WO2011004254A1 - Souche de levure saccharomyces cerevisiae, son utilisation comme inoculant pour la production par fermentation de nourriture, en particulier de vin prosecco, et inoculant relatif - Google Patents

Souche de levure saccharomyces cerevisiae, son utilisation comme inoculant pour la production par fermentation de nourriture, en particulier de vin prosecco, et inoculant relatif Download PDF

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WO2011004254A1
WO2011004254A1 PCT/IB2010/001686 IB2010001686W WO2011004254A1 WO 2011004254 A1 WO2011004254 A1 WO 2011004254A1 IB 2010001686 W IB2010001686 W IB 2010001686W WO 2011004254 A1 WO2011004254 A1 WO 2011004254A1
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strain
wine
yeast
prosecco
fermentation
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PCT/IB2010/001686
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Viviana Corich
Alessio Giacomini
Angiolella Lombardi
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Regione Del Veneto
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12G—WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
    • C12G1/00—Preparation of wine or sparkling wine
    • C12G1/02—Preparation of must from grapes; Must treatment and fermentation
    • C12G1/0203—Preparation of must from grapes; Must treatment and fermentation by microbiological or enzymatic treatment
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C12N1/18—Baker's yeast; Brewer's yeast
    • C12N1/185—Saccharomyces isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/85—Saccharomyces
    • C12R2001/865—Saccharomyces cerevisiae

Definitions

  • This invention regards a yeast strain belonging to the species Saccharomyces cerevisiae suitable for alcoholic fermentation, particularly of grapes. Furthermore, the invention refers to the use of the yeast strain in the fermentative production of food and particularly in the vinification process, especially in the production of Prosecco wine, and a relative inoculant.
  • Selected yeast or selected starter therefore intends a strain characterized by physiological, biochemical, and enological properties that are optimized in relation to the technological needs of varietal fermentation processes (Pretorius, 2000).
  • the first yeasts were selected with the goal of enhancing the technological characteristics (fermentative vigor, alcohol tolerance) so as to obtain products without defects.
  • Today the latest generation yeasts are selected on the basis of characteristics that can improve the quality of the wines through the expression of precursors already present in the musts and the production of secondary metabolites (higher alcohols, esters, ketones, aldehydes).
  • yeasts may also present some non-negligible disadvantages, and could lead to an excessive standardization resulting in a reduction of the biodiversity of the wine yeast strains associated with the winery environment.
  • ecotypic starters are exclusively used in the isolation area and have been selected following the typicality characteristics of the local product. In reality they are the yeasts that originally gave rise to spontaneous fermentation and thus contributed to constructing the wine's typicality characteristics.
  • the fermentative or alcohol-generating power which according to Delfini is the maximum alcohol content (intended as % ethanol) produced by a yeast through fermentation of a must containing excess sugar (Delfini, 1995) is among the principal technological characteristics.
  • Ethanol the principal product of the yeasts' activity under anoxic conditions, is the compound that exerts an inhibitory action on all microorganisms. Since notable performance is required from yeasts in enology (for example the requirement for the production of wines with high alcohol content), the resistance (alcohol tolerance) toward this compound also evidently assumes fundamental importance.
  • the yeasts of southern musts have an alcohol-generating power that is on average higher than that coming from northern musts of the same species (Zambonelli et al., 2000).
  • fermentative vigor expresses the capacity to give rise to ready and rapid fermentation, also in the presence of antiseptics at the doses allowed by law and at temperatures between 2O 0 C and 3O 0 C.
  • Another very important factor is resistance to sulfur dioxide SO 2 , i.e. the capacity to maintain the fermentation velocity unaltered or sufficiently elevated in the presence of the selective doses of SO 2 added to the musts. It is important to select the strains that are most resistant to this antiseptic to avoid start-up problems in the fermentative process.
  • the development mode i.e. the behavior of the yeast cells at the end of the budding process, distinguishing from among powdery development, flocculant development, or development in aggregates.
  • the most common development in S. cerevisiae is the powdery one; this is also the most desirable in a starter selected for primary fermentation.
  • the sedimentation speed is another positive characteristic as it facilitates the subsequent clarification and filtration operations. The sedimentation speed can be measured by the clarification time expressed in days.
  • the strains that make the wine murky in the most annoying and persistent manner are the foaming ones.
  • the foaming power results from the fact that in many strains (20-30%) the cells adhere to the CO 2 bubbles that are produced during fermentation. Instead of breaking once they arrive at the surface, they blend together and increase in volume, giving rise to foams that assume a gray-brown color due to the presence of the cells.
  • the height of the foam is evaluated to measure this parameter.
  • the absence or lack of foam production is surely a positive characteristic in both primary fermentations and in re-fermentation, because it reduces the volume occupied by the must.
  • Another aspect is the film-forming power. At the end of normal alcoholic fermentation, some strains give rise to a film on the surface of the wine, where development continues under oxidizing conditions (flor). The flor strains oxidize the ethanol and form a series of aromatic compounds.
  • Adhesiveness instead is the capacity of the yeast cells to grow adherent to the surfaces of wine vessels; this is a negative characteristic because it complicates the sanitization of the tanks at the end of processing (Vincenzini et al., 2005).
  • the principal quality characteristics i.e. the characteristics relative to the production of substances that are important from an organoleptic point of view, are described below.
  • Glycerol after ethanol and CO 2 , is the compound produced in the greatest quantities during alcoholic fermentation. It greatly influences the "body of the wine”, and contributes to giving wines the characteristics of "fullness” and “sweetness” (perception threshold 5.2 g/l).
  • Saccharomyces of vinification can produce from 2 to 10 g/l as a function of the species and strain of yeast.
  • S. cerevisiae is the yeast that forms the smallest quantities of glycerol and this is the principal reason for its high ethanol yield.
  • Glycerol is appreciated at higher concentrations in red wines, to which it gives the sensation of "aged", and is for the same reason not appreciated in the case of wines that must conserve their original freshness.
  • This compound in reality, is not a secondary product of fermentation because it derives directly from the sugars and its amount of formation, with the same strain, is proportional to their concentration (Zambonelli et al. 2000).
  • acetic acid that derives from the degradation of sugars during fermentation by the oxidation of acetaldehyde (Ribereau-Gayon, 2000).
  • the capacity to produce higher or lower amounts of acetic acid is a characteristic of the species and strain, and is hereditary and stable.
  • This characteristic is currently defined with the term of fermentative purity, and is expressed as the relationship between the volatile acidity formed and the ethanol produced.
  • the best strains are those that have the lowest value of fermentative purity. In principle, for a wine with 10% alcohol, a value of this characteristic less than 0.3 g/l is already considered to be very good.
  • the volatile acidity of wines may have different origins and does not derive only from the activity of the yeast; often the must also has a significant acetic acid content as a consequence of acid rot or the development of acetic acid bacteria on the grapes (Zambonelli, 2003).
  • Acetaldehyde is the most important carbonyl compound formed during fermentation and represents over 90% of the wine's total aldehyde content. It is a normal product of alcoholic fermentation and its content in wine can vary notably (10-300 mg/l); the evaluation of its content is used as an indicator of the degree of oxidation of a wine.
  • a high acetaldehyde level is undesirable and wines containing 500 mg/l are considered unmarketable.
  • White wines generally have an average value of 80 mg/l. It confers a pleasant fruit aroma when present in wine at low levels, but already at concentrations of 100-125 mg/l it liberates a pungent and irritating odor. Sulfur dioxide induces the production of acetaldehyde by the yeast and this seems to be correlated to the resistance of yeast to dioxide itself.
  • the main factor determining the variability of acetaldehyde content is the species of yeast. S. cerevisiae is among the major producers of the molecule (50-120 mg/l) (Vincenzini, et al. 2005).
  • the higher alcohols predominantly represented are: n-propanol, isobutanol, amyl alcohol, and 2-phenyl ethyl alcohol. These compounds are derived from the catabolism of the corresponding amino acids present in the must, but also from the metabolism of glucose without the involvement of amino acid precursors.
  • Sulfur compounds from a qualitative point of view, consist primarily of hydrogen sulfide (H 2 S) and sulfur dioxide (SO 2 ), which derive directly from the reduction of sulfates present in the musts; they are always produced by S. cerevisiae, even if at quantities that vary according to the strain.
  • Malic acid is one of the principal organic acids in musts and wines. Some non-Saccharomyces yeasts can consume it through malolactic fermentation. On the other hand, some strains of Saccharomyces, generally those among the cold-resistant ones, have been identified that can also produce malic acid during the fermentative process (Fatichenti et al., 1981).
  • esters are produced, obtained from the union of acetic acid or other primarily medium-chain fatty acids with ethanol or other higher alcohols present in the wine and also produced by the yeast's metabolism.
  • the influence on the wine's quality can be negative and unappreciated, especially in the case of ethyl acetate, which confers a typical vinegar (or even solvent) smell.
  • Others are instead quite desired, as for example the ester ethyl caproate, especially in those wines characterized by hints of white fruit (e.g. Prosecco).
  • yeasts can modify the composition of wines indirectly, i.e. by mediating the secretion of hydrolytic enzymes into the medium.
  • ⁇ -glucosidase activity acts on the glycosylated aromatic precursors that characterize the different grape varieties, liberating the fragrant molecule responsible for the varietal aroma. It has long been known that these molecules (belonging to the classes of terpenes, benzenes, and norisoprenoids) play a very important role in conferring the taste and smell of the grape and wines. These compounds are primarily present in the form of glycosidic complexes that are inactive from a sensorial point of view.
  • the hydrolysis of the glycosidic complexes and the consequent liberation of monoterpenes can be obtained with physical methods (temperature), chemical methods (acidification), and with biochemical methods (by enzyme); the latter are the most effective.
  • the enzymes in question represented by the class of ⁇ -glucosidases, are widespread in the plant world and, in particular, in the grape itself (Gunata et al., 1985). In S. cerevisiae, ⁇ -glucosidase activity is present in a very limited group of strains, while it is quite frequent in the apiculates (Zambonelli, 2003).
  • the most important nutrient during vinification is readily assimilable nitrogen; it can be present in the form of ammonium or also amino acids.
  • the concentration of nitrogen in the grape decreases during the latter's maturation. Grape must often contains insufficient quantities of readily assimilable nitrogen. In this case it is necessary to add nitrogen, e.g. in the form of diammonium phosphate, during fermentation.
  • EP 1 482 029 A1 describes two strains of ecotypic Saccharomyces cerevisiae isolated in Spain. Particularly favorable characteristics are described, such as a good sugar/ethanol yield and particular fruity and fresh aromas. The document does not however describer the chemical analysis of particular chemical components.
  • the purpose of the invention is to provide a yeast strain of the species Saccharomyces cerevisiae ecotypic of the DOC area of Conegliano and Valdobbiadene (Veneto Region - Italy) that is equipped with technological and enological characteristics that allow a good fermentation of the must obtained, above all with the Prosecco grape, allowing the production of a pleasant wine.
  • Another purpose is to provide a strain that produces higher alcohols in low quantities and that simultaneously has an elevated production of aromas that give the Prosecco wine the typical hints of white fruit and citrus.
  • a particularly important purpose of the invention is to provide a yeast strain of the species Saccharomyces cerevisiae that can also be used in the fermentation of musts with a low content of readily assimilable nitrogen, and that can also preferably be used without the addition of readily assimilable nitrogen.
  • Another purpose of the invention is to provide a yeast strain that can be used in alcoholic fermentation for the production of food in general and a relative inoculate that contains this strain.
  • the yeast strain Saccharomyces cerevisiae according to the invention has optimal enological characteristics, both technological and chemical, and is ecotypic to the DOC area of Conegliano and Valdobbiadene.
  • the strain has been identified with the denomination P301.9 and has been deposited according to the Budapest Treaty on 05/26/2009 at the "Industrial Yeasts Collection" DBVPG in Perugia (Department of Applied Biology, Section of Microbiology, Faculty of Agriculture - University of Perugia, Borgo XX Giugno, 74 - 06121 PERUGIA - IT) with deposit number 24P and protocol number PROT. 521/P/2009.
  • a fundamental characteristic of the selected strain is to represent an optimum compromise between technological characteristics and aspects of typicality linked to Prosecco wine.
  • the isolated strain is selected and overwhelmingly characterized by a very low need for assimilable nitrogen. This fact makes it universally utilizable even in musts with a low content of readily assimilable nitrogen.
  • vinification carried out with the addition of inocula of this yeast does not require the addition of assimilable nitrogen or only at low concentrations.
  • the strain presents a very low production of higher alcohols (1-propanol and 3-methyl-1-butanol) and the absence of the production of 1-butanol. Furthermore, the ethyl acetate concentration is very low. The wine obtained with this yeast is free of pungent solvent odors.
  • the strain according to the invention produces less than the commercial control strain (Premium Prosecco, Enologica Vason SrI, Nassar, Pedemonte, Verona, containing a strain of the species Saccharomyces cerevisiae), which is the most used in the Prosecco area and is considered to be a good enological strain.
  • commercial control strain Premium Prosecco, Enologica Vason SrI, Nassar, Pedemonte, Verona, containing a strain of the species Saccharomyces cerevisiae
  • the P301.9 strain is therefore absolutely superior to currently available strains for its olfactory qualities. It furthermore produces geraniol (citrus aroma); this terpenic alcohol is not found in detectable concentrations in the wine produced with the commercial strain currently rated as the best strain available. These molecules give the typical desired aromas to the Prosecco.
  • the good beta-glucosidase activity of the strain surely plays an important role in the development of the varietal aromas which are essential for defining the typicality as characterizing.
  • the ITS and D1/D2 sequences that genetically characterize the strain are represented in the list of sequences with the sequence identification numbers SEQ ID NOs 19 (D1/D2) and 20 (ITS).
  • the yeast according to the invention has the following physiological characteristics: sporulates on acetate agar, with persistent asci, containing from one to four spherical spores. Does not grow in the presence of 10 mg/l cycloheximide.
  • the yeast subject of this invention has the enological characteristics reported below: glucose consumed after 2 days (fermentative vigor) 3.6 g; glucose consumed after 7 days 15.1 g; glucose consumed at the end of fermentation 18.3 g; 15 days of fermentation, maximum height of the foam produced 18 mm; clarification time 14 days, adhesiveness 2 mm.
  • yeast strain according to the invention has a nitrogen requirement less than 50 mg/l in the tested experimental conditions cited above.
  • the strain has been genetically and enologically characterized.
  • Example 2 describes the genetic characterization of the strain in detail, while example 3 describes the enological characteristics in detail.
  • the strain is preferably stored desiccated, lyophilized, or as a paste. These forms make it suitable for transport and sale.
  • the strain can also be stored at low temperatures or in frozen form.
  • the desiccated strain can be in the form of dust or pellets. Freezing or dehydration occurs through methods well known to the expert.
  • the strain according to the invention is suitable to be used in the production of food obtained through alcoholic fermentation.
  • Foods produced with this yeast include, for example, bread, dairy-based products, cider, beer, sparkling wine, and wine.
  • Other alcoholic beverages obtained by the alcoholic fermentation of other fruits, cereals (such as wheat or rice), or potatoes by using the strain proposed in this invention are however conceivable.
  • Another aspect of the invention therefore regards the use of the strain identified as P301.9 according to the invention as an inoculate for the production of food obtained through alcoholic fermentation.
  • the strain can be used not only in vinification, but also generally in the production of food obtained through alcoholic fermentation.
  • the inoculate is a set of cells, generally selected and genetically homogeneous, that is introduced in the raw material (e.g. must) to produce food (e.g. wine).
  • the food is preferably a wine; alcoholic fermentation is a vinification process and the inoculate is a wine starter.
  • the wine starter is the dose of yeast necessary to activate fermentation.
  • the use of the strain in vinification refers to a white wine produced from Prosecco grapes. This because it produces the apple and citrus aromas that are highly desired in Prosecco. Other white wines, red wines, rose wines, etc. are obviously conceivable.
  • vinification using the strain according to the invention as a starter, occurs without the addition of readily assimilable nitrogen.
  • the Prosecco produced has a concentration of ethyl caproate naturally produced by the strain > 800 ⁇ g/l. These concentrations give the wine a pleasant apple aroma.
  • the Prosecco produced with the strain according to the invention as a starter contains geraniol naturally produced by the strain.
  • the geraniol gives the wine an aroma of rose and citrus.
  • Another important aspect concerns an alimentary inoculate that includes the strain identified as P301.9 (DBVPG 24P).
  • the inoculate is a wine starter.
  • the strain is particularly suited for the production of wines, as it produces pleasant aromas with a low production of higher alcohols.
  • the Prosecco grapes used for the production of Prosecco wine with the strain according to the invention are preferably produced in the DOC zone of
  • the P301.9 strain can easily be cultivated and is therefore highly suited to be used to produce biomass, also at an industrial scale, with noted standard methods. Its cultivation preferably occurs on a YM medium containing 3 g/l yeast extract, 3 g/l malt extract, 5 g/l peptone, and 10 g/l glucose.
  • Its growth preferably occurs at a temperature of 25°C. Cultivation can also occur on a YPD media (10 g/l yeast extract, 20 g/l peptone, 20 g/l glucose) at 25°C. The recommended subculture period is from 24-48 hours. The preferred storage conditions are in glycerol (YM media containing 40% v/v glycerol) at -80 0 C. Dangerous characteristics for the health of people, animals, or for the environment are not known; the strain is not pathogenic. The type of coupling is not known. Sporulation preferably occurs on PRE5 media (Codon et al., 1995, Applied Environ. Microbiol. 61 , p.
  • the colony counting method on a Petri dish, using the YM growth medium, can be used to check for the presence of living cells.
  • the wine is obtained in this process without the addition of readily assimilable nitrogen.
  • readily assimilable nitrogen makes it particularly suitable for this process, thus avoiding a possible overdose of artificial nitrogen sources.
  • This strain is therefore particularly suitable for fermentations in which the fermentation media is poor in readily assimilable nitrogen.
  • a Prosecco wine obtained through the alcoholic fermentation of Prosecco must with the strain according to the invention comports many advantages, including elevated concentrations of ethyl caproate (apple flavor) and low concentrations of higher alcohols (absence of pungent odors).
  • the very low ethyl acetate content makes it superior to wines produced with other yeasts.
  • the strain according to the invention has optimal enological qualities, both chemical and technological, making it particularly suited to the production of a good Prosecco wine.
  • Vinification can be executed in an optimal way by introducing the strain in the form of an inoculate prepared according to the protocols described in example 1 , e.g. at point 1.9, which regards microvinification.
  • the method allows ecotypic strains with olfactory, sensorial, technological, and chemical qualities suitable for the vinification of Prosecco grapes to be selected.
  • the technological, chemical, and olfactory parameters examined can vary according to the characteristics that are required for the strain and also depend on the type of wine that must be produced with the selected strain.
  • the selection of the most appropriate strains for individual phases is preferably a combined selection, considering all of the results of all of the analyses carried out.
  • the choice of the combination of analyses to be carried out is determined by the yeast's desired characteristics. Olfactory/sensorial parameters also represent an important factor for defining a reduction in the number of strains in the pool to be considered in the next phase and to choose the strain that also satisfies aromatic requirements from among many strains with comparable technological characteristics.
  • Example 1 describes the different phases occurring during the isolation of strains suited to vinification.
  • strain according to the invention was chosen on the basis of its readily assimilable nitrogen requirement (see example 3, point 3.6: Nitrogen requirement) and elevated production of ethyl caproate and geraniol.
  • the sampling territory in which the strain covered by the invention was isolated constitutes the DOC production zone of Conegliano and Valdobbiadene, which prevalently extends through the hilly portion of the Treviso province (a group of hills with east-west directionality that reach the Alps); only toward Conegliano are the vineyards planted in more low-lying areas.
  • the vines are cultivated only in the sunniest part of the hills, at an altitude between 50 and 500 meters above sea level.
  • Prosecco grapes The strain has been isolated from Prosecco grapes.
  • the ampelographic findings have demonstrated the existence of significant differences relative to 22 characteristics among the biotypes of Prosecco tondo and Prosecco lungo present in the DOC area of Conegliano-Valdobbiadene.
  • Prosecco lungo differs from Prosecco tondo primarily for having a sprout that is a little hairier and more colorful, leaves that are slightly larger, leathery, and with the petiolar sinus less closed, a slightly shorter bunch, and a grape with an elliptical-short shape instead of a spherical one.
  • the Conegliano and Valdobbiadene DOC area was subdivided into 37 micro-areas in which the sampling of the bunches was carried out. Bunches from 11 of the 37 localities were obtained, allowing yeasts of technological interest (Saccharomyces sensu stricto) to be isolated after fermentation.
  • the strain P301.9 comes from the "CoI de Roer" area (in the municipality of Valdobbiadene) in which the highest biodiversity (the 192 isolates coming from this region were grouped into 12 genetic profiles that correspond to as many strains) was found.
  • the Saccharomyces yeasts collected in this area constitute 16.2% of the total of isolates identified as belonging to this group.
  • - Fig. 1 shows the karyotype of yeast strain P301.9 obtained by pulsed electrophoresis
  • Fig. 2 shows the analysis of the microsatellite sequences of strain P301.9
  • - Fig. 3 shows the restriction profile of the mitochondrial DNA of strain P301.9.
  • Fig. 1 shows the result of an electrophoresis carried out with TBE 0.5x buffer at 9°C, agarose at 1.2% concentration, a voltage gradient of 5.1 V/m, a length (run time) of 34 h with an initial switch of 60 seconds and a final switch of 120 seconds.
  • the strain YPH755 is the reference marker.
  • M represents the molecular weight marker (mix of Marker III + Marker Vl, Roche, Milan). They mean:
  • M is the GeneRulerTM 1 kb DNA Ladder (Fermentas Life Sciences,
  • strain P301.9 determines the electrophoretic profiles obtained from the enzymatic digestion of the mitochondrial DNA restriction profile and from the amplification of the microsatellite sequences represented in Fig. 2.
  • the sampling was carried out by collecting individual bunches of grapes within the DOC Prosecco area of Conegliano and Valdobbiadene in the days immediately preceding the harvest.
  • the collection was carried out at each phase by avoiding touching the bunches with the hands and periodically sterilizing the scissors, with the aim of limiting contaminations as much as possible.
  • the salient characteristic of the media used is that it contains a colorant, bromocresol green, which can be variably absorbed by yeast. It is therefore possible to characterize the microorganism on the basis of the colony's morphology and the coloration that it assumes.
  • the pure culture obtained after the last plate passage was withdrawn with a sterile spatula, and the material was inserted in 0.5 ml Eppendorf tubes containing a sterile solution of 20% glycerol in water (approx. 300 ⁇ l) and resuspended through vortex agitation.
  • the tubes containing the yeast were stored at -20 0 C.
  • the method allowed this group of yeast to be discriminated with respect to others present in the enological environment on the basis of differences between nucleotides within the region of DNA encoding for ribosomal RNA (rDNA).
  • the section of DNA that carries the most information relative to the differences between the yeast species is the D1/D2 region of DNA 26S; information on it relative to the sequence characteristics of many yeast species is available in the public nucleotide database GenBank.
  • yeast belonging to the group Saccharomyces sensu stricto gave rise to the amplification of two DNA fragments (with lengths of 460 and
  • the aforementioned amplified fragments can easily be visualized as distinct bands through gel electrophoresis on agarose gel.
  • the DNA sample for the amplification was prepared by resuspending a single yeast colony of 1-2 mm in diameter in 20 ⁇ l of sterile deionized water with a tip.
  • DNA amplification mixture Lysis of the cells occurs during the first step of the heat protocol reported below, by heating at 94°C for 5 minutes.
  • the PCR tests were carried out in reaction volumes of 25 ⁇ l.
  • SAC26F with a length of 22 nt corresponds to SEQ ID NO: 1
  • SAC26R with a length of 27 nt corresponds to SEQ ID NO: 2
  • SAC18F with a length of 23 nt corresponds to SEQ ID NO: 3
  • SAC18R with a length of 25 nt corresponds to SEQ ID NO: 4.
  • each of the 308 yeast collected were submitted to fermentation tests using synthetic must (0.1 g/l CaCb, 0.1 g/l NaCI, 1 g/l KH 2 PO 4 , 0.5 g/l MgSO 4 -7H 2 O, 3 g/l tartaric acid, 0.2 mg/l NaMoO 4 -2 H 2 O, 0.004 mg/l ZnSO 4 -7H 2 O, 0.5 mg/l H 3 BO 3 , 0.04 mg/l CuSO 4 -5 H 2 O, 0.1 mg/l KJ, 0.004 mg/l FeCI 3 -6H 2 O, 0.4 mg/l MnSO 4 H 2 O, 400 ⁇ g/l pyroxidine hydrochloride, 400 ⁇ g/l thiamine hydrochloride, 2000 ⁇ g/l inosite, 20 ⁇ g/l biotin, 400 ⁇ g/l calcium pantothenate, 400 ⁇ g/l nicotinic amide, 200 ⁇ g/l/l
  • the inoculate was prepared by diluting the film obtained from a culture grown in GPY media (10 g/l yeast extract, 20 g/l peptone, 20 g/l glucose, 20 g/l agar) in 10 ml of synthetic must until achieving an optical density at 620 nm (OD 62 o) of 1.5. With this procedure, a final concentration in the must of approximately 1.5 x 10 6 cells/g is obtained. The OD 62 o was measured with a Pharmacia spectrophotometer mod. Ultrospec 2000 using an aliquot of 1 ml of culture transferred to the appropriate tube.
  • GPY media 10 g/l yeast extract, 20 g/l peptone, 20 g/l glucose, 20 g/l agar
  • the suspension was inoculated in a flask containing 90 ml of synthetic must and incubated at 20 0 C.
  • the decrease in weight due to the loss of CO 2 from the flasks prepared for the fermentation tests was monitored daily, choosing the decrease in weight at 2 days, 7 days, and at the end of fermentation as parameters for comparison between the strains.
  • the clarification time of the media was evaluated as the number of days necessary for the sedimentation of the yeast beginning at the moment of inoculation.
  • the capacity of the cells to grow adherent to the walls of the flask was determined for each isolate grown in synthetic must.
  • the height (in mm) of the ring of cell growth from the bottom of the flask was measured. The measurement was carried out at the end of fermentation.
  • each isolate was inoculated on Biggy agar media so as to obtain the growth of an individual colony.
  • the plates were incubated at 25°C for 4 days, after which the coloration of the individual colonies developed was evaluated.
  • the quantity of hydrogen sulphide is proportional to the intensity of coloration, due to the development of bismuth sulphite (dark brown if present in significant quantities).
  • the fermented material was submitted to an olfactory evaluation to identify the presence of abnormal odors.
  • the following DNA extraction procedure was carried out for each of the 308 isolates.
  • the film obtained from a culture grown in Petri dishes containing YM media for 48 h at 25°C was resuspended in 1 ml of sterile water and then centrifuged at 14000 rpm for 3 minutes in an Eppendorf microcentrifuge.
  • the cells were resuspended again in 500 ml of a solution containing lytic enzyme obtained from Rhizoctonia solani (25 mg/ml in 1M sorbitol, 0.1 M EDTA, pH 7.5). The suspension was briefly agitated with a vortex. The samples were incubated at 45°C for two hours during which time they were periodically agitated to maintain the cells in suspension. At the end of incubation, the samples were centrifuged at 14000 rpm for 5 minutes and the supernatant was removed.
  • lytic enzyme obtained from Rhizoctonia solani
  • the cells were then resuspended in 500 ml of TE (50 mM Tris-HCI, 20 mM EDTA at pH 7.4) and 50 ⁇ l of 10% SDS (sodium dodecyl sulfate) were added.
  • the samples were incubated in a water bath at 65°C for 30 minutes. At the end, 200 ⁇ l of 5M potassium acetate were added and the samples were left in ice for 30 minutes.
  • the tubes were centrifuged at 14000 rpm for 5 minutes and the supernatant was transferred to a 1.5 ml Eppendorf tube. After having added 600 ⁇ l of cold isopropanol, the samples were kept at ambient temperature for 5 minutes, mixing them by inverting the tubes, and then centrifuged at 14000 rpm for 10 minutes.
  • the supernatant was eliminated and 500 ⁇ l of 70% ethanol was added. After centrifugation at 14000 rpm for 5 minutes and removal of the supernatant, the pellet was dried for 1 h at 37°C.
  • the samples were resuspended in 50 ⁇ l of sterile water, to which 1.5 ⁇ l of RNases (10 mg/ml) were added, and left at ambient temperature for 20 minutes.
  • the samples obtained in this way were stored at -20 0 C.
  • the enzymatic digestion of whole DNA was carried out in reaction volumes of 15 ⁇ l containing 10 ⁇ l of DNA and 10 U of H/nfl enzyme. The reactions were conducted at 37°C for 2 hours. The linear DNA fragments were separated through electrophoresis on 1% agarose gel.
  • the selection criteria in addition to the best fermentation performances, also included the genetic profile and the geographical origin of the isolate so as to represent, also in subsequent tests, the microbial biodiversity collected from both a genetic point of view and from the point of view of representativeness of the territory.
  • the 66 samples chosen were submitted to a further genetic investigation to determine the species of membership among those included in the group Saccharomyces sensu stricto.
  • the section of DNA between those encoding the subunits 18S and 26S, encoding the rRNA of subunit 5.8S and containing the two adjacent zones denominated ITS (Internal Transcribed Spacers), particularly interesting because they demonstrate a much higher sequence polymorphism than that associated with the genes encoding the rRNA of the 18S and 26S subunits (Cai et al., 1996; James et al., 1996) was taken into consideration.
  • This variability is extremely high, if organisms belonging to different species are considered, while it drops dramatically when strains of the same species are considered.
  • This intra-specific polymorphism can be revealed through amplification by PCR of the ITS region and subsequent analysis of the restriction profile using opportune enzymes (Esteve-Zarzoso et al., 1999). It is a fast and easy method for the identification of yeast that has the advantage of providing information relative not only to strain to be recognized but also to the rest of the microbial population.
  • the primers used are ITS1 with a length of 19 bp with SEQ ID NO: 5 and ITS4 with a length of 20 bp with SEQ ID NO: 6 (White T. J. et al. 1990).
  • the amplification products of the ITS1-5, 8S-ITS2 region of the rDNA were digested with the enzymes Haelll and Mae ⁇ .
  • the digestions were carried out in reaction volumes of 20 ⁇ l containing 10 U of enzyme and 10 ⁇ l of amplified product.
  • the reactions were conducted at 37°C for 16 hours.
  • the digestion products were visualized on a 1.5% agarose gel.
  • Technological characterization of the chosen isolates in Prosecco must The 66 strains chosen on the basis of the fermentations in synthetic must were further assayed through growth tests in flasks, this time using Prosecco must. For this analysis, the characteristics taken into consideration were the decrease in weight at 2 days (fermentative vigor) and at 7 days, the duration of the fermentation, the height of the foam produced and the velocity of clarification of the fermented product.
  • the inoculate was prepared by diluting the film obtained from a culture grown in GPY media in 23 ml of 18% sucrose solution until the achievement of an OD 62 o of 1.6, which corresponds to approx. 3.5 x 10 6 cells/ml.
  • the suspension was centrifuged at 8000 rpm for 10 minutes, the pellet resuspended with 10 ml of natural must, and inoculated in a flask containing 190 ml of the same must.
  • the culture was incubated at 25 0 C.
  • the product (approx. 200 ml of basic wine) was transferred into two 100 ml bottles, closed with a crown cap and stored at 10 0 C.
  • the wines obtained in this way were subjected to a sensorial evaluation by a panel composed of 8 tasters between 25 and 50 years of age. Expert tasters, technicians in the sector, and experts in Prosecco production technology were chosen.
  • the samples were presented anonymously, and the wine was served at a temperature of approximately 10 0 C.
  • the specially prepared evaluation form asked for a judgment expressed numerically with a scale from 1 to 10, in relation to the following parameters: typicality, presence of any defects, presence of pleasing organoleptic notes.
  • a judgment of preference was also requested, numerically reordering the samples beginning with that with the best characteristics.
  • microvinification tests were carried out with the aim of further evaluating the 9 strains with the best results during tasting together with a control consisting of a strain of commercial yeast (Premium Prosecco, Enologica Vason SrI, containing a strain of Saccharomyces cerevisiae).
  • the 10 yeast proposed for the microvinification were cultivated on modified Malt agar (30 g/l malt extract, 5 g/l plant peptone, 15 g/l agar, pH 5.4), using the plant peptone instead of the traditional meat protein extract, in view of the use of the wines obtained for tasting.
  • a definite quantity of yeast was withdrawn from these plates to inoculate 20 ml of an 18% sucrose solution in order to have an OD 6 oo equal to 1, corresponding to about 10 7 cell/ml.
  • CFU colony-forming units
  • the microvinification tests were conducted using pre-sulphited Prosecco must stored at 15°C (50 mg/l total SO 2 ) transported in 100 I bins to the processing room, which was maintained at 10 0 C for the inoculate. The must was subdivided into 10 carboys (container volume 34 I) with 30 I in each and brought to ambient temperature for approximately 12 hours so as to bring the temperature to 16-18°C.
  • the inoculation was carried out by adding 3 I of yeast culture to 30 I of must. In conjunction with the inoculate, an activator was added (ammonium phosphate 59.88%, thiamine hydrochloride 0.6%, cellulose 39.52%) at a concentration of 10 g/hl. The day after the inoculation, 100 ml of material were sampled for the chemical analyses. The sampling for these analyses was carried out daily in the first fermentation period and then every 2-3 days.
  • the SO 2 level was checked again and brought to a value of 1.2 mg/l.
  • WineScan is a spectrophotometer dedicated to the analysis of liquid matrices and is prevalently used for the analysis of wines, musts, beer, and balsamic vinegars.
  • the instrument operates in the infrared spectral range, using an IR source as the light beam and a special optical-electronic device called an interferometer as the system to measure the wavelength; the latter exploits the principal of the interference of light waves, and can measure the various wavelengths of the individual components of the liquid matrix.
  • the instrument In the case of the analysis of musts and wines, the instrument is equipped with respective calibration curves that can interpret the measured values in a specific way.
  • the must was sampled at intervals of approximately 2-3 days and the principal chemical parameters were determined: pH, sugar content, alcohol content, volatile acidity, concentration of malic acid, lactic acid, tartaric acid, total acidity, and glycerol.
  • the microvinifications were blocked when the concentration of sugars was less than 1 g/l.
  • the 10 wines obtained were stabilized, filtered, and bottled, and conserved at ambient temperature. They were then subjected to sensorial analysis.
  • the evaluations were carried out analogously to the sensorial profile procedure (ISO13299), using a form with points structured in the following way: the descriptors (odor intensity, flavor intensity, sweet, salty, acid, bitter, pleasantness, typicality) were evaluated through the use of a continuous scale from 0 to 10 points, while the olfactory characteristics (acacia, wisteria, rose, green apple, ripe apple, pear, lemon, other citrus fruits, banana, pineapple, peach, apricot, bread, yeast, honey, spices) were detected through a discontinuous scale of points which included three intensities, absent, weak, and medium-high, in correspondence to 0, 5, and 10 points.
  • the 4 most interesting strains, along with a commercial control strain were produced "in a paste" according to standard methods known to producers specialized in the preparation of commercial starters.
  • the inoculate was calibrated with the purpose of obtaining an initial concentration of yeast of approximately 10 6 cell/ml in the fermentation tanks containing 50 hi of Prosecco grape must.
  • the vinifications were conducted according to corporate protocols.
  • the must was sampled at intervals of approximately 2-3 days and the principal chemical parameters were determined: pH, sugar content, alcohol content, volatile acidity, concentration of malic acid, lactic acid, tartaric acid, total acidity, and glycerol.
  • the vinifications were blocked when the concentration of sugars was less than 1 g/l.
  • the 5 wines obtained were stabilized, filtered, and bottled, and stored at ambient temperature. They were then subjected to sensorial analysis following the method described above.
  • PFGE Pulsed-Field Gel Electrophoresis
  • the karyotype of the yeast strain can be obtained with this particular separation system by gel electrophoresis as this system, unlike traditional electrophoresis in which the direction of the electric field is constant, varies the electric field's direction over time thanks to a system of electrodes, allowing the separation of large DNA molecules such as those of the yeast chromosomes.
  • the DNA extraction protocol involves the collection by centrifugation of the proper quantity of cells beginning with an overnight culture in YM media incubated at 28 0 C 1 with agitation at 120 rpm.
  • the cells are then washed with 1.5 ml of cold (5°C) distilled water and then with 50 mM cold EDTA pH 8.0. The pellet is gently resuspended in 0.9 ml of
  • SPG buffer (10 mM NaH 2 PO 4 in 50% glycerol) containing 25 mg/ml of lytic enzyme from Rhizoctonia solani and incubated in a water bath at 30 0 C for
  • the spheroplasts (yeast cells without their walls) obtained in this way are incubated in a thermostat at 37°C for
  • the suspension is left to cool at 0-4 0 C for 30 min to allow the agarose to solidify.
  • the agarose blocks obtained from each mold are first incubated at 30 0 C for 4 hours in 3 ml of LET buffer (500 mM EDTA, 10 mM Tris, pH 7.5) and then washed with 50 mM cold EDTA pH 8.0.
  • LET buffer 500 mM EDTA, 10 mM Tris, pH 7.5
  • the agarose blocks were incubated overnight at 50 0 C in 1.5 ml of NDS buffer (500 mM EDTA, 500 mM Tris, 1% laurylsarcosine, pH 7.5) containing 2 mg/ml of K proteinases.
  • NDS buffer 500 mM EDTA, 500 mM Tris, 1% laurylsarcosine, pH 7.5
  • proteolytic treatment was concluded with three washes using a solution of 50 mM cold EDTA at pH 8.0. Before loading the blocks on the gel, they were cut into 2-3 mm thick slices with a sterile scalpel and stored at 4°C in 500 mM EDTA at pH 9.0.
  • microsatellites are small sequences of DNA repeated in tandem (from one to six bases) that vary in the number of repetitions and can also be localized within encoding genomic regions (Legras et al., 2005).
  • the analysis of the polymorphism of the microsatellites is a highly reproducible method because specific primers at high annealing temperatures are used for their amplification.
  • the DNA sample for the amplification is prepared through the method already described above in example 1 , point 1.6 (Genetic characterization of the strains).
  • the PCR tests were carried out in reaction volumes of 25 ⁇ l.
  • the various components of the reaction mixture were used at the final concentrations reported in the following Table 5:
  • the six pairs of primers used are (sequence 5'-3'): C4 F (SEQ ID NO: 7), C4 R (SEQ ID NO: 8), C5 F (SEQ ID NO: 9), C5 R (SEQ ID NO: 10), C8 F (SEQ ID NO: 11), C8 R (SEQ ID NO: 12), C11 F (SEQ ID NO: 13), C11 R (SEQ ID NO: 14), ScYOR267c F (SEQ ID NO: 15) and ScYOR267c R (SEQ ID NO: 16).
  • This method provides for the determination of the restriction profile of the mitochondrial DNA through enzymatic digestion of whole DNA.
  • rDNA 5.8S subunit of the ribosomal RNA
  • ITS1 and ITS2 denominated Internal Transcribed Spacers
  • ITS1 length 19 bp, SEQ ID NO: 5
  • ITS4 length 20 bp, SEQ ID NO: 6
  • the heat protocol used is reported in Table 4 of chapter 1.7 Characterization at the level of species.
  • BLAST Basic Local Alignment Search Tool
  • the genomic region D1/D2 located in the initial section of the gene encoding the large subunit (LS) is used by modern taxonomy for the identification of species or groups of species of yeast, as yeast belonging to the same group are characterized by the same sequence in the D1/D2 section.
  • the DNA sample for the amplification was prepared through the method described in example 1, point 1.6 (Characterization of the strains).
  • the sequence of the pair of primers used is as follows: NL-1 (SEQ ID NO: 17) and NL-4
  • D1/D2 sequence is composed of 507 nucleotides and is represented in the sequence listing with SEQ ID NO: 19.
  • the yeast covered by this patent has the enological characteristics reported below. Some were determined through growth in synthetic must (the characteristics of the test were described in example 1 , point 1.5).
  • the alcohol-generating power was evaluated by growth in synthetic must in which the glucose concentration (300 g/l) was varied.
  • a commercial control strain (Premium Prosecco, Enologica Vason SrI, containing a strain of Saccharomyces cerevisiae) used by many producers that operate in the DOC Prosecco area of Conegliano and Valdobbiadene was also tested. The tests were conducted in triplicate.
  • the alcohol-generating power of strain P301.9 is 16.4% while the commercial strain has an alcohol-generating power of 16.1%.
  • This activity is linked to the yeast's capacity to liberate the varietal aromas present in the must that are primarily present in the glycosylated form and thus odorless.
  • a fluorimetric method based on the emission of a fluorescent signal by the substrate 4-methylumbelliferyl ⁇ -D-glucopyranoside once this molecule is cleaved by the ⁇ -glucosidase enzyme was carried out.
  • the activity was assayed during the growth of the strain in synthetic must (the tests were conducted in triplicate) and was measured at pH 5 and pH 3.5 (enological situation).
  • the strain showed to possess a good activity, especially at pH 3.5.
  • a commercial control strain (Premium Prosecco, Enologica Vason SrI, containing a strain of Saccharomyces cerevisiae) used by many producers that operate in the DOC Prosecco area of Conegliano and Valdobbiadene was also tested.
  • the fermentation was conducted by maintaining the fermentation speed constant at 0.7 g I ⁇ V 1 through the addition of amino acidic nitrogen beginning with 5 g I "1 of CO 2 released up to 75 g I "1 of CO 2 .
  • the fermentation was monitored by automatically measuring the drop in weight every twenty minutes.
  • the strain P301.9 has a nitrogen requirement of 49 mg/l, while the commercial one shows a higher requirement (79 mg/l).
  • the strain was assayed in the laboratory by setting up an in-flask vinification
  • the wines produced were submitted to sensorial analyses as described in example 1 , point 1.9 (Microvinification).
  • the wine produced with the P301.9 strain has a high aromatic intensity (greater than that detected for other experimental yeasts). It is distinct with respect to that obtained with the commercial yeast for an equilibrium between the aromatic intensity and the characteristics of the olfactory attributes. Furthermore, good characteristics of typicality were encountered with hints of acacia and wisteria and in particular green apple, ripe apple, and citrus.
  • the strain next to a commercial control was tested in vinification trials in Prosecco must in 50 hectoliter wine makers, as described in example 1, point 1.10 (Vinification).
  • the concentration of the P301.9 strain in the paste was equal to (5.97 ⁇ 0.57)*10 9 CFU/ml while that of the control yeast was (8.60 ⁇ 0.6) * 10 9 CFU/ml.
  • the inoculate for both was 2.5*10 6 CFU/ml.
  • the SEQ ID Nos 1-18 regard primers with their respective names.

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Abstract

Cette invention concerne une souche de Saccharomyces cerevisiae identifiée par la désignation P301.9 et déposée au DBVPG sous le numéro de dépôt 24P. La souche a été isolée à partir de grappes de Prosecco dans la zone DOC de Conegliano et de Valdobbiadene dans la province italienne de Trévise. Cette souche représente un compromis optimum entre des caractéristiques technologiques et des aspects associés généralement au vin Prosecco. De plus, elle diffère de souches analogues en ce que ses besoins en azote sont très faibles. De plus, l’invention concerne l’utilisation de ladite souche comme inoculat dans la production de nourriture obtenue par fermentation alcoolique, en particulier dans le cadre de la vinification, et un inoculat relatif.
PCT/IB2010/001686 2009-07-10 2010-07-08 Souche de levure saccharomyces cerevisiae, son utilisation comme inoculant pour la production par fermentation de nourriture, en particulier de vin prosecco, et inoculant relatif WO2011004254A1 (fr)

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Cited By (7)

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Publication number Priority date Publication date Assignee Title
EP2634247A1 (fr) * 2012-02-29 2013-09-04 Guserbiot S.L.U. Souche de Saccharomyces cerevisiae et son utilisation dans la production de boissons alcoolisées
MD4203C1 (ro) * 2012-04-06 2013-09-30 Общественное Учреждение Научно-Практический Институт Садоводства И Пищевых Технологий Tulpină de levuri Saccharomyces cerevisiae pentru producerea vinurilor albe seci
ITUB20160092A1 (it) * 2016-01-25 2017-07-25 Univ Degli Studi Padova CEPPO DI LIEVITO Saccharomyces cerevisiae DBVPG35P, IL SUO USO PER LA PRODUZIONE FERMENTATIVA DI ALIMENTI, PARTICOLARMENTE DI VINO LISON E LISON CLASSICO, UN RELATIVO PRODOTTO E UN METODO PER LA SELEZIONE DEL CEPPO
CN110343625A (zh) * 2019-07-25 2019-10-18 江西师范大学 一种酿酒酵母菌株及其应用
WO2021134138A1 (fr) * 2019-12-31 2021-07-08 Universidad De Santiago De Chile Souches de levure saccharomyces cerevisiae non transgéniques améliorées génétiquement utiles dans la production de boissons alcoolisées
CN114149932A (zh) * 2021-11-08 2022-03-08 泸州老窖股份有限公司 酿酒酵母lj-2及其应用
CN114149933A (zh) * 2021-11-08 2022-03-08 泸州老窖股份有限公司 酿酒酵母lj-1及其应用

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2634247A1 (fr) * 2012-02-29 2013-09-04 Guserbiot S.L.U. Souche de Saccharomyces cerevisiae et son utilisation dans la production de boissons alcoolisées
MD4203C1 (ro) * 2012-04-06 2013-09-30 Общественное Учреждение Научно-Практический Институт Садоводства И Пищевых Технологий Tulpină de levuri Saccharomyces cerevisiae pentru producerea vinurilor albe seci
ITUB20160092A1 (it) * 2016-01-25 2017-07-25 Univ Degli Studi Padova CEPPO DI LIEVITO Saccharomyces cerevisiae DBVPG35P, IL SUO USO PER LA PRODUZIONE FERMENTATIVA DI ALIMENTI, PARTICOLARMENTE DI VINO LISON E LISON CLASSICO, UN RELATIVO PRODOTTO E UN METODO PER LA SELEZIONE DEL CEPPO
CN110343625A (zh) * 2019-07-25 2019-10-18 江西师范大学 一种酿酒酵母菌株及其应用
WO2021134138A1 (fr) * 2019-12-31 2021-07-08 Universidad De Santiago De Chile Souches de levure saccharomyces cerevisiae non transgéniques améliorées génétiquement utiles dans la production de boissons alcoolisées
CN114149932A (zh) * 2021-11-08 2022-03-08 泸州老窖股份有限公司 酿酒酵母lj-2及其应用
CN114149933A (zh) * 2021-11-08 2022-03-08 泸州老窖股份有限公司 酿酒酵母lj-1及其应用

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