FIELD OF THE ART

The present invention concerns a strain of yeast belonging to the speciesSaccharomyces bayanussubsp.uvarum, and a selection method for yeasts for oenological use from flowers. Furthermore, the invention refers to the use of the yeast strain in the fermentative production of foods.

State of the Art

The use of starter yeast cultures in wine production is a fundamental practice allowing the control and steering of the fermentative process.

The term selected yeast or selected starter is used to mean a yeast strain characterised by physiological, biochemical and oenological properties that are optimised with regard to the technological demands of the fermentation processes in terms of purity (Pretorius, 2000).

The use of selected yeasts ensures the prompt initiation of fermentation since the quality and quantity of yeast added to the must are carefully selected; it allows greater control of the fermentative process by the operator opting to conduct fermentation without said process being left to yeasts naturally present in grapes that do not always have positive characteristics; it reduces the problems of arrest or slowing-down of the process, typical of spontaneous fermentation processes; it achieves good sugar transformation yields into alcohol, reducing the possibility of other microorganisms becoming established in the wine, thus damaging it (for example acetic bacteria); it reduces or eliminates anomalous organoleptic characteristics and, by no means last, it contributes towards the standardisation of production of a given wine, thus allowing it to be recognisable by the consumer year after year (Fleet and Heard, 1993).

Predominantly, the yeast selection occurs by means of the isolation of yeast strains from must or grapes, that are subjected to fermentation and oenological characterisation testing. Said tests are very laborious and require a great deal of time, in that they envisage the study of numerous physiological and metabolic characteristics for the yeast. In addition, musts and grapes can contain multiple copies of the same strain, whereby there is a risk of isolating said multiple copies and thus conducting duplicate tests.

The important oenological characteristics can be subdivided into two groups: those with technological importance, influencing the progression of the vinification process, and those affecting quality, influencing the chemical and sensory characteristics of the wines (Zambonelli, 2003). The term technological characteristics is understood to mean the characteristics associated with the fermentation activity of the yeasts (absolute alcoholigenic power, fermentative force, fermentation initiation rate), while the quality characteristics include those characteristics contributing towards the aroma, and more generally, to the organoleptic quality of the wine, such as for example the production of glycerol, volatile substances and sulphurated compounds (Pretorius, 2000; Fleet, 2008).

The major technological characteristics include the fermentative or alcoholigenic power, which, according to Delfini, is the maximum alcohol level (understood as % ethanol) produced by a given yeast by means of the fermentation of a must containing excess sugar (Delfini, 1995). The main ethyl alcohol produced by the activity of yeasts under anoxic conditions is the compound exercising an inhibitory action on all microorganisms. Since, for oenological use, yeasts require significant performance characteristics, for example, for the production of wines with high alcohol content, also the resistance (alcohol tolerance) to ethyl alcohol has, evidently, a fundamental importance.

An additional important technological characteristic is the fermentative force (or rate of fermentation), expressing the rate at which the yeast initiates fermentation, even in the presence of antiseptics at legally permitted doses and at temperatures between 15° C. and 30° C. Rapid initiation of the fermentative process is one of the characteristics an oenological yeast must possess regardless of its type of use (fresh or dried) and therefore must be considered a fundamental requirement in the selection of yeasts for vinification.

An additional, very important characteristic is resistance to sulphur dioxide SO2, i.e. the ability to maintain unaltered or sufficiently high the fermentation rate in the presence of selective doses of SO2added to musts in order to avoid oxidation and achieve a suitable microbiological stability. Where SO2supplemented wines are produced, it is therefore important to have strains available that are resistant to this antiseptic, in order to avoid initiation problems affecting the fermentation process.

Another important aspect concerns the methods of growth, i.e. the behaviour of the yeast cells on completion of the budding process, since said characteristic can affect management of the fermentation process. Yeast growth may be distinguished between pulverulent, flocculent or aggregate growth. The sedimentation rate of the strain is thus an additional positive characteristic, since it aids wine clarification and filtration operations.

The strains that cloud wine in the most annoying and persistent manner are the foam generating strains. Foam generating capacity is a strain-dependent characteristic, associated with the hydrophobicity of the cell wall. In many strains (20-30%) the cells adhere to the CO2bubbles produced during fermentation, and these bubbles, upon arrival at the surface, rather than breaking up, fuse together and increase in volume, giving rise to foams which take on a grey-brown colour due to the presence of the cells. The absence or reduced production of foam is a positive characteristic, both in primary fermentations and in refermentations, since it reduces the volume occupied by the must.

With regard to the qualitative characteristics, to be subsequently described in detail, the main considerations include:low or zero production of hydrogen sulphide;the production or degradation of malic acid (as a function of the must and the oenological vintage);high glycerol production;low acetic acid production;high aromatic potential.

From the qualitative viewpoint, the sulphurated compounds mainly consist of hydrogen sulphide (or H2S) and sulphur dioxide (SO2); said compounds are always produced by theS. cerevisiaestrains commercially available as wine starters, even though in different and variable quantities depending on the strain.

Vinification using non-producing or low H2S producing yeasts is particularly advantageous for those varieties that are peculiarly predisposed to “reduction”. Indeed, hydrogen sulphide is the main compound of light mercaptans, and has the drawback of having the smell of rotten eggs. Wines displaying olfactive defects traced to the presence of H2S have concentrations of the latter varying between 0.8 and 80 μg/l (Suzzi and Tofalo, 2014). H2S can also react with other compounds present in wine, altering the aromatic profile. This is mainly produced during alcoholic fermentation, as a result of yeast action.

Hence, use of a yeast that does not produce, or produces little hydrogen sulphide, not only allows cleaner fermentations and more fragrant wines, but also delays and/or avoids racking on completion of fermentation, allowing the wine to remain longer on the lees, with consequent enrichment of mannoproteins and other substances resulting from yeast lysis. It is known that H2S production inSaccharomyces cerevisiaeranges from 0 μg/litre to 300 μg/litre and that the quantity produced is a strain-dependent characteristic. Furthermore, data from the literature shows that only about 1% of oenological strains is incapable of producing H2S (Zambonelli et al., 1984).

Also, SO2production by yeasts shows broad variability. Certain strains that produce significant quantities of SO2(100-120 mg/l) also have a tendency to produce significant quantities of acetaldehyde (to defend against said antiseptic) and the result, on completion of fermentation, is a high concentration of bound SO2, which can compromise wine quality. Hence, during oenological starter strain selection operations, preference should be given to strains that produce low levels of SO2(maximum of 10-20 mg/l) (Vincenzini et al., 2005).

With regard to the production of malic acid during alcoholic fermentation, this represents an advantage in the majority of vinification processes involving quality white grapes, in that the presence of high quantities of malic acid gives an increased sensation of freshness to the resulting wine, in particular an acid sensation, considered more intense, in comparison to tartaric acid.

An additional advantage is that the production of malic acid allows lower pH, with resulting increased sulphur dioxide activity and, in general, increased microbiological stability in addition to increased overall acidity. Said production by yeasts of oenological interest has been described in the known art for certain cryotolerant yeasts, capable of producing up to 1 g/l of malic acid during the fermentation process (Castellari et al., 1994).

Another secondary product of fermentation by yeasts is glycerol.

After ethanol and CO2, glycerol is the compound produced in greatest quantities during alcoholic fermentation. This significantly influences the “body” of the wine, contributing towards giving the wines the characteristics of “fullness” and “sweetness”, with a perception threshold corresponding to about 5.2 g/l. The concentration of glycerol present in the wine depends on the concentration of sugars present in the must, the fermentation conditions and the strain of yeast.

In particular, in vinification processes,Saccharomycescan produce from 2 to 10 g/l, depending on the yeast species and strain. Generally,S. cerevisiaeis the yeast producing the lowest quantities of glycerol, and this is the main reason for its high ethanol yield. In reality, this compound is not a secondary fermentation product since it is derived directly from the sugars, and, strain for strain, the quantity produced is proportional to their concentration (Zambonelli et al., 2000).

Among the secondary products, even though negative, a very important role is played by acetic acid, which, during fermentation, is derived from degradation of the sugars due to the oxidation of acetaldehyde (Ribéreau-Gayon, 2000). Besides the species, the ability to produce greater or reduced quantities of acetic acid is a characteristic that varies as a function of the strain.

Said ability to produce acetic acid is actually defined by the term fermentative purity, and is expressed as the ratio between volatile acidity formed and ethyl alcohol produced. The best strains are those that have the lowest fermentative purity value and, generally, the yeasts used for oenological ends should not produce quantities of acetic acid higher than 0.4 g/l.

However, the volatile acidity of wines can be the result of several origins, and is not only the result of yeast activity. Indeed, the final concentration of acetic acid in wine also depends on the quality of the grapes and the vinification conditions. Acetaldehyde is the most important carbonyl compound formed during fermentation, and represents over 90% of the total wine aldehyde content. It is a normal product of alcoholic fermentation, and its content in wine can vary significantly (10-300 mg/l) depending on the yeast strain and also the vinification conditions, and the concentration of sulphur dioxide, to which the acetaldehyde is bound. Evaluation of the acetaldehyde content is used as an indicator of the degree of oxidation of the wine. A low concentration of said compound in wine gives the latter a pleasant fruity aroma; however, at increasing concentrations, it gives rise to a pungent and oxidised odour and a herb-like flavour, so as to make the wine no longer marketable when the acetaldehyde concentration exceeds 500 mg/l.

For white wines, the mean value is generally 80 mg/l. It is known that one of the main factors determining acetaldehyde content variability is the strain of yeast. It is likewise also known that sulphur dioxide can induce the production of acetaldehyde by yeasts due to the correlation between the resistance of the yeasts themselves to sulphur dioxide.

The higher alcohols, generally represented in wine by n-propanol, isobutanol, amyl alcohol and 2-phenyl ethyl alcohol, are secondary yeast fermentation products, and are compounds that can significantly influence the aroma of wine. Said compounds are predominantly derived from metabolism of the aminoacids present in the must, but also from the metabolism of glucose without the involvement of precursor aminoacids. Indeed, there is no direct correlation between the quantities of aminoacids present in the must and the higher alcohols present in the wine. The production of said compounds is mainly due to the action of the yeast strains that can produce from 100 to 500 mg/l, depending on the composition of the medium, the availability of oxygen, the source of nitrogen and the initial sugar concentration. Generally, when their total concentration is below 300 mg/l, they lend a positive contribution to the complexity of the aroma, while when present in concentrations higher than 400 mg/l, they lend a negative quality to the product (Suzzi and Tofalo, 2014). The exception is 2-phenylethanol, which releases a rose-scented fragrance, and even at high concentrations contributes positively to the aroma of the wine (Vincenzini, 2005).

During fermentation, yeasts can produce esters resulting from the condensation of acetic acid and fatty acids, predominantly medium chain, with ethanol or other alcohols present in the wine, and themselves also produced by yeast metabolism. Yeast esterase activity is very important in the vinification process since it has a powerful effect on the sensory characteristics of the wine, producing the majority and most important of the fermentation aromas. The influence of esters on wine quality can be negative and unwelcome, particularly in the case of ethyl acetate, which confers the typical vinegar (or even solvent) scent. On the other hand, the production of isoamyl acetate or isobutyl acetate is positive, as it is responsible for a fruity character.

DESCRIPTION OF THE INVENTION

The scope of the invention is to provide a strain of yeast from the speciesSaccharomyces bayanussubsp.uvarum, equipped with the technological and oenological characteristics allowing good fermentation of the must, allowing the production of a pleasing wine with increased and balanced aromatic complexity.

An additional scope is to provide a strain that produces malic acid and glycerol in high quantities and, at the same time, does not produce hydrogen sulphide during alcoholic fermentation.

A further scope of the present invention is to identify a strain of yeast suitable for use as a parent strain for obtaining yeast hybrids with oenological potential. The provision of a yeast autolysate to be used as a fermentation activator is also a scope of the present invention.

By no means the final scope of the invention is the optimization of a perfected and rapid method for the selection of yeast strains for oenological use, particularly yeasts from the speciesSaccharomyces cerevisiaeandbayanusisolated from flowers.

The aforementioned scopes are achieved by means of the strain of yeast as characterised in claim1, and precisely by a strain ofSaccharomyces bayanussubsp.uvarumidentified as SERIUS, and deposited at the DBVPG with deposit number 36P, and by the selection method for yeast strains for oenological use, the characteristics of which are set out subsequently, and precisely, by a selection method for yeast strains of the speciesSaccharomyces cerevisiae, Saccharomyces bayanusandSaccharomyces bayanussubsp.uvarum, adapted for alcoholic fermentation, particularly of grapes, isolated from flowers.

The yeast strainSaccharomyces bayanussubsp.uvarumaccording to the invention possesses optimal oenological characteristics, both technological and qualitative, and has been isolated from flowers in an area of the Veneto region with high grape-wine producing activity. The strain has been identified by the name SERIUS, and has been deposited in accordance with the Treaty of Budapest, on 24 May 2016 with the “Industrial Yeasts Collection” DBVPG in Perugia, with deposit number DBVPG36P.

The strain has also been selected for its essentially non-existent hydrogen sulphide production, a very rare characteristic in yeast strains of the genusSaccharomyces. Indeed, on plates of Biggy agar culture medium, preferably used to verify the ability to produce H2S, the colonies are white, thus demonstrating the strain's inability to produce this metabolite.

Another positive aspect observed with this yeast concerns the production of glycerol. Indeed, in must, the strain produces glycerol in greater concentrations with respect to commercially availableS. cerevisiaestrains used as control.

Advantageously, the quantity of glycerol produced has been analysed in white berry grape musts and red berry grape musts and, with both types of grape, the quantity of glycerol naturally produced was greater with respect to the corresponding must obtained with the commercially available reference strain.

Advantageously, the wine produced using the strain according to the invention has a glycerol concentration naturally produced by the strain higher than about 8.5 g/l, preferably higher than about 9.5 g/l, more preferably higher than about 10 g/l.

By the term “produced by the strain” (herein and in similar contexts) it is understood that the compound has been produced as a result of fermentation using the strain, and has not been added artificially.

The strain according to the invention has also been selected for its unexpected ability to produce malic acid.

Advantageously, the strain of the invention has the ability to produce a wine with a quantity of malic acid higher than about 0.5 g/l, preferably higher than about 1 g/l, more preferably higher than about 2 g/l. This molecule is particularly advantageous in vinification processes, particularly for quality white grape processes, since it gives the wine an increased sensation of freshness.

An additional and advantageous aspect is given by the fact that a high quantity of malic acid makes it possible to have wine with lower pH, a characteristic allowing greater sulphur dioxide activity.

Still advantageously, the strain of the invention with its unexpectedly high malic acid production makes it possible to obtain increased microbiological stability for the wine, with increased total acidity.

Due to its technological qualities, the SERIUS strain is therefore markedly superior to the commercial strains currently available.

Advantageously, the strain has good fermentation capacity, with mean weight loss values analogous to those observed in commercial yeasts of the speciesSaccharomyces cerevisiaeand with fermentation closure times that vary as a function of the type of must used and the fermentation temperature, with durations, at optimal fermentation temperature, of between 10 to 15 days.

Still advantageously, the strain of the invention has good fermentative capacity, even at low temperatures, particularly at temperatures between 12° C. and 15° C., thus also allowing its use for the fermentation of wines at low temperatures, and hence increasing the possibility of activating specific known biochemical reactions leading to the production of aromatic compounds, adapted to improving wine quality.

For the strain of the invention, the ability to produce acetic acid in natural white and red berry must has also been assessed, and the results obtained demonstrate that the strain SERIUS is characterised by the ability to maintain very low volatile acidity values; in particular, the values obtained are generally lower compared to the values obtained from comparative fermentations with commercially availableS. cerevisiaestrains.

The strain of the present invention has also been selected based on its so-called killer activity, namely, the ability to produce toxins that cause the death of other yeasts; indeed, in the case where mixtures of strains are used, the strains to be used must necessarily be compatible with one another, namely, incapable of producing the killer toxins that can inhibit the other strains present in the mixture. From the analysis performed, it was advantageously shown that the strain SERIUS is not capable of inhibiting other yeasts and, even more advantageously, the strain has not in turn been inhibited by any of the yeasts tested.

The strain has been taxonomically classified at the species level by means of sequencing of the ITS (Internal Transcribed Spacer) region of the ribosomal operon, and subsequent compared by means of alignment with the homologous region available for the phylogenetically more related species of the genusSaccharomycesMeyen ex Rees (1870), in accordance with Vaughan-Martini&Martini; said molecular analysis has allowed identification of the strain SERIUS asSaccharomyces bayanussubsp.uvarum.

The strain has also been subjected to genotypic characterisation by means of analysis of the mitochondrial DNA restriction profiles using the enzyme Hinf I, as described by Zilio et al. and reported in detail in example 1, point 1.6 and, further, by means of the use of particular molecular markers, known as microsatellites, in accordance with a method, also described in detail in example 1, point 1.6.

If transferred to suitable culture medium under limiting nutrient conditions, the strain according to the invention is capable of sporulation. Improved conditions for inducing sporulation of the strain SERIUS are obtained in SP medium (1% potassium acetate; 0.1% yeast extract; 0.05% glucose; 1.8% agar) incubated at 25° C. for 7-10 days.

Other positive aspects found for this yeast have been observed during growth in synthetic and natural must (example 2).

The yeast forming the subject of this patent has the oenological characteristics reported below: sugars consumed after 2 days (fermentative vigour): about 4-5 g/100 ml; sugars consumed after 7 days: about 15-17 g/100 ml; 10-15 days: duration of fermentation at optimal temperature; low foam production.

A particularly advantageous positive aspect observed with this yeast concerns the production of H2S, which is substantially absent.

Furthermore, the strain is resistant to an SO2concentration of at least 50, preferably 100, even more preferably 200 and ulteriorly preferably 300 ppm.

The strain also has high resistance to copper, in particular it is resistant to a copper concentration of 300 μmol/l. Advantageously, said copper concentration has also been shown to be superior to the copper resistance of commercially available yeast strains, equal to about 50-100 μmol/l. Preferably, the strain is stored in frozen form (−80° C.) in the presence of appropriate cryoprotective agents (40% w/w glycerol) or preserved by means of lyophilisation/desiccation. Freezing or lyophilisation/desiccation are achieved using methods known to those skilled in the art.

The strain according to the invention may be advantageously industrialised in a cream form, desiccated form, lyophilized form, or in the form of a paste, depending on the requirements of the user.

The strain according to the invention is adapted to being used as a food inoculum in the production of foods, obtained through alcoholic fermentation. Foods that are producible with this yeast include for example bread, milk-based products, cider, bear, spumante and wine. Other alcoholic beverages obtained from the alcoholic fermentation of other fruits or cereals, such as wheat or rice, are also however conceivable. The strain is also adapted to being used as a probiotic in dietary supplements or in foods. Preliminary characterisation tests suggest this strain is resistant to acidity and bile salts, and therefore has the possibility of surviving gastric transit.

Advantageously, the strainS. bayanussubsp.uvarumSERIUS of the invention has been particularly suitable for the production of a yeast autolysate to be used as a fermentation activator in the production of wine and other alcoholic beverages.

According to one preferred executive variant of the invention, the strain is used as an inoculum in vinification in order to obtain wines, for example red wines, rosé wines or white wines. In addition, it can also be used in both primary fermentation and in refermentation.

Advantageously, use of the strain of the invention in a vinification process allows the production of a wine with pleasing and balanced aromas, with a substantially non-existent H2S concentration, high glycerol concentration and high malic acid concentration.

Advantageously, the wine is a wine produced from grapes selected from white berry grapes and red berry grapes. In addition, said grapes may be derived from both native vine varieties and from international vine varieties.

In particular, vinification tests performed on grapes selected from garganega, kerner, trebbiano, merlot, nerello mascalese, nero d'avola and traminer have made it possible to obtain wines with pleasing aroma and balanced flavour.

According to the preferred embodiment of the invention, the strain SERIUS is used for the production of wine containing glycerol and malic acid, produced naturally by the strain in the following concentrations: glycerol higher than about 8.5 g/l, preferably higher than about 9.5 g/l, more preferably higher than about 10 g/l; malic acid higher than about 0.5 g/l, preferably higher than about 1 g/l, more preferably higher than about 2 g/l. The strain SERIUS can be easily cultivated and is therefore particularly suited to being used to produce biomass, even on an industrial scale, using known standard methods.

Preferably, biomass production using the strain SERIUS occurs on a synthetic medium containing dextrose, yeast extract, vitamins and mineral salts, with batch-type fermentation (first 24 hours) followed by fed-batch type fermentation for a further 20-24 hours. Still preferably, growth occurs at a temperature of between 29 and 31° C. The strain produces ethanol and is therefore particularly applicable in food fermentation. The strain is not pathogenic for humans or animals.

Another aspect of the invention concerns a food inoculum consisting of the strain ofSaccharomyces bayanusidentified as SERIUS (DBVPG 36P). Preferably, the food inoculum is a wine starter.

An additional aspect of the invention concerns a wine obtainable by means of use according to the invention. Advantageously, the wine is a wine obtained from the fermentation of grapes selected from white berry grapes and red berry grapes. Advantageously, said grapes may come from both native vine varieties and from international vine varieties.

Cultures of the yeastSaccharomyces bayanussubsp.uvarumSERIUS, obtainable by means of the reproduction and/or multiplication of the strain SERIUS of the present invention, capable of being used as food inoculates in the production of foods obtained through alcoholic fermentation, are also to be understood as being protected by the present invention. According to the preferred executive variant of the present invention, said strains are adapted to being used in alcoholic fermentation processes for the production of wine, i.e. in vinification processes.

Particularly preferred are cultures obtainable from the reproduction or multiplication of the strain SERIUS of the invention, adapted to being used for the production of wine produced from grapes selected from white berry grapes and red berry grapes, wherein said grapes may come from both native vine varieties and from international vine varieties.

Preferably, said cultures are adapted to being used for the production of a wine containing glycerol and malic acid, produced naturally by the strains in the following concentrations: glycerol higher than about 8.5 g/l, preferably higher than about 9.5 g/l, more preferably higher than about 10 g/l; malic acid higher than about 0.5 g/l, preferably higher than about 1 g/l, more preferably higher than about 2 g/l.

Independently of their species, it is known that yeasts can reproduce sexually in order to ensure the survival of the species, creating genetic diversity, and hybridisation is an essential part of this sexual reproduction cycle, which can exist in nature as it is or be induced in the laboratory. The aforementioned hybridisation allows the attainment of novel yeast hybrids originating from a parental strain, according to a process that is absolutely non-GMO. Consequently, the establishment of hybrid strains is a mode of evolution for natural yeasts.

Hence, the yeast strains obtained through hybridisation of the strain SERIUS with a yeast strain belonging to the same species, or rather aSaccharomycesstrain, by means of a so-called intraspecies hybridisation, also form a part of the present invention.

However, it is not excluded that according to executive variants of the invention, strains may be obtained by hybridisation of the strain SERIUS with yeast strains from other species, by means of interspecies hybridisation.

Another aspect of the invention concerns a selection method for yeast strains from flowers, particularlySaccharomyces cerevisiae, Saccharomyces bayanusandSaccharomyces bayanussubsp.uvarumstrains, adapted to alcoholic fermentation, particularly of grapes. Obviously, sampling from flowers may be also transferred to other strain isolation matrices (for example fruit, medicinal herbs).

The selection method according to the present invention preferably consists of a step for yeast isolation from flowers, preferably a yeast identification step, a seeding phase for the isolated yeasts on plates, in order to identify strains that are non-producers or low producers of H2S, and an inoculation phase in must and the vinification of grapes with the purpose of studying their technological and quality characteristics.

Preferably, the grapes used are grapes selected from white berry grapes or red berry grapes.

According to the preferred embodiment of the method of the invention, the wine obtained during the aforementioned vinification step is analysed and the concentrations of glycerol and malic acid present therein are determined. A sensory evaluation is also performed with the aim of identifying unpleasant odours traceable to the presence of sulphurated compounds. Particular attention is paid to the identification of reduction defects due to the presence of H2S. Subsequently, strains are selected with enhanced fermentative vigour, producing wines where the concentrations of glycerol and malic acid meet the following limits: glycerol higher than about 8.5 g/l, preferably higher than about 9.5 g/l, more preferably higher than about 10 g/l; malic acid higher than about 0.5 g/l, preferably higher than about 1 g/l, more preferably higher than about 2 g/l; furthermore, sensory examination must give no olfactory perception of reduction defects due to the presence of H2S, i.e. preferably less than 1-1.5 g/l.

Advantageously, the analysis of this specific combination of compounds has been shown to be very suitable for the replacement of further and laborious characterisation processes, and the finding of strains producing wines with pleasing and balanced aroma.

In one preferred variant of the invention, the selection method for yeast strains from the speciesSaccharomycespreviously specified as adapted to alcoholic fermentation, particularly of grapes, consists of the following steps:a) sampling of flowers, preferably in spring and in areas of high grape-wine producing activity, and even more preferably away from wineries, in order to avoid contamination with commercial yeasts used in wineries that may be present not just in the wineries, but also in the adjoining areas;b) fermentation of the samples on suitable substrates and cultivation of the fermented samples on isolation media, preferably containing bromocresol green;c) if the isolation medium contains bromocresol green, the identification of presumed yeast strains of the genusSaccharomycesbased on colony morphology and colour, and their discrimination from non-Saccharomycesyeasts of lesser oenological interest;d) identification of isolates belonging to the genusSaccharomycesby means of genetic analysis of the yeast colonies isolated in step b) or step c); advantageously, said genetic analysis, performed using molecular methods, allows both identification of the species of origin and identification of the various strains, or rather individuals, present within a given species;e) laboratory fermentation of the strains selected in step d) on synthetic must and/or on natural must, and subsequent selection of the strains with the most interesting technological and oenological characteristics, and at the same time seeding the strains in Biggy agar medium in order to identify those that are non-producers or low producers of H2S;f) the winery vinification of the strains selected in step e) and selection of the most suitable strains based on the results obtained through chemical analysis of the wine obtained, preferably including pH, residual sugars, alcohol, total acidity, volatile acidity parameters, and, in particular, the quantity of glycerol, malic acid and preferably hydrogen sulphide, where the latter is assessed by olfactory means.

Preferably the collection of flowers and their subsequent processing is performed by avoiding touching the flowers with the hands and with a periodic sterilisation of the sampling equipment, for steps a) and b), with the aim of limiting contamination as much as possible.

By way of non-limiting example, on arrival in the laboratory, the flowers are transferred into sterile flasks containing 200 ml of synthetic must or other culture medium capable of sustaining the growth of any yeasts present. The addition of an antibiotic to each sample, in order to avoid the proliferation of any bacteria, has been shown to be particularly useful.

Advantageously, the preparations are overlaid with sterile paraffin oil in order to avoid the growth of surface mould.

Samples with obvious fermentation underway are processed for isolation of the yeasts in the subsequent step b) or c). A very suitable and preferred isolation medium for said step is WL agar isolation medium (4.0 g/l yeast extract, 5.0 g/l tryptone, 50 g/l glucose, 550 mg/l potassium phosphate monobasic, 425 mg/l potassium chloride, 125 mg/l calcium chloride, 125 mg/l magnesium sulphate, 2.5 mg/l ferrous chloride, 2.5 mg/l manganese sulphate, 22 mg/l bromocresol green, 20 g/l agar, final pH 5.5±2). The salient characteristic of this medium is that it contains a dye, bromocresol green, which is capable of being absorbed by yeasts to a varying extent. It is therefore possible to characterise the microorganism based on colony morphology and the colour it assumes. Strains belonging to the genusSaccharomycesabsorb bromocresol green poorly, and therefore their colonies assume a colour that varies from cream to green, allowing their discrimination from any non-Saccharomycesyeasts.

Advantageously, the use of bromocresol green and the execution of step c) allows simple reduction, and in an initial step of the method, the number of strains to be subjected to the subsequent step d).

Still advantageously, according to one executive variant of the method of the invention, prior to step d), the colonies to be analysed are subjected to purification by means of one or more reseeding passes on suitable medium. Preferably, any reseeding occurs on WL agar growth medium. Advantageously, the procedure is repeated in order to guarantee that each isolate definitely originates from a single colony.

Step c) is already somewhat selective for the isolation of strains belonging to the groupSaccharomyces, but in addition toSaccharomycesthere are also other strains that, in this culture medium, may display morphology and colouring very similar to those of theSaccharomycesyeasts. Therefore, in order to eliminate these strains from the samples and give definitive identification to the isolates, it is preferable to perform step d) which, by means of a rapid and simple to execute genetic method, makes it possible to perform screening on a large number of isolates, thus eliminating yeasts not belonging to the genusSaccharomycesand identifying any strains (=individuals) that should be present within the framework of a given species.

Advantageously, said preliminary genetic evaluation reduces the risk of performing the technological characterisation on yeasts that are potentially not adapted to the production of wine and, equally advantageously, allows the exclusion of any multiple copies of the same strain that may be present in a given sample.

Still advantageously, said preliminary analysis avoids the need to technologically and chemically test strains not belonging to the genusSaccharomyces.

Further advantageously, said analysis makes it possible to perform the technological and oenological characterisation only on truly diverse strains, and not on any possible multiple copies of the same strain.

In order to confirm the origin of the genusSaccharomycesand hence exclude yeasts belonging to different genuses, and hence uninteresting from the oenological viewpoint, it is preferable to use genetic characterisation envisaging the use of PCR technique. Both specific PCR techniques, capable of recognising the various yeast species, and RAPD-PCR (Random Amplified Polymorphic DNA analysis) techniques, known to those skilled in the sector, may be used for this purpose.

Advantageously, in cases where the use of PCR methods does not allow reliable strain identification, it is possible to resort to potential analysis of the rDNA sequence; this analysis makes it possible to distinguish betweenS. cerevisiae, S. bayanusandS. bayanussubsp.uvarum, species that are phylogenetically closely related and with high oenological interest, with such metabolic characteristics as to allow them to survive and grow in wine. Preferably, sequencing concerns the ITS (Internal Transcribed Spacer) region of the ribosomal operon.

According to the preferred embodiment of the method of the invention, the genetic characterisation at strain level in step d) is performed by means of analysis of the mitochondrial DNA restriction profile, obtained by enzyme digestion of total DNA. Enzyme digestion of total DNA is preferably performed using the enzyme Hinf I. Genetic analysis of this type makes it possible to bring yeasts together into groups, each of which with a different genetic profile, common to all isolates belonging to that grouping. Based on the genetic data collected, it is then possible to perform screening on the yeasts collected, avoiding the selection of multiple copies of the same strain.

According to one executive variant of the method of the invention, genetic characterisation by means of restriction polymorphism analysis of mitochondrial DNA is used in association with microsatellite analysis. Microsatellites are repetitive sequences of non-coding DNA, consisting of very short repeat units (1-5 bp) arranged as tandem repeats that can be used as molecular markers of genetic loci. According to the preferred embodiment of the method of the invention, microsatellite analysis for the speciesSaccharomyces bayanussubsp.uvarumis performed on the eight most variable loci, described by Masneuf-Pomarede et al. in 2016. The analysis conditions are subsequently reported in detail (example 1, point 1.6). Generally, a reaction is performed for each locus named: SuYIL130W (16 allelic forms), SuYHR102W (8 allelic forms), SuYKR045C (10 allelic forms), SuHTZ1PLB3 (7 allelic forms), SuARS409 (5 allelic forms), SuYHR042-043 (5 allelic forms), SuYBR049C (5 allelic forms), SuYGC170W (4 allelic forms). Subsequently, for definition of the profile and characterisation of the strains, amplification of PCR products relating to the eight aforementioned loci is performed in addition to agarose gel electrophoresis migration, in accordance with known methods.

Advantageously, according to the method of the invention, subsequently to step d), optionally, for characterisation of the yeastsS. bayanussubsp.uvarum, a further analysis of the eight loci described above is performed by means of multiplex PCR, preferably two multiplex-PCR reactions, defined as multiplex PCR1 and multiplex PCR2. By means of said multiplex PCR, it is possible to simultaneously amplify more than a locus at a time. In particular, multiplex PCR1 and multiplex PCR2 allow the amplification of three and five loci respectively. In detail, multiplex PCR1 allows analysis of the DNA-microsatellite profile for the loci SuYIL130W, SuYHR102W and SuYGC170W, while multiplex-PCR2 makes it possible to obtain the DNA-microsatellite profile for the loci SuYKR045C, SuHTZ1PLB3, SuARS409, SuYHR042-043 and SuYBR049C.

Optionally, according to the preferred embodiment of the method of the invention, to further characterise the yeastsS. bayanussubsp.uvarum, prior to step e), it is possible to perform further genetic analysis by means of capillary electrophoresis based on amplification of the four loci characterised by a greater number of allelic forms, selected in accordance with data reported in the literature.

Advantageously, said characterisation makes it possible to define the exact size of the PCR products for the loci considered.

According to the present invention, the loci considered are the following: SuYIL130W, SuYHR102W, SuYKR045C and SuHTZ1PLB3.

Preferably, for the characterisation of step e) at the technological level, the following parameters are evaluated: fermentative vigour, understood to be the quantity of sugar consumed after 2 and after 7 days, and the duration of fermentation, the foam production capacity, type of growth, hydrogen sulphide production (on Biggy Agar medium), glycerol production, volatile acidity production, the ability to produce or degrade malic acid, the olfactory profile of the fermentates.

With regard to the progression of the fermentations, these are monitored on samples of synthetic must and/or natural must, preferably on both types of must. The main parameters monitored preferably concern: pH, residual sugars, alcohol level, total acidity, volatile acidity, fermentative vigour, glycerol production, production/degradation of malic acid, olfactory profile. The in-winery vinification tests for step f) are preferably performed on musts from white berry grapes and musts from red berry grapes.

Advantageously, analysis of fermentation progression for the strains on musts from both white berry and red berry makes it possible to check progression and evaluate the activity of a strain for the production of white, rosé and red wines, according to specific needs.

Particularly advantageously, the vinification phase of the method according to the invention is performed on grapes from both native vine varieties and international vine varieties. Preferably, the concentrations of glycerol, malic acid and hydrogen sulphide are determined in the wine obtained, and wine-producing strains are selected where the concentrations meet the following limits: glycerol higher than about 8.5 g/l, preferably higher than about 9.5 g/l, more preferably higher than about 10 g/l; malic acid higher than about 0.5 g/l, preferably higher than about 1 g/l, more preferably higher than about 2 g/l; and hydrogen sulphide below the olfactory perception level, i.e. less than about 1-1.5 μg/l. Advantageously, the wine produced using the strain SERIUS is characterised by the presence of high quantities of glycerol and malic acid, while the quantity of hydrogen sulphide is particularly reduced. In particular, the wine produced by the strain of the invention has glycerol in quantities exceeding 9 g/l, malic acid in quantities exceeding 1.1 g/l and H2S in quantities less than the olfactory level of perception.

The technological and chemical parameters examined can vary depending on the characteristics required for the strain and also depend on the type of wine to be produced using the strain selected. Selection of the most suitable strains at the end of the individual steps is preferably a combined selection considering all the results of all analyses performed.

This selection method guarantees the screening of multiple yeast isolates originating from flowers.

Particular examples of execution of the individual steps have been described in examples 1, 2 and 3. The individual steps should be considered as being independent from one another, i.e. the execution of step d) in a defined manner does not automatically imply, for example, the execution of step e) precisely as reported in the examples. The parameters within a step (e.g. the types of technological parameters under test) may be altered independently of the other steps.

