Strain of yeast <i>Saccharomyces bayanus </i>subsp. <i>uvarum </i>DBVPG36P, its use in the fermentative production of foods and a method for the selection of the strain
11525109 · 2022-12-13
Assignee
Inventors
Cpc classification
C12G2200/05
CHEMISTRY; METALLURGY
Y02E50/10
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
International classification
C12G1/02
CHEMISTRY; METALLURGY
Abstract
The invention concerns a strain of Saccharomyces bayanus subsp. uvarum identified as SERIUS and deposited at the DBVPG with deposit number 36P. The invention further concerns the use of the strain of Saccharomyces bayanus subsp. uvarum identified as SERIUS (DBVPG 36P) as a food inoculate in the production of foods obtained through alcoholic fermentation, and a selection method for yeast strains of the group Saccharomyces adapted to alcoholic fermentation, particularly of grape, consisting of an isolation step for such yeasts from flowers.
Claims
1. A method of using a strain of Saccharomyces bayanus subsp. uvarum identified as SERIUS (DBVPG 36P) and deposited at the DBVPG with deposit number 36P, the method comprising: inoculating a food with the strain; and allowing the food to undergo alcoholic fermentation, wherein the food is a grape juice and the alcoholic fermentation is a vinification process that converts the grape juice to a wine, and wherein the Saccharomyces bayanus subsp. uvarum generates more malic acid as compared to a vinification process of the same grape juice using a Saccharomyces cerevisiae strain.
2. The method according to claim 1, wherein the strain is in a cream form, desiccated form, lyophilized form, or in the form of a paste when inoculating the food.
3. The method according to claim 1, wherein the grape juice is produced from grapes selected from white berry grapes or red berry grapes.
4. The method according to claim 1, wherein the wine contains glycerol and malic acid naturally produced by the strain in the following concentrations: glycerol higher than about 8.5 g/l; malic acid higher than about 0.5 g/l.
5. The method according to claim 1, wherein the wine contains glycerol higher than about 9.5 g/l and malic acid higher than about 1 g/l.
6. The method according to claim 1, wherein the wine contains glycerol higher than about 10 g/l and malic acid higher than about 2 g/l.
7. The method according to claim 1, wherein the wine contains malic acid at a concentration of about 2 g/l or more.
8. The method according to claim 1, wherein the wine contains malic acid at a concentration of about 2.5 g/l or more.
9. The method according to claim 1, wherein the wine contains malic acid at a concentration of about 3 g/l or more.
10. A method of using a strain of Saccharomyces bayanus subsp. uvarum identified as SERIUS (DBVPG 36P) and deposited at the DBVPG with deposit number 36, the method comprising: producing a dietary supplement or food product containing the strain, wherein the strain itself functions as a probiotic within the dietary supplement or food product independent of other components of the dietary supplement or food product.
11. The method according to claim 10, wherein the food product is formed from a single alcoholic fermentation with the Saccharomyces bayanus subsp. uvarum.
Description
BRIEF DESCRIPTION OF THE FIGURES
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EXAMPLE 1—ISOLATION AND SELECTION STEPS FOR THE STRAIN ACCORDING TO THE INVENTION
(5) 1.1 Sampling
(6) Sampling has been conducted by collecting flowers during springtime, in an area of the Veneto region with high grape-wine producing activity.
(7) Collection has been performed by avoiding touching the flowers with bare hand at all stages, and periodically sterilising the scissors, with the aim of limiting contamination as much as possible.
(8) Flowers have been harvested in sufficient quantities to fill an appropriately sealed sterile bag. Upon arrival in the laboratory, the flowers have been transferred into sterile flasks containing 200 ml of synthetic must, previously prepared in distilled water, adjusted to pH 3.2 using KOH, and sterilised by filtration, the composition of which is reported in table 1.
(9) TABLE-US-00001 TABLE 1 Component category Component Dose per litre Macroelements CaCl.sub.2 0.1 g NaCl 0.1 g KH.sub.2PO.sub.4 1 g MgSO.sub.4 × 7H.sub.2O 0.5 g Tartaric acid 3 g Microelements NaMoO.sub.4 × 2H.sub.2O 0.2 mg ZnSO.sub.4 × 7H.sub.2O 0.4 mg H.sub.3BO.sub.3 0.5 mg CuSO.sub.4 × 5H.sub.2O 0.04 mg KI 0.1 mg FeCl.sub.3 × 6H.sub.2O 0.4 mg MnSO.sub.4 × H.sub.2O 0.4 mg Vitamins Pyridoxine 400 μg hydrochloride Thiamine hydrochloride 400 μg Inosite 2000 μg Biotin 20 μg Calcium pantothenate 400 μg Nicotinamide 400 μg p-Aminobenzoic acid 200 μg Varying components (NH.sub.4).sub.2SO.sub.4 0.3 g (NH.sub.4).sub.2HPO.sub.4 0.3 g Glucose 200 g Casein hydrolysate 0.2 g Malic acid 2 g
(10) In addition, the synthetic must has been supplemented with the antibiotic, oxytetracycline, at a concentration of 0.1 mg/ml, so as to avoid any bacterial proliferation. The preparations have then been overlaid with sterile paraffin oil in order to avoid the formation of mould. The flasks have been sealed using a bored silicone bung, into which a Pasteur pipette, curved at the upper end, has been inserted. This has the purpose of avoiding potential contamination with microorganisms present in the environment, and allowing CO.sub.2 to vent with no loss of water vapour. The flasks have been placed in an incubator at a constant temperature of between 22° C. and 25° C.
(11) Progress of the fermentation has been monitored by measuring the drop in weight for each flask (an indicator of fermentation of the sugars to CO.sub.2 and hence the progress of the fermentation).
(12) 1.2 Isolation of the Strains from the Samples
(13) On completion of fermentation, 10 ml of product have been removed from each flask, a first 1:10 serial dilution has been performed, from which 1 ml has been removed and 5 subsequent serial dilutions (1:10) in Ringer's solution have been performed; 100 μl of the final three dilutions have then been seeded by spreading on WL agar 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 iron chloride, 2.5 mg/l manganese sulphate, 22 mg/l bromocresol green, 20 g/l agar) suitably sterilised by autoclave and adjusted to a final pH of 5.5±2. The plates have then been incubated at 28° C. for 3 days under aerobic conditions. In this medium, yeast colonies of the genus Saccharomyces appear with varying colour ranging from cream to light green, with a smooth-opaque surface and a creamy consistency. Yeast counting has revealed a Saccharomyces concentration of about 10.sup.7 CFU/ml, with the presence of both white and green colonies, with a smooth-opaque surface and a creamy consistency.
(14) 1.3 Purification and Storage of the Isolates
(15) Colonies picked from the isolation plates have been reseeded on WL agar growth medium, the composition of which has been specified at point 1.2 of example 1. The procedure has been repeated at least twice, so as to be certain that each isolate originates from a single colony. The pure cultures obtained following the final passage on plates have been picked using a sterile loop and then cryopreserved at −80° C. following the addition of glycerol to 40% (w/w). On plates, the strain, subsequently identified as Saccharomyces bayanus subsp. uvarum, appears as small colonies, with an intense green colour, while strains attributable to the species Saccharomyces cerevisiae come from larger colonies, with a creamy consistency, coloured white or tending towards light green.
(16) 1.4 Identification of the Isolates at the Species and Strain Level
(17) The presumed colonies of the yeasts Saccharomyces have been identified at the species level by means of RAPD-PCR using the M13 primer, according to the method described by Andrighetto et al. in 2000. In the analysis and subsequent processing of amplification profiles for the yeasts isolated from flowers, Type and reference strains from various yeast species of oenological and non-oenological interest have also been included. Comparison of the profiles has made it possible to recognise yeasts attributable to the species S. cerevisiae and S. bayanus.
(18) To be able to discriminate at the strain level, two techniques have been used:
(19) 1) Analysis of mitochondrial DNA restriction profiles;
(20) 2) Analysis of microsatellites;
(21) which shall be described in detail in the executive examples in section 1.6.
(22) It is not excluded that the implementation of said genetic techniques may be performed using methods in themselves known to those skilled in the sector and that, therefore, the technical indications described herein may vary from that reported in the aforementioned examples.
(23) 1.5 Technological Characterisation of the Strains Isolated
(24) In order to assess the fermentation characteristics, each of the yeasts selected has been subjected to fermentation testing, using natural or synthetic must according to the composition described in table 1.
(25) The inoculum has been prepared in synthetic or natural must (depending on whether the fermentative vigour evaluation test was performed on synthetic must or natural must), starting from a wire loop pick taken from a slant or plate growth culture, and has been then incubated at 25° C. for 24 hours. Subsequently, this has been inoculated in the region of 10% in synthetic or natural must with a final volume of 200 ml, in flasks sealed with a centre-bored silicone bung, into which has been inserted a Pasteur pipette, bent so as to allow the venting of carbon dioxide with no loss of water vapour.
(26) The drop in weight due to the loss of CO.sub.2 from the flasks, set up for fermentation testing, has been monitored daily until completion of fermentation, corresponding to the phase where the drop in weight has remained constant for several days. For each isolate grown, the ability to produce foam during fermentation has been assessed by means of visual evaluation. Again, by means of visual evaluation, the growth method has been assessed for the strain during fermentation (pulverulent or flocculent).
(27) Finally, for the evaluation of hydrogen sulphide production, each isolate has been seeded on Biggy agar medium (Bismuth Glucose Glycine Yeast Agar: 5 g/l bismuth ammonium citrate, 3 g/l sodium sulphite, 10 g/l glucose, 10 g/l glycine, 1 g/l yeast extract, 16 g/l agar, pH 6.8±0.2) and incubated at 25° C. for 5 days, after which, the colour of the colonies grown on the plates has been assessed. The quantity of hydrogen sulphide is directly proportional to the intensity of the colour, due to the creation of bismuth sulphite (dark brown if present in significant quantities). The technological characterisation of the isolates from flowers has made it possible to identify certain Saccharomyces yeast strains, including the strain Saccharomyces bayanus subsp. uvarum SERIUS, endowed with promising oenological characteristics.
(28) 1.6 Genetic Characterisation at the Strain Level
(29) In order to achieve characterisation at the strain level, two techniques have been used: Analysis of mitochondrial DNA restriction profiles; Analysis of microsatellites.
(30) Analysis of restriction profiles has been performed by following the method reported in Zilio et al. in 1998, using the restriction enzyme Hinf I. Separation of the linear DNA fragments has been obtained through electrophoresis on 1% agarose gel. Comparison of the restriction profiles has been performed using the software GelComparII (Applied Maths, Belgium) which, by means of the construction of a matrix, is capable of calculating the level of similarity between profiles and expressing the results graphically as a dendrogram. The mitochondrial DNA restriction profile for the strain SERIUS of the invention, obtained using the enzyme Hinf I, is shown in
(31) Microsatellite analysis envisages the use of PCR technique with primers that are complimentary to regions of the DNA known as microsatellites, which are small sequences of tandem repeat DNA (from one to six bases) varying in the number of repetitions, and may also be localised inside genomic coding regions (Legras, 2005). They are extremely variable in length, as a result of DNA replication errors, and therefore display a certain degree of polymorphism between individuals in the same species. Microsatellite polymorphism analysis is a highly reproducible method because specific primers and high annealing temperatures are used for their amplification. For characterisation of the strain S. bayanus subsp. uvarum SERIUS, the eight most variable loci described by Masneuf-Pomarede et al. in 2016 have been considered, and a reaction has been 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).
(32) The reaction has been conducted in a volume of 20 μl using 1 U of DNA polymerase in 1× buffer, with the addition of magnesium chloride (MgCl.sub.2) at a concentration of 1.5 mM, nucleotide triphosphates (dNTPs) at a concentration of 200 μM and 1 μM for each of the primers for each reaction.
(33) Amplification has been performed in a Mastercycler Nexus Gradient (Eppendorf) with a unique thermal program for all amplification reactions, with the exception of the annealing temperature (Ta). The thermal protocol used is reported in table 2 below (the number of repetitions for each cycle is reported in parentheses).
(34) TABLE-US-00002 TABLE 2 Amplification protocol cycle temperature duration Cycle1 94° C. 5 min (1x) Cycle2 94° C. 30 s (30x) Ta ° C. 30 s 72° C. 30 s Cycle3 72° C. 10 min (1x)
(35) The Ta used has been set at 55° C. for PCR reactions concerning the loci SuYIL130W, SuYHR102W and SuYGC170W; while the aforementioned Ta has been set at 50° C. for the PCR reactions concerning the loci SuYKR045C, SuHTZ1PLB3, SuARS409, SuYHR042-043 and SuYBR049C.
(36) Amplification and agarose gel electrophoresis migration of the PCR products relating to the eight most variable loci in S. bayanus var. uvarum, have made it possible, for the strain of Saccharomyces bayanus subsp. uvarum SERIUS, to define the unique and characteristic profile reposted in
(37) In addition to the individual PCR reaction, the eight aforementioned loci have also been analysed by means of two multiplex reactions, capable of the simultaneous amplification of three and five loci, respectively.
(38) In particular, two multiplex-PCR reactions have been used: multiplex-PCR1 allows analysis of the DNA-microsatellite profile for loci SuYIL130W, SuYHR102W and SuYGC170W, and multiplex-PCR2 allows attainment of the DNA-microsatellite profile for loci SuYKR045C, SuHTZ1PLB3, SuARS409, SuYHR042-043 and SuYBR049C.
(39) The two multiplex-PCR reactions have been conducted in a volume of 20 μl, using 1.5 U and 1 U of DNA polymerase respectively in 1× buffer with the addition of magnesium chloride (MgCl.sub.2) at a concentration of 1.5 mM and nucleotide triphosphates (dNTPs) at a concentration of 200 μM. The concentration of the primers has been optimised as follows.
(40) In the case of multiplex-PCR1, the two primers for locus SuYIL130W have been added at a concentration of 0.25 μM, the two primers for locus SuYHR102W at a concentration of 0.75 μM, while the two primers for locus SuYGC170W at a concentration of 1 μM.
(41) In the case of multiplex-PCR2, the two primers for loci SuYKR045C, SuHTZ1PLB3, SuARS409, SuYHR042-043 and SuYBR049C have been applied at a concentration of 0.5 μM.
(42) Amplification has been performed in a Mastercycler Nexus Gradient (Eppendorf) with a unique thermal program for all amplification reactions, with the exception of the annealing temperature (Ta). The thermal program used is reported in table 3 below (the number of repetitions for each cycle is reported in parentheses).
(43) TABLE-US-00003 TABLE 3 Amplification protocol cycle temperature duration Cycle 1 94° C. 5 min (1x) Cycle 2 94° C. 30 s (30x) Ta ° C. 30 s 72° C. 30 s Cycle 3 72° C. 10 min (1x)
(44) In the case of multiplex-PCR1 the Ta used has been 55° C., while in the case of multiplex-PCR 2 it has been 50° C.
(45) The profiles obtained from the aforementioned analysis are reported in
(46) Furthermore, characterisation of the strain of the invention has also been performed by means of capillary electrophoresis based on amplification of the four loci which, according to data in the literature, are characterised by a greater number of allelic forms.
(47) Advantageously, said characterisation has made it possible to define the exact size of the PCR products for the 4 loci considered.
(48) The loci considered are the following: SuYIL130W, SuYHR102W, SuYKR045C and SuHTZ1PLB3. Amplification has been performed according to the protocol reported previously, with the single variant consisting of modification of the 5′ end of the forward primers with a fluorescent molecule. In particular, SuYIL130W-FW and SuYKR045C-FW have been labelled with Hexachloro-fluorescein (HEX), while SuHTZ1PLB3-FW and SuYHR102W-FW have been labelled with 6-carboxyfluorescein (FAM).
(49) Analysis of the capillary profile has been performed using the D filter, with internal reference ROX. Interpretation has been performed using the software Peak Scanner 2.0, with the following parameters: “size standard: GS500 (−250)” and “analysis method: sizing default—NPP”. The results obtained, namely the sizes of the fragments, are reported in table 4 and are expressed in base pairs (bp) with an error of +/−1 bp.
(50) TABLE-US-00004 TABLE 4 Strain SuYIL130W SuYHR102W SuYKR045C SuHTZ1PLB3 Saccharomyces 179 219 300 259 bayanus var. uvarum SERIUS
(51) It is not excluded that the implementation of said genetic techniques may be performed using methods in themselves known to those skilled in the sector and that, therefore, the aforementioned technical indications may vary from that reported.
EXAMPLE 2—TECHNOLOGICAL CHARACTERISATION OF THE YEAST
(52) 2.1 Production of Hydrogen Sulphide
(53) Production of hydrogen sulphide by the strain SERIUS has been evaluated by means of incubation at 25° C. for 5 days in Biggy Agar medium (1 g/l yeast extract, 10 g/l glycine, 10 g/l dextrose, 5 g/l bismuth ammonium citrate, 3 g/l sodium sulphite, 16 g/l agar). On completion of incubation, in this medium, the colonies appear with different colours, based on the quantity of hydrogen sulphide they produce. Colonies that are dark brown in colour indicate high hydrogen sulphide production, colonies that are clear brown-beige in colour indicate moderate production, while white colonies are characterised in that they are incapable of producing it. Following incubation on Biggy agar medium, colonies of the strain of the invention appear white in colour, thus, under the conditions tested, the strain is incapable of producing H.sub.2S. Testing has also been repeated by analysis of hydrogen sulphide production in Lead Acetate Agar medium (15 g/l peptone, 5 g/l proteose peptone, 1 g/l dextrose, 0.2 g/l lead acetate, 0.08 g/l sodium thiosulphate, 15 g/l agar) incubated at 25° C., and the results obtained have been analogous to those obtained with Biggy agar medium.
(54) 2.2 Fermentative Vigour
(55) The fermentative vigour of the strain of the invention has been evaluated in both synthetic must, the characteristics of which are described in example 1, point 1.1, and in various types of natural white berry grapes and red berry grapes must. The inoculum has been prepared in synthetic or natural must (depending on whether the fermentative vigour evaluation test was performed on synthetic must or natural must), starting from a wire loop pick taken from a slant or plate growth culture, and has been then incubated at 25° C. for 24 hours. Subsequently, this has been inoculated in the region of 10% in synthetic or natural must with a final volume of 200 ml, in flasks sealed with a centre-bored silicone bung, into which has been inserted a Pasteur pipette, bent so as to allow the venting of carbon dioxide with no loss of water vapour. This way it has been possible to follow the progress of the fermentation by means of monitoring the drop in weight due to the production of CO.sub.2, an indicator of fermentation of the sugars. Incubation has been conducted at a constant temperature of 25° C., and evaluation of the weight loss has been performed every 1-2 days, until achieving constant weight. Using the data collected, growth curves have been prepared, and the fermentative vigour of the strain Saccharomyces bayanus var. uvarum SERIUS has been compared with the fermentative vigour of commercial strains of Saccharomyces bayanus and cerevisiae. In the various fermentation tests, the strain of the invention has displayed a good ability for fermentation, with mean loss of weight values at 2 and 7 days that are analogous to those observed with commercial yeasts of the species Saccharomyces cerevisiae, and with fermentation completion times varying depending on the type of must used (from 10 to 15 days).
(56) The fermentative vigour of the strain has also been evaluated in natural must obtained from Trebbiano grapes, by means of incubation at temperatures of 12, 15 and 24° C. Analysis reveals that the strain has good fermentative ability, even at temperatures of 12° C. and 15° C.
(57) Advantageously, this latter characteristic of the strain of the invention allows its use in wine fermentations at low temperatures, thus increasing the possibility of activating specific biochemical reactions leading to the production of aromatic compounds having a positive influence on the organoleptic qualities of the wine produced.
(58) 2.3 Production of Glycerol
(59) Glycerol production has been evaluated by means of HPLC (Jasco RI 930 detector, Rezex™ ROA-Organic Acid H+8% column (300×7.8 mm) in natural white berry grapes and red berry grapes must inoculated with the strain SERIUS, with incubation at 25° C. until completion of fermentation.
(60) In addition, comparative tests have been set up where a commercial strain of Saccharomyces cerevisiae has been inoculated. The results obtained are reported in table 5, where the quantity of glycerol produced is expressed in g/l. It may be observed that the strain of the invention shows significant glycerol production, higher on average than the quantity of glycerol produced in the same must by commercial yeast used as a comparison.
(61) TABLE-US-00005 TABLE 5 Red berry musts White berry musts Nerello Nero Strain Garganega Kerner Trebbiano Merlot Mascalese d'Avola SERIUS 8.8 10.6 8.7 9.1 9.8 9.5 Commercial 6.8 7.5 5.8 8.1 8.1 6.5 S. cerevisiae
2.4 Production of Malic Acid
(62) The strain of the invention has been tested for the ability to produce malic acid in natural white berry and red berry must, following incubation at 25° C. until fermentation completion. In particular, the quantity of malic acid present in the various musts before and after fermentation using the aforementioned strain has been assessed by means of HPLC Jasco UV 975 detector, Resex™ ROA-Organic Acid H+8% column (300×7.8 mm). Analogously to example 2.3, comparative tests have been conducted with an inoculum of the commercial strain of Saccharomyces cerevisiae used previously. The results obtained are reported in table 6 where the quantity of malic acid is expressed in g/l. It may be observed that the strain SERIUS is capable of producing malic acid, even though with varying efficiency, in 5 of the 6 musts tested, unlike the commercial strain, which is characterised by the ability to partially degrade malic acid.
(63) TABLE-US-00006 TABLE 6 End of fermentation Prior to End of fermentation commercial Must fermentation SERIUS S. cerevisiae Garganega 1.3 3.9 1.2 Kerner 3.0 4.0 2.1 Trebbiano 1.8 1.3 1.5 Merlot 2.3 3.0 1.2 Nerello 2.8 3.1 1.4 Mascalese Nero d'Avola 1.5 2.5 1.2
2.5 Production of Volatile Acidity
(64) The ability to produce acetic acid has been evaluated by means of HPLC Jasco RI 930 detector, Rezex™ ROA-Organic Acid H+8% (300×7.8 mm) column in natural white berry and red berry must inoculated with the strain SERIUS, incubated at 25° C. until completion of fermentation. Comparative tests inoculated with a commercial strain of Saccharomyces cerevisiae have also been set up. The results obtained are reported in table 7, where the acetic acid production values, expressed in g/l, are reported, demonstrating that the strain of the invention is characterised by the ability to produce little volatile acidity.
(65) TABLE-US-00007 TABLE 7 End of fermentation Prior to End of fermentation commercial Must fermentation SERIUS S. cerevisiae Garganega 0 0.11 0.29 Kerner 0 0.22 0.18 Trebbiano 0 0.16 0.23 Merlot 0 0.30 0.39 Nerello 0 0.22 0.46 Mascalese Nero d'Avola 0 0.1 0.18
2.6 Killer Activity
(66) The presence of killer activity in the yeast SERIUS has been studied using YEPD agar medium, prepared by means of the prior inoculation and overnight incubation of a culture of a sensitive indicator strain. Operatively, after having left said plates including the indicator strain to dry, 10 μl of a fresh culture of the strain SERIUS has been deposited on them and the same plates have been left to incubate for 48 hours at 25° C. Subsequently, on completion of the incubation period, killer activity has been detected by the appearance of “inhibition halos” (an inhibition halo is understood as being an area with the absence of growth) around the colony of the strain under test.
(67) With regard to the strain SERIUS, killer activity has been tested in relation to 10 oenological yeast strains belonging to the species S. cerevisiae and S. bayanus respectively.
(68) The analysis has shown that none of the yeasts tested has been inhibited by the strain of the invention. In addition, none of the 10 yeasts tested has demonstrated the ability to inhibit the strain SERIUS.
(69) 2.7 Resistance to Copper and Resistance to Sulphur Dioxide
(70) Copper resistance has been assayed by means of growth in synthetic YNB (Yeast Nitrogen Base) medium, characterised by the absence of aminoacids and the presence of 6.7 g/l sulphate, 20 g/l glucose and 20 g/l agar, supplemented with various concentrations of CuSO.sub.4 (50, 100, 200, 300 μmol/l respectively). With regard to sulphur dioxide resistance, this has been evaluated by means of growth in agarised must supplemented with potassium metabisulphite in such concentrations as to give a final SO.sub.2 concentration essentially corresponding to 50, 100, 200 and 300 ppm. Both analyses described above have been performed by means of incubation of the strain SERIUS in previously prepared media for 48 hours at 26° C., and a visible growth has been evaluated on plates.
(71) The results obtained are reported in table 8, expressed as MTC (Maximum Tolerated Concentration, of copper or sulphur dioxide respectively). As may be observed from the following table, the strain SERIUS demonstrates the ability to grow in the presence of 300 ppm SO.sub.2 and 300 μmol/l copper.
(72) By way of comparison, the aforementioned tests have also been conducted on 5 commercially available strains of S. cerevisiae for oenological use.
(73) TABLE-US-00008 TABLE 8 Strain MTC SO.sub.2 (ppm) MTC CuSO.sub.4 (μmol/l) SERIUS 300 300 Commercial 300 50 S. cerevisiae 1 Commercial 300 50 S. cerevisiae 2 Commercial 50 50 S. cerevisiae 3 Commercial 300 100 S. cerevisiae 4 Commercial 300 100 S. cerevisiae 5
2.8 Tolerance to Ethanol
(74) Ethanol tolerance has been assayed using agarised must including varying concentrations of ethanol, corresponding to 12, 14, 16 and 18% respectively. The strain SERIUS has been incubated in said medium for 48 hours at 26° C., on completion of which, visible growth has been evaluated on plates.
(75) The analysis shows that the strain SERIUS has an MTC value for ethanol equal to 16% vol/vol.
(76) 2.9 Carbon Source Use
(77) The ability of the strain SERIUS to assimilate/ferment various carbon source compounds has been evaluated by means of growth of the aforementioned strain in media containing yeast extract (10 g/l), peptone (20 g/l) and sugar/organic acid at a concentration of 20 g/l.
(78) The analysis has shown that the strain SERIUS is capable of using such organic compounds as galactose, raffinose, maltose and gluconic acid.
(79) 2.10 Volatile Compound Production and Aromatic Profile
(80) The production of acetaldehyde, methyl acetate, ethyl acetate and higher alcohols has been evaluated on completion of fermentation in samples of Trebbiano must inoculated and fermented using the strain SERIUS, and in samples of the same must inoculated and fermented with a commercial Saccharomyces cerevisiae yeast. Determination of the volatile compounds has been conducted by means of gas chromatography, injecting samples of suitably distilled wine directly into a ZB-WAX Plus polar column (stationary phase of polyethylene glycol, FID—Flame Ionization Detector).
(81) The results obtained, expressed in mg/l, are reported in Table 9.
(82) TABLE-US-00009 TABLE 9 Metabolite (mg/l) SERIUS COMM 1 Acetaldehyde 52 42 Methyl acetate <2 <2 Ethyl acetate 41 25 Methanol 0.05 0.04 2-Butanol <2 <2 N-Propanol 55 23 Isobutanol 43 40 Isoamyl acetate <2 <2 N-Butanol <2 <2 Isoamyls 69 120
(83) The strain SERIUS produces slightly higher quantities of acetaldehyde with respect to those produced by the commercial yeast used for comparison, concentrations however that are not compromising for the quality of the wines. Indeed, it is known that a low concentration of said compound in wine gives a pleasing fruity aroma, while at increasing concentrations, the wine has a tendency to release a pungent and irritant odour, so as to become unmarketable when the acetaldehyde concentration exceeds 500 mg/l.
(84) With regard to higher alcohols, the strain SERIUS produces N-propanol in greater quantities compared to the commercial reference strain, while it produces similar quantities of isobutanol; however, these alcohols do not play a significant olfactory role in wine.
(85) On the other hand, in comparison to the commercial S. cerevisiae strain, the strain SERIUS produces a lower quantity of isoamyl alcohol; the latter higher alcohol plays an important role as it results in the so-called “amylic” odour, which is considered very negative from an olfactory viewpoint.
(86) The aromatic profile of the strain SERIUS has been evaluated by means of the solid phase extraction (SPE) technique used on Trebbiano grapes must, obtained following incubation with the aforementioned strain at 25° C. for a length of time sufficient to reach the completion of fermentation. Furthermore, analogously, two commercial S. cerevisiae strains have been assayed by way of comparison.
(87) Operatively, 10 ml of each strain sampled (SERIUS, and the two commercial strains) have been diluted with 30 ml of water and spiked with the internal standard (1-heptanol) and absorbed onto the SPE C18 column. Elution has been performed using dichloromethane, and the eluate, reduced to a small volume, has been injected into the Shimadzu GC2010/QP2010 GC/MS system. Identification of the resulting compounds has been performed by means of searching the latest versions of the Whiley and NBS libraries available at the time of preparation of the present application.
(88) From the present analysis, it is demonstrated that the strain of the invention allows the attainment of a wine with concentrations of fatty acids (isovaleric acid, hexanoic acid, octanoic acid, butanoic acid, decanoic acid) that are on average 2-3 fold less than those detected in wines obtained with the two commercial strains. It is known that certain medium and long-chain fatty acids (C6, C8, C10, C12) can have inhibitory effects with regard to the fermentative activity of yeasts. In particular, even at low concentrations (a few milligrams/litre), decanoic acid (C10) shows significant antagonistic activity with regard to fermentation processes.
(89) An additional characteristic differentiating the strain SERIUS from the two commercial strains used by way of comparison is the significantly higher production (30 fold greater on average) of phenyl ethyl esters of octanoic acid, volatile compounds giving the aroma typical of green cocoa. Finally, the wine obtained with the yeast SERIUS shows reduced concentrations of pyrazine and piperazine, molecules responsible for herb and plant aromas.
EXAMPLE 3—OENOLOGICAL CHARACTERISATION OF THE YEAST, VINIFICATION TESTS
(90) The strain of the invention has been tested by means of winery vinification tests on red berry grapes and white berry grapes musts.
(91) The production process used has been the fed-batch type, followed by the concentration of the culture by means of centrifugation. Then, a yeast biomass has been prepared in cream form with a dry substance content essentially equal to 22%, and a yeast concentration of about 10 billion CFU/g.
(92) For subsequent inoculation of the must, the biomass has been resuspended in a vinous suspension consisting of yeast, must and activators.
(93) In particular, for use in a 5000-litre tank, 1 kg of cream-form biomass of the yeast SERIUS has been suspended at a temperature of 25° C. in 200 litres of must, supplemented with activators at the working concentrations recommended by the manufacturer, and known commercially. The suspension has been kept in motion using a stirrer or racking pump for 2/4 hours. Subsequently, said suspension has been added to the must by means of racking.
(94) 3.1 Test Using Traminer Grape Must with Must Acidification
(95) The yeast of the invention has been tested with Traminer (white berry) grape must in a winery in a 50 hl tank.
(96) The yeast, prepared in fresh cream form, has been used at a dose of 20 g/hl.
(97) The addition to the must of 100 g/hl of yeast autolysate and 140 g/hl of tartaric acid has been followed by inoculation with the strain SERIUS, prepared previously according to the method described in the general section. Fermentation has been conducted at 13° C. for a period of 19 days.
(98) Table 10 reports the analytical data concerning the must prior to yeast inoculation and the wine on completion of fermentation. It may be observed how the strain produces advantageously high quantities of malic acid and glycerol, and at the same time, shows low volatile acidity production.
(99) With regard to the aromatic profile, on completion of fermentation, greater floral complexity is noted, synergistic with the terpene profile, in comparison to commercial yeast.
(100) TABLE-US-00010 TABLE 10 Must Wine Total acidity g/l 4.80 6.57 pH 3.47 3.46 Malic acid g/l 1.09 1.92 Citric acid g/l 0.12 0.2 Lactic acid g/l 0.10 0.22 Glucose g/l 88.1 0.4 Fructose g/l 106.9 6.5 Succinic acid g/l 0.07 0.99 Glycerol g/l 3.47 11.45 Acetic acid g/l 0.21 0.09 Ethanol % 2.82 14.11
3.2 Test Using Traminer Grape Must without Must Acidification
(101) The yeast SERIUS has been tested with Traminer (white berry) grape must in a winery in a 100 hl tank. The yeast has been prepared in fresh form (cream) and used at a dose of 20 g/hl. Preparation of the inoculum has been carried out as described previously. Prior to inoculation, 90 g/hl of yeast autolysate have been added to the must. The must has not been acidified with tartaric acid. Fermentation has been conducted at 17° C. for a period of 7 days.
(102) Table 11 reports the analytical data concerning the must prior to yeast inoculation and the wine on completion of fermentation.
(103) The results obtained highlight the ability of the strain SERIUS to produce high quantities of glycerol and low volatile acidity. With respect to the previous example in Traminer must, wherein malic acid production has been observed, in this case, malic acid production is not observed. It should be noted that the must in example 3.1 had been acidified with tartaric acid, while the must in this example has not been subjected to supplementing with tartaric acid. Hence, the production of malic acid by the strain SERIUS seems to be correlated with tartaric acid acidification of the must.
(104) Olfactory analysis confirms the results highlighted in the example described at point 3.1.
(105) TABLE-US-00011 TABLE 11 Must Wine Total acidity g/l 6.70 6.29 pH 3.26 3.30 Malic acid g/l 1.24 1.10 Citric acid g/l 0.16 0.27 Lactic acid g/l 0.23 Glucose g/l 81.5 0.3 Fructose g/l 100.3 5.7 Succinic acid g/l 0.55 Glycerol g/l 3.54 10.15 Acetic acid g/l 0.08 0.15 Ethanol % 2.72 13.10
3.3 Test Using Garganega Grape Must with Must Acidification
(106) The strain SERIUS has been tested with Garganega (white berry) grape must in a winery in a 150 hl tank. The yeast has been prepared in fresh form (cream) and used at a dose of 20 g/hl. Preparation of the inoculum has been carried out as described above. Prior to inoculation, 120 g/hl of yeast autolysate and 120 g/hl of tartaric acid have been added to the must. Fermentation has been conducted at 16° C. for a duration of 15 days.
(107) The results obtained are reported in table 12. The ability of the strain SERIUS to produce malic acid, high quantities of glycerol and low volatile acidity is highlighted.
(108) With regard to the aromatic profile, marked floral notes with a hint of glycine are reported.
(109) TABLE-US-00012 TABLE 12 Must Wine Total acidity g/l 4.72 8.85 pH 3.53 3.27 Malic acid g/l 0.97 2.70 Citric acid g/l 0.16 0.23 Lactic acid g/l 0.01 0.24 Glucose g/l 80.6 0.3 Fructose g/l 90.4 9.2 Succinic acid g/l 0.02 1.36 Glycerol g/l 1.55 10.34 Acetic acid g/l 0.02 0.16 Ethanol % 1.21 11.23
3.4 Testing Using Garganega Grape Must without Must Acidification
(110) The strain SERIUS has been tested with Garganega (white berry) grape must in a winery in a 100 hl tank. The yeast has been prepared in fresh form (cream) and used at a dose of 20 g/hl. Preparation of the inoculum has been carried out as described above. Prior to inoculation, 120 g/hl of yeast autolysate have been added to the must. The must has been acidified with tartaric acid. Fermentation has been conducted at 16° C. for a period of 12 days.
(111) Table 13 reports the results obtained with the must prior to inoculation, and of the wine on completion of fermentation.
(112) It may be observed how the strain has the ability to produce high quantities of glycerol with low volatile acidity production. Again, in this case, in the absence of acidification of the must with tartaric acid, no malic acid production is reported.
(113) The aromatic profile confirms that observed in example 3.3, namely the presence of marked floral notes with a hint of glycine.
(114) TABLE-US-00013 TABLE 13 Must Wine Total acidity g/l 6.36 6.44 pH 3.47 3.59 Malic acid g/l 2.86 2.64 Citric acid g/l 0.40 0.46 Lactic acid g/l 0.02 0.13 Glucose g/l 96.9 0.3 Fructose g/l 105.8 3.7 Succinic acid g/l 0.08 0.56 Glycerol g/l 1.68 9.74 Acetic acid g/l 0.07 0.25 Ethanol % 1.35 13.60
3.5 Testing in Syrah, Petit Verdot, Cabernet Grapes Must with Must Acidification
(115) The strain SERIUS has been tested with Syrah, Petit Verdot, Cabernet (red berry) grape must in a winery in a 100 hl tank. The yeast has been prepared in fresh form (cream) and used at a dose of 20 g/hl. Preparation of the inoculum has been carried out as described above. Prior to inoculation, 150 g/hl of yeast autolysate and 100 g/hl of tartaric acid have been added to the must. Fermentation has been conducted at 20° C. for a period of 10 days.
(116) Table 14 reports the results obtained with the must prior to inoculation, and of the wine on completion of fermentation. Again, in this case, the ability of the strain SERIUS to produce malic acid and high quantities of glycerol is observed.
(117) With regard to the aromatic profile, the wine produced using the strain SERIUS has distinct blueberry, raspberry and wild strawberry notes.
(118) TABLE-US-00014 TABLE 14 Must Wine Total acidity g/l 4.85 7.48 pH 3.78 3.74 Malic acid g/l 1.37 3.70 Citric acid g/l 0.58 0.63 Lactic acid g/l 0.4 0.44 Glucose g/l 125.7 1.6 Fructose g/l 138.3 16 Succinic acid g/l 0.04 0.80 Glycerol g/l 2.2 12.46 Acetic acid g/l 0.19 0.15 Ethanol % 1.4 16.62
EXAMPLE 4—HYBRIDS OBTAINED FROM THE YEAST S. BAYANUS SUBSP. UVARUM SERIUS
(119) The strain S. bayanus subsp. uvarum SERIUS may be used as the parental strain in order to obtain yeast hybrids with additional and improved oenological characteristics. The direct hybridisation technique has been chosen and a standard spore-spore conjugation protocol with slight modifications has been followed to obtain hybrids (Solieri et al., 2008). Initially, a sporulation study has been performed on the strains selected for hybridisation. Several culture media have been tested for this purpose. The spore viability and sporulation efficiency study has allowed the selection of SP medium (1% potassium acetate; 0.1% yeast extract; 0.05% glucose; 1.8% agar). Growth of the strain on SP at 25° C. for 7-10 days has been followed by partial digestion of the cell wall in order to allow subsequent separation of the spores from the ascii using a micromanipulator needle. The ascii, with the semi-digested cell wall, have been placed on one side of a YPD plate (2% glucose; 2% peptone; 1% yeast extract; 2% agar) and then subjected to micromanipulation. The spores of the two parental strains have been placed randomly next to one another. Continuous observation has allowed the identification of successful conjugation. Following incubation at 25° C. for 2-3 days, the colonies selected have been re-streaked on YPD medium. The following step has been the stabilisation of the strains (also performed on WL medium). Hybrids have then been confirmed by means of molecular analysis and have then been subjected to fermentation testing in must along with phenotypic analysis in order to verify their physiological characteristics and oenological potential.
EXAMPLE 5—PRODUCTION OF YEAST AUTOLYSATES STARTING FROM THE STRAIN S. BAYANUS SUBSP. UVARUM SERIUS
(120) The strain S. bayanus subsp. uvarum SERIUS may be used to produce a yeast autolysate to be used as a fermentation activator in the production of wine and other alcoholic beverages. To this end, the yeast cream, prepared as described above, is subjected to appropriate heat treatment (40-60° C. for 12-48 hours). The autolysate obtained may be used as it is or further processed by means of appropriate filtration/centrifugation/decanting with the aim of separating the solid or liquid phases for subsequent suitable applications.
(121) Hence, based on the above, the present invention has achieved all pre-set objectives.
(122) In particular, the goal of providing a yeast strain of the species Saccharomyces bayanus subsp. uvarum, equipped with the technological and oenological characteristics allowing good fermentation of the must, allowing the production of a pleasing wine, is achieved.
(123) Another goal achieved is that of providing a strain allowing the attainment of a wine characterised by high quantities of malic acid and glycerol, and at the same time not producing hydrogen sulphide during alcoholic fermentation.
(124) Another goal achieved by the present invention concerns the possibility of using the strain SERIUS as the parental strain to obtain hybrid yeasts with novel oenological potential.
(125) A further goal achieved concerns the possibility of using the strain forming the subject of the invention to obtain a yeast autolysate to be used as an activator of fermentation.
(126) By no means a final goal of the invention achieved is the optimization of a perfected and rapid method for the selection of yeast strains for oenological use, particularly yeasts from the species Saccharomyces bayanus isolated from flowers.
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