YEAST STRAINS FOR FERMENTED DRINKS, PARTICULARLY WINE

Abstract

The present invention relates to a yeast strain likely to be obtained by hybridizing a strain S1 with a strain S2, said yeast strain presenting, according to test A, the following characteristics: fermentation kinetics from 15 to 22 days at a temperature of 24? C.; and resistance with an alcoholic strength of more than or equal to 15% v/v; and a nitrogen requirement of less than or equal to 200 ppm,
to the method of producing this strain and to its use.

Claims

1. A yeast strain obtained by hybridizing a strain S1, which is the yeast strain deposited on 9 Sep. 2015 at the National Collection of Microorganism Cultures (Collection Nationale de Cultures de MicroorganismesCNCM)Institut Pasteur, 25 rue du docteur Roux F75724 Paris under number I-5011, with a strain S2, which is the yeast strain deposited on 9 Sep. 2015 at the National Collection of Microorganism Cultures (Collection Nationale de Cultures de MicroorganismesCNCM)Institut Pasteur, 25 rue du docteur Roux F75724 Paris under number I-5012, said yeast strain presenting, according to test A, the following characteristics: fermentation kinetics from 15 to 22 days at a temperature of 24? C.; and resistance with an alcoholic strength of more than or equal to 15% v/v; and a nitrogen requirement of less than or equal to 200 ppm, where test A corresponds to an analysis of the fermentation activity of said yeast strain seeded in the amount of 2.10.sup.6 CFU/ml on a synthetic must standardized by the International Organisation of Vine and Wine adjusted to 250 g/l of fermentable sugars with an initial nitrogen concentration of 200 ppm by fermenting at a constant temperature of 24? C.

2. The yeast strain according to claim 1, said strain being the strain deposited on 5 May 2015 at the National Collection of Microorganism Cultures (Collection Nationale de Cultures de MicroorganismesCNCM)Institut Pasteur, 25 rue du docteur Roux F75724 Paris pursuant to the Budapest Treaty, under number I-4975.

3. The yeast strain according to claim 1, said strain being the strain deposited on 5 May 2015 at the National Collection of Microorganism Cultures (Collection Nationale de Cultures de MicroorganismesCNCM)Institut Pasteur, 25 rue du docteur Roux F75724 Paris pursuant to the Budapest Treaty, under number I-4977.

4. Yeast isolated from a yeast strain according to claim 1.

5. A method of obtaining an oenological yeast strain, said method comprising the following steps: a) hybridizing a yeast strain S1, which is the yeast strain filed on 9 Sep. 2015 at the National Collection of Microorganism Cultures (Collection Nationale de Cultures de MicroorganismesCNCM)Institut Pasteur, 25 rue du docteur Roux F75724 Paris under number I-5011, with a yeast strain S2, which is the yeast strain filed on 9 Sep. 2015 at the National Collection of Microorganism Cultures (Collection Nationale de Cultures de MicroorganismesCNCM)Institut Pasteur, 25 rue du docteur Roux F75724 Paris under number 1-5012; b) isolation of the strains obtained in step a); c) seeding of the strains isolated in step b) on a synthetic must standardized by the International Organisation of Vine and Wine [Organisme International de la Vigne et du VinOIV]; d) studying the fermentation kinetics of said strains ; and e) selecting the strains presenting the following three characteristics : i) fermentation kinetics from 15 to 22 days at a temperature of 24? C.; and ii) tolerance to an alcoholic strength of more than or equal to 15% v/v; and iii) a nitrogen requirement of less than or equal to 200 ppm.

6. The method of obtaining an oenological strain according to claim 5, said method also comprising a step in which strains presenting fermentation kinetics at least 15%, preferably at least 30%, more preferentially at least 40% greater than the parent strain S1 and at least 15%, preferably at least 30%, more preferentially at least 40% lower than the parent strain S2 on a synthetic OIV must are selected.

7. A method for producing wine, said method comprising using a yeast strain according to claim 1.

8. The method according to claim 7, wherein the wine is red wine.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0065] FIG. 1 is a graph representing the fermentation kinetics of strains 51-062 (H), 51-135 (F), S1 (D) and S2 (E) represented by measuring the weight/initial weight ratio as a function of time.

EXAMPLES

Example 1

Hybridization of Strains S1 with Those From S2

[0066] Sporulation of two parent strains and development of operational conditions for sporulating strain S1.

[0067] The sporulation conditions were applied to both S2 and S1 strains. The sporulation rates were calculated and are presented in Table 1.

TABLE-US-00001 TABLE 1 Strain % ascospores Wine strain S2 13% Wine strain S1 54% [0068] Isolation of spores by micromanipulation and study of the self-diploidization of spores isolted from strains S2 and S1.

[0069] The sporulation conditions were applied on a larger scale on the two wine strains. After treating asci and visualizing tetrads by microscope, a dissection consisting of breaking tetrads, removing spores and then depositing them on precise locations of the agar has been performed. The enumeration of deposited spores and germinated spores is presented in Table 2 below.

TABLE-US-00002 TABLE 2 Number of Number of Number of dissected deposited germinated Germination Strain tetrads spores spores Rates S2 50 200 73 36% S1 33 132 95 72%

[0070] To be able to hybridize between strain S2 and S1, sexual type spores clearly differentiated a or alpha must be obtained for each strain. The sexual sign of each spore previously obtained was studied by PCR mating type (Table 3).

TABLE-US-00003 TABLE 3 Number of germinated Sign Strain spores Sign a Sign alpha a/alpha No result S2 39 20 14 4 1 S1 95 0 0 95 0

[0071] Table 3 shows that sexual type spores differentiated a or alpha could not be obtained for strain S1. Therefore it appears that strain S1 is homothallic. Under these conditions, contemplating a conventional hybridization program between strains S2 and S1 is not possible. [0072] Developing hybridization operating conditions specific to the S1?S2 crossing, isolating the new strains and studying their genetic profile to select the hybrids obtained from the crossing between S1 and S2.

[0073] The inventors, having no collection of sexual type a or alpha spores for strain S1 available, have hybridized spores from S1 and S2 following the dissection of tetrads from each strain via the micromanipulator. The new hybrid can therefore be the result of crosses between a spore from S1 with a spore from S2 or rather the result of crosses between a spore from S1 with a spore from S1 in which the sexual type has changed during replication. in order to select hybrids obtained from the cross between a spore from S1 and a spore from S2, the genetic profiles of the new hybrids were analyzed by PCR Ty, and these profiles were then compared to that of the parent strains. Finally, 68 hybrids presenting mixed profiles at different strengths in relation to the parent strain profile were retained. The oenological qualities of these 68 strains were then evaluated. Strains 51-062 and 51-135 were selected as follows.

Example 2

Development of Hybrid Selection Techniques

[0074] Several selection techniques were used:

[0075] a) Selection in synthetic conditions by studying fermentable sugar dehydration, fermentation kinetics and the production of fermentation compounds of interest in hybrids and parent strains on synthetic Lesaffre wine must (250 g/L sugars and 250 ppm available nitrogen)=test A and synthetic OIV must.

[0076] A first selection, done on the kinetics criterion, enabled the selection of 11 hybrids presenting better properties than S1 in terms of fructose dehydration.

[0077] Among these hybrids, strains 51-062 and 51-135 were selected because they present average kinetics between S1 and S2 and at the end of fermentation, these strains produce intermediate fermentation compounds that are different from the two parent strains.

[0078] b) Selection in real conditions by studying fermentation kinetics and ester production on natural musts of hybrids and parent strains:

[0079] The selection criterion was the analysis of fermentation kinetics, control of oenological parameters and principal ester analysis during microvinifications on natural Cabernet Sauvignon must. This must is obtained by hot pre-fermentation maceration of very ripe cabernet sauvignon grapes. The must from this year's harvest has been stored at ?60? C. in the freezer.

[0080] Before fermentation, the must was processed in the following manner: 1) Thawing at room temperature, 2) Clarification by centrifugal separation 3) Separation of the must into 5 homogeneous batches.

[0081] Before fermentation, the must was analyzed by Fourier transform infrared spectroscopy+colorimetric SO.sub.2 determination. The results obtained are summarized in Table 4 below:

TABLE-US-00004 TABLE 4 Total Glucose + acidity Malic Tartaric Citric fructose (g eq acid acid acid K NH3 NOPA SO.sub.2L SO.sub.2 T (g/L) H2SO4/L) pH (g/L) (g/L) (g/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) 246 4.30 3.34 3.38 1.8 0.66 670 42 103 0 0

[0082] Following the must analysis, the absence of alcohol and acetic acid before fermentation were also observed. 246 g of Glucose+fructose corresponding to a probable strength of 14.6% vol (yield of 16.83). [0083] Preparations of yeasts on agar tubes: strains 51-135 (F) and 51-062 (H)

[0084] Preparation of the medium to be incubated on Day 0: Yeast extract 20 g, Sucrose 100 g, Mineral solution (100 g MgSO4+100 g KH2PO4+Demineralized water QS 1 L) 10 mL, Tap water QS 1 L.

[0085] On Day 3inoculation of test tubes A (for each strain tested): A monosaccharide is removed from the agar and deposited in 10 ml of medium to be incubated for 24 hours at 26? C.

[0086] On Day 4preparation of test tubes B 1 ml from tube A in 9 ml of medium to be incubated 24 hours at 26? C.

[0087] On Day 5: Pour each tube B into 90 ml of medium to be incubated 16 hours+/?2 hours at 26? C. with good agitation.

[0088] On Day 6: Measuring the population with a Thoma cell.

[0089] Seeding jars (Table 5):

TABLE-US-00005 TABLE 5 Yeasts/ Volume Estimated large Yeasts/ Yeasts/ seeded population Preparation square mlx1.6.10.sup.5 100 mL in a jar cell/L F (strain 152 2.107 2.10.sup.9 100 mL 2.10.sup.8 51-135) H (strain 173 3.10.sup.7 3.10.sup.9 100 mL 3.10.sup.8 51-062)

[0090] Control of the population in jars (Table 6):

TABLE-US-00006 TABLE 6 Preparation Yeasts/Large square Cell population/L F (strain 51-135) 13 2.109 H (strain 51-062) 16 3.109 [0091] Preparation of dry yeasts: strains D (strain S1) and E (strain S2):

[0092] On Day 6: Hydration of yeasts in sterile water: 1 g in 10 mL and then ultrasonic agitation for 10 minutes, incubation at 30? C. for 20 minutes, ultrasonic agitation for 15 minutes, rest 20 minutes at ambient temperature.

[0093] The jars were seeded at 2 ml of preparation in 1 L of must. The population was verified with a Thoma cell: 10.sup.10 cell/L (Table 7):

TABLE-US-00007 TABLE 7 Preparation Yeasts/Large square Cell population /L D (strain S1) 35 6.10.sup.9 E (strain S2) 34 5.10.sup.9

[0094] Fermentation Analyses

[0095] Fermentations are conducted in a climate-controlled room with a temperature of 18? C. The fermentation kinetics are analyzed by weighing jars on a +/?0.1 g scale, approximately 2 times /day. Fermentation started on Day 7 and stopped on Day 19.

[0096] The results are presented in FIG. 1.

[0097] At the end of fermentation: Racking and sulfuring around 60 mg/L, bottling and storage at 8? C.

[0098] Analyses on Resulting Wines

[0099] Analysis by Fourier transform infrared spectroscopy and colorimetric SO2 determination on D20:

TABLE-US-00008 TABLE 8 Total Total alcoholic Glucose + alcoholic Total Acetic Free Total Malic Lactic strength by fructose strength by acidity acid SO2 SO2 acid acid volume % v (g/L) volume % v (gH.sub.2SO.sub.4/L) (g/L) (mg/L) (mg/L) pH (g/L) g/L D 15.13 18 16.2 4.94 0.62 10 72 3.51 2.1 0 E 12.74 54 15.9 4.81 0.6 10 58 3.34 2.0 0 F 14.64 23 16 5.12 0.71 12 74 3.51 2.4 0 H 15.39 12.9 16.2 4.81 0.48 10 44 3.51 2.1 0

[0100] Depending on the yeast strain, the following is observed: [0101] 0.5% volume variance on the total alcoholic strength by volume. [0102] After 19 days, residual sugars are present for all strains. [0103] total acidity varies from 4.69 to 5.12 g eqH2SO4/L [0104] volatile acidity varies from 0.48 to 0.71 g eqH2SO4/L [0105] The malolactic fermentation did not begin.

[0106] Polyphenol color analysis (Table 9)

TABLE-US-00009 Color intensity Shade DO280 D 13.8 0.58 54 E 17.9 0.67 59 F 13.5 0.61 56 H 14.8 0.60 55

[0107] Ester Analysis

TABLE-US-00010 Ethyl-2- Ethyl Ethyl-2- Ethyl Ethyl Ethyl Ethyl Ethyl Ethyl Hydroxy-4- Isoamyl butyrate methylbutyrate Isobutyrate Levulinate Isovalerate Hexanoate Octanoate Decanoate methylpentanoate Acetate Sample (?g/L) (?g/L) (?g/L) (?g/L) (?g/L) (?g/L) (?g/L) (?g/L) (?g/L) (?g/L) D 170.2 <5 <5 <20 <5 254.5 349.8 339.3 168.3 609.4 E 128.6 <5 <5 <20 <5 253.3 466 473.8 84.5 484 F 219.9 <5 5 <20 <5 279 441.3 366.1 201 436.2 H 276 <5 7.1 <20 <5 386.5 567.5 447.6 211.5 523

CONCLUSION

[0108] In general, we observe no significant difference between the results produced by dry yeast D and those produced by agar tube yeasts.

TABLE-US-00011 Active dry yeasts Agar tube yeasts Total alcoholic strength No significant difference Residual sugars Presence of residual sugars <30 g/L Stop fermentation Volatile acidity No significant difference except with H = 0.48 Malolactic fermentation Not done Color intensity 13.8 to 14.8 Shade 0.58 to 0.65 DO280 54 to 56

[0109] The hybrids confirm faster kinetics than S1 but 2 groups are formed: [0110] 51-135 shows intermediate fermentation kinetics compared to S2 and S1, residual contents of sugar and total SO2 (high therefore negative) and an influence on color close to S2 and an ester production slightly higher but close to the parent yeasts. [0111] 51-062 shows faster kinetics than S2 but with an equivalent slope, residual sugar content, volatile acidity and SO2 lower than S2 and higher ester production than the parent strains. This yeast does not appear to correspond to the kinetic criterion but presents very interesting characteristics that should be subject to further study.