Low-diacetyl yeast offspring strains

Abstract

The present invention relates to a progeny yeast strain having the useful characteristic of producing low levels of diacetyl during fermentation. The progeny yeast strain may be of the Saccharomyces cerevisiae species. The invention also provides a method for producing these progeny yeast strains, a method for producing a malt and / or cereal-based beverage using these strains, and the beverage produced by this method.

Description

Technical Field

[0001] The present invention relates to the field of beer production, particularly to the field of ale beer production. More specifically, the present invention provides a low-diacetyl progeny yeast strain that is particularly useful for rapid and efficient fermentation during beer production, particularly in ale beer production.

Background Art

[0002] Ale beer is often characterized by a sweet and full-bodied fruity flavor. Diacetyl contributes to the flavor of many fermented products. However, its typical buttery flavor is considered an off-flavor in many types of beer, and the removal of this compound has a significant impact on the time and energy consumption in the brewery.

[0003] Ale beer is usually prepared by fermenting wort (a liquid rich in carbohydrates) with ale yeast. Generally, ale yeast differs from lager yeast in several respects. Ale yeast generally belongs to the species Saccharomyces cerevisiae. Since ale yeast remains in a suspended state during fermentation, it is often also called "top-fermenting yeast". Yeast sedimentation or flocculation can also affect the processing time because the yeast needs to settle sufficiently until it can be harvested for the next brewing. In the case of ale yeast with low flocculence (slowly settling at the top), cooling is required, resulting in additional processing time. Furthermore, ale yeast is generally optimally used at a temperature of 12 - 24°C. In contrast to lager yeast, which generally belongs to the species Saccharomyces pastorianus, ale yeast does not have the ability to use melibiose as the sole carbon source and can typically still grow even at a temperature of 37°C.

[0004] During fermentation, diacetyl is produced by the non-enzymatic oxidation of acetolactate released from yeast cells, but during maturation, diacetyl is reabsorbed and metabolized by the yeast cells. Part of fermentation management is performed to ensure that the diacetyl content in the finished beer is below a set threshold. The problem of reducing the diacetyl content in the finished beer is less pronounced in ale beers compared to lager beers, which are fermented at lower temperatures, but a "diacetyl" rest period is often required at the end of fermentation to allow for the reabsorption and metabolism of the diacetyl produced by the ale yeast. This rest is particularly important because, at higher brewing temperatures for ale beers compared to lager beers, diacetyl metabolism may proceed relatively quickly, and during this period, the precursor acetolactate is also spontaneously converted to diacetyl. If the yeast is removed too early at the end of fermentation, excessively high diacetyl levels due to the spontaneous conversion of acetolactate will still be present in the final beer.

[0005] The lower limit of diacetyl taste in beer is generally considered to be 50 ppb. Therefore, it is necessary to reduce the diacetyl concentration in the finished beer to this level and to ensure that the precursor acetolactate is completely converted to diacetyl from the beginning. Traditionally, this process included adding the extra time required for ale maturation before the brewing process was complete. [Overview of the project]

[0006] As described above, when wort is fermented at temperatures between 12°C and 24°C using conventional Saccharomyces pastorianus or Saccharomyces cerevisiae yeast strains, the diacetyl level in the wort far exceeds the taste threshold of 50 ppb. Consequently, conventionally, it was necessary to add yeast maturation days to ensure that the precursor acetolactate present in the wort was sufficiently converted to diacetyl, and that the diacetyl level subsequently decreased sufficiently so that the diacetyl content of the finished beer fell below the set threshold.

[0007] Interestingly, the present invention provides novel progeny yeast strains that produce low levels of diacetyl and / or rapidly consume said diacetyl during fermentation, resulting in beers produced by these strains requiring little to no maturation time. Specifically, when wort is fermented with the yeast strains of the present invention, the total diacetyl level is less than 50 ppb at any point during sugar fermentation, i.e., below the taste threshold. Therefore, the present invention allows brewers to harvest the beer at any desired time without having to worry about diacetyl levels. Thus, the present invention makes it possible to skip maturation and harvest the beer immediately after fermentation. Furthermore, in the production of low-alcohol and non-alcoholic beverages, it may be necessary or beneficial to stop fermentation at an early stage to ensure a low alcohol level. In fermentation using the progeny yeasts of the present invention, the diacetyl level is always less than 50 ppb, so fermentation can be stopped at any time. This is particularly useful when the progeny yeast strains are maltose-negative or otherwise do not produce high alcohol levels.

[0008] Specifically, the present invention demonstrates that when a progeny yeast strain is generated from a first parent strain, e.g., M49, that produces a total diacetyl level of less than 50 ppb during fermentation, the progeny yeast strain may inherit this ability. Furthermore, the progeny yeast strain may inherit other useful phenotypes from the second parent strain.

[0009] In one embodiment, a first parent yeast strain and a descendant yeast strain of the second parent yeast strain are provided, where a. The first parent yeast strain is of the species Saccharomyces cerevisiae, and also, b. The first parent yeast strain and the progeny yeast strain each have the ability to produce a fermentation test solution when cultured in the test solution, the test solution being an extract of malt and / or cereals, and the fermentation test solution does not reach a total diacetyl level of more than 50 ppb, for example more than 40 ppb, or for example more than 30 ppb at any point during fermentation.

[0010] In some embodiments, a method for producing offspring yeast strains is provided, and this method is a. To provide spores of the first parent yeast strain described herein. b. Cross the above spores with the spores of the second parent yeast strain described herein. The process includes the step in which at least one spore of the first parent yeast strain has a different mating type from at least one spore of the second parent yeast strain.

[0011] Therefore, in some embodiments, a method for producing a fermented aqueous extract is provided, and this method is i) Provides aqueous extracts of malt and / or cereals. ii) to provide a progeny yeast strain (wherein the progeny yeast strain is as described herein), and The step includes fermenting the aqueous extract provided in step i) with the yeast strain from step ii) to obtain a fermented aqueous extract.

[0012] In some embodiments, fermented aqueous extracts prepared by the methods described herein are also provided.

[0013] In some embodiments, a method for producing a beverage is provided, and the method is i. Prepare the fermented aqueous extract described herein, and ii. Further process the above fermented aqueous extract into a beverage. Includes steps. [Brief explanation of the drawing]

[0014] [Figure 1] Evaluation of propanol-to-isobutanol ratios of yeasts described in WO2016101960 and other yeasts with breeding potential from the yeast bank. The Y-axis shows the propanol:isobutanol ratio measured in the indicated yeasts. The results are further described in Example 2. [Figure 2]Total diacetyl, propanol, and propanol:isobutanol ratios of yeast M49 and two previously described yeasts of WO2016 / 101960. Results are further described in Example 3. [Figure 3] Fermentation rate measured by decrease in specific gravity over time of three different yeasts in a 50 L test at 15°C. Results are further described in Example 14. [Figure 4] Total diacetyl measured over time for three different yeasts in a 50 L test at 15°C in 9 Plato AFB wort. Results are further described in Example 14.

Mode for Carrying Out the Invention

[0015] Definitions As used herein, "a" may mean one or more, depending on the context in which it is used.

[0016] As used herein, the term "about" means ±10%, preferably ±5%, more preferably ±2%.

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

[0018] As used herein, the term "deworting" refers to diluting a beverage or beverage base, such as beer, with water. The water can be, for example, tap water, demineralized water, or a mixture of both. The purpose of deworting is usually to adjust the alcohol content to a predetermined low level.

[0019] As used herein, the term "diacetyl" refers to a compound of the following formula:

Chemical formula

[0020] The concentration of diacetyl in the sample may be measured by gas chromatography according to EBC 9.24.2.

[0021] As used herein, the term "total diacetyl" refers to a diacetyl concentration measurement obtained by gas chromatography according to the European Brewing Convention method EBC 9.24.2, including a period of incubation of the sample at 60°C for 90 minutes. Total diacetyl reflects the sum of precursor acetolactic acid and free diacetyl.

[0022] Throughout this disclosure, when describing diacetyl produced by the yeast strains of this disclosure, the term "diacetyl" refers to "total diacetyl" unless otherwise specified. Therefore, the diacetyl concentration of a sample refers to the total diacetyl concentration, i.e., the sum of precursor acetolactate and free diacetyl.

[0023] As used herein, the term “cereals” refers to grasses that produce edible grains such as wheat, millet, rice, barley, oats, rye, rye wheat, sorghum, and maize.

[0024] As used herein, the term “grain” refers to the seed of a cereal, including the caryopsis, also known as the endocarp. In addition, the grain may include the exocarp and endocarp. In most barley varieties, the exocarp and endocarp remain attached to the caryopsis and become part of the grain after threshing. However, there are also varieties of hulless barley, in which the caryopsis is separated from the exocarp and endocarp and threshed freely like wheat. The terms “grain” and “kernel” are used interchangeably herein.

[0025] The term “wort” refers to the liquid extract of malt and / or cereal grains, and optionally, additional additives. Wort is generally obtained by grinding, optionally followed by “spraying,” a process in which residual sugars and other compounds are extracted from the spent grains after grinding using hot water. Spraying is usually carried out in a filtration tank, wort filter, or another apparatus for separating the extracted water from the spent grains. The wort obtained after grinding is generally called “first wort,” and the wort obtained after spraying 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 be called “sweet wort,” while wort boiled with hops may be called “boiled wort” or simply wort.

[0026] As used herein, the term “aqueous extract” refers to any aqueous extract of malt and / or cereal grains. Therefore, a non-limiting example herein is a wort containing a predetermined amount of fermentable sugars.

[0027] As used herein, the term “fermented aqueous extract” refers to any aqueous extract fermented with a microorganism such as a yeast strain. In some embodiments, “fermented aqueous extract” is an aqueous extract from which sugar fermentation has been completed. Fermented aqueous extract may be, for example, a fermented malt and / or cereal-based extract.

[0028] Sugar fermentation of an aqueous extract, test solution, or wort is considered complete when the sugar content, as measured by plateau degrees, no longer decreases significantly during fermentation. Preferably, sugar fermentation may be considered complete when the sugar content does not change by more than 0.5 plateau degrees in 24 hours, or when the sugar content does not change by more than 0.25 plateau degrees in 12 hours. Alternatively, completion of fermentation may be determined by measuring the amount of gas produced, for example, by measuring the cumulative pressure in the container. Fermentation is considered complete when the pressure does not change significantly, for example, when the cumulative pressure does not rise by more than 30 PSI in 24 hours.

[0029] As used herein, the term “test solution” refers to any aqueous liquid or solution. A test solution may contain a predetermined level of a specific compound. Preferably, the test solution is a wort having a predetermined sugar content.

[0030] As used herein, the term “fermentation test solution” refers to a test solution cultured with microorganisms, such as a yeast strain, in which sugar fermentation has been completed.

[0031] As used herein, the term "plateau" refers to density measured on a plateau scale. A plateau scale is an empirically derived hydrometer scale that measures the density of beer or wort as a weight percentage of the extract. The plateau scale expresses density in grams of extract per 100g of wort. Plateaus can be measured, for example, with an Anton Paar Alcolyzer or a portable instrument.

[0032] As used herein, the term "apparent extract" refers to the density of beer or wort measured at a plateau. Since density is primarily determined by sugar content, the apparent extract serves as an indicator of the sugar content of a solution or extract. The apparent extract of a solution can be measured, for example, using a portable Anton-PAAR, serial number DM.

[0033] As used herein, the term "alcohol content (ABV)" means the amount of alcohol (ethanol) contained 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 with an alcoholizer.

[0034] As used herein, the term "RDF" or "attenuation" refers to the degree to which the sugars in the wort have fermented into alcohol in the beer. RDF represents the percentage of the fermented extract. An RDF of 50-60% means that more than 40% of the original extract remains unfermented, resulting in a full-bodied beer. An RDF of 80% or higher means that less than 20% of the original extract remains unfermented, resulting in a highly fermented beer. The mouthfeel is greatly influenced by the RDF%, with higher RDF percentages resulting in lighter and drier beers.

[0035] As used herein, the term “aggregation” refers to the process by which fine particles, such as yeast cells, aggregate to form clumps. These clumps may then float on top of a liquid (creaming), sink to the bottom of the liquid (sedimentation), or be easily filtered out of the liquid. Yeast experts and brewers often classify yeast agglutination behavior as “high,” “medium,” or “low” depending on the degree of agglutination observed in the yeast strain during the fermentation process. Highly agglutinating strains can produce lighter-colored beers with less floating yeast and facilitate filtration. Aggregation is promoted at lower temperatures, so low-aggregating yeasts may require an additional cooling step after fermentation is complete. Therefore, highly agglutinating yeasts can produce lighter, more easily filtered beers without requiring cooling, thus reducing processing time compared to low-aggregating yeasts. Agglutination may 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 a fermentation aqueous extract or test solution.

[0036] As used herein, the term “fermentation” refers to culturing an aqueous extract or test solution with microorganisms such as a yeast strain.

[0037] As used herein, the term “malt” refers to germinated grains of cereals. The term “germination” means a process that includes soaking and germinating grains in a process carried out under controlled environmental conditions, optionally followed by a drying step. The drying step may preferably involve oak drying of the germinated grains at a raised temperature. Green malt that has not been oak-dried may also be used, particularly malt obtained by the process described in WO2018 / 001882. The term “green malt” refers to germinated grains of cereals that have not been oak-dried. In some embodiments, green malt is crushed green malt. As used herein, the term “oak-dried malt” refers to germinated grains of cereals that have been dried by oak drying. In some embodiments, oak-dried malt is crushed oak-dried malt. Generally, the grains of cereals were germinated under controlled environmental conditions.

[0038] As used herein, the term “carbon source” refers to any organic molecule that can provide energy to yeast and provide carbon for cellular biosynthesis. Specifically, 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 include, but are not limited to, glucose, fructose, maltose, maltotriose and sucrose.

[0039] As used herein in relation to yeast, the term “proliferation” refers to the process by which yeast cells multiply. Therefore, when yeast cells multiply, the number of yeast cells increases. The number of yeast cells may be determined by any useful method.

[0040] As used herein, the term “capable of utilizing” refers to the ability of yeast to utilize certain compounds as carbon and / or nitrogen sources for cellular biosynthesis.

[0041] As used herein, the term “yeast offspring strain” refers to offspring from two parent yeast strains, for example, the first and second parent yeast strains disclosed herein. This term refers to both direct offspring and progeny. Yeast offspring strains may have any ploidy level. In some embodiments, yeast offspring strains are haploid. In some embodiments, yeast offspring strains are diploid. Parent strains may also have any ploidy level. For example, each parent strain may be individually haploid or diploid. If one or both parent strains are diploid, the strains generally spore-form and produce haploids before mating to produce offspring. Diploid offspring of two haploid parent strains may spore-form, and new diploids may be produced from the mating of spores of the opposite mating type. Such “progeny” diploids, and haploids produced by spore-formation of diploid offspring produced from the mating of two parent yeast strains, are also included in the term “yeast offspring strain” as used herein. The terms "hybrid yeast" and "yeast hybrid" are also sometimes used to refer to yeast offspring or yeast offspring strains resulting from the cross between two parent yeast strains with different genotypes. The two parent yeast strains may be of the same species, for example, Saccharomyces cerevisiae.

[0042] As used herein, the term “sequence identity” describes the relationship between two amino acid sequences or two nucleotide sequences based on a pairwise alignment between a candidate sequence (e.g., a mutant sequence) and a reference sequence (e.g., a wild-type sequence). 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.Mol.Biol.48:443-453), preferably version 5.0.0 or later (available at https: / / www.ebi.ac.uk / Tools / psa / emboss_needle / ), implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.). The parameters used are a gap-open penalty of 10, a gap-extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. Needle's output labeled "longest identity" (obtained using the -nobrief option) is used as the identity percentage and is calculated as follows: (Same residue × 100) / (Alignment length - Total number of alignment gaps).

[0043] The Needleman-Wunsch algorithm is also used to determine whether a given amino acid in a sequence other than the reference sequence (e.g., a native mutant or monoploid of SEQ ID NO: 1) corresponds to a given position in SEQ ID NO: 1 (the reference sequence). For example, if a native mutant has two additional amino acids at its N-terminus, position 70 of the native mutant corresponds to position 68 of SEQ ID NO: 1.

[0044] For the purposes of this invention, sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra), preferably version 5.0.0 or later, implemented 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). The parameters used are a gap-open penalty of 10, a gap-elongation penalty of 0.5, and a DNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The Needle output labeled "Longest Identity" (obtained using the -nobrief option) is used as the identity percentage and is calculated as follows: (100 identical deoxyribonucleotides) / (alignment length - total number of alignment gaps).

[0045] As used herein, a functional homolog refers to a polypeptide that exhibits at least some sequence identity with a reference polypeptide and retains at least one aspect of the original function of the reference polypeptide.

[0046] The term "corresponding amino acid at position X" is used herein to describe an amino acid of a given polypeptide (e.g., a variant of SEQ ID NO: 1) in relation to an amino acid of a reference polypeptide (e.g., SEQ ID NO: 1). After alignment between the polypeptide and the reference polypeptide, an amino acid corresponds to X if it is at the same position as X in the aforementioned alignment. For example, amino acid 498 of SEQ ID NO: 1 may correspond to the amino acid at position 455 of the variant sequence of SEQ ID NO: 1. The aforementioned alignment is preferably carried out as described herein.

[0047] Descendant yeast strains This disclosure relates to a progeny yeast strain, such as a Saccharomyces cerevisiae progeny yeast strain, that, when cultured in malt and / or cereal extracts, produces low levels of total diacetyl during fermentation and / or rapidly consumes diacetyl. Therefore, at any point in fermentation using the progeny yeast strain of the present invention, the total diacetyl level will not exceed the taste threshold of 50 ppb.

[0048] Various types of yeast are used in beer production, but the most noteworthy are Saccharomyces pastorianus and Saccharomyces cerevisiae. Ale beers are typically fermented using Saccharomyces cerevisiae yeast. Therefore, the Saccharomyces cerevisiae according to the present invention may be any yeast useful for the production of ale beers, for example. Specifically, Saccharomyces cerevisiae may be a top-fermenting yeast strain.

[0049] The offspring yeast strains of the present invention are offspring of first and second parent yeast strains. In some embodiments, the offspring yeast strains are offspring of two S. cerevisiae parent strains. The offspring of two S. cerevisiae parent strains may be considered S. cerevisiae yeast in themselves.

[0050] In one embodiment, a first parent yeast strain and a descendant yeast strain of the second parent yeast strain are provided, where a. The first parent yeast strain is of the species Saccharomyces cerevisiae, and also, b. The first parent yeast strain and the progeny yeast strain each have the ability to produce a fermentation test solution when cultured in the test solution, the test solution being an extract of malt and / or cereals, and the fermentation test solution does not reach a total diacetyl level of more than 50 ppb, for example more than 40 ppb, or for example more than 30 ppb at any point during fermentation.

[0051] In other words, during the fermentation of the test solution using the first parent strain or the offspring yeast strain of the present invention, the level of total diacetyl in the test solution shall not exceed 50 ppb, for example 40 ppb or for example 30 ppb, at any point during fermentation.

[0052] Accordingly, in some embodiments, the offspring yeast strain according to the present invention has a “low diacetyl” phenotype, i.e., the offspring yeast strain, when cultured in a test solution, has the ability to produce a fermentation test solution, where the test solution is an extract of malt and / or cereals, and where the fermentation test solution does not reach a total diacetyl level of more than 50 ppb, e.g., more than 45 ppb, e.g., more than 40 ppb, e.g., more than 35 ppb, or e.g., more than 30 ppb at any point during fermentation. This phenotype may be obtained from a first parent yeast, which preferably also has the aforementioned “low diacetyl” phenotype.

[0053] In some embodiments, the offspring yeast strain harbors a mutation in the gene encoding ILV2, where ILV2 is as described in Sequence ID No. 1, where the aforementioned mutation results in an amino acid substitution at position 498 of Sequence ID No. 1.

[0054] In some embodiments, the offspring yeast strain harbors a mutation in the gene encoding ILV2, where ILV2 is a functional homolog of SEQ ID NO: 1 having at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 95%, e.g., at least 98%, e.g., at least 99% sequence identity to SEQ ID NO: 1, where the aforementioned mutation results in an amino acid substitution at the position of the aforementioned functional homolog corresponding to position 498 of SEQ ID NO: 1.

[0055] In some embodiments, the aforementioned mutation results in a substitution from a nonpolar amino acid, such as glycine (G), to a charged amino acid, such as a negatively charged amino acid.

[0056] In some embodiments, the aforementioned mutations result in the substitution of a nonpolar amino acid, such as glycine (G) with aspartic acid (D) or glutamic acid (E).

[0057] In some embodiments, the aforementioned mutation results in a substitution of a nonpolar amino acid, such as glycine (G) to glutamic acid (E).

[0058] Nonpolar amino acids may be selected from the group consisting of alanine (A), valine (V), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), tyrosine (Y), tryptophan (W), and glycine (G), more preferably from the group consisting of alanine (A), valine (V), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), tryptophan (W), and glycine (G).

[0059] In some embodiments, the aforementioned mutation results in a glycine (G) to glutamic acid (E) substitution, such as the G498E substitution. Thus, in some embodiments, the progeny yeast strain includes the mutant ILV2 described in SEQ ID NO: 2, or a functional homolog of SEQ ID NO: 2 having at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 95%, e.g., at least 98%, e.g., at least 99% sequence identity to SEQ ID NO: 2, provided that the functional homolog contains the amino acid corresponding to position 498 of SEQ ID NO: 2.

[0060] In some embodiments, the aforementioned offspring yeast strains harbor a mutation in the ILV2 gene, as described in Sequence ID No. 3, where the mutation is a substitution of guanine (G) to adenine (A) at position 1493 of Sequence ID No. 3.

[0061] In some embodiments, the aforementioned offspring yeast strains possess a mutation in the ILV2 gene, which is a homolog of SEQ ID NO: 3 having at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 95%, e.g., at least 98%, e.g., at least 99%, sequence identity to SEQ ID NO: 3, wherein the mutation is a substitution of guanine (G) corresponding to the guanine at position 1493 of SEQ ID NO: 3 with adenine (A).

[0062] Therefore, in some embodiments, the offspring yeast strain contains the ILV2 gene described in SEQ ID NO: 4, or a homolog thereof having at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 95%, e.g., at least 98%, e.g., at least 99% sequence identity to SEQ ID NO: 4, provided that the homolog contains the nucleotide corresponding to position 1493 of SEQ ID NO: 4.

[0063] Therefore, in some embodiments, offspring yeast strains of a first parent yeast strain and a second parent yeast strain are provided, where, a. The first parent yeast strain is of the species Saccharomyces cerevisiae, and also, b. The first parent yeast strain and the progeny yeast strain each have the ability to produce a fermentation test solution when cultured in the test solution, the test solution being an extract of malt and / or cereals, and the fermentation test solution does not reach a total diacetyl level of more than 50 ppb at any point during fermentation.

[0064] In some embodiments, a first parent yeast strain and a second parent yeast strain are provided, where, a. The first parent yeast strain is of the species Saccharomyces cerevisiae, and also, b. The first parent yeast strain and the progeny yeast strain each have the ability to produce a fermentation test solution when cultured in the test solution, the test solution being an extract of malt and / or cereals, and the fermentation test solution does not reach a total diacetyl level of more than 40 ppb at any point during fermentation.

[0065] In some embodiments, a first parent yeast strain and a second parent yeast strain are provided, where, a. The first parent yeast strain is of the species Saccharomyces cerevisiae, and also, b. The first parent yeast strain and the progeny yeast strain each have the ability to produce a fermentation test solution when cultured in the test solution, the test solution being an extract of malt and / or cereals, and the fermentation test solution does not reach a total diacetyl level of more than 30 ppb at any point during fermentation.

[0066] In some embodiments, the aforementioned fermentation occurs at a maximum temperature of 18°C. In some embodiments, the aforementioned fermentation occurs at a maximum temperature of 16°C. In some embodiments, the aforementioned fermentation occurs at a temperature in the range of 12°C to 18°C.

[0067] In some embodiments, the progeny yeast strain according to the present invention is a) Provide a test solution (wherein the test solution is an extract of malt and / or cereals having an apparent extract of at least 9 plateau degrees), b) The aforementioned progeny yeast strains are cultured together with the above test solution at a temperature of 18°C ​​or lower, preferably 12-18°C, more preferably 16°C, and c) Measure the diacetyl level at least every 24 hours from the start of step b) until the end of fermentation. When tested by a method including step c), it has the ability to produce a fermentation test solution, wherein the test solution contains at most 50 ppb of total diacetyl, for example at most 40 bbp of total diacetyl, or for example at most 30 ppb of total diacetyl at any point in time during fermentation as measured in step c).

[0068] In some embodiments, the above fermentation occurs after inoculation of 7,000,000 to 20,000,000,000 viable yeast cells per 1 mL of test solution.

[0069] In some embodiments, the culture in the test solution is carried out for a maximum of 6 days. In some embodiments, the culture in the test solution is carried out for a maximum of 5 days. In some embodiments, the culture in the test solution is carried out for a maximum of 4 days. In some embodiments, the culture in the test solution is carried out for a maximum of 3 days. In some embodiments, the culture in the test solution is carried out for a maximum of 2 days.

[0070] In some embodiments, the culture in the test solution is carried out for 2 to 6 days. In some embodiments, the culture in the test solution is carried out for 2 to 4 days.

[0071] In some embodiments, the offspring yeast strain has the ability to produce a fermentation test solution after culturing the yeast strain in a test solution, where the test solution is a malt and / or cereal extract having an apparent extract in the range of at least 9 plateau degrees, e.g., 9 to 12 plateau degrees, and the fermentation test solution contains at most 50 ppb of diacetyl at any point during the cultivation with the yeast strain for a maximum of 6 days. Specifically, the fermentation test solution may contain at most 50 ppb of diacetyl at any point during the cultivation with the yeast strain for a maximum of 5 days. Specifically, the fermentation test solution may contain at most 50 ppb of diacetyl at any point during the cultivation with the yeast strain for a maximum of 4 days.

[0072] In some embodiments, the offspring yeast strain has the ability to produce a fermentation test solution after culturing the yeast strain in a test solution, wherein the test solution is a malt and / or cereal extract having an apparent extract in the range of at least 9 plateau degrees, e.g., 9 to 12 plateau degrees, and the fermentation test solution contains at most 50 ppb of diacetyl at any point during culturing with the yeast strain for a maximum of 5 days at a maximum temperature of 18°C. Specifically, the fermentation test solution may contain at most 50 ppb of diacetyl at any point during culturing with the yeast strain for a maximum of 4 days at a maximum temperature of 18°C.

[0073] The offspring yeast strains of the present invention may have additional advantageous phenotypes in addition to the low-diacetyl phenotype. For example, these additional advantageous phenotypes may be inherited from a second parent strain. Therefore, the second parent strain may be selected depending on which phenotype is desired.

[0074] In some embodiments, the yeast strain exhibits high cohesiveness. Cohesiveness may be determined, for example, by the number of cells suspended after fermentation. "Suspended cells" are generally determined by counting the yeast cells in a sample taken from the upper three-quarters, e.g., the upper two-thirds, such as the upper half of a container containing the fermentation test solution. If fermentation is carried out in a conical cylindrical tank, the aforementioned sample is preferably taken from the top of the cone. A low number of cells in the solution after fermentation indicates high cohesiveness.

[0075] The offspring yeast strains according to the present invention may be useful in the production of low-alcohol or non-alcoholic beverages. In such cases, the second parent strain may be a yeast strain that produces low levels of alcohol or does not produce alcohol at all.

[0076] Therefore, in some embodiments, the fermentation test solution has an alcohol content of at most 1.0% ABV. In some embodiments, the fermentation test solution has an alcohol content of at most 0.9% ABV. In some embodiments, the fermentation test solution has an alcohol content of at most 0.8% ABV. In some embodiments, the fermentation test solution has an alcohol content of at most 0.7% ABV. In some embodiments, the fermentation test solution has an alcohol content of at most 0.6% ABV.

[0077] In some embodiments, the progeny yeast strains either lack the ability to grow using maltose as the sole carbon source, or, using maltose as the sole carbon source, grow significantly slower than other yeasts, preferably compared to Weihenstephan 34 / 70 yeast and / or Safale US5 (both commercially available). In this context, "significantly slower" preferably means that the second parent strain has a doubling time at least twice, e.g., at least three times, e.g., at least five times, e.g., at least ten times higher than M49. Thus, in some embodiments, the progeny yeast strains are maltose intolerant. In some embodiments, the progeny yeast strains lack the ability to completely convert maltose to ethanol.

[0078] In some embodiments, the offspring yeast strains lack the ability to grow using maltotriose as the sole carbon source. Therefore, in some embodiments, the offspring yeast strains are maltotriose intolerant. In some embodiments, the offspring yeast strains lack the ability to completely convert maltotriose to ethanol.

[0079] In some embodiments, the offspring yeast strains, when cultured in an aqueous solution containing p-coumaric acid, lack the ability to convert more than 25% of p-coumaric acid to 4-ethylphenol.

[0080] In preferred embodiments, the yeast strains, such as the progeny yeast strain, first parent yeast strain, or second parent yeast strain, as disclosed herein, are non-GMO organisms. Therefore, in preferred embodiments, the yeast strains, such as the progeny yeast strain, first parent yeast strain, or second parent yeast strain, have not undergone any genetic engineering steps.

[0081] Parent yeast strain The offspring yeast strains of the present invention are offspring of the first and second parent yeast strains.

[0082] The first parent yeast strain may be of the species Saccharomyces cerevisiae.

[0083] The first parent yeast strain described above, when cultured in malt and / or cereal extracts, produces remarkably low levels of diacetyl and / or rapidly consumes diacetyl during fermentation.

[0084] In some embodiments, the first parent yeast strain according to the present invention is a) Provide a test solution (wherein the test solution is an extract of malt and / or cereals having an apparent extract of at least 9 plateau degrees), b) The aforementioned progeny yeast strains are cultured together with the above test solution at a temperature of 18°C ​​or lower, preferably 12-18°C, more preferably 16°C, and c) Measure the diacetyl level at least every 24 hours from the start of step b) until the end of fermentation. When tested by a method including step c), it has the ability to produce a fermentation test solution, wherein the test solution contains at most 50 ppb of total diacetyl, for example at most 40 bbp of total diacetyl, or for example at most 30 ppb of total diacetyl at any point in time during fermentation as measured in step c).

[0085] In preferred embodiments, the first parent yeast strain disclosed herein is a non-GMO organism. Therefore, in preferred embodiments, the first parent yeast strain has not undergone any genetic engineering steps.

[0086] In some embodiments, the first parent yeast strain described above is M49, deposited with DSMZ under acceptance number DSM34496. M49 is a yeast strain of the species Saccharomyces cerevisiae, deposited on January 12, 2023, at Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (hereinafter referred to as DSMZ), Inhoffenstrasse 7B, D-38124 Braunschweig, Germany, and received acceptance number DSM34496.

[0087] In some embodiments, the first parent yeast strain has a mutation in the gene encoding ILV2, where ILV2 is as described in Sequence ID No. 1, where the aforementioned mutation results in an amino acid substitution at position 498 of Sequence ID No. 1.

[0088] In some embodiments, the first parent yeast strain has a mutation in the gene encoding ILV2, where ILV2 is a functional homolog of SEQ ID NO: 1 having at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 95%, e.g., at least 98%, e.g., at least 99% sequence identity to SEQ ID NO: 1, where the aforementioned mutation results in an amino acid substitution at the position of the aforementioned functional homolog corresponding to position 498 of SEQ ID NO: 1.

[0089] In some embodiments, the aforementioned mutation results in a substitution from a nonpolar amino acid, such as glycine (G), to a charged amino acid, such as a negatively charged amino acid.

[0090] In some embodiments, the aforementioned mutations result in the substitution of a nonpolar amino acid, such as glycine (G) with aspartic acid (D) or glutamic acid (E).

[0091] In some embodiments, the aforementioned mutation results in the substitution of a nonpolar amino acid, such as glycine (G) with glutamic acid (E).

[0092] Nonpolar amino acids may be selected from the group consisting of alanine (A), valine (V), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), tyrosine (Y), tryptophan (W), and glycine (G), more preferably from the group consisting of alanine (A), valine (V), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), tryptophan (W), and glycine (G).

[0093] In some embodiments, the aforementioned mutations result in a substitution from glycine (G) to glutamic acid (E), such as the G498E substitution.

[0094] Therefore, in some embodiments, the first parent yeast strain includes the mutant ILV2 described in SEQ ID NO: 2, or a functional homolog thereof having at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 95%, e.g., at least 98%, e.g., at least 99% sequence identity to SEQ ID NO: 2. However, the aforementioned functional homolog contains the amino acid corresponding to position 498 of SEQ ID NO: 2.

[0095] In some embodiments, the first parent yeast strain described above has a mutation in the ILV2 gene described in SEQ ID NO: 3, the mutation being a substitution of guanine (G) to adenine (A) at position 1493 of SEQ ID NO: 3.

[0096] In some embodiments, the first parent yeast strain has a mutation in the ILV2 gene, which is a homolog of SEQ ID NO: 3 having at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 95%, e.g., at least 98%, e.g., at least 99%, sequence identity to SEQ ID NO: 3, where the mutation is a substitution of guanine (G) to adenine (A) corresponding to the guanine at position 1493 of SEQ ID NO: 3.

[0097] Therefore, in some embodiments, the first parent yeast strain contains the ILV2 gene described in SEQ ID NO: 4, or a homolog thereof having at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 95%, e.g., at least 98%, e.g., at least 99% sequence identity to SEQ ID NO: 4, provided that the homolog contains the nucleotide corresponding to position 1493 of SEQ ID NO: 4.

[0098] The offspring yeast strains produced from the cross of the first and second parent yeast strains preferably have the low-diacetyl phenotype described herein. The second yeast strain may have one or more additional desirable phenotypes in its offspring yeast strains that can be usefully combined with the low-diacetyl phenotype described herein.

[0099] The second parent yeast strain may be any yeast strain capable of mating with the first parent yeast strain. Specifically, it is preferable that the spores of the second parent yeast strain are capable of mating with the spores of the first parent yeast strain. In some embodiments, the spores of the second parent yeast strain are haploid. In some embodiments, the spores of the first parent yeast strain are haploid. In some embodiments, the spores of the first and second parent yeast strains are haploid. Preferably, the spores of the first and second parent yeast strains are of different mating types.

[0100] In some embodiments, the second parent yeast strain is of the genus Saccharomyces. In some embodiments, the second parent yeast strain is not of the species Saccharomyces cerevisiae. In some embodiments, the second parent yeast strain is of the genus Saccharomyces, but not of the species Saccharomyces cerevisiae.

[0101] When a descendant yeast strain having the species Saccharomyces cerevisiae is desired, it is preferable that both the first and second parent yeast strains are of the species Saccharomyces cerevisiae. Therefore, in some embodiments, the second parent yeast strain is of the species Saccharomyces cerevisiae. In some embodiments, both the first and second parent yeast strains are of the species Saccharomyces cerevisiae.

[0102] In some embodiments, the second parent yeast strain is PPU121, deposited with DSMZ under acceptance number DSM34497. PPU121 is a yeast strain of the species Saccharomyces cerevisiae, deposited on January 12, 2023, at Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (hereinafter referred to as DSMZ), Inhoffenstrasse 7B, D-38124 Braunschweig, Germany, and received acceptance number DSM34497. In some embodiments, the second parent yeast strain, compared to other yeasts, preferably Weihenstephan34 / 70 yeast and / or Safale US5, either lacks the ability to grow using maltose as its sole carbon source or grows significantly slower.

[0103] In this context, "significantly slow" preferably means that the second parent strain has a doubling time at least twice, for example, at least three times, for example, at least five times, for example, at least ten times higher than M49. Therefore, in some embodiments, the second parent yeast strain is maltose intolerant. In some embodiments, the second parent yeast strain does not have the ability to completely convert maltose to ethanol.

[0104] In preferred embodiments, the second parent yeast strain disclosed herein is a non-GMO organism. Therefore, in preferred embodiments, the second parent yeast strain has not undergone any genetic engineering steps.

[0105] In preferred embodiments, the first and second parent yeast strains, as disclosed herein, are non-genetically modified organisms. Therefore, in preferred embodiments, the first and second parent yeast strains have not undergone any genetic engineering steps.

[0106] Test solution In some embodiments, the test solution is wort having an apparent extract of at least 7 plateau degrees. In some embodiments, the test solution is wort having an apparent extract of at least 8 plateau degrees. In some embodiments, the test solution is wort having an apparent extract of at least 9 plateau degrees. In some embodiments, the test solution is wort having an apparent extract of at least 12 plateau degrees. In some embodiments, the test solution is wort having an apparent extract of at least 15 plateau degrees. In some embodiments, the test solution is wort having an apparent extract of at least 15 plateau degrees. In some embodiments, the test solution is wort having an apparent extract in the range of 5 to 20 plateau degrees. In some embodiments, the test solution is wort having an apparent extract in the range of 8 to 15 plateau degrees.

[0107] In some embodiments, the test solution is wort having an apparent extract of about 16 plateau degrees. The test solution may be wort in particular.

[0108] In some embodiments, the test solution contains at least 40 g / kg of maltose, for example, in the range of 40 to 60 g / kg.

[0109] In some embodiments, the test solution contains 1.0 to 3.5 g of fructose per liter of test solution, for example, 1.5 to 3.0 g of fructose per liter of test solution, or for example, 2.0 to 2.5 g of fructose per liter of test solution. In some embodiments, the test solution contains approximately 2.2 g of fructose per liter of test solution.

[0110] In some embodiments, the test solution contains 5.0 to 8.0 g of glucose per liter of test solution, for example, 5.5 to 7.5 g of glucose per liter of test solution, or for example, 6.0 to 7.0 g of glucose per liter of test solution. In some embodiments, the test solution contains approximately 6.5 g of glucose per liter of test solution.

[0111] In some embodiments, the test solution contains 1.0 to 5.0 g of sucrose per liter of test solution, for example, 1.5 to 4.0 g of sucrose per liter of test solution, or for example, 2.0 to 3.0 g of sucrose per liter of test solution. In some embodiments, the test solution contains about 2.5 g of sucrose per liter of test solution.

[0112] In some embodiments, the test solution contains at most 3500 mg / L of amino acids. In some embodiments, the test solution contains at most 3000 mg / L of amino acids. In some embodiments, the test solution contains at most 2500 mg / L of amino acids. In some embodiments, the test solution contains amino acids in the range of 500 to 2500 mg / L.

[0113] The test solution may preferably have an apparent extract in the range of 8 to 10 plateau degrees, for example, 9 plateau degrees. The test solution may further contain zinc in the range of 0.10 mg / L to 0.20 mg / L, and the pH may be adjusted to the range of 4.0 to 5.0.

[0114] An example of a test solution includes glucose in the range of 3-9 g / L, e.g., about 6.5 g / L; maltose in the range of 40-50 g / L, e.g., about 45 g / L; maltotriose in the range of 10-15 g / L, e.g., about 12 g / L; zinc in the range of 0.10 mg / L-0.20 mg / L, e.g., about 0.15 mg / L; free α-amino nitrogen (FAN) in the range of 110-250 mg / L, e.g., about 160 mg / L; and a valine / FAN ratio of 0.5-0.7, e.g., about 0.6.

[0115] The test solution may be a wort suitable for brewing alcohol-free beer, such as debrewed wort. In some embodiments, the test solution is as described in Example 5.

[0116] In some embodiments, the test solution is glucose wort, as described in Example 4. As described in the examples, glucose wort may be produced by enzymatic treatment, converting maltose and maltotriose in standard wort into glucose, thereby achieving a high glucose concentration in the wort and a corresponding decrease in maltose and maltotriose concentrations.

[0117] In some embodiments, the test solution is glucose wort containing at least 40 g / L of glucose, for example, at least 50 g / L of glucose, for example, at least 60 g / L of glucose, for example, at least 70 g / L of glucose, for example, at least 80 g / L of glucose, for example, at least 90 g / L of glucose, for example, at least 100 g / L of glucose, or for example, at least 110 g / L of glucose. In some embodiments, the test solution is glucose wort containing 40 to 1000 g / L of glucose, for example, 50 to 500 g / L of glucose.

[0118] In some embodiments, the test solution is glucose wort and contains at most 10 g / L of maltose, for example, at most 8 g / L of maltose, for example, at most 6 g / L of maltose, for example, at most 4 g / L of maltose. In some embodiments, the test solution is glucose wort and contains 0 to 10 g / L of maltose, for example, 0 to 8 g / L of maltose, for example, 0 to 6 g / L of maltose, for example, 0 to 4 g / L of maltose.

[0119] In some embodiments, the test solution is glucose wort and contains at most 10 g / L of maltotriose, for example, at most 8 g / L of maltotriose, for example, at most 6 g / L of maltotriose, for example, at most 4 g / L of maltotriose, for example, at most 2 g / L of maltotriose, for example, at most 1 g / L of maltotriose. In some embodiments, the test solution is glucose wort and contains 0 to 10 g / L of maltotriose, for example, 0 to 8 g / L of maltotriose, for example, 0 to 6 g / L of maltotriose, for example, 0 to 4 g / L of maltotriose, for example, 0 to 2 g / L of maltotriose, for example, 0 to 1 g / L of maltotriose.

[0120] In some embodiments, the test solution is glucose wort and contains 0.5 to 8.0 g of fructose per liter of test solution, for example, 1.0 to 5.0 g of fructose per liter of test solution, or for example, 2.0 to 3.5 g of fructose per liter of test solution. In some embodiments, the test solution contains about 3 g of fructose per liter of test solution.

[0121] In some embodiments, the test solution is glucose wort and contains 0.1 to 8.0 g of sucrose per liter of the test solution, for example, 0.5 to 6.0 g of sucrose per liter of the test solution, or for example, 1.0 to 5.0 g of sucrose per liter of the test solution. In some embodiments, the test solution contains about 2 g of sucrose per liter of the test solution.

[0122] In some embodiments, the test solution may be prepared from pilsner malt and may include additional barley additives.

[0123] Method for generating offspring yeast strains In some embodiments, a method for producing offspring yeast strains is provided, and this method is a. Provide spores of a first parent yeast strain (wherein the first parent yeast strain may be, for example, one of the first parent yeast strains described in the “Parent Yeast Strains” section of this specification), b. Cross the aforementioned spores with spores from the second parent yeast strain. The process includes the step in which at least one spore of the first parent yeast strain has a different mating type from at least one spore of the second parent yeast strain.

[0124] The second parent yeast strain may be any yeast strain, for example, a yeast strain having desirable properties. In some embodiments, the second parent yeast strain may be one of the strains described in the “Parent Yeast Strains” section of this specification.

[0125] In some embodiments, the method does not require and / or does not include GMO technology.

[0126] It is preferable that the spores of the first parent yeast strain and the spores of the second parent yeast strain are used for cross-pollination. In some embodiments, the spores of the first parent yeast strain and the spores of the second parent yeast strain are used for cross-pollination, where the second parent yeast strain belongs to the genus Saccharomyces.

[0127] Preferably, spores of a first parent yeast strain having a predetermined mating type are crossed with spores of a second parent yeast strain having the opposite mating type. Therefore, if the spores of the first parent yeast strain are mating type a, it is preferable that the spores of the second parent yeast strain are mating type α, and vice versa.

[0128] Specifically, the mating may involve the mating of haploid spores of the first parent yeast strain with spores of the second parent yeast strain, where the spores of the second parent yeast strain have the opposite mating type to those of the haploid spores of the first parent yeast strain.

[0129] In some embodiments, both the spores of the first parent yeast strain and the spores of the second parent yeast strain are haploid. In such cases, the resulting offspring yeast strain may be diploid.

[0130] However, in other embodiments, different ploidy levels of spores may be used, in which case the resulting progeny yeast cells may be polyploid. In some embodiments, the ploidy level of the spores is unknown. This may be the case, for example, in some embodiments where the second parent yeast strain is Saccharomyces pastorianus.

[0131] The spores of the first and second parent yeast strains described above may be produced by any useful means, for example, by culturing the yeast in a medium that induces spore formation. Numerous useful media for spore formation are known to those skilled in the art. In this regard, see, for example, Lundblad and Struhl, 2008, Dunham et al., 2015, Mertens et al., 2017, and Examples 6 and 11 below.

[0132] Spores may be isolated, for example, using a dissecting microscope, and the spores may be propagated before mating.

[0133] The mating type of spores may be determined by any conventional means, such as a halo assay of mating pheromone-sensitive yeast on a lawn (see, e.g., Kempf et al., 2017 Microbiological Research 200:53-63, section 2), or by means such as those described in Dohlman et al., 1996.

[0134] Once the spores have been propagated, spores from the opposite mating types of the first and second parent yeast strains may be crossed with each other. Crossing may be carried out by any conventional means. Typically, the spores are cultured together in a culture medium supporting yeast growth, or on a solid or semi-solid culture medium supporting yeast growth. The spores are generally supplied in approximately equal amounts. The spores are cultured together for a suitable time, e.g., 6–24 hours, e.g., 10–14 hours. Preferred methods for crossing yeast cells are described, for example, in Treco and Winston, 2008, and in Examples 6, 11, and 12 below.

[0135] Once useful offspring yeast strains have been identified, these offspring yeast strains may be subjected to further crossbreeding. Therefore, spore clones may be generated from the offspring yeast strains and crossbred to produce further offspring yeast strains. Such further offspring yeast strains are also considered in this specification to be offspring yeast strains of the first and second parent strains.

[0136] In preferred embodiments, the offspring yeast strain is prepared as described in Example 6, Example 11, or Example 12 below in this specification, and more preferably as described in Example 6 below in this specification.

[0137] In some embodiments, the method further includes the step of selecting a progeny yeast strain that retains one or more phenotypes of interest of a first parent yeast strain and / or a second parent yeast strain.

[0138] In a preferred embodiment, the offspring yeast strain retains the low-diacetyl phenotype of the first parent yeast strain. Other useful phenotypes include: Maltose intolerance • Increased production of certain esters, such as isoamyl acetate, phenylethyl acetate, ethyl hexanoate and / or ethyl acetate, compared to the first parent yeast strain. • When cultured in an aqueous solution containing p-coumaric acid, it does not have the ability to convert more than 25% of the p-coumaric acid to 4-ethylphenol. • Having the ability to produce high levels of propanol, which is higher than isobutanol. Specifically, it is preferable that the beverage prepared by fermentation using the offspring yeast strain of the present invention has a propanol:isobutanol ratio of at least 6.0, for example, at least 8.0, for example, at least 10.0, for example, at least 12.

[0139] Methods for selecting and / or determining whether a progeny yeast strain retains a desired phenotype from the parent yeast strain are known to those skilled in the art.

[0140] Malt and / or cereal-based fermented aqueous extract and method for producing the same The present invention provides yeast offspring strains described in the above section, as well as a method for preparing malt and / or cereal-based fermentation aqueous extracts using said yeast strains.

[0141] Therefore, in some embodiments, a method for producing a fermented aqueous extract is provided, and this method is i) Provides aqueous extracts of malt and / or cereals. ii) to provide a progeny yeast strain (wherein the progeny yeast strain is as described herein), and iii) The aqueous extract provided in step i) is fermented with the yeast strain from step ii) to obtain a fermented aqueous extract. Includes steps.

[0142] The aqueous extract may be any aqueous extract of malt and / or cereal grains. A non-limiting example of this is wort. The aqueous extract may be prepared, for example, by preparing a malt extract by grinding and, optionally, by spraying, as described later herein.

[0143] Malt is germinated grain of cereals, such as barley grain. The term "germination" means a process that includes soaking and germinating of grain in a process carried out under controlled environmental conditions, optionally followed by a drying step. The drying step may preferably involve oak drying of the germinated grain at a raised temperature. Green malt that has not been oak dried may also be used, particularly malt obtained by the process described in WO2018 / 001882.

[0144] Germination is crucial for the synthesis of numerous enzymes that modify the grain, primarily by depolymerizing the starch and cell walls of the dead endosperm to mobilize the grain's nutrients and activate other depolymerizing enzymes. In the subsequent drying process, flavor and color are produced, at least partially, by chemical browning reactions.

[0145] Immersion may be carried out by any conventional method known to those skilled in the art. A non-limiting example involves immersion with alternating periods of dry and wet conditions at a temperature in the range of 10°C to 25°C. Germination may be carried out by any conventional method known to those skilled in the art. A non-limiting example involves germination with varying temperatures at a temperature in the range of 10°C to 25°C, arbitrarily ranging from 1 to 4 hours. Immersion and germination may also be carried out in a combined manner, as described, for example, in international patent application WO2018 / 001882.

[0146] Oven drying may be omitted. If performed, oven drying may 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 may 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, such as methods for producing specialty malt, including, but not limited to, methods for roasting malt, may also be used in conjunction with the present invention.

[0147] Malt may be further processed, for example, by grinding. Grinding can be carried out in a dry state; that is, the malt is ground in a dry state, or, if green malt is used, in a wet state.

[0148] Malt, for example, crushed malt, may be ground to prepare an aqueous extract of the malt. The starting liquid for preparing a beverage may be an aqueous extract of malt, for example, an aqueous extract of malt prepared by grinding.

[0149] Accordingly, 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 grinding malt and optionally additional additives. The grinding step may optionally include spraying, and accordingly, the grinding step may be a grinding step including a spraying step or a grinding step excluding a spraying step.

[0150] Generally, the production of aqueous extracts begins with the grinding of malt and / or grains. If additional additives are added, they may also be ground depending on their properties. If the additives are grains, they may be ground, for example, but syrups, sugars, etc., are generally not ground. Grinding makes it easier for water to penetrate the grains during the grinding stage. During grinding, enzymatic depolymerization of the substrate that began during germination may continue.

[0151] Generally, aqueous extracts are prepared in the grinding process by mixing and culturing ground malt with water. During grinding, carbohydrate-rich auxiliary compositions, such as ground barley, corn, or rice additives, may be added to the malt / liquid composition. Since ungerminated cereal additives usually contain little to no active enzymes, it is important to add malt or exogenous enzymes to supply the enzymes necessary for polysaccharide depolymerization and other processes.

[0152] During grinding, the ground malt and / or ground grains, and optionally additional additives, are cultured with a liquid fraction such as water. The culture temperature is generally kept constant (isothermal grinding) or gradually increased, e.g., continuously. In either case, soluble substances in the malt / grain / additives are released into the liquid fraction. Subsequent filtration separates the aqueous extract from the residual solid particles (the latter also called "spent grain"). The resulting aqueous extract is sometimes referred to as "first wort." Additional liquids, such as water, may be added to the spent grains during a process also called spraying. After spraying and filtration, a "second wort" may be obtained. Further worts may be prepared by repeating this procedure. Non-limiting examples of suitable procedures for wort preparation are described in Briggs et al. (1981) and Hough et al. (1982).

[0153] As described above, aqueous extracts may also be prepared by grinding unsprouted grains alone, or a mixture of sprouted and unsprouted grains. Unsprouted grains lack or contain only limited amounts of enzymes beneficial to wort production, such as enzymes capable of breaking down cell walls or enzymes capable of depolymerizing starch into sugars. Therefore, in embodiments of the present invention in which up to 80%, for example 90%, or even 100%, of unsprouted grains, such as barley grains, are used for grinding, it is preferable that one or more suitable external brewing enzymes be added to the mash. Suitable enzymes may include lipolytic enzymes, 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 proteolytic enzymes, or enzyme mixtures containing one or more of the aforementioned enzymes, such as Cereflo, Ultraflo, or Ondea Pro (Novozymes). However, the present invention also includes the addition of the above enzymes even when using only malt.

[0154] Aqueous extracts may also be prepared using a mixture of germinated and ungerminated grains, in which case one or more suitable enzymes may be added during preparation. Enzymes may also be added even in embodiments in which malt is used. More specifically, grains may be used for grinding with malt in any combination, for example, but not limited to, grain:malt ratios of about 100:0, or about 75:25, or about 50:50, or about 25:75, with or without external brewing enzymes.

[0155] The aqueous extract obtained after grinding is sometimes called "wort." In traditional methods, the wort is boiled with or without hops, and is then sometimes called boiled wort.

[0156] The aqueous extract may be heated or boiled before fermentation with the yeast of the present invention. In one embodiment of the present invention, the second malt and further malt may be combined and subsequently subjected to heating or boiling. The aqueous extract may be heated or boiled for any preferred time, for example, in the range of 60 to 120 minutes.

[0157] The results of fermentation aqueous extracts based on malt and / or cereals depend heavily on the amount and type of fermentable sugars present in the aqueous extract of malt and / or cereals, as well as the characteristics of the yeast strain used during fermentation.

[0158] In some embodiments of the present invention, the aqueous extract has an apparent extract with at least 6 plateau degrees, for example, at least 9 plateau degrees, for example, at least 12 plateau degrees, for example, at least 15 plateau degrees, for example, plateau degrees in the range of 5 to 20, for example, plateau degrees in the range of 9 to 15, for example, plateau degrees in the range of 6 to 12. For the production of low-alcohol beverages or alcohol-free beverages, it may be preferable that the aqueous extract has an apparent extract with plateau degrees in the range of 6 to 12, for example, plateau degrees in the range of 6 to 9.

[0159] In some embodiments, the aqueous extract is fermented with the yeast strain for a maximum of 6 days, for example, a maximum of 5 days, for example, a maximum of 4 days, for example, a maximum of 3 days.

[0160] In some embodiments, the aqueous extract contains at most 3500 mg / L of amino acids, for example, at most 3000 mg / L, or for example, at most 2500 mg / L.

[0161] Therefore, aqueous extracts, such as wort, may be prepared as described above. Fermented aqueous extracts based on malt and / or grains may be prepared by fermenting the aqueous extract with the yeast offspring strain described above according to the present invention.

[0162] In some preferred embodiments, the fermented aqueous extract is green beer.

[0163] Generally speaking, alcoholic fermented aqueous extracts such as beer may be produced from germinated and / or ungerminated grains. Malt, in addition to hops and yeast, contributes to the flavor and color of beverages such as beer. Furthermore, malt serves as a source of fermentable sugars and enzymes. Non-exclusive descriptions of suitable methods for germination and brewing can be found, for example, in publications by Briggs et al. (1981) and Hough et al. (1982). There are numerous regularly updated methods for the analysis of grains, malt, and beer products, including, but are not limited to, those of the American Association of Cereal Chemists (1995), the American Society of Brewing Chemists (1992), the European Brewery Convention (1998), and the Institute of Brewing (1997). It is recognized that many specific procedures are employed in a given brewery, and that there are most important variations related to the preferences of local consumers. Such a beer manufacturing method may be used in conjunction with the present invention.

[0164] The first step in producing beer from wort preferably involves heating the wort, then cooling the wort, and optionally performing a swirl pause, as described above herein.

[0165] The method of the present invention includes the step of fermenting an aqueous extract of malt and / or cereal grains with a yeast strain according to the present invention. The fermentation may be of an unfermented aqueous extract or of a fermented aqueous extract that still contains sugars fermentable by the yeast. Thus, in some embodiments, the fermentation may be carried out substantially immediately after the completion of grinding or after heating of the wort.

[0166] Fermentation may be carried out in a fermentation tank containing a descendant yeast strain according to the present invention, i.e., a descendant yeast strain having one or more of the characteristics described herein.

[0167] During fermentation, which lasts for several days, flavor compounds are produced. If the yeast strain does not have the ability to convert certain compounds, these will still be present after fermentation step iii).

[0168] In some embodiments, fermentation step iii) occurs at one of the temperatures specified below, the aqueous extract is cultured with one of the yeast cell counts described below, the fermented aqueous extract contains the highest level of diacetyl described below, and fermentation is completed after a maximum of 5 days, for example, a maximum of 4 days, for example, a maximum of 3 days. Specifically, fermentation is completed after a maximum of 4 days.

[0169] The above fermentation is preferably carried out at a temperature of at most 18°C, for example, in the range of 12°C to 18°C. In particular, the above fermentation may be carried out at a temperature of approximately 16°C.

[0170] The maximum level of diacetyl described above is preferably at most 50 ppb diacetyl at any point during fermentation, for example, at most 40 ppb diacetyl, for example, at most 30 ppb diacetyl. Specifically, the level of diacetyl described above at any point during fermentation is at most 50 ppb.

[0171] The above fermentation is preferably carried out by culturing the aqueous extract with at least 6 million viable yeast cells per milliliter, for example, at least 10 million viable yeast cells per milliliter, for example, at least 14 million viable yeast cells per milliliter, for example, in the range of 7 to 8 million viable yeast cells per milliliter, for example, in the range of 14 to 16 million viable yeast cells per milliliter. Specifically, the above fermentation may be carried out by culturing the aqueous extract with approximately 15 million yeast cells per milliliter.

[0172] In some embodiments, the fermented aqueous extract has an alcohol content of at most 1.0% ABV, for example, at most 0.9% ABV, for example, at most 0.8% ABV, for example, at most 0.7% ABV, for example, at most 0.7% ABV, or for example, at most 0.6% ABV.

[0173] In some embodiments, fermented aqueous extracts prepared by the methods described above are also provided herein.

[0174] Malt and / or grain-based beverages and methods for producing the same The malt and / or cereal-based fermented aqueous extracts described herein may be further processed into beverages.

[0175] Therefore, in some embodiments, a method for producing a beverage is provided, and the method is i. Prepare the fermented aqueous extract described herein, and ii. Further process the above fermented aqueous extract into a beverage. Includes steps.

[0176] In some embodiments of the present invention, a malt and / or cereal-based beverage is diluted with a liquid such as water.

[0177] Optionally, water may 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 ratio of water to malt and / or cereal-based beverage may range from 0.1 to 5 parts water to 1 part malt and / or cereal-based beverage.

[0178] 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, malt and / or grain-based beverages, such as beer, may be pasteurized and / or filtered before being packaged (e.g., bottled or canned).

[0179] In some embodiments, the processing steps are: i. Filtration, ii. Carbonation iii. Aging, or iv. Bottling Includes one or more of the following.

[0180] The processing steps may further include one or more steps to reduce the alcohol content of the fermented aqueous extract before bottling the beer. In some embodiments, the step of reducing the alcohol content is distillation, an alcohol reduction method using a membrane, rectification, or debrewing.

[0181] The present invention also provides a malt and / or cereal-based beverage prepared by the method described above.

[0182] In a preferred embodiment, the beverage is beer. In some embodiments, the beer has a lager-like flavor. In some embodiments, the beer has an ale-like flavor.

[0183] The beverage may be a beverage with reduced alcohol content, such as a low-alcohol beverage. In some embodiments, the beverage is an alcohol-free beverage. In preferred embodiments, the low-alcohol beverage is a low-alcohol beer. In preferred embodiments, the alcohol-free beverage is an alcohol-free beer.

[0184] In some embodiments, the alcohol content of the alcohol-free beverage is less than 0.5 vol%. In some embodiments, the alcohol content of the alcohol-free beverage is 0.05 vol% or less, for example, 0.049% or less, for example, 0.0%. In some embodiments, the alcohol content of the low-alcohol beverage is 0.5 vol% to 1.2 vol%. In some embodiments, the alcohol content of the low-alcohol beverage is 0.5 vol% to 2.8 vol%.

[0185] In one aspect of the present invention, a malt and / or cereal-based beverage produced by fermenting an aqueous extract with the aforementioned offspring yeast strain according to the present invention has a pleasant taste.

[0186] The taste of malt and / or grain-based beverages produced by fermentation using the yeast according to the present invention may be analyzed, for example, by a beer taste tester. Preferably, the tester is trained in tasting and describing the flavors of beer, with particular focus on aldehydes, light flavor, aged flavor, esters, higher alcohols, fatty acids, and sulfur components.

[0187] Generally, a taste panel consists of 3 to 30 people, for example, 5 to 15, preferably 8 to 12. The taste panel may evaluate various flavors, such as blandness, oxidation, staleness, and off-flavors like bread, as well as the presence of esters, higher alcohols, sulfur compounds, and the body of the beer. The overall taste of a beer is generally determined by the taste panel's evaluation of several different characteristics on a scale from 1 to 9, with an average rating of 5 or higher indicating an acceptable taste.

[0188] item 1. A yeast hybrid or offspring yeast strain that is the offspring of a first parent yeast strain and a second parent yeast strain, where, a. The first parent yeast strain is of the species Saccharomyces cerevisiae, and also, b. The first parent yeast strain and the hybrid or progeny yeast strain each have the ability to produce a fermentation test solution when cultured in the test solution, the test solution being an extract of malt and / or cereals, and the fermentation test solution does not reach a total diacetyl level of more than 50 ppb, e.g., more than 40 ppb, or e.g., more than 30 ppb at any point during fermentation.

[0189] 2. The yeast mixture or offspring yeast strain described in item 1, in which the above fermentation occurs at a maximum temperature of 18°C.

[0190] 3. A yeast mixture or offspring yeast strain described in any one of the preceding items, in which the above fermentation occurs at a maximum temperature of 16°C.

[0191] 4. A yeast mixture or offspring yeast strain described in any one of items 1 or 2, in which the above fermentation occurs at a temperature in the range of 12°C to 18°C.

[0192] 5. A yeast mixture or progeny yeast strain described in any one of the preceding items, wherein the culture in the above test solution is carried out for a maximum of 6 days, for example, a maximum of 5 days, for example, a maximum of 4 days.

[0193] 6. A yeast mixture or offspring yeast strain described in any one of items 1 to 4, wherein the culture in the above test solution is carried out for 2 to 6 days, or for example, 2 to 4 days.

[0194] 7. A yeast mixture or offspring yeast strain described in any one of the preceding items, in which the above fermentation occurs after inoculation of 7,000,000 to 20,000,000 viable yeast cells per 1 mL of test solution.

[0195] 8. A yeast mixture or offspring yeast strain described in any one of the preceding items, wherein the above test solution contains at least 3500 mg / L of amino acids, for example, at least 3000 mg / L, or for example, at least 2500 mg / L.

[0196] 9. The above test solution contains at least 40 g / kg of maltose, and is a yeast mixture or offspring yeast strain as described in any one of the preceding items.

[0197] 10. A yeast composite or progeny yeast strain described in any one of the preceding items, wherein the above test solution has an apparent extract of at least 9 plateau degrees.

[0198] 11. A yeast composite or progeny yeast strain according to any one of the preceding items, wherein the above test solution has an apparent extract of at least 12 plateau degrees, for example, at least 15 plateau degrees.

[0199] 12. A yeast mixture or progeny yeast strain described in any one of the preceding items, wherein the above test solution is wort.

[0200] 13. A yeast mixture or offspring yeast strain described in any one of the preceding items, wherein the above test solution is a wort having an apparent extract of about 16 plateau degrees.

[0201] 14. A yeast mixture or offspring yeast strain described in any one of the preceding items, wherein the first parent yeast strain described above is M49 deposited with DSMZ under receipt number DSM34496.

[0202] 15. A yeast mixture or offspring yeast strain described in any one of the preceding items, wherein the second parent yeast strain described above belongs to the genus Saccharomyces.

[0203] 16. A yeast mixture or offspring yeast strain described in any one of the preceding items, wherein the second parent yeast strain is of the species Saccharomyces cerevisiae.

[0204] 17. A yeast mixture or offspring yeast strain described in any one of the preceding items, wherein the second parent yeast strain is maltose intolerant, such as lacking the ability to completely convert maltose to ethanol.

[0205] 18. The yeast mixture or offspring yeast strain described in any one of the preceding items, wherein the second parent yeast strain is maltose intolerant, for example, the second parent yeast strain does not have the ability to grow using maltose as its sole carbon source, or grows at a significantly slower rate using maltose as its sole carbon source compared to other yeasts, preferably compared to Weihenstephan34 / 70 yeast and / or Safale US5.

[0206] 19. The yeast mixture or offspring yeast strain described in any one of the preceding items, wherein the offspring yeast strain or mixture is maltose intolerant, for example, the offspring yeast strain or mixture does not have the ability to completely convert maltose to ethanol.

[0207] 20. The yeast mixture or progeny yeast strain described in any one of the preceding items, wherein the progeny yeast strain or the second parent yeast strain is maltose intolerant, for example, the progeny yeast strain or the second parent yeast strain does not have the ability to grow using maltose as its sole carbon source, or grows at a significantly slower rate using maltose as its sole carbon source compared to other yeasts, preferably compared to Weihenstephan 34 / 70 yeast and / or Safale US5.

[0208] 21. The yeast mixture or offspring yeast strain described in any one of the preceding items, wherein the offspring yeast strain or mixture is maltotriose intolerant, for example, the offspring yeast strain or mixture does not have the ability to completely convert maltotriose to ethanol.

[0209] 22. The above fermentation test solution has an alcohol content of at most 1.0% ABV, for example, at most 0.8% ABV, or for example, at most 0.6% ABV, and is a yeast mixture or offspring yeast strain as described in any one of the preceding items.

[0210] 23. The yeast mixture or progeny yeast strain described in any one of the preceding items, wherein, after being cultured in the test solution, the yeast mixture or progeny yeast strain produces at most 1.0% ABV, for example at most 0.8% ABV, or for example at most 0.6% ABV.

[0211] 24. A yeast mixture or offspring yeast strain described in any one of the preceding items, wherein the second parent yeast strain described above is PPU121 deposited with DSMZ under receipt number DSM34497.

[0212] 25. The yeast mixture or offspring yeast strain described in any one of the preceding items, wherein the offspring yeast strain or mixed yeast strain does not have the ability to convert more than 25% of p-coumaric acid to 4-ethylphenol when cultured in an aqueous solution containing p-coumaric acid.

[0213] 26. A yeast mixture or offspring yeast strain according to any one of the preceding items, wherein the second parent strain and / or the offspring yeast strain, or the mixed yeast strain, does not have the ability to convert more than 25% of p-coumaric acid to 4-ethylphenol when cultured in an aqueous solution containing p-coumaric acid.

[0214] 27. A descendant yeast strain of the first parent yeast strain and the second parent yeast strain, where, a. The first parent yeast strain is yeast strain M49 deposited with DSMZ under acceptance number DSM34496, and also, b. The second parent yeast strain, i. PPU121 deposited in DSMZ with acceptance number DSM34497, or ii. X2180-1A, or iii.PE-2 This is a descendant yeast strain of the first parent yeast strain and the second parent yeast strain.

[0215] 28. A method for producing a yeast mixture or a progeny yeast strain, the method being: a. Provide spores of a first parent yeast strain (wherein the first parent yeast strain is defined in any one of the preceding items). b. Cross the aforementioned spores with spores from the second parent yeast strain. The process includes the step in which at least one spore of the first parent yeast strain has a different mating type from at least one spore of the second parent yeast strain.

[0216] 29. The method according to item 28, wherein the second parent yeast strain is as defined in any one of items 15 to 27.

[0217] 30. A method for producing a fermented aqueous extract, wherein the method is i) Provides aqueous extracts of malt and / or cereals. ii) Provide a progeny yeast strain or hybrid yeast strain described in any one of items 1 to 27, and iii) The aqueous extract provided in step i) is fermented with the yeast strain from step ii) to obtain a fermented aqueous extract. A method for producing a fermented aqueous extract, including the steps.

[0218] 31. The method according to item 30, wherein the aqueous extract is wort.

[0219] 32. The method according to any one of items 30 to 31, wherein the aqueous extract has an apparent extract of up to 16 plateau degrees, for example in the range of 6 to 16 plateau degrees, for example in the range of 8 to 16 plateau degrees, preferably in the range of 6 to 12 plateau degrees, for example in the range of 8 to 10 plateau degrees, for example about 9 plateau degrees.

[0220] 33. A fermented aqueous extract prepared by any one of the methods described in items 30-32.

[0221] 34. A method for manufacturing a beverage, the method is i. Prepare a fermented aqueous extract according to item 30, and ii. Process the above fermented aqueous extract into a beverage. A method for producing a beverage, including the steps involved.

[0222] 35. The processing step is, i. Filtration, ii. Carbonation iii. Aging, or iv. Bottling The method described in item 34, which includes one or more of the following.

[0223] 36. The method according to items 34-35, further comprising the step of reducing the alcohol content of the fermented aqueous extract.

[0224] 37. The method according to item 36, wherein the step of reducing the alcohol content is rectification or debrewing.

[0225] 38. A beverage prepared by any one of items 34 to 37.

[0226] 39. The beverage described in item 38, wherein the beverage is a beverage with reduced alcohol content, such as a low-alcohol beverage.

[0227] 40. The beverages listed in item 38, for which the above beverages are alcohol-free beverages.

[0228] 41. The beverage described in any one of items 38-40, wherein the beverage is, for example, a low-alcohol beer or, for example, an alcohol-free beer.

[0229] 42. The beverage according to any one of items 38 to 41, wherein the beverage has a propanol:isobutanol ratio of at least 6.0, for example at least 8.0, for example at least 10.0, for example at least 12.

[0230] [Table 1] JPEG2026510245000003.jpg220159JPEG2026510245000004.jpg101159 [Examples]

[0231] Example 1 - Analysis of the compound The sample was centrifuged at 1900 g for 10 minutes, and the supernatant was stored in a -20°C freezer until further use. The inventors measured the total diacetyl by gas chromatography according to the European Brewing Convention method EBC9.24.2, which includes culturing the sample at 60°C for 90 minutes to determine the total diacetyl, which reflects the sum of precursor acetolactic acid and free diacetyl.

[0232] Alcohol and plateau levels were measured using the Anton Paar Alcolyzer beer analysis system (Colomer et al, 2020).

[0233] The volatile substances propanol and isobutanol were measured using gas chromatography-CS2 extraction with minimal modifications previously described (Egan S, 1972). Octanol was used as the internal standard. An Agilent 6890A gas chromatograph with split / splitless injection and an FID detector was used, employing a DBWAX column, J&W 123-7032 (30 m × 0.32 mm × 0.25 μm).

[0234] Example 2 - Discovery of M49 yeast by evaluation The inventors evaluated the yeast as described above (Sanchez RG et al, 2012), except that they added only 0.5 g / l of yeast extract (Difco) instead of 10 g / l to wort made from commercially available granular malt (spray-dried malt) called GranMalt. This medium had an initial pH of 5.4. The inventors decided to compare the previously described yeast (WO2016101960) with several yeasts from their yeast collection that were promising for breeding.

[0235] Fermentation of 150 ml was carried out in an ANKOM Gas Pressure module, as described above (Colomer et al, 2020), using GranMalt wort supplemented with 0.5 g / l of yeast extract as the culture medium, at 16°C for 7 days, with an input rate of 10 million cells per ml. The inventors had previously shown that a high propanol-to-isobutanol ratio correlated with low diacetyl production in beer fermentation (WO2022 / 002960), so these fusel alcohol metabolites were measured at harvest on the 7th day. Surprisingly, the yeast called M49 (acceptance number DSM34496) had the highest propanol / isobutanol ratio (Figure 1). Furthermore, the M49 yeast was unable to completely ferment the maltose in the wort, resulting in a high concentration of 4VG (clove-like odor). M49 is deposited with DSMZ under acceptance number DSM34496.

[0236] Sequencing of M49 revealed that M49 has a mutation in the ILV2 gene, in which the G at position 1493 of SEQ ID NO: 3 is replaced with an A, resulting in the ILV2 gene encoding a mutant ILV2 protein with the G498E substitution. Therefore, the ILV2 gene of M49 encodes a mutant ILV2 protein of the sequence provided herein as SEQ ID NO: 2.

[0237] Example 3 - Results of diacetyl and propanol in ANKOM fermentation using M49 yeast A second fermentation was performed using the ANKOM Gas Pressure system, and this time, total diacetyl was also analyzed. The fermentation data is shown in Figure 2. The propanol / isobutanol ratio of M49 (acceptance number DSM34496) was again very high compared to the ratio of the other two yeasts, which was less than 1. The total diacetyl of M49 on day 7 was also very low, well below the taste threshold of 50 ppb.

[0238] Example 4 - 50L fermentation test of M49 yeast in glucose wort The M49 yeast (acceptance number DSM34496) was evaluated on a 50L scale, and its performance over time was compared with that of yeast mixture 7. Since it was observed that the M49 yeast had difficulty fermenting maltose, the inventors decided to enzymatically convert standard 14-plateau Pilsner wort to convert all maltose and maltotriose into glucose (Gutierrez et al., 2018). In this way, the inventors produced so-called glucose wort.

[0239] Fermentation was carried out with 14-plateau glucose wort at 16°C and an input rate of 10 million cells / ml. The results are shown in Tables 1 and 2. The fermentation rate of M49 yeast was slightly slower than that of mixture 7, but it was demonstrated that it produced diacetyl levels of less than 50 ppb throughout the entire fermentation period. This extremely low diacetyl characteristic was considered very interesting for further breeding. Unfortunately, M49 produced off-odor in 4VG because the PAD1 and FDC1 genes were active.

[0240] Table 1. Results of a 50L glucose wort test at 16°C using yeast mixture 7. [Table 2]

[0241] Table 2 Results of a 50L glucose wort test at 16°C using yeast M49 [Table 3]

[0242] Example 5 - Preparation of low-alcohol 9-plateau wort Nine-plateau wort for the growth and fermentation of PPU191 and FY121 was prepared as follows. First, a standard twelve-plateau wort containing 87% Pilsner malt and 13% Munich malt was prepared using the grinding outline in Table 3 (Grinding). Bitter hops were added at the start of boiling, and the wort was boiled at 105°C for 50 minutes. The pH was adjusted to pH 4.4 with phosphoric acid. The resulting twelve-plateau wort was debrewed to nine plateau wort to obtain so-called AFB wort with the following composition. [Table 4]

[0243] Table 4 shows the detailed sugar, amino acid, and mineral composition of the wort.

[0244] Table 3.12 Summary of grinding before defermentation of plateau wort to 9 plateau degree [Table 5]

[0245] Table 4.12 Low-alcohol wort composition when plateau wort is reduced to 9 plateaus. [Table 6] JPEG2026510245000010.jpg24159

[0246] Example 6 - Preparation of low-alcohol fermentation yeast PPU191 (diacetyl-free S. cerevisiae ale yeast) The main concept of the S. cerevisiae PPU191 strain was to create a diploid yeast that could be used as a basis for further development of novel brewing yeasts that could be used in the production of non-alcoholic, lager-like brewed beverages. Meanwhile, this strain had to possess the most important characteristics of a typical lager yeast, such as good flocculation, low diacetyl production, and low production of 4-vinylguaiacol (4-VG), one of the major phenolic off-flavors (POF-). However, at the same time, this strain must not ferment maltose and maltotriose (Mal-) to limit alcohol production. This is clearly an important characteristic for a yeast used in the fermentation of non-alcoholic beverages. Furthermore, this yeast must produce a low amount of diacetyl during fermentation. To achieve this, the inventors decided to combine the maltose and POF-negative characteristics of the PPU121 strain, which has been used in the production of non-alcoholic and low-alcohol beers, with the haploid yeast M49 (acceptance number DSM34496), which has very low diacetyl production, as described in previous examples. PPU121 is deposited with DSMZ under acceptance number DSM34497.

[0247] As a first step, diploid S. cerevisiae strain PPU121 was spore-forming, and its haploid derivative was prepared. For this purpose, PPU121 was grown overnight in YPD at 30°C. The cells were harvested and spun-washed twice with sterile water to remove the complex medium. Finally, the cells were placed on a standard S. cerevisiae SPOR plate (Dunham et al., 2015), and the plate was cultured at 30°C for 3–7 days, or until asci / 4 molecules were visible. To isolate individual spores, small cell fragments from the SPOR patch were suspended in 500 μL of sterile water in a 1.5 ml reaction tube and spun-washed twice with sterile water. The cells were suspended in 450 μL of 100 mM sodium phosphate buffer (pH 7.0), 50 μL of zymolyase (20T; 10 mg / mL stock solution) was added, and the cells were cultured at 30°C for 10–30 minutes to loosen the asci cell walls. Subsequently, monospores were isolated on YPD agar plates using a Singer MSM400 dissecting microscope. The mating type of individual spore clones was determined using a pheromone-based halo assay (Dohlmann et al., 1996) as follows: Two ml of YPD cultures of mating type testers BY4741 Δsst2(MATa) and BY4742 Δsst2(MATα), as well as the target strain, were grown overnight at 30°C. The test strains grown overnight were first subjected to approximately 0.1 OD. 600 The strain was diluted to [specific concentration]. Approximately 200 μl of the diluted test strain was added to 4 ml of YPD bilayer agar medium (agar medium containing 0.5% agar, one medium per test strain), and the agar medium was spread onto YPD agar plates and allowed to solidify. 2-5 μl of the target strain was taken from an overnight culture and placed on each test strain plate, and the test strain plates were cultured overnight at 30°C. The next day, the presence or absence of areas without growth around each arrangement was scored. The test strains in the turf stopped growing in response to the OPPOSITE mating type pheromone and formed a ring around the target arrangement.

[0248] In the next step, a MATa spore clone (AFB6) from PPU121 was crossed with MATα strain M49. First, strains AFB6 and M49 were grown overnight in 2 ml of YPD at 30°C. The strains were then grown at high cell density (OD).600 >10) The cells were mixed in a ratio of approximately 1:1 and placed on a new YPD plate. After culturing at 30°C for 4-5 hours, the cell patches were examined under a microscope for zygote formation. The zygotes were isolated using a Singer MSM400 dissecting microscope. The diploid nature of the isolated strains was then demonstrated by their ability to spore on SPOR plates (see above for the spore formation protocol).

[0249] Next, one of the diploid yeasts created, AFB17, was allowed to form spores as described above (see above). However, this time, in order to isolate a large number of spores, the spore-forming patch was treated with zymolyase for several hours to remove as many plant cells as possible. The spores were plated onto a standard YPD plate to obtain single colonies (ideally about 100-200 per plate). For further analysis, potentially spore clones were collected in a 96-well plate containing 200 μl of YPD medium using a Molecular Devices QPix 460 microbial colony collector. In order to finally create a diploid yeast with the above characteristics, it was necessary to identify the respective MATa and ATα strains from the pool of haploid spore clones.

[0250] Subsequently, the aforementioned characteristics were analyzed using approximately 550 spore clones, with PPU121, M49, and a standard POF-negative brewer's yeast as controls. Fermentation rate (growth curve) and flocculation (visual test) were analyzed using 500 μl of low-alcohol wort (Table 4) with an EnzyScreen CR9001 Growth Profiler at 16°C. The supernatants from these small-scale fermentations were also used for ethanol analysis using the Megazyme Ethanol Assay Kit, according to the manufacturer's instructions. The ability of spore clones to produce phenolic off-odors was analyzed using a previously described absorbance-based rapid assessment method for phenolic off-odors (Mertens et al, 2017). For this purpose, spore clones were grown for 3–5 days at 16°C and 750 rpm in 150 μl of liquid YPD supplemented with 100 μg / ml ferulic acid in a 96-well plate using a Heidolph Titramax 1000 shaker. Subsequently, 100 μl of the supernatant was analyzed for residual ferulic acid concentration at 325 nm using a Tecan Infinite 200PRO plate reader and compared with POF-negative control strains. Spore clones were also placed on standard SC-ILV plates (synthetic complex media lacking branched-chain amino acids isoleucine, leucine, and valine), and growth of the low-diacetyl phenotype exhibiting ILV supplementation was analyzed. The mating type of the selected spore clones was analyzed using the halo assay based on the pheromone reaction described above.

[0251] As described above, since the main objective of this study was to create strains for the production of non-alcoholic or low-alcohol beverages, the most important criterion for selecting candidates for further breeding was alcohol production. In the initial screening of the spore clone library, strains with an alcohol content (ABV) of 0.5 - 0.6% were considered interesting for further analysis. The selected spore clones were re-analyzed for fermentation performance to confirm the screening results. However, this time, 160 ml of low-alcohol wort was placed in 250 ml glass bottles and fermented at 16 °C while monitoring CO2 production with an ANKOM RF Gas Production System. At the end of fermentation, the degree of aggregation was visually measured again, the alcohol content was analyzed using an Anton Paar Alcolyzer beer analysis system, and the vicinal diketone (diacetyl / butane-2,3-dione; pentane-2,3-dione) levels were analyzed using headspace / GC-ECD. Based on these analyses, four spore clones of two MATa strains (AFB94, AFB96) and two MATα strains (AFB92, AFB90) were selected for further breeding efforts.

[0252] To create the target diploid strains, these four isolated haploid strains were mated in all possible combinations. The mating reaction was set up as described above, and once zygote formation began, the mating reaction products were plated onto single colonies on YPD plates. After culturing at 30 °C for 3 - 4 days, assuming that diploid strains usually grow into colonies of larger size than the haploid parent strains, large colonies were picked into 96-well plates. Then, approximately 1200 strains were analyzed for fermentation performance, ethanol production, ILV auxotrophy, flocculability, and phenolic off-flavors, similar to the selection of haploid spore clones.

[0253] From this initial bulk screening, the PPU191 strain, which was born from the mating of the AFB90 strain (Ilv-, Mal-, POF-, Flocc-, Matα) and the AFB96 strain (Ilv+, Mal-, POF-, Flocc+, Mata), was considered a good candidate as the base strain, and as described above for the recheck of the breeding haploid spore clones, all important characteristics were rechecked using ANKOM fermentation.

[0254] Example 7 - Propagation of PPU191 and FY112 for Low-Alcohol Fermentation Two maltose-negative yeasts, PPU191 and FY112, suitable for low-alcohol beer production can be propagated in standard lager wort at 12 - 16 Plato degrees. However, when using the wort with the highest Plato for propagation, there is a risk that alcohol accumulates to more than 1% ABV. This alcohol may, in some cases, be carried over to the fermentation tank at the input stage after propagation. Therefore, to avoid the carry-over of ethanol, the inventors propagated the yeast in 9 Plato degree wort (the same wort as for subsequent low-alcohol fermentation). The propagation was carried out in 9 Plato degree wort using standard procedures and wort aeration similar to standard lager / ale yeast.

[0255] Example 8 - Fermentation of 50 L Scale 9 Plato Degree Wort for Low-Alcohol Beer Using Yeast PPU121 and PPU191 Yeast at a rate of 5 - 10 million cells per ml was inoculated into the 9 Plato degree wort. The fermentation was carried out at 15°C. The fermentation outlines of yeast PPU121 (accession number DSM34497) and PPU191 are as follows.

[0256] The fermentation was carried out until the total diacetyl (free diacetyl + precursor acetolactic acid) was less than 50 pbb, and the fermented product was cooled to harvest the beer.

[0257] Tables 5 - 9 below show the results of these fermentations.

[0258] Table 5. 50 L Test of PPU191 at 15°C and Inoculation Rate of 9 Million Cells / ml [Table 7]

[0259] Table 6. 50L test of PPU191 at 15°C and an input rate of 7 million units / ml [Table 8]

[0260] Table 7. 50L test of PPU191 at 15°C and an input rate of 9 million units / ml. [Table 9]

[0261] Table 8. 50L test of PPU121 at 15°C and an input rate of 10 million units / ml. [Table 10]

[0262] As can be seen from the table above, during fermentation using PPU191, the total diacetyl level never exceeds the taste threshold of 50 ppb.

[0263] Example 9 - Fermentation of 9 plateau wort for low-alcohol beer at 16°C on a 900HL scale using yeast PPU121 Table 9 below shows the results of 900HL fermentation at 16°C using PPU121 (acceptance number DSM34497).

[0264] Table 9 [Table 11]

[0265] As can be seen from Table 9, in the case of yeast PPU121, at an important time for the harvest of low-alcohol beer, when the alcohol reaches ABV 0.5-0.6, the level of diacetyl exceeds the taste threshold of 50 ppb. This may lead to a situation where the alcohol becomes too high before the diacetyl level drops below 50 ppb. That is, in the case of yeast such as PPU121, since the endpoint of diacetyl is also affected by pH and temperature, it means that it is a process that is difficult to control.

[0266] Example 10 - Fermentation of 9 Plato wort for low-alcohol beer using yeast PPU191 at 14°C on a 1500 HL scale The 1500 HL fermentation at 14°C using PPU191 is shown in Table 10 below.

[0267] Table 10

Table 12

[0268] As can be seen from Table 10 above, during the fermentation using PPU191, the total diacetyl level does not exceed the taste threshold of 50 ppb.

[0269] Example 11 - Second breeding to generate ultra-low diacetyl yeast by mating M49 and bioethanol yeast PE2 The purpose of creating the M49×PE-2 hybrid was to verify that the low-diacetyl characteristics of M49 could be combined with a different strain background than those used in previous examples. For this purpose, yeast PE-2, a diploid S. cerevisiae strain commonly used in the Brazilian bioethanol industry for high-concentration alcohol production, was selected. To combine the low-diacetyl characteristics of M49 with the PE-2 background, the inventors first spore-formed PE-2 and examined haploid, phenol-negative (POF) spore clones as described above (Mertens et al, 2017). One such POF-negative spore clone obtained from PE2 was PE-2-ST13, which the inventors used for further crosses with M49.

[0270] Therefore, the POF-negative spore clone PE2-ST13 (mating type A) was crossed with M49 yeast (Matα) as follows. First, both strains were cultured overnight in 2 mL of YPD at 30°C while rotating. Both strains were then cultured at high cell density (OD). 600 >10) The cells were mixed in a ratio of approximately 1:1 and placed on a YPD plate. After culturing at 30°C for 4-5 hours, the cell mixture was examined under a microscope to confirm zygote formation. The cell mixture was further cultured at 30°C for 20-24 hours to allow the maximum number of zygotes to form. The cells were scraped and plated onto a standard S. cerevisiae SPOR plate and cultured at 20°C for 3-5 days, or until asci / 4 molecules were visible. To isolate spores, large-scale spore isolation was performed using zymolyase, as in the example using PPU191. Finally, the spores were plated onto a YPD plate to form single colonies (ideally about 100-200 per plate), and for further analysis, potentially spore clones were collected into a 96-well plate containing 200 μl of YPD medium using a Molecular Devices QPix 460 microbial colony collector.

[0271] To demonstrate that the reduction in diacetyl in M49 is combined with the PE-2 background, isolated spore clones were subsequently analyzed for low diacetyl formation and lack of POF production, using M49 and PE2-ST13 as controls. Furthermore, the inventors examined these spore clones obtained from spore formation of M49XPE2-ST13 crosses for mating type and inability to form spores, confirming that the spore clones were haploid. Examination of low diacetyl (due to branched-chain amino acid orthotrophy), POF production, and mating type was performed in the same manner as described above. After culturing at 20°C for 7 days, the absence of spore formation on SPOR plates further demonstrated their polyploid nature.

[0272] The main objective of this study was to confirm that the low diacetyl characteristics of M49 could be introduced into a PE-2 background. Therefore, several spore clones identified in the initial screening were selected for further analysis (Table 11). These spore clones were analyzed for fermentation performance in 150 mL of 70% barley malt / 30% barley-added (70 / 30) wort, with CO2 production monitored using an ANKOM RF Gas Production System at 16°C in 250 mL glass bottles. At the end of fermentation, 4-vinylguaiacol (4VG / POF) and vicinal diketone (diacetyl / butane-2,3-dione) were analyzed using headspace GS-MSMS and GC-ECD, respectively. As described in previous examples, the liquid was also analyzed for propanol levels using headspace GC-FID. Four spore clones, ST55, ST56, ST57, and ST59, showed low diacetyl formation ability along with low POF production. Furthermore, all four spore clones corresponding to the spore clone with the lowest total diacetyl showed high propanol production or a high propanol-to-isobutanol ratio. Spore clone ST46, which produced high levels of diacetyl, also had the lowest propanol production level and the lowest propanol-to-isobutanol ratio. Therefore, spore clone ST46 was considered undesirable due to its phenolic properties and high diacetyl content.

[0273] These results confirm that the low diacetyl characteristic of M49 can be combined with the PE-2 strain background, in addition to the strain background used in the example with PPU191.

[0274] Table 11. Summary of total diacetyl, POF(4-VG), and propanol from several types of spores obtained from spore formation of M49×PE-2 hybrid (M49×ST13). The liquid was sampled from fermentation carried out at 16°C via an ANKOM RF Gas Production System in 150 mL of 70 / 30 wort. [Table 13]

[0275] Example 12 - Third cross - Cross between M49 and yeast X2180-1A The low-diacetyl phenotype of strain M49 allows for easy cross-breeding with other strains. This example demonstrates the cross-breeding of M49 (MATα His- ilv2) (acceptance number DSM34496) with the commonly available S288C derivative X2180-1A (MATa SUC2 Mel- Mal- Gal- CUP1) (acceptance numbers DSM4266; NCYC956; ATCC26786). For this purpose, both strains were first grown overnight in liquid YPD medium at 30°C. The following day, 20 μl of each culture medium was mixed, and the cells were placed on YPD agar plates, which were then cultured at 30°C. After 4-5 hours, small samples of the placed cells were taken and microscopic examination was performed to confirm zygote formation. Subsequently, zygotes were isolated onto new YPD plates using a Singer MSM4004 molecular dissection microscope. One of the zygotes obtained from the cross between M49 and X2180-1A was cultured into colonies on a YPD plate at 30°C for 3 days, and this diploid yeast was called hybrid M49X2180.

[0276] Mixed M49X2180 colonies were smeared onto standard yeast spore-forming plates, and the plates were cultured at 30°C for several days. During this time, cell patches were periodically checked for spore formation / tetramolecular formation under a microscope. Spores were isolated onto YPD agar plates from one of the patches that successfully formed spores, again using a Singer MSM4004 molecular dissection microscope. The obtained spore clones (spores growing on YPD medium) were finally re-seed onto YPG lactose, SC-His, and SC-Ile / Leu / Val agar plates, and marker isolation was analyzed. Spore clones showing recombinant marker isolation (His+ ilv2 or Gal- ilv2) were then fermented on a laboratory scale, and diacetyl formation was analyzed.

[0277] Example 13 - Laboratory-scale fermentation using parent strain, new hybrid M49X2180, and spore clones of the new hybrid. The parent plant M49 (acceptance number DSM34496), as well as X2180-1A (acceptance numbers DSM4266; NCYC956; ATCC26786), the diploid hybrid strain hybrid M49X2180, and spore clones generated from spore formation of hybrid M49X2180 were fermented on a laboratory scale.

[0278] Cells were first pre-grown in 2 ml of liquid YPD overnight at room temperature, and then transferred to a shaking flask containing 40 ml of 16.6 plateau brewing wort with the composition shown in Table 12. Culturing was carried out for 72 hours over the weekend. 10 ml of cells were collected from each shaking flask by centrifugation and washed with sterile water. Finally, 5 ml of washed cells were resuspended in 5 ml of wort and added to 145 ml of fresh wort, and fermentation was carried out at 16°C in the same wort (Table 12). Laboratory-scale fermentation was performed in a 250 ml tall glass graduated cylinder (331 × 39 × 39 mm; Duran) containing 150 ml of fermentation medium, sealed with an inverted glass beaker (Duran) to allow carbon dioxide to escape and facilitate sampling for analysis. Fermentation was carried out with continuous stirring at 130 rpm, and weight loss and diacetyl, indicating the fermentation rate, were tracked over time (Tables 13 and 14). As can be seen, all spore clones resulting from spore formation of the diploid hybrid M49X21 had diacetyl levels below 50 ppb at all time points measured, while the parent strain X2180-1a and the diploid hybrid M49X2180 had total diacetyl levels exceeding 100 ppb on day 2. The specific gravity of plateau units at the end of fermentation is shown in Table 15. This indicates that the low-diacetyl characteristic, along with other genetic markers from X2180-1A, was transferred from M49 to the new spore clones. Both M49 and X2180-1A are maltose and maltotriose-negative yeasts, and the wort contained considerable amounts of maltose and maltotriose, so the wort could only partially ferment from 16.6 plateaus to 11.6 plateaus.

[0279] Table 12. Wort used for laboratory-scale fermentation of spore clones of M49, X2180-1A, diploid, and hybrid M49X2180. The starting specific gravity of this wort was 16.6 plateau. [Table 14]

[0280] Table 13. Weight loss in grams over time, reflecting the fermentation rate due to carbon dioxide loss during alcoholic fermentation. [Table 15]

[0281] Table 14. Total diacetyl over time in experimental full-scale fermentation of parent yeast M49, X2180-1A, new hybrid M49X2180, and selected spore clones of the new hybrid. [Table 16]

[0282] Table 15. Specific gravity of the starting wort beer after fermentation. Since all yeasts were maltose-negative, fermentation did not fall below plateau 11. [Table 17]

[0283] Example 14 - 50L test example using low-alcohol wort with yeasts PPU191, M49, and LONA The yeast was pre-grown in the same wort used for fermentation, namely the 9 Plateau AFB wort. The yeast was grown at room temperature in a 3L blue-capped bottle filled with sterile air using a magnetic stirrer.

[0284] Yeast was recovered by centrifugation. The desired cell quantity was calculated by the weight of the cell paste and using a nucleocounter. Fermentation was carried out on a 50L scale at 15°C with a target cell input rate of 7 to 10 million cells / mL. Three types of yeast, M49 (acceptance number DSM34496), PPU191, and Lallemand's maltose-negative low-alcohol yeast called LONA, were compared simultaneously. Nine plateaus of wort were fermented at 15°C. The fermentation summary for yeasts M49, PPU191, and Lallemand's low-alcohol yeast LONA is as follows (Tables 16-18).

[0285] Table 16. 50L test using yeast M49, 15°C, infusion rate 7 million to 10 million cells / ml [Table 18]

[0286] Table 17. 50L test using yeast LONA, 15°C, infusion rate 7 million to 10 million cells / ml [Table 19]

[0287] Table 18. 50L test using yeast PPU191, 15°C, infusion rate 7 million to 10 million cells / ml. [Table 20]

[0288] As can be seen from Figures 3 and 4, the fermentation rate due to plateau descent was similar in all cases, except for M49, which exhibited slower fermentation. In the two yeasts, M49 and PPU191, diacetyl levels were consistently below 45 ppb throughout the entire fermentation period. This diacetyl-free characteristic facilitates the control of the low-alcohol fermentation process.

[0289] Furthermore, the lower cell count of the PPU191 strain (38 × 10^6 cells / mL) compared to LONA (50 × 10^6 cells / mL) on day 4 indicates the high cohesiveness of the PPU191 strain. Since the sample port for cell count measurement is located one-third of the way up the container above the sedimentation cone, a lower cell count means a higher cell count in the cone at the end of fermentation, which in turn means more sediment and higher cohesiveness.

[0290] References Briggs,DEet al.Malting and Brewing science.1981 Colomer,M.S.,Funch,B.,Solodovnikova,N.,Hobley,T.J.,&Forster,J.(2020).Biotransformation of hop derived compounds by Brettanomyces yeast strains.Journal of the Institute of Brewing,126(3),280-288.https: / / doi.org / 10.1002 / jib.610 Dohlman HG,Song J,Ma D,Courchesne WE,Thorner J.Sst2,a negative regulator of pheromone signaling in the yeast Saccharomyces cerevisiae: expression,localization,and genetic interaction and physical association with Gpa1(the G-protein alpha subunit).Mol Cell Biol.1996 Sep;16(9):5194-209.doi:10.1128 / MCB.16.9.5194.PMID:8756677;PMCID:PMC231520 Dunham MJ,Gartenberg MR,Brown GW.(2015)Methods in yeast genetics and genomics: a Cold Spring Harbor Laboratory course manual Egan S.Reproducibility of some gas chromatographic methods used in beer flavour analysis.Brygmesteren 1971:28:191-199 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.Journal of the Institute of Brewing 2018:DOI 10.1002 / jib.512 Hough,J.S.et al.Malting and Brewing science: Hopped Wort and Beer,Volume 2.1982 Kempf C,Lengeler K,Wendland J,2017.Differential stress response of Saccharomyces hybrids revealed by monitoring Hsp104 aggregation and disaggregation.Microbiological Research 200:53-63 Lundblad,V and Struhl,K,2008.Yeast.Current Protocols in Molecular Biology 82(1): 1934-3639.https: / / doi.org / 10.1002 / 0471142727.mb1300s82 Mertens S,Steensels J,Gallone B,Souffriau B,Malcorps P,Verstrepen KJ.Rapid Screening Method for Phenolic Off-Flavor(POF)Production in Yeast.Journal of the American Society of Brewing Chemists 2017:75:318-323.https: / / doi.org / 10.1094 / ASBCJ-2017-4142-01 Sanchez RG,Solodovnikova N,Wendland J.Breeding of lager yeast with Saccharomyces cerevisiae improves stress resistance and fermentation performance.Yeast 2012:29:343-355 Treco,D.A.,Winston,F.,2008.Growth and manipulation of yeast.Curr Protoc Mol Biol Chapter 13,Unit 13.2.https: / / doi.org / 10.1002 / 0471142727.mb1302s82

Claims

1. This is a descendant yeast strain, which is a descendant of the first parent yeast strain and the second parent yeast strain, and here, The first parent yeast strain is of the species Saccharomyces cerevisiae, and also, The first parent yeast strain described above is a) M49 deposited with DSMZ under receipt number DSM34496, and / or b) A gene encoding ILV2 having a mutation, where ILV2 is as described in SEQ ID NO: 1, or a functional homolog of SEQ ID NO: 1 having at least 90%, e.g., at least 95%, e.g., at least 98% sequence identity to SEQ ID NO: 1, wherein the mutation results in an amino acid substitution at position 498 of SEQ ID NO: 1, or an amino acid substitution at the corresponding position of the functional homolog. The first parent yeast strain and the progeny yeast strain each have the ability to produce a fermentation test solution when cultured in a test solution, wherein the test solution is an extract of malt and / or cereals, and the fermentation test solution does not reach a total diacetyl level of more than 50 ppb, for example more than 40 ppb, or for example more than 30 ppb at any point during fermentation. The aforementioned offspring yeast strain.

2. The offspring yeast strain according to claim 1, wherein the fermentation occurs at a maximum temperature of 18°C.

3. The descendant yeast strain according to any one of the prior claims, wherein the fermentation occurs at a maximum temperature of 16°C.

4. The offspring yeast strain according to any one of claims 1 to 3, wherein the fermentation occurs at a temperature in the range of 12°C to 18°C.

5. The offspring yeast strain according to any one of the prior claims, wherein the culture in the test solution is carried out for a maximum of six days, for example, a maximum of five days, for example, a maximum of four days.

6. The offspring yeast strain according to any one of claims 1 to 4, wherein the culture in the test solution is carried out for 2 to 6 days or 2 to 4 days.

7. The progeny yeast strain according to any one of the prior claims, wherein the fermentation occurs after inoculation of 7,000,000 to 20,000,000 viable yeast cells per 1 mL of test solution.

8. The offspring yeast strain according to any one of the prior claims, wherein the test solution contains at least 3500 mg / L, for example, at least 3000 mg / L, or for example, at least 2500 mg / L of amino acids.

9. The progeny yeast strain according to any one of the prior claims, wherein the test solution contains at least 40 g / kg of maltose.

10. The progeny yeast strain according to any one of the prior claims, wherein the test solution contains 1.0 to 3.5 g of fructose per liter of test solution, for example, 1.5 to 3.0 g of fructose per liter of test solution, or for example, 2.0 to 2.5 g of fructose per liter of test solution, for example, about 2.2 g of fructose per liter of test solution.

11. The progeny yeast strain according to any one of the prior claims, wherein the test solution contains 5.0 to 8.0 g of glucose per liter of test solution, for example, 5.5 to 7.5 g of glucose per liter of test solution, or for example, 6.0 to 7.0 g of glucose per liter of test solution, for example, about 6.5 g of glucose per liter of test solution.

12. The offspring yeast strain according to any one of claims 1 to 10, wherein the test solution contains at least 40 g / L of glucose, for example, at least 50 g / L of glucose, for example, at least 60 g / L of glucose, for example, at least 70 g / L of glucose, for example, at least 80 g / L of glucose, for example, at least 90 g / L of glucose, for example, at least 100 g / L of glucose, or for example, at least 110 g / L of glucose.

13. The progeny yeast strain according to any one of the prior claims, wherein the test solution contains 1.0 to 5.0 g of sucrose per liter of test solution, for example, 1.5 to 4.0 g of sucrose per liter of test solution, or for example, 2.0 to 3.0 g of sucrose per liter of test solution, for example, about 2.5 g of sucrose per liter of test solution.

14. The progeny yeast strain according to any one of the prior claims, wherein the test solution has an extract with an apparent degree of at least 9 plateaus.

15. The progeny yeast strain according to any one of the prior claims, wherein the test solution has an extract with an apparent degree of at least 12 plateaus, for example, at least 14 plateaus, for example, at least 15 plateaus.

16. The progeny yeast strain according to any one of the prior claims, wherein the test solution is wort.

17. The progeny yeast strain according to any one of the prior claims, wherein the test solution is a wort having an apparent extract of about 16 plateau degrees.

18. The first parent yeast strain and the second parent yeast strain are descendant yeast strains, where, The first parent yeast strain is of the species Saccharomyces cerevisiae, and also, a) M49 deposited with DSMZ under receipt number DSM34496, and / or b) A gene encoding ILV2 having a mutation, where ILV2 is as described in SEQ ID NO: 1, or a functional homolog of SEQ ID NO: 1 having at least 90%, e.g., at least 95%, e.g., at least 98% sequence identity to SEQ ID NO: 1, wherein the mutation results in an amino acid substitution at position 498 of SEQ ID NO: 1, or an amino acid substitution at the corresponding position of the functional homolog. and, The first parent yeast strain and the progeny yeast strain each have the ability to produce a fermentation test solution when cultured in the test solution. Here, the test solution is wort. • Having an apparent extract with a plateau degree of 9 to 16 plateau degrees, • Contains 40-60 g / kg of maltose, The test solution contains 2.0g to 3.0g of fructose per liter. The test solution contains 6.0g to 7.0g of glucose per liter. The test solution contains 2.0g to 3.0g of sucrose per liter. • Contains 500-2500 mg / L of amino acids, Here, the culture in the test solution is carried out at a temperature of 14°C to 16°C for 2 to 6 days. The culture in the aforementioned test solution is carried out after inoculating a range of 7,000,000 to 10,000,000 viable yeast cells per 1 mL of the test solution. The aforementioned offspring yeast strain.

19. The progeny yeast strain according to any one of the prior claims, wherein the mutation in the first parent yeast strain results in the substitution of a nonpolar amino acid with a charged amino acid, for example, a negatively charged amino acid.

20. The progeny yeast strain according to any one of the prior claims, wherein the mutation in the first parent yeast strain results in the substitution of the nonpolar amino acid aspartic acid (D) or glutamic acid (E).

21. The progeny yeast strain according to any one of the prior claims, wherein the mutation in the first parent yeast strain results in the substitution of the nonpolar amino acid glutamic acid (E).

22. The progeny yeast strain according to any one of the prior claims, wherein the mutation in the first parent yeast strain results in the substitution of glycine (G) for glutamic acid (E), for example, the mutation results in the substitution of G498E.

23. The offspring yeast strain according to any one of the prior claims, wherein the second parent yeast strain is of the genus Saccharomyces.

24. The offspring yeast strain according to any one of the prior claims, wherein the second parent yeast strain is of the species Saccharomyces cerevisiae.

25. The progeny yeast strain according to any one of the prior claims, wherein the second parent yeast strain is maltose intolerant, for example, the second parent yeast strain does not have the ability to completely convert maltose to ethanol.

26. The progeny yeast strain according to any one of the prior claims, wherein the second parent yeast strain is maltose intolerant, for example, the second parent yeast strain does not have the ability to grow using maltose as its sole carbon source, or its growth using maltose as its sole carbon source is significantly slower than that of other yeasts, preferably compared to Weihenstephan 34 / 70 yeast and / or Safari US5.

27. The offspring yeast strain according to any one of the prior claims, wherein the offspring yeast strain is maltose intolerant, for example, the offspring yeast strain does not have the ability to completely convert maltose to ethanol.

28. The progeny yeast strain according to any one of the prior claims, wherein the progeny yeast strain is maltose intolerant, for example, it does not have the ability to grow using maltose as its sole carbon source, or its growth using maltose as its sole carbon source is significantly slower than that of other yeasts, preferably compared to Weihenstephan 34 / 70 yeast and / or Safari US5.

29. The progeny yeast strain according to any one of the prior claims, wherein the progeny yeast strain is maltotriose intolerant, for example, the progeny yeast strain does not have the ability to completely convert maltotriose to ethanol.

30. The offspring yeast strain according to any one of the prior claims, wherein the fermentation test solution has an alcohol content of at most 1.0% ABV, for example, at most 0.8% ABV, or at most 0.6% ABV.

31. The offspring yeast strain according to any one of the prior claims, wherein the offspring yeast strain produces at most 1.0% ABV, for example, at most 0.8% ABV, or at most 0.6% ABV after being cultured in the test solution.

32. The progeny yeast strain according to any one of the prior claims, wherein the second parent yeast strain is PPU121 deposited with DSMZ under acceptance number DSM34497.

33. The offspring yeast strain according to any one of the prior claims, wherein when the offspring yeast strain is cultured in an aqueous solution containing p-coumaric acid, it does not have the ability to convert more than 25% of p-coumaric acid to 4-ethylphenol.

34. The progeny yeast strain according to any one of the prior claims, wherein the second parent strain and / or the progeny yeast strain does not have the ability to convert more than 25% of p-coumaric acid to 4-ethylphenol when cultured in an aqueous solution containing p-coumaric acid.

35. A method for producing offspring yeast strains, wherein the method is - Provide spores of a first parent yeast strain (wherein the first parent yeast strain is defined in any one of the prior claims), - Cross the aforementioned spores with spores from a second parent yeast strain. The process includes the step, wherein at least one spore of the first parent yeast strain has a different mating type than at least one spore of the second parent yeast strain. The aforementioned method.

36. The method according to claim 35, wherein the second parent yeast strain is as defined in any one of claims 23 to 26.

37. A method for producing a fermented aqueous extract, wherein the method is i) Provide an aqueous extract of malt and / or cereals. ii) To provide a progeny yeast strain according to any one of claims 1 to 34, iii) The aqueous extract provided in step i) is fermented with the yeast strain from step ii) to obtain a fermented aqueous extract. The method, including the step.

38. The method according to claim 37, wherein the aqueous extract is wort.

39. The method according to any one of claims 37 to 38, wherein the extract has an apparent extract of at most 16 plateau degrees, for example in the range of 6 to 16 plateau degrees, for example in the range of 8 to 16 plateau degrees, preferably in the range of 6 to 12 plateau degrees, for example in the range of 8 to 10 plateau degrees, for example in the range of about 9 plateau degrees.

40. A fermented aqueous extract prepared by the method described in any one of claims 37 to 39.

41. A method for manufacturing a beverage, wherein the method is a) Prepare a fermented aqueous extract according to claim 36, and b) Processing the fermented aqueous extract into a beverage. The method, including the step.

42. The processing step described above is a) Filtration, b) Carbonation c) Aging, or d) Bottling The method according to claim 41, comprising one or more of the above.

43. The method according to claims 41 to 42, further comprising the step of reducing the alcohol content of the fermented aqueous extract.

44. The method according to claim 43, wherein the step of reducing the alcohol content is rectification or debrewing.

45. A beverage prepared by the method described in any one of claims 41 to 44.

46. The beverage according to claim 45, wherein the beverage is a beverage with reduced alcohol content, such as a low-alcohol beverage.

47. The beverage according to claim 45, wherein the beverage is an alcohol-free beverage.

48. The beverage according to any one of claims 45 to 47, wherein the beverage is, for example, a beer such as a reduced-alcohol beer or, for example, an alcohol-free beer.

49. The beverage according to any one of claims 45 to 48, wherein the beverage has a propanol:isobutanol ratio of at least 6.0, for example, at least 8.0, for example, at least 10.0, for example, at least 12.

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