DESCRIPTION

Field of the invention The present invention generally relates to the field of oenological productions, and more precisely it refers to a yeast strain of the Schizosaccharomyces japonicus species that, once thermally inactivated, has been shown to be able to significantly improve protein stability of wine and is therefore useful for preparing additive products for wine-making State of the Art

Proteins in wine, although present in low concentrations, have a significant influence on its stability and clarity. A wine that is not stable in terms of proteins, particularly if subjected to thermal stress, could be subject to clouding phenomena after bottling. To avoid this type of problem, two different approaches have so far been used: eliminating the proteins present in the wine (subtractive treatment) or preventing the proteins present in the wine from precipitating by adding stabilising agents (additive treatment).

Currently, the treatment most commonly used in wineries is a subtractive treatment and consists of adding bentonite, a cation exchange clay used in oenology since 1930. Bentonite, by binding to proteins present in wine through electrostatic interactions, forms complexes that can then be removed by filtration. This treatment, although very effective, has several drawbacks: i) removal of important aromatic compounds, ii) decrease in colour, iii) loss of wine, estimated at between 3 and 5% of the volume treated and at a cost of around $1 billion per year, iv) cost of disposing of the spent bentonite.

Because of the many negative implications associated with the use of bentonite, various alternatives to its use have been explored over the years, some of which are still being tested: examples thereof are proteases, capable of degrading the proteins present; carrageenan, a polymer extracted from red algae and negatively charged and therefore capable of flocculating and precipitating proteins, positively charged at the pH of the wine; chitin, chitosan and zirconium oxide, capable of adsorbing unstable proteins. To date, however, none of the aforesaid alternatives has been found to be satisfactory. In fact, there are several drawbacks deriving from their possible use: carrageenan and other polysaccharides, such as agar and alginic acid, can in fact have a negative impact on the filterability of wines; furthermore, possible carrageenan residues can cause clouding phenomena in the wine. Chitin, on the other hand, despite its proven efficacy in removing proteins from wine, has been reported in some studies to have a negative impact on the aromatic component of wine. The use of zirconium oxide in large quantities in a wine can also limit its marketing; furthermore, some studies have shown a decrease in the aromatic component of the wine due to the use of this product, which has, among other things, the drawback of being necessarily used in discontinuous mode, periodically removed to be regenerated and reused. The addition of proteases also proved not to be very effective, especially at room temperature, due to the high protease resistance of the proteins responsible for the protein instability of the wine, mainly chitinases and thaumatins. This is why commercially available protease-based enzyme preparations are able to degrade unstable proteins of wine only after heat treatment, which denatures proteins and makes them more easily attacked by the proteolytic enzymes present in commercial preparations. Obviously, there is great resistance on the part of producers to allowing heating treatments on wines, both because of the cost associated with increased energy consumption and the possible negative impact that the heating could have on the sensory characteristics of the wine.

Mannoproteins derived from yeast of the genus Saccharomyces have also been proposed to increase protein stability of wines. Mannoproteins represent the main source of polysaccharides that can be found in wines and are naturally released from the yeast cell wall during alcoholic fermentation following cell multiplication. During aging of the wines "sur lies " (from the French for "on the lees"), an increase in the concentration of mannoproteins in the respective wines can also be observed, but in this case as a consequence of cell lysis. Several scientific works have in fact highlighted multiple advantages associated with these compounds, including: reduction of protein and tartaric instability, improvement of the mouth-feel, increase in sweetness and roundness, decrease in astringency, prevention of aggregation and precipitation of tannins, increased complexity and aromatic persistence, colour stabilisation of red wines and foam stability in sparkling wines. The yeast Saccharomyces cerevisiae, which is naturally present in wines and the main player in alcoholic fermentation, releases a quantity of mannoproteins into the wine, normally between 50 and 150 mg/litre. However, this quantity is not sufficient to guarantee the various positive effects mentioned above and associated with these proteins. Therefore, in order to increase the mannoprotein content in wine, several producers have started to add commercial preparations containing polysaccharides derived from the cell walls of yeasts of the genus Saccharomyces to wine. Preparations of this type, already available on the market, have different activities depending on the type of extracted and purified glycoproteins. For example, some researchers have identified, in the cell wall of a yeast of the genus Saccharomyces, a mannoprotein having a molecular weight of 40 kDa, which is particularly effective at tartaric stabilisation of wine, i.e. at preventing the crystalline formations of potassium tartrates that can form in wines. Today, this mannoprotein is extracted from the yeast cell wall by enzymatic treatment, purified and marketed for the tartaric stabilisation of wines. Among the glycoproteins released by S. cerevisiae, an invertase fragment with a molecular weight of 32KDa was also identified, which appears to have a protective action against the precipitation of proteins or protein casse. However, apart from the efficacy of the latter purified extract, it should be pointed out that its industrial production by enzymatic extraction and subsequent purification was not cost-effective.

In the light of the above, it is clear that there is still a great need to identify an effective treatment for protein stabilisation of wine that is also cost-effective and respects the organoleptic properties of wine. Recently, other yeast derivatives have been proposed, such as autolysates, yeast hulls and inactivated matters of the genus Saccharomyces obtained by different physico-chemical treatments. However, these derivatives contribute only minimally to protein stabilisation or even, in the vast majority of the cases, lead to a deterioration in protein stability, and this would depend on the type of treatment used (thermal, enzymatic, high pressures, etc.) to obtain the derivative itself. Treatments using high temperatures for inactivation were considered negative for the efficacy of the inactivated yeast in winemaking as they would lead to alterations in the tertiary and quaternary structure of the yeast proteins.

Summary of the invention

The inventors have now identified a yeast strain belonging to the Schizosaccharomyces japonicus species which, following thermal inactivation, has been shown to be able to give wines to which it is added a significantly high protein stability, and also to some extent an improved tartaric stability.

In particular, as further described in detail in the experimental part, a thermally inactivated yeast strain of the invention, which is thus added both to grape musts and to some white and rose wines that showed protein instability, has showed a significant improvement in protein stability compared to the same products not treated with the present inactivated yeast, or even treated with commercial products based on inactivated yeasts, identified below.

Therefore, subject of the invention is a yeast strain belonging to the Schizosaccharomyces japonicus species registered on September 30, 2020, with No. 42P in the Industrial Yeast Collection of DBVPG, Department of Agricultural, Food and Environmental Sciences - University of Perugia - Borgo XX Giugno, 74, 06121 Perugia, Italy, as defined in the first of the appended claims.

A further subject of the invention is an additive product for winemaking comprising the aforesaid thermally inactivated yeast strain, in dried, freeze-dried or paste form, whose essential characteristics are defined in the related independent claim appended hereto.

Still a further subject of the invention is the use of the aforesaid additive product in the fermentation of grape must or as an additive in wine prior to bottling; and a winemaking process comprising the addition of the aforesaid additive product comprising the inactivated yeast to grape must prior to fermentation or to wine prior to bottling, as in the related independent claims appended hereto.

Other important characteristics of the yeast strain, the winemaking additive, its use in winemaking, and the winemaking process according to the invention are defined in the dependent claims, and illustrated in the following detailed description.

Brief description of the figures Figure 1 shows the electrophoretic profile of the proteins characterising the soluble component of the thermally inactivated matter, with gels stained with Coomassie G-250 (A) and with Schiff's reagent (B).

Figure 2 is a graph of the percentage of CO2 produced during fermentation trials carried out by adding only the commercial strain EC1118 S. cerevisiae (control) to the must, or also 20 g/hL of the inactivated yeast of the invention (thermally inactivated), or (for comparison) 20 g/hL of a commercial inactivated yeast of the S. cerevisiae species (Surli One) or a commercial yeast hull of the S. cerevisiae species (Enartis Green Nutriente). The data in the figure represent the mean of the three replications ± standard deviation.

Figure 3 shows, in the form of histograms, the ethanol concentrations measured for the 4 types of fermented matters tested, the control with only commercial strain EC1118 of S. cerevisiae (C), or also 20 g/hL of inactivated yeast of the invention (IT), or (for comparison) 20 g/hL of commercial inactivated yeast of the species S. cerevisiae Surli One (CS) and of yeast hull of the species S. cerevisiae Green Nutriente (CG). The data in the figure represent the mean of the three replications ± standard deviation.

Figure 4 shows, in the form of histograms, the concentrations of polysaccharides measured for the 4 types of fermented matters tested, the control with only commercial strain EC1118 of S. cerevisiae (C), or also 20 g/hL of inactivated yeast of the invention (IT), or (for comparison) 20 g/hL of a commercial inactivated yeast of the species S. cerevisiae Surli One (CS) and of yeast hull of the S. cerevisiae Green Nutriente (CG). The data in the figure represent the mean of the three replications ± standard deviation.

Figure 5 shows in the form of histograms the ANTU (Nephelometric Turbidity Unit) values measured after the hot test with a nephelometer for each sample tested, of the control (C), of the invention (IT) and of the comparisons (CG, CS). The data in the figure represent the mean of the three replications ± standard deviation.

Figure 6 shows, in the form of histograms, the values of difference in concentration of tartaric acid (before and after the cold treatment) detected for the fermented samples added with the yeast of the invention (IT) or with the commercial additives (CG, CS), and for the control samples without additives (C). The data in the figure represent the mean of the three replications ± standard deviation.

Figure 7 reports, in the form of histograms, the DNTII values obtained after the hot test on samples of Vernaccia white wine added respectively with 5, 10, 20 and 40 g/hL of the yeast of the invention and untreated (control). The data in the figure represent the mean of the three replications ± standard deviation.

Figure 8 shows, in the form of histograms, the DNTII values obtained after the hot test on samples of Trebbiano/Malvasia white wine added with 5, 10, and 20 g/hL of the yeast of the invention, and untreated (control). The data in the figure represent the mean of the three replications ± standard deviation.

Figure 9 shows, in the form of histograms, the DNTII values obtained after the hot test on samples of Vermentino white wine added with 5, 10, and 20 g/hL of the yeast of the invention, and untreated (control). The data in the figure represent the mean of the three replications ± standard deviation. Figure 10 shows the electrophoretic profile of the proteins present in the

Vernaccia wine sample, both hot-tested (C,D) and not hot-tested (A,B), with and without the addition of different concentrations of inactivated yeast. In the plates TQ indicates the wine sample as such, S indicates the supernatant fractions after hot test, P indicates pellet fractions after hot test. A,C are gels stained with Coomassie and B,D with Schiff's reagent.

Figure 11 shows, in the form of histograms, the ANTU values obtained after the hot test with tannin on two samples of Teroldego rose wine added with 15 g/hL of the yeast of the invention (IT) or 15 g/hL of a commercial yeast hull S. cerevisiae Surli Elevage (SE) and untreated (control). Figure 12 shows in the form of histograms the values of dye intensity (IC), calculated as the sum of the optical densities at 420, 520 and 620 nm, and of hue, calculated as the ratio between the optical density at 420 nm and the optical density at 520 nm, of the samples of Teroldego rose, added with 15 g/hL of the yeast of the invention (IT) or with a commercial yeast hull S. cerevisiae Surli Elevage (SE), and of untreated wine (control). Figure 13 shows, in the form of histograms, the percentage values of reduction in protein instability of an untreated Chardonnay wine (control, C) when added with 20 g/hL of the inactivated yeast of this invention (IT), or with 20 g/hL of two inactivated yeasts of Sch. japonicus of the state of the art UCD 2489 and UCD 2096. Figure 14 shows, in the form of histograms, the percentage values of reduction in protein instability of an untreated rose wine (control, C) when added with 20 g/hL of the inactivated yeast of this invention (IT), or with 20 g/hL of two inactivated yeasts of Sch. japonicus of the state of the art UCD 2489 and UCD 2096.

Figure 15 shows, in the form of histograms, the percentage values of reduction in protein instability of an untreated rose wine (control, C) when added with 20 g/hL of the inactivated yeast of this invention, in two different formulations, freeze-dried (LC) and spray-dried (SC).

Detailed description of the invention

The yeast strain belonging to the Sch. japonicus species of this invention was registered on September 30, 2020, with No. 42P in the Industrial Yeast Collection of DBVPG of the Industrial Yeast Collection of the Department of Agricultural, Food and Environmental Sciences of the University of Perugia, Borgo XX Giugni, 74 - 06121 Perugia, Italy. This yeast was isolated by the inventors from grapes in the Chianti area of Tuscany, Italy. A different strain of yeast Sch. japonicus, not inactivated, had been the subject of previous studies by the inventors (see for example Domizio P. et al. Am. J. Enol. Vitic. 69:3 (2018) 266-277), who had used it together with a yeast of the Saccharomyces cerevisiae species as a starter in winemaking trials, with less than encouraging results. Its addition during fermentation had resulted in an increase in compounds with a strong impact on the wine's aroma, such as ethyl acetate and acetaldehyde, and in an unacceptable level of volatile acidity in the wine obtained.

In the context of the present invention, the expression "inactivated yeast" means a yeast consisting essentially of cells that are no longer alive or active, and consequently can no longer produce the effects of live, non-inactivated yeast, such as for example fermentation. The expression "inactivated yeast" is a commonly used expression in particular in the wine sector and codified in monographs and manuals, including the monograph COEI-1-INAYEA by the OIV, International Organization of Vine and Wine, approved by the General Assembly of the OIV with resolution OIV- OENO 459-2013. In general, the inactivation of yeast biomasses can be carried out by means of heat and/or pH changes; in the first case, one speaks about "thermal inactivation", such as the inactivation of the yeast subject matter of the present invention.

Now, once thermally inactivated, the present inactivated yeast strain Sch. japonicus showed a completely different behaviour to the one mentioned above, both following addition in fermentation and when added directly to wines prior to bottling This addition, in fact, not only had significant effects on protein stability and, even if to a lesser extent, tartaric stability, but also showed no negative impact on colour, hue of wine or organoleptic characteristics.

The yeast identified above may be thermally inactivated to obtain the yeast of the invention in any suitable type of reactor equipped with heating means, for example in a sterilisation autoclave. In an embodiment of the invention, the yeast is inactivated by heating at a temperature ranging from 80 to 121 °C, for a time ranging from 1 to 4 hours. Preferably, thermal inactivation of the yeast according to the invention is carried out by heating at about 121 °C for about 60 minutes.

Protein stabilisation of wines by using the thermally inactivated matter of Sch. japonicus is more economically viable than that obtainable by using polysaccharides released by Sch. japonicus in the culture broth, as proposed in the state of the art, e.g. in Millarini V. et al. Foods, vol. 9, no. 10, (2020), 1407. In the latter case, in fact, the polysaccharides must be recovered from the culture medium by ultrafiltration, thus involving an additional costly step in the production process. Upstream of inactivation, the production of the yeast biomass of the invention can be carried out using for example as a growth substrate the medium consisting of yeast extract, peptone and D- glucose or dextrose, known as YPD medium (Yeast extract - Peptone - Dextrose). The growth temperature may range for example between about 25°C and about 30°C, and preferably is of about 27°C. The yeast cells thus grown can be recovered by centrifugation, washed and suspended in a suitable liquid, for example in phosphate buffer, before being subjected to thermal inactivation under the conditions outlined above.

The inactivated yeast of this invention is then used to prepare an additive product for winemaking, which may comprise, in addition to yeast, further active ingredients/excipients. The additive product comprising the inactivated yeast of the invention may be in dried or freeze-dried form or in paste form. In a preferred aspect of the present invention, the additive product comprising the inactivated yeast of the invention is in a spray-dried form.

In one aspect of the invention, the above additive product may be used by adding it prior to fermentation to the grape must or it may be added directly to the wine prior to bottling.

Particularly significant results of improved protein stability were achieved by the inactivated yeast of this invention when added to white grape must, or to white or rose wine. In one aspect, the winemaking process is the subject of this invention wherein the additive product comprising the inactivated yeast is added to the grape must prior to fermentation.

According to the invention, the additive product comprising the present thermally inactivated yeast may be added typically in an amount of 5 to 40 g of thermally inactivated yeast per hl_ of grape must or wine. Any person skilled in the art with ordinary knowledge of the sector will in any case be able to choose different or specific optimal quantities within the range indicated above, depending on the type of wine or grape to be treated.

The advantages linked to the present invention are manifold. Firstly, as mentioned above, and demonstrated in the experimental part below, the thermally inactivated matter of the present yeast resulted in a significant stabilising effect at the protein level, both when added to the grape must before alcoholic fermentation and when added to the wine prior to bottling. This has been experimentally verified by the inventors on different types of wine, even on wines with medium to high instability.

The addition of the present inactivated yeast also results in a decrease in the tartaric instability of the wines, again both by addition to must and by addition to wine, and on different types of white and red wine.

The inactivated yeast of the invention also showed a positive effect on fermentation kinetics, which was accelerated by its addition. In view of the aforesaid positive effects, the addition of the inactivated yeast of the invention did not show any negative impact either on the colour and hue of the wine or on its organoleptic characteristics.

Last but not least, the advantage linked to the present invention is that it is possible to obtain the yeast ready to be used in winemaking by means of a heat treatment, therefore in a simple way, without the need for expensive plants, thus facilitating the industrial production.

The following experimental examples are provided in an illustrative and non limiting way of the present invention.

EXAMPLES Materials used

The yeast strain of this invention, of the Schizosaccharomyces japonicus species, tested in the experiments described below, was registered in the Industrial Yeast Collection of DBVPG of the University of Perugia, with No. 42P on September 30, 2020. A non-inactivated yeast strain of the Saccharomyces cerevisiae species, marketed by Lallemand under the trade name Lalvin EC1118™, was used in the following experimental trials. In the examples and figures attached here, the trials carried out by adding yeast EC1118 alone (without adding the present inactivated yeast) have been used as a control and therefore also indicated as "C". A commercial product based on inactivated yeasts of the species S. cerevisiae,

Surli One, and a commercial product based on yeast hulls Green Nutriente by Enartis are used as comparison products in the fermentation trials reported below. In the examples and figures attached here, these products have also been referred to as "CS" and "CG", respectively. Surli One is produced starting from a baker's yeast strain of the Saccharomyces cerevisiae species. The strain is multiplied by the same process like the one used to produce an active yeast. When the desired amount of biomass is reached, the yeast suspension is washed and centrifuged to obtain the so-called yeast cream. The cream is then inactivated by heating the mass to approximately 105-110°C for 5-10 minutes. This is followed by drying with a spray-dryer. Prior to its commercialisation as Surli One, the inactivated yeast is added with an amount between 0.25 and 0.5 % of enzyme proteins (endo-1,3(4)-p-glucanase - CAS 62213-14-3) in order to accelerate the lysis of the inactivated yeast at the time of its use in the wine.

Green Nutriente by Enartis is instead composed of yeast hulls as defined in the monograph COEI-1-YEHULL approved by the OIV with Resolution Oeno 497-2013 and obtained starting from a baker's yeast of the Saccharomyces cerevisiae species. The production process is the same as described above for Surli One until the yeast cream is obtained. Once the yeast cream is obtained, it is allowed to stand for 12 to 24 hours at a temperature of around 45-50°C in order to promote cell lysis by the enzymes naturally present in the cell. At the end of the lysis phase, the cream is inactivated by heating at approximately 105-110°C for 5-10 minutes. For subsequent centrifugation, the yeast hulls are separated from the soluble phase and then dried with a spray-dryer.

The tested white and red wines came from wineries based in Tuscany, and were previously subjected to trials to determine their protein and tartaric stability respectively with hot test, under the same conditions as described below for the analysis of fermented products, and with mini-contact test (carried out with Checkstab a2016) and/or cold test (with evaluation of the concentration of tartaric acid before and after the test), also described in detail below for the analysis of fermented products.

The rose wines tested came from wineries based in Trentino Alto Adige, and were preliminarily subjected to trials to determine their protein stability with hot test with tannin described in detail below.

EXAMPLE 1 - Production of biomass and preparation of thermally inactivated matter of the yeast Sch. iaponicus of the invention

The inventors have identified the following optimal conditions for the production of biomass by this yeast, which can then be used for the preparation of inactivated yeast Sch. japonicus.

-Medium: Yeast extract 2%, peptone 2%, Dextrose 10%, pH 4.5 (YPD) -Temperature: 27°C

-Stirring: 150 rpm

Approximately 24 hours after inoculation of Sch. japonicus on YPD medium under the above conditions, the cells Sch. japonicus were harvested by centrifugation at 8800 rpm for 15 minutes. The resulting pellet was washed several times with sterile deionised water (~1:4) and resuspended (1:5) in 50 mM phosphate buffer, pH 7. The suspension was then heat-treated in an autoclave at a temperature of 121°C for 60 minutes (5 psi) in order to inactivate the yeast and, at the same time, to extract the glycoproteins that were not covalently linked to the cell wall by breaking the hydrogen bonds. After cooling, the suspension was frozen and then freeze-dried in a Modulyo freeze-dryer, Edwards Crawley, UK. After freeze-drying, the dry weight of inactivated yeast obtained per litre of culture was approximately 9.5 g.

The product thus obtained is referred to in the present description as "thermally inactivated matter of Sch. japonicus". EXAMPLE 2 - Characterisation of the thermally inactivated matter of the yeast

Sch. japonicus of the invention

The thermally inactivated product of Sch. japonicus obtained as described above in Example 1 contained both soluble material (e.g. polysaccharides) and insoluble material (e.g. inactivated cells, and cell walls). The polysaccharides present in the soluble fraction of the inactivated matter, released by the thermally inactivated matter of Sch. japonicus in a wine-like solution (12% ethanol, 0.03 M tartaric acid, pH 3.20), were characterised by chemical and electrophoretic analysis. Specifically, 1% of thermally inactivated matter of Sch. japonicus was added to the wine-like solution and kept under stirring for 48 hours at room temperature. The sample thus treated was then centrifuged and then filtered on cellulose acetate filters with 0.45 pm porosity to remove inactivated cells and cell walls (insoluble fraction).

The quantity of polysaccharides (glycoproteins) released into the medium was determined gravimetrically, after their precipitation with acidified ethanol (Usseglio- Tomasset and Castino method, 1975). In particular, the supernatant was added with 4 volumes of cold acidified ethanol for the precipitation of the polysaccharides (glycoproteins), and held at 4°C for approximately 16 hours. After centrifugation, the pellets were recovered, washed with 96% ethanol, dried and weighed. The pellet thus obtained was then used for the subsequent analyses.

The percentage of sugars characterising the glycoproteins was determined after their acid hydrolysis with trifluoroacetic acid (TFA), by ion chromatography (Dionex ICS-2500) with pulsed amperometric detector, Dionex CarboPacPAI column (4.6 x 250 mm, Thermo Scientific). Eluents used were: water (HPLC-grade water) (A), 0.185 M NaOH (B), and 0.488 M sodium acetate (C), mixed as follows: from injection to 20 min A:B = 90:10; from 20 to 30 min B:C = 50:50; from 30 to 60 min, A:B = 90:10. A flow of 1 mL/minute was used for the analysis. Sugars were identified on the basis of the retention time of known standards.

The concentration of the proteins present in the glycoproteins was assessed using the Bradford Protein Assay Bio-Rad kit (Bio-Rad Laboratories S.r.l., Milan). The electrophoretic profile of the proteins was evaluated by SDS-PAGE electrophoresis (Sodium Dodecyl Sulphate PolyAcrylamide Gel Electrophoresis) with the Mini PROTEAN II system (Bio-Rad Laboratories S.r.l., Milan). The gels were stained with both Bio-Safe Coomassie G-250 (Bio-Rad) and Schiff's reagent and periodic acid (PAS reaction).

For the determination of the thiol compounds of the soluble component of the inactivated matter, the method of Gallardo-Chacon et al. (2010) was used. In particular, following the reaction of 4,4'-dithiodipyridine (DTDP) with the thiols, compounds detectable by spectrophotometric reading at 324 nm are formed. The results were expressed in mg/L in relation to a calibration straight line made with different dilutions starting from a glutathione stock solution. The percentage antiradical activity (AAR%) was determined using the Molyneux method (Molyneux et al., 2004), using the free radical DPPH (1,1 -diphenyl-2- picrylhydrazyl radical). Spectrophotometric readings were taken by measuring absorbance at 517 nm. Anti-radical activity was assessed in relation to the decrease in absorbance after 20 minutes, i.e. after reaching a stable plateau. The percentage antiradical activity (AAR%) was calculated by applying the following formula:

A.R.% — (Ato-At2o/Ato) x 100 where A« is the DPPH reading and A _t 2o is the sample reading.

The results of the above-mentioned analyses made it thus possible to characterise the polysaccharides from the inactivated matter, released in solution. In particular, the quantity of polysaccharides (glycoproteins) was equal to 434.46 ± 30 mg/L. The glucose component of the glycoproteins is represented by: 47.61% mannose, 32.55% galactose, 19.16% glucose, 0.67% glucosamine. The protein content of the glycoproteins was equal to 0.5%. The electrophoretic profile of the proteins released in the hydroalcoholic solution showed mainly the presence of glycoproteins with a molecular weight > 250 KDa (Figure 1). The content of the thiol groups of the soluble component of the thermally inactivated matter was equal to 5 mg/g of inactivated matter. The percentage antiradical activity (AAR%) was equal to 56.73%.

The thermally inactivated matter of Sch. japonicus subject of the present invention was analysed by infrared - NIR spectroscopy and allowed to obtain the following characterisation:

Dry substance: 95.6%; Ash: 6.93%; Total Nitrogen: 8.45%; Ammoniacal Nitrogen: 0.16%; Carbohydrates: 32.5%; Total Fat: 4.91%; Saturated Fat: 0.94%; Monounsaturated Fat: 3.97%; Sterols: 0.65%; Ergosterol: 0.27 %

The content of the thiol groups present in the pellet of thermally inactivated matter is equal to 3.7 mg/g.

In the trials carried out and described in the following Examples, this thermally inactivated matter was added:

-to a white grape must, to evaluate the effect of the present yeast on the fermentation kinetics and on protein stabilisation of the respective wines obtained; -to different types of wine, which had different levels of protein and tartaric instability, in order to assess its impact on wine stability.

EXAMPLE 3 - Evaluation of the impact of the present thermally inactivated yeast strain Sch. japonicus on the fermentation of a white qrape must and on the protein stabilisation of the wines obtained To evaluate the impact of the thermally inactivated matter of the yeast Sch. japonicus of the invention on fermentation kinetics, 20 g/hL of this thermally inactivated matter was added to a must of Trebbiano/Malvasia grapes.

In Table 1 below, the chemical characteristics of Trebbiano/Malvasia’s must, as used in this test, are summarised.

Table 1

After inoculation with 25 g/hL of commercial active dry yeast Lalvin EC1118™ rehydrated according to the manufacturer's instructions, the must was divided into 250 ml aliquots and distributed in sterile 300 ml flasks. 20 g/hL of thermally inactivated matter of the yeast of the invention were added to each flask. In parallel, samples were also prepared to which, instead of the yeast of the invention, a commercial product based on inactivated yeast and a yeast hull S. cerevisiae were added in the same quantities. Surli One and Green Nutriente by Enartis were used as commercial products for these comparison trials. Three flasks, without any inactivated yeast added, were used as control trials.

The fermentations, set up in triplicate, were carried out at 25°C. The evolution of fermentation was assessed by recording the decrease in weight of the flasks, following CO2 loss until the end of fermentation (i.e. when a constant weight was detected for two consecutive days). Figure 2 shows the fermentation kinetics of the trials carried out with the commercial strain EC1118 of S. cerevisiae alone (control, C), or with it added with 20 g/hL of inactivated yeast of the invention, or added with commercial inactivated yeasts (Surli One, CS, and Green Nutriente, CG). As can be seen from the figure, compared to the control, the fermentation carried out with the yeast of the invention has shown faster kinetics since the first days of alcoholic fermentation, comparable to that of the trials carried out with the other two commercial inactivated yeasts. At the end of alcoholic fermentation, an aliquot was taken from each flask to determine the dry weight. The dry weight of the biomass obtained, after 14 days of fermentation, was very similar to the control for the samples with the yeast of the invention added. The remaining quantity of fermented matter in the flasks was centrifuged and filtered with 0.45 mGP cellulose acetate filters and used for subsequent analyses. In particular, as described in detail below, analyses were carried out to assess the basic chemical parameters, the concentration of proteins and total polysaccharides, and to evaluate the electrophoretic profile of the fermented matters. Finally, the protein and tartaric stability of the resulting wine was evaluated. Analysis of basic chemical parameters

At the end of fermentation, each sample was analysed for determining basic chemical parameters (alcohol, residual sugars, pH, total acidity, volatile acidity) using Winescan (FOSS, Denmark).

Figure 3 shows, in the form of histograms, the ethanol concentrations measured for the 4 types of samples, and indicates a higher alcohol content, although not significantly different, for the sample with the yeast of the invention compared to control C.

Determination of protein content

The protein content analysis was carried out on the fermentation products using the Bradford Protein Assay Bio-Rad kit (Bio-Rad Laboratories S.r.l., Milan) and a protein concentration in the different fermented samples tested was found to be similar to each other and ranged from 540 to 577 mg/L.

Electrophoretic profile of the fermented matters

The electrophoretic profile of each fermented matter was evaluated by SDS- PAGE electrophoresis (Sodium Dodecyl Sulphate PolyAcrylamide Gel Electrophoresis) using the Mini PROTEAN II system (Bio-Rad Laboratories S.r.l., Milan). The wine samples obtained at the end of alcoholic fermentation were precipitated with acidified cold ethanol (96%). The gels were stained both with Bio-Safe Coomassie G-250 (Bio- Rad) and with Schiff's reagent and periodic acid (PAS reaction), obtaining similar profiles for the control fermented matter containing only the commercial yeast of the strain S. cerevisiae and for the fermented matter with the yeast of the invention added, with the same bands present in the two different profiles, likely belonging already to the starting must.

Determination of total polysaccharide content

The concentration of total polysaccharides in the fermented matters was determined by high-performance liquid chromatography (HPLC Varian Inc., USA) with refractometric detector, TSK-GEL G-Oligo-PW column (Supelco 808031) and TSK- GEL Oligo guard column (Supelco 808034), using a NaCI solution (0.2 M) as eluent with a flow of 0.8 mL/minute, at a temperature of 65°C. A calibration straight line, constructed using a commercial mannane preparation (Sigma Aldrich), was used to calculate the concentrations. Figure 4 shows, in the form of histograms, the concentrations found for the 4 different samples, which all show similar concentrations of polysaccharides, but higher for the fermented matters with the yeast of the invention added.

Protein stability of the fermented matters The protein stability of the fermented matters was evaluated by means of a hot test carried out in the following ways: temperature of 80°C for 2 hours; temperature of 4°C for 16 hours; and room temperature for 2 hours. Turbidity was assessed by nephelometric reading, using the using the HI83749 turbidimeter (Hanna Instruments Italia S.r.l.) Figure 5 shows the ANTU (Nephelometric Turbidity Unit) values observed after the hot test, indicating the turbidity measured by nephelometer for each sample tested, of the invention (IT) and of control (C) and of reference (CG, CS). Control C showed an ANTU value equal to 5.3, while the three replications with the sample of the invention showed values equal to about 3. The samples with the two commercial inactivated matters CG and CS instead showed ANTU values of 4.0 and 4.6, respectively.

This figure therefore indicates that the addition of the yeast of the invention during fermentation significantly contributed to protein stabilisation of wine obtained, whereas the addition of commercial products based on inactivated strains of S. cerevisiae contributed to a lesser extent to protein stabilisation. The result obtained with the yeast of this invention is interesting if one also considers that wines with DNTII values of less than 2 are considered stable from a protein point of view.

Tartaric stability of the fermented matters The tartaric stability of the fermented matters was assessed by calculating the difference in the concentration of tartaric acid and potassium before and after the cold test (0°C, for 7 days) for the various samples, the results of which are shown as histograms in Figure 6 attached herein. According to what literature reports, wines with a tartaric acid concentration delta higher than 0.2 can be considered as tartarically unstable. Although values higher than 0.2 were observed for all the samples of fermented matters tested, it should nevertheless be pointed out that the wine obtained from the trials added with the yeast of the invention has a lower - although not significant - delta in the concentration of tartaric acid than that observed for the control with no inactivated yeast-based additives added, as can be seen in Figure 6. EXAMPLE 4 - Evaluation of the efficacy of the present thermally inactivated yeast strain Sch. iaponicus on protein and tartaric stability of wines

In order to evaluate the efficacy of the thermally inactivated matters of Sch. japonicus on the stability of wines, various wine samples from several wineries based in Tuscany were analysed. In particular, for the current trials, white wines with medium- high values of protein and tartaric instability and red wines with high tartaric instability were identified. Table 2 below shows the types of wine used with the respective values of both protein and tartaric instability found in the tests indicated in the table, carried out under standard conditions, as described above in the fermentation analysis methods. Table 2

Different amounts of inactivated matter of the yeast of the invention were added to the wines to evaluate their effect on protein stability. After the addition, the wines were kept under gentle stirring for 48 hours. The wines were then centrifuged and filtered with 0.45 mGP cellulose acetate membranes before performing the hot test. In parallel, samples of the same wines not treated with the yeast of the invention were used as controls.

Figure 7 shows the DNTII values obtained after the hot test of Vernaccia added with 5, 10, 20 and 40 g/hL of the yeast of the invention (IT), compared to those of the same untreated wine (control). The results obtained highlight, compared to the untreated control, a decrease in protein instability equal to about 30%, 54%, 57% and 62% following the addition of 5, 10, 20 and 40 g/hL respectively of the inactivated yeast of the invention.

Figures 8 and 9 show the DNTII values obtained after the hot test on Trebbiano/Malvasia wine and Vermentino wine respectively, added with 5, 10, 20 g/hL of yeast of the invention (IT).

In Vernaccia wine, a decrease in protein instability proportional to doses of 5 and 10 g/hL is observed. However, the further increase in the yeast dose in an amount of 20 and 40 g/hL does not correspond to a proportional decrease in instability.

Compared to the untreated wine used as a control, the 10 g/hL dose resulted in a decrease in instability of approximately 50% in the case of Vernaccia, and 30% and 15% in Trebbiano/Malvasia and Vermentino wines, respectively. In the latter two wines, increasing the dose of added yeast of the invention to 20 g/hL, an even greater protein stabilisation of the wine was observed, with a decrease in instability of 40% and 30%, respectively. The glycoproteins of the thermally inactivated matter, released into the wines after treatment, remain soluble in the wine even after heat treatment (temperature of 80°C x 2 hours) (Figure 10). This highlights their particular resistance to heat.

The protein profiles of Vernaccia wine as such (TQ) and of Vernaccia wine added with 30 g/hL of inactivated matter, both of which were subjected to heat treatment, are similar (Figure 10). Therefore, the inventors, although not wishing to bind themselves to a theory, believe that the protective effect of the thermally inactivated matter towards protein instability would not be a consequence of adsorption of the PR proteins on the wall of the inactivated matters, but is probably attributable to the soluble component of the inactivated matter itself.

With regard to the evaluation of tartaric stability, the selected white and red wines were added with the thermally inactivated matter of the yeast Sch. japonicus of the invention (10 g/hL and 30 g/hL) and subjected to cold test (7 days at 0°C), in parallel with samples of the same untreated wines as controls. At the time of yeast addition and at the end of 7 days at 0°C, the treated and control wines were analysed with the FOSS Analytics instrument (FOSS Italia S.r.l.) to determine the tartaric acid content. In addition, after 20 days, the samples were observed to assess the formation of potassium bitartrate (KHT) crystals.

The variation (delta) of concentration in tartaric acid between before and after cold treatment of the wines added with the thermally inactivated matter of the present yeast was lower or similar to that of the relative controls.

Table 3 below shows the results obtained in terms of the presence or absence of potassium tartrate crystals in treated wines compared to samples of the same untreated wines (control); if crystals are present, the quantity is indicated in the table.

Table 3

++ = presence of crystals in large amount + = presence of crystals in small amount - = crystals not present A decrease in the quantity of crystals was observed in all the white wines tested, added with the thermally inactivated matter of the present yeast. When the wines were treated with the yeast of the invention, although the amount of crystals present was comparable to that of the control, the crystals present were however much smaller in the treated wines. In order to evaluate the efficacy of the thermally inactivated matters of Sch. japonicus on protein stability of rose wines, two wine samples from wineries based in Trentino Alto Adige were analysed. In particular, rose wines with average protein instability values were identified for the current trials. The protein stability of the wines was evaluated by means of a hot test with tannin carried out as follows: 1 ml_ of 5% gallic tannin solution is added in 20 ml_ of wine filtered with cellulose acetate membrane with 0.45 mhi porosity and T1 turbidity is measured; the sample is heated to 80°C for 30 minutes, allowed to cool and then T2 turbidity is measured. Wine is considered stable if T2-T1 is less than 10 NTU. Turbidity was assessed by nephelometric reading, using the using the HI83749 turbidimeter (Hanna Instruments Italia S.r.l.)

Figure 11 shows the DNTII (Nephelometric Turbidity Unit) values observed after the hot test, indicating the turbidity measured by nephelometer for each sample tested, of the invention (IT) and of control (C), and of reference (SE). It is observed that in both wines, the addition of the yeast of the invention resulted in a decrease in protein instability between 27 and 29%, whereas the addition of the yeast hull S. cerevisiae contributed to a lesser extent. EXAMPLE 5 - Evaluation of the effect of the present thermally inactivated yeast strain Sch. japonicus on wine colour

Trials were carried out to assess the impact of the addition of the yeast of the invention on the wine with respect to the natural colour of red wine, white wine and rose wine treated and indicated above, and the results did not show any significant differences either in the intensity and in the hue of the colour detected.

The trials on white and red wine were carried out by keeping the wine samples added with the yeast at three different concentrations for each sample, namely 5, 10 and 20 g/hL, under slow stirring for 48 hours. The samples were then centrifuged and filtered on cellulose acetate filters with 0.45 mhi porosity, and several parameters were analysed: dye intensity (1C = DO420nm + DO520nm + DO620nm), hue (DO420nm: DO520nm), total polyphenol index, CIELab coordinates. The results obtained showed no significant differences in the colour parameters of the wines analysed.

The trials on rose wine were carried out by keeping the wine samples added with 15 g/hL of inactivated yeast of the invention (IT) under slow stirring for 48 hours in comparison with the same dose of a commercial yeast hull S. cerevisiae Surli Elevage (SE) and the untreated control. The impact of the treatment on colour was assessed by spectrophotometric measurements at 420 nm, 520 nm and 620 nm of wine samples, previously filtered with a cellulose acetate filter with 0.45 mhi porosity, and subsequent calculation of dye intensity (1C = DO420nm + DO520nm + DO620nm) and of hue

(DO420nm: DO520nm). Figure 12 shows the intensity (IC) and hue values found on wine as such, treated with the inactivated matter of the invention (IT) and treated with the commercial yeast hull S. cerevisiae showing that the treatment with (IT) has no impact on the colour of the wines. EXAMPLE 6 - Evaluation of the efficacy of the present thermally inactivated yeast strain Sch. japonicus on the protein stability of two different wines in comparison with two state-of-the-art yeast strain Sch. japonicus

Table 4 below shows the characteristics of the two wines tested in protein stabilisation trials: Table 4

Each of the two wines was tested after addition, in an amount of 20 g/hL, of the inactivated yeast of the invention and of two state-of-the-art yeast strains Sch. japonicus belonging to the collection UCDAVIS, UCD 2489 and UCD 2096, to evaluate the effect of these additions on the protein stability of the two wines. In parallel, a sample of the same two untreated wines was used as a control. The hot test carried out on the wines envisaged the following scheme: heating at 80°C for 2 hours, then holding at 4°C for a further 16 hours and finally room temperature for 2 hours.

The NTU values obtained after the above-mentioned hot test of the two wines added with the different yeast strains Acompared to those of the same untreated wines (control) are shown in Figures 13 and 14 annexed therein. The data in Figures 13 and 14 represent the mean of three replications ± standard deviation.

The results obtained highlight the greater ability of the thermally inactivated matter of Sch.japonicus of the invention (IT) to stabilise wine with a percentage reduction in protein instability corresponding to DNTII values ranging between 41% and 47%, in the two different types of wine compared to untreated wine. For the two inactivated yeast derivatives of Sch. japonicus belonging to the collection UCDAVIS (#2096 and 2489), the percentage of reduction in protein instability showed significantly lower values, of ANTU ranging between 25 and 29%, in the two different types of wine. EXAMPLE 7 - Evaluation of the efficacy of two different formulations of the present thermally inactivated yeast strain Sch. japonicus on protein stability of wines Table 5 below shows the characteristics of the rose wine tested in protein stabilisation trials:

Table 5 This rose wine was tested in a hot test after addition, in an amount of 20 g/hL, of the inactivated yeast of the invention in two different formulations, freeze-dried and spray-dried, to assess the effect of such additions on protein stability of wine, whether it was the same or different for the two different formulations. The freeze-dried formulation was obtained as described above. Furthermore, in order to obtain the spray-dried formulation, the suspension of inactivated yeast, produced in the same ways adopted in the preparation of the suspension intended for freeze-drying and described above, was then dried using the Mini Spray Dryer B290, Buchi. The cream flow rate in the feed was adjusted between 5 and 20 mL/minute. The air temperature at the inlet was 130-150°C while the product temperature inside the cyclone was 60- 80°C. The flow rate of the atomising air (at standard temperature and pressure) was 800 - 1700 litres/hour. The dried product thus obtained had a dry matter content > 92%.

In parallel, a sample of the same untreated wine was used as a control. The hot test carried out provided for the same temperature and timing scheme as reported above in Example 6.

Figure 15 shows the DNTII values obtained after the above-mentioned hot test of rose wine added with the two different formulations compared to those of the same untreated wine (control). The data in Figure 15 represent the mean of three replications ± standard deviation.

The results obtained show that both freeze-drying and spray-drying techniques of the inactivated yeast strain of Sch.japonicus of the invention allow, with the same concentration of derivative, to reach similar levels of stabilisation of the rose wine (similar values of decrease in percentage of DNTII compared to the respective untreated wine C). This is a very important aspect as, compared to freeze-drying, the spray-drying process involves considerable cost savings in the production of the formulation. Table 6 below shows the values of the spectrophotometric readings and CiELAB parameters, as defined by the International Commission CIE, related to rose wine treated with the two different formulations (freeze-dried and spray-dried) of the thermally inactivated matter of the invention (LC and SC, respectively) and those of their respective untreated control (C). The data reported represent the mean of three replications. There is no significant difference between freeze-dried thermally inactivated matter and spray dried thermally inactivated matter.

Table 6