Production of fruit based or other types of alcoholic beverages

Abstract

The present invention relates to methods for cider production, specifically sake yeast strains of the species Saccharomyces cerevisiae and stopped fermentation. The invention is based on the surprising finding that use of Sake yeasts in the production of cider leads to beverages with good organoleptic properties. In particular, the beverages 5 have good fruitiness / fruity aromas, which is in general desirable in ciders.

Description

[0001] Production of fruit based or other types of alcoholic beverages

[0002] Technical field

[0003] The present invention relates to the field of beverage, such as cider production. More specifically, the invention is based on the surprising finding that use of Sake yeasts in the production of fruit or malt based beverages, such as cider, leads to beverages with good organoleptic properties. In particular, the beverages have good fruitiness / fruity aromas, which is in general desirable in e.g. ciders.

[0004] Background

[0005] Cider is an alcoholic beverage, a fruit wine made from apple extracts by alcoholic fermentation. In countries such as Spain, UK and France such a fruit wine will be called a cider, other countries such as Germany and Switzerland call it an apple wine, and in the US and Canada it will be called a hard cider.

[0006] Cider fermentations can be made in different ways for example by fermentation of apple juice from pressed apples or from apple juice concentrate. Modern, industrial cider making also uses chaptalization, otherwise called fortification, that is the addition of sugars or syrups to unfermented grape must to supplement carbohydrates and increase the alcohol content of the cider fermentation (Lea, A. 2015).

[0007] The use of apple juice concentrate has increased considerably because of several advantages (price, storage stability and storage of the concentrate for months or years without spoilage / microbial infection and / or spontaneous fermentation) leading to less dependency to specific apple cultivars for cider making. The disadvantage is that most apple juice concentrates have a low intrinsic apple flavour, as these flavours are removed in the vacuum distillation step to produce the concentrate. Thus flavour houses / commercial food laboratories typically add flavours to the concentrates or the final ciders after the wine fermentation step.

[0008] In general, industrial cider fermentation consists of a wine fermentation, followed by a purification step or wine cleaning step, and finally a cider mixing step to blend the wine with fruit juice, sugars, and flavours and carbonized water to obtain the final cider. Wine fermentation is typically conducted in similar manner as grape wine fermentation but without the fruit skin. Typical pitching rates of wine yeast S. cerevisiae is a few million cells per ml and fermentation temperatures around 20 Celsius.

[0009] With fermentation of chaptalized ciders, wherein the original "juice base" has a high sugar content, to obtain a final high alcohol by volume content in the end fermented wine, some extra yeast food and active aeration are required at the start of fermentation to get a strong and vital yeast. Further, a higher pitching rate of yeast is beneficial.

[0010] The traditional method of making cider relies on the indigenous microflora of apple to carry out spontaneous natural fermentation. Modern cider production is also made with inoculation of specific microorganisms, the majority with yeast. Some of the yeasts have been isolated from the cider factories. Wine yeasts are used by cider manufacturers and widespread use of active dried wine yeasts has occurred since 1980s.

[0011] Particular wine yeast strains produce a specific range of compounds that affect the aroma and taste of cider. Examples of wine yeasts commonly used are within the Saccharomyces genera e.g. S. cerevisiae, S. bayanus, S. uvarum or hybrids of Saccharomyces.

[0012] The choice of yeast strain affects the quality of the cider and the production of metabolites that affect the taste and flavour (e.g. esters, higher alcohols, organic acids). Low production of SO₂ and H₂S are also desired. The wine produced by a typical wine yeast often has some off flavours like fusel alcohol or sulphury components. Such components can be negatively affect the aroma and taste of the final wine and, can be minimized by choosing a wine yeast known to be more neutral and clean in flavours profile, for example Red Star Premier Cuvee wine yeast, or low in sulphur metabolism, such as Chardonnay White wine yeast WLP730, or CEG Uvaferm.

[0013] During the wine cleaning stage of cider production, the fusel alcohols can be removed by various filtration steps, such as active carbon. This results in a pure white wine made from apples and sugars, which contains minimal flavours.

[0014] During the cider mixing stage, the pure white wine (15-16% in ABV) is mixed with apple juice concentrate with apple flavours or other flavours (e.g., other fruits, such as pear) from flavours houses / commercial food laboratories and sparkling water to obtain a cider of 4.5% ABV. Finally, additional sugars are added to the mix to increase the sweetness of the cider.

[0015] The choice of yeast can strongly influence the flavours profile of the cider, and some may negate the requirement for some of the aforementioned processes of cider fermentation, such as cider filtration or mixing due to their intrinsic capacity to produce a specific range of compounds that positively affect the aroma and taste of cider (Grumezescu & Holban.

[0016] 2019; Kosseva et al., 2017)

[0017] Sake yeast strains, such as S. cerevisiae var. sake, are typically used to produce sake in Japan, which is a rice wine of 18-20% ABV, that is often reduced to 15% ABV before packaging. However, less is known regarding their usefulness in the production of cider.

[0018] Summary

[0019] The present invention is based on the surprising finding that Sake yeasts are particularly useful for production of fruit or malt based beverages, such as cider. Thus, fermentation of e.g. a cider base with Sake yeast results in ciders with extraordinarily good organoleptic properties. In particular, the ciders have good fruitiness / fruity aromas, which is in general desirable in ciders. Thus, beverages, such as ciders produced by fermentation with Sake yeasts have high levels of terpenes, organic acids and esters advantageous for fruitiness, whereas they produce low levels of undesirable fusel alcohols. Further, as a result of said extraordinarily good organoleptic properties, fermentation of beverage bases, such as cider bases with Sake yeast negates the requirement to use active carbon filtration during cider filtration stage.

[0020] The invention furthermore provides S. cerevisiae yeasts, preferably Sake yeasts, comprising genes encoding BIO1 of SEQ ID NO: 16 and / or BIO6 of SEQ ID NO: 17 or functional homologues thereof sharing at least 90% sequence identity therewith. Such yeast are particularly useful for production of ciders with extraordinary high levels of terpenes (e.g. citronellol), organic acids (e.g. octanoic acid and decanoic acid) and esters (e.g. ethyl hexanoate and ethyl decanoate) advantageous for fruitiness, whereas they produce low levels of undesirable fusel alcohols. The invention also provides methods for production of fruit or malt bases beverages, such a cider, comprising fermenting a beverage base, such as a cider base, with a sake yeast and stopping fermentation before fermentation is complete. Such method is also referred to as “stopped fermentation” herein.

[0021] The invention is further described in the claims attached hereto.

[0022] Description of Drawings

[0023] Figure 1. Maximum-likelihood phylogenetic tree constructed by comparison of Single-Nucleotide Polymorphism (SNPs), called using Saccharomyces cerevisiae S288C as reference, of the yeasts investigated. Node shapes indicate the origin of the yeasts.

[0024] Figure 2. Phylogenetic tree constructed based on single-copy orthologs of the investigated yeast strains.

[0025] Figure 3. Sensory evaluation of the resultant cider’s sensory profile following (A) stopped or (B) dry fermentation of control vs sake yeast.

[0026] Detailed description

[0027] Definitions

[0028] The term “degree Brix” (abbreviated as “°Bx”) is a measure of dissolved solids in an aqueous composition and is based on measurement of the specific gravity. °Bx is in particular useful as an approximate measure of dissolved sugar. In a solution of sucrose in water, one °Bx corresponds to 1 g of sucrose in 100 g said solution. Other measures for specific gravity include the Plato scale (abbreviated as °Plato). Whereas °Bx and °Plato are different measures of specific gravity and thus differ from each other, they are typically in the same range, and can therefore be used interchangeably for an approximate indication of the specific gravity.

[0029] The term “beverage base” as used herein refers to an aqueous composition, which is useful for preparing a beverage by fermentation. A “cider base” is an aqueous composition, which is useful for preparing cider by fermentation Thus, a cider may be prepared by fermenting a cider base with yeast. A “cider base” comprises apple juice and / or apple juice concentrate and water, but may also comprise additional compounds such as sugar. In some cases, a ‘cider base’ also comprises pear juice and / or pear juice concentrate. It is preferred that at least 90%, such as least 95% of the cider base is made up of apple juice, apple juice concentrate, water and sugar.

[0030] The term “dry fermentation” as used herein refers to a fermentation carried out until the essentially all of the fermentable sugars have been converted and the maximum potential ABV (%) has been achieved. Preferably, the residual concentration of fermentable sugars after dry fermentation is at the most 0.2 g / L.

[0031] The term “fruit juice” as used herein refers to a liquid made from the extraction or pressing out of the liquid naturally contained in fruits; the term “fruit juice” may also refer to a fruit juice concentrate, obtained after removal of water from a fruit juice.

[0032] " Organoleptic properties" means properties of beverages as detected by the human olfactory and taste senses. These may be analysed, for example, by a trained, specialized taste panel.

[0033] The term “degree Plato” (abbreviated as “°Plato”) is a measure of specific gravity of an aqueous composition and measures the density of the aqueous composition compared to the density of pure water. °Plato is in particular useful as an approximate measure of dissolved sugar. Other measures for specific gravity include the Brix scale (abbreviated as °Bx). Whereas °Bx and °Plato are different measures of specific gravity and thus differ from each other, they are typically in the same range, and can therefore be used interchangeably for an approximate indication of the specific gravity.

[0034] The term “RTD” as used herein in relation to juice refers to “ready to drink”. Juice may be provided in the form of a concentrate, which must be diluted in order to arrive at a “ready to drink” juice. Typically, an RTD juice has a specific gravity in the range of 5 to 20°P, such as in the range of 5 to 15°P.

[0035] A "specialist taste panel" within the meaning of the present application is a panel of specialists extensively trained in tasting and describing flavours. Although a number of analytical tools exist for evaluating flavour components, the relative significance of flavour-active components are difficult to assess analytically. However, such complex properties can be evaluated by taste specialists. Their continuous training includes tasting and evaluation of standard samples.

[0036] The term “stopped fermentation” as used herein refers to a fermentation, which is stopped before all fermentable sugars has been converted.

[0037] Methods for producing a beverage

[0038] The present disclosure provides methods for producing a beverage, such as a cider, using a sake yeast strain of the species Saccharomyces cerevisiae, which surprisingly produces a beverage, such as a cider with extraordinary good organoleptic properties comprising a more positive perception of fruitiness / fruity aromas, mouthfeel, naturalness and / or sensation of freshness, related to desirable concentrations of flavour compounds. Any sake yeast may be used with the methods, in particular any of sake yeasts described herein below in the section “Sake yeast”. The beverages, such as ciders produced according to the methods of the invention in general comprise reduced levels of undesirable fusel alcohols and volatile organic acids, increased ester, terpenes and nonvolatile organic acid concentrations. In addition, they may have increased sweetness, which may be due to higher residual fructose concentrations compared with beverages, such as ciders produced using non-sake yeasts and / or traditional beverage, such as cider production methods.

[0039] In some aspects is provided a method for producing a beverage, said method comprising;

[0040] i) providing a beverage base comprising fruit juice or an aqueous extract of malt for fermentation, and

[0041] ii) providing a sake yeast strain of the species Saccharomyces cerevisiae, wherein said yeast strain comprises an OYE3 gene encoding full length OYE3 protein, preferably OYE3 of SEQ ID NO: 2 or SEQ ID NO: 18, or a polypeptide with at least 80%, such as at least 85%, such as at least 90%, such as at least 95% sequence identity to SEQ ID NO:s 2 or 18,

[0042] iii) fermenting the beverage base provided in step i) with said yeast strain of step ii), iv) thereby obtaining a fermented beverage base,

[0043] v) processing the fermented beverage base into a beverage.

[0044] In one aspect of the present invention, said method comprises the following steps: i) providing a cider base comprising fruit juice for fermentation, and ii) providing a sake yeast strain of the species Saccharomyces cerevisiae, iii) fermenting the cider base provided in step i) with said yeast strain of step ii),

[0045] iv) stopping fermentation before fermentation is complete,

[0046] v) thereby obtaining a cider.

[0047] In some aspects is provided a method for producing a cider, said method comprising;

[0048] i) providing a cider base comprising fruit juice for fermentation, and

[0049] ii) providing a sake yeast strain of the species Saccharomyces cerevisiae, wherein said yeast strain comprises an OYE3 gene encoding full length OYE3 protein, preferably OYE3 of SEQ ID NO: 2 or SEQ ID NO: 18, or a polypeptide with at least 80%, such as at least 85%, such as at least 90%, such as at least 95% sequence identity to SEQ ID NO:s 2 or 18,

[0050] iii) fermenting the cider base provided in step i) with said yeast strain of step ii), iv) stopping fermentation before fermentation is complete,

[0051] v) thereby obtaining a fermented cider base,

[0052] vi) processing the fermented cider base into a cider.

[0053] Apple juice for e.g. producing a cider may be prepared by washing the fruits, optionally milling the fruits into pulp and extracting (preferably by pressing) the juice of the fruits without the pulp. After pressing, the apple juice may be concentrated by conventional means. The cider base is prepared by mixing the ingredients thereof, for example fruit juice and / or fruit juice concentrate, and optionally water and sugar and further optionally additional ingredients. Fermentation of the cider base is normally carried out by controlled fermentation by addition of sake yeast.

[0054] The product obtained after step iv) may herein also be referred to as a “fermented beverage base” or “fermented cider base”. The fermented beverage or cider base may be subjected to one or more further processing steps before arriving at the final beverage or cider.

[0055] In some aspects of the present disclosure is also provided a method for producing a beverage, such as a cider, said method comprising;

[0056] i) providing a beverage base, such as a cider base, for fermentation, and II) providing a sake yeast strain as described herein,

[0057] iii) fermenting the beverage base, such as the cider base, provided in step i) with said yeast strain of step ii),

[0058] iv) thereby obtaining a fermented beverage base, such as a fermented cider base, v) processing the fermented beverage base, such as the fermented cider base, into a beverage, such as a cider.

[0059] Thus, in specific embodiments wherein the sake yeast as described herein, e.g. in the sections ‘sake yeast’ or ‘preferred sake yeast’, is used, the method of producing a beverage, such as a cider, wherein the fermentation is performed until completion. In some embodiments, said fermentation is a dry fermentation.

[0060] In some aspects of the present invention is also provided a method for producing a beverage, said method comprising;

[0061] i) providing a beverage base for fermentation, and

[0062] ii) providing a sake yeast strain of the species Saccharomyces cerevisiae, wherein said yeast strain comprises

[0063] a) a gene encoding BIO1 of SEQ ID NO: 16 or SEQ ID NO: 21, or a polypeptide sharing at least 70% sequence identity with SEQ ID NO:s 16 or 21, such as at least 80% sequence identity therewith, such as at least 90% sequence identity with SEQ ID NO:s 16 or 21, such as at least 95% sequence identity with SEQ ID NO:s 16 or 21, such as at least 98% sequence identity with SEQ ID NO:s 16 or 21, wherein said gene encoding BIO1 is expressed from its endogenous promoter, and;

[0064] b) a gene encoding BIO6 of SEQ ID NO: 17 or 22, or a polypeptide sharing at least 70% sequence identity with SEQ ID NO:s 17 or 22, such as at least 80% sequence identity with SEQ ID NO:s 17 or 22, such as at least 90% sequence identity with SEQ ID NO:s 17 or 22, such as at least 95% sequence identity with SEQ ID NO:s 17 or 22, such as at least 98% sequence identity with SEQ ID NO:s 17 or 22, wherein said gene encoding BIO6 is expressed from its endogenous promoter,

[0065] iii) fermenting the beverage base provided in step i) with said yeast strain of step ii), iv) thereby obtaining a fermented beverage base,

[0066] v) processing the fermented beverage base into a beverage. Preferred method of producing beverages (stopped fermentation)

[0067] The length of fermentation can be managed as a way of managing the residual sugars and maximum ABV (%) content in the finished beverage or cider, which contributes to an agreeable taste. Stopped fermentation is a preferred method due to the high levels of residual sugar and lower ABV (%) in the resultant beverage or cider, and can be achieved by techniques such as rapidly cooling the fermentation broth.

[0068] The present invention relates to methods of producing a beverage, such as a cider, preferred methods as related to the invention are described in this section. In one aspect of the invention, fermentation is stopped when the fermented beverage or cider base has an ABV in the range of 8 to 17%. In one embodiment of the invention, fermentation is stopped when the fermented beverage or cider base has an ABV in the range of 8 to 16%. In one embodiment of the invention, fermentation is stopped when the fermented beverage or cider base has an ABV in the range of 9 to 15%. In one embodiment of the invention, fermentation is stopped when the fermented beverage or cider base has an ABV in the range of 9 to 14%. It may be preferred that fermentation is stopped when the fermented beverage or cider base has an ABV in the range of 9 to 13%. It may also be preferred that fermentation is stopped when the fermented beverage or cider base has an ABV in the range of 10 to 13%. It may also be preferred that fermentation is stopped when the fermented beverage or cider base has an ABV in the range of 10 to 12%. It may also be preferred that fermentation is stopped when the fermented beverage or cider base has an ABV in the range of 10 to 11%. In one aspect of the invention, fermentation is stopped when the fermented beverage or cider base has a maximum ABV of 12.5%. The final beverage or cider may be prepared from the fermented beverage or cider base by adding blending liquids and / or compounds. In particular, the alcohol content of the final beverage or cider may be adjusted by adding water to the fermented beverage or cider base. In some embodiments, the fermentation is allowed to complete.

[0069] In one aspect of the invention, fermentation is stopped when the fermented beverage or cider base has a specific gravity in the range of 0.5 to 5° Plato. It may be preferred that fermentation is stopped when the fermented beverage or cider base has a specific gravity in the range of 0.5 to 4.5°Plato. It may also be preferred that fermentation is stopped when the fermented beverage or cider base has a specific gravity in the range of 0.5 to 4°Plato. It may also be preferred that fermentation is stopped when the fermented beverage or cider base has a specific gravity in the range of 0.7 to 3.8 Plato. In one aspect of the invention, fermentation is stopped when the fermented beverage or cider base has a specific gravity of at least 0.7° Plato.

[0070] In one aspect of the invention, fermentation is stopped when the fermented beverage or cider base has a concentration of remaining fermentable sugars in the range of 10-60g / L. It may be preferred that fermentation is stopped when the fermented beverage or cider base has a concentration of remaining fermentable sugars in the range of 15-55g / L. It may also be preferred that fermentation is stopped when the fermented beverage or cider base has a concentration of remaining fermentable sugars in the range of 15-50g / L. It may also be preferred that fermentation is stopped when the fermented beverage or cider base has a concentration of remaining fermentable sugars in the range of 20-50g / L. In one aspect of the invention, fermentation is stopped when the fermented beverage or cider base has a concentration of remaining fermentable sugars of at least 20g / L.

[0071] In one aspect of the invention, fermentation is performed at a temperature in the range of 15 to 30°C. It may be preferred that fermentation is performed at a temperature in the range of 17 to 25°C. In some embodiments, fermentation is performed at a temperature in the range of 19 to 23°C.

[0072] Beverage base

[0073] The present invention provides methods for producing beverages, such as cider, as aforementioned in section ‘methods for producing a beverage’, by fermentation of a beverage or cider base. It may be preferred that the beverage or cider base comprises apple juice or apple juice concentrate diluted with water. In addition, the beverage or cider base may comprise sugars, preferably sucrose. If the beverage being produced is a malt-based beverage, such as a beer, the beverage base may comprise or consist of an extract of malt, such as an aqueous extract of month. In some embodiments, the beverage base is a non-sake beverage base.

[0074] It is may also preferred that the beverage or cider base has a specific gravity in the range of 15 to 30°Plato, or more preferably in the range of 20 to 28°Plato, or more preferably in the range of 20 to 25° Plato. It may also be preferred that the beverage or cider base has a specific gravity in the range of 15 to 30°Bx, or more preferably in the range of 20 to 28 Bx, or more preferably in the range of 20 to 25 Bx, for example in the range of 23 to 24° Bx.

[0075] Including the preferred concentrations of fermentable sugars within the beverage or cider base in the aforementioned methods of producing a beverage or cider may be relevant for ensuring the desired sweetness flavour profile of the resultant beverage or cider. For example, inclusion of sucrose into the cider base may be preferable, because following fermentation, the resultant fermented beverage or cider base in general comprises higher concentrations of residual fructose. It may be preferred that the beverage or cider comprises high levels of fructose, because fructose provides high levels of perceived sweetness.

[0076] Accordingly, in reference to the aforementioned methods for producing a beverage or cider it may be preferred that the beverage or cider base comprises sucrose. It may also be preferred that the beverage or cider base comprises in the range of 162 to 225 g / L sucrose.

[0077] Further, in reference to the aforementioned methods for producing a beverage or cider, it may be preferred that the beverage or cider base is inoculated with said yeast at a concentration of in the range of 5 to 40 million cells / ml, such as in the range of 15 to 25 million cells / ml.

[0078] In some embodiments, the beverage or cider base is inoculated with said yeast in freeze-dried form.

[0079] Residual sugar concentrations in the fermented cider base

[0080] As aforementioned in the section ‘methods for producing beverages’, the present invention provides methods for a producing a beverage or cider using a sake yeast strain and preferably stopping the fermentation before fermentation is complete. Further, as aforementioned in the previous section ‘preferred method of producing a beverage (stopped fermentation)’, the method of the present invention is preferred due to the high levels of residual sugars, such as fructose, in the resultant beverage or cider which contribute to a desirable sweetness flavour profile. Accordingly, in reference to the aforementioned methods for producing a beverage, such as a cider, it may be preferred that the sake yeast strain is capable of producing a fermented beverage or cider base comprising increased relative average levels of total fermentable sugars such as fructose and / or glucose of at least 180%, such as at least 190%, such as at least 200%, such as at least 210%, such as at least 220%, such as at least 230%, such as at least 240%, such as at least 250%, such as at least 260% such as in the range of 180-500%, such as in the range of 190-400% compared to a reference fermented beverage or cider base prepared in the same manner but fermented with the S. cerevisiae yeast strain; Uvaferm CEG.

[0081] In reference to the aforementioned methods for producing a beverage, such as a cider, it may also be preferred that the sake yeast strain is capable of producing a fermented beverage or cider base comprising increased relative average levels of fructose of at least 210%, such as at least 220%, such as at least 230%, such as at least 240%, such as at least 250%, such as at least 260%, such as at least 270%, such as at least 280%, such as at least 290%, such as at least 300%, such as in the range of 210-500%, such as in the range of 220-450% compared to a reference fermented beverage or cider base prepared in the same manner but fermented with the S. cerevisiae yeast strain; Uvaferm CEG.

[0082] In reference to the aforementioned methods for producing a beverage, such as a cider, it may also be preferred that the sake yeast strain is capable of producing a fermented beverage or cider base comprising increased relative average levels of sorbitol of at least 101%, such as at least 110%, such as at least 120%, such as at least 130%, such as in the range of 101-400%, such as in the range of 101-350% compared to a reference fermented beverage or cider base prepared in the same manner but fermented with the S. cerevisiae yeast strain; Uvaferm CEG.

[0083] Processing beverage or cider base into a beverage

[0084] After fermentation, the fermented beverage or cider base prepared as described herein above may be further processed before arriving at a beverage, such as a cider.

[0085] In one aspect of the invention providing methods of producing a beverage or, such as a cider, said methods comprise the steps of: i) preparing a fermented beverage or cider base as described herein above in the sections “Methods for producing a beverage” and “Preferred method of producing beverages (stopped fermentation)”, and

[0086] ii) further processing said fermented beverage or cider base into a beverage, such as a cider.

[0087] It may be preferred that that the aforementioned methods of producing a beverage, such as cider further comprises one of more of the following steps:

[0088] i) Maturation,

[0089] ii) Blending,

[0090] iii) Stabilisation, or

[0091] iv) Packaging

[0092] It may also be preferred that the method of producing a beverage, such as a cider further comprises blending the fermented beverage or cider base with a blending liquid. In particular, it may be preferred that the method of producing a beverage, such as a cider further comprises blending the fermented beverage or cider base with a blending liquid comprising or consisting of water and / or fruit juice, such as apple juice.

[0093] It may be preferred that the aforementioned method of producing a beverage, such as a cider further comprises blending the fermented beverage or cider base with a blending liquid wherein said blending liquid is added in an amount, so that the mixture of fermented beverage or cider base and blending liquid has an alcohol content in the range of 3 to 8% (vol / vol). It may also be preferred that the method of producing a beverage, such as a cider further comprises blending the fermented beverage or cider base with a blending liquid wherein said blending liquid is added in an amount, so that the mixture of fermented beverage or cider base and blending liquid has an alcohol content in the range of 3 to 6% (vol / vol). It may also be preferred that the method of producing a beverage, such as a cider further comprises blending the fermented beverage or cider base with a blending liquid wherein said blending liquid is added in an amount, so that the mixture of fermented beverage or cider base and blending liquid has an alcohol content in the range of 4 to 5% (vol / vol). The blending liquid may preferably be water.

[0094] In one embodiment of the present invention, no artificial flavours or aromas are added to the beverage or cider base, the blending liquid or to the beverage, such as the cider. In one embodiment of the present invention, no apple juice, apple juice concentrate or apple aromas are added to the blending liquid, to the fermented beverage base or cider base or to the beverage, such as the cider.

[0095] However, in other embodiments, one or more aromas or artificial flavours, such as apple aromas may be added to the blending liquid, to the fermented beverage or cider base or to the beverage, such as the cider. In some embodiments, apple juice, apple juice concentrate or apple aromas are added to the blending liquid, to the fermented beverage or cider base or to the beverage, such as the cider. In some embodiments, apple juice is added to reach a minimum content of 14%, such as 15%, such as 16%, such as 17%, such as 18%, such as 19%, such as 20% apple juice in said beverage or cider. In a specific embodiment, apple juice is added to reach a minimum content of 16% apple juice in said beverage or cider. Any type of juice, aromas or flavours, such as floral or fruity flavours, may be added in the blending liquid.

[0096] In some embodiments, the blending liquid is a mix of water and one or more juices, such as apple juice or pear juice.

[0097] Sake yeast

[0098] A wide variety of yeast can be used to ferment the beverage or cider base. Conventionally, wine yeasts or Saccharomyces yeasts isolated from cider production sites have frequently been used to inoculate yeast in modern cider making.

[0099] The selection of yeast used for beverage, such as cider production is critical to the final quality of the resultant beverage or cider. When evaluating yeast for making beverages, such as ciders, the following characteristics are important to consider: flavour profile, alcohol tolerance, temperature range, nutrient requirements, and flocculation. For example, many beer yeast strains will produce a completely dry cider with low concentrations of residual sugar.

[0100] The present invention provides the use of sake yeast for production of beverages, such as ciders. The sake yeast utilised in the present invention can be any of those disclosed in this section. The present disclosure relates to methods of producing beverages, such as ciders utilising a Sake yeast strain of the species Saccharomyces cerevisiae, that surprisingly produces a beverage, such as a cider with extraordinary good organoleptic properties comprising a more positive perception of fruitiness / fruity aromas, mouthfeel, and naturalness, related to desirable concentrations of flavour compounds. These compounds include reduced levels of undesirable fusel alcohols, increased ester, terpenes and non-volatile organic acid concentrations, as well as increased sweetness due to higher fructose concentrations which results in a reduced calorie content, compared with non-sake yeasts and / or traditional beverage, such as cider production methods. Preferred methods as related to the invention are described in this section.

[0101] The term sake yeast as used herein refers to the type of yeast typically used for production of sake.

[0102] In one aspect of the invention, the sake yeast is a yeast strain, which clusters closer with other sake yeasts, compared to conventional cider yeasts or wine yeasts in a phylogenetic analysis.

[0103] It may be preferred that the sake yeast strain clusters closer with other sake yeasts, compared to conventional cider yeast strain; Uvaferm CEG, in a phylogenetic analysis.

[0104] It may also be preferred that the sake yeast strain clusters closer with other sake yeasts, compared to conventional cider yeast strain; English cider yeast WLP775, in a phylogenetic analysis.

[0105] It may also be preferred that the yeast strain is phylogenetically distant from wine, bread, ale and / or Kveik yeast strains.

[0106] It may also be preferred that the sake yeast strain clusters with sake yeast strain; NCYC 479, in a phylogenetic analysis.

[0107] It may also be preferred that the sake yeast strain clusters with sake yeast strain; PPU4512 in a phylogenetic analysis. It may also be preferred that the sake yeast strain clusters with sake yeast strain; PPU4514 in a phylogenetic analysis.

[0108] The phylogenetic analysis may be performed by any conventional method known to the skilled person. Preferably, the phylogenetic analysis is performed as disclosed in the ‘Phylogeny analysis’ section within the examples of the present application.

[0109] Consumer interest towards alcoholic beverages produced by the yeast species Saccharomyces cerevisiae, such as cider, is mainly linked to their production of a desirable complex aroma. Many chemical components are involved in these aromas, including esters, terpenes and organic acids. The synthesis of said compounds is under the control of various genes, such as those disclosed below.

[0110] OYE2 and OYE3 are the two isoenzymes of Old Yellow Enzyme, their physiological roles in yeast may include detoxification of unsaturated metabolites (Odat etal., 2007). Terpenes provide a positive perception of floral and fruity aromas of a cider.

[0111] It may be preferred that the yeast strain useful in the aforementioned methods for producing beverages, such as ciders comprises an OYE3 gene encoding full length OYE3 protein, preferably OYE3 of SEQ ID NO: 1 or a functional homologue thereof. In some embodiments, of the above method, said yeast strain does not comprise an OYE3 gene comprising a frame shift mutation, for example the yeast strain does not comprise an OYE3 gene encoding a Glu259 frame shift mutation, corresponding to amino acid position 259 of SEQ ID NO: 2. In some embodiments, of the above method, said yeast strain comprises an OYE3 gene, which does not encode an OYE3 protein comprising a Val114lle mutation, corresponding to amino acid position 114 of SEQ ID NO: 2 or SEQ ID NO: 18. In some embodiments, of the above method, said yeast strain comprises an OYE3 gene encoding an lle182Thr mutation, preferably OYE3 of SEQ ID NO: 3. The OYE3 gene is preferably of SEQ ID NO: 1 or a functional homologue thereof, and the aforementioned mutation are mutations of OYE3 of SEQ ID NO: 2 or SEQ ID NO: 18.

[0112] Thus, a sake yeast as used herein may in some embodiments be defined as a yeast strain expressing a full-length OYE3 protein. A sake yeast strain may also be defined as a yeast strain that does not comprise a frameshift mutation in the OYE3 gene that causes a C-terminal truncation of the encoded OYE3 protein compared to the wild type OYE3 protein, preferably wherein said wild type OYE3 protein is a polypeptide of SEQ ID NO: 2 or SEQ ID NO: 18, or a polypeptide sharing at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 98% sequence identity with SEQ ID NO: 2. In particular, it is preferable that the sake yeast strain expresses an OYE3 protein comprising the amino acid sequence from position 259 to 400 of SEQ ID NO: 2 or SEQ ID NO: 18.

[0113] It may be preferred that the yeast strain useful in the aforementioned methods for producing beverages, such as ciders comprises an OYE2 gene, preferably of SEQ ID NO: 4, encoding full length OYE2 protein, preferably of SEQ ID NO: 5 or a polypeptide sharing at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 98% sequence identity with SEQ ID NO: 5. In some embodiments, of the above method, said yeast strain carries an Ala59Ser and / or a Ser193Gly mutation in the gene encoding OYE2. In some embodiments, of the above method, said yeast strain carries an Ala59Ser mutation in the gene encoding OYE2, preferably OYE2 of SEQ ID NO: 6. In some embodiments, of the above method, said yeast strain carries an Ala59Ser mutation in the gene encoding OYE2, preferably OYE2 of SEQ ID NO: 7. OYE2 is preferably OYE2 of SEQ ID NO: 4, and the aforementioned mutations are mutations of OYE2 of SEQ ID NO: 5.

[0114] The genes ERG20 and ATF1 both play a role in the terpene synthesis signalling pathway in the yeast species Saccharomyces cerevisiae.

[0115] Accordingly, it may be preferred that the yeast strain carries a Lys290Asn mutation in the gene encoding ERG20, preferably ERG20 of SEQ ID NO: 8.

[0116] It may also be preferred that the yeast strain carries a Thr84lle mutation in the gene encoding ATF1, preferably ATF1 of SEQ ID NO: 9 or SEQ ID NO: 19. Said ATF1 may also be a functional homologue of ATF1 of SEQ ID NO: 9 or SEQ ID NO: 19 sharing at least 80%, such as at least 85%, such as at least 90%, such as at least 95% sequence identity to SEQ ID NO: 9 or 19, with the proviso that said functional homologue carries said Thr84lle mutation. Said mutant ATF1 may have lower or abrogated enzymatic activity compared to the wildtype ATF1 not comprising said Thr84lle mutation. Without being bound by theory, a yeast strain, such as a sake yeast strain, expressing ATF1 of SEQ ID NO: 9 may produce less acetate esters, such as less isoamyl acetate and 2- phenylethyl acetate, than a yeast strain, such as a cider yeast strain, expressing wild type ATF1.

[0117] It may be preferred that the yeast strain carries a Lys54STOP mutation in the gene encoding FDC1, corresponding to amino acid position 54 of SEQ ID NO: 10. It may therefore be preferred that the yeast strain comprises an FDC1 gene encoding an FDC1 protein as set forth in SEQ ID NO: 20 or a polypeptide with at least 80%, such as at least 85%, such as at least 90%, such as at least 95% sequence identity to SEQ ID NO: 20.

[0118] It may also be preferred that the yeast strain carries a Ser116Asn mutation in the gene encoding FAS2, preferably FAS2 of SEQ ID NO: 12.

[0119] It may also be preferred that the yeast strain carries a Val18Met mutation in the gene encoding PEX22, preferably PEX22 of SEQ ID NO: 14.

[0120] It may be preferred that the yeast strain does not comprise an FAS2 gene comprising a mutation selected from Ser565Asn, corresponding to amino acid position 564 of SEQ ID NO: 11, Glu1475Lys, corresponding to amino acid position 1475 of SEQ ID NO: 11 and / or Ser1800Asn, corresponding to amino acid position 1800 of SEQ ID NO: 11.

[0121] It may also be preferred that the yeast strain does not comprise a VID24 gene comprising a mutation selected from Phe209Tyr, corresponding to amino acid position 209 of SEQ ID NO: 15, and / or Arg124Lys, corresponding to amino acid position 124 of SEQ ID NO: 15.

[0122] It may also be preferred that the yeast strain does not comprise a PEX22 gene comprising a mutation selected from Gly103Glu, corresponding to amino acid position 103 of SEQ ID NO: 13, Arg129Lys, corresponding to amino acid position 129 of SEQ ID NO: 13 and / or Val170lle, corresponding to amino acid position 170 of SEQ ID NO: 13.

[0123] It may be preferred that the sake yeast strain useful in the aforementioned methods for producing a beverage, such as a cider is a Kyokai 7, Kyokai 6, or Kyokai 9. It may be preferred that the sake yeast strain useful in the aforementioned methods for producing a beverage, such as a cider is a Kyokai 7. It may also be preferred that the sake yeast strain is a Kyokai 6. It may also be preferred that the sake yeast strain is a Kyokai 9. It may also be preferred that the sake yeast strain clusters closer with other Kyokai 7 yeasts in a phylogenetic analysis.

[0124] The genes; BIO1 and BIO6, are involved in the synthesis of biotin, a water-soluble vitamin which is significant for yeast metabolism. It may be preferred that the yeast strain of the present invention, which is useful in the aforementioned methods for producing cider comprises a gene encoding BIO1 of SEQ ID NO: 16 or SEQ ID NO: 21, or a polypeptide sharing at least 70% sequence identity with SEQ ID NO:s 16 or 21, such as at least 80% sequence identity with SEQ ID NO:s 16 or 21, such as at least 90% sequence identity therewith, such as at least 95% sequence identity with SEQ ID NO:s 16 or 21, such as at least 98% sequence identity with SEQ ID NO:s 16 or 21. It may also be preferred that the sake yeast strain comprises a gene encoding BIO6 of SEQ ID NO: 17 or SEQ ID NO: 22 or a polypeptide sharing at least 70% sequence identity with SEQ ID NO:s 17 or 22, such as at least 80% sequence identity with SEQ ID NO:s 17 or 22, such as at least 90% sequence identity with SEQ ID NO:s 17 or 22, such as at least 95% sequence identity with SEQ ID NO:s 17 or 22, such as at least 98% sequence identity with SEQ ID NO:s 17 or 22.

[0125] It is preferred that the genes encoding BIO1 and / or BIO6 are endogenous genes to the sake yeast strain as described herein, i.e. that BIO1 and / or BIO6 are not heterologous genes and that the expressed BIO1 and / or BIO6 proteins are not heterologous proteins. Thus, in preferred embodiments, said gene(s) encoding BIO1 and / or BIO6 are expressed from their endogenous promoters. In some embodiments, the genes encoding BIO1 and BIO6 are expressed from their endogenous promoters.

[0126] Flavour profile

[0127] Consumer interest towards alcoholic beverages produced by the yeast species Saccharomyces cerevisiae, such as cider, is mainly linked to their production of a desirable complex aroma and thus, flavour profile. The present invention relates to methods of producing beverages, such as cider as described above, wherein in one aspect, fermentation of the beverage or cider base with said yeast strain improves the flavour profile of said beverage, such as said cider compared to a beverage, such as a cider produced by fermenting said beverage or cider base with a conventional used for beverage production, such as a cider yeast. In another aspect of the invention, fermentation of the beverage or cider base with said yeast strain enhances the positive perception of any one of the following flavour characteristics of said cider: fruitiness, mouthfeel, floral, fragrant, sweetness, naturalness and / or fullness.

[0128] Fusel alcohols and volatile acids

[0129] As aforementioned in previous sections, high levels of fusel alcohols and volatile acids in a beverage such as a cider are negatively perceived due to a harsh finish and aftertaste, and therefore it is desirable to utilise a yeast useful in methods for producing beverages, such as ciders with the capacity to produce lower levels of said compounds. The preferred yeast as related to the present invention can be any as described in this section, and said yeast can further have any genotype as described in the section ‘sake yeast’ or ‘preferred sake yeast’.

[0130] Accordingly, in one aspect of the present invention, the yeast strain useful in the aforementioned methods for producing beverages, such as ciders is capable of producing a fermented beverage or cider base comprising decreased average levels of one or more of the following compound(s) compared to a reference fermented beverage or cider base prepared in the same manner but fermented with the S. cerevisiae yeast strain; Uvaferm CEG; wherein said compounds are decanoic acid, isobutanol, and / or total fusel alcohols such as butanol, 2-methyl butanol and / or 3-methyl butanol.

[0131] It may be preferred that the sake yeast strain is capable of producing a fermented beverage or cider base comprising a relative concentration of at the most 90%, such as at the most 80%, such as at the most 70%, such as at the most 60%, such as at the most 50%, such as at the most 40%, such as at the most 30%, such as in the range of 0-90%, such as in the range of 0-80% of isobutanol compared to a reference fermented beverage or cider base prepared in the same manner but fermented with the S. cerevisiae yeast strain; Uvaferm CEG.

[0132] It may be preferred that the sake yeast strain is capable of producing a fermented beverage or cider base comprising a relative concentration of at the most 80%, such as at the most 70%, such as at the most 60%, such as at the most 50%, such as at the most 40%, such as at the most 30%, such as in the range of 0-80%, such as in the range of 0-70% of decanoic acid compared to a reference fermented beverage or cider base prepared in the same manner but fermented with the S. cerevisiae yeast strain; Uvaferm CEG.

[0133] It may be preferred that the sake yeast strain is capable of producing a fermented beverage or cider base comprising a relative concentration of at the most 90%, such as at the most 80%, such as at the most 70%, such as at the most 60%, such as at the most 50%, such as at the most 40%, such as in the range of 0-90%, such as in the range of 0-85% of total fusel alcohols compared to a reference fermented beverage or cider base prepared in the same manner but fermented with the S. cerevisiae yeast strain; Uvaferm CEG.

[0134] It may be preferred that the sake yeast strain is capable of producing a fermented beverage or cider base comprising a relative concentration of at the most 90%, such as at the most 80%, such as at the most 70%, such as at the most 60%, such as at the most 50%, such as at the most 40%, such as in the range of 0-90%, such as in the range of 0-85% of total fusel alcohols such as isobutanol, butanol, 2-methyl butanol and / or 3-methyl butanol compared to a reference fermented beverage or cider base prepared in the same manner but fermented with the S. cerevisiae yeast strain; Uvaferm CEG.

[0135] Esters, Terpenes and non-volatile organic acids

[0136] As aforementioned in previous sections, high levels of esters, terpenes and non-volatile organic acids, such as Malate, in a beverage such as a cider are positively perceived and therefore it is desirable to utilise a yeast useful in methods for producing beverages, such as ciders with the capacity to produce higher levels of said compounds. The preferred yeast as related to the present invention can be any as described in this section, and said yeast can further have any genotype as described in section ‘sake yeast’ or ‘preferred sake yeast’.

[0137] Accordingly, in one aspect of the present invention, the yeast strain useful in the aforementioned methods for producing beverages, such as ciders is capable of producing a fermented beverage or cider base comprising increased average levels of one or more of the following compound(s) compared to a reference fermented beverage or cider base prepared in the same manner but fermented with the S. cerevisiae yeast strain; Uvaferm CEG; wherein said compounds are ethyl 3-hydroxybutanoate, total levels of terpenes such as linalol, nerol, geraniol, citronellol and / or alpha terpineol, total levels of nerol, gernamol and citronellol, nerol, geraniol, citronellol, total fermentable sugars such as fructose and / or glucose, sorbitol, and / or malate.

[0138] Esters

[0139] Esters are perceived as fruity aromas or flavours, and may include apple, banana, pear, grape, strawberry, citrus, melon, or others flavours. As such, they crucially contribute to a significant proportion of the flavour profile added by yeast to a beverage such as cider, and vary enormously by yeast strain in variety and concentrations produced. Therefore it is desirable to utilise a yeast useful in methods for producing beverages, such as ciders with the capacity to produce higher levels of esters. The preferred yeast as related to the present invention can be any as described in this section, and said yeast can further have any genotype as described in section ‘sake yeast’ or ‘preferred sake yeast’.

[0140] It may be preferred that the yeast strain useful in the aforementioned methods for producing beverages, such as ciders is capable of producing a fermented beverage or cider base comprising decreased average levels of 2-phenylethyl acetate compared to a reference fermented beverage or cider base prepared in the same manner but fermented with the S. cerevisiae yeast strain; Uvaferm CEG. It may also be preferred that said sake yeast strain is capable of producing a fermented beverage or cider base comprising a relative concentration of at the most 90%, such as at the most 75%, such as at the most 50%, such as at the most 20%, such as in the range of 0-90%, such as in the range of 0-80% of 2-phenylethyl acetate compared to a reference fermented beverage or cider base prepared in the same manner but fermented with the S. cerevisiae yeast strain; Uvaferm CEG.

[0141] It may be preferred that said sake yeast strain useful in the aforementioned methods for producing beverages, such as ciders is capable of producing a fermented beverage or cider base comprising increased average levels of ethyl 3-hydroxybutanoate of at least 125%, such as at least 150%, such as at least 175%, such as at least 200%, such as at least 225%, such as at least 250%, such as at least 275%, such as at least 300%, such as in the range of 125-500%, such as in the range of 150-400% compared to a reference fermented beverage or cider base prepared in the same manner but fermented with the S. cerevisiae yeast strain; Uvaferm CEG. It may be preferred that said sake yeast strain useful in the aforementioned methods for producing beverages such as cider is capable of producing a fermented beverage or cider base comprising increased average levels of ethyl hexanoate of at least 120%, such as at least 135%, such as at least 150%, such as at least 175%, such as at least 200%, such as at least 225%, such as at least 250%, such as in the range of 130-400%, such as in the range of 140-300% compared to a reference fermented beverage or cider base prepared in the same manner but fermented with the S. cerevisiae yeast strain; Uvaferm CEG.

[0142] It may be preferred that said sake yeast strain useful in the aforementioned methods for producing beverages, such as cider is capable of producing a fermented beverage or cider base comprising increased average levels of ethyl octanoate of at least 110%, such as at least 125%, such as at least 150%, such as at least 175%, such as at least 200%, such as in the range of 110-350% compared to a reference fermented beverage or cider base prepared in the same manner but fermented with the S. cerevisiae yeast strain; Uvaferm CEG.

[0143] Terpenes

[0144] Terpenes provide a positive perception of floral and fruity aromas of a beverage, such as a cider, either directly or through synergistic effects. As such, they crucially contribute to a significant proportion of the flavour profile added by yeast to beverages, such as cider, and vary enormously by yeast strain in variety and concentrations produced. Therefore it is desirable to utilise a yeast useful in methods for producing beverages, such as ciders with the capacity to produce higher levels of terpenes. The preferred yeast as related to the present invention can be any as described in this section, and said yeast can further have any genotype as described in section ‘sake yeast’ or ‘preferred sake yeast’.

[0145] Accordingly, it may be preferred that the yeast strain useful in the aforementioned methods for producing beverage, such as ciders is capable of producing a fermented beverage or cider base comprising increased relative average levels of total levels of terpenes of at least 105%, such as at least 110%, such as at least 120%, such as at least 130%, such as at least 140%, such as in the range of 105-350%, such as in the range of 105-300% compared to a reference fermented beverage or cider base prepared in the same manner but fermented with the S. cerevisiae yeast strain; Uvaferm CEG. It may also be preferred that the yeast strain useful in the aforementioned methods for producing beverages, such as ciders is capable of producing a fermented beverage or cider base comprising increased relative average levels of total levels of terpenes such as linalol, nerol, geraniol, citronellol and / or alpha terpineol of at least 105%, such as at least 110%, such as at least 120%, such as at least 130%, such as at least 140%, such as in the range of 105-350%, such as in the range of 105-300% compared to a reference fermented beverage or cider base prepared in the same manner but fermented with the S. cerevisiae yeast strain; Uvaferm CEG.

[0146] It may also be preferred that the yeast strain useful in the aforementioned methods for producing beverages, such as ciders is capable of producing a fermented beverage or cider base comprising increased relative average levels of total levels of nerol, geraniol and citronellol of at least 120%, such as at least 130%, such as at least 140%, such as at least 150%, such as at least 160%, such as at least 170%, such as at least 180%, such as in the range of 120-400%, such as in the range of 120-300% compared to a reference fermented beverage or cider base prepared in the same manner but fermented with the S. cerevisiae yeast strain; Uvaferm CEG.

[0147] It may also be preferred that the yeast strain useful in the aforementioned methods for producing beverages, such as ciders is capable of producing a fermented beverage or cider base comprising increased relative average levels of terpenes selected from the group of nerol, geraniol, and / or citronellol of at least 120%, such as at least 130%, such as at least 140%, such as at least 150%, such as at least 160%, such as at least 170%, such as at least 180%, such as in the range of 120-400%, such as in the range of 120-300% compared to a reference fermented beverage or cider base prepared in the same manner but fermented with the S. cerevisiae yeast strain; Uvaferm CEG

[0148] It may also be preferred that the yeast strain useful in the aforementioned methods for producing beverages, such as ciders is capable of producing a fermented beverage or cider base comprising increased relative average levels of nerol of at least 110%, such as at least 120%, such as at least 130%, such as at least 140%, such as at least 150%, such as at least 160%, such as in the range of 110-400%, such as in the range of 110-300% compared to a reference fermented beverage or cider base prepared in the same manner but fermented with the S. cerevisiae yeast strain; Uvaferm CEG. It may also be preferred that the yeast strain useful in the aforementioned methods for producing beverages, such as ciders is capable of producing a fermented beverage or cider base comprising increased relative average levels of geraniol of at least 101%, such as at least 110%, such as at least 120%, such as at least 130%, such as at least 140%, such as at least 150%, such as in the range of 101-350%, such as in the range of 110-400% compared to a reference fermented beverage or cider base prepared in the same manner but fermented with the S. cerevisiae yeast strain; Uvaferm CEG.

[0149] It may also be preferred that the yeast strain useful in the aforementioned methods for producing beverages, such as ciders is capable of producing a fermented beverage or cider base comprising increased relative average levels of citronellol of at least 140%, such as at least 150%, such as at least 160%, such as at least 170%, such as at least 180%, such as at least 190%, such as at least 200%, such as at least 210%, such as in the range of 140-400%, such as in the range of 150-350% compared to a reference fermented beverage or cider base prepared in the same manner but fermented with the S. cerevisiae yeast strain; Uvaferm CEG.

[0150] Non-volatile organic acids

[0151] As aforementioned in previous sections, non-volatile organic acids are positively perceived in the complex aroma profile of a cider. For example, a higher concentration of the non-volatile, organic acid; malate, is crucial in providing the sensation of freshness and / or naturalness (Negoro et al. 2022; Kitagaki et al. 2013). Therefore it is desirable to utilise a yeast useful in methods for producing beverages, such as cider with the capacity to produce higher levels of non-volatile organic acids. The preferred yeast as related to the present invention can be any as described in this section, and said yeast can further have any genotype as described in section ‘sake yeast’ or ‘preferred sake yeast’.

[0152] Accordingly, it may also be preferred that the yeast strain useful in the aforementioned methods for producing beverages, such as ciders is capable of producing a fermented beverage or cider base comprising increased relative average levels of malate of at least 101%, such as at least 110%, such as at least 120%, such as at least 130%, such as at least 140%, such as at least 150%, such as in the range of 100-400%, such as in the range of 100-350% compared to a reference fermented beverage or cider base prepared in the same manner but fermented with the S. cerevisiae yeast strain; Uvaferm CEG. Relative concentrations of flavour compounds

[0153] As aforementioned in previous sections, the desirable complex aroma and thus flavour profile of a beverage, such as a cider with extraordinary good organoleptic properties is dependent on the delicate balance between the various above-mentioned flavour compounds produced during fermentation. A higher concentration of positively perceived flavour compounds, such as terpenes or esters, relative to the concentration of negatively perceived flavour compounds, such as fusel alcohols, will result in a beverage, such as a cider with more positively perceived organoleptic properties. Therefore it is desirable to utilise a yeast useful in methods for producing beverages, such as ciders which has the capacity to produce said relative concentrations of flavour compounds. The preferred yeast as related to the present invention can be any as described in this section, and said yeast can further have any genotype as described in section ‘sake yeast’ or ‘preferred sake yeast’.

[0154] Accordingly, it may be preferred that the yeast strain useful in the aforementioned methods for producing a beverage, such as a cider is capable of producing a fermented beverage or cider base comprising a ratio of the concentrations of at least 0.75, such as at least 0.8, such as at least 0.9, such as at least 1, such as at least 1.1, such as at least 1.2, such as in the range of 0.75-3, such as in the range of 0.75-2.5 of citronellol vs geraniol.

[0155] It may also be preferred that the yeast strain useful in the aforementioned methods for producing beverages, such as ciders is capable of producing a beverage or fermented cider base comprising a ratio of the concentrations of at least 0.2, such as at least 0.3, such as at least 0.4, such as at least 0.5, such as in the range of 0.2-2, such as in the range of 0.3-1.5 of citronellol vs total levels of terpenes.

[0156] It may also be preferred that the yeast strain useful in the aforementioned methods for producing beverages, such as ciders is capable of producing a beverage or fermented cider base comprising a ratio of the concentrations of one or more of the following compounds; at least 0.0003, such as at least 0.0004, such as at least 0.0005, such as at least 0.0006, such as at least 0.0007, such as in the range of 0-1, such as in the range of 0-0.05 of the total levels of terpenes, such as linalol, nerol, geraniol, citronellol and alpha terpineol, vs total levels of fusel alcohols. It may also be preferred that the yeast strain useful in the aforementioned methods for producing beverages, such as ciders is capable of producing a beverage or fermented cider base comprising a ratio of the concentration of at least one of the following compounds; at least 0.01, such as at least 0.012, such as at least 0.014, such as at least 0.016, such as at least 0.018, such as at least 0.02, such as at least 0.04, such as in the range of 0-1, such as in the range of 0-0.075 of the total levels of mid-chain fatty acid esters, such as ethyl decanoate, ethyl hexanoate, ethyl octanoate and / or ethyl butanoate, vs total levels of fusel alcohols.

[0157] Preferred sake yeast

[0158] As aforementioned in the section ‘sake yeast’, the selection of yeast used for beverage, such as cider, production is critical to the final quality of the resultant beverage, such as cider. Further, the chemical components produced by yeast during the fermentation process are fundamental to the desirable complex aroma and flavour profile of the resultant beverage, such as cider. The preferred yeast as related to the present invention can be any as described in this section.

[0159] In one aspect of the invention, the sake yeast strain of the invention is of the species Saccharomyces cerevisiae, wherein said yeast comprises;

[0160] a gene encoding BIO1 of SEQ ID NO: 16 or SEQ ID NO: 21, or a polypeptide sharing at least 70% sequence identity therewith, such as at least 80% sequence identity with SEQ ID NO:s 16 or 21, such as at least 90% sequence identity with SEQ ID NO:s 16 or 21, such as at least 95% sequence identity with SEQ ID NO:s 16 or 21, such as at least 98% sequence identity with SEQ ID NO:s 16 or 21 and / or

[0161] a gene encoding BIO6 of SEQ ID NO: 17 or SEQ ID NO: 22, or a polypeptide sharing at least 70% sequence identity with SEQ ID NO:s 17 or 22, such as at least 80% sequence identity with SEQ ID NO:s 17 or 22, such as at least 90% sequence identity with SEQ ID NO:s 17 or 22, such as at least 95% sequence identity with SEQ ID NO:s 17 or 22, such as at least 98% sequence identity with SEQ ID NO:s 17 or 22.

[0162] In one aspect of the invention, the sake yeast strain of the invention is of the species Saccharomyces cerevisiae, wherein said yeast comprises;

[0163] a gene encoding BIO1 of SEQ ID NO: 16 or SEQ ID NO: 21, or a polypeptide sharing at least 70% sequence identity with SEQ ID NO:s 16 or 21, such as at least 80% sequence identity therewith, such as at least 90% sequence identity with SEQ ID NO:s 16 or 21, such as at least 95% sequence identity with SEQ ID NO:s 16 or 21, such as at least 98% sequence identity with SEQ ID NO:s 16 or 21, wherein said gene encoding BIO1 is expressed from its endogenous promoter, and / or

[0164] a gene encoding BIO6 of SEQ ID NO: 17 or 22, or a polypeptide sharing at least 70% sequence identity with SEQ ID NO:s 17 or 22, such as at least 80% sequence identity with SEQ ID NO:s 17 or 22, such as at least 90% sequence identity with SEQ ID NO:s 17 or 22, such as at least 95% sequence identity with SEQ ID NO:s 17 or 22, such as at least 98% sequence identity with SEQ ID NO:s 17 or 22, wherein said gene encoding BIO6 is expressed from its endogenous promoter.

[0165] The preferred sake yeast comprising said genes, preferably is capable of producing a fermented beverage or cider base as described below in the sections “Preferred sake yeast - Fusel alcohols & volatile acids”, “Preferred sake yeast – Esters, Terpenes and non-volatile acids”, “Preferred sake yeast – Esters”, “Preferred sake yeast - Terpenes”, “Preferred sake yeast - non-volatile organic acids”.

[0166] Preferred sake yeast – Esters, Terpenes and non-volatile organic acids

[0167] As aforementioned in previous sections, high levels of esters, terpenes and organic acids, such as malate, in a beverage such as a cider are positively perceived and therefore it is desirable to utilise a yeast useful in methods for producing beverages, such as ciders with the capacity to produce higher levels of said compounds. The preferred yeast as related to the present invention can be any as described in this section, and said yeast can further have any genotype as described in section ‘sake yeast’ or ‘preferred sake yeast’.

[0168] Accordingly, it is preferred that the sake yeast strain is capable of producing a fermented beverage or cider base comprising increased average levels of one or more of the following compound(s) compared to a reference fermented beverage or cider base prepared in the same manner but fermented with the Kyokai yeast strain yeast strain; NCYC 479; wherein said compounds are isoamyl acetate, 2-phenylethyl acetate, ethyl hexanoate, ethyl octanoate, ethyl butanoate, ethyl 2-hydroxyisocaproate, total levels of medium-chain fatty acid esters such as ethyl decanoate, ethyl hexanoate, ethyl octanoate, and / or ethyl butanoate, citronellol, butanol, total levels of 2-methyl butanol and 3-methyl butanol, butanoic acid, hexanoic acid, total levels of fusel alcohols such as isobutanol, butanol, 2-methyl butanol and / or 3-methyl butanol, malate, and / or total levels of medium-chain fatty acid esters, such as ethyl decanoate, ethyl hexanoate, ethyl octanoate and / or ethyl butanoate, and terpenes, such as linalol, nerol, geraniol, citronellol and alpha terpineol.

[0169] Preferred sake yeast – Esters

[0170] As aforementioned in the ‘Esters’ section above, Esters are as fruity aromas or flavours, and thus contribute to a significant proportion of the flavour profile added by yeast to a beverage, such as a cider. Therefore it is desirable to utilise a yeast useful in methods for producing beverages, such as ciders with the capacity to produce higher levels of esters during fermentation. The preferred yeast as related to the present invention can be any as described in this section, and said yeast can further have any genotype as described in section ‘sake yeast’ or ‘preferred sake yeast’.

[0171] Accordingly, it is also preferred that the sake yeast strain is capable of producing a fermented beverage or cider base comprising increased relative average levels of total levels of medium-chain fatty acid esters, such as ethyl decanoate, ethyl hexanoate, ethyl octanoate and / or ethyl butanoate, and terpenes, such as linalol, nerol, geraniol, citronellol and alpha terpineol, of at least 100%, such as at least 110%, such as at least 120%, such as at least 130%, such as at least 140%, such as at least 150%, such as at least 160%, such as at least 170%, such as in the range of 100-400%, such as in the range of 100-350% compared to a reference fermented beverage or cider base prepared in the same manner but fermented with the Kyokai yeast strain; NCYC 479.

[0172] It is also preferred that the sake yeast strain is capable of producing a fermented beverage or cider base comprising increased relative average levels of isoamyl acetate of at least 130%, such as at least 150%, such as at least 170%, such as at least 190%, such as at least 200%, such as at least 210%, such as in the range of 130-400%, such as in the range of 140-350% compared to a reference fermented beverage or cider base prepared in the same manner but fermented with the Kyokai yeast strain; NCYC 479.

[0173] It is also preferred that the sake yeast strain is capable of producing a fermented beverage or cider base comprising increased relative average levels of 2-phenylethyl acetate of at least 110%, such as at least 120%, such as at least 140%, such as at least 160%, such as at least 180%, such as at least 200%, such as at least 220%, such as in the range of 110-400%, such as in the range of 120-350% compared to a reference fermented beverage or cider base prepared in the same manner but fermented with the Kyokai yeast strain; NCYC 479.

[0174] It is also preferred that the sake yeast strain is capable of producing a fermented beverage or cider base comprising increased relative average levels of ethyl hexanoate of at least 105%, such as at least 110%, such as at least 125%, such as at least 140%, such as at least 150%, such as at least 160%, such as at least 175%, such as in the range of 105-400%, such as in the range of 110-350% compared to a reference fermented beverage or cider base prepared in the same manner but fermented with the Kyokai yeast strain; NCYC 479.

[0175] It is also preferred that the sake yeast strain is capable of producing a fermented beverage or cider base comprising increased relative average levels of ethyl octanoate of at least 110%, such as at least 125%, such as at least 140%, such as at least 150%, such as at least 160%, such as at least 175%, such as in the range of 110-400%, such as in the range of 110-350% compared to a reference fermented beverage or cider base prepared in the same manner but fermented with the Kyokai yeast strain; NCYC 479.

[0176] It is also preferred that the sake yeast strain is capable of producing a fermented beverage or cider base comprising increased relative average levels of ethyl butanoate of at least 101%, such as at least 140%, such as at least 160%, such as at least 170%, such as at least 190%, such as at least 210%, such as at least 230%, such as in the range of 101-400%, such as in the range of 101-350% compared to a reference fermented beverage or cider base prepared in the same manner but fermented with the Kyokai yeast strain; NCYC 479.

[0177] It is also preferred that the sake yeast strain is capable of producing a fermented beverage or cider base comprising increased relative average levels of ethyl 2-hydroxyisocaproate of at least 101%, such as at least 140%, such as at least 160%, such as at least 170%, such as at least 190%, such as at least 210%, such as at least 230%, such as in the range of 101-400%, such as in the range of 101-350% compared to a reference fermented beverage or cider base prepared in the same manner but fermented with the Kyokai yeast strain; NCYC 479. It is also preferred that the sake yeast strain is capable of producing a fermented beverage or cider base comprising increased relative average levels of total levels of medium-chain fatty acid esters of at least 110%, such as at least 120%, such as at least 130%, such as at least 140%, such as at least 150%, such as at least 160%, such as at least 170%, such as in the range of 110-400%, such as in the range of 120-350% compared to a reference fermented beverage or cider base prepared in the same manner but fermented with the Kyokai yeast strain; NCYC 479.

[0178] It is also preferred that the sake yeast strain is capable of producing a fermented beverage or cider base comprising increased relative average levels of total levels of medium-chain fatty acid esters such as ethyl decanoate, ethyl hexanoate, ethyl octanoate, and / or ethyl butanoate of at least 110%, such as at least 120%, such as at least 130%, such as at least 140%, such as at least 150%, such as at least 160%, such as at least 170%, such as in the range of 110-400%, such as in the range of 120-350% compared to a reference fermented beverage or cider base prepared in the same manner but fermented with the Kyokai yeast strain; NCYC 479.

[0179] It is also preferred that the sake yeast strain is capable of producing a fermented beverage or cider base comprising increased relative average levels of total levels of medium-chain fatty acid esters such as ethyl decanoate, ethyl hexanoate, and / or ethyl octanoate of at least 110%, such as at least 120%, such as at least 130%, such as at least 140%, such as at least 150%, such as at least 160%, such as at least 170%, such as in the range of 110-400%, such as in the range of 120-350% compared to a reference fermented beverage or cider base prepared in the same manner but fermented with the Kyokai yeast strain; NCYC 479.

[0180] It is also preferred that the sake yeast strain is capable of producing a fermented beverage or cider base comprising increased relative average levels of total levels of medium-chain fatty acid esters such as ethyl hexanoate and / or ethyl decanoate of at least 110%, such as at least 120%, such as at least 130%, such as at least 140%, such as at least 150%, such as at least 160%, such as at least 170%, such as in the range of 110-400%, such as in the range of 120-350% compared to a reference fermented beverage or cider base prepared in the same manner but fermented with the Kyokai yeast strain; NCYC 479. Preferred sake yeast - Terpenes

[0181] As above-mentioned in the ‘terpenes’ section - terpenes provide a positive perception of floral and fruity aromas in a beverage, such as a cider, and thus contribute to a significant proportion of the flavour profile added by yeast to a beverage, such as cider. Therefore it is desirable to utilise a yeast useful in methods for producing beverages, such as ciders with the capacity to produce higher levels of terpenes during fermentation. The preferred yeast as related to the present invention can be any as described in this section, and said yeast can further have any genotype as described in section ‘sake yeast’ or ‘preferred sake yeast’.

[0182] Accordingly, it is preferred that the sake yeast strain is capable producing a fermented beverage or cider base comprising increased relative average levels of citronellol of at least 105%, such as at least 110%, such as at least 115%, such as at least 120%, such as at least 125%, such as at least 130%, such as at least 135%, such as at least 140%, such as in the range of 105-400%, such as in the range of 105-350% compared to a reference fermented beverage or cider base prepared in the same manner but fermented with the Kyokai yeast strain; NCYC 479.

[0183] It is preferred that the sake yeast strain is capable of producing a fermented beverage or cider base comprising increased relative average levels of total levels of mediumchain fatty acid esters and terpenes of at least 100%, such as at least 110%, such as at least 120%, such as at least 130%, such as at least 140%, such as at least 150%, such as at least 160%, such as at least 170%, such as in the range of 100-400%, such as in the range of 100-350% compared to a reference fermented beverage or cider base prepared in the same manner but fermented with the Kyokai yeast strain; NCYC 479.

[0184] Preferred sake yeast - Non-volatile organic acids

[0185] As above-mentioned in the ‘non-volatile organic acids’ section - non-volatile organic acids in a beverage such as cider are positively perceived in the complex aroma profile of beverages, such as ciders, and thus contribute to a significant proportion of the flavour profile added by yeast to a beverage, such as cider. Therefore it is desirable to utilise a yeast useful in methods for producing beverages, such as ciders with the capacity to produce higher levels of non-volatile organic acids during fermentation. The preferred yeast as related to the present invention can be any as described in this section, and said yeast can further have any genotype as described in section sake yeast or ‘preferred sake yeast’.

[0186] Accordingly, it is preferred that the sake yeast strain is capable of producing a fermented beverage or cider base comprising increased relative average levels of malate of at least 100%, such as at least 105%, such as at least 110%, such as at least 115%, such as at least 120%, such as at least 125%, such as at least 130%, such as in the range of 100-400%, such as in the range of 100-350% compared to a reference fermented beverage or cider base in the same manner but fermented with the Kyokai yeast strain; NCYC 479.

[0187] Beverages

[0188] In one aspect of the present disclosure is provided a beverage produced by the method as described herein.

[0189] In another aspect of the present disclosure is provided a beverage produced by the sake yeast strain as described herein, e.g. in the sections ‘sake yeast’ or ‘preferred sake yeast’.

[0190] In some embodiments, the beverage is a cider, a perry, a mead, a beer, or a fruit wine, such as apple, pear, cherry or plum wine. In some embodiments, the beverage is a fruit based beverage, such as a cider, a perry, or a fruit wine, such as apple, pear, cherry or plum wine. In some embodiments, the beverage is a malt based beverage, such as beer. In preferred embodiments, the beverage is a cider.

[0191] Items

[0192] 1. A method for producing a fruit or malt based beverage, said method comprising;

[0193] i) providing a beverage base comprising fruit juice or an aqueous extract of malt for fermentation, and

[0194] ii) providing a sake yeast strain of the species Saccharomyces cerevisiae, iii) fermenting the beverage base provided in step i) with said yeast strain of step ii),

[0195] iv) thereby obtaining a fermented beverage base,

[0196] v) processing the fermented beverage base into a fruit based or malt based beverage. 2. The method according to item 1, wherein said fermentation is stopped before fermentation is complete.

[0197] 3. A method for producing a cider, said method comprising;

[0198] i) providing a cider base comprising fruit juice for fermentation, and ii) providing a sake yeast strain of the species Saccharomyces cerevisiae, iii) fermenting the cider base provided in step i) with said yeast strain of step ii),

[0199] iv) stopping fermentation before fermentation is complete,

[0200] v) thereby obtaining a fermented cider base,

[0201] vi) processing the fermented cider base into a cider.

[0202] 4. The method according to any one of the preceding items, wherein the sake yeast strain clusters with other sake yeasts in a phylogenetic analysis.

[0203] 5. The method according to any one of the preceding items, wherein the sake yeast strain clusters closer with other sake yeasts, compared to conventional cider yeasts in a phylogenetic analysis.

[0204] 6. The method according to any one of the preceding items, wherein the sake yeast strain clusters closer with other sake yeasts, compared to conventional cider yeast strain; Uvaferm CEG, in a phylogenetic analysis.

[0205] 7. The method according to any one of the preceding items, wherein the sake yeast strain clusters closer with other sake yeasts, compared to conventional cider yeast strain; English cider yeast WLP775, in a phylogenetic analysis.

[0206] 8. The method according to any one of the preceding items, wherein said yeast strain is phylogenetically distant from wine, bread, ale and / or Kveik yeast strains.

[0207] 9. The method according to any one of the preceding items, wherein the sake yeast strain clusters with sake yeast strain; NCYC 479, in a phylogenetic analysis.

[0208] 10. The method according to any one of the preceding items, wherein the sake yeast strain clusters with sake yeast strain; PPU4512 in a phylogenetic analysis. 11. The method according to any one of the preceding items, wherein the sake yeast strain clusters with sake yeast strain; PPU4514 in a phylogenetic analysis.

[0209] 12. The method according to any one of the preceding items, wherein said yeast strain comprises an OYE3 gene encoding full length OYE3 protein, preferably OYE3 of SEQ ID NO: 2 or SEQ ID NO: 18, or a polypeptide with at least 80%, such as at least 85%, such as at least 90%, such as at least 95% sequence identity to SEQ ID NO:s 2 or 18.

[0210] 13. The method according to any one of the preceding items, wherein said yeast strain does not comprise an OYE3 gene comprising a frame shift mutation, for example the yeast strain does not comprise an OYE3 gene comprising a Glu259 frame shift mutation, corresponding to amino acid position 259 of SEQ ID NO: 2 or SEQ ID NO: 18.

[0211] 14. The method according to any one of the preceding items, wherein said yeast strain comprises an OYE3 gene encoding an OYE3 protein, which does not comprise a Val114lle mutation in the amino acid corresponding to amino acid position 114 of SEQ ID NO: 2 or SEQ ID NO: 18.

[0212] 15. The method according to any one of the preceding items, wherein said yeast strain comprises an OYE3 gene encoding an OYE3 protein comprising an I Ie182Thr mutation, preferably encoding an OYE3 of SEQ ID NO: 3 or SEQ ID NO: 18.

[0213] 16. The method according to any one of the preceding items, wherein said yeast strain comprises an ERG20 gene encoding an ERG20 protein comprising a Lys290Asn mutation, preferably encoding an ERG20 of SEQ ID NO: 8.

[0214] 17. The method according to any one of the preceding items, wherein said yeast strain comprises an ATF1 gene encoding an ATF1 protein comprising a Thr84lle mutation, preferably encoding ATF1 of SEQ ID NO: 9 or SEQ ID NO: 19. 18. The method according to any one of the preceding items, wherein said yeast strain comprises an OYE2 gene encoding full length OYE2 protein, preferably OYE2 of SEQ ID NO: 5.

[0215] 19. The method according to any one of the preceding items, wherein said yeast strain carries comprises an OYE2 gene encoding an OYE2 protein comprising an Ala59Ser and / or a Ser193Gly mutation.

[0216] 20. The method according to any one of the preceding items, wherein said yeast strain comprises an OYE2 gene encoding an OYE2 protein comprising an Ala59Ser mutation, preferably encoding an OYE 2 of SEQ ID NO: 6.

[0217] 21. The method according to any one of the preceding items, wherein said yeast strain comprises an OYE2 gene encoding an OYE2 protein comprising a Ser193Gly mutation, preferably OYE2 of SEQ ID NO: 7.

[0218] 22. The method according to any one of the preceding items, wherein said yeast strain carries a premature stop codon in the gene encoding FDC1.

[0219] 23. The method according to any one of the preceding items, wherein said yeast strain carries a premature stop codon in the gene encoding FDC1, wherein said mutant gene encodes a truncated FDC1 comprising at the most 54 or at the most 53 consecutive amino acids of FDC1 of SEQ ID NO: 10.

[0220] 24. The method according to any one of the preceding items, wherein said yeast strain carries a Lys54STOP mutation in the gene encoding FDC1, corresponding to amino acid position 54 of SEQ ID NO: 10.

[0221] 25. The method according to any one of the preceding items, wherein said yeast strain comprises an FDC1 gene encoding an FDC1 protein as set forth in SEQ ID NO: 20 or a polypeptide with at least 80%, such as at least 85%, such as at least 90%, such as at least 95% sequence identity to SEQ ID NO: 20. 26. The method according to any one of the preceding items, wherein said yeast strain comprises a FAS2 gene encoding a FAS2 protein comprising a Ser116Asn mutation, preferably encoding a FAS2 of SEQ ID NO: 12.

[0222] 27. The method according to any one of the preceding items, wherein said yeast strain comprises a PEX22 gene encoding a PEX22 protein comprising a Val18Met mutation, preferably encoding a PEX22 of SEQ ID NO: 14.

[0223] 28. The method according to any one of the preceding items, wherein said yeast strain does not comprise an FAS2 gene encoding a FAS2 protein comprising a mutation selected from Ser565Asn, corresponding to amino acid position 564 of SEQ ID NO: 11, Glu1475Lys, corresponding to amino acid position 1475 of SEQ ID NO: 11 and / or Ser1800Asn, corresponding to amino acid position 1800 of SEQ ID NO: 11.

[0224] 29. The method according to any one of the preceding items, wherein said yeast strain does not comprise a VID24 gene encoding a VID24 protein comprising a mutation selected from Phe209Tyr, corresponding to amino acid position 209 of SEQ ID NO: 15, and / or Arg124Lys, corresponding to amino acid position 124 of SEQ ID NO: 15.

[0225] 30. It may also be preferred that the yeast strain does not comprise a PEX22 gene encoding a PEX22 protein comprising a mutation selected from Gly103Glu, corresponding to amino acid position 103 of SEQ ID NO: 13, Arg129Lys, corresponding to amino acid position 129 of SEQ ID NO: 13 and / or Val170lle, corresponding to amino acid position 170 of SEQ ID NO: 13.

[0226] 31. The method according to any one of the preceding items, wherein the sake yeast strain is a Kyokai 7, a Kyokai 6 or a Kyokai 9.

[0227] 32. The method any one of the preceding items, wherein the sake yeast strain is a Kyokai 7.

[0228] 33. The method according to any one of the preceding items, wherein the sake yeast strain clusters closer with other Kyokai 7 yeasts in a phylogenetic analysis. The method according to any one of the preceding items, wherein said sake yeast strain comprises a gene encoding BIO1 of SEQ ID NO: 16 or SEQ ID NO: 21, or a polypeptide sharing at least 70% sequence identity with SEQ ID NO:s 16 or 21, such as at least 80% sequence identity with SEQ ID NO:s 16 or 21, such as at least 90% sequence identity with SEQ ID NO:s 16 or 21, such as at least 95% sequence identity with SEQ ID NO:s 16 or 21, such as at least 98% sequence identity with SEQ ID NO:s 16 or 21.

[0229] The method according to any one of the preceding items, wherein said sake yeast strain comprises a gene encoding BIO6 of SEQ ID NO: 17 or SEQ ID NO: 22, or a polypeptide sharing at least 70% sequence identity with SEQ ID NO:s 17 or 22, such as at least 80% sequence identity with SEQ ID NO:s 17 or 22, such as at least 90% sequence identity with SEQ ID NO:s 17 or 22, such as at least 95% sequence identity with SEQ ID NO:s 17 or 22, such as at least 98% sequence identity with SEQ ID NO:s 17 or 22.

[0230] The method according to any one of the preceding items, wherein said sake yeast strain comprises a gene encoding BIO2 of SEQ ID NO: 24, or a polypeptide sharing at least 70% sequence identity with SEQ ID NO: 24, such as at least 80% sequence identity with SEQ ID NO: 24, such as at least 90% sequence identity with SEQ ID NO: 24, such as at least 95% sequence identity with SEQ ID NO: 24, such as at least 98% sequence identity with SEQ ID NO: 24.

[0231] The method according to any one of the preceding items, wherein said sake yeast strain comprises a gene encoding BIO3 of SEQ ID NO: 25, or a polypeptide sharing at least 70% sequence identity with SEQ ID NO: 25, such as at least 80% sequence identity with SEQ ID NO: 25, such as at least 90% sequence identity with SEQ ID NO: 25, such as at least 95% sequence identity with SEQ ID NO: 25, such as at least 98% sequence identity with SEQ ID NO: 25.

[0232] The method according to any one of the preceding items, wherein said sake yeast strain comprises a gene encoding BIO4 of SEQ ID NO: 26, or a polypeptide sharing at least 70% sequence identity with SEQ ID NO: 26, such as at least 80% sequence identity with SEQ ID NO: 26, such as at least 90% sequence identity with SEQ ID NO: 26, such as at least 95% sequence identity with SEQ ID NO: 26, such as at least 98% sequence identity with SEQ ID NO: 26.

[0233] 39. The method according to any one of the preceding items, wherein said sake yeast strain comprises a gene encoding BIO5 of SEQ ID NO: 27, or a polypeptide sharing at least 70% sequence identity with SEQ ID NO: 27, such as at least 80% sequence identity with SEQ ID NO: 27, such as at least 90% sequence identity with SEQ ID NO: 27, such as at least 95% sequence identity with SEQ ID NO: 27, such as at least 98% sequence identity with SEQ ID NO: 27.

[0234] 40. The method according any one of the preceding items, wherein the beverage is a cider, and wherein fermentation of the cider base with said yeast strain improves the flavour profile of said cider compared to a cider produced by fermenting said cider base with a conventional cider yeast.

[0235] 41. The method according to any one of the preceding items, wherein fermentation of the beverage or cider base with said yeast strain enhances the positive perception of any one of the following flavour characteristics of said beverage or cider: fruitiness, body, mouthfeel, floral, fragrant, sweetness, freshness, naturalness and / or fullness.

[0236] 42. The method according to any one of the preceding items, wherein fermentation of the beverage or cider base with said yeast strain reduces the negative perception of alcoholic notes.

[0237] 43. The method according to any one of the preceding items, wherein said sake yeast strain is capable of producing a fermented beverage or cider base comprising decreased average levels of one or more of the following compound(s) compared to a reference fermented beverage or cider base prepared in the same manner but fermented with the S. cerevisiae yeast strain; Uvaferm CEG; wherein said compounds are decanoic acid, isobutanol, and / or total fusel alcohols such as butanol, 2-methyl butanol and / or 3-methyl butanol.

[0238] The method according to any one of the preceding items, wherein said sake yeast strain is capable of producing a fermented beverage or cider base comprising a relative concentration of at the most 90%, such as at the most 80%, such as at the most 70%, such as at the most 60%, such as at the most 50%, such as at the most 40%, such as at the most 30%, such as in the range of 0-90%, such as in the range of 0-80% of isobutanol compared to a reference fermented beverage or cider base prepared in the same manner but fermented with the S. cerevisiae yeast strain; Uvaferm CEG.

[0239] The method according to any one of the preceding items, wherein the beverage is a cider, and wherein said sake yeast strain is capable of producing a fermented cider base comprising a relative concentration of at the most 80%, such as at the most 70%, such as at the most 60%, such as at the most 50%, such as at the most 40%, such as at the most 30%, such as in the range of 0-80%, such as in the range of 0-70% of decanoic acid compared to a reference fermented cider base prepared in the same manner but fermented with the S. cerevisiae yeast strain; Uvaferm CEG.

[0240] The method according to any one of the preceding items, wherein the beverage is a cider, and wherein said sake yeast strain is capable of producing a fermented cider base comprising a relative concentration of at the most 90%, such as at the most 80%, such as at the most 70%, such as at the most 60%, such as at the most 50%, such as at the most 40%, such as in the range of 0-90%, such as in the range of 0-85% of total fusel alcohols compared to a reference fermented cider base prepared in the same manner but fermented with the S. cerevisiae yeast strain; Uvaferm CEG.

[0241] The method according to any one of the preceding items, wherein the beverage is a cider, and wherein said sake yeast strain is capable of producing a fermented cider base comprising a relative concentration of at the most 90%, such as at the most 80%, such as at the most 70%, such as at the most 60%, such as at the most 50%, such as at the most 40%, such as in the range of 0- 90%, such as in the range of 0-85% of total fusel alcohols such as isobutanol, butanol, 2-methyl butanol and / or 3-methyl butanol compared to a reference fermented cider base prepared in the same manner but fermented with the S. cerevisiae yeast strain; Uvaferm CEG.

[0242] 48. The method according to any one of the preceding items, wherein the beverage is a cider, and wherein said sake yeast strain is capable of producing a fermented cider base comprising decreased average levels of 2-phenylethyl acetate compared to a reference fermented cider base prepared in the same manner but fermented with the S. cerevisiae yeast strain; Uvaferm CEG.

[0243] 49. The method according to any one of the preceding items, wherein the beverage is a cider, and wherein said sake yeast strain is capable of producing a fermented cider base comprising a relative concentration of at the most 90%, such as at the most 75%, such as at the most 50%, such as at the most 20%, such as in the range of 0-90%, such as in the range of 0-80% of 2-phenylethyl acetate compared to a reference fermented cider base prepared in the same manner but fermented with the S. cerevisiae yeast strain; Uvaferm CEG.

[0244] 50. The method according to any one of the preceding items, wherein the beverage is a cider, and wherein said sake yeast strain is capable of producing a fermented cider base comprising increased average levels of one or more of the following compound(s) compared to a reference fermented cider base prepared in the same manner but fermented with the S. cerevisiae yeast strain; Uvaferm CEG; wherein said compounds are ethyl 3-hydroxybutanoate, total levels of terpenes such as linalol, nerol, geraniol, citronellol and / or alpha terpineol, total levels of nerol, gernaniol and citronellol, nerol, geraniol, citronellol, total fermentable sugars such as fructose and / or glucose, sorbitol, and / or malate.

[0245] 51. The method according to any one of the preceding items, wherein the beverage is a cider, and wherein said sake yeast strain is capable of producing a fermented cider base comprising increased relative average levels of total levels of medium-chain fatty acid esters of at least 110%, such as at least 120%, such as at least 130%, such as at least 140%, such as at least 150%, such as at least 160%, such as at least 170%, such as in the range of 110-400%, such as in the range of 120-350% compared to a reference fermented cider base prepared in the same manner but fermented with but fermented with the S. cerevisiae yeast strain; Uvaferm CEG..

[0246] 52. The method according to any one of the preceding items, wherein the beverage is a cider, and wherein said sake yeast strain is capable of producing a fermented cider base comprising increased average levels of ethyl 3- hydroxybutanoate of at least 125%, such as at least 150%, such as at least 175%, such as at least 200%, such as at least 225%, such as at least 250%, such as at least 275%, such as at least 300%, such as in the range of 125-500%, such as in the range of 150-400% compared to a reference fermented cider base prepared in the same manner but fermented with the S. cerevisiae yeast strain; Uvaferm CEG.

[0247] 53. The method according to any one of the preceding items, wherein the beverage is a cider, and wherein said sake yeast strain is capable of producing a fermented cider base comprising increased average levels of ethyl hexanoate of at least 120%, such as at least 135%, such as at least 150%, such as at least 175%, such as at least 200%, such as at least 225%, such as at least 250%, such as in the range of 130-400%, such as in the range of 140-300% compared to a reference fermented cider base prepared in the same manner but fermented with the S. cerevisiae yeast strain; Uvaferm CEG.

[0248] 54. The method according to any one of the preceding items, wherein the beverage is a cider, and wherein said sake yeast strain is capable of producing a fermented cider base comprising increased average levels of ethyl octanoate of at least 110%, such as at least 125%, such as at least 150%, such as at least 175%, such as at least 200%, such as in the range of 110-350% compared to a reference fermented cider base prepared in the same manner but fermented with the S. cerevisiae yeast strain; Uvaferm CEG.

[0249] 55. The method according to any one of the preceding items, wherein the beverage is a cider, and wherein said sake yeast strain is capable of producing a fermented cider base comprising increased relative average levels of total levels of terpenes of at least 105%, such as at least 110%, such as at least 120%, such as at least 130%, such as at least 140%, such as in the range of 105-350%, such as in the range of 105-300% compared to a reference fermented cider base prepared in the same manner but fermented with the S. cerevisiae yeast strain; Uvaferm CEG.

[0250] The method according to any one of the preceding items, wherein the beverage is a cider, and wherein said sake yeast strain is capable of producing a fermented cider base comprising increased relative average levels of total levels of terpenes such as linalol, nerol, geraniol, citronellol and / or alpha terpineol of at least 105%, such as at least 110%, such as at least 120%, such as at least 130%, such as at least 140%, such as in the range of 105-350%, such as in the range of 105-300% compared to a reference fermented cider base prepared in the same manner but fermented with the S. cerevisiae yeast strain; Uvaferm CEG.

[0251] The method according to any one of the preceding items, wherein the beverage is a cider, and wherein said sake yeast strain is capable of producing a fermented cider base comprising increased relative average levels of total levels of nerol, geraniol and citronellol of at least 120%, such as at least 130%, such as at least 140%, such as at least 150%, such as at least 160%, such as at least 170%, such as at least 180%, such as in the range of 120-400%, such as in the range of 120-300% compared to a reference fermented cider base prepared in the same manner but fermented with the S. cerevisiae yeast strain; Uvaferm CEG.

[0252] The method according to any one of the preceding items, wherein the beverage is a cider, and wherein said sake yeast strain is capable of producing a fermented cider base comprising increased relative average levels of terpenes selected from the group of nerol, geraniol, and / or citronellol of at least 120%, such as at least 130%, such as at least 140%, such as at least 150%, such as at least 160%, such as at least 170%, such as at least 180%, such as in the range of 120-400%, such as in the range of 120-300% compared to a reference fermented cider base prepared in the same manner but fermented with the S. cerevisiae yeast strain; Uvaferm CEG. The method according to any one of the preceding items, wherein the beverage is a cider, and wherein said sake yeast strain is capable of producing a fermented cider base comprising increased relative average levels of nerol of at least 110%, such as at least 120%, such as at least 130%, such as at least 140%, such as at least 150%, such as at least 160%, such as in the range of 110-400%, such as in the range of 110-300% compared to a reference fermented cider base prepared in the same manner but fermented with the S. cerevisiae yeast strain; Uvaferm CEG.

[0253] The method according to any one of the preceding items, wherein the beverage is a cider, and wherein said sake yeast strain is capable of producing a fermented cider base comprising increased relative average levels of geraniol of at least 101%, such as at least 110%, such as at least 120%, such as at least 130%, such as at least 140%, such as at least 150%, such as in the range of 101-350%, such as in the range of 110-400% compared to a reference fermented cider base prepared in the same manner but fermented with the S. cerevisiae yeast strain; Uvaferm CEG.

[0254] The method according to any one of the preceding items, wherein the beverage is a cider, and wherein said sake yeast strain is capable of producing a fermented cider base comprising increased relative average levels of citronellol of at least 140%, such as at least 150%, such as at least 160%, such as at least 170%, such as at least 180%, such as at least 190%, such as at least 200%, such as at least 210%, such as in the range of 140-400%, such as in the range of 150-350% compared to a reference fermented cider base prepared in the same manner but fermented with the S. cerevisiae yeast strain; Uvaferm CEG.

[0255] The method according to any one of the preceding items, wherein the beverage is a cider, and wherein said sake yeast strain is capable of producing a fermented cider base comprising increased relative average levels of total fermentable sugars such as fructose and / or glucose of at least 180%, such as at least 190%, such as at least 200%, such as at least 210%, such as at least 220%, such as at least 230%, such as at least 240%, such as at least 250%, such as at least 260% such as in the range of 180-500%, such as in the range of 190-400% compared to a reference fermented cider base prepared in the same manner but fermented with the S. cerevisiae yeast strain; Uvaferm CEG.

[0256] The method according to any one of the preceding items, wherein the beverage is a cider, and wherein said sake yeast strain is capable of producing a fermented cider base comprising increased relative average levels of fructose of at least 210%, such as at least 220%, such as at least 230%, such as at least 240%, such as at least 250%, such as at least 260%, such as at least 270%, such as at least 280%, such as at least 290%, such as at least 300%, such as in the range of 210-500%, such as in the range of 220-450% compared to a reference fermented cider base prepared in the same manner but fermented with the S. cerevisiae yeast strain; Uvaferm CEG.

[0257] The method according to any one of the preceding items, wherein the beverage is a cider, and wherein said sake yeast strain is capable of producing a fermented cider base comprising increased relative average levels of sorbitol of at least 101%, such as at least 110%, such as at least 120%, such as at least 130%, such as in the range of 101-400%, such as in the range of 101-350% compared to a reference fermented cider base prepared in the same manner but fermented with the S. cerevisiae yeast strain; Uvaferm CEG.

[0258] The method according to any one of the preceding items, wherein the beverage is a cider, and wherein said sake yeast strain is capable of producing a fermented cider base comprising increased relative average levels of malate of at least 101%, such as at least 110%, such as at least 120%, such as at least 130%, such as at least 140%, such as at least 150%, such as in the range of 100-400%, such as in the range of 100-350% compared to a reference fermented cider base prepared in the same manner but fermented with the S. cerevisiae yeast strain; Uvaferm CEG.

[0259] The method according to any one of the preceding items, wherein the beverage is a cider, and wherein said sake yeast strain is capable of producing a fermented cider base comprising a ratio of the concentrations of one or more of the following compound(s); at least 0.75, such as at least 0.8, such as at least 0.9, such as at least 1, such as at least 1.1, such as at least 1.2, such as in the range of 0.75-3, such as in the range of 0.75-2.5 of citronellol vs geraniol;

[0260] at least 0.2, such as at least 0.3, such as at least 0.4, such as at least 0.5, such as in the range of 0.2-2, such as in the range of 0.3-1.5 of citronellol vs total levels of terpenes;

[0261] at least 0.0003, such as at least 0.0004, such as at least 0.0005, such as at least 0.0006, such as at least 0.0007, such as in the range of 0-1, such as in the range of 0-0.05 of total levels of terpenes, such as linalol, nerol, geraniol, citronellol and alpha terpineol, vs total levels of fusel alcohols; and / or,

[0262] at least 0.01, such as at least 0.012, such as at least 0.014, such as at least 0.016, such as at least 0.018, such as at least 0.02, such as at least 0.04, such as in the range of 0-1, such as in the range of 0-0.075 of total levels of mid-chain fatty acid esters, such as ethyl decanoate, ethyl hexanoate, ethyl octanoate and / or ethyl butanoate, vs total levels of fusel alcohols.

[0263] 67. The method according to any one of the preceding items, wherein fermentation is stopped when the fermented beverage or cider base has;

[0264] i) an ABV of in the range of 8 to 17%, such as in the range of 9 to 14%, preferably in the range of 9 to 13%, or more preferably in the range of 10 to 13%, or more preferably in the range of 10 to 12%, or more preferably in the range of 10 to 11%; and / or,

[0265] ii) a specific gravity in the range of 0.5 to 5°Plato, or more preferably in the range of 0.5 to 4.5°Plato, or more preferably in the range of 0.5 to 4°Plato, or more preferably in the range of 0.7 to 3.8°Plato, and / or

[0266] iii) a concentration of remaining fermentable sugars in the range of 10-60g / L, or more preferably 15-55g / L, or more preferably 15-50g / L, or more preferably 20-50g / L.

[0267] 68. The method according to any one of the preceding items, wherein fermentation is stopped when the fermented beverage or cider base has a maximum ABV of 12.5%. 69. The method according to any one of the preceding items, wherein fermentation is stopped when the fermented beverage or cider base has a specific gravity of at least 0.7°Plato.

[0268] 70. The method according to any one of the preceding items, wherein fermentation is stopped when the fermented beverage or cider base has a concentration of remaining fermentable sugars of at least 20g / L.

[0269] 71. The method according to any one of the preceding items, wherein the beverage or cider base comprises apple juice or apple juice concentrate diluted with water.

[0270] 72. The method according to any one of the preceding items, wherein the beverage or cider base has a specific gravity in the range of 15 to 30°Plato, or more preferably in the range of 20 to 28°Plato, or more preferably in the range of 20 to 25°Plato.

[0271] 73. The method according to any one of the preceding items, wherein the beverage or cider base has a specific gravity in the range of 15 to 30° Bx, or more preferably in the range of 20 to 28° Bx, or more preferably in the range of 20 to 25°Bx, for example in the range of 23 to 24°Bx.

[0272] 74. The method according to any one of the preceding items, wherein the beverage or cider base comprises sucrose.

[0273] 75. The method according to any one of the preceding items, wherein the beverage or cider base comprises in the range of 162 to 225 g / L sucrose.

[0274] 76. The method according to any one of the preceding items, wherein said beverage or cider base is inoculated with said yeast at a concentration of in the range of 10 to 40 million cells / ml, such as in the range of 15 to 25 million cells / ml.

[0275] 77. The method according to any one of the preceding items, wherein said beverage or cider base is inoculated with said yeast in freeze-dried form. 78. The method according to any one of the preceding items, wherein fermentation is performed at a temperature in the range of 15 to 30°C, such as in the range of 17 to 25°C, for example in the range of 19 to 23°C.

[0276] 79. The method according to any one of the preceding items wherein the method further comprises one or more of the following steps:

[0277] i) Maturation,

[0278] ii) Blending,

[0279] iii) Stabilisation, or

[0280] iv) Packaging

[0281] 80. The method according to any one of the preceding items, wherein the method further comprises blending said fermented beverage or cider base with a blending liquid.

[0282] 81. The method according to item 80, wherein the blending liquid comprises or consists of water and / or fruit juice, such as apple juice.

[0283] 82. The method according to any one of items 80 to 81, wherein said blending liquid is added in an amount, so that the mixture of fermented beverage or cider base and blending liquid has an alcohol content in the range of 3 to 8%, preferably in the range of 3 to 6%, even more preferably in the range of 4 to 5% (vol / vol).

[0284] 83. The method according to any one of the preceding items, wherein one or more aromas, such as apple aroma or apple juice, is added to the beverage or cider base, the blending liquid or to the beverage or cider, such as wherein apple juice is added to reach a minimum content of 16% apple juice in said beverage or cider.

[0285] 84. The method according to any one of items 1 to 82, wherein no artificial flavours or aromas are added to the beverage or cider base, the blending liquid or to the beverage or cider. 85. The method according to any one of the preceding items, wherein said beverage is a cider, a perry, a mead, a beer, or a fruit wine, such as apple wine, pear wine, cherry wine or plum wine.

[0286] 86. A sake yeast strain of the species Saccharomyces cerevisiae, wherein said yeast comprises;

[0287] i) a gene encoding BIO1 of SEQ ID NO: 16 or SEQ ID NO: 21 or a polypeptide sharing at least 70% sequence identity with SEQ ID NO:s 16 or 21, such as at least 80% sequence identity therewith, such as at least 90% sequence identity with SEQ ID NO:s 16 or 21, such as at least 95% sequence identity with SEQ ID NO:s 16 or 21, such as at least 98% sequence identity with SEQ ID NO:s 16 or 21, and / or;

[0288] ii) a gene encoding BIO6 of SEQ ID NO: 17 or SEQ ID NO: 22, or a polypeptide sharing at least 70% sequence identity with SEQ ID NO:s 17 or 22, such as at least 80% sequence identity with SEQ ID NO:s 17 or 22, such as at least 90% sequence identity with SEQ ID NO:s 17 or 22, such as at least 95% sequence identity with SEQ ID NO:s 17 or 22, such as at least 98% sequence identity with SEQ ID NO:s 17 or 22.

[0289] 87. A sake yeast strain of the species Saccharomyces cerevisiae, wherein said yeast comprises;

[0290] i) a gene encoding BIO1 of SEQ ID NO: 16 or SEQ ID NO: 21 or a polypeptide sharing at least 70% sequence identity with SEQ ID NO:s 16 or 21, such as at least 80% sequence identity therewith, such as at least 90% sequence identity with SEQ ID NO:s 16 or 21, such as at least 95% sequence identity with SEQ ID NO:s 16 or 21, such as at least 98% sequence identity with SEQ ID NO:s 16 or 21, wherein said gene encoding BIO1 is expressed from its endogenous promoter, and;

[0291] ii) a gene encoding BIO6 of SEQ ID NO: 17 or SEQ ID NO: 22, or a polypeptide sharing at least 70% sequence identity with SEQ ID NO:s 17 or 22, such as at least 80% sequence identity with SEQ ID NO:s 17 or 22, such as at least 90% sequence identity with SEQ ID NO:s 17 or 22, such as at least 95% sequence identity with SEQ ID NO:s 17 or 22, such as at least 98% sequence identity with SEQ ID NO:s 17 or 22, wherein said gene encoding BIO6 is expressed from its endogenous promoter. 88. The sake yeast strain according to item 87, wherein said genes encoding BIO1 and BIO6 are not heterologous to said yeast strain.

[0292] 89. The sake yeast strain according to any one of items 86 to 88, wherein said yeast strain is capable of producing a fermented beverage or cider base comprising increased average levels of one or more of the following compound(s) compared to a reference fermented beverage or cider base prepared in the same manner but fermented with the Kyokai yeast strain yeast strain; NCYC 479; wherein said compounds are isoamyl acetate, 2-phenylethyl acetate, ethyl hexanoate, ethyl octanoate, ethyl butanoate, ethyl 2-hydroxyisocaproate, total levels of mediumchain fatty acid esters such as ethyl decanoate, ethyl hexanoate, ethyl octanoate, and / or ethyl butanoate, citronellol, butanol, total levels of 2-methyl butanol and 3-methyl butanol, butanoic acid, hexanoic acid, total levels of fusel alcohols such as isobutanol, butanol, 2-methyl butanol and / or 3-methyl butanol, malate, and / or total levels of medium-chain fatty acid esters, such as ethyl decanoate, ethyl hexanoate, ethyl octanoate and / or ethyl butanoate, and terpenes, such as linalol, nerol, geraniol, citronellol and alpha terpineol.

[0293] 90. The sake yeast strain according to any one of items 86 to 89 wherein said sake yeast strain is capable of producing a fermented beverage or cider base comprising increased relative average levels of isoamyl acetate of at least 130%, such as at least 150%, such as at least 170%, such as at least 190%, such as at least 200%, such as at least 210%, such as in the range of 130-400%, such as in the range of 140-350% compared to a reference fermented beverage or cider base prepared in the same manner but fermented with the Kyokai yeast strain; NCYC 479.

[0294] 91. The sake yeast strain according to any one of items 86 to 90, wherein said sake yeast strain is capable of producing a fermented beverage or cider base comprising increased relative average levels of 2-phenylethyl acetate of at least 110%, such as at least 120%, such as at least 140%, such as at least 160%, such as at least 180%, such as at least 200%, such as at least 220%, such as in the range of 110-400%, such as in the range of 120-350% compared to a reference fermented beverage or cider base prepared in the same manner but fermented with the Kyokai yeast strain; NCYC 479.

[0295] The sake yeast strain according to any one of items 86 to 91, wherein said sake yeast strain is capable of producing a fermented beverage or cider base comprising increased relative average levels of ethyl hexanoate of at least 105%, such as at least 110%, such as at least 125%, such as at least 140%, such as at least 150%, such as at least 160%, such as at least 175%, such as in the range of 105-400%, such as in the range of 110-350% compared to a reference fermented beverage or cider base prepared in the same manner but fermented with the Kyokai yeast strain; NCYC 479.

[0296] The sake yeast strain according to any one of items 86 to 92, wherein said sake yeast strain is capable of producing a fermented beverage or cider base comprising increased relative average levels of ethyl octanoate of at least 110%, such as at least 125%, such as at least 140%, such as at least 150%, such as at least 160%, such as at least 175%, such as in the range of 110-400%, such as in the range of 110-350% compared to a reference fermented beverage or cider base prepared in the same manner but fermented with the Kyokai yeast strain; NCYC 479.

[0297] The sake yeast strain according to any one of items 86 to 93, wherein said sake yeast strain is capable of producing a fermented beverage or cider base comprising increased relative average levels of ethyl butanoate of at least 101%, such as at least 140%, such as at least 160%, such as at least 170%, such as at least 190%, such as at least 210%, such as at least 230%, such as in the range of 101-400%, such as in the range of 101-350% compared to a reference fermented beverage or cider base prepared in the same manner but fermented with the Kyokai yeast strain; NCYC 479.

[0298] The sake yeast strain according to any one of items 86 to 94, wherein said sake yeast strain is capable of producing a fermented beverage or cider base comprising increased relative average levels of ethyl 2-hydroxyisocaproate of at least 101%, such as at least 140%, such as at least 160%, such as at least 170%, such as at least 190%, such as at least 210%, such as at least 230%, such as in the range of 101-400%, such as in the range of 101-350% compared to a reference fermented beverage or cider base prepared in the same manner but fermented with the Kyokai yeast strain; NCYC 479.

[0299] 96. The sake yeast strain according to any one of items 86 to 95, wherein said sake yeast strain is capable of producing a fermented beverage or cider base comprising increased relative average levels of total levels of medium-chain fatty acid esters of at least 110%, such as at least 120%, such as at least 130%, such as at least 140%, such as at least 150%, such as at least 160%, such as at least 170%, such as in the range of 110-400%, such as in the range of 120-350% compared to a reference fermented beverage or cider base prepared in the same manner but fermented with the Kyokai yeast strain; NCYC 479.

[0300] 97. The sake yeast strain according to any one of items 86 to 96, wherein said sake yeast strain is capable of producing a fermented beverage or cider base comprising increased relative average levels of total levels of medium-chain fatty acid esters such as ethyl decanoate, ethyl hexanoate, ethyl octanoate, and / or ethyl butanoate of at least 110%, such as at least 120%, such as at least 130%, such as at least 140%, such as at least 150%, such as at least 160%, such as at least 170%, such as in the range of 110-400%, such as in the range of 120- 350% compared to a reference fermented beverage or cider base prepared in the same manner but fermented with the Kyokai yeast strain; NCYC 479.

[0301] 98. The sake yeast strain according to any one of items 86 to 97, wherein said sake yeast strain is capable of producing a fermented beverage or cider base comprising increased relative average levels of total levels of medium-chain fatty acid esters such as ethyl decanoate, ethyl hexanoate, and / or ethyl octanoate of at least 110%, such as at least 120%, such as at least 130%, such as at least 140%, such as at least 150%, such as at least 160%, such as at least 170%, such as in the range of 110-400%, such as in the range of 120-350% compared to a reference fermented beverage or cider base prepared in the same manner but fermented with the Kyokai yeast strain; NCYC 479.

[0302] 99. The sake yeast strain according to any one of items 86 to 98, wherein said sake yeast strain is capable of producing a fermented beverage or cider base comprising increased relative average levels of total levels of medium-chain fatty acid esters such as ethyl hexanoate and / or ethyl decanoate of at least 110%, such as at least 120%, such as at least 130%, such as at least 140%, such as at least 150%, such as at least 160%, such as at least 170%, such as in the range of 110-400%, such as in the range of 120-350% compared to a reference fermented beverage or cider base prepared in the same manner but fermented with the Kyokai yeast strain; NCYC 479.

[0303] 100. The sake yeast strain according to any one of items 86 to 99, wherein said sake yeast strain is capable of producing a fermented beverage or cider base comprising increased relative average levels of citronellol of at least 105%, such as at least 110%, such as at least 115%, such as at least 120%, such as at least 125%, such as at least 130%, such as at least 135%, such as at least 140%, such as in the range of 105-400%, such as in the range of 105-350% compared to a reference fermented beverage or cider base prepared in the same manner but fermented with the Kyokai yeast strain; NCYC 479.

[0304] 101. The sake yeast strain according to any one of items 86 to 100, wherein said sake yeast strain is capable of producing a fermented beverage or cider base comprising increased relative average levels of total levels of fusel alcohols such as isobutanol, butanol, 2-methyl butanol and / or 3-methyl butanol of at least 100%, such as at least 105%, such as at least 115%, such as at least 125%, such as at least 135%, such as at least 145%, such as at least 155%, such as at least 165%, such as at least 175%, such as in the range of 100-400%, such as in the range of 100-350% compared to a reference fermented beverage or cider base prepared in the same manner but fermented with the Kyokai yeast strain; NCYC 479.

[0305] 102. The sake yeast strain according to any one of items 86 to 101, wherein said sake yeast strain is capable of producing a fermented beverage or cider base comprising increased relative average levels of malate of at least 100%, such as at least 105%, such as at least 110%, such as at least 115%, such as at least 120%, such as at least 125%, such as at least 130%, such as in the range of 100-400%, such as in the range of 100-350% compared to a reference fermented beverage or cider base in the same manner but fermented with the Kyokai yeast strain; NCYC 479.

[0306] 103. The sake yeast strain according to any one of items 86 to 102, wherein said sake yeast strain is capable of producing a fermented beverage or cider base comprising increased relative average levels of total levels of mediumchain fatty acid esters and terpenes of at least 100%, such as at least 110%, such as at least 120%, such as at least 130%, such as at least 140%, such as at least 150%, such as at least 160%, such as at least 170%, such as in the range of 100-400%, such as in the range of 100-350% compared to a reference fermented beverage or cider base prepared in the same manner but fermented with the Kyokai yeast strain; NCYC 479.

[0307] 104. The sake yeast strain according to any one of items 86 to 103, wherein said sake yeast strain is capable of producing a fermented beverage or cider base comprising increased relative average levels of total levels of mediumchain fatty acid esters, such as ethyl decanoate, ethyl hexanoate, ethyl octanoate and / or ethyl butanoate, and terpenes, such as linalol, nerol, geraniol, citronellol and alpha terpineol, of at least 100%, such as at least 110%, such as at least 120%, such as at least 130%, such as at least 140%, such as at least 150%, such as at least 160%, such as at least 170%, such as in the range of 100-400%, such as in the range of 100-350% compared to a reference fermented beverage or cider base prepared in the same manner but fermented with the Kyokai yeast strain; NCYC 479.

[0308] 105. The sake yeast strain according to any one of items 86 to 104, wherein dry fermentation of the beverage or cider base with said yeast strain improves the flavour profile of the resultant beverage or cider compared to a beverage or cider produced by dry fermenting said beverage or cider base with a conventional cider yeast.

[0309] 106. The sake yeast strain according to any one of items 86 to 105, wherein dry fermentation of the beverage or cider base with said yeast strain reduces the negative perception of alcoholic notes, astringency and dryness of the resultant beverage or cider compared to a beverage or cider produced by dry fermenting said beverage or cider base with a conventional cider yeast.

[0310] 107. The sake yeast strain according to any one of items 86 to 106, wherein stopped fermentation of the beverage or cider base with said yeast strain improves the flavour profile of the resultant beverage or cider compared to a beverage or cider produced by stopped fermentation of said beverage or cider base with a conventional cider yeast.

[0311] 108. The sake yeast strain according to any one of items 86 to 107, wherein dry fermentation of the beverage or cider base with said yeast strain enhances the positive perception of any one of the following flavour characteristics of said cider: fruitiness, body, mouthfeel, floral, fragrant, sweetness, freshness, naturalness and / or fullness of the resultant beverage or cider compared to a beverage or cider produced by stopped fermentation of said beverage or cider base with a conventional cider yeast.

[0312] 109. The sake yeast strain according to any one of items 86 to 108, wherein said yeast strain further has the genotype as defined in the method according to any one of items 12 to 39.

[0313] 110. The method according to any one of items 1 to 85, wherein the sake yeast strain is the sake yeast strain according to any one of items 86 to 109.

[0314] 111. A method for producing a beverage, such as a cider, said method comprising;

[0315] i) providing a beverage base, such as a cider base, and

[0316] ii) providing a sake yeast strain according to any one of items 86 to 109, iii) fermenting the beverage base, such as the cider base, provided in step i) with said yeast strain of step ii),

[0317] iv) thereby obtaining a fermented beverage base, such as a fermented cider base,

[0318] v) processing the fermented beverage base, such as the fermented cider base, into a beverage, such as a cider. 112. The method according to item 111, wherein the method is performed as detailed in any one of items 1 to 85.

[0319] 113. A beverage produced by the method or the sake yeast strain according to any one of the preceding items.

[0320] 114. The beverage according to item 113, wherein said beverage is a cider, a perry, a mead, a beer, or a fruit wine, such as an apple wine, pear wine, cherry wine or plum wine.

[0321] 115. The beverage according to any one of items 113 to 114, wherein said beverage is a cider.

[0322] Examples

[0323] Materials and Methods

[0324] Cider base preparation

[0325] The cider base was made from a mix of non-flavoured apple juice concentrate, a sucrose syrup, yeast food ingredients and water, mixed to a density of 23°Plato. The yeast was propagated in a shake flask at a target inoculation of 20M cells / ml. The shake flasks for propagation were oxygenated by agitation at 125 rpm in a shake incubator. Fermentation was carried out at 20°C in a total volume of 500 ml, in shake flasks (laboratory scale) continuous agitation at 130 rpm. Fermentations were monitored daily. During fermentations, the following parameters of were quantified: BRIX via a digital refractometer, Plato via an Anton Parr alcoholizer, and fermentable sugars via HPLC.

[0326] This example describes preparation of fermented cider base in a laboratory scale. The skilled person will be able to upscale the conditions. For example, C-flasks may be used for propagation of yeast for production scale.

[0327] Fermentation Dry fermentation

[0328] Fermentations were monitored daily and were carried out until the majority of the fermentable sugars had been converted and the maximum potential ABV (%) was achieved, resulting in a fermented cider base (wine) with a maximum residual concentration of fermentable sugars of 0.14 g / L, and an ABV of >13.5%.

[0329] Stopped fermentation

[0330] Fermentations were monitored daily and stopped by cooling to 0°C at the point when the fermented cider base had an ABV (%) in the range of 12.13-12.40%.

[0331] The fermented cider base after stopped fermentation had:

[0332] • Fermentable sugar concentration in the range of 20.63 - 49.9 g / L.

[0333] • A specific gravity (which is a measure for the concentration of dissolved solids) in the range of 0.79-3.62°Plato.

[0334] Yeast strains

[0335] PPU4512 Saccharomyces cerevisiae is a sake kyokai 6 yeast.

[0336] PPU4513 Saccharomyces cerevisiae is a sake kyokai 7 yeast.

[0337] NCYC479 Saccharomyces cerevisiae is a commercially available (New York yeast collection; NCYC479, USA) sake kyokai 7 yeast.

[0338] PPU4514 Saccharomyces cerevisiae is a sake kyokai 9 yeast.

[0339] Uvaferm CEGTMis a hybrid wine yeast of Saccharomyces cerevisiae and Saccharomyces kudriavzevii yeast (Erny et al. 2012), and is commercially available (E491; Lallemand; 10050-06-02, France). Uvaferm CEGTMis used as a reference to the yeast of the current invention.

[0340] WLP775 English cider yeast Saccharomyces bayanus is a commercially available (White Labs; WLP775, USA) yeast. WLP775 is used as a reference to the yeast of the current invention.

[0341] Analyses of fermented cider base samples

[0342] The concentration of volatile aromatic compounds were analysed in fermented cider base samples were by targeted GC-MS in Nyseos, France, using an Agilent 5975 Mass Spectrometer coupled to an Agilent 6890 N Gas Chromatograph (Agilent, Santa Clara, USA). In total, 30 volatile aroma compounds (fusel alcohols, esters, terpenes and organic acids) were quantified. A Carbowax type polar capillary column was used for separation of the compounds (Agilent; 30m x 0.25 mm ID, 0.25 µM film thickness). Working parameters were as follows: injector temperature, 240°C; MS source, 230°C; MS Quad, 150°C; and transfer line, 280°C. The initial temperature was 40°C, 3 min and then increased to 180°C at a rate of 3°C / min. The temperature was subsequently raised to 260°C at a rate of 20°C / min, and maintained at 260°C for 10 min. The carrier gas was Helium (He) with a flow rate of 1.5 mL / min. Samples were injected using the splitless mode. A mass range of 50-400m / z was recorded at one scan per second. Five-point calibration curves were constructed for quantification of standard compounds, utilising an internal standard. Extraction of volatile compounds was performed three times for each fermented cider base and then each extract was injected into GC / MS. The relative peak area (in the total ion chromatogram) of each analyte from the fermented cider base to the internal standard was compared to those obtained for the standards. Dilution of the samples were taken into consideration. Concentrations of volatile compounds for which there was no pure reference were quantified using the calibration curve of one of the standard compounds with the most similar chemical structure. The concentrations of sugars (fructose, glucose, sucrose and sorbitol) and alcohols of the fermented cider base samples were quantified by HPLC, and the concentrations of organic acids (citrate and malic acid) of the fermented cider base samples were quantified via ionic chromatography. Both analyses were conducted at the Carlsberg Central Lab in Obernai, France, methods are summarized in Table 1.

[0343] Table 1. HPLC and Ionic chromatography methods utilised for quantification of sugar, alcohols and organic acid concentrations of fermented cider base samples.

[0344] Organic acids Sugars and ethanol Technique Ionic chromatography HPLC Detection Conductimetric Ri

[0345] Mobile phase Ultra-pure water / KOH Acetonitrile / Water 75 / 25 Gradient (KOH addition by the use

[0346] Mode Isocratic

[0347] of eluent generator)

[0348] Flow 0,25 ml / min 1 ml / min Dionex guard column AG11 +

[0349] Column Asahipak NH2P-50 4E analytical column AS11

[0350] Filtration and then dilution 1 / 10 in

[0351] Sample preparation Filtration

[0352] ultra-pure water

[0353]

[0354] DNA extraction and generation of sequencing data

[0355] For genomic DNA extraction, yeast cells were grown to saturation in 100 ml shake flasks in Yeast Extract-Peptone-Dextrose (YPD) medium. Biomass was collected by centrifugation (4000 xG, 10 minutes at 4oC), washed with sterile water and stored at -20oCin 70% ethanol. DNA extraction, short insert size library preparation (<800 bp), 150 bp paired-end sequencing on DNBseq was performed by BGI Genomics.

[0356] Paired-end sequencing data for the yeast strain; WLP775 was obtained from the NCBI Sequencing Read Archive (https: / / www.ncbi. nlm.nih.gov / sra / ?term=SRR2968043), with accession number SRR2968043.

[0357] Read mapping and variant calling

[0358] Prior to read mapping, reads were trimmed for quality using trimmomatic software (v0.39) with SLIDINGWINDOW:4:20. For each yeast strain, the reads were mapped to the S. cerevisiae S288C reference genome (version R64-3-1) using the Burrows- Wheeler Aligner (BWA v0.7.17), using default parameters and the resulting alignment file was converted to bam format and sorted using samtools (v1.15). Duplicated reads were marked and removed using GATK (v4.2.2.0). Base quality scores were recalibrated, and variants were called using GATK HaplotypeCaller with default parameters. Single nucleotide polymorphisms (SNPs) were selected using GATK SelectVariants and filtered using GATK VariantFiltration, keeping variants with quality depth > 2.0, mapping quality > 40, depth > 5.0 and genotype quality > 30. SnpEff (v5.1) was used to annotate and predict the effect of the variants. Variants were visualized using IGV (v2.13.2).

[0359] Genome assembly and annotation

[0360] For each of the yeast strains, genome assembly was performed using multiple assemblers. Abyss software (v2.3.4) was used testing combinations of parameter kc value 2 and 3 and k in the range of 40-130, with a step size of 10. Spades assembler (v3.15.3) was used with default parameters. Velvet assembler (v1.2.10) was used with hash length set to 21,131,10. The genome assembly quality was evaluated using Quast (v5.0.2) and the most complete assembly was selected based on N50, genome size and largest contig size. AUGUSTUS (v3.4.0) was used to predict genes from the assembled genomes.

[0361] The parameters used included: --strand=both, --genemodel=complete, --gff3=on, --cds=on, --codingseq=on,

[0362] --stopCodonExcludedFromCDS=false, --protein=true,

[0363] --species=saccharomyces_cerevisiae_S288C.

[0364] The nucleotide and proteins sequences of the predicted genes were obtained from the resulting GFF file using the utility scripts getAnnoFasta.pl. The function of each gene was annotated from similarity to Saccharomyces cerevisiae S288C determined by BlastP searches for the predicted protein sequence against the S288C proteome using the NcbiblastpCommandline module of BioPython (v1.79).

[0365] Phylogeny analysis

[0366] A maximum-likelihood SNP tree was created based on the read mapping and variant calling data using the Microbial Genomics Module of CLC Genomics Workbench (version 21) with default parameters.

[0367] Single-copy orthologs shared between the six strains specified above were identified using OrthoFinder (v2.5.4) with default parameters, except for parameter -msa to create a species tree based on the single-copy orthologs. For creating the species tree, MAFFT (v7.505) and FastTree (v2.1.11) were used for alignment and maximum likelihood tree inference, respectively. The resulting tree was visualized using FigTree (v1.4.4).

[0368] Gene presence / absence analysis

[0369] For each of the genes reported to be specifically found in Sake yeasts, the complete set of protein sequences available for S. cerevisiae, including strain Kyokai 7, for each gene were downloaded from the UniProt database. Each of the sequences were then compared to the predicted and annotated proteome of the strains involved in the comparison using BlastP, executed through the NcbiblastpCommandline module of BioPython (v1.79). For defining a gene as present in the strain analysed, a cut-off of 80% sequence identity of a full length, unique hit was used.

[0370] Statistical analysis

[0371] Statistical data analyses were performed using Xlstat (Addinsoft, France). Analyses of volatile aromatic compound concentrations were conducted in triplicate. Model analysis of the interaction between yeast strain and fermentation was conducted via PCA and 3-way ANOVA with Tukey’s post-hoc analyses at a 95% confidence interval. Statistical significance was defined as p<0.05. Phylogenetic analyses were performed by hierarchical cluster analysis based on the generated spectral dataset, using euclidean distance measure and ward, and presented as Dendrograms. D2 agglomeration method compiled in R script. The dendrograms were further compared using tanglegram analysis.

[0372] Example 1 - Comparison of ABV (%) and fermentable sugar concentrations of fermented cider base following dry vs stopped fermentation.

[0373] To test the difference in final ABV (%), sum of fermentable sugar concentrations and concentration of fructose of the fermented cider base following dry vs stopped fermentation, 500mL fermented cider base was prepared as described in the material and methods section, data are presented in Table 2. Stopped fermentation of the cider base resulted in a significantly decreased ABV (%), increased total concentration of fermentable sugars due to increased concentration of fructose for all yeasts compared with dry fermentation. There were no significant differences in ABV (%) or concentration of fructose between all sake yeasts and non-sake yeasts tested following stopped fermentation. However, the concentration of fructose was significantly greater in the fermented cider base following fermentation with sake yeast compared with wine (Uvaferm CEGTM) or traditional cider (WLP775) yeasts.

[0374] Conclusion

[0375] Stopping fermentation decreases the final ABV (%) and increases the total levels of fermentable sugars and the concentration of fructose in the fermented cider base compared with dry fermentation for all yeasts. Stopped fermentation of the cider base with sake yeasts results in a fermented cider base with a higher concentration of fructose compared with other non-sake yeasts. The stopped fermented cider base with sake yeast has an improved mouthfeel compared with the fermented cider base following stopped fermentation with non-sake yeasts, due to the higher concentration of fructose.

[0376] Table 2. Stopped fermentation reduces final ABV (%) and increases concentration of fermentable sugars compared with dry fermentation. Data are average of triplicate samples. Different lowercase letters (a-d) in the same column indicate significant differences between conditions (ANOVA with Tukey’s post hoc testing; p < 0.05). Total concentration of Fructose Yeast Fermentation Final ABV (%) fermentable sugars

[0377] (g / L) (g / L)

[0378] Uvaferm

[0379] Dry 14.668 a 0.000 d 0.000 d CEGTM

[0380] Uvaferm

[0381] Stopped 12.505 b 20.633 c 17.567 c CEGTMNCYC

[0382] Dry 14.364 a 0.133 d 0.000 d 479

[0383] NCYC

[0384] Stopped 11.090 c 45.467 ab 45.467 a 479

[0385] PPU

[0386] Dry 14.111 a 0.000 d 0.000 d 4513

[0387] PPU

[0388] Stopped 10.701 c 47.300 a 46.900 a 4513

[0389] PPU4512 Dry 14.487 a 0.000 d 0.000 d 40.300 PPU4512 Stopped 11.301 c 40.300 ab

[0390] ab PPU4514 Dry 14.309 a 0.000 d 0.000 d PPU4514 Stopped 10.448 c 49.933 a 49.567 a WLP775 Dry 13.878 a 0.000 d 0.000 d 26.967 WLP775 Stopped 11.453 be 30.133 be

[0391] be Pr > F(Model) <0,0001 <0,0001 <0,0001 Significance YES YES YES Pr > F(Yeast*Fermentation) 0.008 0.001 0.001 Significance YES YES YES

[0392]

[0393] Example 2 - Comparison of ester concentrations of fermented cider base following dry vs stopped fermentation.

[0394] To test the difference in ester concentrations of the fermented cider base following dry vs stopped fermentation, fermented cider base was prepared as described in the material and methods section, data are presented in Table 4. Previously reported olfactory description and perception thresholds are shown in Table 3. Stopped fermentation of the cider base resulted in significant differences in the concentrations of ethyl hexanoate, ethyl butanoate, ethyl 2-hydroxypropanoate, ethyl 3-hydroxybutanoate, ethyl 2-methylpropanoate, and ethyl 2-hydroxyisocaproate compared with dry fermentation for all yeasts.

[0395] Table 3. Olfactory description and perception thresholds (ug / L) of esters.

[0396] Olfactory Olfactory perception

[0397] Ester

[0398] description Threshold Base References Isoamyl Banana, pear, (San Juan et al.

[0399] 30 Wine

[0400] Acetate fruit 2012) 2-Phenyl Ethyl (Ferreira et al.

[0401] Flowery, roses 250 Wine

[0402] Acetate 2000) Ethyl Green apple, (Bingman et al.

[0403] 14 Wine

[0404] Hexanoate fruity, strawberry 2020)

[0405] (Ferreira et al. Ethyl Candy,

[0406] 5-580 Wine 2012, Bingman et Octanoate pineapple

[0407] al. 2020) Apple, Model

[0408] Ethyl (San Juan et al.

[0409] pineapple, 125 solution

[0410] Butanoate 2012)

[0411]

[0412] tropical with ethanol

[0413] Concentrations of isoamyl acetate in the fermented cider base were significantly decreased following stopped fermentation with sake yeasts compared with traditional cider yeast (WLP775). Concentrations of 2-phenylethyl acetate in the fermented cider base were significantly decreased following stopped fermentation with sake yeasts compared with traditional cider yeast (WLP775). The concentrations of ethyl hexanoate, ethyl octanoate, and the total concentration of medium-chain fatty acid esters (ethyl decanoate, ethyl hexanoate, ethyl octanoate, and ethyl butanoate) were greater in the fermented cider base following stopped fermentation with sake yeasts compared with wine yeast (Uvaferm CEGTM). Concentrations of ethyl 3-hydroxybutanoate were greater in the fermented cider base following stopped fermentation with sake yeasts compared with wine (Uvaferm CEGTM) or traditional cider (WLP775) yeasts.

[0414] Concentrations of the esters ethyl hexanoate, ethyl octanoate, and ethyl butanoate were greater in the fermented cider base following dry fermentation with sake yeast; PPU4513, compared with all other sake yeasts (PPU4512, NCYC479, and PPU4514). Conclusion

[0415] Higher concentrations of esters, in particular medium-chain fatty acid estersgive a more positive perception of fruitiness / fruity aromas. For both stopped and dry fermentations, sake yeasts are able to produce higher levels of esters compared with wine (Uvaferm CEGTM) yeasts and similar levels of esters compared with traditional cider (WLP775) yeasts. Further, for dry fermentation, sake yeast; PPU4513, produced more esters compared with all other sake yeasts (PPU4512, NCYC479, and PPU4514).

[0416] Stopped fermentation of the cider base with sake yeasts increased the concentrations of esters in the fermented cider base, especially medium-chain fatty acid esters, compared with stopped fermentation with wine yeasts. Thus, stopped fermentation with sake yeasts results in a fermented cider base with a more positive perception of fruitiness / fruity aromas, compared with stopped fermentation of wine yeasts. These include a more positive perception of green apple flavour (ethyl hexanoate), pineapple and candy flavours (ethyl octanoate) and apple, pineapple and tropical flavours (ethyl butanoate). Table 4. Impact of stopped fermentation on concentrations of esters of fermented cider base. Data are average of triplicate samples. Different lowercase letters (a-h) in the same column indicate significant differences between conditions (ANOVA with Tukey’s post hoc testing; p < 0.05). MCFA; medium¬ chain fatty acid.

[0417] 2- Ethyl 2- Ethyl 3- Ethyl 2- Ethyl 2- Ethyl Ethyl Ethyl Ethyl SUM Isoamyl phenyl hydrox hydrox methyl hydrox Ethyl Fermenta decane hexane octane butane MCFA Yeast acetate ethyl ypropa ybutan propan yisocap acetate

[0418] bon ate ate ate ate esters acetate noate oate oate roate (POflJ (pglL) (P0 / L) (P0 / L) (pgflj (PO / L) (P8 / L) (Pfl / L) WQ (Pfl / L)

[0419] Uvafer

[0420] 248.151 190.869 220.104 256.486 276.138 145.877 615.256 642.530 5.325 41718.5 898.60 m Stopped 0.000 d

[0421] b b ab fl c d bed fgh de 21 c 5c CEG™

[0422] Uvafer

[0423] 397.777 297.815 247.326 311.867 492.113 256.023 1024.05 1128.28 42.038 12.611 66512.0 1307.3 m Dry

[0424] b b ab fg abc abc 4 a 1 def abc a 39 abc 29 c CEG™

[0425] NCYC 390.601 152.731 148.962 370.254 423.507 141.311 466.615 1005.76 11.711 5.615 57094.4 10840

[0426] Stopped

[0427] 479 b b ab be d cd 7efg cd cde 82 be 34 c NCYC 312.292 184.861 173.347 453.423 485.845 171.575 707.320 1649.03 51.641 10.412 61823.6 12841

[0428] Dry

[0429] 479 b b ab defg be cd be 6 cd ab ab 40 abc 90 c PPU 271.614 118.257 220.102 452.332 427.717 157.204 400.957 1463.30 50697.9 1257.3

[0430] Stopped 0.000 d 4.924 e

[0431] 4513 b b ab defg be cd d 8 ode 49 c 55c PPU 475.444 236.050 174.874 697.430 612.925 326.746 633.870 2697.46 64.824 10.551 97885.6 1811.9

[0432] Dry

[0433] 4513 b b ab abc ab a bed 0 a ab ab 99 ab 75 c

[0434]

[0435] - - -! \;826276 PP6 2763756 4997864570 454 53273 924 20305836208530 24889U44144 41 11 1 1..........

[0436] I Sd 5066 Itoeppe!

[0437] ; i 3662d 6 9 b 1dd bd b b c c ce ce c ce a:

[0438] ! I i _ _ _ _

[0439] !:37 570 607063 835 72776 207953870 28552370575702 PP5610801 1 412U4 1414 1......

[0440] D iry §

[0441] 53 90d 9 bdbfb 12 b b b d be cee ae ace a i

[0442] J J i J _ o

[0443] 522266224 50907 5310364 55807769 39043 7228294111 111 141 11 | i;.........

[0444] 6 Sd 53 4t eoppe

[0445] 8 537fd ddd4 b ’e c c c c ce c ■

[0446] J » i J _ _ 8

[0447] jo

[0448] 7873698 777 5732602 3695 553 6263208 27055 88 PPU45 81 3866858628919765859541 114 11:,......... o $ Dry

[0449] 628bb b b b1b b 4b b bd iee c ace a a a a >

[0450] I J _ _ O ON ONN ’ ' I659203 3848676 580 802305 25903 908 693265000 53326 235983 6036611444 141;..........

[0451] ! 3

[0452] I 5720 hb 5dbbddb l a a ce c ae aa a cc a d

[0453] CO •o1': i958 70278207 280226 7650 778652 320935 10353887141 i893 15920 10 9082 1111411411 1::...........

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[0456] I V

[0457] P0000000000 FM00dl111 () < < < >oer,..

[0458] L.........

[0459] YES YESifi YES YES YES YES YES YES YES YES YES YES Sngcancei

[0460] PF>r

[0461] 007 037 0165 0161 0023 0001 0023 005 066 008 02YFi0414411’ttt (easmenaoner........... o

[0462] V

[0463] o YES Sifi YES YES YES YES YESgnicance o o

[0464] d

[0465] V

[0466] qe

[0467] 0000 P'

[0468] Izd us SfrdrDi I I g o

[0469] o

[0470] V

[0471] Cj

[0472] o

[0473] o 5 o

[0474] §

[0475] o

[0476] V

[0477] co

[0478] 0>

[0479] o>

[0480] o §

[0481] co o

[0482] CM -Q V

[0483] "3

[0484] g* &

[0485]

[0486] Example 3 - Comparison of terpene concentrations of fermented cider base following dry vs stopped fermentation.

[0487] To test the difference in terpene concentrations of the fermented cider base following dry vs stopped fermentation, fermented cider base was prepared as described in the material and methods section, data are presented in Table 6. Previously reported olfactory description and perception thresholds are shown in Table 5.

[0488] Table 5. Olfactory description and perception thresholds (ug / L) of terpenes for beer and wine bases.

[0489] Olfactory perception threshold Terpene Olfactory description

[0490] Beer base Wine base Lavender, Flowery,

[0491] Linalool 5 15-25

[0492] muscat

[0493] Nerol Floral, citrus 500 400 Geraniol Rose 6 30 Citronellol Lemon, lime 8 18

[0494] a- terpineol Lilac, piney, iris 2000 250

[0495] (Noguerol-Pato R et (Haslbeck K et al. al. 2009,

[0496] (Haslbeck K et al.

[0497] References 2018, Noguerol-Pato R Ferreira et al. 2000,

[0498] 2018)

[0499] et al. 2009) Ribereau-Gavon P.

[0500] et al. 1975)

[0501]

[0502] Fermentation of the cider base with sake yeasts resulted in greater concentrations of citronellol, total levels of nerol, geraniol, and total levels of terpenes (nerol, geraniol, citronellol, linalool and alpha terpinol), compared with wine (Uvaferm CEGTM) or traditional cider (WLP775) yeasts.

[0503] Additionally, stopped fermentation of the cider base with sake yeasts resulted in greater concentrations of nerol, geraniol and citronellol, and total levels of terpenes (nerol, geraniol, citronellol, linalool and alpha terpinol), compared with stopped fermentation of wine (Uvaferm CEGTM) or traditional cider (WLP775) yeasts. Further, stopped fermentation of the cider base with sake yeasts resulted in a higher ratio of citronellol vs geraniol concentrations, as well as citronellol vs total terpenes concentrations (nerol, geraniol, citronellol, linalool and alpha terpinol) compared with stopped fermentation of wine (Uvaferm CEG™) or traditional cider (WLP775) yeasts.

[0504] Concentrations of the terpene; citronellol, were greater in the fermented cider base following dry fermentation with sake yeast; PPU4513, compared with all other sake yeasts (PPU4512, NCYC479, and PPU4514). Concentrations of the terpene; nerol, were greater in the fermented cider base following stopped fermentation with sake yeast; PPU4513, compared with all other sake yeasts (PPU4512, NCYC479, and PPU4514).

[0505] Conclusion

[0506] Higher concentrations of terpenes give a more positive perception of fruitiness / fruity aromas. Fermentation using sake yeasts results in a fermented cider base with higher concentrations of terpenes compared with non-sake yeasts. Stopped fermentation further enhances this, with higher concentrations of terpenes in the fermented cider base following stopped fermentation with sake vs non-sake yeasts. Therefore, stopped fermentation with sake yeasts results in a fermented cider base with a more positive perception of fruitiness / fruity aromas, compared with stopped fermentation with non-sake yeasts. The sake yeast strain; PPU4513, produces higher concentrations of citronellol and nerol following dry and stopped fermentation, respectively, compared with other sake yeasts. Therefore, sake yeast strain; PPU4513 has the capacity to further enhance the positive perception of fruitiness / fruity aromas in a fermented cider base compared with other sake yeasts. ble 6. Impact of stopped fermentation on concentrations of terpenes of fermented cider base. Data are average of triplicate samples. fferent lowercase letters (a-d) in the same column indicate significant differences between conditions (ANOVA with Tukey’s post hoc testing; <0.05).

[0507] Alpha terpineol Sum Terpenes Yeast Fermentation Linalol (pg / L) Nerol (pg / L) Geraniol (pg / L) Citronellol (pg / L)

[0508] (pg / L) (pg / L) vafermCEG™ Dry 14.599 a 1.183 bc 5.034 c 11.958 bc 3.379 a 36.152 a vafermCEG™ Stopped 12.755 a 1.256 abc 10.530 abc 7.634 cd 3.206 ab 35.381 a CYC479 Dry 7.284 cd 1.089 bc 14.263 a 13.319 ab 3.186 ab 39.141 a CYC479 Stopped 7.528 cd 1.709 ab 15.452 a 12.254 abc 2.797 cd 39.740 a PU 4513 Dry 7.307 cd 1.369 abc 8.919 abc 17.156 a 2.971 bcd 37.723 a PU 4513 Stopped 7.337 cd 1.864 a 12.186 abc 12.792 ab 2.833 bcd 37.013 a PU4512 Dry 7.398 cd 1.407 ab 13.694 ab 15.833 ab 3.049 abc 41.381 a PU4512 Stopped 7.356 cd 1.859 a 14.675 a 14.477 ab 2.659 d 41.025 a PU4514 Dry 10.318 b 1.185 bc 14.916 a 15.351 ab 3.113 abc 44.883 a PU4514 Stopped 8.405 bc 1.533 ab 15.144 a 14.851 ab 2.881 bcd 42.813 a LP775 Dry 5.648 d 0.000 d 4.921 c 6.241 d 2.628 d 19.438 a LP775 Stopped 6.077 cd 0.739 c 6.458 bc 4.741 d 2.632 d 20.646 a r> F (Model) <0,0001 <0,0001 <0,0001 <0,0001 <0,0001 <0,0001 ignificance YES YES YES YES YES YES r >f (Yeast'Fermentation) 0.054 0.196 0.514 0.241 0.106 0.993 ignificance NO NO NO NO NO NO

[0509]

[0510] Example 4 - Comparison of fusel alcohol, volatile acid and organic acid concentrations of fermented cider base following dry vs stopped fermentation.

[0511] To test the difference in concentrations of fusel alcohols in the fermented cider base following dry vs stopped fermentation, fermented cider base was prepared as described in the material and methods section. The concentrations of fusel alcohols, and volatile and organic acids in the fermented cider base following dry vs stopped fermentation are presented in Table 7 and Table 8, respectively. Stopped fermentation of the cider base resulted in a significantly decreased concentration of butanol, and significantly different concentrations of butanoic acid and decanoic acid, compared with dry fermentation for all yeasts.

[0512] Stopped fermentation of the cider base with sake yeasts resulted in lower concentrations of isobutanol, decanoic acid and the total levels of fusel alcohols (propanol, isobutanol, butanol, 2-methyl butanol and 2-methyl butanol) compared with wine (Uvaferm CEGTM) or traditional cider (WLP775) yeasts. Additionally, stopped fermentation of the cider base with sake yeasts resulted in lower total concentrations of 2-methyl butanol and 3-methyl butanol compared with traditional cider (WLP775) yeasts. Further, stopped fermentation of the cider base with sake yeasts resulted in greater concentrations of malate compared with stopped fermentation of wine (Uvaferm CEGTM) or traditional cider (WLP775) yeasts.

[0513] Table 7. Impact of stopped fermentation on concentrations of fusel alcohols of fermented cider base. Data are average of triplicate samples. Different lowercase letters (a-d) in the same column indicate significant differences between conditions (ANOVA with Tukey’s post hoc testing; p < 0.05)

[0514] Sum 2-methyl Propanol Isobutanol butanol and 3- Yeast Fermentation Butanol (pg / L)

[0515] (pg / L) (pg / L) methyl butanol (pg / L) Uvaferm

[0516] Dry 25704.369 a 39012.563 a 0.593 e 133095.577 a CEGTM

[0517] Uvaferm

[0518] Stopped 25549.805 a 31430.735 ab 0.000 e 92481.822 abc CEGTMNCYC 479 Dry 26820.474 a 33347.100 a 502.362 ab 93245.174 abc 25569.015

[0519] NCYC 479 Stopped 30694.128 a 362.760 be 68665.546 be abed

[0520] PPU 4513 Dry 25745.111 a 28906.393 abc 680.938 a 104542.126 ab

[0521]

[0522] PPU 4513 Stopped 24398.035 a 19264.071 bed 284.875 cd 81315.097 be 26126.256

[0523] PPU4512 Dry 24551.418 a 374.965 be 86219.365 be abed

[0524] PPU4512 Stopped 29028.650 a 17168.763 cd 231.248 cd 63399.343 be PPU4514 Dry 27444.128 a 28500.366 abc 335.786 bed 94376.417 abc PPU4514 Stopped 24304.671 a 14355.565 d 148.915 de 58280.463 c WLP775 Dry 23441.743 a 31672.887 ab 0.000 e 90731.156 abc 27789.501

[0525] WLP775 Stopped 26181.064 a 0.000 e 78530.874 be abed

[0526] Pr > F (Model) 0.124 <0,0001 <0,0001 0.000 Significance NO YES YES YES Pr > F (Yeast*Fermentation) 0.135 0.600 0.001 0.638 Significance NO NO YES NO

[0527]

[0528] Table 8. Impact of stopped fermentation on concentrations of volatile and non-volatile organic adds of fermented cider base. Data are average of triplicate samples. Different lowercase letters (a-e) in the same column indicate significant differences between conditions (ANOVA with ukey’s post hoc testing; p < 0.05).

[0529] Butanoic acid Hexanoic Octanoic Decanoic Malate

[0530] Yeast Fermentation

[0531] (µg / L) acid (pg / L) acid (pg / L) acid (pg / L) (pg / L)

[0532] Uvaferm

[0533] 4172.933 c

[0534] CEG™ Dry 735.245 be 1323.438 e 1981.659 c 724040 de

[0535] Uvaferm

[0536] 4127.533 c

[0537] CEG™ Stopped 540.864 c 1058.648 e 2121.488 c 2398.730 a

[0538] NCYC479 Dry 721.184 be 1944.840 d 2148.200 c 558.347 e 5771.567 a NCYC479 Stopped 879.226 abc 1944.880 d 2631.644 c 1257.610 be 5215.300 ab PPU 4513 Dry 1296.769 a 2716.566 abc 2533.642 c 516.148 e 5792.500 a PPU4513 Stopped 795.315 be 2251.068 cd 2769.232 bc 1493.235 be 5348.933 a PPU4512 Dry 916.572 abc 2344.592 cd 2176.579 c 554.539 e 5694.800 a PPU4512 Stopped 986.166 ab 2251.565 cd 2663.767 c 1495.423 be 5251.600 a PPU4514 Dry 1020.437 ab 2532.288 be 1824.329 c 276.999 e 5401.000 a PPU4514 Stopped 983.997 ab 2296.026 cd 2228.305 c 1092.362 cd 4544.933 be WLP775 Dry 1297.812 a 3181.453 a 3654.152 ab 602.631 de 4325.867 c WLP775 Stopped 1118.491 ab 2954.377 ab 4148.374 a 1697.475 b 4232.367 c Pr > F (Model <0,0001 <0,0001 <0,0001 <0,0001 <0,0001 Significance YES YES YES YES YES

[0539] Pr > F (Yeast*Fermentation) 0.010 0.379 0.897 0.002 0.059 Significance YES NO NO YES NO

[0540]

[0541] Conclusion

[0542] Stopped fermentation reduces the concentration of isobutanol in the fermented cider base compared with dry fermentation for all yeasts. Stopped fermentation of sake yeasts specifically result in decreased levels of fusel alcohols compared with non-sake yeasts. Low fusel alcohols improve the perception of fruity aromas from esters and terpenes. A higher concentration of the non-volatile, organic acid; malate, is important in providing the sensation of freshness and / or naturalness. Fermentation of sake yeasts results in the fermented cider base with higher concentrations of malate compared with non-sake yeasts. Therefore, a fermented cider base produced via stopped fermentation with sake yeasts has a more positive perception of fruity aromas and a greater sensation of freshness and / or naturalness.

[0543] Overall, these data demonstrate that stopped fermentation of a cider base with sake yeasts results in a fermented base with a decreased ABV (%), increased total levels of fermentable sugars, an increased concentration of fructose, increased concentrations of esters, terpenes and malate, and decreased concentrations of fusel alcohols and volatile acids, compared with stopped fermentation with non-sake yeasts. This results in the production of a fermented cider base with a more positive perception of mouthfeel, sweetness, fruitiness, freshness and / or naturalness.

[0544] Example 5 - Genotyping of yeast strains

[0545] The genotypic differences between strains as described in the methods and materials section were investigated to elucidate the genotypes associated with the various yeasts. Relevant genes and mutations for each yeast respectively are presented in Table 9, wherein “yes” denotes presence of the indicated gene or mutation, and “no” denotes lack of indicated gene or mutation. By way of example, if a gene carries a Lys290Asn mutation in the ERG20 gene, it is denoted “yes” and otherwise “no”.

[0546] Table 9. Yeast strain genotypes. Yes; presence of indicated mutation, No; denotes lack of indicated mutation. *Gene, yes indicates presence of gene, no indicates lack of gene.

[0547] Gene and PPU NCYC

[0548] PPU4512 PPU4514 WLP775 CEG mutation 4513 479

[0549] ERG20

[0550] Yes Yes Yes Yes No No (Lys290Asn)

[0551]

[0552] ATF1 (Thr84lle) Yes Yes Yes Yes No No OYE2

[0553] Yes Yes Yes Yes No No (Ala59Ser)

[0554] OYE2

[0555] Yes Yes Yes Yes Yes Yes (Asn108Lys)

[0556] OYE2

[0557] Yes Yes Yes Yes No No (Ser193Gly)

[0558] OYE2

[0559] Yes Yes Yes Yes Yes No (Lys311Glu)

[0560] OYE3

[0561] Yes Yes Yes Yes Yes#Yes# (Ser267Ala)

[0562] OYE3

[0563] Yes Yes Yes Yes No No (lle182Thr)

[0564] OYE3

[0565] No No No No Yes Yes (Glu259fs)

[0566] OYE3

[0567] No No No No Yes Yes (Val114Ile)

[0568] FDC1

[0569] Yes Yes Yes Yes No No (Lys54STOP)

[0570] FAS2

[0571] Yes Yes Yes Yes No No (Ser116Asn)

[0572] FAS2

[0573] No No No No Yes Yes (Ser565Asn)

[0574] FAS2

[0575] No No No No Yes Yes (Glu1475Lys)

[0576] FAS2

[0577] No No No No Yes Yes (Ser1800Asn)

[0578] VID24

[0579] No No No No Yes Yes (Phe209Tyr)

[0580] VID24

[0581] No No No No Yes Yes (Arg124Lys)

[0582] PEX22

[0583] Yes Yes Yes Yes No No (Val18Met)

[0584]

[0585] PEX22

[0586] No No No No Yes Yes (Gly103Glu)

[0587] PEX22

[0588] No No No No Yes Yes (Arg129Lys)

[0589] PEX22

[0590] No No No No Yes Yes (Val170lle)

[0591] BIO1* Yes No Likely No No No Yes BIO6* Yes No No No No Yes

[0592]

[0593] #

[0594]

[0595] : As WLP775 and CEG comprise an Glu259fs mutation, they do not express the full length of OYE3, i.e. Ser267 is not expressed as part of this protein in these strains. However, the method used for variant calling and annotation simply identifies the changes in the sequence compared to the reference sequence and annotates the individual mutation, hence these strains are annotated as having the Ser267Ala mutation although Ser267 is not part of the expressed protein.

[0596] The sake yeast strain PPU4513 was determined by sequencing to encode the OYE3 protein according to SEQ ID NO: 18, the ATF1 protein according to SEQ ID NO: 19, the FDC1 protein according to SEQ ID NO: 20, the BIO1 protein according to SEQ ID NO: 21, and the BIO6 protein according to SEQ ID NO: 22.

[0597] We observed that all sake yeasts had the following mutations: ERG20 (Lys290Asn), ATF1 (Thr84lle), OYE2 (Ala59Ser), OYE2 (Ser193Gly), OYE3 (lle182Thr), FDC1 (Lys54STOP), FAS2 (Ser116Asn), and PEX22 (Val18Met), whereas non-sake yeasts (Uvaferm CEG™ and WLP775) yeast did not. Additionally, we observed that all sake yeasts did not have the following mutations: OYE3 (Glu259fs), OYE3 (Val114lle), VID24 (Phe209Tyr), VID24 (Arg124Lys), PEX22 (Gly103Glu), PREX22 (Arg129Lys) and PEX22 (Val170lle).

[0598] We observed an increase in total concentration of terpenes following fermentation of all sake yeasts compared with non-sake yeasts, all of which carry a mutation in the OYE2 gene. Thus, these OYE2 mutations may assist in the greater production of terpenes in the fermented cider base, and account for the more positive perception of fruitiness.

[0599] Additionally, we observed that sake yeast strain; PPU4513, contained the BIO1 and BIO6 genes, which may be involved in biotin biosynthesis, whereas other sake yeast strains did not. We observed a greater production of esters, including ethyl hexanoate and higher ethyl octanoate, in the fermented cider base following dry fermentation with the sake yeast strain PPU4513. Thus, the expression of BIO1 and BIO6 in sake yeast strain PPU4513 in combination with the other genotypic hallmarks of this Sake yeast, may drive the greater production of esters and thus the more positive perception of fruity aromas in the fermented cider base.

[0600] We observed that all sake yeast strains possessed the ATF1 (Thr84lle) mutation and that stopped fermentation of all sake yeasts resulted in decreased concentrations of isoamyl acetate and 2-phenylethyl acetate in the fermented cider base compared with traditional cider yeast (WLP775), and both wine (Uvaferm CEGTM) or traditional cider (WLP775) yeasts, respectively. Thus, the presence of the ATF1 (Thr84lle) mutation in sake yeasts may drive the reduction in acetate ester production during fermentation.

[0601] Example 6- Sensory evaluation of the resultant cider’s sensory profile following dry or stopped fermentation of control vs sake yeast.

[0602] In aim to determine the effect and benefits of sake yeast on the resultant cider’s sensory profile, sensory evaluations were made using a specialist taste panel comprising 11 taste specialist. Samples were served blind, and panellists were instructed to evaluate the samples for 16 pre-selected attributes via the check all that apply method. Samples were served in a fixed order with the reference (CEG) being served first to avoid carry-over effects in both sets. Four ciders were evaluated: dry or stopped fermented with either CEG yeast as the reference, or sake PPU4513 Saccharomyces cerevisiae yeast - as detailed in Table 10.

[0603] Table 10. Ciders sampled in sensory evaluations.

[0604] Fermentation ABV Cider product Yeast

[0605] method (%) Reference (12%) Uvaferm CEG™ Stopped 12 Reference (16%) Uvaferm CEG™ Dry 16 PPU4513 Saccharomyces

[0606] Sake yeast (12%) Stopped 12 cerevisiae

[0607] PPU4513 Saccharomyces

[0608] Sake yeast (16%) Dry 16 cerevisiae

[0609]

[0610] Following stopped fermentation with sake yeast, the resultant cider s sensory profile was characterised by increased apple notes, fruitiness and body, and decreased alcoholic notes in comparison to stopped fermentation with reference non-sake yeast (Figure 3A).

[0611] Following dry fermentation with sake yeast, the resultant cider’s sensory profile was characterised by decreased alcoholic notes and was less dry and astringent in comparison to stopped fermentation with reference non-sake yeast (Figure 3B).

[0612] Overall, the resultant cider’s sensory profile following dry fermentation was characterised by higher levels of off-flavours, alcoholic notes and levels of astringency, compared with stopped fermentation.

[0613] Sequence overview

[0614] SEQ ID NO: 1 - OYE3 (WT gene, Saccharomyces cerevisiae strain S288c) ATGCCATTTGTAAAAGGTTTTGAGCCGATCTCCCTAAGAGACACAAACCTTTTTGAACCA ATTAAGATTGGTAACACTCAGCTTGCACATCGTGCGGTTATGCCCCCATTGACCAGAATG AGGGCCACTCACCCCGGAAATATTCCAAATAAGGAGTGGGCTGCTGTGTATTATGGTCAG CGTGCTCAAAGACCTGGTACCATGATCATCACGGAAGGTACGTTTATTTCCCCTCAAGCC GGCGGCTATGACAACGCCCCTGGGATTTGGTCTGATGAGCAGGTCGCTGAGTGGAAGAAT ATCTTTTTAGCCATCCATGATTGTCAGTCGTTCGCGTGGGTACAACTTTGGTCTTTAGGC TGGGCATCCTTCCCAGACGTATTGGCAAGAGACGGGTTACGCTATGACTGTGCATCTGAC AGAGTGTATATGAATGCTACGTTACAAGAAAAGGCCAAAGATGCGAATAATCTCGAACAT AGTTTGACTAAAGACGACATTAAACAGTATATCAAGGATTACATCCATGCGGCTAAGAAT TCTATCGCGGCTGGCGCCGATGGTGTAGAAATTCATAGCGCCAATGGGTACTTGTTGAAT CAGTTCTTGGATCCACATTCTAATAAGAGGACCGACGAATACGGCGGAACGATCGAAAAC AGGGCCCGCTTTACACTGGAGGTTGTCGATGCTCTTATCGAAACTATCGGTCCTGAACGG GTGGGTTTGAGGTTGTCGCCGTACGGCACTTTTAACAGTATGTCTGGGGGTGCTGAACCA GGTATTATCGCTCAATATTCGTATGTTTTGGGTGAATTAGAGAAGAGGGCAAAGGCTGGT AAGCGTTTGGCCTTTGTGCACCTCGTTGAACCACGTGTCACGGACCCATCGTTGGTGGAG GGCGAAGGAGAATATTCCGAGGGTACTAACGATTTTGCCTACTCTATATGGAAGGGTCCA ATCATCAGAGCTGGTAATTACGCTCTTCATCCAGAAGTGGTTAGAGAACAAGTAAAGGAT CCCAGAACCTTGATAGGCTATGGTAGATTCTTCATCTCTAACCCAGATTTAGTCTACCGT TTAGAAGAGGGCCTGCCATTGAACAAGTATGACAGAAGTACCTTCTACACCATGTCCGCG GAAGGTTATACCGACTACCCAACATATGAAGAGGCAGTAGATTTAGGTTGGAACAAGAAC TGA

[0615] SEQ ID NO: 2 - OYE3 (WT protein, Saccharomyces cerevisiae strain S288c) MPFVKDFKPQALGDTNLFKPIKIGNNELLHRAVIPPLTRMRAQHPGNIPNRDWAVEYYAQ RAQRPGTLIITEGTFPSPQSGGYDNAPGIWSEEQIKEWTKIFKAIHENKSFAWVQLWVLG WAAFPDTLARDGLRYDSASDNVYMNAEQEEKAKKANNPQHSITKDEIKQYVKEYVQAAKN SIAAGADGVEIHSANGYLLNQFLDPHSNNRTDEYGGSIENRARFTLEWDAWDAIGPEK VGLRLSPYGVFNSMSGGAETGIVAQYAYVLGELERRAKAGKRLAFVHLVEPRVTNPFLTE GEGEYNGGSNKFAYSIWKGPIIRAGNFALHPEWREEVKDPRTLIGYGRFFISNPDLVDR LEKGLPLNKYDRDTFYKMSAEGYIDYPTYEEALKLGWDKN

[0616] SEQ ID NO: 3 - OYE3 (protein, Saccharomyces cerevisiae strain S288c, lle182Thr mutation) MPFVKGFEPISLRDTNLFEPIKIGNTQLAHRAVMPPLTRMRATHPGNIPNKEWAAVYYGQ RAQRPGTMIITEGTFISPQAGGYDNAPGIWSDEQVAEWKNIFLAIHDCQSFAWVQLWSLG WASFPDVLARDGLRYDCASDRVYMNATLQEKAKDANNLEHSLTKDDIKQYIKDYIHAAKN

[0617] S [TJAGADGVEIHSANGYLLNQFLDPHSNKRTDEYGGTIENRARFTLEWDALIETIGPER VGLRLSPYGTFNSMSGGAEPGIIAQYSYVLGELEKRAKAGKRLAFVHLVEPRVTDPSLVE GEGEYSEGTNDFAYSIWKGPIIRAGNYALHPEWREQVKDPRTLIGYGRFFISNPDLVYR LEEGLPLNKYDRSTFYTMSAEGYTDYPTYEEAVDLGWNKN

[0618] SEQ ID NO: 4 - OYE2 (WT gene, Saccharomyces cerevisiae strain S288c) ATGCCATTTGTTAAGGACTTTAAGCCACAAGCTTTGGGTGACACCAACTTATTCAAACCA ATCAAAATTGGTAACAATGAACTTCTACACCGTGCTGTCATTCCTCCATTGACTAGAATG AGAGCCCAACATCCAGGTAATATTCCAAACAGAGACTGGGCCGTTGAATACTACGCTCAA CGTGCTCAAAGACCAGGAACCTTGATTATCACTGAAGGTACCTTTCCCTCTCCACAATCT GGGGGTTACGACAATGCTCCAGGTATCTGGTCCGAAGAACAAATTAAAGAATGGACCAAG ATTTTCAAGGCTATTCATGAGAATAAATCGTTCGCATGGGTCCAATTATGGGTTCTAGGT TGGGCTGCTTTCCCAGACACCCTTGCTAGGGATGGTTTGCGTTACGACTCCGCTTCTGAC AACGTGTATATGAATGCAGAACAAGAAGAAAAGGCTAAGAAGGCTAACAACCCACAACAC AGTATAACAAAGGATGAAATTAAGCAATACGTCAAAGAATACGTCCAAGCTGCCAAAAAC TCCATTGCTGCTGGTGCCGATGGTGTTGAAATCCACAGCGCTAACGGTTACTTGTTGAAC CAGTTCTTGGACCCACACTCCAATAACAGAACCGATGAGTATGGTGGATCCATCGAAAAC AGAGCCCGTTTCACCTTGGAAGTGGTTGATGCAGTTGTCGATGCTATTGGCCCTGAAAAA GTCGGTTTGAGATTGTCTCCATATGGTGTCTTCAACAGTATGTCTGGTGGTGCTGAAACC GGTATTGTTGCTCAATATGCTTATGTCTTAGGTGAACTAGAAAGAAGAGCTAAAGCTGGC AAGCGTTTGGCTTTCGTCCATCTAGTTGAACCTCGTGTCACCAACCCATTTTTAACTGAA GGTGAAGGTGAATACAATGGAGGTAGCAACAAATTTGCTTATTCTATCTGGAAGGGCCCA ATTATTAGAGCTGGTAACTTTGCTCTGCACCCAGAAGTTGTCAGAGAAGAGGTGAAGGAT CCTAGAACATTGATCGGTTACGGTAGATTTTTTATCTCTAATCCAGATTTGGTTGATCGT TTGGAAAAAGGGTTACCATTAAACAAATATGACAGAGACACTTTCTACAAAATGTCAGCT GAGGGATACATTGACTACCCTACGTACGAAGAAGCTCTAAAACTCGGTTGGGACAAAAAT TAA

[0619] SEQ ID NO: 5 - OYE2 (WT protein, Saccharomyces cerevisiae strain S288c) MPFVKDFKPQALGDTNLFKPIKIGNNELLHRAVIPPLTRMRAQHPGNIPNRDWAVEYYAQ RAQRPGTLIITEGTFPSPQSGGYDNAPGIWSEEQIKEWTKIFKAIHENKSFAWVQLWVLG WAAFPDTLARDGLRYDSASDNVYMNAEQEEKAKKANNPQHSITKDEIKQYVKEYVQAAKN SIAAGADGVEIHSANGYLLNQFLDPHSNNRTDEYGGSIENRARFTLEWDAWDAIGPEK VGLRLSPYGVFNSMSGGAETGIVAQYAYVLGELERRAKAGKRLAFVHLVEPRVTNPFLTE GEGEYNGGSNKFAYSIWKGPIIRAGNFALHPEWREEVKDPRTLIGYGRFFISNPDLVDR LEKGLPLNKYDRDTFYKMSAEGYIDYPTYEEALKLGWDKN

[0620] SEQ ID NO: 6- OYE2 (protein, Saccharomyces cerevisiae strain S288c, Ala59Ser mutation)

[0621] MPFVKDFKPQALGDTNLFKPIKIGNNELLHRAVIPPLTRMRAQHPGNIPNRDWAVEYY [S] Q RAQRPGTLIITEGTFPSPQSGGYDNAPGIWSEEQIKEWTKIFKAIHENKSFAWVQLWVLG WAAFPDTLARDGLRYDSASDNVYMNAEQEEKAKKANNPQHSITKDEIKQYVKEYVQAAKN SIAAGADGVEIHSANGYLLNQFLDPHSNNRTDEYGGSIENRARFTLEWDAWDAIGPEK VGLRLSPYGVFNSMSGGAETGIVAQYAYVLGELERRAKAGKRLAFVHLVEPRVTNPFLTE GEGEYNGGSNKFAYSIWKGPIIRAGNFALHPEWREEVKDPRTLIGYGRFFISNPDLVDR LEKGLPLNKYDRDTFYKMSAEGYIDYPTYEEALKLGWDKN SEQ ID NO: 7 - OYE2 (protein, Saccharomyces cerevisiae strain S288c, Ser193Gly mutation) MPFVKDFKPQALGDTNLFKPIKIGNNELLHRAVIPPLTRMRAQHPGNIPNRDWAVEYYAQ RAQRPGTLIITEGTFPSPQSGGYDNAPGIWSEEQIKEWTKIFKAIHENKSFAWVQLWVLG WAAFPDTLARDGLRYDSASDNVYMNAEQEEKAKKANNPQHSITKDEIKQYVKEYVQAAKN SIAAGADGVEIH [G] NGYLLNQFLDPHSNNRTDEYGGSIENRARFTLEWDAWDAIGPEK VGLRLSPYGVFNSMSGGAETGIVAQYAYVLGELERRAKAGKRLAFVHLVEPRVTNPFLTE GEGEYNGGSNKFAYSIWKGPIIRAGNFALHPEWREEVKDPRTLIGYGRFFISNPDLVDR LEKGLPLNKYDRDTFYKMSAEGYIDYPTYEEALKLGWDKN

[0622] SEQ ID NO: 8 - ERG20 (protein, Saccharomyces cerevisiae strain S288c, Lys290Asn mutation) MASEKEIRRERFLNVFPKLVEELNASLLAYGMPKEACDWYAHSLNYNTPGGKLNRGLSW DTYAILSNKTVEQLGQEEYEKVAILGWCIELLQAYFLVADDMMDKSITRRGQPCWYKVPE VGEIAINDAFMLEAAIYKLLKSHFRNEKYYIDITELFHEVTFQTELGQLMDLITAPEDKV DLSKFSLKKHSFIVTFKTAYYSFYLPVALAMYVAGITDEKDLKQARDVLIPLGEYFQIQD DYLDCFGTPEQIGKIGTDIQDNKCSWVINKALELASAEQRKTLDENYGK [N] DSVAEAKCKK IFNDLKIEQLYHEYEESIAKDLKAKISQVDESRGFKADVLTAFLNKVYKRSK

[0623] SEQ ID NO: 9- ATF1 (protein, Saccharomyces cerevisiae strain S288c, Thr84lle) MNEIDEKNQAPVQQECLKEMIQNGHARRMGSVEDLYVALNRQNLYRNFCTYGELSDYCTR DQLTLALREICLKNPTLLHIVLP [I] RWPNHENYYRSSEYYSRPHPVHDYISVLQELKLSGV VLNEQPEYSAVMKQILEEFKNSKGSYTAKIFKLTTTLTIPYFGPTGPSWRLICLPEEHTE KWKKFIFVSNHCMSDGRSSIHFFHDLRDELNNIKTPPKKLDYIFKYEEDYQLLRKLPEPI EKVIDFRPPYLFIPKSLLSGFIYNHLRFSSKGVCMRMDDVEKTDDWTEIINISPTEFQA IKANIKSNIQGKCTITPFLHVCWFVSLHKWGKFFKPLNFEWLTDIFIPADCRSQLPDDDE MRQMYRYGANVGFIDFTPWISEFDMNDNKENFWPLIEHYHEVISEALRNKKHLHGLGFNI QGFVQKYVNIDKVMCDRAIGKRRGGTLLSNVGLFNQLEEPDAKYSICDLAFGQFQGSWHQ AFSLGVCSTNVKGMNIWASTKNWGSQESLEELCSIYKALLLGP

[0624] SEQ ID NO: 10 - FDC1 (WT protein, Saccharomyces cerevisiae strain S288c) MRKLNPALEFRDFIQVLKDEDDLIEITEEIDPNLEVGAIMRKAYESHLPAPLFKNLKGAS KDLFSILGCPAGLRSKEKGDHGRIAHHLGLDPKTTIKEIIDYLLECKEKEPLPPITVPVS SAPCKTHILSEEKIHLQSLPTPYLHVSDGGKYLQTYGMWILQTPDKKWTNWSIARGMWD DKHITGLVIKPQHIRQIADSWAAIGKANEIPFALCFGVPPAAILVSSMPIPEGVSESDYV GAILGESVPWKCETNDLMVPATSEMVFEGTLSLTDTHLEGPFGEMHGYVFKSQGHPCPL YTVKAMSYRDNAILPVSNPGLCTDETHTLIGSLVATEAKELAIESGLPILDAFMPYEAQA LWLILKVDLKGLQALKTTPEEFCKKVGDIYFRTKVGFIVHEIILVADDIDIFNFKEVIWA YVTRHTPVADQMAFDDVTSFPLAPFVSQSSRSKTMKGGKCVTNCIFRQQYERSFDYITCN FEKGYPKGLVDKVNENWKRYGYK

[0625] SEQ ID NO: 11 - FAS2 (WT protein, Saccharomyces cerevisiae strain S288c) MKPEVEQELAHILLTELLAYQFASPVRWIETQDVFLKDFNTERWEIGPSPTLAGMAQRT LKNKYESYDAALSLHREILCYSKDAKEIYYTPDPSELAAKEEPAKEEAPAPTPAASAPAP AAAAPAPVAAAAPAAAAAEIADEPVKASLLLHVLVAHKLKKSLDSIPMSKTIKDLVGGKS TVQNEILGDLGKEFGTTPEKPEETPLEELAETFQDTFSGALGKQSSSLLSRLISSKMPGG FTITVARKYLQTRWGLPSGRQDGVLLVALSNEPAARLGSEADAKAFLDSMAQKYASIVGV DLSSAASASGAAGAGAAAGAAMIDAGALEEITKDHKVLARQQLQVLARYLKMDLDNGERK FLKEKDTVAELQAQLDYLNAELGEFFVNGVATSFSRKKARTFDSSWNWAKQSLLSLYFEI IHGVLKNVDREWSEAINIMNRSNDALIKFMEYHISNTDETKGENYQLVKTLGEQLIENC KQVLDVDPVYKDVAKPTGPKTAIDKNGNITYSEEPREKVRKLSQYVQEMALGGPITKESQ PTIEEDLTRVYKAI SAQADKQDISSSTRVEFEKLYSDLMKFLESSKEIDPSQTTQLAGMD VEDALDKDSTKEVASLPNKSTISKTVSSTIPRETIPFLHLRKKTPAGDWKYDRQLSSLFL DGLEKAAFNGVTFKDKYVLITGAGKGSIGAEVLQGLLQGGAKVWTTSRFSKQVTDYYQS IYAKYGAKGSTLIWPFNQGSKQDVEALIEFIYDTEKNGGLGWDLDAIIPFAAIPEQGIE LEHIDSKSEFAHRIMLTNILRMMGCVKKQKSARGIETRPAQVILPMSPNHGTFGGDGMYS ESKLSLETLFNRWHSESWANQLTVCGAIIGWTRGTGLMSANNIIAEGIEKMGVRTFSQKE MAFNLLGLLTPEWELCQKSPVMADLNGGLQFVPELKEFTAKLRKELVETSEVRKAVSIE TALEHKWNGNSADAAYAQVEIQPRANIQLDFPELKPYKQVKQIAPAELEGLLDLERVIV VTGFAEVGPWGSARTRWEMEAFGEFSLEGCVEMAWIMGFISYHNGNLKGRPYTGWVDSKT KEPVDDKDVKAKYETSILEHSGIRLIEPELFNGYNPEKKEMIQEVIVEEDLEPFEASKET AEQFKHQHGDKVDI FEI PETGEYSVKLLKGATLYI PKALRFDRLVAGQI PTGWNAKTYGI SDDIISQVDPITLFVLVSWEAFIASGITDPYEMYKYVHVSEVGNCSGSGMGGVSALRGM FKDRFKDEPVQNDILQESFINTMSAWVNMLLISSSGPIKTPVGACATSVESVDIGVETIL SGKARICIVGGYDDFQEEGSFEFGNMKATSNTLEEFEHGRTPAEMSRPATTTRNGFMEAQ GAGIQIIMQADLALKMGVPIYGIVAMAATATDKIGRSVPAPGKGILTTAREHHSSVKYAS PNLNMKYRKRQLVTREAQIKDWVENELEALKLEAEEIPSEDQNEFLLERTREIHNEAESQ LRAAQQQWGNDFYKRDPRIAPLRGALATYGLTIDDLGVASFHGTSTKANDKNESATINEM MKHLGRSEGNPVIGVFQKFLTGHPKGAAGAWMMNGALQILNSGIIPGNRNADNVDKILEQ FEYVLYPSKTLKTDGVRAVSITSFGFGQKGGQAIWHPDYLYGAITEDRYNEYVAKVSAR EKSAYKFFHNGMIYNKLFVSKEHAPYTDELEEDVYLDPLARVSKDKKSGSLTFNSKNIQS KDSYINANTIETAKMIENMTKEKVSNGGVGVDVELITSINVENDTFIERNFTPQEIEYCS AQPSVQSSFAGTWSAKEAVFKSLGVKSLGGGAALKDIEIVRVNKNAPAVELHGNAKKAAE EAGVTDVKVS I SHDDLQAVAVAVSTKK

[0626] SEQ ID NO: 12 - FAS2 (protein, Saccharomyces cerevisiae strain S288c, Ser565Asn) MKPEVEQELAHILLTELLAYQFASPVRWIETQDVFLKDFNTERWEIGPSPTLAGMAQRT LKNKYESYDAALSLHREILCYSKDAKEIYYTPDPSELAAKEEPAKEEAPAPTPAASAPAP AAAAPAPVAAAAPAAAAAEIADEPVKASLLLHVLVAHKLKKSLDSIPMSKTIKDLVGGKS TVQNEILGDLGKEFGTTPEKPEETPLEELAETFQDTFSGALGKQSSSLLSRLISSKMPGG FTITVARKYLQTRWGLPSGRQDGVLLVALSNEPAARLGSEADAKAFLDSMAQKYASIVGV DLSSAASASGAAGAGAAAGAAMIDAGALEEITKDHKVLARQQLQVLARYLKMDLDNGERK FLKEKDTVAELQAQLDYLNAELGEFFVNGVATSFSRKKARTFDSSWNWAKQSLLSLYFEI IHGVLKNVDREWSEAINIMNRSNDALIKFMEYHISNTDETKGENYQLVKTLGEQLIENC KQVLDVDPVYKDVAKPTGPKTAIDKNGNITYSEEPREKVRKLSQYVQEMALGGPITKESQ PTIEEDLTRVYKAISAQADKQDIS [N] STRVEFEKLYSDLMKFLESSKEIDPSQTTQLAGMD VEDALDKDSTKEVASLPNKSTISKTVSSTIPRETIPFLHLRKKTPAGDWKYDRQLSSLFL DGLEKAAFNGVTFKDKYVLITGAGKGSIGAEVLQGLLQGGAKVWTTSRFSKQVTDYYQS IYAKYGAKGSTLIWPFNQGSKQDVEALIEFIYDTEKNGGLGWDLDAIIPFAAIPEQGIE LEHIDSKSEFAHRIMLTNILRMMGCVKKQKSARGIETRPAQVILPMSPNHGTFGGDGMYS ESKLSLETLFNRWHSESWANQLTVCGAIIGWTRGTGLMSANNIIAEGIEKMGVRTFSQKE MAFNLLGLLTPEWELCQKSPVMADLNGGLQFVPELKEFTAKLRKELVETSEVRKAVSIE TALEHKWNGNSADAAYAQVEIQPRANIQLDFPELKPYKQVKQIAPAELEGLLDLERVIV VTGFAEVGPWGSARTRWEMEAFGEFSLEGCVEMAWIMGFISYHNGNLKGRPYTGWVDSKT KEPVDDKDVKAKYETSILEHSGIRLIEPELFNGYNPEKKEMIQEVIVEEDLEPFEASKET AEQFKHQHGDKVDI FEI PETGEYSVKLLKGATLYI PKALRFDRLVAGQI PTGWNAKTYGI SDDIISQVDPITLFVLVSWEAFIASGITDPYEMYKYVHVSEVGNCSGSGMGGVSALRGM FKDRFKDEPVQNDILQESFINTMSAWVNMLLISSSGPIKTPVGACATSVESVDIGVETIL SGKARICIVGGYDDFQEEGSFEFGNMKATSNTLEEFEHGRTPAEMSRPATTTRNGFMEAQ GAGIQIIMQADLALKMGVPIYGIVAMAATATDKIGRSVPAPGKGILTTAREHHSSVKYAS PNLNMKYRKRQLVTREAQIKDWVENELEALKLEAEEIPSEDQNEFLLERTREIHNEAESQ LRAAQQQWGNDFYKRDPRIAPLRGALATYGLTIDDLGVASFHGTSTKANDKNESATINEM MKHLGRSEGNPVIGVFQKFLTGHPKGAAGAWMMNGALQILNSGIIPGNRNADNVDKILEQ FEYVLYPSKTLKTDGVRAVSITSFGFGQKGGQAIWHPDYLYGAITEDRYNEYVAKVSAR EKSAYKFFHNGMIYNKLFVSKEHAPYTDELEEDVYLDPLARVSKDKKSGSLTFNSKNIQS KDSYINANTIETAKMIENMTKEKVSNGGVGVDVELITSINVENDTFIERNFTPQEIEYCS AQPSVQSSFAGTWSAKEAVFKSLGVKSLGGGAALKDIEIVRVNKNAPAVELHGNAKKAAE EAGVTDVKVS I SHDDLQAVAVAVSTKK SEQ ID NO: 13 - PEX22 (WT protein, Saccharomyces cerevisiae strain S288c) MPPPSRSRINKTRTLGIVGTAIAVLVTSYYIYQKVTSAKEDNGARPPEGDSVKENKKARK SKCIIMSKSIQGLPIKWEEYAADEWLLVPTSHTDGSMKQAIGDAFRKTKNEHKIIYCDS MDGLWSCVRRLGKFQCILNSRDFTSSGGSDAAWPEDIGRFVKFWDSDVEDVLIDTLCN SEQ ID NO: 14 - PEX22 (protein, Saccharomyces cerevisiae strain S288c, Val170Met) MPPPSRSRINKTRTLGIVGTAIAVLVTSYYIYQKVTSAKEDNGARPPEGDSVKENKKARK SKCIIMSKSIQGLPIKWEEYAADEWLLVPTSHTDGSMKQAIGDAFRKTKNEHKIIYCDS MDGLWSCVRRLGKFQCILNSRDFTSSGGSDAAWPEDIGRFVKFWDSD [M] EDVLIDTLCN

[0627] SEQ ID NO: 15 - VID24 (WT protein, Saccharomyces cerevisiae strain S288c) MINNPKVDSVAEKPKAVTSKQSEQAASPEPTPAPPVSRNQYPITFNLTSTAPFHLHDRHR YLQEQDLYKCASRDSLSSLQQLAHTPNGSTRKKYIVEDQSPYSSENPVIVTSSYNHTVCT NYLRPRMQFTGYQISGYKRYQVTVNLKTVDLPKKDCTSLSPHLSGFLSIRGLTNQHPEIS TYFEAYAVNHKELGFLSSSWKDEPVLNEFKATDQTDLEHWINFPSFRQLFLMSQKNGLNS TDDNGTTNAAKKLPPQQLPTTPSADAGNISRIFSQEKQFDNYLNERFIFMKWKEKFLVPD ALLMEGVDGASYDGFYYIVHDQVTGNIQGFYYHQDAEKFQQLELVPSLKNKVESSDCSFE FA

[0628] SEQ ID NO: 16 - BIO1 (WT protein, Saccharomyces cerevisiae strain Kyokai 7) MNTKSLDFYEPLEIDGQKYIKMTKKEDQGVYEAGVTQEAFTAKDKYDYKGIVENLERYGLCIVPSFIEPS KCDQILEELDPHFYRHDAWQGSPFPKETTWTRAVLHSPTVLKEWSDRLFCDMANHFLNEKNYFLTGNV IRKCSSGIQLNSGIVYKVGAGAGDQGYHREDHIHHTIHQACDSFQYGKETLLGVGVAFTDMNKANGATRV IIGSHLWGPHDSCGKFDKRMEFHVNAAKGDAVLFLGSLYHAASANHTLEDRIAGYFFMSQGYLKQEENLH FGTDPEFFKDMPLETLKLLGLTTSEPYCGHIDYKSPGHIANPSLFENEVEKGYYGETIKIIYDDKE

[0629] SEQ ID NO: 17 - BIO6 (WT protein, Saccharomyces cerevisiae strain Kyokai 7) MLEHQLTQEDLEFDKKHIWHPYTSITTPLKVYPITKAEGSYLYLDNGTKWDGMASWWCVQQGYNNHRLN AAAISQINKMSHVMFGGITHKAGIDFCKKLLALLPDTLECALLADSGSISVDIAMKMALRYHHSLGNTTK KRFLTIKKGYHGDAFGAVSVCDPVNSRHNTYNGFLAENIFCKAPEVRFDCREKDVEKLVEELDVKPFAEI IDKHHSEISGWMESIVQGAGGLRMYHPYFLKRVRALCNDFNILLILDEVAVGLGRTGMLFGFEHAGIVP DIVCLGKTLTAGYLTLSATVTTREIGDQISSGPEGCFMHGQTYMANPLACAVASENLSILMEGKWKSQVR QIEVQLKKELVPLLEHPIVADVRILGAIGWEVTKRVNVEVLQEKFIKAGAWIRPFGNIIYILPPYIITS EELTVLTEAIRSVLDFI SEQ ID NO: 18 - OYE3 (PPU4513) MPFVKGFEPISLRDTNLFEPIKIGNTQLAHRAVMPPLTRMRATHPGNIPNKEWAAVYYGQRAQRPGTMII TEGTFISPQAGGYDNAPGIWSDEQVAEWKNIFLAIHDCQSFAWVQLWSLGWASFPDVLARDGLRYDCASD RVYMNATLQEKAKDANNLEHSLTKDDIKQYIKDYIHAAKNSTAAGADGVEIHSANGYLLNQFLDPHSNKR TDEYGGTIENRARFTLEWDALIETIGPERVGLRLSPYGTFNSMSGGAEPGIIAQYAYVLGELEKRAKAG KRLAFVHLVEPRVTDPSLVEGEGEYSEGTNDFAYSIWKGPIIRAGNYALHPEWREQVKDPRTLIGYGRF FISNPDLVYRLEEGLPLNKYDRSTFYTMSAEGYTDYPTYEEAVDLGWNKN

[0630] SEQ ID NO: 19-ATF1 (PPU4513) MNEIDEKNQAPVQQECLKEMIQNGHARRMGSVEDLYVALNRQNLYRNFCTYGELSDYCTRDQLTLALREI CLKNPTLLHIVLPIRWPNHENYYRSSEYYSRPHPVHDYISVLQELKLSGWLNEQPEYSAVMKQILEEFK NSKGSYTAKIFKLTTTLTIPYFGPTGPSWRLICLPEEHTEKWKKFIFVSNHCMSDGRSSIHFFHDLRDEL NNIKTPPKKLDYIFKYEEDYQLLRKLPEPIEKVIDFRPPYLFIPKSLLSGFIYNHLRFSSKGVCMRMDDV EKTDDWTEIINISPTEFQAIKANIKSNIQGKCTITPFLHVCWFVSLHKWGKFFKPLNFEWLTDIFIPAD CRSQLPDDDEMRQMYRYGANVGFIDFTPWISEFDMNDNKEKFWPLIEHYHEVISEALRNKKHLHGLGFNI QGFVQKYVNIDKVMCDRAIGKRRGGTLLSNVGLFNQLEEPDAKYSICDLAFGQFQGSWHQAFSLGVCSTN VKGMNIWASTKNWGSQESLEELCSIYKALLLGP

[0631] SEQ ID NO: 20 - FDC1 expressed protein (PPU4513) MRKLNPALEFRDFIQVLKDEDDLIEITEEIDPNLEVGAIMRKAYESHLPAPLF SEQ ID NO: 21 - BIO1 (WT protein, PPU4513) MNTKSLDFYEPLEIDGQKYIKMTKKEDQGVYEAGVTQEAFTAKDKYDYKGIVENLERYGLCIVPSFIEPS KCDQILEELDPHFYRHDAWQGSPFPKETTWTRAVLHSPTVLKEWSDRLFCDMANHFLNEKNYFLTGNV IRKCSSGIQLNSGIVYKVGAGAGDQGYHREDHIHHTIHQACDSFQYGKETLLGVGVAFTDMNKANGATRV IIGSHLWGPHDSCGKFDKRMEFHVNAAKGDAVLFLGSLYHAASANHTLEDRIAGYFFMSQGYLKQEENLH FGTDPEFFKDMPLETLKLLGLTTSEPYCGHIDYKSPGHIANPSLFENEVEKGYYGETIKIIYDDKE SEQ ID NO: 22 - BIO6 (WT protein, PPU4513) MCEHQLTQEDLEFDKKHIWHPYTSITTPLKVYPVTKAEGSYLYLDNGTKWDGMASWWCVQQGYNNHRLN AAAISQINKMSHVMFGGITHKAGIDFCKKLLALLPDTLECALLADSGSISVDIAMKMALRYHHSLGNTTK KRFLTIKKGYHGDAFGAVSVCDPVNSRHNTYNGFLAENIFCKAPEVRFDCREKDVEKLVEELDVKPFAEI IDKHHSEISGWMESIVQGAGGLRMYHPYFLKRVRALCNDFNILLILDEVAVGLGRTGMLFGFEHAGIVP DIVCLGKTLTAGYLTLSATVTTREIGDQISSGPEGCFMHGQTYMANPLACAVASENLSILMEGKWKSQVR QIEVQLKKELVPLLEHPIVADVRILGAIGWEVTKRVNVEVLQEQFIKAGAWIRPFGNIIYILPPYIITS EELTVLTEAIRSVLDFI SEQ ID NO: 23 - Expressed FDC1 compared to full-length (PPU4513) MRKLNPALEFRDFIQVLKDEDDLIEITEEIDPNLEVGAIMRKAYESHLPAPLF*NLKGASKDLFSILGCP AGLRSKEKGDHGRIAHHLGLDPKTTIKEIIDYLLECKEKEPLPPITVPVSSAPCKTHILSEEKIHLQSLP TPYLHVSDGGKYLQTYGMWILQTPDKKWTNWSIARGMVVDDKHITGLVIKPQHIRQIADSWAAIGKANEI PEAL CFGVPPAA IL VSSMPIPEGVSESD YVGA IL GES VPWKCE TNDLMVPA TSEMVFEGTLSL ID THLE GPFGEMHGYVFKSQGHPCPLYTVKAMSYRDNAILPVSNPGLCTDETHTLIGSLVATEAKELAIESGLPIL DAFMPYEAQAL WL ILKVDLKGLQALKTTPEEFCKKVGDIYFR TKVGFIVHEIIL VADDIDIFNFKEVIWA YVTRHTPVADQMAFDDVTSFPLAPFVSQSSRSKTMKGGKCVTNCIFRQQYERSFDYITCNFEKGYPKGLV DKVNDNWKR YGYK

[0632] *: Stop

[0633] Italic sequence after stop. Not expressed due to premature termination of protein SEQ ID NO: 24 - BIO2 (WT protein, PPU4513) MMSTIYRHLSTARPALTKYATNAAVKSTTASSEASTLGALQYALSLDEPSHSWTKSQLKEIYHTPLLELT HAAQLQHRKWHDPTKVQLCTLMNIKSGGCSEDCKYCAQSSRNDTGLKAEKMVKVDEVIKEAEEAKRNGST RFCLGAAWRDMKGRKSAMKRIQEMVTKVNDMGLETCVTLGMVDQDQAKQLKDAGLTAYNHNIDTSREHYS KVITTRTYDDRLQTIKNVQESGIKACTGGILGLGESEDDHIGFIYTLSNMSPHPESLPINRLVAIKGTPM AEELADPKSKKLQFDEILRTIATARIVMPKAIIRLAAGRYTMKETEQFVCFMAGCNSIFTGKKMLTTMCN GWDEDKAMLAKWGLQPMEAFKYDRS SEQ ID NO: 25 - BIO3 (WT protein, PPU4513) MSQEISYTPDVAELLDFDKKHIWHPYTSLSSPLNVYPVKSAHGCKLVLDTDSPVDVEVIDAMSSWWCVIH GYNNPELNEALTKQMLKFSHVLLGGFTHKGAVNLVQKLLKVIDEPSLQYCFLADSGSVAVEVALKMALQS NMSGEATKNRTKFLTIKNGYHGDTFGAMSVCDPENSMHHIYNDRLSENIFSQAPSIVDGLPTSQNGFEDH WNAEEVTDLKKQFELHSDEICAVILEPILQGAGGLRPYHPQFLIEVQKLCNQYDVLFIMDEIATGFGRTG EIFAFKHCQKYQDQRGISPSDQIKWPDILCVGKGLTSGYMTMSAVWNDKVASRISSPNSPTGGCFMHG PTFMGNALACSVAEKSMDILLRGEWRKQVSAIEDQIYRELYQYIKNPDNGLIGTWKRVSVIGAVGIVEL YKKTDPEWFQKKFISKGVHIRPFNCLCYIMPPYVITTEELTKVNQVLIEVLHEWKSHINQ SEQ ID NO: 26 - BIO4 (WT protein, PPU4513) MNSKSQQQEQQPIVFVTGTDTDVGKTFVSTLLVHKWKAAYWKPVQTGIESDQGDSETLKNFKIAASTWQP PIFTPTYALQKPLSPLQAMEYEPNVDIRLLDFWPEEWSAENPLWEGAGGVCVPITRKLEITTDLIKHL IETSGHPVYWWARSGLGTLNHTLLTWNHLCDNGLRSHLFGVILNGEPNEGNVQALKKFGVNIMAQVAQ CTTAHDQDMALHELPSVESLMTQQDVE SEQ ID NO: 27 - BIO5 (WT protein, PPU4513) MSSSERSEVKFDKHFNWWSLLGIAFSLSCSWVGISASMAVGIASGGPLLIIYGLIIAAFFSLMCGISLGD FAAILPNSSGGSFWVLKMLEQESVTLKTPEYEDPSDDDEEVFLENYCQTVNVEVSSKFQKVSSMWGLLN YFGAI FTTASICSSLSMSCIGIHKLLHPDYELKHWHVFVGYECINAVLTLFNIYSTPLPYISQFGLYTSL LSFAMTFIICIVSRSDNTVDPWPKASNIFGSFDNQTGWNSSGMAFWGLVNPIWAFVGIDSATHMIDEVG YSKSRFLVPKVIITTIIVGFVTSFIYCVGLFFCITDQTAWESILPIVEIFHQATGNRNLSVFLQCMCIT TGFVSGIASGTWQSRILQSFAKSYAPFYKEGSLGNKSLKKLAMLTPGFKSPLYAHFLSQICVTIIGCIFM GSSTAFNAIITACITLLLMSYAVPSFIFLFVIKKEKFIHRIESDVNCVSRPNRRRMSMIPHIICILWTLF CLVFLSFPYTLPVTAGNMNYTSWYAWFCIISIWFPTCI

[0634] References

[0635] Odat et al., (2007) ‘Old yellow enzymes, highly homologous FMN oxidoreductases with modulating roles in oxidative stress and programmed cell death in yeast’, Journal of Biological Chemistry, 282(49), pp. 36010-36023. doi:10.1074 / jbc.m704058200.

[0636] Haslbeck K, Bub S, von Kamp K, Michel M, Zarnkow M, Hutzler M, et al. The influence of brewing yeast strains on monoterpene alcohols and esters contributing to the citrus flavour of beer. Journal of the Institute of Brewing [Internet], John Wiley and Sons Inc.; 2018 [cited 2020 Mar 25]; 124:403-15. Available from: http: / / doi.wiley.com / 10.1002 / jib.523

[0637] Noguerol-Pato R, Gonzalez-Barreiro C, Cancho-Grande B, Simal-Gandara J.

[0638] Quantitative determination and characterisation of the main odourants of Mencia monovarietal red wines. Food Chemistry. 2009;117:473-84.

[0639] Ferreira V, Lopez R, Cacho JF. Quantitative determination of the odorants of young red wines from different grape varieties. Journal of the Science of Food and Agriculture.

[0640] 2000;80. Ribereau-Gayon, P., Boidron, J. N., & Terrier, A. (1975). Aroma of Muscat Grape Varieties. Journal of Agricultural and Food Chemistry, 23(6).

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[0643] Hiroaki Negoro, Hiroki Ishida, Development of sake yeast breeding and analysis of genes related to its various phenotypes, FEMS Yeast Research, Volume 22, Issue 1, 2022, foac057, https: / / doi.org / 10.1093 / femsyr / foac057

[0644] Kitagaki H, Kitamoto K. Breeding research on sake yeasts in Japan: history, recent technological advances, and future perspectives. Annu Rev Food Sci Technol.

[0645] 2013;4:215-35. doi: 10.1146 / annurev-food-030212-182545. PMID: 23464572.

[0646] Erny C, Raoult P, Alais A, Butterlin G, Delobel P, Matei-Radoi F, Casaregola S, Legras JL. Ecological success of a group of Saccharomyces cerevisiae / Saccharomyces kudriavzevii hybrids in the northern european wine-making environment. Appl Environ Microbiol. 2012 May;78(9):3256-65. doi: 10.1128 / AEM.06752-11. Epub 2012 Feb 17. PMID: 22344648; PMCID: PMC3346444.

[0647] Lea, A. (2018) Craft Cider making. Marlborough: The Crowood Press.

[0648] Kosseva, M., Joshi, V. and Panesar, P. (2017) ‘Specific Features of Table Wine Production Technology’, in Science and Technology of Fruit Wine Production. London, United Kingdom: Academic Press, pp. 295-461.

[0649] Grumezescu, A., & Holban, A. M. (Eds.). (2019). Fermented Beverages. Volume 5. The Science of Beverages (1st ed.).

Claims

Claims1. A method for producing a fruit or malt based beverage, said method comprising;i) providing a beverage base comprising fruit juice or an aqueous extract of malt for fermentation, andii) providing a sake yeast strain of the species Saccharomyces cerevisiae, wherein said yeast strain comprises an OYE3 gene encoding full length OYE3 protein, preferably OYE3 of SEQ ID NO: 2 or SEQ ID NO: 18, or a polypeptide with at least 80%, such as at least 85%, such as at least 90%, such as at least 95% sequence identity to SEQ ID NO:s 2 or 18, iii) fermenting the beverage base provided in step i) with said yeast strain of step ii),iv) stopping fermentation before fermentation is complete,v) thereby obtaining a fermented beverage base,vi) processing the fermented beverage base into a fruit or malt based beverage.

2. A method for producing a cider, said method comprising;i) providing a cider base comprising fruit juice for fermentation, and ii) providing a sake yeast strain of the species Saccharomyces cerevisiae, wherein said yeast strain comprises an OYE3 gene encoding full length OYE3 protein, preferably OYE3 of SEQ ID NO: 2 or SEQ ID NO: 18, or a polypeptide with at least 80%, such as at least 85%, such as at least 90%, such as at least 95% sequence identity to SEQ ID NO:s 2 or 18, iii) fermenting the cider base provided in step i) with said yeast strain of step ii),iv) stopping fermentation before fermentation is complete,v) thereby obtaining a fermented cider base,vi) processing the fermented cider base into a cider.

3. The method according to any one of the preceding claims, wherein said yeast strain comprises an OYE3 gene encoding full length OYE3 protein, preferably OYE3 of SEQ ID NO: 2 or SEQ ID NO: 18, or a polypeptide with at least 80%, such as at least 85%, such as at least 90%, such as at least 95% sequence identity to SEQ ID NO:s 2 or 18.

4. The method according to any one of the preceding claims, wherein the sake yeast strain is a Kyokai 7, a Kyokai 6 or a Kyokai 9.

5. The method any one of the preceding claims, wherein the sake yeast strain is a Kyokai 7.

6. The method according to any one of the preceding claims, wherein said sake yeast strain comprises a gene encoding BIO1 of SEQ ID NO: 16 or SEQ ID NO: 21, or a polypeptide sharing at least 70% sequence identity therewith, such as at least 80% sequence identity with SEQ ID NO:s 16 or 21, such as at least 90% sequence identity therewith, such as at least 95% sequence identity with SEQ ID NO:s 16 or 21, such as at least 98% sequence identity with SEQ ID NO:s 16 or 21.

7. The method according to any one of the preceding claims, wherein said sake yeast strain comprises a gene encoding BIO6 of SEQ ID NO: 17 or 22, or a polypeptide sharing at least 70% sequence identity with SEQ ID NO:s 17 or 22, such as at least 80% sequence identity with SEQ ID NO:s 17 or 22, such as at least 90% sequence identity therewith, such as at least 95% sequence identity therewith, such as at least 98% sequence identity with SEQ ID NO:s 17 or 22.

8. The method according any one of the preceding claims, wherein the beverage is a cider, and wherein fermentation of the cider base with said yeast strain improves the flavour profile of said cider compared to a cider produced by fermenting said cider base with a conventional cider yeast.

9. The method according to any one of the preceding claims, wherein the beverage is a cider, and wherein said sake yeast strain is capable of producing a fermented cider base comprising decreased average levels of one or more of the following compound(s) compared to a reference fermented cider base prepared in the same manner but fermented with the S. cerevisiae yeast strain; Uvaferm CEG; wherein said compounds are decanoic acid, isobutanol, and / or total fusel alcohols such as butanol, 2-methyl butanol and / or 3-methyl butanol.

10. The method according to any one of the preceding claims, wherein the beverage is a cider, and wherein said sake yeast strain is capable of producing a fermented cider base comprising increased average levels of one or more of the following compound(s) compared to a reference fermented cider base prepared in the same manner but fermented with the S. cerevisiae yeast strain; Uvaferm CEG; wherein said compounds are ethyl 3-hydroxybutanoate, total levels of terpenes such as linalol, nerol, geraniol, citronellol and / or alpha terpineol, total levels of nerol, gernaniol and citronellol, nerol, geraniol, citronellol, total fermentable sugars such as fructose and / or glucose, sorbitol, and / or malate.

11. The method according to any one of the preceding claims, wherein the beverage is a cider, and wherein said sake yeast strain is capable of producing a fermented cider base comprisingi) increased average levels of ethyl 3-hydroxybutanoate of at least 125%, such as at least 150%, such as at least 175%, such as at least 200%, such as at least 225%, such as at least 250%, such as at least 275%, such as at least 300%, such as in the range of 125-500%, such as in the range of 150-400% compared to a reference fermented cider base prepared in the same manner but fermented with the S. cerevisiae yeast strain; Uvaferm CEG, and / or;ii) increased average levels of ethyl hexanoate of at least 120%, such as at least 135%, such as at least 150%, such as at least 175%, such as at least 200%, such as at least 225%, such as at least 250%, such as in the range of 130-400%, such as in the range of 140-300% compared to a reference fermented cider base prepared in the same manner but fermented with the S. cerevisiae yeast strain; Uvaferm CEG, and / or; iii) increased average levels of ethyl octanoate of at least 110%, such as at least 125%, such as at least 150%, such as at least 175%, such as at least 200%, such as in the range of 110-350% compared to a reference fermented cider base prepared in the same manner but fermented with the S. cerevisiae yeast strain; Uvaferm CEG.

12. The method according to any one of the preceding claims, wherein the beverage is a cider, and wherein said sake yeast strain is capable of producing afermented cider base comprising increased relative average levels of citronellol of at least 140%, such as at least 150%, such as at least 160%, such as at least 170%, such as at least 180%, such as at least 190%, such as at least 200%, such as at least 210%, such as in the range of 140-400%, such as in the range of 150-350% compared to a reference fermented cider base prepared in the same manner but fermented with the S. cerevisiae yeast strain; Uvaferm CEG.

13. The method according to any one of the preceding claims, wherein the beverage is a cider, and wherein said sake yeast strain is capable of producing a fermented cider base comprising increased relative average levels of total fermentable sugars such as fructose and / or glucose of at least 180%, such as at least 190%, such as at least 200%, such as at least 210%, such as at least 220%, such as at least 230%, such as at least 240%, such as at least 250%, such as at least 260% such as in the range of 180-500%, such as in the range of 190-400% compared to a reference fermented cider base prepared in the same manner but fermented with the S. cerevisiae yeast strain; Uvaferm CEG.

14. The method according to any one of the preceding claims, wherein fermentation is stopped when the fermented beverage base has;i) an ABV of in the range of 8 to 17%, such as in the range of 9 to 14%, preferably in the range of 9 to 13%, or more preferably in the range of 10 to 13%, or more preferably in the range of 10 to 12%, or more preferably in the range of 10 to 11%; and / or,ii) a specific gravity in the range of 0.5 to 5°Plato, or more preferably in the range of 0.5 to 4.5°Plato, or more preferably in the range of 0.5 to 4°Plato, or more preferably in the range of 0.7 to 3.8°Plato, and / oriii) a concentration of remaining fermentable sugars in the range of 10-60g / L, or more preferably 15-55g / L, or more preferably 15-50g / L, or more preferably 20-50g / L.

15. The method according to any one of the preceding claims, wherein said beverage is a cider, a perry, or a fruit wine, such as apple wine, pear wine, cherry wine or plum wine, preferably wherein said beverage is a cider.

16. A sake yeast strain of the species Saccharomyces cerevisiae, wherein said yeast comprises;i) a gene encoding BIO1 of SEQ ID NO: 16 or SEQ ID NO: 21, or a polypeptide sharing at least 70% sequence identity with SEQ ID NO:s 16 or 21, such as at least 80% sequence identity therewith, such as at least 90% sequence identity with SEQ ID NO:s 16 or 21, such as at least 95% sequence identity with SEQ ID NO:s 16 or 21, such as at least 98% sequence identity with SEQ ID NO:s 16 or 21, wherein said gene encoding BIO1 is expressed from its endogenous promoter, and; ii) a gene encoding BIO6 of SEQ ID NO: 17 or 22, or a polypeptide sharing at least 70% sequence identity with SEQ ID NO:s 17 or 22, such as at least 80% sequence identity with SEQ ID NO:s 17 or 22, such as at least 90% sequence identity with SEQ ID NO:s 17 or 22, such as at least 95% sequence identity with SEQ ID NO:s 17 or 22, such as at least 98% sequence identity with SEQ ID NO:s 17 or 22, wherein said gene encoding BIO6 is expressed from its endogenous promoter.

17. The method or sake yeast strain according to any one of the preceding claims, wherein said yeast straini) comprises an OYE3 gene encoding full length OYE3 protein, preferably OYE3 of SEQ ID NO: 2 or SEQ ID NO: 18, or a polypeptide with at least 80%, such as at least 85%, such as at least 90%, such as at least 95% sequence identity to SEQ ID NO:s 2 or 18; and / orii) does not comprise an OYE3 gene comprising a frame shift mutation, for example the yeast strain does not comprise an OYE3 gene comprising a Glu259 frame shift mutation, corresponding to amino acid position 259 of SEQ ID NO: 2 or SEQ ID NO: 18; and / oriii) comprises an OYE3 gene encoding an OYE3 protein, which does not comprise a Val114I le mutation in the amino acid corresponding to amino acid position 114 of SEQ ID NO: 2 or SEQ ID NO: 18; and / or iv) comprises an OYE3 gene encoding an OYE3 protein comprising an lle182Thr mutation, preferably encoding an OYE3 of SEQ ID NO: 3 or SEQ ID NO: 18.

18. The method or sake yeast strain according to any one of the preceding claims, wherein said yeast strain comprises an ATF1 gene encoding an ATF1 protein comprising a Thr84lle mutation, preferably encoding ATF1 of SEQ ID NO: 9 or SEQ ID NO: 19, or a polypeptide with at least 80%, such as at least 85%, such as at least 90%, such as at least 95% sequence identity to SEQ ID NO:s 9 or 19.

19. The method or sake yeast strain according to any one of the preceding claims, wherein said yeast strain carries a premature stop codon in the gene encoding FDC1, wherein said mutant gene encodes a truncated FDC1 comprising at the most 54 consecutive amino acids or at the most 53 consecutive amino acids of FDC1 of SEQ ID NO: 10, such as wherein said yeast strain carries a Lys54STOP mutation in the gene encoding FDC1, corresponding to amino acid position 54 of SEQ ID NO: 10, preferably wherein said mutant gene encodes the truncated FDC1 as set forth in SEQ ID NO: 20.

20. The sake yeast strain according to any one of claims 16 to 19, wherein said yeast strain is capable of producing a fermented cider base comprising increased average levels of one or more of the following compound(s) compared to a reference fermented cider base prepared in the same manner but fermented with the Kyokai yeast strain yeast strain; NCYC 479; wherein said compounds are isoamyl acetate, 2-phenylethyl acetate, ethyl hexanoate, ethyl octanoate, ethyl butanoate, ethyl 2-hydroxyisocaproate, total levels of medium-chain fatty acid esters such as ethyl decanoate, ethyl hexanoate, ethyl octanoate, and / or ethyl butanoate, citronellol, butanol, total levels of 2-methyl butanol and 3-methyl butanol, butanoic acid, hexanoic acid, total levels of fusel alcohols such as isobutanol, butanol, 2-methyl butanol and / or 3-methyl butanol, malate, and / or total levels of medium-chain fatty acid esters, such as ethyl decanoate, ethyl hexanoate, ethyl octanoate and / or ethyl butanoate, and terpenes, such as linalol, nerol, geraniol, citronellol and alpha terpineol.

21. A method for producing a beverage, such as a cider, said method comprising;i) providing a beverage base, such as a cider base, comprising fruit juice or an aqueous extract of malt for fermentation, andii) providing a sake yeast strain according to any one of claims 16 to 20,iii) fermenting the beverage base, such as the cider base, provided in step i) with said yeast strain of step ii),iv) thereby obtaining a fermented beverage base, such as a fermented cider base,v) processing the fermented beverage base, such as the fermented cider base, into a beverage, such as a cider.

22. A beverage, such as a cider, produced by the method or sake yeast strain according to any one of the preceding claims.

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