedure

In the following examples, the analysis of organic acids in the samples is was performed by reversed-phase HPLC (RP-HPLC) on a Hypersil ODS-2 reversed-phase column, 5 ?M, 250 mm x 4.6 mm. Samples were diluted 10-fold in 5 mM potassium phosphate, pH 2.60, and centrifuged for 10? at 16000g; supernatants were used for HPLC runs. Runs were performed with a flow rate of 0.5 ml/min of 5 mM potassium phosphate, pH 2.60, at 30 ?C for 60 minutes. The detection? occurred by reading the absorbance at 210 nm. The quantification of acids, malic and acetic, is was carried out by setting up two calibration lines with quantities? standard of the two acids, also diluted in 5 mM potassium phosphate, pH 2.60 (Fig.1).

Example 1

Use of exogenous enzymes for the degradation of organic acids

In this experiment? An exogenous malate dehydrogenase enzyme, MDH, added to an S. Cerevisiae yeast starter was used as an adjuvant for the degradation of the organic acids of interest.

To amplify the effect of the enzyme malate dehydrogenase? A second enzyme, lactate dehydrogenase (LDH), was also added, capable of regenerating the NAD+ cofactor starting from the NADH generated by the primary enzyme, according to the reaction:

pyruvate NADH H+ ? (L)-lactate NAD+

The MDH and LDH enzymes were obtained in the laboratory from native microorganisms or purified from the yeast itself and then added exogenously at the desired concentrations.

The conditions tested are summarized in Table 5 below.

Table 5

The yeast starter added with the exogenous MDH 10 U/ml and LDH 10 U/ml enzymes led to a reduction in acetic acid, compared to the untreated yeast itself, of 54%, going from a concentration of 5.7 g/ l to a rate of 3 g/l and an almost total removal of malic acid (Fig.2).

Example 2

Induction of endogenous malate dehydrogenase in yeast starter

Oleic acid (0.015 -0.5 g/l), polysorbate (polyoxyethylenesorbate monooleate, 1-10 g/l) and ethanol (20g/l) were administered both to a S. Cerevisiae yeast starter and to a wine red (?Carignano 2019?) inoculated with 0.2 g/l of the yeast starter, so? to simulate the wine fermentation process.

The tests were conducted in shake flasks incubated in different operating conditions.

The experimental matrices were incubated at different temperatures, from 20°C to 30°C, with static or shaking incubation for a time between 2 and 6 days.

Example 3

Production process of the wine product

Yes ? first isolated a strain of S. Cerevisiae yeast by plating serial dilutions of the yeast starter obtained in example 2 on a selective medium for yeasts and in the presence of a bacteriostatic antibiotic to inhibit the growth of contaminated bacteria (Sabouraud Dextrose Agar with Chloramphenicol). Yeast colonies were then isolated and used to produce pure inocula of the yeast monoculture.

In a first test, the yeast ? was first grown on a medium rich with glucose as a carbon source and subsequently the biomass, recovered by centrifugation, was was transferred to minimal medium in the presence of sodium acetate.

In a second test, the yeast ? was grown directly in organic soils free of sugars and with sodium acetate as the sole carbon source.

The different culture media were prepared using the compounds in Table 1.

At the end of these tests, culture conditions 1-3 were selected, reported in table 6 below.

Table 6

The yeast ? been cultivated in these conditions, then the yeast cells were recovered by centrifugation and used as they were (active biomass), or lysed and then using the lysate, to catalyze the degradation of malic and acetic acid, in a starter substrate of the same yeast and in the wine.

The parameters evaluated at the end of the fermentation process, in the different conditions, are:

- the biomass yield, to determine the productivity? in terms of quantity? of cells produced

- the activity? malate dehydrogenase enzyme analysis to determine the degree of expression of this enzyme

- the quantity? of total proteins in the enzyme lysate to determine the percentage of the enzyme of interest on the yeast protein expression profile and the performance of the cell lysis process.

The yield and activity data? obtained by cultivating the yeasts in the conditions of table 3 are shown in table 7 below:

Table 7

Growth in organic medium (Condition 2) supplemented with a sugar (glucose) generates a greater quantity? of biomass and greater enzymatic expression. On the other hand, growth in organic medium supplemented with sodium acetate (Condition 3) allows achieving a lower biomass yield but a much higher enzyme-to-protein ratio. advantageous.

At the end of the enzymatic evaluation tests, the lysates (here indicated as 1, 2 or 3 depending on the Growth Conditions 1, 2 or 3 of table 7) were used as biocatalysts for the degradation of organic acids from yeast starters and from Carignano 2019 wine.

In particular, the lysates obtained were added at a concentration of 20 g/l to a yeast starter or to wine, so the reactions were as follows? prepared incubated at 25 ?C for 24 hours.

The addition of lysate 2 to the wine led to a 20% reduction in the malic acid contained in it in 24 hours, going from 3.7 g/l to 2.9 g/l (Fig.3).

The addition of the lysates to the yeast starter led to a notable reduction in the acetic acid it contained within 24 hours, as shown in Table 8 and Fig.4.

Table 8

Example 4

Development of the Fed-batch production process

? The Fed-Batch fermentation process for the production of the wine product was developed here.

During the process, the following parameters were monitored and kept constant via set-points (Table 9):

Table 9

Oxygenation? was maintained at the values indicated by increasing the agitation, with a cascade system on the regulation of the engine revolutions.

The first phase of biomass growth? was conducted while maintaining the pH constant through a pH-stat system. The pH correction occurs by basification with NH3. In the induction phase of the endogenous enzymes, by adding acetate salt to the soil, the pH correction occurs through acidification with H3PO4, which is necessary since in this phase a shift in the yeast metabolism occurs which leads to a reduction in the pH.

Example 5

Acid degradation in wine with whole cells

The wine ? was subjected to degradation using a comparison yeast (Comp. Yeast) and the oenological product according to the invention (Induced Yeast), obtained through the fermentation process of example 3 with condition 3.

The tests were conducted under the same conditions:

- Yeast inoculation: 0.1% of the volume of wine

- T= 22 ?C

- Static Incubation

- Duration = 5 days

At the end of the indicated time, the treated wine is was subjected to HPLC analysis to determine the content of malic acid and acetic acid.

From the results of the degradation tests (Table 10) it can be seen:

- a 50% degradation of acetic acid with the product of the invention compared to a 23% degradation with the comparison yeast.

- 100% degradation of malic acid with the product of the invention compared to a 40% degradation with the comparison yeast.

Table 10

Example 6

Acid degradation in wine with starter lysate

The biomass obtained from Fed-Batch fermentation according to the process of the invention of example 4, stored at 4°C, is was subjected to cell lysis by continuous sonication for approximately 2 hours at 50 ml/min with a sonicator (Hielscher UP200ST laboratory unit, power 100% pressure 40 atm T? 20?C).

The degree of lysis? was monitored through observation under an optical microscope as shown in Figure 7.

The lysate? It was then used as a biocatalyst to degrade organic acids from both the yeast starter itself and the wine.

In this test? was a quantity used? of lysate corresponding to an inoculum of 0.1% of whole cells, on the volume of starter or wine.

The addition of the lysate to the starter led to a reduction of 80% of acetic acid and 20% of malic acid in the wine in 24 hours (Fig.5).

Example 7

Acid degradation in red and white must with whole yeast cells

Following Fed-Batch fermentation of the yeast according to the process of the invention of example 4, it is The biotransformation of musts, red must and white must was carried out, with the resulting wine product.

The red must? been preserved at 4? C and at -20? C, the white must at -20? C for the control analysis of the untreated at the end of fermentation.

For the biotransformation 2L glass bottles were used and in each? 1L of red must and white must were placed.

The must? was inoculated at 0.1% weight/volume with the fermentation product. On top of the bottles? a foam cap was placed and they were left at room temperature in static conditions for 6 days.

At the end of the biotransformation? A sample was taken from each bottle to perform HPLC analysis.

The results of the HPLC analysis above highlighted the following concentrations of malic acid and acetic acid (Table 11, Fig.6 A and B).

Table 11

The results show that during the biotransformation of both the white must and the red must, the concentration of acetic acid was ? below 0.1 g/l. The concentration of malic acid, however, is ? higher in the red must, around 0.3 g/l while in the white must it is? very reduced.

Example 8

Production process of the wine product

Yeast of the S. Cerevisiae species? was placed to grow in a culture medium comprising: 15 g/L casein peptone, 10 g/L malt extract and 10 g/L yeast extract.

The culture? was stirred at 400-500 rpm, with the addition of oxygen at 10-15 mg/l/day (40% dissolved oxygen), at a temperature of approximately 28°C, for 2 days.

At the end of the yeast growth phase (when the growth plateau is reached due to the exhaustion of organic sources), the soil is ? sodium acetate (10 g/L) was added to induce the metabolic pathways of interest (induction phase). This induction phase? lasted for 2 days.

At the end of the induction phase, the yeast is been recovered by centrifugation (active yeast) and part of it? been dried and lysed (lysed inactive yeast). The active, inactive yeast and/or the lysate individually or together constitute the oenological product of the invention.

Active yeast, optionally together with inactive yeast and lysate, is intended for the fermentation of musts and wines, to activate alcoholic fermentation and to reduce or eliminate malic, lactic and acetic acids.

The inactive and lysed yeast? also intended for the fermentation of musts and wines, as an activator and as a corrector of organoleptic defects during the maturation, refinement and aging phases of the wine. It, can? can also be used added to active yeast to improve the activities? metabolic processes of this product.

The product of the present invention allows to obtain a very important reduction of the analyzed acids, both in the solution of the strain itself and on the treated products (must and wine), even in conditions considered prohibitive. This surprisingly solves many of the problems that arise in the cellar (degradation and transformations of organic acids into harmless compounds), improving the general performance of the fermentation and the wine produced.

Claims (11)

1. Process for the preparation of an oenological product comprising a yeast, or a yeast derivative, said process comprising in succession the phases of: a) provide a yeast, preferably of the S. Cerevisiae species; b) cultivating said yeast in a culture medium comprising an acetic acid salt, preferably at a concentration of 5-20 g/l of medium, c) recover the yeast. 2. The process of claim 1, wherein said acetic acid salt is? an acetate salt of an alkali metal, preferably sodium acetate. 3. The process of any of claims 1-2, in which step b) ? conducted at a pH between 5 and 7, preferably approximately equal to 6. 4. The process of any of claims 1-3, in which in phase b) oxygenation is greater than or equal to 40% dissolved oxygen (dO2). 5. The process of any of claims 1-4, wherein step b) is? preceded by a first phase b0) of yeast growth in a first medium comprising at least one sugar, preferably glucose, and in which the yeast biomass obtained at the end of phase b0)? collected and transferred to a second medium, said second medium being the medium comprising the acetic acid salt and not including sugars. 6. The process of any of claims 1-5, further comprising: d) add at least one of the following to the culture medium: acetyl-coA synthase enzyme, malate dehydrogenase enzyme, lactate dehydrogenase enzyme. 7. The process of any of claims 1-6, further comprising a step of incubating the yeast with a solution capable of inducing the yeast's endogenous malate dehydrogenase enzyme, preferably comprising at least one of a long-chain carboxylic acid, polysorbate and ethanol, and in which phase of incubating the yeast is? preceding or contemporary with step b) of cultivating the yeast in the medium comprising an acetic acid salt. 8. The process of any of claims 1-7, further comprising the step of: d) lyse the yeast recovered in step c), preferably by sonication. 9. Oenological product comprising yeast, preferably of the S. Cerevisiae species, or a derivative of said yeast, obtainable at the end of the process of any of claims 1-8. 10. Use of the oenological product of claim 9 in a winemaking process. 11. Winemaking process comprising adding the oenological product of claim 9 to the must, or to the wine, optionally together with a further yeast.