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Title:
A YEAST STRAIN AND USES THEREOF
Document Type and Number:
WIPO Patent Application WO/2017/214673
Kind Code:
A1
Abstract:
The present invention relates generally to a novel yeast strain deposited with the National Measurement Institute and assigned Accession No. V16/014811, and cultures thereof. The present disclosure also relates generally to the use of yeast strain V16/014811, cultures thereof and yeast cells derived therefrom, in the manufacture of fermented products, including fermented beverages and food.

Inventors:
ANGELUCCI, Constanza (4 Wilhelmina Avenue, West Launceston, Tasmania 7250, 7250, AU)
HOWARD, Daryl (311 Lum Road, Wheelers Hill, Victoria 3150, 3150, AU)
THURROWGOOD, David (2 Invermay Road, Launceston, Tasmania 7250, 7250, AU)
BORNEMAN, Anthony (c/ - The Australian Wine Research Institute, Hartley Grove cnr Paratoo RoadUrrbrae, South Australia 5064, AU)
DILLON, Simon (c/ - The Australian Wine Research Institute, Hartley Grove cnr Paratoo RoadUrrbrae, South Australia 5064, AU)
Application Number:
AU2017/050596
Publication Date:
December 21, 2017
Filing Date:
June 14, 2017
Export Citation:
Click for automatic bibliography generation   Help
Assignee:
LAUNCESTON CITY COUNCIL (PO Box 403, Launceston, Tasmania 7250, 7250, AU)
THE AUSTRALIAN WINE RESEARCH INSTITUTE (Hartley Grove cnr Paratoo Road, Urrbrae, South Australia 5064, 5064, AU)
ANGELUCCI, Constanza (4 Wilhelmina Avenue, West Launceston, Tasmania 7250, 7250, AU)
HOWARD, Daryl (311 Lum Road, Wheelers Hill, Victoria 3150, 3150, AU)
International Classes:
C12N1/16; A21D8/04; A21D10/00; C12C11/00; C12G3/02; C12P7/06; C12R1/85
Foreign References:
JPH0889236A1996-04-09
GB1152286A1969-05-14
Other References:
LIBKIND, D. ET AL.: "Microbe domestication and the identification of the wild genetic stock of lager-brewing yeast", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, vol. 108, no. 35, 2011, pages 14539 - 14544, XP055450070
NGUYEN, H. V. ET AL.: "Deciphering the hybridisation history leading to the lager lineage based on the mosaic genomes of Saccharomyces bayanus strains NBRC1948 and CBS380T.", PLOS ONE, vol. 6, no. 10, 2011, pages e25821, XP055450071
ALMEIDA, P. ET AL.: "A Gondwanan imprint on global diversity and domestication of wine and cider yeast Saccharomyces uvarum", NATURE COMMUNICATIONS, vol. 5, 2014, pages 1 - 12, XP055450072
KROGERUS, K. ET AL.: "New lager yeast strains generated by interspecific hybridization.", JOURNAL OF INDUSTRIAL MICROBIOLOGY & BIOTECHNOLOGY, vol. 42, no. 5, 2015, pages 769 - 778, XP035480650
NGUYEN, H. V. ET AL.: "Characterization of Saccharomyces uvarum (Beijerinck, 1898) and related hybrids: assessment of molecular markers that predict the parent and hybrid genomes and a proposal to name yeast hybrids", FEMS YEAST RESEARCH, vol. 17, no. 2, 3 March 2017 (2017-03-03), pages 1 - 19, XP055465876
Attorney, Agent or Firm:
DAVIES COLLISON CAVE PTY LTD (1 Nicholson Street, Melbourne, Victoria 3000, 3000, AU)
Download PDF:
Claims:
CLAIMS:

1. An isolated yeast strain deposited with the National Measurement Institute with Accession No. V 16/01481 1.

2. A culture of yeast cells comprising the yeast strain of claim 1.

3. A composition comprising a yeast cell from the culture of claim 2 and at least one additional ingredient.

4. The composition of claim 3, wherein the at least one additional ingredient is a beverage ingredient.

5. The composition of claim 3, wherein the at least one ingredient is a food ingredient.

6. A method of preparing a fermented product, said method comprising contacting a yeast cell from the culture of claim 2 with a source of sugar under fermentation conditions to produce a fermented product.

7. The method of claim 6, wherein the fermented product is a beverage.

8. The method of claim 7, wherein the beverage is selected from the group consisting of beer, sake, wine, cider, mead, root-beer, ginger-beer, kefir and kumis.

9. The method of claim 6, wherein the fermented product is a food.

10. The method of claim 9, wherein the food is selected from the group consisting of a food supplement, a dough and a bread.

11. A fermented product obtained by the method of claim 6.

12. A fermented beverage obtained by the method of claim 7 or claim 8.

13. A fermented food obtained by the method of claim 9 or claim 10.

14. A fermented product comprising a yeast cell from the culture of claim 2.

15. The fermented product of claim 14, wherein the product is a beverage.

16. The fermented product of claim 15, wherein the beverage is selected from the group consisting of beer, sake, wine, cider, mead, root-beer, ginger-beer, kefi r and kumis.

17. The fermented product of claim 14, wherein the product is a food.

18. The fermented product of claim 17, wherein the food is selected from the group consisting of a food supplement, a dough and a bread.

19. A method of producing ethanol from sugar, the method comprising culturing a yeast cell from the culture of claim 2 in a solution comprising sugar under conditions such that the yeast strain converts the sugar to ethanol

Description:
. l .

[0001] The present disclosure relates generally to a novel yeast strain deposited with the National Measurement Institute and assigned Accession No. V16/01481 1, and cultures thereof. The present disclosure also relates generally to the use of the yeast strain deposited as VI 6/01481 1, cultures thereof and veast cells derived therefrom, in the manufacture of a fermented product, including a fermented beverage and a fermented food,

BACKGROUND OF THE INVENTION

[0002] Yeasts are unicellular eukaryotic microorganisms of the fungus kingdom, of which there are more than 1,500 known species. Yeast sizes can vary greatly, depending on species and environment, though typically measuring 3-4 μηι in diameter. Most yeasts reproduce asexualiy by mitosis, and many do so by the asymmetric division process known as budding. Yeasts do not form a single taxonomic or phylogenetic grouping. Their phylogenetic diversity is shown by their placement in two separate phyla: the Ascomycota and the Basidiomycota. Typically, the budding form of yeasts are classif ed in the order Saccharomycetales.

[0003] Yeast is used in many diverse industrial and commercial applications, in particular for the fermentation of sugars. Yeast is also used as an important organism in research, noting that it is one of the most thoroughly researched eukaryotic microorganisms. Yeast also has utility in the generation of electricity in microbial fuel cells, in the production of biofuels {e.g., ethanol) and in probiotics.

[0004] In this context of fermentation, many types of yeasts are used for making fermented beverages and food, such as baker's yeast in bread production, brewer's yeast in beer fermentation, and yeast in wine fermentation, where yeast species such as Saccharomyces cerevisiae are used to convert carbohydrates (sugars) to carbon dioxide and alcohols.

[0005] Methods of brewing beer, for example, will be familiar to persons skilled in the art and will typically depend on the style of beer to be produced (e.g., an ale, a lager, a stout). In an illustrative example, the brewing process typically begins by producing a wort, which is fonned by the addition of water to malted and unrnalted crushed grain such as, but not limited to, barley or mash. Subsequently, the liquid in the wort is drained from the mash slurry or mash tun and directed to a brew kettle where hops are added. The hopped liquid is then boiled in the brew kettle to produce a hopped wort. The final step in the brewing process involves the addition of a yeast for fermentation, whereby the yeast convert the sugars in the hopped wort into alcohol,

[0006] Over the years, the process of brewing fermented beverages such as beer, wine and cider has been modified by brewers to produce beverages having different characteristics, examples of which include flavour, colour, clarity and alcohol content. These characteristics can be modified, for example, by changes in brewing pressures, temperatures, grains and fermentation times. These characteristics can also be modified by the type of yeast strain (or combination of yeast strains) that are employed for fermentation.

SUMMARY OF THE INVENTION

[0007] In a first aspect disclosed herein, there is provided an isolated yeast strain deposited with the National Measurement Institute with Accession No. VI 6/014811.

[0008] In a second aspect disclosed herein, there is provided a culture of yeast cells comprising a yeast strain deposited with the National Measurement Institute with Accession No. V16/01481 1. In an embodiment, the culture comprises a substantially pure population of yeast strain V16/014811.

[0009] In a third aspect disclosed herein, there is provided a composition comprising a yeast cell from the culture of yeast cells, as herein described, and at least one additional ingredient. In an embodiment, the at least one additional ingredient is a beverage ingredient. In another embodiment, the at least one additional ingredient is a food ingredient.

[0010] In a fourth aspect disclosed herein, there is provided a method of preparing a fermented product, said method comprising contacting a yeast cell from the yeast ceil culture disclosed herein with a source of sugar under fermentation conditions to produce a fermented product. In an embodiment, the fermented product is a beverage. In an embodiment, the beverage is selected from the group consisting of beer, sake, wine, cider, mead, root-beer, ginger-beer, kefir and kumis. In another embodiment, the fermented product is a food. In an embodiment, the food is selected from the group consisting of a food supplement, a dough and a bread.

[0011] In a fifth aspect disclosed herein, there is provided a fermented product obtained by the methods disclosed herein. In an embodiment, the fennented product is a fermented beverage. In another embodiment, the fermented product is a fermented food.

[0012] In a sixth aspect disclosed herein, there is provided a fermented product comprising a yeast cell from the yeast cell culture disclosed herein. In an embodiment, the fermented product is a fermented beverage. In another embodiment, the fermented product is a fermented food. In an embodiment, the fermented product is a beverage. In an embodiment, the beverage is selected from the group consisting of beer, sake, wine, cider, mead, root-beer, ginger-beer, kefir and kumis. In another embodiment, the fermented product is a food. In an embodiment, the food is selected from the group consisting of a food supplement, a dough and a bread.

[0013] In a seventh aspect disclosed herein, there is provided a method of producing ethanol from sugar, the method comprising culturing a yeast cell from the yeast cell culture disclosed herein in a solution comprising sugar under conditions such that the yeast strain converts the sugar to ethanol. Also disclosed herein is ethanol produced by the methods disclosed herein.

BRIEF DESCRIPTION OF FIGURES

[0014] Figure 1 shows the chromosomal location of the genomic sequence of the novel Saccharomyces strain deposited with the National Measurement Institute and assigned Accession No. V 16/014811. These data trace portions of its genomi c sequence back to S. cerevisiae (blue; light grey), S. uvarum (brown; dark grey) and S. eiihayanus (red; black). jQOlS] Figure 2 shows the location of the S. cerevisiae portion of the genomic sequence of the novel Saccharomyces strain, Accession No. VI 6/014811 on a phylogeny tree of hundreds of known yeast strains. Wine strains exist within the green area and ale strains within the red area. The novel Saccharomyces strain, Accession No. VI 6/01481 1 (blue; see arrow), exists outside of these two different industrial groups, showing that it is not a classical ale or wine yeast strain.

DETAILED DESCRIPTION OF THE INVENTION

[0016] Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

[0017] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

[0018] All publications mentioned in this specification are herein incorporated by- reference in their entirety.

[0019] It must be noted that, as used in the subject specification, the singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "yeast cell" includes a single yeast cell, as well as two or more yeast cells; reference to "composition" includes a single composition, as well as two or more compositions; and so forth.

Yeast strain VI 6/014811

[0020] The present invention is predicated on the inventors' surprising finding of a novel yeast strain, which has been isolated and deposited at the National Measurement Institute (Victoria, Australia) on 10 June 2016 and assigned Accession No. V16/014811. Thi s deposit will be maintained under the terms of the Budapest Treaty on the International _ ^ _

Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. The deposit will be maintained without restriction in the National Measurement Institute for a period of 30 years, or for the enforceable life of the patent, whichever is longer, and will be replaced if it ever becomes nonviable during that period.

[0021] Genomic analysis of this yeast strain, the results of which are shown in Figures 1 and 2 herein, suggests that yeast strain VI.6/01481 1 was derived from multiple hybridization events between three species of Saccharomyces: S. cerevisiae, S, iivarum and S. eubayanus. However, for the purpose of brevity, the novel strain V16/01481 1 is also referred to herein as a strain of Saccharomyces uvarum.

[0022] Thus, in a first aspect disclosed herein, there is provided an isolated yeast strain deposited with the National Measurement Institute with Accession No. VI 6/014811.

[0023] The expression "yeast strain" typically denotes a relatively homogeneous population of yeast cells and, unless otherwise stated, the terms "strain" and "cell" are used interchangeably herein.

[0024] The terms "yeast ceil", "yeast", "yeasts", "yeast ceils" are used interchangeably herein to denote yeast obtained by culturing starting from the yeast strain with Accession No. VI 6/014811.

[0025] The term "isolated", as used herein, typically refers to a yeast strain that has been separated or otherwise removed from its natural environment. In an embodiment, the yeast strain is isolated from its natural environment such that it can be grown as a culture or otherwise used in accordance with the methods disclosed herein.

[0026] Yeast strain V16/014811 can be used, stored or otherwise disposed of in any- suitable form. In an embodiment, the yeast strain is in the form of a fresh yeast (e.g., cream yeast, pressed yeast), of a dry yeast or of a deep-frozen yeast.

[0027] Fresh easts are typically characterized by a high water content in comparison with dry yeasts. Fresh yeasts typically encompass cream yeasts and pressed yeasts. Cream yeasts, also called "liquid yeasts", are aqueous suspensions of yeast ceils having a cream- type viscosity. The term "cream yeast" is understood to mean a liquid suspension, typicaily an aqueous suspension, of live yeast cells, wherein the suspension comprises a dry matter content of preferably at least 12% by mass, preferably from about 12% to about 50% by mass. Preferably, a cream yeast comprises a dry matter content comprised from about 12% to about 25% by mass, preferably from about 14% to about 22% by mass.

[0028] Among the pressed yeasts, a distinction is made between pressed yeasts as a compact block, also called "yeast cakes", which comprise a dry matter content in the range of about 26% to about 35%, and pressed yeasts as granules, which comprises a water content in the range of about 21% to about 35%. Dry yeasts typically comprise a dry matter content of greater than about 92%.

[0029] The deep-frozen yeasts typically comprise a dry matter content in the range of about 74% to about 80%.

[0030] The yeast according to the invention therefore has the characteristics desired for a yeast intended for producing alcohol, namely a reliable quality and an effective alcohol production.

Yeast cell cultures

[0031] In a second aspect disclosed herein, there is provided a culture of yeast cells comprising a yeast strain deposited with the National Measurement Institute with Accession No. V16/01481 1.

[0032] In an embodiment, the culture comprises a substantially pure population of yeast strain V16/014811. The expression "substantially pure population", as used herein, means a population of yeast cells that are predominantly of yeast strain ¥16/0148 1. In an embodiment, at least 60% of the yeast cells in the culture are of yeast strain V16/014811, preferably at least 70% of the yeast cells in the culture are of yeast strain VI 6/01481 1, preferably at least 80% of the yeast ceils in the culture are of yeast strain V16/01481 1, preferably at least 90% of the yeast cells in the culture are of yeast strain V16/014811 , preferably at least 95%> of the yeast ceils in the culture are of yeast strain V16/014811, more preferably at least 98% of the yeast cells in the culture are of yeast strain V16/01481 1, even more preferably at least 99% of the yeast ceils in the culture are of yeast strain V16/01481 1, and most preferably 100% of the yeast in the culture are of yeast strain V16/01481 1.

[0033] Yeast is an anaerobe that can survive and grow in the presence (aerobic) or absence (anaerobic) of oxygen, although typically its survival, growth and metabolism is optimal in the presence of oxygen. Moreover, the conditions and methodologies used for propagating and maintaining yeast need not be identical to those used for fermentation. In an embodiment disclosed herein, the yeast strain V16/014811 can be propagated and/or maintained by culturing the yeast strain in a culture medium suitable for the growth of the yeast strain. Suitable culture medium will be familiar to persons skilled in the art, illustrative examples of which include the a liquid medium or an agar medium,

[0034] A variety of growth media and growth conditions will be familiar to persons skilled in the art for culturing yeast strain V16/01481 1, preferably under sterile conditions.

[0035] In an illustrative example, a one mi lliliter CryoTube IM (Nunc, Inc.) containing yeast strain V16/014811 is partially thawed and a 10 , u,L aliquot removed using a sterile inoculation loop. Strain V16/01481 1 is then added to 50 mL of sterile malt extract broth (50 g/L) and incubated at 28°C. for 24 hours. The yeast suspension is then transferred to a 5L flask containing 1L of malt extract broth and incubated at 28°C while shaking for 48 hours. The broth i s decanted and the remaining yeast sediment (approximately 100 milliliters) is used as an inoculum for 1 1.3L in an 18.9L bottle. The yeast is incubated with shaking for 48-72 hours at 28°C and grown to concentrations of, for example, 10 s or greater (e.g., l-5xl Q 8 /mL or >5xl0 8 /mL, such as lxl0 9 /mL). After the incubation, the yeast is allowed to sediment and the sediment or portion thereof is used as the pitch for the fermentation of a fermented beverage or food product in a tank (e.g., using 8 barrel or 12 barrel fermentation tanks). The yeast sediment or portion thereof can also be also used as an inoculum (e.g., a starter culture) in methods for the preparation of a yeast-based product, including a fermented beverage or a fermented food, as herein described.

[0036] In another exemplar}' method, a one milliliter CryoTube™ (Nunc, Inc.) containing yeast strain V16/01481 1 is partially thawed and a 10 μΐ. aliquot removed using a calibrated, sterile, inoculation loop. Strain VI 6/01481 1 is then streaked for i solation onto a plate of malt extract agar and incubated at 28°C for 48 hours. After incubation, the plate is examined for the presence of characteristic, isolated colonies of yeast strain V16/01481 1. Portions of 2-5 colonies are then picked with a sterile inoculating loop and used to inoculate 5 mL of sterile light malt extract broth with specific gravity of 1.035 in a 15 125mm tube. The tube is incubated for 24 hours at 28°C. After incubation, the tube is examined for evidence of growth and gas formation. The yeast culture is then used to inoculate 50 mL of the sterile light malt extract broth with specific gravity of 1.035 and incubated for 24 hours in an orbital shaker incubator at 28°C. at 200 RPM. The yeast suspension is then transferred to a 5L flask containing 1L of the sterile light malt extract broth with specific gravity of 1.035 and incubated for 48 hours in an orbital shaker incubator at 28°C. at 200 RPM. The yeast suspension is then used to inoculate 10L of the sterile light malt extract broth with a specific gravity of 1 .035 in an 1 1.3L container and incubated for 48 hours in an orbital shaker incubator at 28°C. at 200 RPM. After incubation, the yeast is allowed to sediment and the supernatant fluid siphoned off. An additional 10L of the sterile light malt extract broth with specific gravity of 1.035 were added to the yeast sediment and incubated with shaking for an additional 48 hours. For example, the yeast is grown to concentrations of 10 8 or greater (e.g., l-5x ' 10 8 /ml or >5xT() 8 /ml, such as lxl 0 9 /ml). After the incubation, the yeast is allowed to sediment and the sediment or a portion thereof was used as the pitch for a fermentation process, for example, using 8 barrel or 12 barrel fermentation tanks. The yeast sediment or portion thereof can also be used as an inoculum (e.g., a starter culture) in methods for the preparation of a yeast-based product, such as a fermented product including a fermented beverage or a fermented food, as herein described.

[0037] In another exemplary method, a starter culture (e.g., an active yeast starter) is prepared by streaking an aliquot of yeast strain VI 6/01481 1 onto an agar plate (e.g., malt extract agar), followed by incubation at approximately 28°C. for several hours (e.g., 48 hours). After incubation, portions of a few colonies (e.g., 2-5 colonies) are then picked (e.g., with a sterile inoculating loop) and used to inoculate sterile light malt extract broth (e.g., 5 mL with specific gravity of 1.035) in a tube (e.g., 15x 125 mm tube). Alternatively, a dried yeast, as described elsewhere herein, may be used to inoculate the sterile light malt extract broth. Next, the tube is incubated for several hours (e.g., 24 hours) at approximately 28°C and examined for evidence of growth and gas formation. The yeast culture is then used to inoculate sterile light malt extract broth (e.g., 50 mL with specific gravity of 1.035) and incubated for several hours (e.g., 24 hours) in an orbital shaker incubator (e.g., at approximately 28°C. and 200 RPM). The yeast suspension is then transferred to a flask (e.g., 5L flask) contaimiig sterile light malt extract broth (e.g., 1 L with specific gravity of 1.035) and incubated for several hours (e.g., 48 hours) in an orbital shaker incubator (e.g., at approximately 28°C and 200 RPM). After the incubation, the yeast is allowed to sediment and the sediment or a portion thereof may be used in methods of making products, including, fermented product such as a fermented beverage or a fermented food, as herein described. The yeast may be recovered and kept in lyophilized form or in the form of a powder, granule or tablet, as well as encapsulated (e.g., in capsules such as hard or soft gelatin capsules) or suspended in liquid media to a desired volume.

[0038] In an embodiment disclosed herein, the yeast strain is cultured in simple sugar solution supplemented with a nitrogen source. Illustrative examples of suitable media for growing (propagating) and maintaining yeast include YPD (yeast extract, peptone, dextrose), potato dextrose, and sabouraud dextrose. These media are designed for laboratory yeast and do not contain a complex source of fermentable sugars. Laboratory- media may not be suitable for extensive propagation of yeast for brewing purposes, as this may ultimately affect fermentation performance. Suitable culture media for propagating and maintaining yeast for brewing purposes will be familiar to persons skilled in the art, an illustrative example of which includes MYGP (e.g., 0.3% malt extract, 0.5% peptone, 0.3% yeast extract and 1.0% dextrose) and a composition comprising 1.5% malt extract, 0.078% peptone, 1.275% maltose, 0.275% dextrin, 0.235% glycerol, 0.1% dipotassiumphosphate and 0.1 % ammonium chloride. These media can be used for slants, plates and limited liquid propagation, but are generally not useful for starters. It will be understood by persons skilled in the art that the ingredients in culture media can be modified as desired (e.g., in content and/or concentration and/or proportion) in order to optimize the propagation and/or maintenance of the yeast strain.

[0039] In an embodiment disclosed herein, yeast strain V16/014811 is cultured on an agar medium. In an embodiment disclosed herein, yeast strain V16/01481 1 is cultured at a temperature of from 25°C to 35°C, preferably from 28°C to 32°C. In an embodiment, yeast strain V16/01481 1 is cultured at a temperature of 30°C.

[0040] Other factors that influence the propagation and maintenance of yeast cultures include oxygen, pH and temperature.

[0041] The initial culture of yeast strain VI.6/01481 1 is also referred to herein as the "starting culture" or "yeast starter" (R0). In some embodiments, successive subcultures are carried out from the starting culture of yeast strain V16/01481 1 . For example, all or part of the starting culture corresponding to R0 is inoculated for the following culture which results in a yeast population Gl; then all or part of the culture corresponding to the yeast population Gl is inoculated for the following culture which results in a yeast population G2, and so on, until the culture which results in a yeast population Rn. In an embodiment, the n successive subcultures are carried out at regular time intervals. In an illustrative example, the n successive subcultures are daily subcultures. In another embodiment, the n subcultures are carried out in the same medium and/or at the same temperature and/or with the same amount of inoculated biomass.

[0042] Thus, unless otherwise stated, reference to a yeast cell culture is a reference to a culture of yeast strain VI 6/014811, or a subculture thereof.

[0043] Typically, the purpose of a yeast starter (R0) is to produce a sufficient quantity of yeast for subsequent used, such as fermentation. For example, for fermentation purposes, propagation conditions should be such that a maximal amount of yeast is produced which provides optimal fermentation performance, which can be based on the rate and extent of fermentation, as well as the desired sensory profile {e.g., colour, flavour, aroma).

[0044] The term "biomass" is used herein to denote a population of yeast cells obtained from a culture of yeast strain V16/01481 1. For example, during each subculture, a biomass corresponding to a dry mass of 0.05 mg of yeasts is inoculated, preferably at the surface of an agar medium. If required, the biomass harvested at the end of a subculture can be stored at 4°C. for several days (e.g., 1 to 2 days), before being inoculated for following subcultures, as required. Compositions

[0045] It is to be understood that yeast strain V16/014811 is suitable for many different applications, such as those in which similar strains of yeast can be used (e.g., S. cerevisiae, S. nvarum and S. euhavanus). However, it has been found by the inventors that yeast strain V16/01481 1 is particularly suitable as a fermentation yeast; that is, suitable for the production of fermented products such as fermented beverages and fermented food. Yeast strain V16/014811 and cultures thereof can therefore be used in the manufacture of fermented products such as beverages {e.g. beer, wine, cider), food (e.g., bread) or food intermediates {e.g., bread dough). In the context of fermented beverages, the inventors have surprisingly found that the yeast strain V16/014811 exhibits properties that make it amenable to the manufacture of both lagers and ales. The inventors have also found that when yeast strain V16/014811 is used to produce beer, it imparts colour and flavour characteristics that are considered unique to the beer.

[0046] Thus, in a third aspect disclosed herein, there is provided a composition comprising a yeast cell from a yeast cell culture of yeast strain V16/014811, as disclosed herein, and at least one additional ingredient,

[0047] In an embodiment, at least 10% of the yeast cells in the composition are from a yeast cell culture of yeast strain V16/014811, preferably at least 20% of the yeast cells in the composition are yeast strain V16/01481 1, preferably at least 30% of the yeast cells in the composition are yeast strain V16/01481 1, preferably at least 40%> of the yeast cells in the composition are yeast strain V16/014811, preferably at least 50% of the yeast cells in the composition are yeast strain V16/014811, preferably at least 60% of the yeast cells in the composition are yeast strain V16/014811, preferably at least 70% of the yeast cells in the composition are yeast strain V16/01481 1 , preferably at least 80% of the yeast cells in the composition are yeast strain V16/014811, preferably at least 90% of the yeast cells in the composition are yeast strain V16/014811, preferably at least 95% of the yeast cells in the composition are yeast strain V16/01481 1, and most preferably 100% of the yeast cells in the composition are yeast strain VI 6/014811. [0048] Suitable additional ingredients will be known to persons skilled in the art, the nature of which will depend on the intended use and/or desired outcome. Illustrative examples of additional ingredients include a beverage ingredient and a food ingredient. In an embodiment, the at least one additional ingredient comprises a beverage ingredient. In another embodiment, the at least one additional ingredient comprises a food ingredient.

Methods of preparing a fermented product

[0049] The present disclosure also provides methods for manufacturing or preparing a fermented product, including beverages such as beer.

[0050] Thus, in a fourth aspect disclosed herein, there is provided a method of preparing a fermented product, said method comprising contacting a yeast cell from the yeast cell culture disclosed herein with a source of sugar under fermentation conditions to produce a fermented product.

[0051] Suitable fermented products will be familiar to persons skilled in the art, illustrative examples of which include beverages {e.g., beer, sake, wine, cider, mead, root- beer, ginger-beer, kefir and kumis) and food {e.g., food supplements, dough, bread). In an embodiment, the fermented product is a beverage. In an embodiment, the beverages is selected from the group consisting of a grain-based beverage, a fruit-based beverage, a honey-based beverage, a vegetable-based beverage and a dairy-based beverage. Exemplary grain-based beverages may include beer, sake, vodka and malt whiskey. Exemplary fruit- based beverages may include wine, cider and brandy. Exemplary honey-based beverages may include mead. Exemplary vegetable-based beverages may include root beer and ginger beer. Exemplary dairy-based beverages may include kefir and kumis. In an embodiment, the beverage is selected from the group consisting of beer, sake, wine, cider, mead, root-beer, ginger-beer, kefir and kumis. In an embodiment, the beverage is beer.

[0052] In another embodiment, the fermented product is a food. Fermented foods may include, for example, yeast paste {e.g., nutritional yeast paste), yeast extracts, probiotics, food supplements and breads. The food and/or food supplements disclosed herein may also be used to make beverages {e.g., nutritional beverages). In an embodiment, the food is selected from the group consisting of a food supplement, a dough and a bread. [0053] The methods disclosed herein will typically comprise a series of processes, the nature of which will depend on the desired outcome. Suitable process steps for a given product (e.g., a fermented beer or cider, bread dough, bread, etc.) will be familiar to persons. As an illustrative example, a method of producing beer can include (i) a malting process, (ii) a wort production process and (ii) a fermentation process. A malting process is a process in which a grain may be germinated to produce malt. After germination, the malt may be kilned and its root removed. Optionally, the malt may be ground or milled. Alternatively, a malt may be obtained, including purchased from any commercial source, and used directly in a wort production process. In a wort production process, brewing water may be added to the malt (e.g., obtained from a malting process or obtained from a commercial source of malts), thereby producing a mash by permitting enzymes in the malt to convert starch to sugars. In the process of manufacturing fermented beverages (e.g., beer), adjuncts such as rice and starch may also be added with brewing water. Mash may be lautered and then boiled after hops are added. Such boiling-treatment may be performed to inactivate enzymes in the wort, to make the wort clear by precipitating proteins, to extract and isomerize hop components and/or to sterilize the ingredients. Subsequently, the extract of the wort may be adjusted by the addition of water to the wort after boiling. After cooling of the wort obtained in the wort production process, it may be submitted to the fermentation process. In a fermentation process, yeast strain V16/01481 1 , or a yeast cell from a culture/subculture thereof, is added to convert the sugars in the wort to alcohol. Optionally, a conditioning process may be conducted at the end of the fermentation process to allow the fermented beverage (e.g., beer) to mature.

[0054] Styles of beer include that may be manufactured by the methods disclosed herein include, for example, ales and lagers. Changes in grains, kilning time/temperature, water salt content and hops can all contribute, individually and in combination, to the manufacture of different styles of beer. For example, certain grains or commercially available malts, as well as certain hops, are known to be useful for the preparation of ales (including various types of ales), whereas other grains or malts as well as other hops are known to be useful for the preparation of lagers (including various types of lagers).

[0055] Fermented beverages may include those with an alcohol content of about 2% to about 15% by weight. Preferably, the fermented beverage comprises an alcohol content of about 4% to about 8% by weight. The alcohol content can be modified, for example, by adjusting the extract concentration in the wort production process.

[0056] The term "fermentation", as used herein, means the enzymatic and anaerobic breakdown of organic substances by a microorganism; such as the process by which sugars produce ethanol, carbon dioxide (waste product) and cellular energy. Although yeast ferments ethanol without oxygen, it is understood that the process may occur in the presence of oxygen as an aerobic process. Methods of fermentation and other ethanol - producing method steps (including separation of end products, distillation, purification and denaturation of ethanol) are well known in the art. In some embodiments, a fermentation process includes any process for propagating the yeast strain,

[0057] Suitable fermentation conditions will be familiar to persons skilled in the art and generally refer to fermentation media and conditions adjustable with, pH, temperature, levels of aeration, etc., preferably optimum conditions that allow microorganisms to produce carbon-based products of interest. To determine if culture conditions permit product production, the yeast strain can be cultured for about 24 hours to one week after inoculation and a sample can be obtained and analyzed. The cells in the sample or the medium in which the cells are grown are tested for the presence of the desired product.

[0058] The fermentation process is preferably run at a temperature that is optimal for the yeast. In an embodiment, the fermentation process is performed at a temperature which is less than 38°C. For yeast strain V16/014811 , the fermentation process is preferably performed at a temperature which is lower than 37°C, lower than 36°C, lower than 35°C, lower than 34°C, lower than 33°C, lower than 32°C, lower than 31°C, lower than 30°C, lower than 29°C or lower than 28°C and at a temperature which is preferably higher than 20°C, higher than 21°C, higher than 22°C, higher than 23°C, higher than 24°C or higher than 25°C.

[0059] In an exemplary method for preparing a fermented beverage such as sake {e.g., a beverage containing approximately 18-20% ethanol), rice is used in a fermentation process similar to that of beer making, including as described above. Grains, such as rice {e.g., California Pearl, Mochi and Gomi), are milled to remove the hulls, bran and aleurone layers, leaving the starch-rich endosperm consisting of broken kernels. Next, the kernels are ground to particles (e.g., <2 mm) and mashed at 36°C-42°C for a period of time (e.g., variable time) after the addition of malt grist (e.g., amylase and protease source) to liquefy the mash. The mash is then boiled for a period of time (e.g., 15 minutes) to gelatinize the mash and to stop the starch reduction. Next, a starter culture of yeast strain VI 6/01481 is added to the mash with ammonium salts to initiate fermentation. The production of sake from rice involves a single fermentation step at slightly higher temperatures than beer using alcohol tolerant yeast.

[0060] In an exemplary method for preparing a beverage such as vodka, sugars from grain (e.g., rye or wheat), potatoes, or sugar beet molasses are fermented with yeast strain V16/01481 1. The grain (e.g., in the form of wheat) or vegetables is placed inside a mash tub that rotates to break down the grains. Malt grist is then added to the mash for the conversion of the starch to sugars. The mash is then heated unti l it reaches boiling point to sterilize the mash, followed by the addition of acid bacteria to increase the acidity level needed for the vodka fermentation process. The mash is again sterilized by boiling. Next, the mash is then streamed into tanks (e.g., stainless steel tanks), a starter culture of yeast strain VI 6/01481 1 is added and the mash is fermented for a few days (e.g., four days). The ethanol is then distilled using a still (e.g., a column still comprised of vaporization chambers stacked on top of each other or pot still). The alcohol is continuously heated (e.g., with steam) in the still while it cycles up and down in the column. This cycle continues until the vapors created from the heat are released and condense at the top vaporization chambers. The by-products and extracted materials drain into the lower chambers where they can be discarded. The vapors created by the distillation process (e.g., fine spirits), contain between approximately 95%-100% alcohol. The vapors may be made suitable for drinking by adding water and flavorings to the vapors to dilute the alcohol concentration from approximately 100% to approximately 40%.

[0061] In an exemplar}' method for preparing a beverage such as malt whiskey, a grain (e.g., barley or rye) is dampened and allowed to germinate, which changes the starch contained in the grain to sugar to form a malt. The end of the germination is triggered by drying the germinating grain over a fire (e.g., a kiln). When the malt is dry, it is ground to a coarse flour called grist. Next, the grist is mixed (e.g., 1 :4 volume of grist to water) with hot water (e.g., at a temperature between approximately 63-95°C) in a mash tun to extract the sugars, for example, with three successive waters. The sugared water is called wort. Yeast strain V16/01481 1 is then added and the wort fermented (e.g., similar to beer fermentation) to an alcohol percentage of approximately 8%. The fermented product is then disti lled to separate the alcohol from water and other substances contained in the fermented wort. The quality of the distillation depends partially on the type of surface (e.g., copper surface) in contact with the liquids during the distil lation process and other still features (e.g., the shape, the height, the length of the lyne arm). Distinctive flavors may be generated during the aging process where several factors (e.g., the casks used, the nature of the warehouse, the taste of the air) may exert an effect. For example, storage in casks and aging for up to 15-20 years may be used to impart characteristic flavors and aromas.

[0062] In an exemplary method for preparing a beverage such as wine, crushed grapes are fermented using yeast strain V16/01481 1, which ferments sugars found in the grapes and converts them into alcohol. Grapes are harvested, for example, during the cool morning hours, when the sugar content i s at its peak and moved to the winery in open bins. Next, the grapes are transferred to a stemmer/crusher where the stems are removed and the grapes are crushed. After crushing and de-stemming, the juice, now called must, is placed into the fermentation vats and a yeast starter culture is added to start the fermentation. The primary fermentation therefore takes place with free access to the air at a temperature for several days (e.g., red wine at 70-90°F. for 5-10 days and white wine at 55-80°F. for 10-15 days. The young wine is run off into casks or vats which are covered from the air for the secondary fermentation. After this fermentation, the wine is transferred (e.g., racked) into a different vessel. Next, the wine is aged in tanks (e.g., stainless steel tanks) or barrels (e.g., oak barrels) depending upon the style of wine being fermented. After aging and prior to bottling, the wines are fined and filtered to stabilize and clarify them. Next, wines are bottled in a sterile environment, and sealed (e.g., with a cork). Different varieties of grapes and the presence or absence of skin produce various types of wine.

[0063] In an exemplary method for preparing a beverage such as cider, apple juice is fermented by yeast strain V16/014811. In this process, apples (e.g., approximately 1000 pounds) are first washed, then inspected to make sure that they are clean and whole. Next they are fed into a grinding mill that mashes them into a texture resembling that of applesauce but with seeds and skin included. Next, the pulp is pumped or scooped for filtration onto filters. For example, the filters may be a woven cheesecloth placed in a square frame above a wooden rack such as an open checkerboard of thin oak slats crosswise to each other. After each cloth is filled with pulp (e.g., about five gallons), it is folded over and another rack and cloth are placed on top. In addition, for example, a dozen or so racks and cloths are filled and stacked into a pile (e.g., about three feet high)and rolled into a viselike press (e.g., that applies a pressure of 2,000 to 3,000 pounds per square inch to the whole stack). Following filtration, the apple juice in poured into casks or other fermentation vessels. Next, a starter culture of yeast strain V16/014811 is added and the apple juice is allowed to ferment (e.g., at a temperature of 4-16°C). Shortly before the fermentation consumes the sugar, the liquor is racked (e.g., siphoned) into new vats, leaving dead yeast cells and other undesirable material at the bottom of the old vat. Finally, vats are filled completely to exclude air including, to exclude airborne acetic bacteria, and the fermentation is allowed to continue. The remaining available sugar generates a small amount of carbon dioxide that forms a protective layer, reducing air contact. This final fermentation al so creates a small amount of carbonation. Extra sugar may be added specifically for this purpose. Racking is sometimes repeated if the liquor remains too cloudy. The cider is ready to drink after a fermentation period (e.g., three month fermentation period), though more often it is matured in the vats for several years (e.g., up to two or three years),

[0064] In an exemplar}' method for preparing a beverage such as brandy, yeast strain V16/01481 1 is used in the fermentation of a liquid that contains any form of sugar. Sources of sugar may include, for example, grapes or other fruits (e.g., grapes, apples, blackberries), vegetables (e.g., potato), sugar cane, honey, milk, rice, wheat, corn, or rye. French brandies are typically made from the wine of the St. Emiilion, Colombard (e.g., Folle Blanche) grapes. In an exemplary method, brandy may be produced by the alcoholic fermentation of grapes (e.g., white wine grapes) in a process similar to that of wine making. The grapes are allowed to ferment to an alcohol content of approximately 10%. After fermentation the wine is distilled to purify and increase the alcohol concentration to approximately 36-60%. Brandy is usually double-distilled, meaning that the alcohol is concentrated twice. For example, it takes about 34L of wine to make 3.8L of brandy. After a first distillation, which takes about eight hours, the concentrated liquid has an alcohol content of approximately 26-32%. The product of a second distillation has an alcohol content of approximately 72%. The higher the alcohol content the more neutral (e.g., tasteless) the brandy. Next, the brandy is transferred to oak casks and allowed to age. Most brandy consumed today, even fine brandy, is less than six years old. However, some fine brandies are more than 50 years old. As the brandy ages, it absorbs flavors from the oak while its own structure softens, becoming less astringent. Through evaporation, brandy will lose about 1% of is alcohol content per year for the first 50 years or so that it is on oak.

[0065] In an exemplary method for preparing a beverage such as mead, honey and water is fermented with yeast strain V16/01481 1. A must is formed by diluting the honey in water (e.g., 1 :4- 1 :66), adding ammonium salts and growth factors, and steeping for a suitable time and temperature (e.g., 15 minutes at 65°C). After cooling (e.g., to 2 ! "( ' )„ yeast strain VI 6/01481 1 (e.g., as a yeast starter culture) is added and the primary fermentation is allowed to take place for several days (e.g., 10-15 days). Next, the must is racked to a clean fermentor, additional growth supplements added and the temperature reduced (e.g., to 18°C). The must is allowed to stay at this temperature for a period of days (e.g., 120 days), racked into a third fermentation vessel and held at lower temperatures (e.g., temperatures ranging from approximately 2- 16°C) for up to a couple of years (e.g., two years), depending on the desired taste. The alcohol concentration of the final product varies (e.g., from approximately 5 to 14%) and may have a slight carbonation depending of the fermentation conditions. Mead may be flavored with hops and/or spices.

[0066] In another exemplary method for preparing a fermented beverage such as root beer, sassafras roots (e.g., 50 g) and hops (e.g., 28 grams) is steeped in boiling water (e.g., 7.5L) for a period of time (e.g., 20 minutes). The root extract is allowed to cool to at approximately 28-30°C and then strained (e.g., through several layers of cheese cloth) into a sterile, container with cap (e.g., 19L). Alternatively, root beer extracts may be obtained from commercial sources such as Shank's Extracts (Lancaster, Pa.) or Zatarain (New Orleans, La.). Next, sugar (e.g., approximately 2.3 kg) is added and mixed well until it is completely dissolved. When the sugar is dissolved, yeast strain V16/01481 1 (e.g., as a yeast starter culture) is added to a final concentration of lxl 0 8 cells/mL. After the yeast is added, the container is sealed tightly (e.g., with a cork or plastic cap), then stored for a period of time (e.g., 6-8 hours) in a warm place then refrigerated for a period of time (e.g., 24 hours). Alternatively, the sweetened root beer extract and the yeast can be bottled (e.g., in 24 ounce bottles), capped and stored.

[0067] In another exemplary method for preparing a fermented beverage such as ginger beer, raw honey (e.g., 15 mL) is added to warm water (e.g., 1L) in a sterile fermentation vessel (e.g., 3L). Once the honey is dissolved, yeast strain V16/01481 1. (e.g., as a starter culture) is added to a final concentration of 3xl0 8 yeast cells/mL. The container is then capped and the vessel is allowed to stand in a warm pl ace for a period of time (e.g., approximately 2 hours or overnight). Next, fresh ginger (e.g., 1 50 g) is peeled and grated. The juice is then squeezed into a disinfected, fermentation vessel (e.g., 3L) containing water (e.g., 1 L) and honey (e.g., 15 mL). The remaining ginger pulp is steeped by simmering in water (e.g., 250 mL) for a period of time (e.g., 30 minutes). The steeped liquid is al lowed to cool and then filtered (e.g., through several layers of gauze) into the fermentation vessel and the remaining pulp steeped as described above. Alternatively, ginger beer extracts may be obtained from commercial sources. Next, lemon or lime juice (e.g., 30 mL) is added to the fermentation vessel and sufficient water is added to fill the vessel (e.g., to bring the total volume in the vessel to 3 L). After the juice is added, the container is sealed tightly (e.g., with a cork or plastic cap), and then stored for a period of time (e.g., 5 days). Next, rice syrup (e.g., 10 mL) is dissolved in water (e.g., 100 mL) and added to the fermented ginger beer. The ginger beer is then bottled, capped, and stored with refrigeration until used.

[0068] In another exemplary method for preparing a fermented beverage such as kefir, mi lk is fermented by yeast and lactic acid bacteria in a matrix of proteins, lipids and sugars (e.g., kefir grains). The milk is obtained from cows, goats or sheep. An aliquot (e.g., IL) of cow's mil k is mixed with Kefir granules (e.g., 10 mL) containing yeast strain VI 6/01481. 1 (e.g., as a starter culture) and other starter microorganisms in a container (e.g., 2L glass container). The container containing the mixture is then loosely covered (e.g., with plastic wrap or lid) and stored at room temperature for a period of time (e.g., 24 hours). The fermented milk is then filtered (e.g., through several layers of gauze) to remove the kefir grains and stored with refrigeration (e.g., 5°C) until used. The Kefir grains are stored also in a closed container and can be used as starters for the next batch. The Kefir beverage may be slightly carbonated with a low ethanoi content (e.g., 1-2%), depending on the length of the fermentation.

[0069] In another exemplary method for preparing a fermented beverage such as kumis, mare's milk is fermented by a starter culture of yeast strain V16/01481 1 and clabber. Mare's milk (e.g., 1L) is heated to a boil with water (e.g., 250 mL) and sugar (e.g., 5 g). Next, the mixture i s allowed to cool to room temperature and then mixed with clabber (e.g., 25 mL). The mixture is allowed to incubate, for example, at room temperature, until it sours (e.g., breaks up into cured and whey). Next, a suspension of yeast strain V16/01481 1 (e.g., as a starter culture) is added to a final concentration of 1x10 s yeast cells/ml and then incubated for a period of time (e.g., 24-48 hours) until fermentation is completed. The final product is then strained into suitable containers. The kumis beverage appears as a milk product with carbonation and a low alcohol concentration (e.g., 0.5. times.1.0% ethanoi).

[0070] In an exemplary method for preparing a fermented food such as yeast paste (e.g., nutritional yeast paste) or yeast extract (e.g., a food additive or flavoring), a residual yeast slurry resulting from a fermentation using yeast strain V16/01481 1 (e.g., as a starter culture) (e.g., a 7 barrel fermentation) can be collected from the fermentation vessel and stored in a cool place for several days to promote autolysis. Alternatively, the yeast may be hydrolyzed. Autolysis, or self digestion, refers to the destruction of a cell as a result of the hydrolytic action of its own enzymes. Next, the autolyzed or hydrolyzed yeast is pressed to remove excess liquids to produce a yeast paste (e.g., a nutritional yeast paste) or a yeast extract. The yeast paste is then packaged and may be used as a spread, food additive, or flavor enhancer. Alternatively, the yeast paste can be further flavored with spices and/or artificial flavorings and then packaged. The yeast extract may be used as a food additive or a flavoring agent (e.g., flavoring enhancer).

[0071] In an exemplar}' method for preparing a food product, such as a probiotic or a food supplement, yeast strain V16/01481 1 can be manufactured including, in tablets, feed pellets or capsules of pressed yeast cells. A yeast starter culture (e.g., an active yeast starter) of yeast strain VT6/01481 1 is prepared, for example, as described elsewhere herein. A yeast suspension is used to inoculate sterile light malt extract broth (e.g., 10L with specific gravity of 1.035) in an container (e.g., 1 1.3L) and incubated with agitation for several hours (e.g., 48 hours in an orbital shaker incubator at approximately 28°C and 200 RPM). After incubation, the yeast is allowed to sediment and the supernatant fluid is siphoned off. Additional sterile light malt extract broth (e.g., 10L with specific gravity of 1.035) is added to the yeast sediment and incubated with agitation for several hours (e.g., 48 hours). For example, the yeast is grown to concentrations of 10 8 (e.g., l-5xl0 8 /mL or >5xl 0 8 /mL such as l x l( )9 /ml). After incubation, the yeast is allowed to sediment and recovered. The yeast mass is then dried and may be pressed into tablets or encapsulated in capsules containing a desired amount of yeast per tablet or capsule (e.g., approximately 1- 10 mg of yeast per tablet).

[0072] In another exemplary method for preparing food, such as bread, is made by baking leavened dough comprising yeast strain V16/01481 1, water, salt and flour. Yeast participates in the leavening process by fermenting the carbohydrates in the flour to produce carbon dioxide. During bread making, the dough is allowed to rise in a warm place then baked. The ri sing of the dough is a result of the carbon dioxide production by the yeast during fermentation. Yeasts may also impart characteristic flavors and aromas to the bread. In an exemplary method for the manufacture of exemplary leavened bread and in preparation for the baking process a culture of active yeast starter is prepared for the leavening process. A yeast starter culture is dispended into a mixing bowl along with warm water (e.g., 500 raL) and flour (e.g., 3 cups of whole wheat flour) and salt (e.g., 2 teaspoons). This mixture is mixed will until all of the ingredients are combined. Then additional flour (e.g., 1.5 cups) is added in aliquots (e.g., 0.5-cup), mixing well after each addition. When the dough has pulled together, it is turned out onto a floured surface (e.g., lightly floured surface) and kneaded until smooth and elastic. Next, the resulting dough is placed into a large, lightly oiled bowl and turned to coat with oil. The dough is covered (e.g., with a damp cloth) and allowed to rise in a warm place until it has doubled in volume. The dough is then deflated, turned out onto a lightly floured surface, then divided into two equal loaves. Next, the loaves are placed into lightly-oiled pan (e.g., 9x5 inch loaf pans). The loaves are then covered (e.g., with a damp cloth) and allowed to rise until doubled in volume. Next, the bread dough is baked (e.g., at 220°C.) for a period of time (e.g., 30 minutes or until the top is golden brown) and the bottom of the loaf sounds hollow when tapped.

[0073] In some embodiments, the fermentation may be undertaken in the presence of yeast strain V16/01481 1 and one or more additional yeast strains other than V16/01481 1. Persons skilled in the art will understand that the choice of the one or more additional yeast strains will depend on the fermented product to be prepared. For example, where the fermented product is a beer, the one or more additional yeast strains may include Saccharomyces cerevisiae (ale yeast), Saccharomyces pastor ianus, Sacckaromyces. Carlsbergensis, Brettanomyces hriixeUensis and combinations thereof. Other suitable yeast strains that can be used as the second or further yeast strains in the methods disclosed herein include Pichia kluyveri PK- R1 and PK-KR2 strains as deposited on 24 Aug. 2006 at the National Measurement Institute (Victoria, Australia) and given the accession numbers V06/022711 and V06/022712, respectively, and mutants and variants thereof. These strains are described in international patent application published as WO 2009/1 10807,

[0074] In a fifth aspect disclosed herein, there is provided a fermented product obtained by the methods disclosed herein. In an embodiment, the fermented product is a fermented beverage. In another embodiment, the fermented product is a fermented food.

[0075] In a sixth aspect disclosed herein, there is provided a fermented product comprising a yeast cell from the yeast cell culture disclosed herein. In an embodiment, the fermented product is a fermented beverage. In another embodiment, the fermented product is a fermented food. In an embodiment, the fermented product is a beverage. In an embodiment, the beverage is selected from the group consisting of beer, sake, wine, cider, mead, root-beer, ginger-beer, kefir and kumis. In another embodiment, the fermented product is a food. In an embodiment, the food is selected from the group consisting of a food supplement, a dough and a bread.

Method of brewing beer

[0076] The process of brewing beer is well known to the skilled person. In an illustrative example, malt is prepared from dried, germinated cereal grains (mainly barley or wheat) and grounded into a grist which may contain unmalted adjuncts. The grist is mashed (mixed with water and steeped) to allow enzymes in the malt to convert the starch into sugars. The grain particles and adjuncts are separated from the liquid wort in a process called iautering. The malt making and mashing steps can be skipped by adding water to malt extract. After addition of hops and/or other ingredients such as herbs and sugars, the wort is boiled (hops may also be added after boiling), cooled and aerated. The wort is then moved to a fermentation tank and fermented by the addition of a brewer's yeast. The primary fermentation, lasting typically 5 to 10 days, may be followed by a secondary fermentation step using a further brewer's yeast. After fermentation the fresh beer or "green" beer, is conditioned, optionally filtrated and carbonated.

[0077] Hops are typically added to the wort to balance the sweetness of the malt with bitterness and impart onto the beer desirable flavours and aromas. Several hop varieties are used for this purpose, illustrative examples of which include Ahtanum, Amarillo, Apollo, Cascade, Centennial, Chinook, Citra, Cluster, Columbus, Crystal, Eroica, Galena, Glacier, Greenburg, Horizon, Liberty, Millenium, Mount Hood, Mount Rainier, Newport, Nugget, Palisade, Santiam, Simcoe, Sterling, Summit, Tomahawk, Ultra, Vanguard, Warrior, Willamette, Zeus, Admiral, Brewer's Gold, Bullion, Challenger, First Gold, Fuggles, Goldings, Herald, Northdown, Northern Brewer, Phoenix, Pilot, Pioneer, Progress, Target, Whi thread Golding Variety (WGV), Hallertau, Hersbrucker, Saaz, Tettnang, Spelt, Feux- Coeur Francais, Galaxy, Green Bullet, Motueka, Nelson Sauvin, Pacific Gem, Pacific Jade, Pacifica, Pride of Ringwood, Riwaka, Southern Cross, Lublin, Magnum, Perle, Polnischer Lublin, Saphir, Satus, Select, Strisselspalt, Styrian Goldings, Tardif de Bourgogne and Tradition. Further varieties exist including but not limited to Bravo, Calypso, Chelan, Comet, El Dorado, San Juan Ruby Red, Satus, Sonnet Golding, Super Galena, Tillicum, Bramling Cross, Pilgrim. Hallertauer Herkuies, Hailertauer Magnum, Hallertauer Taurus, Merkur, Opal, Sraaragd, Halleratau Aroma, Kohatu, Rakau, Stella, Sticklebract, Summer Saaz, Super Alpha, Super Pride, Topaz, Wai-iti, Bor, Junga, Marynka, Premiant, Sladek, Styrian Atlas, Styrian Aurora, Styrian Bobek, Styrian Celeia, Sybilla and Sorachi Ace.

[0078] The inventors have unexpectedly found that yeast strain V16/014811, when used in a beer brewing process comprising hops, imparts advantageous and characteristics of flavour and colour to the beer. These characteristics are also considered to be unique to a beer brewed using yeast strain VI 6/014811 in the fermentation process.

[0079] In an embodiment, there is provided a method for brewing beer comprising the steps of: a) providing a wort; b) adding at least one hop variety to obtain a hopped wort; and c) fermenting the hopped wort with yeast strain V16/014811 to obtain a beer.

[0080] In other embodiments disclosed herein, the method further comprises the step of d) fermenting the hopped wort with a second yeast strain that is not yeast strain V16/014811. Suitable beer brewing yeast strains that can be used as the second yeast strain will be familiar to persons skilled in the art, illustrative examples of which include top-cropping yeast such as Saccharomyces cerevisiae (ale yeast) and bottom-cropping yeast such as Saccharomyces pastorianus, Saccharomyces. carlshergensis and Brettctnomyces hriixettensis . Other suitable yeast strains that can be used as the second yeast strain in the methods disclosed herein include Pichia kiuyveri PK-KRl and PK-KR2 strains as deposited on 24 Aug. 2006 at the National Measurement Institute (Victoria, Australia) and given the accession numbers V06/022711 and V06/022712, respectively, and mutants and variants thereof. These strains are described in international patent application published as WO 2009/1 10807.

[0081] In an embodiment, the fermentation in step c) is allowed to proceed for at least 12 hours, such as for at least 24 hours, such as for at least 36 hours, such as for at least 48 hours, such as for at least 60 hours, such as at least 72 hours prior to inoculation of the second yeast strain in step d). In a preferred embodiment, the fermentation in step c) is allowed to proceed for at least 36 hours prior to inoculation of the second yeast strain in step d).

[0082] In an embodiment, the fermentation in step c) is carried out at a temperature of between about I 2°C and about 28°C. In a more preferred embodiment the fermentation temperature in step c) is between about 20°C and about 22°C. Most preferably the fermentation temperature in step c) is about 20°C.

[0083] In another embodiment, step c) and step d) are carried out at the same time by co-inoculation of yeast strain V 16/01481 1 of step c) and the second yeast strain of step d). [0084] In an embodiment, the second fermentation step in step d) is allowed to proceed for at least 12 hours, such as at least 24 hours, such as at least 48 hours, such as at least 72 hours, such as at least 96 hours, such as at least 120 hours.

[0085] In a preferred embodiment of the present invention the beer is a low-alcohol, reduced-alcohol or non-alcohol beer.

[0086] In another embodiment disclosed herein, there is provided a beer produced by the methods disclosed herein. In an embodiment, the beer comprises detectable amounts of yeast strain VI 6/01481 1.

[0087] In some embodiments, the yeast is removed from the beer after fermentation. However, it has been found that retaining at least some yeast will give the beer a certain flavour and/or colour and/or aroma profile that is different from a beer in which yeast strain V16/01481 1 has been removed after fermentation.

[0088] In an exemplary method for preparing a fermented beverage such as beer, one or more grains (e.g., barley, wheat, rice and/or corn) are steeped in water for a period of time (e.g., 14-18°C. for approximately 48 hours) to raise the grain's moisture content and then allowed to germinate over several days (e.g., 3-5 days). Next, the germination may be stopped by kilning or heating the grain to high temperature (e.g., temperatures below 1 10°C.) which preserves the amylases and subsequent sugars, thereby producing a malt. The malt may then be ground or milled to fine particles (e.g., by milling). Alternatively, a malt may be obtained from a commercial source and used in the subsequent process steps. Next, the malt (e.g., produced by a malting process or obtained from a commercial source) may be added to water at high temperature (e.g., 65°C.) with well characterized salt profiles (e.g., high calcium is good for ales and low calcium is good for lagers such as pilsners). After approximately 1 hour of heat, the liquid portion (e.g., wort) may be recovered and transferred to a kettle and boiled (e.g., for one hour). This heating may be used to sterilize the wort, precipitate the proteins and remove unpleasant grain flavors. Additionally, during this boil, hops may be added at varying times for bitterness and flavoring. Various hops are commercially available, including hops for beers, ales or lagers. After the boiling, the wort may be cooled, inoculated with yeast strain V16/-1481 1 and aerated to trigger yeast metabolism. Ale fermentation may take a few days at higher temperatures (e.g., approximately 20°C) and lager fermentation may take several weeks at lower temperatures (e.g., approximately 6°C).

[0089] The beer may then be conditioned to settle out the yeast and solids. If desired, additional hops and CO ? , can be added before packaging. For example, an exemplary batch (e.g., 8 barrel batch) of beer (e.g., ale) is prepared as follows. Various malts including, pale malts, Munich malts and wheat malts are added into a mash tun containing water (e.g., water at 180°F) for a period of time (e.g., 10 minutes) and then batch sparged for an additional period of time (e.g., an hour). Next, the wort is pumped into the boil kettle and brought to a boil. Cascade hops are then added at the start of the boil and during the boil (e.g., 30 minutes into the boil). Hallertau hops are additionally added during the boil (e.g., 30 minutes, at 60 minutes, and the end of the boil). The wort (e.g., at a specific gravity of 1.048) is cooled (e.g., to 68°F.) and yeast strain V16/01481 1 is added. Next, the wort is sparged with oxygen, stirred and the fermentation tank sealed. The initial yeast growth is rapid with violent top fermentation, the temperature range is narrow and warm (e.g., between 20-21°C) and is preferably kept in this range for active fermentation. In addition, the yeast flocculates quickly, even with high residual sugar levels and is preferably occasionally agitated and re-circulated during the entire fermentation. In this exemplary 8- barrel batch, the sugar attenuation rate is high (e.g., approximately 80%) and an ale beer is produced with a final specific gravity of 1.014. The beer is then drawn off to a finishing tank and held for several weeks at a temperature (e.g., two weeks at 2-8°C.) and served unfiltered. For another example, an exemplary batch (e.g., 10 barrel batch) of beer (e.g., wheat) is prepared. The process is similar to the process for the ale beer described above but using Pilsner malt, White Wheat malt, and Sterling hops and step mash (e.g., 145°F. for 15 minutes raised to 155°F. over 15 minutes, then mashed at 155°F. for 20 minutes and with the mash out at 168°F.). The fermentation observations are also similar. A wheat beer is produced with a final specific gravity of 1.010.

[0090] As noted elsewhere herein, yeast strain VI 6/0 481 1 is suitable as a fermenting yeast for producing alcohol from sugar. Thus, in a sevent aspect disclosed herein, there is provided a method of producing ethanol from sugar, the method comprising culturing a yeast cell from the yeast cell culture, as herein described, in a solution comprising sugar under conditions such that the yeast strain converts the sugar to ethanol. Also disclosed herein is ethanol produced by the methods disclosed herein.

[0091] The alcoholic fermentation conditions will typically depend on the type of application desired, for example, according to whether it is a brewing, wine-making or distilling fermentation. The alcoholic fermentation conditions can be readily determined by persons skilled in the art. By way of example, reference may be made to the alcoholic fermentation conditions described in the reference manual "Yeast Technology" (2 nd edition, 1991, G. Reed and T. W. Nagodawithana, published by Van Nostrand Reinhold, ISBN 0-442-31892-8). Yeast strain V16/014811 is particularly advantageous for producing drinking alcohol, intended for the production of alcoholic beverages, and/or industrial alcohol, intended, for example, for biofuels or for chemical industries.

[0092] Those skilled in the art will appreciate that the invention described herein in susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications which fall within the spirit and scope. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

EXAMPLES

Example 1: Method of brewing beer

[0093] Three replicate colonies of yeast strain V16/014811 were prepared by streaking conventional agar plate media under sterile laboratory conditions, and growing at room temperature for three days. Individual colonies were selected for subculture and were preserved as single colony subcultures at -80°C and as agar slopes. Individual colonies were studied by sequencing their genetic code, which was subsequently compared to databases of similar microorganisms. As shown in Figures I and 2, the Saccharomyces strain V16/014811 was identified as being unique when compared to reference brewing (baking, wine and beer) yeasts, and was identified as a rare three way hybrid of early brewing heritage. [0094] To brew beer, the yeast was prepared as a live yeast culture. A sterile broth was prepared containing lg/litre Oxoid LP0021 Yeast Extract (or equivalent) and 50g/litre Oxoid CM0057 Malt Extract Broth (or equivalent). A sterile 1L Shott Bottle was used for preparation of the brewing culture. 250 ml of broth was inoculated from an agar slope containing the original strain. A magnetic stir bar was placed into the bottle. The bottle was then sealed and the screw cap wrapped in Para film. The bottle was maintained at 25°C for 48 hours during which yeast growth is observed as a fawn coloured particulate on the base of the bottle. After this period, the yeast may be stored at 4°C prior to use for brewing and remains viable for up to two weeks (tested so far).

[0095] Within 24 hours of brewing, the bottle was brought to room temperature (21°C) and was placed on a magnetic stirrer. The broth was stirred slowly for a period of approximately 8 hours with the bottle lid loosened but not open to allow gas to escape. Significant additional yeast growth was used during this period. At the conclusion of this period, the yeast is suitable for use as a starter for the brewing process. The entire content of the bottle was used as a starter for a brew volume of 25 litres,

[0096] Beer was brewed by creating a 5kg barley mash/25 litre water wort comprising 170g crystal malt barley, 1.5g Calcium sulphate, 50g Kentish Goldings (Hop plant variety). Heating was by conventional methods, for approximately one hour to produce a wort mixture. The approximately ten minutes prior to completion of the heating period, 50grams of Kenti h Goldings was added and I g proprietary seaweed-based flocculating agent to promote clarity. The wort was then artificially cooled as swiftly as practical using a heat transfer method to a temperature of 22°C.

[0097] The cooled wort was then transferred to a brewing vessel, which may be sealed and which incorporates an air lock. The yeast stock solution was added, the container sealed and swirled to mix. A sample of wort was measured for its initial specific gravity for use in final alcohol percentage estimation and fermentation completion.

[0098] The brewing vessel was then left to ferment at approximately 20°C for seven days. The vessel was agitated by swirling approximately every 24 hours. [0099] Sugar (dissolved in water, sterile) was then added to sterile 750 ml brown beer bottles at a rate of 6g per bottle (30 ml solution). Using sterile apparatus, approximately 720ml of brewed mixture was decanted into each brewing bottle. The bottle was then caped and allowed to mature for a minimum of four weeks prior to consumption, to allow bottle fermentation to occur. The bottle was shown to contain live culture in the beverage that is intended for consumption. The yeast was not filtered or pasteurised. The yeast strain VI 6/014811 is carefully managed to minimize or otherwise prevent further evolution or hybridization of the strain. This is achieved by management of a reserve stock of the first successful culture of the yeast strain in a -80°C preservation environment. Yeast is known to evolve rapidly in response to its environment and, for example, yeast used for one brewing process may evolve and produce different flavour characteristics in subsequent brewing processes. The genetic sequence of yeast strain V16/01481 1 is recorded, which allows any genetic variation in the active brewing strain to be monitored. At all times, a preserved original yeast strain is maintained separately to yeast colonies intended for use in brewing processes in order to ensure that the original yeast strain can be revived, should it be required. For this brewing process, all reasonable efforts are made to ensure that the yeast closely resembles its original genetic makeup. Moreover, during preparation of the yeast starter to be used for brewing activities, high purity culture ingredients and sterile conditions are applied in order to ensure minimal genetic variance or contamination of the yeast strain.

Example 2: Method of producing ethanol by distillation

[0100] A possible application of the yeast strain is in the production of ethanol by distillation. An illustrative example is in the production of ethanol for human consumption in distilled beverages. Ethanol produced by fermentation can also be distilled for industrial applications. For distilled beverage production, grain or other plant material based mash is typically fermented, and the resulting ferment is distilled to separate alcohol and trace amounts of aromatic odour and flavour molecules that can contribute to the taste of the final product. For beverage production, it is generally preferred that the yeast is capable of converting the sugar in the mash into ethanol and producing a alcohol level of about 7% to about 10 % w/v, whilst avoiding the production of adverse flavour characteristics. [0101] In this study, yeast strain V16/014811 was benchmarkecl against a Reference Standard (a species of yeast against which all other potential commercial yeasts are compared) and two additional yeast strains (Commercial A and B) that are used commercially for distilled beverage production to assess the potential of yeast strain VI 6/014811 as a suitable yeast strain for such applications. Using a barley mash broth, the yeast strains were allowed to ferment the broth to completion, and the components of the broth were measured by High Pressure Liquid Chromatography HPL:C) to assess the time interval performance of the yeast strain for this application. The use of yeast strain V16/01481 1 in this study resulted in an ethanol yield that was comparable to the commercial yeasts strains. Moreover, yeast strain V16/014811 did not appear to produce excessive chemical species associated with a poor tasting beverage. The testing identified that yeast strain VI 6/014811 has potential to be an effective production species for distilled beverages, such as whiskey. The data from this fermentation study are given in Table 1, below:

Table 1 :

72 hour HPI C results (in % /v, unless stat ed otherwise) dextrin 0.483 0.386 0.433 0,474 maltotriose 0.343 0.244 0.597 0.458 maltose 0, 1 2 0,084 0.408 0,247 d-glucose 0.042 0.055 0.071 0.063

1-lactic acid 0,033 0,035 0.035 0,060 glycerol %w/v 0.268 0.321 0.346 0.351 acetic acid 0,000 0,000 0.013 0,000 ethanol %w/v 7.510 7.557 7.060 7.235 ethanol %v/v 9.511 9.571 8.941 9.163 std dev 0.021 0.01 1 0.013 0.021 Example 3: Method of producing dough and bread

[0102] Another application of yeast strain V16/014811 is in the production of dough for bread and other baked goods. This study was undertaken to assess whether yeast strain V16/01481 1 is a suitable substitute for bread making, The following method was used:

[0103] Under sterile conditions, a yeast prorogation broth was prepared comprising;

(i) 2g Yeast extract (Oxoid LP0021 )

(ii) lOg Sugar (Oxoid LP0039); and

(iii) 100 ml. sterile and de-ionised water,

[0104] Pyrex laboratory glassware was used, a magnetic stir bar was added, and the mixture was heated to 90°C for one hour using a magnetic stirrer. The container was sealed and the mixture allowed to cool for 24 hours.

[0105] Using sterile transfer techniques, the broth was transferred to ten sterile Falcon tubes. Eight of the tubes were inoculated with yeast strain V16/014811, two acting as contamination controls. The inoculated tubes were maintained at 20°C for 72 hours, and manually agitated at approximately 8 hour intervals.

[0106] White ground wheat flour in 50g batches was oven warmed to 60°C for 1 hour, followed by exposure to ultraviolet radiation (UV-C) under clean laboratory conditions in a flat Pyrex container in order to minimise the presence of environmental yeast species within the flour. 50 niL deionised water containing 5g sugar (cool, sterile) was added to the flour along with the yeast culture propagated in the Falcon tube. The yeast starter containers were covered with plastic film to prevent contamination during yeast growth. Eight yeast starter replicates were created using this method. The mixture was stirred periodically and maintained at 25°C for 24 hours, at which time the mixture was observed to have gas pocket bubbles associated with yeast starters used for sour dough bread production. The mixture had a yeast odour, and the plastic film cover expanded periodically indicating the presence of gas being produced by the yeast growth. [0107] This bread starter was added to a mixture comprising three cups of freshly ground white wheat flour (high protein, bread making), 5 grams sugar, 2 g salt (NaCl) and 330ml deionised sterile water, all mixed in a sterile stainless steel container. The mixture was allowed to stand at 25°C for 24 hours, lightly covered in plastic film to allow evolved gas to escape. Over this period the mixture expanded to approximately three times its volume with visible gas bubbles.

[0108] The mixture was kneaded using a dough hook on a clean kitchen mixing machine for 5 minutes at low speed. The mixture was shaped into a bail and placed onto a metal oven tray. It was allowed to rest for 4 hours at approximately 25°C while covered in a loose plastic film. During this time, the mixture was observed to rise (bread proofing) and undertake the typical shape of an unbaked sourdough bread loaf,

[0109] The proofed bread dough was placed into a pre-heated electric commercial kitchen oven at 230°C for 40 minutes, during which time it was observed to further rise and forms a brown crust. When removed from the oven, the mixture was observed to have a characteristic appearance of bread. When cut, the bread had lightly aerated structure and moist appearance, characteristic of sourdough bread loaf with slow yeast growth characteristics. The bread did not have unpalatable odours or flavours, and was observed to have a slightly fragrant/fruity odour that was atypical of sourdough yeast bread. Similar results were observed for each of the eight replicated bread making processes.

Example 4: Method of producing mead

[0110] Yeast strain V16/01481 1 was observed to have properties of vigorous growth in sugar solutions. Following that observation, this study was undertaken to determine whether yeast strain V16/014811 could be used to ferment honey and make mead, noting that honey contains a sugar mixture that many commercial yeast varieties cannot successfully ferment. [0111] Under sterile conditions, a yeast prorogation broth was prepared comprising:

(i) 2g Yeast extract (Oxoid LP0021 );

(ii) l Og Sugar( Oxoid LP0039); and

(iii) 100 mL sterile and de-ionised water,

[0112] Pyrex laboratory glassware was used, a magnetic stir bar was added, and the mixture was heated to 90°C for J hour using a magnetic stirrer. The container was sealed and the mixture allowed to cool for 24 hours,

[0113] Using sterile transfer techniques the broth was transferred to ten sterile Falcon tubes. Eight of the tubes were inoculated with yeast strain V16/01481 1, two acting as a contamination control . The inoculated tubes were maintained at 20"C for 72 hours, and manually agitated at approximately 8 hour intervals.

[0114] A 5000 ml Pyrex conical flask was chemically sterilized. Four litres of sterile de- ionized water was added.

[0115] One kilogram of single origin honey was pasteurised by heating to 90°C for 20 minutes to destroy wild yeast species. The warm honey was added to the conical flask, and the water honey mixture, or "Must", was boiled for 30 minutes to precipitate protein and further sterilise the mixture of bacteria or wild yeast. The solution was allowed to cool to 20°C and its specific gravity measured.

[0116] The contents of the Falcon tube were added to the mixture using laboratory practices that would avoid contamination.

[0117] After the yeast had been "pitched", the conical flask was sealed with a silicon stopper which contained a water air lock device to exclude new air from entering the flask and allow carbon dioxide created during fennentation to escape the flask, The flask, was wrapped in aluminium foil to exclude light and the mixture was allowed to ferment for 8 weeks. During the first and second week the fermentation process was observed to be fast. Fermentation appeared to have stopped after 6 weeks. After 8 weeks, the specific gravity- was measured, suggesting an alcohol content of about 10 to 1 1%. [0118] A second sterile 5000 L conical flask was prepared, and the Mead was "racked" to clear the mead by siphoning the contents of the first flask into the second flask, with the objective of leaving behind yeast sediment in the first flask bottom. A "Camden" tablet was added to the second flask to aid in clumping and precipitation of yeast and other solution residue at the base of the second flask.

[0119] The Mead was aged in the flask in the dark with the airlock fitted for a further 8 weeks to improve and develop its flavour and to allow final fermentation to occur and for remaining yeast particles to drop out of suspension. Aging occurred at room temperature.

[0120] Sterile bottles were prepared and the Mead was transferred by siphoning into the bottles for additional aging of twelve months. The bottles were sealed airtight and stored on their sides. The incompletely aged Mead was observed to have an alcohol content of approximately eleven percent, and to not have adverse flavours or odours. This observation suggested that yeast strain V16/01481 1 was capable of fermenting honey, and that with sufficient aging, the Mead drink produced is likely to have palatable flavours.