Low Diacetyl Yeast

Abstract

The present invention relates to yeast strains of the species Saccharomyces pastorianus that have the useful property of producing low levels of diacetyl during fermentation. Methods for producing malt- and / or grain-based beverages using these strains, as well as the beverages produced thereby, are also provided.

Description

Technical Field

[0001] The present invention relates to the field of beer production, particularly to the field of lager beer production. More specifically, the invention provides a low diacetyl yeast, which is particularly useful for fast and efficient fermentation during beer production, especially in the production of lager beer.

Background Art

[0002] Lager beer is often characterized by a fresh and clean flavor profile. Diacetyl contributes to the flavor profile of many fermented products. However, its typical buttery flavor is considered an off-flavor in lager-style beers, and the removal of this compound has a significant impact on time and energy consumption in breweries.

[0003] Lager beer is usually prepared by the fermentation of wort, a carbohydrate-rich liquid, with lager yeast. Lager yeast generally differs from top-fermenting yeast in several respects. Lager yeast generally belongs to the species Saccharomyces pastorianus. Frequently, lager yeast is also called "bottom-fermenting yeast" because they sediment to the bottom during fermentation. The sedimentation of yeast, or flocculation, can also affect the processing time because the yeast needs to sediment sufficiently to be collected for the next brewing. For lager yeast that does not flocculate (settles gradually), this requires cooling, resulting in additional processing time. Furthermore, lager yeast strains are generally used optimally at temperatures in the range of 7°C to 18°C. In contrast to top-fermenting yeast, which generally belongs to the species Saccharomyces cerevisiae, lager yeast can use melibiose as the sole carbon source and typically cannot grow at 37°C.

[0004] During fermentation, diacetyl is formed by the non-enzymatic oxidation of acetolactate, which is excreted by yeast cells; however, during maturation, diacetyl is reabsorbed and metabolized by yeast cells. Part of fermentation management is undertaken to ensure that the finished beer contains diacetyl below a set threshold. The problem of reducing the diacetyl content in the finished beer is particularly pronounced in lager beers, as fermentation occurs at lower temperatures, resulting in slower reabsorption and metabolism of diacetyl by lager yeast.

[0005] The lower limit of diacetyl's taste perception in beer is generally considered to be 50 ppb, and the need to reduce the diacetyl concentration to this level in the finished beer traditionally adds a considerable amount of extra time required for the maturation of lager beer before the brewing process is complete. [Overview of the project]

[0006] As outlined above, conventional fermentation of wort at temperatures of 7°C to 18°C ​​using the Saccharomyces pastorianus lager yeast strain results in diacetyl levels in the wort that easily exceed the 50 ppb limit for taste perception. Consequently, some additional maturation days by the yeast were required to reduce these levels and ensure that the diacetyl content of the finished beer was below the target threshold.

[0007] Interestingly, the present invention provides novel hybrid variants of Saccharomyces pastorianus yeast strains that produce low levels of diacetyl and / or consume the diacetyl immediately during fermentation at temperatures below 18°C, resulting in beers produced by these strains requiring little to no maturation time. In particular, when wort is fermented with the yeast strains of the invention, the total diacetyl level is ≤60 ppb, preferably ≤50 ppb, at the time when sugar fermentation is complete, i.e., when the sugar level is approximately stable during fermentation. Generally, a reduction of at least one or at least two days in fermentation time is observed compared to current lager strains.

[0008] The present invention also provides a method for producing a beverage that requires little to no maturation time after fermentation at 18°C ​​or below by using the S. pastorianus yeast strain, and which has a pleasant taste.

[0009] In one embodiment, the present invention provides a method for producing a fermented aqueous extract, the method comprising the following steps: i) A step of providing an aqueous extract of malt and / or cereals; ii) A step of providing a yeast strain of the species Saccharomyces pastorianus, wherein the yeast strain can produce a fermentation test solution by incubation in a test solution, the test solution being an extract of malt and / or cereals having an appearance extract content of at least 10° Prato, the fermentation test solution containing a maximum of 60 ppb of diacetyl, preferably a maximum of 50 ppb of diacetyl, at the earliest point when the appearance extract content of the fermentation test solution has not decreased by more than 0.50° Prato in the preceding 24 hours, and the fermentation occurring at a temperature of 18°C ​​or less, preferably 12-16°C; and iii) A step of fermenting the aqueous extract provided in step i) with the yeast strain of step ii), thereby obtaining a fermented aqueous extract.

[0010] In this specification, the term “aqueous extract” is used to refer to an aqueous extract of malt and / or grain, such as wort, which is used to produce beverages, such as beer, while the term “test solution” refers to a solution with a more strictly defined composition used to evaluate the characteristics of microorganisms used for fermentation.

[0011] In another embodiment, the present invention provides a method for producing a beverage, the method comprising the following steps: i. A step of providing an aqueous extract of malt and / or cereals; ii. A step of providing a yeast strain of the species Saccharomyces pastorianus, wherein the yeast strain can produce a fermentation test solution by incubation in a test solution, the test solution being an extract of malt and / or cereals having an appearance extract content of at least 10° Prato, the test solution containing up to 60 ppb of diacetyl, preferably up to 50 ppb of diacetyl, at the earliest point when the appearance extract content of the test solution has not decreased by more than 0.50° Prato in the preceding 24 hours, and the fermentation occurs at a maximum temperature of 18°C; iii. A step of fermenting the aqueous extract provided in step i) with the yeast strain to obtain a fermented aqueous extract; and iv. A step of processing the fermented aqueous extract to make a beverage.

[0012] In another embodiment, the present invention provides a method for producing a beverage, the method comprising the following steps: i. A step of providing an aqueous extract of malt and / or cereals; ii. A step of providing a yeast strain of the species Saccharomyces pastorianus, wherein the yeast strain can produce a fermentation test solution by incubation in a test solution, the test solution being an extract of malt and / or cereals having an appearance extract content of at least 10° Prato, the test solution containing up to 60 ppb of diacetyl, preferably up to 50 ppb of diacetyl, at the earliest point when the appearance extract content of the test solution has not decreased by more than 0.50° Prato in the preceding 24 hours, and the fermentation occurring at a maximum temperature of 18°C; iii. A step of fermenting the aqueous extract provided in step i) with the yeast strain to obtain a fermented aqueous extract; and iv. A process of processing the fermented aqueous extract to make a beverage, wherein the processing step includes one or more of the following steps: 1. Filtration, 2. Carbonation, 3. Aging, or 4. Bottle.

[0013] In another embodiment, the present invention provides a yeast strain that, after incubation of the yeast strain in a test solution having an apparent extract content of at least 10° Prato, can produce a fermentation test solution containing up to 60 ppb of diacetyl, preferably up to 50 ppb of diacetyl, at the earliest point in time when the apparent extract content of the test solution has not decreased by more than 0.50° Prato in the preceding 24 hours after incubation of the yeast strain in the extract. [Brief explanation of the drawing]

[0014] [Figure 1] Figure 1C shows the diacetyl and phosphate levels of wort incubated with the hybrid yeast strain ZDA1 and the control yeast strain Hybrid Yeast 7 from 50L trials at (A) 16°C and (B) 18°C. Figure 1C is the same as Figure 1B, but plotted at 18°C ​​with ZDA1 strain only and using a different axis for total diacetyl. The results are described in Example 1. [Figure 2] Diacetyl levels of wort incubated with hybrid yeast strain ZDA1 and control yeast strain Hybrid Yeast 7 from a 10HL trial at 16° are shown (Figure 2A). °Plate is also shown for hybrid yeast strain ZDA1 (Figure 2B). Results are described in Example 4. [Figure 3] This shows the ratio of propanol per isobutanol in nine different commercially available beers compared to beer prepared with the yeast strain ZDA2 (89-84393 ZDA F1) according to the present invention. All beers are close to 5% ABV. [Figure 4A] This shows a sequence comparison of ILV2 protein sequences between WS34-78, cer (Saccharomyces cerevisiae), eub (Saccharomyces eubyanus), Hybrid7 (Hybrid Yeast 7), ZDA1, and ZDA2. [Figure 4B] This shows a sequence comparison of ILV2 protein sequences between WS34-78, cer (Saccharomyces cerevisiae), eub (Saccharomyces eubyanus), Hybrid7 (Hybrid Yeast 7), ZDA1, and ZDA2. [Figure 4C] This shows a sequence comparison of ILV2 protein sequences between WS34-78, cer (Saccharomyces cerevisiae), eub (Saccharomyces eubyanus), Hybrid7 (Hybrid Yeast 7), ZDA1, and ZDA2. [Figure 4D] This shows a sequence comparison of ILV2 protein sequences between WS34-78, cer (Saccharomyces cerevisiae), eub (Saccharomyces eubyanus), Hybrid7 (Hybrid Yeast 7), ZDA1, and ZDA2. [Figure 4E] This shows a sequence comparison of ILV2 protein sequences between WS34-78, cer (Saccharomyces cerevisiae), eub (Saccharomyces eubyanus), Hybrid7 (Hybrid Yeast 7), ZDA1, and ZDA2. [Figure 4F] This shows a sequence comparison of ILV2 protein sequences between WS34-78, cer (Saccharomyces cerevisiae), eub (Saccharomyces eubyanus), Hybrid7 (Hybrid Yeast 7), ZDA1, and ZDA2. [Figure 5A] This shows a sequence comparison of ILV6 protein sequences between WS34-78, cer (Saccharomyces cerevisiae), eub (Saccharomyces eubyanus), Hybrid7 (Hybrid Yeast 7), ZDA1, and ZDA2. [Figure 5B] This shows a sequence comparison of ILV6 protein sequences between WS34-78, cer (Saccharomyces cerevisiae), eub (Saccharomyces eubyanus), Hybrid7 (Hybrid Yeast 7), ZDA1, and ZDA2. [Figure 5C] This shows a sequence comparison of ILV6 protein sequences between WS34-78, cer (Saccharomyces cerevisiae), eub (Saccharomyces eubyanus), Hybrid7 (Hybrid Yeast 7), ZDA1, and ZDA2. [Figure 6A]Sequence comparison of the ILV3 protein sequences between WS34-78, cer (Saccharomyces cerevisiae), eub (Saccharomyces uvarum), Hybrid7 (Hybrid yeast 7), ZDA1 and ZDA2 is shown. [Figure 6B] Sequence comparison of the ILV3 protein sequences between WS34-78, cer (Saccharomyces cerevisiae), eub (Saccharomyces uvarum), Hybrid7 (Hybrid yeast 7), ZDA1 and ZDA2 is shown. [Figure 6C] Sequence comparison of the ILV3 protein sequences between WS34-78, cer (Saccharomyces cerevisiae), eub (Saccharomyces uvarum), Hybrid7 (Hybrid yeast 7), ZDA1 and ZDA2 is shown. [Figure 6D] Sequence comparison of the ILV3 protein sequences between WS34-78, cer (Saccharomyces cerevisiae), eub (Saccharomyces uvarum), Hybrid7 (Hybrid yeast 7), ZDA1 and ZDA2 is shown. [Figure 6E] Sequence comparison of the ILV3 protein sequences between WS34-78, cer (Saccharomyces cerevisiae), eub (Saccharomyces uvarum), Hybrid7 (Hybrid yeast 7), ZDA1 and ZDA2 is shown. [Figure 7A] Sequence comparison of the ILV5 protein sequences between WS34-78, cer (Saccharomyces cerevisiae), eub (Saccharomyces uvarum), Hybrid7 (Hybrid yeast 7), ZDA1 and ZDA2 is shown. [Figure 7B] Sequence comparison of the ILV5 protein sequences between WS34-78, cer (Saccharomyces cerevisiae), eub (Saccharomyces uvarum), Hybrid7 (Hybrid yeast 7), ZDA1 and ZDA2 is shown. [Figure 7C] Sequence comparison of the ILV5 protein sequences between WS34-78, cer (Saccharomyces cerevisiae), eub (Saccharomyces uvarum), Hybrid7 (Hybrid yeast 7), ZDA1 and ZDA2 is shown. [Figure 7D] This shows a sequence comparison of ILV5 protein sequences between WS34-78, cer (Saccharomyces cerevisiae), eub (Saccharomyces eubyanus), Hybrid7 (Hybrid Yeast 7), ZDA1, and ZDA2. [Figure 8A] This shows a sequence comparison of the BAT1 protein sequences between WS34-78, cer (Saccharomyces cerevisiae), eub (Saccharomyces eubyanus), Hybrid7 (Hybrid Yeast 7), ZDA1, and ZDA2. [Figure 8B] This shows a sequence comparison of the BAT1 protein sequences between WS34-78, cer (Saccharomyces cerevisiae), eub (Saccharomyces eubyanus), Hybrid7 (Hybrid Yeast 7), ZDA1, and ZDA2. [Figure 8C] This shows a sequence comparison of the BAT1 protein sequences between WS34-78, cer (Saccharomyces cerevisiae), eub (Saccharomyces eubyanus), Hybrid7 (Hybrid Yeast 7), ZDA1, and ZDA2. [Figure 8D] This shows a sequence comparison of the BAT1 protein sequences between WS34-78, cer (Saccharomyces cerevisiae), eub (Saccharomyces eubyanus), Hybrid7 (Hybrid Yeast 7), ZDA1, and ZDA2. [Figure 8E] This shows a sequence comparison of the BAT1 protein sequences between WS34-78, cer (Saccharomyces cerevisiae), eub (Saccharomyces eubyanus), Hybrid7 (Hybrid Yeast 7), ZDA1, and ZDA2. [Figure 9A] This shows a sequence comparison of the BAT2 protein sequences between WS34-78, cer (Saccharomyces cerevisiae), eub (Saccharomyces eubyanus), Hybrid7 (Hybrid Yeast 7), ZDA1, and ZDA2. [Figure 9B]This shows a sequence comparison of the BAT2 protein sequences between WS34-78, cer (Saccharomyces cerevisiae), eub (Saccharomyces eubyanus), Hybrid7 (Hybrid Yeast 7), ZDA1, and ZDA2. [Figure 9C] This shows a sequence comparison of the BAT2 protein sequences between WS34-78, cer (Saccharomyces cerevisiae), eub (Saccharomyces eubyanus), Hybrid7 (Hybrid Yeast 7), ZDA1, and ZDA2. [Figure 9D] This shows a sequence comparison of the BAT2 protein sequences between WS34-78, cer (Saccharomyces cerevisiae), eub (Saccharomyces eubyanus), Hybrid7 (Hybrid Yeast 7), ZDA1, and ZDA2. [Figure 10] The °Plate and diacetyl levels of wort incubated with yeast strain ZDA2 and control yeast strain Hybrid Yeast 7 from a commercial-scale trial at 16°C are shown. The results are described in Example 8. [Modes for carrying out the invention]

[0015] definition In this specification, "one (a)" can mean one or more, depending on the context in which it is used.

[0016] In this specification, the term “approximately” means ±10%, preferably ±5%, and more preferably ±2%.

[0017] The term "beer" as used herein refers to a beverage prepared by the fermentation of wort. Preferably, the fermentation is carried out by yeast.

[0018] In this specification, the term "green beer" refers to the solution obtained immediately after the fermentation of the wort by yeast, preferably. Therefore, "green beer" is obtained before lagering. In other words, green beer is the solution obtained immediately after the completion of sugar fermentation. Generally speaking, beer is no longer considered "green" when it has reached full maturity in flavor and aroma.

[0019] In this specification, the term "diacetyl" refers to the chemical compound of the following formula: [ka]

[0020] The concentration of diacetyl in a sample can be measured by gas chromatography in accordance with European Brewing Convention method EBC 9.24.2, which includes a 90-minute incubation period of the sample at 60°C to determine the total diacetyl, and reflects the sum of precursor acetolactate and free diacetyl. Throughout this disclosure, when describing diacetyl produced by the yeast strains disclosed herein, the term "diacetyl" refers to "total diacetyl" unless otherwise specified. Therefore, the diacetyl concentration of a sample represents the total diacetyl concentration, i.e., the sum of precursor acetolactate and free diacetyl.

[0021] The term “cereals” as used herein refers to any grass species from which edible grains are obtained, such as wheat, foxtail millet, rice, barley, oats, rye, rye wheat, sorghum, and maize.

[0022] The term "grain" as used herein refers to the seed of a cereal, including the cereal floret, also called the inner seed. In addition, the grain may include the outer and inner glumes. In most barley varieties, the outer and inner glumes are attached to the floret and become part of the grain after threshing. However, naked barley varieties also occur. In these, the floret does not have an outer or inner glume and is released after threshing, like wheat. The terms "grain" and "kernel" are used herein with the same meaning.

[0023] The term “wort” refers to the liquid extract of malt and / or grains and optionally additional adjuncts. Wort is generally obtained by mashing, optionally followed by “sparging,” in the process of extracting residual sugars and other compounds from the spent grains after mashing with hot water. Sparging is typically carried out in a filtration tank, mash filter, or other apparatus, allowing for the separation of the extract from the spent grains. The wort obtained after mashing is generally called “first wort,” while the wort obtained after sparging is generally called “second wort.” Unless otherwise specified, the term wort may refer to first wort, second wort, or a combination of both. In conventional beer production, wort is boiled with hops. Wort without hops may also be called “sweet wort,” while wort boiled with hops may be called “boiled wort” or simply wort.

[0024] The term "aqueous extract" as used herein refers to any aqueous extract of malt and / or grain. Thus, this non-limiting example may be wort having a predetermined amount of fermentable sugars.

[0025] The term "fermented aqueous extract" as used herein refers to any aqueous extract obtained after incubation with microorganisms, such as a yeast strain, and completion of sugar fermentation. Sugar fermentation is considered complete when the sugar level, as measured by °Prate, no longer decreases significantly during fermentation. Preferably, sugar fermentation can be considered complete when the sugar level does not change by more than 0.5 °Prate over a 24-hour period, or when the sugar level does not change by more than 0.25 °Prate over a 12-hour period. Fermented aqueous extracts may be, for example, fermented malt and / or cereal extracts.

[0026] The term "test solution" in this specification refers to any aqueous liquid or solution. A test solution may contain a predetermined level of a specific compound. Therefore, this non-limiting example could be a wort having a specific sugar content.

[0027] In this specification, the term "fermentation test solution" refers to any test solution that has been incubated with microorganisms, such as a yeast strain, and in which sugar fermentation has been completed.

[0028] The term "Plato" in this specification refers to density measured on a Plato scale. A Plato scale is an empirically derived hydrometer scale for measuring the density of beer or wort in terms of weight percentage of extract. The scale expresses density as grams of extract per 100 g of wort. Plato can be measured, for example, using an Alcolyzer or handheld device manufactured by Anton Paar.

[0029] "Appearance extract content" in this specification refers to the density of a given beer or wort measured with a °Plat. Since density is primarily determined by sugar content, appearance extract content refers to the sugar content of the solution or extract. The appearance extract content of a solution can be measured, for example, using a handheld Anton-PAAR serial number DM.

[0030] The term "Alcohol Content (ABV)" as used herein refers to the amount of alcohol (ethanol) in a given volume of an alcoholic beverage (expressed as a volume percentage). It is defined as the number of milliliters of pure ethanol present in 100 mL of solution at 20°C. ABV can be measured using an alcoholizer.

[0031] The term "RDF" or "Degree of Fermentation" as used herein refers to the degree to which the sugars in the wort have fermented into alcohol in the beer, and is also called the degree of fermentation. RDF represents the percentage of fermented extract. An RDF of 50-60% represents a full-bodied beer in which more than 40% of their original extract remains unfermented, while an RDF of over 80% represents a highly diluted beer in which less than 20% of their original extract remains unfermented. The texture is largely determined by the RDF percentage; the higher the RDF percentage, the lighter and drier the beer.

[0032] The term “cottony sedimentation” as used herein refers to the process by which fine particles, such as yeast cells, aggregate to form a cottony substance. This cottony substance can then float to the top of the liquid (creaming), settle to the bottom of the liquid (sedimentation), or be easily filtered out of the liquid. Yeast experts and brewers often classify yeast cottony sedimentation behavior as “high,” “medium,” or “low,” depending on the degree of cottony sedimentation observed for a yeast strain during the fermentation process. High cottony strains can produce a brighter beer with less suspended yeast and are easier to filter. Cottony sedimentation can be increased at lower temperatures, and therefore, with low cottony yeasts, an additional cooling step may be required after fermentation is complete. Thus, high cottony yeasts can reduce processing time compared to low cottony yeasts, as cooling is not required to achieve a brighter, more easily filtered beer. The cottony precipitate can be determined, for example, by counting the number of yeast cells in the solution after fermentation, or by counting the number of yeast cells in a sample taken from the top three-quarters of a container containing the fermented aqueous extract or test solution.

[0033] In this specification, the term "fermentation" refers to the incubation of an aqueous extract or test solution with a microorganism, such as a yeast strain.

[0034] The term "malt" as used herein refers to malted grains. The term "green malt" refers to germinated grains that have not been subjected to the kiln-drying process. In some embodiments, green malt is ground green malt. The term "kiln-dried malt" as used herein refers to germinated grains that have been dried by kiln drying. In some embodiments, kiln-dried malt is ground kiln-dried malt. Generally, the grains are germinated under controlled environmental conditions.

[0035] The term “carbon source” as used herein refers to any organic molecule that can provide energy to yeast and provide carbon for cellular biosynthesis. In particular, the carbon source may be a carbohydrate, and more preferably, the carbon source may be a monosaccharide, disaccharide, trisaccharide, tetrasaccharide and / or short oligosaccharide. Carbon sources that can be fermented by yeast are often called fermentable sugars, and examples include, but are not limited to, glucose, fructose, maltose, maltotriose and sucrose.

[0036] In this specification, amino acids may be named using IUPAC one-letter and three-letter codes. Unless otherwise specified, the term "amino acid" refers to a standard amino acid.

[0037] The term “functional gene” as used herein refers to a gene that, when transcribed and translated, expresses a protein that, when transcribed and translated, has at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 95%, e.g., at least 99%, e.g., 100% sequence identity with the protein encoded by the corresponding gene in the wild-type, endogenous gene. The protein expressed by a functional gene must be a functional homolog of the protein encoded by the corresponding gene in the wild-type, endogenous gene; that is, it must have the same type of enzymatic activity, and such activity must be at a similar level to that of the protein encoded by the corresponding gene in the wild-type, endogenous gene. In particular, the term “functional gene” does not include genes that encode truncated protein products that have little to no enzymatic activity. Preferably, the term “gene” as used herein refers to a functional gene.

[0038] In this specification, the term "functional copy number" refers to the total number of functional genes in a yeast strain. In this specification, "functional copy number" is used interchangeably with "active copy number."

[0039] The term "functional homolog" as used herein refers to a polypeptide that shares at least one biological function with a reference polypeptide. Generally, such functional homologs also share considerable sequence identity with the reference polypeptide. Preferably, a functional homolog of a reference polypeptide is a polypeptide that has the same biological function as the reference protein and shares a high level of sequence identity with the reference polypeptide.

[0040] The term "natural promoter" as used herein refers to a promoter whose nucleotide sequence has at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 95%, e.g., at least 99%, e.g., 100% sequence identity with the wild-type promoter, where the gene is endogenous. Thus, a gene expressed under such a natural promoter is typically expressed at endogenous levels.

[0041] The term “sequence identity” as used herein describes the relationship between two amino acid sequences or two nucleotide sequences, i.e., between a candidate sequence (e.g., a mutant sequence) and a reference sequence (e.g., a wild-type sequence), based on their paired sequence comparisons. For the purposes of the present invention, sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mo / . Biol. 48: 443-453), which is executed in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later (available at https: / / www.ebi.ac.uk / Tools / psa / emboss_needle / ). The parameters used are a gap start penalty of 10, a gap elongation penalty of 0.5, and an EBLOSUM62 (EMBOSS version of 30BLOSUM62) substitution matrix. The Needle output labeled "Longest Identity" (obtained using the -nobrief option) is used as the percentage identity and is calculated as follows: (identical residues × 100) / (length of alignment - total number of gaps in alignment). The Needleman-Wunsch algorithm is also used to determine whether a given amino acid in a sequence other than the reference sequence corresponds to a given position in the reference sequence. For the purposes of this invention, sequence identity between two nucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, above), which is run in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later.The parameters used are a gap start penalty of 10, a gap elongation penalty of 0.5, and a DNAFULL (EMBOSS version of NCBINUC4.4) substitute matrix. The output of Needle labeled "Longest Identity" (obtained using the -nobrief option) is used as the percentage identity and is calculated as follows: (identical deoxyribonucleotides × 100) / (length of alignment - total number of gaps in alignment). Sequence identity is always measured in comparison to the full-length reference sequence; i.e., a truncated protein without gaps or mismatches is not considered 100% sequence identical to the reference sequence.

[0042] The term “mutation” as used herein includes insertions, deletions, substitutions, transversions, and point mutations in the coding and non-coding regions of a gene. A point mutation may relate to a change of one base pair and may result in an immature stop codon, a frameshift mutation, a splice site mutation, or an amino acid substitution. A gene containing a mutation may be called a “mutant gene.” If such mutant gene codes for a polypeptide having a sequence different from the wild type, such polypeptide may be called a “mutant polypeptide” and / or a “mutant protein.” A mutant polypeptide may be described as harboring a mutation if it contains an amino acid sequence different from the wild type sequence.

[0043] The term "deletion" as used herein may refer to the deletion of an entire gene, a portion of a gene, or a portion of a chromosome.

[0044] The term “stop codon” as used herein refers to a nucleotide triplet in the genetic code, which in mRNA results in the termination of translation. The term “stop codon” as used herein also refers to a nucleotide triplet in a gene that codes for a stop codon in mRNA. Stop codons in DNA typically have one of the following sequences: TAG, TAA, or TGA.

[0045] The term "proliferation," as used herein in relation to yeast, refers to the process by which yeast cells increase in number. Thus, while yeast cells are proliferating, the number of yeast cells increases. The number of yeast cells can be determined by any useful method.

[0046] The term "available" in this specification refers to the ability of yeast to use a particular compound as a carbon and / or nitrogen source for cellular biosynthesis.

[0047] yeast strain This disclosure relates to a yeast strain, such as Saccharomyces pastorianus, which, when incubated in a malt and / or cereal extract having an appearance extract content of at least 10° Prat, surprisingly produces low levels of total diacetyl and / or immediately consumes diacetyl during fermentation at temperatures below 18°C.

[0048] Different types of yeast are used for beer production, the most notable being Saccharomyces pastorianus and Saccharomyces cerevisiae. Lager beer is typically fermented using Saccharomyces pastorianus yeast. Therefore, Saccharomyces pastorianus according to the present invention may be any yeast useful for lager beer production, for example. In particular, Saccharomyces pastorianus may be a bottom-fermenting yeast strain. Saccharomyces pastorianus is preferably a hybrid between S. cerevisiae and S. eubayanus. It is also preferable that the Saccharomyces pastorianus can utilize maltose and melibiose. Therefore, preferably, Saccharomyces pastorianus is a yeast strain which is a hybrid between S. cerevisiae and S. eubayanus. It is also preferable that the Saccharomyces pastorianus can utilize maltose and melibiose.

[0049] In one embodiment, a Saccharomyces pastorianus yeast strain can produce a fermentation test solution after incubation in a test solution of the yeast strain, the test solution being an extract of malt and / or cereals having an appearance extract content of at least 10° Prato, the fermentation test solution containing up to 60 ppb of diacetyl, preferably up to 50 ppb of diacetyl, at the earliest point when the appearance extract content of the test solution has not decreased by more than 0.50° Prato in the preceding 24 hours after incubation of the yeast strain in the extract, and the fermentation occurs at a maximum temperature of 18°C.

[0050] Therefore, the Saccharomyces pastorianus yeast strain according to the present invention can preferably produce a fermentation test solution when tested in a method including the following steps: a) A step of providing a test solution, wherein the test solution is an extract of malt and / or cereals having an appearance extract content of at least 10° Prat. b) Incubating the Saccharomyces pastorianus yeast strain together with the test solution at a temperature of 18°C ​​or lower, preferably 12-18°C, and more preferably 16°C. c) A step of determining the earliest point in time at which the visible extract content of the test solution did not decrease by more than 0.50° Plato during the preceding 24 hours of incubation, and d) A step of determining the level of diacetyl at the earliest point in time, The test solution contains, at the earliest point, a maximum of 60 ppb of diacetyl, preferably a maximum of 50 ppb of diacetyl.

[0051] In some embodiments, the Saccharomyces pastorianus yeast strain can produce a fermentation test solution after incubation with the yeast strain in a test solution, the test solution may be any of the test solutions described below, preferably a malt and / or cereal extract having an appearance extract content of at least 10° Prato, the fermentation test solution contains the maximum level of diacetyl described below at the earliest point after incubation with the yeast strain in the test solution, in the preceding 24 hours, when the appearance extract content of the test solution has not decreased by more than 0.50° Prato, e.g., not by more than 0.40° Prato, e.g., not by more than 0.30° Prato, e.g., not by more than 0.20° Prato, and the fermentation occurs at the temperature specified below. In particular, the appearance extract content of the test solution did not decrease by more than 0.50° Prato in the preceding 24 hours.

[0052] The maximum level of diacetyl is preferably up to 60 ppb, for example, up to 55 ppb of diacetyl, for example, up to 50 ppb of diacetyl, or for example, up to 45 ppb of diacetyl, at the earliest point in time when the visible extract content of the test solution has not decreased by the specified amount. In particular, the level of diacetyl at the earliest point in time may be up to 45 ppb.

[0053] As described above, when identifying the characteristics of yeast, incubation is preferably carried out at a maximum temperature of 18°C, for example, in the range of 12°C to 18°C ​​or 14°C to 16°C. In particular, the incubation may be carried out at a temperature of approximately 16°C.

[0054] In some embodiments, a Saccharomyces pastorianus yeast strain can produce a fermentation test solution after incubation of the yeast strain in a test solution having an apparent extract content of at least 10° Prato, the fermentation test solution containing up to 60 ppb of diacetyl, preferably up to 50 ppb of diacetyl, at the earliest point in the 12 hours preceding incubation of the yeast strain in the extract at which the apparent extract content of the test solution has not decreased by more than 0.25° Prato, and the fermentation occurs at a maximum temperature of 18°C.

[0055] Therefore, the Saccharomyces pastorianus yeast strain according to the present invention can preferably produce a fermentation test solution when tested in a method including the following steps: a) A step of providing a test solution, wherein the test solution is an extract of malt and / or cereals having an appearance extract content of at least 10° Prat. b) A step of incubating the Saccharomyces pastorianus yeast strain with the test solution at a maximum temperature of 18°C. c) A step of determining the earliest point in time at which the visible extract content of the test solution did not decrease by more than 0.25° Plato during the preceding 12 hours of incubation, and d) A step of determining the level of diacetyl at the earliest point in time, The test solution contains, at the earliest point, a maximum of 60 ppb of diacetyl, preferably a maximum of 50 ppb of diacetyl.

[0056] In some embodiments, the Saccharomyces pastorianus yeast strain can produce a fermentation test solution after incubation in a test solution of the yeast strain, the test solution may be any of the test solutions described below, the fermentation test solution containing the maximum level of diacetyl described below at the earliest point in the 24 hours preceding incubation of the yeast strain in the extract at which the apparent extract content of the test solution did not decrease by more than 0.25° Prate, e.g., not by more than 0.20° Prate, e.g., not by more than 0.15° Prate, e.g., not by more than 0.10° Prate, and the fermentation occurs at the temperature specified below. In particular, the apparent extract content of the test solution did not decrease by more than 0.25° Prate in the preceding 12 hours.

[0057] The maximum level of diacetyl is preferably 60 ppb at the earliest point at which the visible extract content of the test solution has not decreased by the specified amount, for example, 55 ppb, 50 ppb, or 45 ppb. In particular, the maximum level of diacetyl at the earliest point may be 50 ppb.

[0058] The incubation is preferably carried out at a maximum temperature of 18°C, for example, in the range of 12°C to 18°C. In particular, the incubation may be carried out at a temperature of approximately 16°C.

[0059] In some embodiments, the Saccharomyces pastorianus yeast strain can produce a fermentation test solution by incubation in a test solution, the test solution being a malt and / or cereal extract having an apparent extract content of at least 10° Prato, the test solution containing up to 120 ppb of diacetyl, for example up to 60 ppb of diacetyl, at the earliest point before the apparent extract content of the test solution decreases by more than 0.50° Prato in the next 24 hours, and the fermentation occurs at a maximum temperature of 18°C.

[0060] Therefore, when the Saccharomyces pastorianus yeast strain according to the present invention is tested in a method including the following steps, it is preferable to be able to produce a fermentation test solution: a) A step of providing a test solution, wherein the test solution is an extract of malt and / or cereals having an appearance extract content of at least 10° Prat. b) A step of incubating the Saccharomyces pastorianus yeast strain with the test solution at a maximum temperature of 18°C. c) A step of determining the earliest time at which the visible extract content of the test solution does not decrease by more than 0.50° Plato during the incubation in the next 24 hours, and d) A step of determining the level of diacetyl at the earliest point in time, The test solution contains, at the earliest point, up to 120 ppb of diacetyl, for example, up to 60 ppb of diacetyl.

[0061] In some embodiments, the Saccharomyces pastorianus yeast strain can produce a fermentation test solution after incubation in a test solution of the yeast strain, the test solution may be any of the test solutions described below, the fermentation test solution containing the maximum level of diacetyl described below at the earliest point in time when the visible extract content of the test solution does not decrease by more than 0.50° Prato in the next 24 hours after incubation of the yeast strain in the extract, e.g.

[0062] The maximum level of diacetyl is preferably 120 ppb of diacetyl, for example, 115 ppb of diacetyl, for example, 110 ppb of diacetyl, or for example, 60 ppb of diacetyl, at the earliest point in time when the visible extract content of the test solution has not decreased by the specified amount. In particular, the maximum level of diacetyl at the earliest point in time may be 110 ppb. In particular, the maximum level of diacetyl at the earliest point in time may be 55 ppb.

[0063] The incubation is preferably carried out at a maximum temperature of 18°C, for example, in the range of 12°C to 18°C. In particular, the incubation may be carried out at a temperature of approximately 16°C.

[0064] In some embodiments, the Saccharomyces pastorianus yeast strain can produce a fermentation test solution by incubation in a test solution, the test solution being a malt and / or cereal extract having an apparent extract content of at least 10° Prato, the test solution containing up to 120 ppb of diacetyl, for example up to 65 ppb of diacetyl, at the earliest point before the apparent extract content of the test solution decreases by more than 0.25° Prato in the next 12 hours, and the fermentation occurs at a maximum temperature of 18°C.

[0065] Therefore, when the Saccharomyces pastorianus yeast strain according to the present invention is tested in a method including the following steps, it is preferable to be able to produce a fermentation test solution: a) A step of providing a test solution, wherein the test solution is an extract of malt and / or cereals having an appearance extract content of at least 10° Prat. b) A step of incubating the Saccharomyces pastorianus yeast strain with the test solution at a maximum temperature of 18°C. c) A step of determining the earliest time at which the visible extract content of the test solution does not decrease by more than 0.25° Plato in the next 12 hours during the incubation, and d) A step of determining the level of diacetyl at the earliest point in time, The test solution contains, at the earliest point, a maximum of 120 ppb of diacetyl, for example, a maximum of 65 ppb of diacetyl.

[0066] In some embodiments, the Saccharomyces pastorianus yeast strain can produce a fermentation test solution after incubation in a test solution of the yeast strain, the test solution may be any of the test solutions described below, the fermentation test solution containing the maximum level of diacetyl described below at the earliest point in time when the visible extract content of the test solution does not decrease by more than 0.25° Prate in the next 12 hours, e.g., more than 0.20° Prate, e.g., more than 0.15° Prate, e.g., more than 0.10° Prate, and the fermentation occurs at the temperature specified below. In particular, the visible extract content of the test solution does not decrease by more than 0.25° Prate in the next 12 hours.

[0067] The maximum level of diacetyl is preferably 120 ppb of diacetyl at the earliest point when the visible extract content of the test solution has not decreased by the specified amount, for example, 115 ppb of diacetyl, for example, 110 ppb of diacetyl, for example, 105 ppb of diacetyl. In particular, the maximum level of diacetyl at the earliest point may be 110 ppb, for example, 55 ppb of diacetyl.

[0068] The incubation is preferably carried out at a maximum temperature of 18°C, for example, in the range of 12°C to 18°C. In particular, the incubation may be carried out at a temperature of approximately 16°C.

[0069] In some embodiments, the Saccharomyces pastorianus yeast strain can produce a fermentation test solution after incubation in a test solution of the yeast strain, the test solution being an extract of malt and / or cereals having an appearance extract content of at least 10° Prat, the test solution containing up to 265 ppb of diacetyl at any point within 5 days after the start of incubation with the yeast, the incubation being carried out at a maximum temperature of 18°C, and live yeast cells ranging from 7 to 15 million / ml being added to the test solution.

[0070] In some embodiments, the Saccharomyces pastorianus yeast strain can produce a fermentation test solution after incubation in a test solution of the yeast strain, the test solution being an extract of malt and / or cereals having an appearance extract content of at least 10° Prat, and the fermentation test solution contains up to 50 ppb of diacetyl after incubation with the yeast strain for up to 6 days. In particular, the fermentation test solution may contain up to 50 ppb of diacetyl after incubation with the yeast strain for up to 5 days. In particular, the fermentation test solution may contain up to 50 ppb of diacetyl after incubation with the yeast strain for up to 4 days.

[0071] In some embodiments, the Saccharomyces pastorianus yeast strain can produce a fermentation test solution after incubation in a test solution of the yeast strain, the test solution being an extract of malt and / or cereals having an appearance extract content of at least 10° Prat, and the test solution containing up to 50 ppb of diacetyl after incubation with the test solution for up to 5 days at a maximum temperature of 16°C. In particular, the fermentation test solution may contain up to 50 ppb of diacetyl after incubation with the yeast strain for up to 4 days at a maximum temperature of 16°C.

[0072] In some embodiments, the Saccharomyces pastorianus yeast strain can produce a fermentation test solution after incubation in a test solution, the test solution being a malt and / or cereal extract having an appearance extract content of at least 10° Prate, and the yeast strain can produce at least 4.0 mL / L ethanol / ° Prate when incubated in the test solution.

[0073] In some embodiments, the Saccharomyces pastorianus yeast strain can produce a fermentation test solution after incubation in a test solution, the test solution being a malt and / or cereal extract having an appearance extract content of at least 10° Prate, and the yeast strain can produce at least 4.0 mL / L ethanol / ° Prate when incubated in the test solution for a range of 4 to 6 days. In particular, the yeast strain can be incubated in the test solution for approximately 4 days.

[0074] In a very preferred embodiment, the yeast strain is a yeast strain capable of the following: - A fermentation test solution is produced by incubation in a test solution, wherein the test solution is a malt and / or cereal extract having an apparent extract content of at least 10° Prat, and the fermentation test solution contains a maximum of 60 ppb of diacetyl at the earliest point when the apparent extract content of the test solution has not decreased by more than 0.50° Prat in the preceding 24 hours; - A fermentation test solution is produced by incubation in a test solution, wherein the test solution is a malt and / or cereal extract having an apparent extract content of at least 10° Prat, and the fermentation solution contains a maximum of 60 ppb of diacetyl, preferably a maximum of 50 ppb of diacetyl, at the earliest point when the apparent extract content of the test solution has not decreased by more than 0.25° Prat in the preceding 12 hours; -Providing a fermentation test solution by incubation in a test solution, wherein the test solution is a malt and / or cereal extract having an appearance extract content of at least 10° Prat, and the fermentation test solution contains a maximum of 120 ppb of diacetyl, for example, a maximum of 60 ppb of diacetyl, at the earliest point in time when the appearance extract content of the test solution does not decrease by more than 0.50° Prat in the next 24 hours; and - A fermentation test solution is produced by incubation in a test solution, wherein the test solution is a malt and / or cereal extract having an apparent extract content of at least 10° Prat, and the fermentation test solution contains a maximum of 120 ppb of diacetyl, for example, a maximum of 60 ppb of diacetyl, at the earliest point in time when the apparent extract content of the test solution does not decrease by more than 0.25° Prat in the next 12 hours; Fermentation occurs as described in Example 6 of this specification.

[0075] In some embodiments, the yeast strain exhibits a cottony precipitate. This cottony precipitate may, for example, be suspended cells after fermentation. "Suspended cells" are generally determined by counting yeast cells in a sample taken from the top three-quarters, e.g., the top two-thirds, e.g., the upper half, of a container containing the fermentation test solution. If fermentation is carried out in a conical tank, the sample is preferably taken from the top of the cone. A low number of cells in the solution after fermentation will result in a cottony precipitate.

[0076] In some embodiments, a Saccharomyces pastorianus yeast strain can produce a fermentation test solution after incubation in a test solution of the yeast strain, the test solution being an extract of malt and / or cereals having an appearance extract content of at least 10° Prato, and the test solution contains up to 10 million cells / milliliter, e.g., up to 9.5 million cells / milliliter, e.g., up to 9 million cells / milliliter, e.g., up to 8.5 million cells / milliliter, e.g., up to 89 million cells / milliliter, e.g., up to 8.5 million cells / milliliter, e.g., up to 8 million cells / milliliter, e.g., up to 8 million cells / milliliter, e.g., up to 8 million cells / milliliter, e.g., up to 10 million cells / milliliter, e.g., up to 9.5 million cells / milliliter, e.g., up to 8 million cells / milliliter, e.g., up to 8 million cells / milliliter, e.g.

[0077] In some embodiments, the Saccharomyces pastorianus yeast strain can be grown on a medium having melibiose as the sole carbon source.

[0078] In preferred embodiments, the yeast strains disclosed herein are non-genetically modified organisms. Therefore, in preferred embodiments, the yeast strains have not undergone any genetic engineering process.

[0079] Test solution In some embodiments, the test solution is wort having an apparent extract content of approximately 16° Prat. In some embodiments, the test solution contains at least 40 g / kg of maltose.

[0080] The test solution is generally a malt and / or cereal extract having an appearance extract content of at least 10° Prato. Therefore, the test solution may be wort in particular. The test solution may preferably have an appearance extract content in the range of 12-18° Prato, more preferably about 16° Prato. The test solution may further contain zinc in the range of 0.20 mg / L to 0.40 mg / L, and the pH may be adjusted to the range of 4.0-6.0.

[0081] One example of a test solution includes glucose in the range of 12–25 g / L, maltose in the range of 60–80 g / L, maltotriose in the range of 15–20 g / L, zinc in the range of 0.20 mg / L to 0.40 mg / L, free α-amino nitrogen (FAN) in the range of 110–250 mg / L, and a valine / FAN ratio of 0.6–0.8.

[0082] In some embodiments, the test solution is prepared by adding potential oomugia junct to pilsner malt.

[0083] Genotype The Saccharomyces pastorianus yeast strain according to the present invention has a phenotype that can produce a fermentation test solution with reduced diacetyl levels, as described in the section having the heading "Yeast Strain" above.

[0084] In addition to the phenotypic characteristics described above, the yeast strain according to the present invention may preferably have one or more of the genotypes described below herein.

[0085] ILV2, ILV6, ILV5, ILV3, BAT1, and BAT2 are involved in the synthesis of L-valine from pyruvate. Spontaneous production of diacetyl can occur in this process.

[0086] The small acetolactic acid synthase subunit (ILV6) and the catalytic acetolactic acid synthase subunit (ILV2) convert pyruvate to α-acetolactic acid.

[0087] Ketolate reductosomerase (ILV5) catalyzes the conversion of α-acetolactate to 2,3-hydroxyisovaleric acid.

[0088] Dihydroxy acid dehydratase (ILV3) catalyzes the conversion of 2,3-hydroxy-isovaleric acid to 2-keto isolaterate.

[0089] Branched-chain amino acid aminotransferase (BAT1) catalyzes the conversion of 2-keto isolaterate to L-valine in mitochondria, while branched-chain amino acid aminotransferase (BAT2) catalyzes the same reaction in the cytosol. [Table 1] [Table 2]

[0090] If a yeast cell "has" a precise number of genes, it is understood that the cell does not contain more than the indicated number of genes. Similarly, if a yeast cell "has" a range of XX to YY genes, the yeast cell does not contain more than YY genes.

[0091] In some embodiments, the Saccharomyces pastorianus yeast strain has up to 5 genes encoding functional ILV2, for example, up to 4 genes encoding functional ILV2, for example, genes in the range of 1 to 5 encoding functional ILV2, for example, genes in the range of 1 to 4 encoding functional ILV2, and each gene encoding ILV2 encodes ScILV2 of SEQ ID NO:32 or a functional homolog that shares at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 95%, e.g., at least 98% sequence identity therewith, or encoding SeILV2 of SEQ ID NO:38 or a homolog that shares at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 95%, e.g., at least 98% sequence identity therewith. The yeast strain of the invention preferably has up to 2 functional genes encoding ILV2, for example, the yeast strain may have 1 to 2 functional genes encoding ILV2, for example, strictly 2 functional genes. Preferably, the functional gene encodes ScILV2 of SEQ ID NO:32 or SeILV2 of SEQ ID NO:38 or any of the functional homologs that share at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 95%, e.g., at least 98% sequence identity. In one embodiment, the functional gene encodes ScILV2 of SEQ ID NO:32, preferably ScILV. In one embodiment, the functional gene encodes ILV2 of ZDA1, ZDA2 and / or ZDA3, the sequences of ILV2 of ZDA1, ZDA2 and / or ZDA3 are provided in Figure 4.

[0092] In some embodiments, the yeast strain has up to two functional genes encoding ILV2, each of which encodes ScILV2 with SEQ ID NO:32 or SeILV2 with SEQ ID NO:38, or a functional homologue sharing at least 80% sequence identity therewith; it has at least four functional genes encoding ILV3, e.g., at least five functional genes encoding ILV3, e.g., at least six functional genes encoding ILV3, each of which encodes ScILV3 with SEQ ID NO:35 or SeILV3 with SEQ ID NO:41, or a functional homologue sharing at least 80% sequence identity therewith; and it has at least three functional genes encoding ILV5, e.g., at least four functional genes encoding ILV5, e.g., at least five functional genes encoding ILV5, each of which encodes ScILV5 with SEQ ID NO:34 or SeILV5 with SEQ ID NO:40, or a functional homologue sharing at least 80% sequence identity therewith.

[0093] In some embodiments, the yeast strain has up to two functional genes encoding ILV2, each gene encoding ILV2 encoding either ScILV2 with SEQ ID NO:32 or SeILV2 with SEQ ID NO:38 or one of the aforementioned functional homologs sharing at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 95%, e.g., at least 98% sequence identity; and also has a range of 5-9 functional genes encoding ILV3, e.g., a range of 5-7 functional genes encoding ILV3, each gene encoding ILV3 encoding either ScILV3 with SEQ ID NO:35 or SeILV3 with SEQ ID NO:41 or one of the aforementioned functional homologs sharing at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 95%, e.g., at least 98% sequence identity; and also has a range of 4-8 functional genes encoding ILV5, e.g., a range of 4-6 functional genes encoding ILV5, each gene encoding ILV5 encoding It encodes either ScILV5 of NO:34 or SeILV5 of SEQ ID NO:40, or any of the aforementioned functional homologs that share at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 95%, e.g., at least 98% sequence identity with the above.

[0094] In some embodiments, the Saccharomyces pastorianus yeast strain has up to four genes encoding functional ILV6, for example, a range of 2 to 4 genes encoding functional ILV6, each gene encoding SEQ ID NO:33 ScILV6 or a functional homolog that shares at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 95%, e.g., at least 98% sequence identity therewith, or SEQ ID NO:39 SeILV6 or a functional homolog that shares at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 95%, e.g., at least 98% sequence identity therewith.

[0095] In some embodiments, the Saccharomyces pastorianus yeast strain has at least two genes encoding functional ILV5, for example, at least four genes encoding functional ILV5, for example, at least five genes encoding functional ILV5, where each gene encoding ILV5 encodes ScILV5 with SEQ ID NO:34 or a functional homologue sharing at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 95%, e.g., at least 98%, up to 99% sequence identity, or encodes SeILV5 with SEQ ID NO:40 or a functional homologue sharing at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 95%, e.g., at least 98%, up to 99% sequence identity. In some embodiments, the Saccharomyces pastorianus yeast strain has up to 20 genes encoding functional ILV5, for example, up to 15 genes, for example, up to 10 alleles, where the genes encoding functional ILV5 may be any of the above. In some embodiments, the Saccharomyces pastorianus yeast strain has 5 to 20 alleles encoding functional ILV5, for example 5 to 15 alleles, for example 5 to 10 genes, and the gene encoding functional ILV5 may be any of the above.

[0096] In one embodiment, the yeast strain has 4 to 8 functional genes encoding ILV5, for example, 4 to 6 functional genes encoding ILV5, and each gene encoding ILV5 encodes either ScILV5 with SEQ ID NO:34 or SeILV5 with SEQ ID NO:40 or any of the aforementioned functional homologs that share at least 80%, for example, at least 85%, for example, at least 90%, for example, at least 95%, for example, at least 98% sequence identity. In one embodiment, the yeast strain has 4 to 8 functional genes encoding ILV5, for example, 4 to 6 functional genes encoding ILV5, and each gene encodes ILV5 of ZDA1, ZDA2 and / or ZDA3, the sequences of ILV5 of ZDA1, ZDA2 and / or ZDA3 are provided in Figure 7.

[0097] In some embodiments, the Saccharomyces pastorianus yeast strain has at least three genes encoding functional ILV3, for example, at least four genes encoding functional ILV3, for example, at least six genes encoding functional ILV3, where each gene encoding functional ILV3 encodes ScILV3 with SEQ ID NO:35 or a functional homologue sharing at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 95%, e.g., at least 98%, up to 99% sequence identity therewith, or SEQ ID NO:41 SeILV3 or a homologue sharing at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 95%, e.g., at least 98%, up to 99% sequence identity therewith. In some embodiments, the Saccharomyces pastorianus yeast strain has up to 20 alleles encoding ILV3, for example, up to 15 alleles, for example, up to 10 alleles, where each gene encoding functional ILV3 may be any of the above. In some embodiments, the Saccharomyces pastorianus yeast strain has 5 to 20 alleles encoding functional ILV3, for example 5 to 15 alleles, for example 5 to 10 genes, and the gene encoding functional ILV3 may be any of the above.

[0098] In one embodiment, the yeast of the invention has functional genes in the range of 5 to 9 encoding ILV3, for example, 6 functional genes in the range of 5 to 7 encoding ILV3, and each gene encoding ILV3 encodes either ScILV3 of SEQ ID NO:35 or SeILV3 of SEQ ID NO:41 or any of the aforementioned functional homologs that share at least 80%, for example, at least 85%, for example, at least 90%, for example, at least 95%, for example, at least 98% sequence identity. In one embodiment, the yeast of the invention has functional genes in the range of 5 to 9 encoding ILV3, for example, 6 functional genes in the range of 5 to 7 encoding ILV3, and each gene encodes ILV3 of ZDA1, ZDA2 and / or ZDA3, the sequences of ILV3 of ZDA1, ZDA2 and / or ZDA3 are provided in Figure 6.

[0099] In some embodiments, the Saccharomyces pastorianus yeast strain has at least three genes encoding functional BAT1, for example, at least four genes encoding functional BAT1, for example, at least five genes encoding functional BAT1, and each gene encoding functional BAT1 encodes ScBAT1 with SEQ ID NO:36 or a functional homologue that shares at least 80%, for example, at least 85%, for example, at least 90%, for example, at least 95%, for example, at least 98%, up to 99% sequence identity therewith, or encodes SeBAT1 with SEQ ID NO:42 or a functional homologue that shares at least 80%, for example, at least 85%, for example, at least 90%, for example, at least 95%, for example, at least 98%, up to 99% sequence identity therewith. In some embodiments, the Saccharomyces pastorianus yeast strain has up to 20 genes encoding BAT1, for example, up to 15 genes, for example, up to 10 genes, and each gene encoding BAT1 may be any of the above. In some embodiments, the Saccharomyces pastorianus yeast strain has 5 to 20 alleles encoding functional BAT1, for example 5 to 15 alleles, for example 5 to 10 alleles, and each allele encoding functional BAT1 may be any of the above.

[0100] In some embodiments, the Saccharomyces pastorianus yeast strain has at least four genes encoding functional BAT2, for example, at least five genes encoding functional BAT2, for example, at least six genes encoding functional BAT2, each gene encoding functional BAT2 encoding ScBAT2 with SEQ ID NO:37 or a functional homolog that shares at least 80%, for example, at least 85%, for example, at least 90%, for example, at least 95%, for example, at least 98%, up to 99% sequence identity therewith, or encoding SeBAT2 with SEQ ID NO:43 or a functional homolog that shares at least 80%, for example, at least 85%, for example, at least 90%, for example, at least 95%, for example, at least 98%, up to 99% sequence identity therewith. In some embodiments, the Saccharomyces pastorianus yeast strain has up to 20 genes encoding functional BAT2, for example, up to 15 genes, for example, up to 10 genes, and the genes encoding functional BAT2 may be any of the above. In some embodiments, the Saccharomyces pastorianus yeast strain has 5 to 20 genes encoding functional BAT2, for example 5 to 15 genes, for example 5 to 10 genes, and each gene encoding functional BAT2 may be any of the above.

[0101] In some embodiments, the Saccharomyces pastorianus yeast strain harbors one or more mutations or deletions in the ILV2 gene.

[0102] In some embodiments, the Saccharomyces pastorianus yeast strain harbors one or more frameshift mutations in the ILV2 gene.

[0103] In some embodiments, the Saccharomyces pastorianus yeast strain harbors mutations that result in reduced or absent expression of one or more ILV2 genes.

[0104] In some embodiments, the Saccharomyces pastorianus yeast strain harbors one or more mutations or deletions in the ILV6 gene.

[0105] In some embodiments, the Saccharomyces pastorianus yeast strain harbors one or more frameshift mutations in the ILV6 gene.

[0106] In some embodiments, the Saccharomyces pastorianus yeast strain harbors mutations that result in reduced or absent expression of one or more ILV6 genes.

[0107] In some embodiments of the present invention, the sum of the genes encoding functional ILV2 and functional ILV6 is lower than the sum of the genes encoding functional ILV5 and functional ILV3, and functional ILV2, ILV6, ILV3, and ILV5 may be any of the above. In some embodiments of the present invention, the sum of the genes encoding functional ILV2 is lower than the sum of the genes encoding functional ILV5 and functional ILV3, and functional ILV2, ILV5, and ILV3 may be any of the above. In one embodiment, the sum of the genes encoding functional ILV2 and functional ILV6 is lower than the sum of the genes encoding functional ILV5, functional ILV3, functional BAT1, and functional BAT2, and functional ILV2, ILV6, ILV3, ILV5; BAT1, and BAT2 may be any of the above. In other words, in some embodiments, the sum of the genes encoding ILV5 and ILV3 is higher than the sum of the genes encoding ILV2 and ILV6. Thus, in some embodiments, the sum of the genes encoding ILV5 and ILV3 is higher than the sum of the genes encoding ILV2, ILV6, BAT1, and BAT2.

[0108] In some embodiments, the gene ratio of the ILV2 gene to the ILV5 and ILV3 genes is at least 1, for example at least 1.5, for example at least 2, for example at least 2.5, for example at least 3, and the ILV2, ILV5 and ILV3 genes each encode one of the functional ILV2, ILV5 and ILV3, respectively.

[0109] In some embodiments, the gene ratio of ILV2 and ILV6 genes to ILV5 and ILV3 genes is at least 1, for example at least 1.2, for example at least 1.4, for example at least 1.6, for example at least 1.8, or for example at least 2, where the ILV2, ILV6, ILV5, and ILV3 genes each encode one of the functional ILV2, ILV6, ILV5, and ILV3, respectively.

[0110] In some embodiments, the gene ratio of ILV2 to ILV5, ILV3, BAT1, and BAT2 is at least 1, e.g., at least 1.5, e.g., at least 2, e.g., at least 2.5, e.g., at least 3, e.g., at least 4, e.g., at least 5, e.g., at least 6, where the ILV2, ILV5, ILV3, BAT1, and BAT2 genes each encode one of the functional ILV2, ILV5, ILV3, BAT1, and BAT2, respectively.

[0111] In some embodiments, the gene ratio of ILV2 and ILV6 to ILV5, ILV3, BAT1 and BAT2 is at least 1, e.g., at least 1.5, e.g., at least 2, e.g., at least 2.5, e.g., at least 3, e.g., at least 4, where the ILV2, ILV6, ILV5, ILV3, BAT1 and BAT2 genes each encode one of the functional ILV2, ILV6, ILV5, ILV3, BAT1 and BAT2, respectively.

[0112] Fermentation aqueous extracts based on malt and / or cereals and methods for producing the same The invention provides a Saccharomyces pastorianus yeast strain described in the section having the heading “Yeast Strains” above, and a method for preparing a fermentation aqueous extract based on malt and / or cereals using the said yeast strain.

[0113] One aspect of the invention is to provide a method for producing a fermented aqueous extract, the method comprising the following steps: i) A step of providing an aqueous extract of malt and / or cereals; ii) A step of providing a yeast strain, wherein the yeast strain is any of the above and any of the yeast strains described in the section “Yeast Strains”; and iii) A step of fermenting the aqueous extract provided in step i) with the yeast strain of step ii), Therefore, a fermented aqueous extract is obtained.

[0114] The aqueous extract may be any aqueous extract of malt and / or grains. A non-limiting example is wort. The aqueous extract may be prepared, for example, by preparing an extract of malt by mashing and optionally sparging, as described in this section below.

[0115] Malt is malted grain, such as barley grain. The term "malting" is understood to mean a process that involves soaking and germinating kernels in a controlled environmental environment, optionally followed by a drying step. The drying step may preferably be kiln drying of germinated kernels at high temperature. Green malt subjected to kiln drying, particularly malt obtained by the process described in WO2018 / 001882, can also be used.

[0116] Malting is crucial for the synthesis of many enzymes that cause kernel modification, mobilizes kernel nutrients, and primarily depolymerizes the starch and cell wall of the dead endosperm to activate other depolymerases. During the subsequent drying process, flavor and color are at least partially produced due to chemical browning reactions.

[0117] Immersion may be carried out by any conventional method known to those skilled in the art. One non-limiting example includes immersion at temperatures in the range of 10°C to 25°C, alternating between dry and wet conditions. Germination may be carried out by any conventional method known to those skilled in the art. One non-limiting example includes germination at temperatures in the range of 10°C to 25°C, optionally varying the temperature over a range of 1 to 4 hours. Immersion and germination may also be carried out in combination, for example, as described in International Patent Application WO2018 / 001882.

[0118] Kiln drying can be carried out at conventional temperatures, for example, at least 75°C, for example in the range of 80-90°C, for example in the range of 80-85°C. Thus, malt can be produced by any of the methods described, for example, by Briggs et al. (1981) and Hough et al. (1982). However, any other suitable method for producing malt, for example, a method for producing specialty malts, for example, a method of roasting malt, can also be used in conjunction with the present invention.

[0119] Malt can be further processed, for example, by milling. Milling can be carried out in a dry state, that is, the malt is crushed in a dry state, or in a wet state if green malt is used.

[0120] Malt, for example, ground malt, can be mashed to obtain an aqueous extract of the malt. The starting liquid for preparing the beverage may be an aqueous extract of malt, for example, an aqueous extract of malt prepared by mashing.

[0121] Therefore, the method for preparing a fermented aqueous extract based on malt and / or cereals according to the present invention may include a step of producing an aqueous extract, such as wort, by mashing malt and optionally additional adjuncts. The mashing step may also optionally include sparging, and therefore the mashing step may be a mashing step including a sparging step or a mashing step without a sparging step.

[0122] Generally, the formation of aqueous extracts is initiated by milling of malt and / or kernels. If additional adjuncts are added, these may also be milled depending on their properties. If the adjunct is a cereal, it may be milled, for example, while syrups, sugars, etc., are generally not milled. Milling facilitates water access to the kernel particles in the mashing phase. During mashing, enzymatic depolymerization of the substrate, initiated during malting, may continue.

[0123] Generally, aqueous extracts are prepared in the mashing process by combining ground malt and water and incubating them. During mashing, the malt / liquid composition may be supplemented with additional carbohydrate-rich ajunct compositions, such as ground barley, corn, or rice ajunct. Unmalted cereal ajuncts typically contain little to no active enzymes, making it important to supplement them with malt or exogenous enzymes to provide the enzymes necessary for polysaccharide depolymerization and other processes.

[0124] During mashing, the ground malt and / or ground kernel and optionally additional adjunct are incubated with a liquid portion, e.g., water. The incubation temperature is generally maintained constant (isothermal mashing) or gradually increased, e.g., in a continuous manner. In either case, soluble substances in the malt / kernel / adjunct are released into the liquid portion. Subsequent filtration separates the aqueous extract from residual solid particles, the latter also called "kernel residue." The aqueous extract thus obtained may also be called "first wort." Additional liquid, e.g., water, may be added to the kernel residue during a process also called sparging. After sparging and filtration, "second wort" may be obtained. Further worts may be prepared by repeating the procedure. Non-limiting examples of preferred procedures for wort preparation are described by Briggs et al. (1981) and Hough et al. (1982).

[0125] As described above, aqueous extracts can also be prepared by mashing only unmalted kernels. Unmalted kernels lack, or contain only in limited amounts, enzymes beneficial to wort production, such as enzymes that can break down cell walls or enzymes that can depolymerize starch into sugars. Therefore, in embodiments of the invention in which up to 80%, for example 90%, or even 100%, of unmalted kernels, such as barley grains, are used for mashing, it is preferable that one or more suitable, externally brewed enzymes be added to the mash. Suitable enzymes may be lipases, starch-degrading enzymes (e.g., amylase), glucanases [preferably (1-4)- and / or (1-3,1-4)-β-glucanase], and / or xylanases (e.g., arabinoxylanase), and / or proteases, or enzyme mixtures containing one or more of the above enzymes, such as Cereflo, Ultraflo, or Ondea Pro (Novozymes).

[0126] Aqueous extracts can also be prepared by using a mixture of malted and unmalted kernels, in which case one or more suitable enzymes may be added during preparation. Even in embodiments where malt is used, enzymes may also be added. More specifically, kernels can be used with malt in any combination for mashing—with or without external brewing enzymes—for example, but are not limited to, kernel:malt ratios of approximately 100:0, or approximately 75:25, or approximately 50:50, or approximately 25:75.

[0127] The aqueous extract obtained after mashing may also be called "sweet wort." In conventional methods, sweet wort is boiled with or without hops, and the resulting liquid may then be called boiled wort.

[0128] The aqueous extract can be heated or boiled, and then subjected to fermentation by the yeast of the invention. In one embodiment of the invention, the second and further worts can be combined and thus subjected to heating or boiling. The aqueous extract can be heated or boiled for any suitable amount of time, for example, in the range of 60 to 120 minutes.

[0129] The outcome of fermentation aqueous extracts based on malt and / or cereals depends significantly on the amount and type of fermentable sugars present in the aqueous extract of malt and / or cereal grains, as well as the characteristics of the yeast strain used during fermentation.

[0130] In some embodiments of the present invention, the aqueous extract has an apparent extract content of at least 12° praline, for example, at least 15° praline, for example, in the range of 5-15° praline, for example, in the range of 10-20° praline, for example, in the range of 15-25° praline.

[0131] In some embodiments, the aqueous extract is fermented with the yeast strain for up to 6 days, for example, up to 5 days, for example, up to 4 days, for example, up to 3 days.

[0132] An additional advantage of the yeast strains of the present invention is that they may be useful for fermenting wort having low levels of amino acids. Thus, in some embodiments, the aqueous extract contains up to 3500 mg / L of amino acids, for example, up to 3000 mg / L, for example, up to 2500 mg / L.

[0133] Therefore, aqueous extracts, such as wort, can be prepared as described above. Fermentation aqueous extracts based on malt and / or cereals can be prepared by fermentation of the aqueous extract with the yeast strain according to the present invention.

[0134] In some preferred embodiments, the fermented aqueous extract is green beer.

[0135] Generally speaking, alcoholic fermentation aqueous extracts—e.g., beer—can be produced from malted and / or unmalted kernels. Malt, in addition to hops and yeast, contributes to the flavor and color of beverages, such as beer. Furthermore, malt functions as a source of fermentable sugars and enzymes. Non-limiting descriptions of examples of preferred methods for malting and brewing can be found, for example, in publications by Briggs et al. (1981) and Hough et al. (1982). Numerous regularly updated methods for the analysis of kernels, malt, and beer products are available, for example, but not limited to, the American Society of Grain Chemists (1995), the American Brewing Chemists Society (1992), the European Breweries Agreement (1998), and the Brewing Institute (1997). Many specific procedures are used for a given brewery, and it is recognized that the most important modifications relate to the preferences of local consumers. Any such method for producing beer can be used in conjunction with the present invention.

[0136] A first step in producing beer from wort preferably includes heating the wort as described herein, followed by wort cooling and optionally a subsequent whirlpool rest phase.

[0137] The method of the invention comprises the step of fermenting an aqueous extract of malt and / or grains with a yeast strain according to the present invention. The fermentation may be of an unfermented aqueous extract or a fermented aqueous extract that still contains fermentable sugars for the yeast. Thus, in some embodiments, the fermentation may be carried out essentially immediately after the completion of mashing or after heating of the wort.

[0138] Fermentation may be carried out in a fermentation tank containing yeast according to the present invention, that is, yeast having one or more of the characteristics described herein above.

[0139] During the fermentation process, which lasts for several days, flavor compounds are produced. If the yeast strain is unable to convert certain compounds, these will still be present after fermentation step iii).

[0140] In some embodiments, fermentation step iii) occurs at one of the temperatures specified below, the aqueous extract is incubated with one of the yeast cell counts described below, the fermented aqueous extract contains the maximum level of diacetyl described below, and fermentation is completed after up to 5 days, e.g., up to 4 days, e.g., up to 3 days. In particular, fermentation is completed after up to 4 days.

[0141] In certain embodiments, fermentation using the yeast strain of the invention is completed at least 12 hours, for example at least 24 hours, earlier than fermentation carried out using the W-34 / 78 yeast strain under the same conditions, and the diacetyl level is less than 60 ppb, for example 50 ppb.

[0142] The fermentation is preferably carried out at a maximum temperature of 18°C, for example, in the range of 12°C to 18°C. In particular, the fermentation may be carried out at a temperature of approximately 16°C.

[0143] The aforementioned maximum level of diacetyl is preferably 60 ppb of diacetyl at the completion of fermentation, for example, 55 ppb of diacetyl, for example, 50 ppb of diacetyl, for example, 45 ppb of diacetyl. In particular, the aforementioned level of diacetyl at the completion of fermentation is 60 ppb at its maximum.

[0144] The fermentation is preferably carried out by incubating the aqueous extract with at least 6 million live yeast cells / milliliter, for example, at least 10 million live yeast cells / milliliter, for example, at least 14 million live yeast cells / milliliter, for example, in the range of 7-8 million live yeast cells / milliliter, for example, in the range of 14-16 million live yeast cells / milliliter. In particular, the fermentation may be carried out by incubating the aqueous extract with approximately 15 million yeast cells / milliliter.

[0145] In some embodiments, the fermented aqueous extract has an alcohol content of at least 4% ABV, for example, at least 5% ABV, for example, at least 6% ABV, for example, at least 7% ABV.

[0146] In some embodiments, the fermented aqueous extract contains at least 25 mg / L of propanol, for example, at least 30 mg / L of propanol.

[0147] In some embodiments, the fermented aqueous extract contains up to 8 mg / L of isobutanol, for example, up to 7 mg / L of isobutanol, for example, up to 6 mg / L of isobutanol.

[0148] In some embodiments, the fermented aqueous extract contains a beneficial isobutanol-to-propanol ratio. In particular, the isobutanol-to-propanol ratio may be at least 1.5, for example at least 2.0, for example at least 2.5, for example at least 3.0, for example at least 3.5, for example at least 4.0, for example at least 4.5, for example at least 5.0, for example at least 5.5, for example at least 6.0, for example at least 6.5.

[0149] In some embodiments, the fermented aqueous extract obtained using the yeast of the invention has a degree of true fermentation (RDF) that is at least 1.5%, for example 2%, lower than that of fermentation carried out under the same conditions using the W-34 / 78 yeast strain or the Hybrid yeast strain 7 at the same %ABV.

[0150] A beverage based on malt and / or grains and a method for producing the same. Fermented aqueous extracts based on malt and / or cereals as described herein may be further processed and made into beverages.

[0151] One aspect of the invention provides a method for producing a beverage based on malt and / or grains, the method comprising the following steps: i. In this specification, the steps described above for preparing fermented aqueous extracts as described in Section “Fermented Aqueous Extracts Based on Malt and / or Cereals and Methods for Producing the Same”, and ii. A step of further processing the fermented aqueous extract to make a beverage.

[0152] In some embodiments of the present invention, beverages based on malt and / or grains are diluted with a liquid such as water.

[0153] Optionally, water can be used to dilute the malt and / or cereal-based beverage, thereby adjusting, for example, the ethanol content. In one embodiment of the present invention, the water:malt and / or cereal-based beverage ratio can range from 0.1 to 5 parts water to 1 part malt and / or cereal-based beverage.

[0154] Further processes may include, for example, cooling and / or filtering the malt and / or grain-based beverage. Additives may also be added. Furthermore, CO2 may be added. Finally, the malt and / or grain-based beverage, such as beer, may be pasteurized and / or filtered, and then it may be packaged (e.g., bottled or canned).

[0155] In a preferred embodiment, the beverage is beer.

[0156] In one aspect of the present invention, a beverage based on malt and / or grains produced by fermenting an aqueous extract using the yeast strain according to the present invention has a pleasant taste.

[0157] The taste of a malt and / or cereal-based beverage produced by fermentation using the yeast according to the present invention can be analyzed, for example, by an expert beer taste panel. Preferably, the panel is trained to focus particularly on aldehydes, papery taste, aged taste, esters, higher alcohols, fatty acids, and sulfur components in beer flavor tasting and description.

[0158] Generally, a taste panel consists of members ranging from 3 to 30, for example, 5 to 15, preferably 8 to 12. The taste panel can evaluate the presence of various flavors, such as papery, oxidized, aged, and bready flavors, as well as ester, higher alcohol, sulfur component flavors and the body of the beer. The overall taste of the beer is generally rated by the taste panel on a scale of 1 to 9 based on several different characteristics, with an average rating of 5 or higher indicating that the beer has an acceptable taste.

[0159] The present invention also provides a beverage based on malt and / or cereals, prepared by the method described above.

[0160] In some embodiments, the beverage contains at least 25 mg / L of propanol, for example, at least 30 mg / L of propanol.

[0161] In some embodiments, the beverage contains up to 8 mg / L of isobutanol, for example, up to 6 mg / L of isobutanol.

[0162] Generally, a fermented aqueous extract or beverage prepared using the yeast strain of the invention has a specific ratio of isobutanol to propanol, for example, a ratio similar to the ratio shown in Figure 2. Therefore, if a fermented aqueous extract or beverage has this specific ratio of isobutanol to propanol, it indicates that the fermented aqueous extract or beverage was produced by fermentation using the yeast strain of the invention. In particular, the isobutanol to propanol ratio may be at least 1.5, for example at least 2.0, for example at least 2.5, for example at least 3.0, for example at least 3.5, for example at least 4.0, for example at least 4.5, for example at least 5.0, for example at least 5.1, for example at least 5.2, for example at least 5.3, for example at least 5.4, for example at least 5.5, for example at least 6.0, for example at least 6.5.

[0163] item The invention may be further defined by the following items: 1. A method for producing a fermented aqueous extract, comprising the following steps: i) A step of providing an aqueous extract of malt and / or cereals; ii) A step of providing a yeast strain of the species Saccharomyces pastorianus, wherein the yeast strain can produce a fermentation test solution by incubation in a test solution, the test solution being an extract of malt and / or cereals having an appearance extract content of at least 10° Prato, the fermentation test solution containing a maximum of 60 ppb of diacetyl at the earliest point when the appearance extract content of the test solution has not decreased by more than 0.50° Prato in the preceding 24 hours, and the fermentation occurring at a maximum temperature of 18°C; and iii) A step of fermenting the aqueous extract provided in step i) with the yeast strain of step ii), thereby obtaining a fermented aqueous extract.

[0164] 2. Yeast strains that encode the following within their genome a. Functional genes encoding up to two ILV2, each encoding ScILV2 with SEQ ID NO:32 or SeILV2 with SEQ ID NO:38, or a functional homolog sharing at least 80% sequence identity with them; and b. A functional gene comprising at least five functional genes, e.g., at least six functional genes, that encode ILV3, wherein each ILV3-encoding gene encodes ScILV3 with SEQ ID NO:35 or SeILV3 with SEQ ID NO:41, or a functional homolog that shares at least 80% sequence identity with the same.

[0165] 3. A yeast strain capable of producing a fermented aqueous extract by incubation in an aqueous extract of malt and / or cereal, wherein the fermented aqueous extract contains at least an isobutanol-to-propanol ratio of 2.0.

[0166] 4. A yeast strain comprising any one of items 2-3, wherein the yeast strain can produce a fermentation test solution by incubation in a test solution, the test solution being an extract of malt and / or grains having an appearance extract content of at least 10° Prat, the fermentation test solution containing a maximum of 60 ppb of total diacetyl at the earliest point when the appearance extract content of the test solution has not decreased by more than 0.50° Prat in the preceding 24 hours, and the fermentation occurs at a maximum temperature of 18°C.

[0167] 5. A method or yeast strain according to item 1 or 4, wherein the time point is the earliest time at which the visible extract content of the test solution did not decrease by more than 0.40° Plate, e.g., 0.30° Plate, e.g., 0.20° Plate, in the preceding 24 hours.

[0168] 6. A method or yeast strain according to any one of items 1 and 4-5, wherein the test solution contains, at the earliest time, up to 55 ppb of diacetyl, for example, up to 50 ppb of diacetyl, for example, up to 45 ppb of diacetyl.

[0169] 7. A method for producing a fermented aqueous extract, comprising the following steps: i) A step of providing an aqueous extract of malt and / or cereals; ii) A step of providing a yeast strain of the species Saccharomyces pastorianus, wherein the yeast strain can produce a fermentation test solution by incubation in a test solution, the test solution being an extract of malt and / or cereals having an appearance extract content of at least 10° Prato, the test solution containing a maximum of 60 ppb of diacetyl at the earliest point when the appearance extract content of the test solution has not decreased by more than 0.25° Prato in the preceding 12 hours, and the fermentation occurring at a maximum temperature of 18°C; and iii) A step of fermenting the aqueous extract provided in step i) with the yeast strain, thereby obtaining a fermented aqueous extract.

[0170] 8. A yeast strain comprising any one of items 2-6, wherein the yeast strain can produce a fermentation test solution by incubation in a test solution, the test solution being an extract of malt and / or grains having an appearance extract content of at least 10° Prate, the test solution containing a maximum of 60 ppb of total diacetyl at the earliest point when the appearance extract content of the test solution has not decreased by more than 0.25° Prate in the preceding 12 hours, and the fermentation occurs at a maximum temperature of 18°C.

[0171] 9. A method or yeast strain according to any one of items 7-8, wherein the time point is the earliest time at which the visible extract content of the test solution did not decrease by more than 0.20° Plate, e.g., 0.15° Plate, e.g., 0.10° Plate, in the preceding 12 hours.

[0172] 10. A method or yeast strain according to any one of items 7-8, wherein the test solution contains, at the earliest time, up to 55 ppb of diacetyl, for example, up to 50 ppb of diacetyl, for example, up to 45 ppb of diacetyl.

[0173] 11. A method for producing a fermented aqueous extract, comprising the following steps: i) A step of providing an aqueous extract of malt and / or cereals; ii) A step of providing a yeast strain of the species Saccharomyces pastorianus, wherein the yeast strain can produce a fermentation test solution by incubation in a test solution, the test solution being an extract of malt and / or cereals having an appearance extract content of at least 10° Prato, the test solution containing a maximum of 120 ppb of diacetyl, for example, a maximum of 60 ppb of diacetyl, at the earliest point in the next 24 hours at which the appearance extract content of the test solution does not decrease by more than 0.50° Prato, and the fermentation occurring at a maximum temperature of 18°C; and iii) A step of fermenting the aqueous extract provided in step i) with the yeast strain, thereby obtaining a fermented aqueous extract.

[0174] 12. A yeast strain according to any one of items 2-6 and 8-10, wherein the yeast strain can produce a fermentation test solution by incubation in a test solution, the test solution being an extract of malt and / or grains having an appearance extract content of at least 10° Prat, the test solution containing a maximum of 120 ppb of total diacetyl, for example, a maximum of 60 ppb of diacetyl, at the earliest point in the next 24 hours at which the appearance extract content of the test solution does not decrease by more than 0.50° Prat, and the fermentation occurs at a maximum temperature of 18°C.

[0175] 13. A method or yeast strain according to any one of items 11-12, wherein the time point is the earliest time in the next 24 hours at which the visible extract content of the test solution does not decrease by more than 0.40° Prato, e.g., 0.30° Prato, e.g., 0.20° Prato.

[0176] 14. A method for producing a fermented aqueous extract, comprising the following steps: i) A step of providing an aqueous extract of malt and / or cereals; ii) A step of providing a yeast strain of the species Saccharomyces pastorianus, wherein the yeast strain can produce a fermentation test solution by incubation in a test solution, the test solution being an extract of malt and / or cereals having an appearance extract content of at least 10° Prato, the test solution containing a maximum of 120 ppb of diacetyl, for example, a maximum of 65 ppb of diacetyl, at the earliest point before the appearance extract content of the test solution decreases by more than 0.25° Prato in the next 12 hours, and the fermentation occurring at a maximum temperature of 18°C; and iii) A step of fermenting the aqueous extract provided in step i) with the yeast strain, thereby obtaining a fermented aqueous extract.

[0177] 15. A yeast strain according to any one of items 2-6, 8-10, and 12-13, wherein the yeast strain can produce a fermentation test solution by incubation in a test solution, the test solution being an extract of malt and / or grains having an appearance extract content of at least 10° Prat, the test solution containing a maximum of 120 ppb of diacetyl, e.g., a maximum of 65 ppb of total diacetyl, at the earliest point before the appearance extract content of the test solution decreases by more than 0.25° Prat in the next 12 hours, and the fermentation occurs at a maximum temperature of 18°C.

[0178] 16. A method or yeast strain according to any one of items 14-15, wherein the time point is the earliest time at which the visible extract content of the test solution does not decrease by more than 0.20° Plato, e.g., 0.15° Plato, e.g., 0.10° Plato, in the next 12 hours.

[0179] 17. A method or yeast strain according to any one of items 11-15, wherein the test solution contains a maximum of 115 ppb of diacetyl at the earliest time.

[0180] 18. A method or yeast strain according to any one of items 1 and 4-17, wherein the test solution contains, in the earliest solution, up to 10 million cells / milliliter, for example, up to 9.5 million cells / milliliter, for example, up to 9 million cells / milliliter, for example, up to 8.5 million cells / milliliter.

[0181] 19. A method or yeast strain relating to any one of the items described above, wherein the test solution or test solution in step ii) is wort having an appearance extract content of approximately 16° Prat.

[0182] 20. A method or yeast strain relating to any one of the items, wherein the test solution or test solution of step ii) contains at least 40 g / kg of maltose.

[0183] 21. A method or yeast strain comprising any one of the items, wherein the fermentation in step ii) or the fermentation occurs at a temperature in the range of 12°C to 18°C.

[0184] 22. A method or yeast strain comprising any one of the items, wherein the fermentation in step ii) or the fermentation occurs at a maximum temperature of 16°C.

[0185] 23. A method or yeast strain comprising any one of the items, wherein the fermentation in step ii) occurs after inoculation of a test solution ranging from 7,000,000 to 20,000,000 live yeast cells / mL.

[0186] 24. A method or yeast strain comprising any one of the items, wherein the aqueous extract is fermented by the yeast strain for a maximum of 6 days, for example, a maximum of 5 days, for example, a maximum of 4 days.

[0187] 25. A method or yeast strain comprising any one of the items, wherein the aqueous extract has an apparent extract content of at least 12° Prato, for example, at least 15° Prato.

[0188] 26. A method or yeast strain comprising any one of the items, wherein the aqueous extract is wort.

[0189] 27. A method or yeast strain comprising any one of the items, wherein the aqueous extract contains an amino acid at a maximum of 3500 mg / L, for example, at a maximum of 3000 mg / L, for example, at a maximum of 2500 mg / L.

[0190] 28. A method or yeast strain comprising any one of the items, wherein the fermentation aqueous extract contains up to 60 ppb of diacetyl, for example up to 55 ppb of diacetyl, for example up to 50 ppb of diacetyl, for example up to 45 ppb of diacetyl, for example up to 40 ppb of diacetyl.

[0191] 29. A method or yeast strain comprising any one of the items, wherein the fermented aqueous extract contains, in the earliest solution, up to 10 million cells / milliliter, for example, up to 9.5 million cells / milliliter, for example, up to 9 million cells / milliliter, for example, up to 8.5 million cells / milliliter.

[0192] 30. A method or yeast strain comprising any one of the items, wherein the fermentation aqueous extract has an alcohol content of at least 4% ABV, for example, at least 5% ABV.

[0193] 31. A method or yeast strain comprising any one of the items described above, wherein the fermentation aqueous extract contains at least 25 mg / L of propanol, for example, at least 30 mg / L of propanol.

[0194] 32. A method or yeast strain comprising any one of the items, wherein the fermented aqueous extract contains a maximum of 8 mg / L of isobutanol, for example, a maximum of 6 mg / L of isobutanol.

[0195] 33. A method or yeast strain comprising any one of the items described above, wherein the fermented aqueous extract contains an isobutanol-to-propanol ratio of at least 2.0, e.g., at least 2.5, e.g., at least 3.0, e.g., at least 4.0, e.g., at least 5.0, e.g., at least 5.5.

[0196] 34. A method or yeast strain comprising any one of the items described above, wherein the yeast strain is capable of producing a fermentation test solution, the test solution containing up to 265 ppb of diacetyl at any point within 5 days after the start of incubation, and to which 7 to 8 million yeast cells are added.

[0197] 35. A method or yeast strain comprising any one of the items, wherein the yeast strain can produce a fermentation test solution containing up to 50 ppb of diacetyl after incubation in the test solution for up to 6 days, for example, up to 5 days, for example, up to 4 days.

[0198] 36. A method or yeast strain comprising any one of the items, wherein the yeast strain can produce a fermentation test solution containing up to 50 ppb of diacetyl after incubation in the test solution at a maximum temperature of 16°C for a maximum of 5 days, for example, a maximum of 4 days.

[0199] 37. A method or yeast strain comprising any one of the items described above, wherein the yeast strain can produce a first fermentation test solution containing up to 60 ppb of diacetyl after incubation in the test solution for a predetermined time, wherein the predetermined time is at least 12 hours, for example, at least 24 hours less than the time required for the W-34 / 78 yeast strain incubated under the same conditions to produce a second fermentation test solution containing up to 60 ppb of diacetyl after incubation in the test solution.

[0200] 38. A method or yeast strain comprising any one of the items, wherein the yeast strain, when incubated in the test solution, can produce at least 4.0, for example, at least 4.7 mL / L ethanol / °Prate.

[0201] 39. A method or yeast strain comprising any one of the items, wherein the yeast strain can produce at least 4.0, for example, at least 4.7 mL / L ethanol / °Prate when incubated in the test solution for a period of 4 to 6 days, for example, approximately 4 days.

[0202] 40. A method or yeast strain comprising any one of the items, wherein the yeast strain can be grown on a culture medium having melibiose as the sole carbon source.

[0203] 41. A method or yeast strain comprising any one of the items described above, wherein the yeast strain has up to five alleles encoding ILV2, and each gene encoding ILV2 encodes ScILV2 with SEQ ID NO:32 or SeILV2 with SEQ ID NO:38, or a homolog that shares at least 80% sequence identity with the same.

[0204] 42. A method or yeast strain relating to any one of the items described above, wherein the yeast strain has up to four genes encoding ILV2, and each gene encoding ILV2 encodes ScILV2 with SEQ ID NO:32 or SeILV2 with SEQ ID NO:38, or a homolog that shares at least 80% sequence identity with the same.

[0205] 43. A method or yeast strain comprising any one of the items described above, wherein the yeast strain has up to two genes encoding ILV2, and each gene encoding ILV2 encodes ScILV2 with SEQ ID NO:32 or SeILV2 with SEQ ID NO:38, or a functional homolog that shares at least 80% sequence identity with the same.

[0206] 44. A method or yeast strain relating to any one of the items described above, wherein the yeast strain has 1 to 2 functional genes encoding ILV2, for example, strictly 2, and each gene encoding ILV2 encodes ScILV2 with SEQ ID NO:32 or SeILV2 with SEQ ID NO:38, or a functional homologue sharing at least 80% sequence identity therewith.

[0207] 45. A method or yeast strain relating to any one of the items described above, wherein the yeast strain has 1 to 2 functional genes encoding ILV2, for example, strictly 2, and each gene encoding ILV2 encodes ScILV2 with SEQ ID NO:32 or a functional homolog that shares at least 80% sequence identity therewith.

[0208] 46. ​​A method or yeast strain relating to any one of the items described above, wherein the yeast strain has up to four genes encoding ILV6, and each gene encoding ILV6 encodes ScILV6 with SEQ ID NO:33 or SeILV6 with SEQ ID NO:39, or a homolog that shares at least 80% sequence identity with the same.

[0209] 47. A method or yeast strain relating to any one of the items described above, wherein the yeast strain has up to four functional genes encoding ILV6, and each gene encoding ILV6 encodes ScILV6 of SEQ ID NO:33 or SeILV6 of SEQ ID NO:39, or a homolog that shares at least 80% sequence identity with the same.

[0210] 48. A method or yeast strain relating to any one of the items described above, wherein the yeast strain has at least four genes encoding ILV5, for example, at least five genes encoding ILV5, and each gene encoding ILV5 encodes ScILV5 with SEQ ID NO:34 or SeILV5 with SEQ ID NO:40, or a homolog that shares at least 80% sequence identity with the same.

[0211] 49. A method or yeast strain relating to any one of the items described above, wherein the yeast strain encodes at least four functional genes encoding ILV5, for example, at least five functional genes encoding ILV5, and each gene encoding ILV5 encodes ScILV5 with SEQ ID NO:34 or SeILV5 with SEQ ID NO:40, or a functional homolog that shares at least 80% sequence identity with the same.

[0212] 50. A method or yeast strain relating to any one of the items, wherein the yeast strain has up to 15 functional genes encoding ILV5, for example 4 to 10 functional genes encoding ILV5, for example 4 to 5 functional genes encoding ILV5.

[0213] 51. A method or yeast strain relating to any one of the items, wherein the yeast strain has 4 to 8 functional genes encoding ILV5, for example, 4 to 6 functional genes encoding ILV5, for example, 5 functional genes encoding ILV5, and each gene encoding ILV5 encodes ScILV5 with SEQ ID NO:34 or SeILV5 with SEQ ID NO:40 or any of the functional homologs thereof that share at least 80% sequence identity.

[0214] 52. A method or yeast strain relating to any one of the items described above, wherein the yeast strain has at least three genes encoding ILV3, for example, at least four genes, and each gene encoding ILV3 encodes ScILV3 of SEQ ID NO:35 or SeILV3 of SEQ ID NO:41, or a homolog that shares at least 80% sequence identity with the same.

[0215] 53. A method or yeast strain relating to any one of the items, wherein the yeast strain has up to 15 functional genes encoding ILV3, for example 5 to 10 functional genes encoding ILV3, for example 5 to 6 functional genes encoding ILV3.

[0216] 54. A method or yeast strain relating to any one of the items, wherein the yeast strain has 5 to 9 functional genes encoding ILV3, for example, 6 functional genes in the range of 5 to 7 encoding ILV3, and each gene encoding ILV3 encodes ScILV3 of SEQ ID NO:35 or SeILV3 of SEQ ID NO:41 or any of the functional homologs thereof that share at least 80% sequence identity.

[0217] 55. A method or yeast strain relating to any one of the items described above, wherein the yeast strain has at least three genes encoding BAT1, for example, at least four genes, and each gene encoding BAT1 encodes ScBAT1 with SEQ ID NO:36 or SeBAT1 with SEQ ID NO:42, or a homolog that shares at least 80% sequence identity with the same.

[0218] 56. A method or yeast strain relating to any one of the items described above, wherein the yeast strain has at least three functional genes encoding BAT1, for example, at least four functional genes, and each gene encoding BAT1 encodes ScBAT1 with SEQ ID NO:36 or SeBAT1 with SEQ ID NO:42, or a homolog that shares at least 80% sequence identity with the same.

[0219] 57. A method or yeast strain relating to any one of the items described above, wherein the yeast strain has at least four genes encoding BAT2, for example, at least five genes, and each gene encoding BAT2 encodes ScBAT2 with SEQ ID NO:37 or SeBAT2 with SEQ ID NO:43, or a homolog that shares at least 80% sequence identity with the same.

[0220] 58. A method or yeast strain relating to any one of the items described above, wherein the yeast strain has at least four functional genes encoding BAT2, for example, at least five functional genes, and each gene encoding BAT2 encodes ScBAT2 with SEQ ID NO:37 or SeBAT2 with SEQ ID NO:43, or a homolog that shares at least 80% sequence identity with the same.

[0221] 59. A method or yeast strain comprising any one of the items described above, wherein the sum of the genes in the yeast strain encoding ILV2 and ILV6 is less than the sum of the genes encoding ILV5 and ILV3.

[0222] 60. A method or yeast strain comprising any one of the items, wherein the sum of the genes in the yeast strain encoding ILV2 is less than the sum of the genes encoding ILV5 and ILV3.

[0223] 61. A method or yeast strain comprising any one of the items described above, wherein the gene ratio of ILV5 and ILV3 to ILV2 in the yeast strain is at least 1, e.g., at least 1.5, e.g., at least 2, e.g., at least 2.5, or e.g., at least 3.

[0224] 62. A method or yeast strain comprising any one of the items described above, wherein the functional gene ratio of ILV5 and ILV3 to ILV2 in the yeast strain is at least 1, e.g., at least 1.5, e.g., at least 2, e.g., at least 2.5, e.g., at least 3.

[0225] 63. A method or yeast strain comprising any one of the items described above, wherein the gene ratio of ILV5 and ILV3 to ILV2 and ILV6 in the yeast strain is at least 1, e.g., at least 1.2, e.g., at least 1.4, e.g., at least 1.6, e.g., at least 1.8, or e.g., at least 2.

[0226] 64. A method or yeast strain comprising any one of the items described above, wherein the functional gene ratio of ILV5 and ILV3 to ILV2 and ILV6 in the yeast strain is at least 1, e.g., at least 1.2, e.g., at least 1.4, e.g., at least 1.6, e.g., at least 1.8, or e.g., at least 2.

[0227] 65. A method or yeast strain relating to any one of the items described above, wherein the genes encoding ILV2, ILV3 and / or ILV5 are expressed from their natural promoters.

[0228] 66. A method or yeast strain relating to any one of the items described above, wherein the genes encoding ILV2, ILV3, ILV5, ILV6, BAT1 and / or BAT2 are expressed from their natural promoters.

[0229] 67. A method or yeast strain comprising any one of the items described above, wherein the yeast strain possesses one or more mutations or deletions in the ILV2 gene.

[0230] 68. A method or yeast strain comprising any one of the items described above, wherein the yeast strain possesses one or more frameshift mutations in the ILV2 gene.

[0231] 69. A method or yeast strain comprising any one of the items described above, wherein the yeast strain possesses a mutation resulting in reduced or absent expression of one or more ILV2 genes.

[0232] 70. A method or yeast strain comprising any one of the items described above, wherein the yeast strain possesses one or more mutations or deletions in the ILV6 gene.

[0233] 71. A method or yeast strain comprising any one of the items described above, wherein the yeast strain possesses one or more frameshift mutations in the ILV6 gene.

[0234] 72. A method or yeast strain comprising any one of the items described above, wherein the yeast strain possesses a mutation resulting in reduced or absent expression of one or more ILV6 genes.

[0235] 73. A method or yeast strain comprising any one of the items described above, wherein the yeast strain does not contain heterologous DNA.

[0236] 74. A method or yeast strain relating to any one of the items described above, wherein the yeast strain has not undergone a genetic engineering step.

[0237] 75. A fermented aqueous extract prepared by any one of the above-mentioned items.

[0238] 76. A method for producing a beverage, comprising the following steps: i. A step of preparing a fermented aqueous extract according to any one of the items, and ii. A step of processing the fermented aqueous extract to make a beverage.

[0239] 77. A method according to item 76, wherein the processing steps include one or more of the following: i.filtration, ii. Carbonation, iii. Aging, or iv. Bottled.

[0240] 78. A beverage prepared by any one of the methods described in items 76-77.

[0241] 79. A beverage according to item 78, wherein the beverage contains at least 25 mg / L of propanol, for example, at least 30 mg / L of propanol.

[0242] 80. A beverage relating to any one of items 78-79, wherein the beverage contains a maximum of 8 mg / L of isobutanol, for example, a maximum of 6 mg / L of isobutanol.

[0243] 81. A beverage which is one of items 78-80, wherein the beverage is beer.

[0244] 82. A fermented aqueous extract according to item 75 or a beverage according to any one of items 78 - 81, wherein the extract or the beverage has an isobutanol to propanol ratio of at least 3.0, for example at least 4.0, for example at least 5.0, for example at least 5.5.

[0245] 83. A yeast strain that can produce a fermentation test solution after incubation of the yeast strain in a test solution having an appearance extract content of at least 10° Plato, and the test solution has a maximum of 60 ppb of diacetyl, for example a maximum of 50 ppb of diacetyl, at the earliest time point when the appearance extract content of the test solution does not decrease by more than 0.50° Plato in the 24 hours preceding the incubation of the yeast strain in the extract.

[0246] 84. A Saccharomyces pastorianus yeast strain according to item 83, wherein the yeast strain is as defined in any one of items 1 - 72.

[0247] Examples Materials and Methods Wort Preparation Generally, the wort used in the following experimental examples was produced by mashing 70% pilsner malt and 30% barley adjunct at 2.7 L of water / kg malt.

[0248] The mashing regime is shown in Table 1 below:

Table 3

[0249] After mashing, the wort was boiled for 60 minutes. To ensure that the wort is suitable for fermentation, preferably, it should have the following contents within the following ranges: · Glucose 12 - 25 g / L · Maltose 60 - 80 g / L Maltotriose 15-20g / L • Zinc 0.16~0.18 mg / L • Free α-amino nitrogen (FAN) 110-250 mg / L • Valin / FAN 0.6~0.8

[0250] For reference, Table 2 below provides the average content of wort produced from 70% Pilsner malt and 30% Oomgia junk. Worts outside some of the parameters below are still expected to produce good beer. [Table 4]

[0251] To aid in the cottony precipitation of yeast, additional zinc may be added to the wort before fermentation, as described in some of the examples below.

[0252] Droplet PCR Genomic DNA was prepared from diploid yeast strains and haploid spore clones using the MasterPure genomic DNA purification kit from Epicentre. The concentration of each final DNA preparation was adjusted using 50 ng / microliter of DNA with Milli-Q water. DNA was quantified using a NanoDrop2000 spectrophotometer (Thermo Scientific).

[0253] Biorad droplet PCR (QX200) and probes designed to anneal to the ILV2 genes of Saccharomyces eubayanus and Saccharomyces cerevisiae were used. These probes were used to target genomic DNA in haploid spore clones and control diploid yeast strains. Using this method, the inventors were able to quantitatively determine the ratio between two variant ILV2 genes. To distinguish between the S. eubayanus and S. cerevisiae ILV2 alleles, a species-specific digital PCR reaction based on a TaqMan assay was designed. For this purpose, target-specific primers were designed in homologous regions of the ILV2 genes for S. eubayanus and S. cerevisiae, meaning that the same forward and reverse primers could simultaneously amplify both ILV2 species copies. Species-specific probes were designed to be identical except for a single nucleotide, enabling differentiation between the S. eubayanus and S. cerevisiae type ILV2 alleles. The species-specific probe for S. cerevisiae has a T at nucleotide position 1515 in the S. cerevisiae ILV2 wild-type sequence, while the species-specific probe for S. eubyanus has a C at nucleotide position 1515 in the S. eubyanus ILV2 wild-type sequence. The following primers and probes were developed to distinguish between the S. eubyanus ILV2 allele and the S. cerevisiae ILV2 allele: Target-specific ILV2 primers that simultaneously amplify both S. cerevisiae and S. eubayanus ILV2: Target-specific forward primer (5'-GCCAACGACACAGGAAGAC-3') (SEQ ID NO:1) Target-specific reverse primer (5'-GTACCTAAACCACCTGATGT-3') (SEQ ID NO:2). A species-specific probe that enables differentiation between S. cerevisiae and S. eubayanus ILV2 copies: Labeled with S. cerevisiae ILV2 allele-specific probe (5'-TGGGCTGCTCAACAC-3')(SEQID:3)-5'FAM and 3'BHQ1. S. eubayanus ILV2 allele-specific probe (5'-ACGTACGGAATGTGGATT-3') (SEQ ID NO:4)-labeled with 5'HEX and 3'BHQ1.

[0254] ddPCR was performed using a droplet digital PCR QX200 system (Bio-Rad Laboratories) according to the manufacturer's instructions. For analytical purposes, 5 μl of purified gDNA (5 ng / μl DNA concentration) of the yeast strain was added to a 17-μl PCR mixture containing 11 μl of 2x ddPCR Supermix (No. dUTP; Bio-Rad) for the probes, a 900 nM target-specific PCR forward primer, a 900 nM target-specific PCR reverse primer, a 250 nM S. cerevisiae ILV2 allele-specific probe, and a 250 nM S. eubayanus ILV2 allele-specific probe. The reaction mixture was loaded onto an AutoDG droplet generator (Bio-Rad Laboratories), and droplet generation was performed according to the manufacturer's manual. Droplet emulsions were thermally cycled using standard PCR conditions: denaturation at 95°C for 10 minutes, PCR for 40 cycles at 94°C for 30 seconds and 55°C for 1 minute, and final extension at 98°C for 10 minutes, followed by storage in microtiter plates at 8°C. PCR amplification in droplets was confirmed using a QX200 droplet reader (Bio-Rad Laboratories), and the data were analyzed using QuantaSoft software (version v1.7, Bio-Rad Laboratories).

[0255] Detection of total ILV2 gene copy number In the first round, the ratio between S. eubayanus and S. cerevisiae ILV2 was directly analyzed on purified genomic DNA from each single spore clone. Despite originating from the same parent yeast, some spore clones had different ILV2 genetic structures. Some spore clones are thought to possess only 100% S. cerevisiae ILV2 gene spore clones. Since most parent Lager yeasts are triploid or tetraploid (Wahlter et al, 2014), it can be assumed that a 100% S. cerevisiae ILV2 spore clone may have approximately 1-3 copies. For spore clones with both types of ILV2, e.g., 50 / 50, one copy of each type in the spore clone, or 2+2 in the parent yeast, would be assumed. A strain with a ratio close to 67 / 33 would mean one copy of S. cerevisiae and two copies of S. eubayanus ILV2.

[0256] To enable a more accurate estimation of copy number in spore clones with nearly 100% S. cerevisiae ILV2, trials were repeated by adding or spiking in pure exogenous S. eubyanus DNA to the sample to obtain the S. cerevisiae / S. eubyanus distribution after spiking. A ratio ScILV2 / SeILV2 that shifted strongly toward S. eubyanus after spiking would indicate a low number of S. cerevisiae ILV2 copies, while a ratio that shifted little toward S. eubyanus DNA and remained dominated by high S. cerevisiae ILV2 would indicate that the original spore clone had many S. cerevisiae ILV2 copies.

[0257] Spiking was performed by blending each sample of 50 ng / microliter DNA with S. eubyanus DNA at the same concentration of 50 ng / microliter in a 50 / 50 ratio (10 microliters of sample + 10 microliters of S. eubyanus DNA).

[0258] The droplet QPCR estimation method quantitatively reflects the total number of ILV2 copies present, but due to the presence of specific SNPs or frameshift mutations, it cannot clarify whether any inactivated ILV2 alleles are present. Such mutations can first be detected by subsequent genomic DNA sequencing, either by Sanger sequencing or next-generation sequencing.

[0259] Screening for flocculation To evaluate the flocculation ability of yeast spore clones, they were first grown in normal wort at room temperature for several days. For each strain, 2 ml of the culture was transferred to a 2 ml reaction tube, vortexed thoroughly, and the cells were allowed to sediment for 10 minutes. To determine calcium-dependent flocculation, cells were collected from 2 ml of the aforementioned culture, washed twice with 50 mM EDTA solution, and then resuspended in 2 ml of 50 mM Tris, 50 mM succinic acid, and 100 mM KOH, without calcium (Stratford 1996). The pH was adjusted to pH 4.0, which is similar to the final pH value of the wort culture. The cell suspension was vortexed again thoroughly, and the cells were allowed to sediment for 5 minutes. The results were documented by photography.

[0260] Measurement of total diacetyl in spore clones in wort fermentation using a cylinder with magnetic stirring Spore clones and control yeast were pre-grown for 3 days in 2 ml of liquid YPD on a Stuart SB2 rotary unit (www.stuart-equipment.com). 2 ml of the preculture was inoculated into 16 baffled shake flasks containing 25 ml of pasteurized standard pilsner wort and grown at room temperature on a shaking table with intermediate shaking for 5 days to reach a high-density stationary-phase culture.

[0261] Beer fermentation was carried out in a glass cylinder as previously described (Guiterrez et al., 2018). Fermentation was carried out in a 250 mL tall glass graduated cylinder (331 × 39 × 39 mm; Duran) containing 200 mL of pilsner wort, sealed over the top of the cylinder with an inverted glass beaker (Duran) to allow carbon dioxide leakage and easy sampling access for analysis. Fermentation was carried out with continuous stirring at 150 rpm using a magnetic bar on a Variomag stirring platform in a refrigerator (type TS 606-G / 4-i incubator cabinet) at 16°C. Yeast cell count was estimated using a cellometer (https: / / www.nexcelom.com / applications / cellometer), seeding (pitching) was performed at a rate of 15 million cells / ml wort, and fermentation performance was monitored by measuring weight loss due to metabolic CO2 release. 10 ml samples were collected on days 2, 3, and 4 during fermentation for diacetyl analysis. The sample was centrifuged at 1900g for 10 minutes, and the supernatant was stored in a freezer at -20°C until further use. Fermentation was considered complete when no further weight loss of the fermentation cylinder could be measured. We measured total diacetyl by gas chromatography according to European Brewing Commission method EBC 9.24.2, which includes a 90-minute incubation period at 60°C for the sample to determine total diacetyl and reflects the sum of precursor acetolactate and free diacetyl.

[0262] yeast strain Hybrid yeast 7, Saccharomyces pastorianus, is a lager-producing strain that exhibits high cottony sedimentation, allowing for yeast collection at the end of fermentation without additional cooling. Hybrid yeast 7 is used as the standard for yeasts in the present invention.

[0263] ZDA1 is the yeast of invention.

[0264] The ZDA2 is the yeast of invention.

[0265] The ZDA3 is the yeast of invention.

[0266] W-34 / 78 Saccharomyces pastorianus is a commercially available lager yeast strain from Weihenstephan Hefebank (info@hefebank-weihenstephan.de), Germany. This yeast is known to reduce diacetyl quite efficiently and exhibits high cottony precipitate, but still significantly less than Hybrid Yeast Strain 7 when fermented in the wort described above. It is used as the standard for the yeast of the present invention. W-34 / 78 is also referred to herein as WS34 / 78.

[0267] Examples 1-16 and 50L trials using yeast strain ZDA1 and comparative strain Hybrid yeast 7 at 18°C To test the difference in diacetyl levels during fermentation between novel hybrid yeast strains and control yeast strains, 50 L of beer was prepared from wort with a pH of 15–16° as described in the Materials and Methods section. Before fermentation, the wort was adjusted to a pH of 4.9 and zinc was added to a final concentration of 0.30 mg / L. Wort containing 12–15 million live yeast cells / mL was inoculated into the wort. Fermentation was carried out at 16°C or 18°C. Generally, lagers are fermented at 12°C–16°C. If the yeast can tolerate 18°C, this can act to accelerate the fermentation process. Samples were obtained on day 0 and at various points during fermentation as shown in the tables below. The results are shown in Tables 3 and 4 below and plotted in Figures 1A–C. [Table 5] [Table 6]

[0268] Beer is said to be within specs if its diacetyl level is below 50 ppb and the prat is stable (all fermentable sugars are utilized). The advantage is to obtain beer within specs in the shortest possible time while producing at least the same amount of alcohol as the control yeast.

[0269] Diacetyl levels at 18℃ As can be seen from Table 3 and Figures 1B and 1C, when fermented at 18°C, yeast strain ZDA1 was able to produce fermented wort (green beer) within 3 days, with diacetyl levels close to 50 ppb, and all fermentable sugars present in the wort were utilized (this is evident from the fact that the °Prate drop remained below 0.50 over 24 hours). In contrast, the control yeast strain Hybrid yeast 7 started at very high diacetyl levels (Figure 1B) and required 4 days to reach diacetyl levels similar to those of strain ZDA1 on day 3.

[0270] The earliest point at which the visible extract content did not decrease by more than 0.50° in the preceding 24 hours was considered to be day 4 for both strains. Green beer obtained using Hybrid yeast 7 on day 4 contained approximately 56 ppb of diacetyl, while green beer obtained using ZDA1 contained only 26 ppb.

[0271] When fermentation was carried out at 16°C (Table 4 and Figure 1A), the earliest point at which the visible extract content did not decrease by more than 0.50° in the preceding 24 hours was day 5 for both strains. At that time, strain ZDA1 reached the desired 50 ppm diacetyl level, while the control yeast Hybrid Yeast 7 required an additional 2 days of fermentation to reach a diacetyl level of less than 50 ppm.

[0272] Isobutanol vs. propanol and SO2 levels after fermentation at 16°C or 18°C 16℃ 18℃ In addition to diacetyl and praline, propanol and isobutanol levels were measured in the final beer produced from 50L trial beer. The final beer was adjusted to an alcohol content of 5.0 vol% and a CO2 concentration of 5.1 g / L, and bottled in 33cl green bottles. While not strictly theoretical, propanol and isobutanol are considered indicators of restricted pathway flow within the branched-chain amino acid pathway, particularly low ILV2 activity in yeast, and therefore provide a quick way to assess whether a yeast strain is likely to be a low diacetyl producer. A high isobutanol-to-propanol ratio indicates a low-diacetyl-producing yeast. The results are shown in Table 5 below: [Table 7]

[0273] As can be seen from Table 5, at both 16°C and 18°C, green beer obtained from fermentation with hybrid yeast strain ZDA1 showed higher levels of propanol compared to green beer obtained with the control yeast strain Hybrid Yeast 7. Wort incubated with hybrid yeast strain ZDA1 also had lower levels of isobutanol compared to Hybrid Yeast 7, with a difference of at least 6.8 times in the isobutanol to propanol ratio.

[0274] It was also observed that the SO2 levels in green beer obtained using the ZDA1 yeast strain were higher than those in green beer derived from the control strain. This may offer an advantage in terms of preserving the final beer.

[0275] Figure 3 shows the propanol-to-isobutanol ratio of commercially available beers compared to "control bottle 89-84393ZDA2" beer produced using the ZDA2 yeast strain according to the present invention. "Control bottle 89-84393ZDA2" was produced in a 50L trial and debrewed to 5%. All commercially available beers had a lower propanol-to-isobutanol ratio.

[0276] Example 2 - 50L trial using yeast strains ZDA2 and ZDA3 at 16°C Two further yeast hybrid strains with favorable diacetyl profiles were identified and compared to the ZDA1 strain of Example 1. 50 L of beer was prepared from wort having 16° plutonium, as described in the Materials and Methods section. Prior to fermentation, the wort was adjusted to a pH of 4.9 and zinc was added to a final concentration of 0.30 mg / L. 14 million live yeast cells / ml of wort were inoculated into the wort. Yeast for inoculation was obtained from the 50 L first fermentation, and yeast cells were collected at the end of fermentation and used to repitch to the second 50 L fermentation trial. This is similar to the method used in large-scale beer production. Fermentation was carried out at 16°C. Samples were obtained on day 0 and during fermentation, as shown in the table below. The results are shown in Table 6 below. [Table 8]

[0277] As shown in Table 6, all three candidates (ZDA1, ZDA2, and ZDA3) showed diacetyl levels of less than 50 ppb on day 4, which corresponds to the time it took for the visible extract content to reach a plateau. Furthermore, ZDA3 already had a diacetyl level of less than 50 ppb on day 3.

[0278] Example 3 - Taste Results The fermented wort obtained in Example 2 was further processed to obtain the final beer by adjusting the alcohol content to 5% by volume and the CO2 to 5.1 g / L, and bottled in standard Danish green 33cl bottles. The beer was evaluated by a professional beer taste panel consisting of 10 tasters. Each taste panel member was well-trained, and in particular, each member was skilled in evaluating the taste characteristics of esters, higher alcohols, sulfur components, and the body of the beer. Each taste panel member evaluated different flavor notes. In particular, the overall taste was rated on a scale of 1 to 9, and is provided as “Main Results” in Table 7 below. [Table 9]

[0279] As shown in Table 7, beer samples produced by all yeast strains were rated at least 5.7 (satisfactory).

[0280] Example 4 - 10HL beer trial using yeast strain ZDA1 and comparative strain Hybrid yeast 7 at 16°C The objective of this example was to investigate whether the hybrid yeast strain ZDA1 exhibited the same favorable diacetyl profile as observed in Example 1 on a 50 L scale. 10 HL beer was prepared from wort having approximately 16° plutonium, as described in the Materials and Methods section. Prior to fermentation, the wort was adjusted to a pH of 4.95 and zinc was added to a final concentration of 0.8 mg / L. The wort was inoculated with either the hybrid yeast strain ZDA1 or the control lager yeast strain Hybrid Yeast 7 at a concentration of at least 15 million live yeast cells / mL of wort, followed by fermentation at approximately 16°C. Yeast for inoculation was obtained from the first brew described in Example 2. Samples were obtained on day 0 and on the days shown in Results Table 8 below. The results are shown in Table 8 and illustrated in Figures 2A and B. [Table 10]

[0281] Yeast strain ZDA1 produced relatively low initial levels of diacetyl, and the green beer obtained using hybrid yeast strain ZDA1 reached near the diacetyl taste threshold (50 ppb) on days 4 and 5, before dropping to 28 ppb. However, the green beer obtained using the control yeast strain Hybrid Yeast 7 only reached this threshold on day 7 (Figure 2A). Figure 2B shows that the total diacetyl level in the green beer obtained using yeast hybrid ZDA1 reached approximately 50 ppb around the time the fermentable sugars were almost completely fermented, i.e., on day 4.

[0282] Example 5 - Taste Results The fermented wort obtained in Example 4 was further processed to adjust the alcohol content to 4.6% by volume and the CO2 to 5.4 g / L to produce the final beer, which was then bottled in 33cl amber bottles. The beer was evaluated by a bot of 10 expert beer tasters from Denmark and France. Each taste panel member was well-trained and particularly adept at evaluating the taste characteristics of esters, higher alcohols, sulfur components, and the body of the beer. Each taste panel member evaluated different flavor notes. In particular, the overall taste was rated on a scale of 1 to 9 and is provided as the "main result" in Table 9 below. [Table 11]

[0283] As shown in Table 9, the beer samples produced using yeast strain ZDA1 were rated 6.6 (satisfactory), which slightly surpasses the beer produced using the control strain Hybrid yeast 7.

[0284] Example 6 - Trial at 16°C including three control lager yeast strains This experiment was conducted to compare three novel yeast strains from Example 2 with additional commercially available yeast strains, as well as controls used in Examples 1, 2, and 4. 40 L of beer was prepared from the wort described in the Materials and Methods section, using a small variation of an initial holding time of 30 minutes at 52°C. For fermentation, the temperature was set to 15-16°C. Before fermentation, the wort was adjusted to a pH of 4.9 and zinc was added to a final concentration of 0.30 mg / L. The wort was inoculated with hybrid yeast strains ZDA1, ZDA2, and ZDA3 at 7.5 million live yeast cells / ml, as well as the control lager yeast strain Hybrid Yeast 7 and the control lager yeast strain W-34 / 78 (commercially available from Weihenstephan, Germany). The wort was fermented at approximately 16°C. Samples were obtained on day 1 and on the days shown in the results table below. The results are shown in Table 10. [Table 12] TIFF2023535281000015.tif100161

[0285] In this trial, all three low-diacetyl strains (ZDA1, ZDA2, and ZDA3) showed diacetyl levels below 50 ppb on day 5, which corresponds to the time at which the visible extract content reached a plateau for all tested yeast strains, while none of the control strains showed diacetyl levels below 110 ppb on the same day. The earliest point at which the visible extract content did not decrease by more than 0.50° in the preceding 24 hours is considered to be day 6 for all of these trials. On day 6, all low-diacetyl strains (ZDA1, ZDA2, and ZDA3) had diacetyl levels below 45 ppb (24 ppb, 25 ppb, and 18 ppb, respectively), while the control strains (Hybrid Yeast 7, W-34 / 70, and W-34 / 78) had higher diacetyl levels (115 ppb, 62 ppb, and 62 ppb, respectively).

[0286] These results are quite noteworthy, considering that Weihenstephan yeast does not precipitate as cottony as the Hybrid Yeast 7 strains and the inventive strain. The number of cells in the liquid at the end of fermentation (above the yeast pellet) is a good measure of cottony precipitation. These data are shown in Table 10 above as cells in the liquid (lower values ​​indicate more cottony yeast). High cottony precipitation is advantageous if the yeast is to be collected from the production tank to be inoculated into the next wort for beer production. Low cottony precipitation of yeast requires cooling of up to 24 hours for green beer. On the other hand, high cottony precipitation may result in less contact between the yeast and the wort during fermentation, which may reduce the rate at which the yeast can consume diacetyl and lengthen the fermentation time.

[0287] Therefore, the examples described herein provide yeast strains that can reduce diacetyl to less than 50 ppb approximately at the same time (or even before) the fermentable sugars are consumed, and at the same time, due to their high cottony precipitation at 16°C, no additional cooling of the green beer is required to collect the yeast for the next beer production.

[0288] Furthermore, it was found that low-diacetyl yeast strains could be easily identified by the isobutanol-to-propanol ratio, as they were all greater than 6, while all control strains were less than 1.4.

[0289] Example 7 - Genotyping of yeast strains Genome construction We developed a tandem analysis approach using NGS data from Illumina and Pacbio platforms for four of five genomes (Hybrid Yeast 7, ZDA1, ZDA2, and W34-78). Quality control, filtration, and construction were performed according to an in-house designed pipeline. All genomes were de novo constructed using Canu1.9 software with data generated by the Pacbio platform. Subsequently, reads generated by the Illumina platform were mapped to assemblies generated by Canu1.9 (Pilon1.22) to improve the consensus accuracy of the draft assemblies. For genome ZDA3, due to the lack of Illumina data, only Canu1.9 was used without further construction improvements.

[0290] The resulting constructed genome was used in the BLAST query.

[0291] Hit Blast and Sequence Comparison For each of the genes (ILV2, ILV3, ILV5, ILV6, BAT1, BAT2), the corresponding amino acid sequence of the S. cerevisiae gene was used as a query sequence for each of the five genomes using the TBLAStN algorithm. Finally, hits were filtered by retaining those that had at least 65-80% sequence identity with the query sequence or occupied at least 40% of its length. The amino acid sequence alignments of ILV2, ILV3, ILV5, ILV6, BAT1, and BAT2 between WS34-78, cer (Saccharomyces cerevisiae), eub (Saccharomyces eubyanus), Hybrid7 (Hybrid Yeast 7), ZDA1, and ZDA2 are shown in Figure 4-9. BLAST analysis was not repeated for the S. eubyanus corresponding gene. This is because, in the final stage of filtering, the hits were able to achieve a low sequence identity of 65%, and therefore, in this setup, all hits originating from both S. cerevisiae and S. eubyanus would be captured due to their high similarity. All hits identified by BLAST analysis were aligned using the MUSCLE(3.8) multiple sequence alignment tool.

[0292] Ploidy identification analysis Ploidy identification analysis was performed by deploying an improved version of the sppIDer algorithm. Short reads from four of the five genomes on the Illumina platform were mapped against the combined reference genomes of S. cerevisiae and S. eubyanus, reads with mapping quality (MQ) > 3 were retained and classified into the order of the combined reference genome; then, coverage across the combined reference genome was calculated. A custom script then calculated the average coverage for each, and the combined reference genome was divided into windows.

[0293] Technical shortcomings of BLAST analysis The use of BLAST to study the effects of genomic differences on strain phenotypes suffers from several drawbacks, the most important of which is: i) Inability to assess duplication at the gene and / or chromosome level. ii) Susceptibility to errors or ambiguities in assembly

[0294] All genome assembly methods have problems producing meaningful assemblies in the presence of ploidy. This results in multiple regions from different chromosome copies collapsing into a single scaffold. In principle, the situation is not so bad when gene duplication events occur, as they can be identified using specific assembly methods and appropriate experimental setups. However, gene duplication is still quite likely to be poorly detected at the assembly stage, especially when there are no or limited genomic differences between the different copies. Regarding the second point, BLAST can, in principle, identify more detailed information about different variants, such as functional SNPs and non-functional ORFs. However, errors or ambiguities in assembly can lead to the generation of numerous false demands—e.g., pseudoSNPs and obscure gene architectures.

[0295] Therefore, we decided to perform simple BLAST identification of variants in parallel with estimation of chromosomal ploidy and the observed ploidy of each genomic region in the sequencing data, due to gene duplication or deletion.

[0296] Overall, the generation of haplotype-resolved de novo assemblies remains a major challenge today. While NGS technologies (Pacbio and Illumina) generate large amounts of data, distinguishing between errors and true sequence variants remains technically difficult. Furthermore, if the sample is polyploid, true variants must be assigned to different genomic copies. The assemblies in these analyses have been performed using haplotype-incompatible algorithms, and therefore the final assemblies represent the broken haplotype genome.

[0297] result The results are shown in Table 11 below. [Table 13] TIFF2023535281000017.tif111160

[0298] The numbers in parentheses represent the modal ploidy of the chromosome. The numbers without parentheses represent the ploidy of locus determination on that chromosome.

[0299] Example 8 - Commercial-scale brewing trial using yeast strain ZDA2 and comparative strain Hybrid yeast 7 The objective of this example is to investigate whether the hybrid yeast strain ZDA2 exhibits the same favorable diacetyl profile observed in the previous example when applied in a commercial-scale brewing environment (approximately 1800 HL), where the yeast is collected and re-pitched.

[0300] 1800HL beer was prepared from wort containing malt and adjunct obtained by conventional injection mashing, with a pH of approximately 16°. The wort was adjusted to a pH of 4.9–5.0 before fermentation. The wort was refitched with both ZDA2 and the control lager yeast strain Hybrid Yeast 7, generation 8, and fermented at approximately 16°C. Samples were taken on day 0 and on the dates shown in Table 12 and Figure 10 below. [Table 14]

[0301] The yeast strain ZDA2 produced relatively low levels of diacetyl throughout fermentation, and the green beer obtained using the hybrid yeast strain ZDA2 reached the taste threshold for diacetyl (50 ppb) as early as day 6. At that time, the prat level did not decrease by more than 0.5 over the last 24 hours. The green beer obtained using the control yeast strain Hybrid Yeast 7 did not reach the taste threshold for diacetyl for 2 days, after the prat level did not decrease by more than 0.5 over 24 hours on day 7 (Figure 10). As a result, the specification for green beer was reached at least 2 days earlier when using the ZDA2 yeast compared to the reference yeast Hybrid Yeast 7, in terms of diacetyl and prat levels.

[0302] Interestingly, the ZDA2 yeast reached true fermentation (RDF), which was 2% lower than that of Hybrid yeast 7, while the alcohol percentage was almost identical in both green beers. As a result, the ZDA yeast was able to produce the same amount of alcohol at a lower true fermentation, which resulted in a beer with a better texture. This trend of 1.5-2.5% lower RDF at the same alcohol level for the ZDA2 yeast was observed across all fermentation generations preceding the 8th generation in this example (see Table 13). [Table 15]

[0303] Example 9 - Illumina sequencing and bioinformatics evaluation of brewing strains to calculate the copy number of hybrid nanopore-related genes The strains were sequenced using the Illumina platform as previously described. For nanopore sequencing, ultra-high molecular weight DNA was prepared using the method outlined by Denis et al.

[0304] To prepare HMW gDNA for sequencing, samples were treated using a Zymo Genomic Clean and Concentrator column, and then checked for quality and quantity using a deNovix dsDNA Broad Range fluorescence quantitative assay.

[0305] All samples were duplicated, and libraries were prepared using SQK-LSK109 Chemistry and Oxford Nanopore Technologies' Native Barcode Extension packs EXP-NBD104 and EXP-NBD114. All necessary cleansing steps were performed using Clean NA magnetic beads for NGS. Genome sequencing was performed on MinIOn FlowCells FLO-MIN106D for 48–72 hours.

[0306] The raw sequencing data was base-called using bonito (https: / / github.com / nanoporetech / bonito) and demultiplexed using guppy (https: / / nanoporetech.com / nanopore-sequencing-data-analysis). Hybrid genome assembly was performed using Marsurca (https: / / github.com / alekseyzimin / masurca) in combination with Nanopore and Illumina fastq files, and the constructed genome was annotated using prokka. (https: / / github.com / tseemann / prokka). Sequence coverage and copy number of target genes were calculated using qualimap (http: / / qualimap.conesalab.org / ) and visualized using Integrative Genomics Viewer (https: / / software.broadinstitute.org / software / igv / ).

[0307] Read mapping and SNP detection were performed using the CLC Genomics Work Bench (version 11). In short, trimmed reads were mapped against an "in silico" yeast hybrid genome (including the ligation of S. eubayanus (acceptance number: GCA_0012986.25.1) and S. cerevisiae (GCA_000146045.2) genomes). Single nucleotide polymorphisms (SNPs) and insertions / deletions (indels) were detected using a basic variant detection tool. Here, the functional copy number of the ILV2 gene can be estimated based on the diagnostic SNP present on a single allele within ZDA yeast.

[0308] In the S. cerevisiae ILV2 gene, a C' frameshift is present, which inactivates the resulting protein (Leu575fs) due to a T nucleotide insertion within this codon. Approximately 33% of Illumina reads mapped at this frequency suggested that one of the three ILV2sc copies was inactivated. Furthermore, in the Eubayanus ILV2 allele, a T nucleotide deletion was observed to result in a frameshift (Leu304fs) that leads to truncation inactivation. Knowing the copy number (CN) of the ILV2 cerevisiae gene allowed for the calculation of the copy numbers of other genes based on the previously described read mapping. The estimation of the WS34 / 78 CN was also based on read mapping-based estimation, based on previous evidence that the highly similar strain (WS34 / 70) is a tetraploid yeast with equal S. cerevisiae and S. Eubayanus genome content (Walther et al. 2014). Furthermore, in Example 10, it was observed that when the ILV2 euybanus allele was removed, WS34 / 78 actually contained two copies of the ILV2 euybanus allele (including a total of four ILV2 alleles and cerevisiae alleles). These results are summarized in Table 14. [Table 16]

[0309] The reference strain WS3478 has four functional copies of the ILV2 gene, while ZDA1 and ZDA2 each have only two functional copies of this gene. In addition, WS3478 has four functional copies of ILV3 and three functional copies of ILV5, while these numbers correspond to 6 and 5 for ZDA1 and ZDA2 strains, respectively.

[0310] Example 10 - Gene copy number matching in model brewer yeast W-34 / 78 The overall objective of this experiment was to adjust the copy number of the relevant genes in a model yeast strain (Saccharomyces pastorianus subspecies karsbergensis strain (WS34 / 78)) to reflect the genotypes of ZDA1 and ZDA2. WS34 / 78 is commercially available from Weihenstephan, Germany, and is also referred to herein as W-34 / 78. To do so, the genes in strain WS34 / 78 had to be inactivated or, in addition, expressed, as shown in Table 15. [Table 17]

[0311] Inactivation was carried out with the help of a resistance-constituting antibiotic-cassette targeting the coding sequence of the target gene (i.e., ILV2). Here, the coding DNA sequence (cds) was looped via homologous recombination, leaving only 99 bp of the N-terminal region of the gene and 107 bp of the C-terminal region. Overexpression of the genes (i.e., ILV3 and ILV5) was carried out in a manner that preserved the true genomic context, i.e., a cassette containing approximately 1 kbp upstream of cds, cds, and 0.5 kbp downstream of cds was cloned. Thus, the cassette contains the innate promoter and terminator sequences, and the expression of additional gene copies is presumably under natural control. Furthermore, yeast single-copy plasmids were used so that the copy number was under control.

[0312] Transformation of WS34 / 78 cells Cells of Saccharomyces pastorianus subspecies karsbergensis strain WS34 / 78 were made competent for DNA uptake according to the method described by Gietz and Schiestel (DOI: 10.1038 / nprot.2007.17). Routinely, cells were transformed with 1 μg of plasmid DNA or PCR-amplicon. The duration of heat shock treatment was reduced to 15 minutes while maintaining the temperature at 42°C.

[0313] Generation of expression cassettes Based on the DNA sequence information of strain WS34 / 78 from Example 9, expression cassettes were constructed by PCR amplification of the natural WS34 / 78 DNA sequence, including approximately 1000 bp upstream of the cds and 500 bp downstream of the cds, thus containing the natural promoter and terminator sequences. The exact length of the amplicon can be estimated from the individual primer sequences given in the example. The 1000 bp or 500 bp cds-adjacent region should allow for true / natural control of the target gene. Cloned PCR-amplicons (integrated into plasmids / chromosomes) were controlled by Sanger-DNA sequencing, using isolated plasmid DNA as a template for the sequencing reaction.

[0314] Chromosomal integration of expression cassettes In the first step, an expression cassette was constructed by fusing a gene expression module with an antibiotic resistance module that enabled positive selection of integration events. The cassette was fused to a PCR-amplicon encoding a DNA stretch for homologous recombination with the WS34 / 78 chromosome. The site for integration was the PAD1 locus of WS34 / 78, because this gene is non-functional in this yeast, and therefore, substitution of this gene has no effect on the flavor profile. The adjacent region used for homologous recombination was approximately 500 bp in length. Yeast clones grown in the presence of antibiotics were analyzed by PCR (i.e., colony PCR) to confirm the insertion of the cassette. Positive clones were further analyzed for mutations in the expression module. The amplicon containing the expression module was controlled by Sanger-DNA sequencing.

[0315] Cloning of the ILV- gene in plasmid pYESVIII Plasmid pYESVIII(BRAIN;) is an E. coli-yeast shuttle vector that replicates at a high copy number in E. coli and is maintained as a single-copy plasmid in yeast cells for the CEN6 replication origin. The expression cassette was routinely inserted into the multicloning site using a Gibson assembly.

[0316] When two copies of the same allele had to be inserted, preferably one copy of the target gene (GOI) had the true DNA sequence, while the cds of the second copy were altered in a way that left the native protein sequence unchanged. This was achieved by chemical gene synthesis employing an algorithm that optimizes gene expression in yeast cells (codon use optimization). This was done to reduce alterations to the recombination event during plasmid transformation into yeast cells.

[0317] A tandem construct is built for the expression of the ILV3 gene from the plasmid (bisistronic expression). Here, a single promoter controls the expression of two ILV3 coding sequences (one native and one synthetic). The two coding sequences are joined by a 2A-peptide linker, which enables ribosome skipping, so that both cds are translated under the control of the native ILV3 promoter. The 2A-peptide technique modifies the C-terminus of the first protein by adding a 3-amino acid tag (NPG), while the N-terminus of the subsequent protein starts with proline (Liu, Z. et al., 2017; DOI: 10.1038 / s41598-017-02460-2).

[0318] E. coli was transformed with in vitro constructed plasmids, the plasmids were isolated from the transformants, controlled by restriction digestion and DNA sequencing, and subsequently transformed into competent WS34 / 78 cells. [Table 18]

[0319] Example: Mutant variant of S. pastorianus WS34 / 78 Recombinant stocks Generation of strain #2 Template for PCR: Plasmid pYES5-Cen6-Sh.ble(sc)2.0(BRAIN) encodes the Sh.ble cassette. gDNAS. Pastorianus WS34 / 78, strain #1 (Table 16) Primer used : CPA43:SEQ ID NO:44 CPA44:SEQ ID NO:45 CPA49:SEQ ID NO:46 CPA50:SEQ ID NO:47 CPA51:SEQ ID NO:48 CPA52:SEQ ID NO:49 The sh.ble cassette (fragment 1) was amplified from plasmid pYES5-Cen6-Sh.ble (Saccharomyces cerevisiae (sc)) 2.0 (BRAIN Biotech AG) using primers CPA43 / 44. Homology arms were generated by amplification of the ILV2 (Saccharomyces eubayanus (se)) facultative region from gDNA strain #1 using allele-specific primers CPA49 / 51 (5'-facultative region; fragment 2) and CPA50 / 52 (3'-facultative region; fragment 3). HR templates were generated with the help of overlap extension PCR using primers CPA49 / 50 and fragments 1+2+3 as templates. Allele-specific knockout of ILV2(se) by homologous recombination was assisted by BRAIN's proprietary NUCLEASE technology. This yielded numerous strains, which had single or double embeddings at the ILV2(se) locus and were visible on agarose gels. Strains with double deletions of ILV2(se) were selected for downstream analysis. ILV2(se) knockout cassette sequence: SEQ ID NO: 50

[0320] Generating stock #3 Template for PCR: gDNA S. pastorianus WS34 / 78, strain #1 (Table 16) NTC-DNA Cassette (Nourseothricin); BRAIN Biotech AG Primers used: CPA70:SEQ ID NO:51 CPA71:SEQ ID NO:52 CPA91:SEQ ID NO:53 CPA96:SEQ ID NO:54 CPA111:SEQ ID NO:55 CPA112:SEQ ID NO:56 CPA113:SEQ ID NO:57 CPA114:SEQ ID NO:58 CPA128:SEQ ID NO:59 CPA129:SEQ ID NO:60 The ILV3(se) expression cassette (product 1) was amplified from gDNA strain #1 using allele-specific primers CPA70 / 71. Product 1 was then further amplified using primer CPA70 / 129 to obtain product 2. The NTC-expression cassette (product 3) was amplified using primers CPA91 / 96 and an NTC-DNA string as a template. Product 4 was generated by amplifying product 3 using primer CPA91 / 128. Products 3 and 4 were constructed via the NEBuilder® HiFi DNA Assembly kit and amplified using primers CPA70 / 91 to produce fragment 1. Homology arms were generated by amplifying the PAD1 adjacent region derived from gDNA strain #1 using primers CPA111 / 112 (5'-adjacent region; fragment 2) and CPA113 / 114 (3'-adjacent region; fragment 3). The final ILV3-NTC embedded cassette was generated by constructing fragments 1+2+3, followed by amplification using primers CPA111 / 114. To generate strain #3, strain #2 was transformed with the final ILV3-NTC embedded cassette. ILV3(se) sequence - NTC embedded cassette: SEQ ID NO: 61

[0321] Recombinant plasmids Plasmid #1 composition Vector preparation Plasmid #0 (pYESVIII, BRAIN Biotech AG) was linearized using PmeI present in the multicloning region. The hydrolysis vector was dephosphorylated before purification.

[0322] Generating Vector Inserts Template for PCR: gDNA S. pastorianus WS34 / 78, strain #1 (Table 16) Primers used: CPA74:SEQ ID NO:62 CPA75:SEQ ID NO:63 CPA86:SEQ ID NO:64 CPA87:SEQ ID NO:65 A natural ILV5-expression cassette (sc) was amplified from WS34 / 78 gDNA using allele-specific primers CPA74 / 75. The vector insert was then generated by amplification of the resulting PCR product using primers CPA86 / 87.

[0323] An ILV5 expression vector (plasmid #1) was constructed via NEBuilder® HiFi DNA Assembly, and E. coli DH10β was transformed with the reaction product. After plasmid preparation, the expression cassette was analyzed by Sanger DNA sequencing. ILV5(sc) expression cassette sequence: SEQ ID NO: 66

[0324] Example 11 - Further gene copy number fitting in the model brewer's yeast WS34 / 78 The objective of this experiment was the same as in Example 10, namely, to generate a further genetically modified WS34 / 78 yeast strain in order to adjust the copy number of the relevant genes to reflect the genotypes of ZDA1 and ZDA2.

[0325] PCR of the natural Saccharomyces cerevisiae gene The S. cerevisiae genes ILV3, BAT1, BAT2, and ILV6, along with their native promoter and terminator regions, were amplified from Weihenstephan34 / 78 (W34 / 78) lager yeast using primers SEQ ID no. 67 to SEQ ID no. 74 and Phusion® High-Fidelity DNA polymerase (New England BioLabs). Following the previous literature (Mumberg et al, 1995), the regions targeting the native promoter and terminator were selected, approximately 1000 base pairs upstream of the start codon (promoter) and 400 base pairs downstream of the stop codon (terminator). Genomic DNA used as a template for the PCR reaction was extracted from W34 / 78 yeast using Lucigen's MasterPure Yeast DNA purification kit fact No. MPY80200. Primers used to amplify the desired S. cerevisiae gene upstream and downstream: ScILV3_F:SEQ ID NO:67 ScILV3_R:SEQ ID NO:68 ScBAT1_F:SEQ ID NO:69 ScBAT1_R:SEQ ID NO:70 ScBAT2_F:SEQ ID NO:71 ScBAT2_R:SEQ ID NO:72 ScILV6_F:SEQ ID NO:73 ScILV6_R:SEQ ID NO:74

[0326] Cloning of PCR fragments into plasmid vectors using Gibson Assembly and Transformation in E. coli cells. Pure PCR fragments were cloned into a centromere single-copy vector containing the kanMXG418 resistance marker. In some cases, genes were combined to create plasmids containing more than one gene. pSH67 from Euroscarf (http: / / www.euroscarf.de / plasmid_details.php?accno=P30673) was linearized using PvuII-HF (New England BioLabs) at 37°C for 60 minutes to remove the Cre-expression construct. The linearized plasmid was then ligated with T7 DNA ligase (New England BioLabs) overnight at 25°C. The ligated plasmid was transformed into NEB5α-competent E. coli cells (New England BioLabs) and plated on Lysogeny broth (LB) agar plates containing ampicillin (100 μg / mL). This plasmid was then used as a backbone for insertion of the target gene.

[0327] PvuII-HF was digested into a vector, and the PCR-amplified insert was constructed using NEBuilder® HiFi DNA Assembly (New England BioLabs). Using a 3:1 insert-to-vector ratio, the reaction was incubated at 50°C for 60 minutes. The reaction was then transformed into NEB 5α-E. coli competent cells (New England BioLabs) and plated on LB agar plates containing ampicillin (100 μg / mL). Clones were grown on LB liquid medium with ampicillin (100 μg / mL), and plasmids were extracted using the Monarch Plasmid MiniPrep kit (New England BioLabs).

[0328] The single-copy plasmids produced in this study were as follows: 1. pSH67-ScILV3 2. pSH67-ScILV3-ScBAT1 3. pSH67-ScILV3-ScBAT2 4. pSH67-ScILV6 5. pSH67-ScBAT2

[0329] Yeast transformation by vectors, and the resulting yeast Weihenstephan34 / 78 and strain #2 from Example 10 (Weihenstephan34 / 78, also known as WS34 / 78[2xILV2eu]::ZeoR, possessing two deleted S. eubyanus ILV2 gene copies) were transformed by plasma electroporation according to the protocol outlined by Benatuil et al. 2010. Several minor modifications were made: the yeast was grown at 25°C, harvested 2-3 hours after electroporation, and the cells were plated on YPD agar plates containing Genethecin G418 (200 μg / mL).

[0330] The yeast produced in this study is shown in Table 17 below. [Table 19]

[0331] Example 12 - Evaluation of fermentation characteristics of modified WS34 / 78 strain The strain was fermented in a cylindrical container as previously described. The cells were grown overnight in 10 ml of liquid YPD containing 25 μg / ml gentamicin (G418), and then transferred to 20 ml of pilsner wort containing 100 μg / ml G418 as previously described. Cell pitting was performed in pilsner wort containing 100 μg / ml G418 as previously described. Plato, diacetyl, and volatility measurements were performed on days 3, 4, and 5 of fermentation. Carbohydrate and ester profile samples were obtained on day 5.

[0332] The genotypes of the tested yeast strains are shown in Table 18 below. [Table 20]

[0333] The results of fermentation using each yeast strain are summarized in Tables 19 and 20 below. [Table 21] [Table 22]

[0334] It should be noted that the genetically modified strains in this example were fermented in the presence of antibiotics, and therefore the results above may differ slightly if the strains were fermented industrially without antibiotics. In particular, the strains may have a faster growth rate, and thus the fermentation would proceed more rapidly. Similarly, the reduction of the initially produced diacetyl is also expected to proceed more rapidly.

[0335] On the fifth day, the strain with the lowest amount of total diacetyl in the test solution was a strain derived from a single-copy plasmid (FGMO0094) in which two copies of the ILV2 gene were deleted, an extra copy of ILV3 was genomically incorporated, and an extra copy of ILV5 was expressed. [Table 23] TIFF2023535281000028.tif163162

[0336] References Lorenzo Benatuil, Jennifer M. Perez, Jonathan Belk, Chung-Ming Hsieh, An improved yeast transformation method for the generation of very large human antibody libraries, Protein Engineering, Design and Selection, Volume 23, Issue 4, April 2010, Pages 155-159, https: / / doi.org / 10.1093 / protein / gzq002 Briggs, D. E. et al. Malting and Brewing science. 1981. Denis E, Sanchez S, Mairey B et al. Extracting high molecular weight genomic DNA from Saccharomyces cerevisiae, Protocol Exchange DOI 10.1038 / protex.2018.076 Hough, J. S. et al. Malting and Brewing science: Hopped Wort and Beer, Volume 2. 1982. Gutierrez A, Boekhout T, Gojkovic Z, Katz M. Evaluation of non‐Saccharomyces yeasts in the fermentation of wine, beer and cider for the development of new beverages J Inst Brew 2018 DOI 10.1002 / jib.512 Mumberg D, Muller R, Funk M Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds Gene 1995:156:119-22. doi: 10.1016 / 0378-1119(95)00037-7 Walther A, Hesselbart A, Wendland J. Genome Sequence of Saccharomyces Carlsbergensis, the World’s First Pure Culture Lager Yeast G3 (Bethesda) 2014:4:783-93.

Claims

1. Within that genome, a. up to two functional genes encoding ILV2, wherein each gene encoding ILV2 encodes ScILV2 of SEQ ID NO: 32 or SeILV2 of SEQ ID NO: 38, or a functional homologue sharing at least 80% sequence identity therewith; and b. At least five functional genes, e.g., at least six functional genes, encoding ILV3, wherein each gene encoding ILV3 encodes ScILV3 of SEQ ID NO: 35 or SeILV3 of SEQ ID NO: 41, or a functional homologue sharing at least 80% sequence identity therewith. Yeast strains encoding

2. The yeast strain of claim 1, wherein the yeast strain encodes at least four functional genes encoding ILV5 within its genome, for example at least five functional genes encoding ILV5, and each gene encoding ILV5 encodes ScILV5 of SEQ ID NO: 34 or SeILV5 of SEQ ID NO: 40, or a functional homologue sharing at least 80% sequence identity therewith.

3. 3. The yeast strain of claim 1, wherein the genes encoding ILV2, ILV3 and / or ILV5 are expressed from their native promoters.

4. The yeast strain of any one of claims 1 to 3, wherein the genes encoding ILV2, ILV3, ILV5, ILV6, BAT1 and / or BAT2 are expressed from their native promoters.

5. the yeast strain does not contain heterologous DNA, and / or The yeast strain according to any one of claims 1 to 4, wherein said yeast strain has not been subjected to a process of genetic engineering.

6. The yeast strain according to any one of claims 1 to 5, wherein the yeast strain has up to 15 functional genes encoding ILV3, such as 5 to 10 functional genes encoding ILV3, such as 5 to 6 functional genes encoding ILV3.

7. The yeast strain according to any one of claims 1 to 6, wherein the yeast strain has up to 15 functional genes encoding ILV5, such as 4 to 10 functional genes encoding ILV5, such as 4 to 5 functional genes encoding ILV5.

8. 8. The yeast strain of claim 1, wherein the yeast strain has up to four functional ILV6-encoding genes, and each ILV6-encoding gene encodes ScILV6 of SEQ ID NO: 33 or SeILV6 of SEQ ID NO: 39, or a homologue sharing at least 80% sequence identity therewith.

9. 9. The yeast strain of any one of claims 1 to 8, wherein the yeast strain has at least three functional genes encoding BAT1, such as at least four functional genes, and each gene encoding BAT1 encodes ScBAT1 of SEQ ID NO: 36 or SeBAT1 of SEQ ID NO: 42, or a homologue sharing at least 80% sequence identity therewith.

10. 10. The yeast strain of any one of claims 1 to 9, wherein the yeast strain has at least four functional genes encoding BAT2, such as at least five functional genes, and each gene encoding BAT2 encodes ScBAT2 of SEQ ID NO: 37 or SeBAT2 of SEQ ID NO: 43, or a homologue sharing at least 80% sequence identity therewith.

11. The yeast strain according to any one of claims 1 to 10, wherein the sum of functional genes in said yeast strain encoding ILV2 and ILV6 is lower than the sum of functional genes encoding ILV5 and ILV3.

12. 12. The yeast strain of any one of claims 1 to 11, wherein the functional gene ratio of ILV2 to ILV5 and ILV3 in said yeast strain is at least 1, such as at least 1.5, such as at least 2, such as at least 2.5, or such as at least 3.

13. 13. The yeast strain of any one of claims 1 to 12, wherein the functional gene ratio of ILV2 and ILV6 to ILV5 and ILV3 in said yeast strain is at least 1, such as at least 1.2, such as at least 1.4, such as at least 1.6, such as at least 1.8 or such as at least 2.

14. the yeast strain carries a mutation or deletion in one or more ILV2 genes, and / or the yeast strain carries a frameshift mutation in one or more ILV2 genes, and / or The yeast strain of any one of claims 1 to 13, wherein the yeast strain carries a mutation that results in reduced or no expression of one or more ILV2 genes.

15. 15. The yeast strain of any one of claims 1 to 14, wherein the yeast strain is capable of producing a fermentation test liquid upon incubation in a test liquid, the test liquid being an extract of malt and / or cereals having an apparent extract content of at least 10° Plato, the fermentation test liquid containing at most 60 ppb of diacetyl at the earliest time point at which the apparent extract content of the test liquid has not decreased by more than 0.50° Plato in the preceding 24 hours, and the fermentation occurs at a temperature of at most 18°C.

16. 16. The yeast strain of claim 15, wherein the yeast strain is capable of producing at least 4.0 mL / L ethanol / °Plate when the yeast strain is incubated in the test solution.

17. 17. The yeast strain according to any one of claims 1 to 16, wherein the yeast strain is capable of growing on a medium having melibiose as the sole carbon source.

18. 18. The yeast strain according to any one of claims 15 to 17, wherein the fermented aqueous extract comprises at most 60 ppb diacetyl, such as at most 55 ppb diacetyl, such as at most 50 ppb diacetyl, such as at most 45 ppb diacetyl, for example at most 40 ppb diacetyl.

19. The yeast strain according to any one of claims 15 to 18, wherein the fermented aqueous extract comprises at least 25 mg / L of propanol, such as at least 30 mg / L of propanol.

20. 20. The yeast strain according to any one of claims 15 to 19, wherein the fermented aqueous extract comprises at most 8 mg / L isobutanol, such as at most 6 mg / L isobutanol.

21. 21. The yeast strain according to any one of claims 15 to 20, wherein the fermented aqueous extract comprises an isobutanol to propanol ratio of at least 2.0, such as at least 2.5, for example at least 3.0, such as at least 4.0, for example at least 5.0, such as at least 5.

5.

22. 1. A method for producing a fermented aqueous extract, comprising: i) providing an aqueous extract of malt and / or grain; ii) providing a yeast strain of the species Saccharomyces pastorianus, said yeast strain being as defined in any one of claims 1 to 21; and iii) fermenting the aqueous extract provided in step i) with said yeast strain of step ii), thereby obtaining a fermented aqueous extract. A method comprising:

23. 1. A method for producing a beverage, comprising: i. preparing the fermented aqueous extract of claim 22, and ii. Processing the fermented aqueous extract into a beverage. A method comprising:

24. 24. The method of claim 23, wherein the step of processing comprises one or more of the following: i. filtration, ii. carbonation; iii. Aging, or iv. Bottling.

25. A beverage prepared by the method of claim 23 or 24, wherein the beverage contains at least 25 mg / L of propanol, for example at least 30 mg / L of propanol, and / or at most 8 mg / L of isobutanol, for example at most 6 mg / L of isobutanol.

26. The beverage described in claim 25, wherein the beverage is beer.

27. ​​A beverage as described in claim 25 or 26, wherein the beverage has an isobutanol to propanol ratio of at least 3.0, such as at least 4.0, such as at least 5.0, such as at least 5.5.

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