Method for producing fermented malt liquid

Abstract

Provided are: a method for producing a fermented malt liquid having a low ethanol concentration and a much less unpleasant odor originating from wort; and a fermented malt beverage produced from the fermented malt liquid obtained by the production method. The present invention pertains to: a method for producing a fermented malt beverage, the method comprising a step for inoculating, with yeast, a fermentation raw material liquid containing a saccharified product of malt and fermenting the same to prepare a fermented malt liquid, wherein the yeast is RAG- phenotype Kluyveromyces lactis, and the ethanol concentration per Brix 5.0 mass% of the fermented malt liquid is less than 0.05 v / v%; and a method for producing a fermented malt beverage, wherein a fermented malt liquid is produced by the method for producing a fermented liquid, and thereafter, a fermented malt beverage is produced using the obtained fermented malt liquid as a raw material.

Description

Method for producing malt fermentation liquid

[0001] The present invention relates to a method for producing a fermented liquid using malt saccharification as a fermentation raw material, which has a low ethanol concentration and a reduced wort odor, and a method for producing a beverage containing the fermented liquid produced by the method as an ingredient. This application claims priority based on Japanese Patent Application No. 2023-109981, filed in Japan on July 4, 2023, the contents of which are incorporated herein by reference.

[0002] In recent years, a wide variety of alcoholic beverages have been released on the market in response to diversifying consumer preferences. For example, there is growing demand for alcoholic beverages with a relatively low alcohol content, as they are easier to drink. On the other hand, in the case of fermented alcoholic beverages, in addition to ethanol, various aroma components are produced by yeast during fermentation, which contribute to the flavor and aroma of the beverage.

[0003] In fermented alcoholic beverages, various aroma components produced during fermentation depend on the type of yeast used in fermentation. For example, the yeast most widely used in brewing beer and wine is Saccharomyces, but in recent years, beers with flavors different from those of conventional beers have been produced by using yeasts other than Saccharomyces.

[0004] For example, a method for producing beer with a high content of isoamyl acetate (IAAT) and ethyl acetate by fermenting wort under aerobic conditions using yeasts of the genus Pichia, such as Pichia kluyveri, is known (Patent Document 1). Another method for producing a fermented beverage with an alcohol concentration of less than 1% by volume is known (Patent Document 2). Furthermore, for the production of low-alcohol beer, Saccharomycodes ludwigii, which has low ethanol productivity, has been used as a yeast for producing non-alcoholic beer (Non-Patent Document 1).

[0005] On the other hand, several yeast mutants with low ethanol production have been reported. For example, in Kluyveromyces lactis, a phenotype in which growth is impossible when respiration is inhibited (RAG) has been reported. － It has been reported that mutant strains with a RAG phenotype exhibit lower ethanol production than wild-type strains on YPD medium (Non-Patent Document 2). In addition, wild-type strains of Kluyveromyces lactis were subjected to mutation treatment, and poor growth in a medium containing antimycin A, a respiratory inhibitor, was used as an indicator to evaluate the RAG phenotype. － It has also been reported that mutant strains with a specific phenotype can be selected (Non-Patent Document 3).

[0006] International Publication No. 2014 / 202564 Japanese Patent Application Laid-Open No. 60-094077

[0007] Bellut, et al., Journal of the American Society of Brewing Chemists, 2019, DOI: 10.1080 / 03610470.2019.1569452Wesolowski-Louvel, et al., Molecular and General Genetics, 1992, vol.233(1-2), p.89-96.Wesolowski-Louvel, et al., Yeast, 1992, vol.8, p.711-719.

[0008] In the production of fermented alcoholic beverages, suppressing the degree of fermentation to keep the ethanol concentration low can also reduce the content of aroma components and can leave an unpleasant wort-derived odor due to poor fermentation. The present invention aims to provide a method for producing a malt fermentation liquor that has a low ethanol concentration and little wort odor remaining due to poor fermentation, a method for producing a fermented malt beverage using the malt fermentation liquor produced by the method, and a fermented malt beverage produced by the method.

[0009] As a result of intensive research to solve the above problems, the present inventors have found that the fermentation of a fermentation raw material liquid containing a saccharified product of malt is carried out by the RAG method. －The present inventors have found that by using the phenotypic Kluyveromyces lactis, fermentation is carried out sufficiently, but ethanol production is greatly suppressed, and a malt fermentation liquid can be produced that has a sufficiently low ethanol concentration and little unpleasant odor derived from the wort, and have completed the present invention.

[0010] The method for producing a malt fermentation liquid, the method for producing a fermented malt beverage, and the fermented malt beverage according to the present invention are as follows: [1] A method for producing a malt fermentation liquid by inoculating a fermentation raw material liquid containing a saccharified product of malt with yeast and fermenting the liquid, the method comprising the steps of: － [2] A method for producing a malt fermentation liquor according to [1], wherein the phenotype is Kluyveromyces lactis, and the malt fermentation liquor has an ethanol concentration of less than 0.05 v / v% per Brix 5.0% by mass. [3] A method for producing a malt fermentation liquor according to [1], wherein the malt fermentation liquor has a methional concentration of 3.0 ppb or less per Brix 5.0% by mass. [4] A method for producing a malt fermentation liquor according to [1] or [2], wherein the methional concentration of the malt fermentation liquor is 15% by mass or less of the methional concentration of the fermentation raw material liquid. [4] A method for producing a malt fermentation liquor according to [1], wherein the bacterial concentration of the malt fermentation liquor at the end of fermentation is 3.0 x 10 ６ [5] The method for producing a malt fermentation liquor according to any one of [1] to [3], wherein the bacterial concentration of the malt fermentation liquor at the start of fermentation is 2.0 × 10 cells / mL or more. ４[6] The method for producing a malt fermentation liquor according to any of [1] to [4], wherein the dissolved oxygen concentration in pure water is 6.0 ppm to 8.0 ppm, and the dissolved oxygen concentration is 8.0 ppm or more. [7] The method for producing a malt fermentation liquor according to any of [1] to [5], wherein the fermentation is carried out under oxygen-providing conditions, and the oxygen-providing conditions are conditions in which the time required to increase the dissolved oxygen concentration in pure water from 6.0 ppm to 8.0 ppm at 18°C ​​and atmospheric pressure is more than 0 hours and not more than 5 hours. [8] The method for producing a malt fermentation liquor according to any of [1] to [7], wherein the fermentation is carried out at 1°C or higher. [9] The method for producing a malt fermentation liquor according to any of [1] to [8], wherein the fermentation is carried out for 4 hours or more.

[10] The method for producing a malt fermentation liquor according to any of [1] to [8], wherein the fermentation is carried out for 24 hours or more.

[11] A method for producing a fermented malt beverage, comprising producing a malt fermentation liquor by the method for producing a malt fermentation liquor according to any one of [1] to

[10] above, and then using the resulting malt fermentation liquor as a raw material to produce a fermented malt beverage.

[12] A method for producing the fermented malt beverage according to

[11] above, comprising a step of diluting the malt fermentation liquor.

[13] A method for producing the fermented malt beverage according to

[11] or

[12] above, comprising producing a fermented malt beverage containing 3 to 7% by mass of original wort extract.

[14] A method for producing the fermented malt beverage according to any one of

[11] to

[13] above, wherein the fermented malt beverage is a beer-like sparkling beverage.

[15] A fermented malt beverage having an ethanol concentration per 5.0% Brix by mass of less than 0.05 v / v% and a methional concentration per 5.0% Brix by mass of 3.0 ppb or less.

[16] The fermented malt beverage according to

[15] above, which is a beer-like sparkling beverage.

[0011] In the method for producing malt fermentation liquid according to the present invention, RAG, which has a very low ethanol production capacity and can sufficiently remove unpleasant odors derived from wort, is used. － Fermentation is performed using the phenotype Kluyveromyces lactis. Therefore, the method for producing a malt fermentation liquor according to the present invention can provide a malt fermentation liquor having a low ethanol concentration and a sufficiently low unpleasant odor derived from wort. Furthermore, by using the malt fermentation liquor, a malt fermented beverage having a low ethanol concentration and a reduced wort odor can be provided.

[0012] FIG. 1 shows the results of measuring the dissolved oxygen concentration over time in pure water incubated at 18° C. and normal pressure under various oxygen-providing conditions in Reference Example 1.

[0013] In the present invention and the present specification, "X １ ~X ２ (I, X １ <X ２ is a real number that satisfies １ More than X ２ In the present invention and this specification, "ppb" and "ppm" mean "ppb by mass" and "ppm by mass", respectively.

[0014] In the present invention and the present specification, "RAG － (Resistance to Antimycin A on Glucose - The term "respiratory inhibitor-sensitive phenotype" refers to a trait of sensitivity to a respiratory inhibitor in the presence of glucose, i.e., a trait of being subject to growth inhibition in the presence of glucose and a respiratory inhibitor. The respiratory inhibitor is not particularly limited as long as it is a substance that has the effect of inhibiting any of the oxidative phosphorylation reactions in mitochondria. Examples include electron transport inhibitors such as antimycin A (CAS RN: 1397-94-0), rotenone (CAS RN: 83-79-4), and thenoyltrifluoroacetone (CAS RN: 326-91-0), and ATP synthase inhibitors such as oligomycin A (CAS RN: 579-13-5).

[0015] In the present invention and the present specification, "RAG － "Phenotype Kluyveromyces lactis" refers to a Kluyveromyces lactis strain that is sensitive to a respiratory inhibitor, i.e., a strain whose normal growth is inhibited in the presence of a respiratory inhibitor. Wild-type Kluyveromyces lactis can grow without problems in the presence of glucose even in the presence of a respiratory inhibitor, but some mutant strains can grow without problems in the presence of a respiratory inhibitor, such as RAG. － The phenotype is that Kluyveromyces lactis is a RAG. －The distinction of whether or not the phenotype is RAG can be carried out, for example, by culturing in a culture medium containing glucose as a carbon source and in a culture medium containing a respiratory inhibitor in addition to glucose. － It is phenotypically identified as Kluyveromyces lactis.

[0016] RAG used in the present invention － The genotype of the phenotype of Kluyveromyces lactis is not particularly limited as long as it is sensitive to a respiratory inhibitor in the presence of glucose. － Examples of the phenotype of Kluyveromyces lactis include a mutant of Kluyveromyces lactis that lacks a function of the RAG1 gene that encodes the glucose transporter protein RAG1. － Kluyveromyces lactis that is not a phenotype can be subjected to mutation treatment, for example, by treating it with a drug such as EMS (Ethyl Methanesulfonate) or by irradiating it with ultraviolet light, and from the resulting mutants, mutants that cannot be cultured in a culture medium containing a respiratory inhibitor but can be cultured in a culture medium not containing a respiratory inhibitor can also be selected. － The phenotype Kluyveromyces lactis can be obtained. － The phenotype of Kluyveromyces lactis can be, for example, a strain that is sensitive to a respiratory inhibitor and can be appropriately selected from strains of Kluyveromyces lactis available from strain collection institutions such as the National Biotechnology Center of the National Institute of Technology and Evaluation (NBRC) and the National Collection of Yeast Cultures (NCYC) or from distributors. － The phenotype of Kluyveromyces lactis may be one type or two or more types. － The phenotype Kluyveromyces lactis may be inoculated in combination with other yeasts.

[0017] The method for producing a malt fermentation liquid according to the present invention includes a step of inoculating a fermentation raw material liquid containing a saccharified product of malt with yeast and fermenting the liquid to prepare a malt fermentation liquid, and the yeast is selected from RAG, － The phenotype is characterized by the use of Kluyveromyces lactis. － The phenotype Kluyveromyces lactis has sufficient ability to produce aroma compounds through fermentation, but has low ability to produce ethanol through fermentation. － Fermentation with the phenotype Kluyveromyces lactis makes it possible to produce a malt fermentation liquid that contains various aroma components but has a sufficiently reduced ethanol concentration.

[0018] The method for producing a malt fermentation liquor according to the present invention makes it possible to produce a malt fermentation liquor having an ethanol concentration of less than 0.05 v / v% per 5.0% Brix by mass. The ethanol concentration per 5.0% Brix by mass of the malt fermentation liquor produced by the present invention is not particularly limited as long as it is less than 0.05 v / v%, but is preferably 0.025 v / v% or less, more preferably 0.010 v / v% or less, even more preferably 0.008 v / v% or less, and even more preferably 0.005 v / v% or less. The lower limit of the ethanol concentration per 5.0% Brix by mass of the malt fermentation liquor produced by the present invention is not particularly limited, and can be, for example, 0.001 v / v% or more, or may be 0.002 v / v% or more. The methional concentration per Brix 5.0% by mass of the malt fermentation liquor produced by the present invention is not particularly limited as long as it is 3.0 ppb or less, but is preferably 1.0 ppb or less. The lower limit of the methional concentration per Brix 5.0% by mass of the malt fermentation liquor produced by the present invention is not particularly limited, and can be, for example, 0.10 ppb or more, or may be 0.20 ppb or more.

[0019] In the present invention and this specification, the ethanol concentration of a malt fermentation liquid or a beverage can be measured by, for example, an enzymatic method in which the amount of NADH (reduced nicotinamide adenine dinucleotide) produced using ADH (alcohol dehydrogenase) and AlDH (aldehyde dehydrogenase) is measured by spectroscopy, an enzymatic method using a multifunctional biosensor BF-5 (manufactured by Oji Scientific Instruments), a distillation-density (specific gravity) method, a gas chromatographic (GC) analysis method, an oxidation method (oxidation-reduction titration method), or the like.

[0020] In the method for producing a malt fermentation liquid according to the present invention, RAG － Fermentation using phenotypic Kluyveromyces lactis not only suppresses ethanol production in malt fermentation liquor, but also suppresses the lingering unpleasant odor derived from wort. For example, one of the unpleasant odors derived from wort is methional. The method for producing a malt fermentation liquor according to the present invention can produce a malt fermentation liquor having an ethanol concentration of less than 0.05 v / v% per 5.0% Brix by mass and a methional concentration of 3.0 ppb or less per 5.0% Brix by mass. Methional is an aroma component that causes wort odor and decreases as fermentation progresses. A methional concentration of 3.0 ppb or less per 5.0% Brix by mass means that fermentation has progressed and the wort odor has been sufficiently reduced. In other words, the present invention can produce a malt fermentation liquor in which both the ethanol concentration and the methional concentration are sufficiently reduced.

[0021] In the present invention and the present specification, the methional concentration of a malt fermentation liquor or beverage can be determined from the peak area of ​​a chromatograph obtained by, for example, GC (gas chromatography) analysis, GC-MS analysis (gas chromatography-mass spectrometry), or solid-phase microextraction (SPME)-GC-MS / MS analysis (gas chromatography-triple quadrupole mass spectrometry). The method for quantification from the peak area is not particularly limited, and examples include the area percentage method, the internal standard method, the standard addition method, and the absolute calibration curve method. Specifically, the methional concentration can be determined by the method described in the Examples below.

[0022] In the method for producing a malt fermentation liquid according to the present invention, RAG － Fermentation using the phenotype Kluyveromyces lactis can also reduce the methional concentration of the malt fermentation liquor sufficiently below the methional concentration of the fermentation raw material liquid. In the present invention, the methional concentration of the malt fermentation liquor can be reduced to preferably 15% by mass or less, more preferably 10% by mass or less, and even more preferably 5% by mass or less of the methional concentration of the fermentation raw material liquid. In other words, the method for producing a malt fermentation liquor according to the present invention can provide a highly palatable malt fermentation liquor that is low in ethanol but also has a low methional concentration and a suppressed wort odor.

[0023] In the present invention, RAG － The fermentation raw material liquid to be inoculated with the phenotype Kluyveromyces lactis and fermented contains a saccharified product of malt, and RAG － There are no particular limitations on the source of carbon or nitrogen as long as it contains a carbon source or nitrogen source that can be assimilated by the phenotype Kluyveromyces lactis. For example, it may consist solely of a saccharified product of malt, or it may contain a carbon source or nitrogen source other than a saccharified product of malt.

[0024] In the present invention and this specification, "malt saccharification product" refers to a product obtained by saccharifying sugars in malt with various enzymes. The enzymes used in the saccharification process may be enzymes that are naturally contained in malt, or may be enzymes derived from sources other than malt. For example, by adding water to malt or its pulverized product and maintaining the mixture at 50 to 60°C, a saccharification product can be obtained using malt-derived amylase. In this process, a saccharifying enzyme derived from a source other than malt may be further added, or an enzyme other than a saccharifying enzyme, such as a protease, may be used in combination.

[0025] In the present invention, for example, wort or malt extract can be used as the saccharified product of malt. Malt extract is a concentrated wort, and its main component is malt sugar. Malt extract is used as a beverage by directly mixing it with carbonated water, or as a raw material for food and beverage products. Wort can be concentrated by a conventional method. Concentration under reduced pressure is preferred because it minimizes the impact on the components in the wort, but concentration by heating to remove the solvent is also possible.

[0026] Wort is prepared by preparing a mixture containing malt and raw water, heating the mixture, and saccharifying the starch in the malt. The malt is preferably used as malt pulverized product obtained by pulverizing the malt. The malt pulverization can be carried out by a conventional method. The malt pulverized product may be subjected to conventional treatments before or after the pulverization. Examples of such treatments include root removal.

[0027] The mixture containing malt and raw material water may contain a fermentation raw material other than malt. The fermentation raw material may be a grain raw material or a carbohydrate raw material. Examples of grain raw materials other than malt include wheat and barley, rice, corn, soybeans, and other pulses, and potatoes. The grain raw material can also be used as a ground grain product, grain syrup, grain extract, etc. Ground grain products may be those that have been subjected to conventional treatments before or after grinding, such as corn starch and corn grits. Examples of such conventional treatments include removal of proteins, husks, and germs, and dehydration. Examples of carbohydrate raw materials include sugars such as liquid sugar.

[0028] The wort may be used as is as a fermentation raw material liquid, but is preferably subjected to a boiling treatment before inoculating with yeast. This boiling treatment enables sterilization and safer fermentation. When performing the boiling treatment, it is preferable to remove insoluble solids by filtration or the like before the boiling treatment. The filtration treatment is not particularly limited, and can be appropriately selected from solid-liquid separation treatments commonly used in the production process of foods and beverages, such as diatomaceous earth filtration and filter filtration. Alternatively, instead of the filtrate of this sugar solution, a malt extract to which warm water has been added may be used and boiled. The boiling method and conditions can be determined appropriately.

[0029] By adding herbs and the like as appropriate before or during the boiling treatment, a malt fermentation liquor having a desired flavor can be produced. In particular, it is preferable to add hops before or during the boiling treatment. By performing the boiling treatment in the presence of hops, the flavor and aroma components of the hops can be efficiently extracted. The amount of hops to be added, the manner of addition (e.g., adding them in several batches), and the boiling conditions can be determined as appropriate.

[0030] It is preferable to remove dregs such as proteins generated by precipitation from the boiled wort before inoculating the yeast. The dregs can be removed by any solid-liquid separation process, but typically a tank called a whirlpool is used to remove the sediment. The temperature of the boiled wort at this time should be 15°C or higher, and is generally about 50 to 80°C. The clear wort (filtrate) after dregs removal is cooled to an appropriate fermentation temperature using a plate cooler or the like. This wort after dregs removal becomes the fermentation raw material liquid.

[0031] In the present invention, RAG － The Brix of the fermentation raw material liquid to be inoculated with the phenotype Kluyveromyces lactis is preferably 4% to 20%, more preferably 8% to 18%, and even more preferably 12% to 16%. － The pH of the fermentation raw material liquid to be inoculated with the phenotype Kluyveromyces lactis is preferably 3.0 to 6.0, more preferably 3.5 to 5.5.

[0032] In the present invention and the present specification, the Brix value of the fermentation raw material liquid can be measured by a conventional method using a measuring device such as a refractometer (RX-5000α, manufactured by Atago Co., Ltd.).

[0033] Add RAG to the cooled fermentation liquid. － The phenotype Kluyveromyces lactis is inoculated and fermentation is carried out. The cooled fermentation raw material liquid may be subjected to fermentation as it is, or may be subjected to fermentation after being adjusted to a desired extract concentration. － The amount of phenotypic Kluyveromyces lactis is not particularly limited. －The amount of phenotypic Kluyveromyces lactis was determined when the bacterial concentration in the malt fermentation broth at the start of fermentation was 2.0 × 10 ４ The amount is preferably 2.0 x 10 cells / mL or more. ４ ~2.0 x 10 ７ It is more preferable that the amount be 1000 cells / mL.

[0034] RAG － If the number of bacteria of the phenotype Kluyveromyces lactis reached at the end of fermentation is too high, the amount of ethanol produced may be too high. On the other hand, if the number of bacteria reached at the end of fermentation is too low, the fermentation is likely to be insufficient, and it may be difficult to sufficiently reduce unpleasant odors derived from the wort, such as methional. In order to easily obtain a malt fermentation liquid with a desired low ethanol concentration and low unpleasant odor, the bacterial concentration of the malt fermentation liquid at the end of fermentation is set to 3.0 × 10 ６ cells / mL or more, and preferably 3.0 x 10 ６ ~1.4 × 10 ８ It is more preferable that the concentration is 10 ...

[0035] In the method for producing a malt fermentation liquid according to the present invention, － The conditions for fermentation performed by inoculating phenotypic Kluyveromyces lactis are not particularly limited. The fermentation temperature may be 1°C or higher, and for example, is preferably 1 to 35°C, which is a temperature range in which yeast can generally survive, more preferably 1 to 28°C, even more preferably 1 to 25°C, and even more preferably 1 to 20°C. Furthermore, fermentation may be carried out at a constant temperature from the start to the end of fermentation, or the fermentation temperature may be varied as appropriate. The fermentation time can be determined appropriately taking into consideration the fermentation temperature, the desired product quality, and the like, but is preferably 4 hours or more, as this is expected to sufficiently reduce unpleasant odors derived from wort, such as methional. In particular, the fermentation time is preferably 4 to 168 hours, more preferably 4 to 96 hours, and even more preferably 4 to 72 hours. Furthermore, the fermentation time may be adjusted to 100% by weight of the fermented liquor. － The time when the phenotype of Kluyveromyces lactis reaches a target bacterial concentration can also be determined as the end of fermentation.

[0036] RAG －In fermentation with Kluyveromyces lactis, RAG － The phenotype of Kluyveromyces lactis may be in a growth phase, or may be fermented in a state where it is barely growing. － Although the phenotype Kluyveromyces lactis grows only slightly, there is a sufficient amount of RAG in the fermentation raw material liquid. － By inoculating the phenotype Kluyveromyces lactis, a malt fermentation liquid with a desired low ethanol concentration and low unpleasant odor can be obtained. On the other hand, even if the amount of inoculation into the fermentation raw material liquid is small, the RAG － By fermenting in an environment in which the phenotype Kluyveromyces lactis can grow, the desired malt fermentation liquid with a low ethanol concentration and a low unpleasant odor can be obtained.

[0037] RAG － In order to sufficiently grow the phenotypic Kluyveromyces lactis, fermentation in an anaerobic environment is preferable to a completely anaerobic environment. Here, "under an anaerobic environment" refers to a condition where a certain oxygen supply condition is maintained, but the amount of oxygen supplied is less than the amount of oxygen consumed by the growth of the bacteria, resulting in a dissolved oxygen concentration of zero in the liquid. Furthermore, "under a completely anaerobic environment" refers to an environment where no new oxygen is supplied, i.e., "under conditions without oxygen supply." A completely anaerobic environment can be achieved by supplying oxygen-free gases such as nitrogen gas or carbon dioxide gas to the fermentation tank.

[0038] For example, if the number of bacteria inoculated into the fermentation raw material liquid is 3 × 10 ６ In the case of less than 3.0 × 10 cells / mL, fermentation is carried out while maintaining an anaerobic environment under constant oxygen supply conditions, and the cell density is reduced to 3.0 × 10 cells / mL at the end of fermentation. ６ cells / mL or more, preferably 3.0 x 10 ６ ~1.4 × 10 ８ RAG up to cells / mL － The phenotype Kluyveromyces lactis can be grown, and the desired malt fermentation liquid with a low ethanol concentration and a low unpleasant odor can be obtained. ６When the RAG concentration is 1000 cells / mL or more, fermentation may be carried out in an anaerobic environment. Even when fermentation is carried out in a completely anaerobic environment, the desired low ethanol concentration and low unpleasant odor malt fermentation liquid can be obtained by adjusting the fermentation time. － The inoculation number of phenotype Kluyveromyces lactis was 3 × 10 ６ By fermenting the malt fermentation liquid at a temperature of 1 to 35°C, preferably 1 to 28°C, more preferably 8 to 28°C, and even more preferably 12 to 24°C for 24 hours or more, it is possible to obtain the desired low ethanol concentration and low unpleasant odor malt fermentation liquid even when fermented in a completely anaerobic environment.

[0039] RAG － When phenotypic Kluyveromyces lactis is fermented in an anaerobic environment, it is preferable to carry out the fermentation under oxygen-adding conditions such that the time required to increase the dissolved oxygen concentration in pure water from 6 ppm to 8 ppm at 18°C ​​and atmospheric pressure is more than 0 hours and not more than 5 hours, for example, 15 minutes to 5 hours, so that the oxygen concentration can be adjusted within an appropriate range. Fermentation under such oxygen-adding conditions can be carried out by fermenting the fermentation liquor while stirring it in a fermentation tank filled with an oxygen-containing gas such as air, rather than by directly mixing oxygen into the fermentation liquor. Specifically, when fermentation is carried out in a fermentation tank, the oxygen-adding conditions can be achieved by stirring the fermentation liquor while supplying air to the empty space in the fermentation tank, or by pressurizing the fermentation tank at 0.026 MPa and supplying air at 10 NL / h per 1 kL of fermentation liquor. Furthermore, when fermentation is carried out in a small-volume fermentation vessel at the laboratory level, the above-mentioned oxygen-adding conditions can be achieved, for example, by stirring the fermentation liquid with a pump, by placing 400 mL of wort in a 500 mL Erlenmeyer flask and stirring it at 60 rpm using a shaking culture device, or by placing 400 mL of wort in a 500 mL jar fermenter and rotating the stirring blades at 50 rpm without actively supplying air, with a 0.2 μm filter attached to the exhaust port. Alternatively, the above-mentioned oxygen-adding conditions can be achieved by leaving the fermentation liquid stationary in a fermentation vessel with a large gas-liquid contact surface.

[0040] The malt fermentation liquor obtained in the present invention can be used as a raw material for foods and beverages as it is. Foods and beverages produced using the malt fermentation liquor as a raw material are not particularly limited. As foods and beverages produced using the malt fermentation liquor obtained in the present invention as a raw material, beverages are preferred, malt fermented beverages are more preferred, and malt-fermented beer-like sparkling beverages are even more preferred.

[0041] In the present invention and this specification, the term "fermented malt beverage" refers to a beverage produced by using malt or a processed malt product as a raw material through a fermentation process using yeast. The fermented malt beverage may be an alcoholic beverage, or may be a so-called non-alcoholic beverage or low-alcohol beverage having an ethanol concentration of less than 1.0 v / v%. The fermented malt beverage produced using the malt fermentation liquid obtained in the present invention as a raw material preferably has an ethanol concentration of less than 0.05 v / v%, preferably 0.025 v / v% or less, more preferably 0.01 v / v% or less, even more preferably 0.008 v / v% or less, and even more preferably 0.005 v / v% or less.

[0042] Examples of fermented malt beverages include beer-like sparkling beverages. In the present invention and the present specification, the term "beer-like sparkling beverage" refers to a sparkling beverage having a flavor, taste, and texture equivalent to or similar to that of beer, and having a beer-like flavor (a taste reminiscent of beer in terms of aroma). Specific examples of malt-fermented beer-like sparkling beverages include beer and happoshu (low-malt beer) made from malt. Other examples include liqueurs obtained by blending a malt fermentation liquid with an ethanol-containing distillate. The ethanol-containing distillate is a solution containing ethanol obtained by distillation, and may be, for example, raw alcohol (ethanol), such as whiskey, brandy, vodka, rum, tequila, gin, shochu, and other distilled spirits.

[0043] For example, the obtained malt fermentation liquor can be subjected to solid-liquid separation to remove yeast and the like, thereby obtaining a fermented malt beverage. The solid-liquid separation may be performed by any method capable of removing yeast and the like, such as centrifugation, diatomaceous earth filtration, or filter filtration using a filter with an average pore size of approximately 0.4 to 0.5 μm, or a combination of these methods. Hops, hop extract, flavorings, and the like may be added to the obtained malt fermentation liquor to adjust the flavor of the fermented malt beverage. The flavor adjustment may be performed before or after the solid-liquid separation. The malt fermentation liquor after the solid-liquid separation may also be stored and aged under low-temperature conditions of approximately 0°C. To produce a fermented malt beverage, further treatments such as pH adjustment and heat treatment may be performed according to conventional methods.

[0044] The obtained malt fermentation liquor can be appropriately diluted to produce a malt fermented beverage having an extract concentration and the like within a desired range. The solution used for dilution is not particularly limited, but examples include water or carbonated water. The dilution ratio is not particularly limited, and can be, for example, 2 to 10 times, more preferably 2 to 6 times, and even more preferably 2 to 4 times. Furthermore, a malt fermented beverage can also be produced by diluting the malt fermentation liquor so that the original wort extract concentration is within a desired range, for example, 3 to 7% by mass. The dilution step may be performed after the fermentation step is completed, or after the yeast and the like are removed, or on the malt fermentation liquor after maturation.

[0045] The original wort extract concentration of malt fermentation liquid or malt fermented beverage can be measured according to the analytical method published by the Brewing Society of Japan ("8.5 Extract-Related Measurement Methods" in "BCOJ Beer Analysis Methods (2013 Revised Edition) (edited by the International Technical Committee (Analysis Committee) of the Brewers Association of Japan)." Specifically, it can be measured from the alcohol concentration and true extract concentration.

[0046] The true extract concentration of malt fermentation liquid or malt fermented beverage can be measured according to the method specified in "8.4.3 Alcolyzer Method" of "BCOJ Beer Analysis Methods (2013 revised edition) (edited by the International Technical Committee (Analysis Committee) of the Brewers Association of Japan)."

[0047] The ethanol concentration of the fermented malt beverage obtained using the malt fermentation liquor obtained in the present invention as a raw material is not particularly limited. By using the malt fermentation liquor obtained in the present invention as a raw material, a fermented malt beverage with a low ethanol concentration and reduced wort odor can be produced. As the fermented malt beverage, for example, a fermented malt beverage having an ethanol concentration of 0.05 v / v% or less per 5.0 mass% Brix is ​​preferred, a fermented malt-like sparkling beverage having an ethanol concentration of 0.05 v / v% or less and a methional concentration of 3.0 ppb or less per 5.0 mass% Brix is ​​more preferred, a fermented malt-like sparkling beverage having an ethanol concentration of 0.01 v / v% or less and a methional concentration of 3.0 ppb or less per 5.0 mass% Brix is ​​even more preferred, and a fermented malt-like sparkling beverage having an ethanol concentration of 0.01 v / v% or less and a methional concentration of 1.0 ppb or less per 5.0 mass% Brix is ​​even more preferred.

[0048] The present invention will now be described in more detail with reference to examples and reference examples, but the present invention is not limited to the following examples.

[0049] <Measurement of Ethanol Concentration> In the following experiments, unless otherwise specified, the ethanol concentration in the malt fermentation liquid or beverage was measured by the following method. The sample was diluted with distilled water to an ethanol concentration of 0.5 to 2.0 v / v%, and the solution was measured using a multifunctional biosensor (BF-5, manufactured by Oji Instruments). The value obtained by multiplying the measured value by the dilution factor was taken as the ethanol concentration (v / v%) in the sample.

[0050] The multifunctional biosensor detects hydrogen peroxide (H O ... ２ O ２ This is a device that electrochemically detects methanol and ethanol, and can measure the total concentration of methanol and ethanol. Since yeast does not biosynthesize methanol, malt fermentation broth contains only trace amounts of methanol that are within the margin of error. Therefore, the measurement value obtained by this multifunctional biosensor is a measurement of the amount of ethanol in the malt fermentation broth sample.

[0051] For samples with an ethanol concentration of less than 0.5 v / v%, this multifunctional biosensor was used to measure the ethanol concentration of a solution diluted with the sample to an alcohol concentration of 0.01 to 0.04 v / v%, using a highly sensitive measurement method with a sodium azide-free buffer solution (SL04-0015, manufactured by Oji Instruments Co., Ltd.).

[0052] <Analysis of Methional> In the following experiments, unless otherwise specified, the methional concentration in the fermentation raw material liquid, malt fermentation liquid, or beverage was quantified by GC-MS analysis. The MRM transitions used for the analysis are shown in Table 1.

[0053]

[0054] Specifically, measurements were performed using solid-phase microextraction (SPME)-GC-MS / MS as follows. 100 μL of o-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine.HCl (PFBOA), prepared at 6 g / L, was added to a vial containing 10 mL of hexane wash water. This was used as a derivatization reagent. A SPME fiber (PDMS / DVB: 65 μm, 1 cm, 57327-U 24 gauge StableFlex, manufactured by SPELCO) was exposed to the headspace of the vial containing the PFBOA solution at 50°C for 10 minutes. Next, the SPME fiber with PFBOA trapped was exposed to the headspace of a vial containing 10 g of sample and 50 μL of 5 mg / L methional-d3 dissolved in ethanol at 50°C for 60 minutes. The derivatized methional was analyzed under the following GC-MS / MS conditions.

[0055] <GC-MS / MS Conditions> Apparatus: GC-MS / MS (7890A / 7000C, manufactured by Agilent) equipped with a multifunction autosampler MPS2 system (manufactured by Gerstel) Column: HP-5MS UI 30 m x 0.25 mm x 0.25 μm (manufactured by Agilent) Carrier gas: Helium Flow rate: 1 mL / min Heating conditions: 80°C (5 min) → 15°C / min → 150°C → 5°C / min → 220°C → 50°C / min → 300°C (5 min) Injection method: Splitless Purge flow: 50 mL / min for 1 min Injection port temperature: 250°C Pressure: 9.3825 psi Transfer line: 320°C Ionization: Electron ionization method (70 eV) Ion source temperature: 250°C Quadrupole temperature: 150°C He quench gas flow rate: 2.25 mL / min N collision gas flow rate: 1.5 mL / min MRM transitions: (see Table 1)

[0056] <Brix Measurement> In the following experiments, unless otherwise specified, the Brix of the fermentation raw material liquid, malt fermentation liquid, or beverage was measured using a digital refractometer (RX-5000α, manufactured by Atago Co., Ltd.).

[0057] <pH Measurement> In the following experiments, unless otherwise specified, the pH of the fermentation raw material liquid, malt fermentation liquid, or beverage was measured using a pH meter (LAQUAtwin-pH-33, manufactured by Horiba).

[0058] <Preparation of Worts A and D> Worts A and D were produced using a 200 L-scale brewing facility. First, 40 kg of ground malt and starchy auxiliary materials as fermentation raw materials and 160 L of raw material water were charged into a brewing tank. Next, the mixture in the brewing tank was heated and saccharified according to a conventional method, and the resulting saccharified liquid was filtered. Hops were added to the resulting filtrate, which was then boiled. The boiled filtrate was transferred to a settling tank to separate and remove precipitates, after which the pH was adjusted with phosphoric acid and the mixture was cooled to obtain worts A and D. The analytical values ​​of the resulting worts A and D were as shown in Table 2.

[0059]

[0060] <Preparation of Worts B and C> Commercially available malt extracts shown in Table 3 were diluted with water and mixed to prepare wort B or wort C having the Brix and pH shown in Table 3, respectively.

[0061]

[0062] Reference Example 1 Pure water was incubated at 18° C. and atmospheric pressure under the following oxygen supply conditions, and the dissolved oxygen concentration was measured.

[0063] Oxygenation condition 1: 400 mL of pure water was placed in a 500 mL Erlenmeyer flask and stirred at 60 rpm in a shaking incubator. Oxygenation condition 2: 400 mL of pure water was placed in a 500 mL jar fermenter, and with a 0.2 μm filter attached to the exhaust port, no active air supply was provided, and the stirring blades were rotated at 50 rpm to stir. Oxygenation condition 3: 100 L of pure water was placed in a 200 L fermentation tank, pressurized to 0.026 MPa, and air was supplied at 0.67 NL / min per 100 L of pure water. Oxygenation condition 4: 400 mL of pure water was placed in a 500 mL jar fermenter, and with a 0.2 μm filter attached to the exhaust port, nitrogen gas was blown in, and the stirring blades were rotated at 50 rpm to stir (fully anaerobic conditions).

[0064] The results of measuring the dissolved oxygen concentration over time under each condition are shown in Figure 1. As a result, under oxygen-adding conditions 1 to 3, the time required to increase the dissolved oxygen concentration of pure water from 6.0 ppm to 8.0 ppm at 18°C ​​and normal pressure was more than 0 hours and not more than 5 hours. On the other hand, under oxygen-adding condition 4, no oxygen was supplied, so the dissolved oxygen concentration of the pure water was maintained at 0 ppm.

[0065] Example 1 Wort was fermented in a flask using various yeasts, and the bacterial concentration, ethanol concentration, and methional concentration of the resulting fermented liquid were examined.

[0066] <Yeast> Kluyveromyces lactis strain NBRC648, Kluyveromyces lactis strain NBRC1267, Kluyveromyces lactis strain NBRC1090TS, Lipomyces starkeyi strain NBRC10381TS, Lipomyces lipofer strain NBRC1288TS, Yarrowia lipolytica strain NBRC717, Rhodotorula gluntis strain NBRC1125TS, Saccharomycodes ludwigii strain NBRC798TS, and Torulaspora delbrueckii strain NBRC955TS were used. Strains with "TS" at the end of their names are type strains. Of the three strains of Kluyveromyces lactis used, Kluyveromyces lactis NBRC648 is a RAG strain. － The phenotype of Kluyveromyces lactis NBRC1267 strain and Kluyveromyces lactis NBRC1090TS strain is RAG. － It has no phenotype and can grow even in the presence of respiratory inhibitors.

[0067] <400 mL Scale Test> Wort A was used as the fermentation raw material liquid. First, each yeast was pre-cultured overnight in wort diluted with water to a Brix of 5% by mass. After washing with water once, 400 mL of wort (Brix 12.0%) was placed in a 500 mL Erlenmeyer flask and the initial cell count of the yeast was 1.0 x 10 per mL of wort. ６ The yeast-inoculated wort was then shake-cultured in an incubator at 60 rpm, 18°C, and atmospheric pressure for 3 days, and fermented. The bacterial concentration, ethanol concentration, and methional concentration of the resulting fermentation broth were then quantified.

[0068] When 400 mL of pure water was placed in a 500 mL Erlenmeyer flask and incubated at 18°C ​​and atmospheric pressure in an incubator with stirring at 60 rpm, it took 135 minutes for the dissolved oxygen concentration of the pure water to increase from 6.0 ppm to 8.0 ppm.

[0069] The quantitative results of the bacterial concentration, ethanol concentration, and methional concentration of the fermentation broth are shown in Table 4. In the table, "KL" represents Kluyveromyces lactis, "LS" represents Lipomyces starchii, "LL" represents Lipomyces lipofus, "YL" represents Yarrowia lipolytica, "RG" represents Rhodotorula gluntis, "SL" represents Saccharomycodes ludwigii, and "TB" represents Torulaspora delbrueckii. Furthermore, "achieved bacterial count (cells / mL)" indicates the bacterial concentration in the fermentation broth at the end of fermentation, "ethanol (% ABV)" indicates the ethanol concentration (v / v%) in the fermentation broth at the end of fermentation, "ethanol / Brix 5% equivalent (% ABV)" indicates the ethanol concentration (v / v%) per Brix 5.0 mass% in the fermentation broth at the end of fermentation, "methional (ppb)" indicates the methional concentration (ppb) in the fermentation broth at the end of fermentation, and "methional / Brix 5% equivalent (ppb)" indicates the methional concentration (ppb) per Brix 5.0 mass% in the fermentation broth at the end of fermentation. However, in the "No inoculation" column, "Methional concentration (ppb)" indicates the methional concentration (ppb) of the fermentation raw material liquid (wort before inoculation), and "Methional / Brix 5% equivalent (ppb)" indicates the methional concentration (ppb) per Brix 5.0% by mass of the fermentation raw material liquid. Also, "Methional reduction rate (%)" indicates the reduction rate of the methional concentration from the fermentation raw material liquid (100% - [methional concentration (ppb) per Brix 5.0% by mass of the fermentation liquid at the end of fermentation] / [methional concentration (ppb) per Brix 5.0% by mass of the fermentation raw material liquid] × 100%).

[0070]

[0071] As shown in Table 4, all of the yeasts produced fermentation broths with low ethanol concentrations, similar to those of Saccharomyces ludwigii, which is commercially used in the production of non-alcoholic beer. However, the ethanol concentrations per Brix 5.0% were particularly low, at less than 0.05 v / v%, for Kluyveromyces lactis NBRC648, Lipomyces starkeyi NBRC10381TS, Lipomyces lipofus NBRC1288TS, Yarrowia lipolytica NBRC717, and Rhodotorula gluntis NBRC1125TS. Of these, the fermentation broths fermented with Kluyveromyces lactis NBRC648 and Lipomyces lipofus NBRC1288TS showed a methional reduction rate of 80% or more, indicating that fermentation was carried out satisfactorily.

[0072] The fermented liquids fermented with the three strains of Kluyveromyces lactis all had a methional reduction rate of 90% or more, indicating that fermentation was sufficient. However, the ethanol concentration (v / v%) per Brix 5.0 mass% was reduced to 0.05 v / v% or less by Rag. － The only phenotype was Kluyveromyces lactis strain NBRC648.

[0073] <Sensory Evaluation> Sensory evaluation was performed by five experienced panelists using a 2.56-fold diluted solution of each fermentation liquid (fermented malt beer-like sparkling beverage) as the evaluation sample using the semantic differential method (SD) on a scale of 1 to 7 points (Frey, The SAGE Encyclopedia of Educational Research, Measurement, and Evaluation, "Semantic Differential Scaling", 2018, pp. 1504-1507). The evaluation items were (a) the intensity of the wort odor and (b) the palatability. (a) was evaluated by aroma, and (b) by taste. Each evaluation sample was 20 mL, and the order of evaluation was randomized so that it varied depending on the panelist.

[0074] For (a), evaluation was performed using water as a standard with a score of 1 and wort A with a score of 7. For (b), wort A was used as a control (score of 3) and the evaluation was performed.

[0075]

[0076] The results of the sensory evaluation are shown in Table 5. The diluted fermentation liquids of Kluyveromyces lactis NBRC648 strain, Kluyveromyces lactis NBRC1267 strain, Kluyveromyces lactis NBRC1090TS strain, Lipomyces lipofus NBRC1288TS strain, and Rhodotorula gluntis NBRC1125TS strain, which had low methional concentrations (ppb) per 5.0 mass% Brix of the fermentation raw material liquid, had a weak wort odor and were highly rated for palatability. In particular, the diluted solution of the fermented liquid fermented with Kluyveromyces lactis NBRC648 strain had a high palatability rating of 5 or more, similar to the diluted solution of the fermented liquid fermented with Saccharomycodes ludwigii, and had an extremely low ethanol concentration of 0.004 v / v% per Brix of 5.0 mass%, making it a mild, refreshing, and easy-to-drink beer-like sparkling beverage.

[0077] Example 2: Various RAGs － Wort was fermented in a flask using the phenotype Kluyveromyces lactis, and the bacterial concentration, ethanol concentration, and methional concentration of the resulting fermented broth were examined.

[0078] <Yeast> RAG － As the phenotype Kluyveromyces lactis, strains shown in Table 6, which were bred from Kluyveromyces lactis strain NBRC648 and / or NBRC1267, were used. In the table, "KL" is an abbreviation for "Kluyveromyces lactis."

[0079] <Obtaining KL No. 86> 90 mL of wort was placed in a 100 mL Erlenmeyer flask, and 2 × 10 NBRC648 strain (pre-cultured cells) was added. ６ The resulting cells were inoculated at 1000 cells / mL and cultured for 2 days at 18°C ​​using a rotary centrifuge at 60 rpm, and this was repeated five times. The resulting cells were smeared on a YM agar medium, placed in a sealed container together with Anaeropack Microaerobic (manufactured by Mitsubishi Gas Chemical Company, Inc.), and cultured at 25°C for 5 days. Colonies that grew rapidly were selected from the colonies that appeared on the agar medium.

[0080] <Obtaining KL P13 and KL P15> Add 1x10 KL NBRC648 to pure water. ８ The bacteria were suspended at 100 μL / mL to obtain a suspension. 100 μL of the suspension was inoculated onto a parafluorophenylalanine-containing agar medium (0.67% Yeast Nitrogen Base with or without Amino Acids, 2% Glucose, 1.0 mg / mL parafluorophenylalanine, 2% agar) and cultured at 25°C for 4 days. From the colonies that formed on the agar medium, strains that produced large amounts of an aroma component (β-phenethyl alcohol) were selected.

[0081] <Acquisition of KL 648x1267-2C, KL 648x1267-4A, and KL 648x1267-5B> NBRC648 strain and RAG － Among the spores obtained by mating the phenotype-free KL NBRC1267 strain, RAG － Strains with the phenotype were selected.

[0082] <Obtaining KL 1267-R6 and KL 1267-R33> The KL NBRC1267 strain was treated with 0.3% ethyl methanesulfonate at 30°C for 60 minutes to achieve a viability of 38%. The treated cells were inoculated onto YPD agar medium to produce approximately 100 colonies, and replica-plated onto antimycin A-containing agar medium (0.5% yeast extract, 1% peptone, 5% glucose, 5 μM antimycin A, 2% agar). Strains that could grow on YPD agar medium but could not grow on antimycin A-containing agar medium were selected.

[0083]

[0084] <400 mL scale test> In the same manner as in Example 1, the pre-cultured yeast was cultured at an initial cell count of 1.0 × 10 per mL of wort.６ The wort was inoculated with the yeast so that the number of bacteria reached 100, and the yeast was fermented by shaking at 18°C ​​under normal pressure for 3 days. The bacterial concentration (achieved bacterial count), ethanol concentration, and methional concentration of the resulting fermented broth were quantified. The results are shown in Table 7.

[0085]

[0086] The fermentation broths obtained from all the breeding strains had an ethanol concentration of less than 0.05 v / v% per Brix 5.0% by mass, and the methional concentration was reduced by 85% by mass or more compared to wort A. Furthermore, the methional concentration per Brix 5.0% by mass was 3 ppb or less. － It was confirmed that if Kluyveromyces lactis had the phenotype, a malt fermentation liquid with a low alcohol concentration and a low methional concentration could be prepared.

[0087] [Example 3] Worts with different compositions and Brix were used as fermentation raw materials and RAG － Fermentation was carried out using the phenotype Kluyveromyces lactis, and the bacterial concentration, ethanol concentration, and methional concentration of the resulting fermentation broth were examined. － As the phenotype Kluyveromyces lactis, Kluyveromyces lactis NBRC648 strain was used.

[0088] (1) The same procedure as in Example 1 was repeated except that wort A was replaced with the wort of Brix shown in Table 8, and the pre-cultured yeast was brewed to an initial cell count of 1.0 x 10 per mL of wort. ６ The wort was inoculated with the yeast so that the number of bacteria reached 100, and the yeast was fermented by shaking at 18°C ​​under normal pressure for 3 days. The bacterial concentration (achieved bacterial count), ethanol concentration, and methional concentration of the resulting fermented broth were quantified. The results are shown in Table 8.

[0089]

[0090] Whether wort B or wort C was used, as with wort A, the ethanol concentration per 5.0% Brix by mass was less than 0.05 v / v%, and the methional concentration was reduced by 85% by mass or more compared to the wort before yeast inoculation.Furthermore, a malt fermentation liquid with a methional concentration of 3 ppb or less per 5.0% Brix by mass was prepared.

[0091] (2) In place of wort A, wort B was used, which was adjusted to the Brix shown in Table 9. The same procedure as in (1) was repeated to add pre-cultured yeast to the wort so that the yeast cell count was 1.0 x 10 per mL of wort. ６ The mixture was inoculated so that the number of cells reached 100, and the mixture was fermented by shaking at 18°C ​​under normal pressure for 3 days. The bacterial concentration (achieved bacterial count), ethanol concentration, and methional concentration of the resulting fermentation broth were quantified. The results are shown in Table 9.

[0092]

[0093] Regardless of the Brix value of the fermentation raw material liquid, the resulting malt fermentation liquid had an ethanol concentration of less than 0.05 v / v% per Brix 5.0% by mass, and the methional concentration was reduced by 85% by mass or more compared to the wort before yeast inoculation.Furthermore, the methional concentration per Brix 5.0% by mass was 3 ppb or less.

[0094] [Example 4] RAG to be inoculated into fermentation raw material liquid － The number of phenotype Kluyveromyces lactis bacteria and the number of bacteria reached were varied, and the bacterial concentration, ethanol concentration, and methional concentration of the resulting fermentation broth were examined. － The Kluyveromyces lactis NBRC648 strain was used as the phenotype Kluyveromyces lactis, and wort A was used as the fermentation raw material liquid.

[0095] The pre-cultured yeast was cultured in the same manner as in Example 1 except that the culture time, culture scale, and initial cell count were as shown in Table 10, so that the initial cell count was 1.0 × 10 per mL of wort. ６The wort in a conical flask was inoculated with the fermented yeast so that the total number of bacteria reached 100. The inoculated yeast was then cultured in an incubator at 60 rpm under normal pressure at 18°C ​​with shaking for fermentation. The bacterial concentration (achieved bacterial count), ethanol concentration, and methional concentration of the resulting fermented broth were quantified. The results are shown in Table 10.

[0096] In the table, "160 / 200 mL" and "160 / 500 mL" in the "Cultivation Scale" column indicate that 160 mL of wort was placed in a 200 mL Erlenmeyer flask or a 500 mL Erlenmeyer flask, respectively, and the wort was inoculated with yeast. When 160 mL of pure water was placed in a 500 mL Erlenmeyer flask and incubated at 18°C ​​and atmospheric pressure in an incubator with stirring at 60 rpm, it took 1 hour for the dissolved oxygen concentration of the pure water to increase from 6.0 ppm to 8.0 ppm. When 160 mL of pure water was placed in a 200 mL Erlenmeyer flask and incubated at 18°C ​​and atmospheric pressure in an incubator with stirring at 60 rpm, it took 160 minutes for the dissolved oxygen concentration of the pure water to increase from 6.0 ppm to 8.0 ppm.

[0097]

[0098] In Table 10, the column with a culture time of 0 days shows the measurement results of the fermentation raw material liquid (wort) before inoculation with yeast. As shown in Table 10, the number of bacteria reached was 3.0 x 10 ６ In the fermentation liquid with a cell count of less than 1.4 × 10 cells / mL, the reduction rate of methional was 60% or less, and the fermentation was insufficient. ８ In the fermentation liquid with a cell count of more than 3.0 × 10 cells / mL, the ethanol concentration per Brix 5.0 mass% was more than 0.05 v / v%, and the amount of ethanol produced was large. ６ ~1.4 × 10 ８ In the fermentation liquor with a concentration of 0.05 v / v% or less per Brix 5.0 mass% and a methional reduction rate of 90% or more, regardless of the initial cell count or the number of days of fermentation, a good malt fermentation liquor with low alcohol and low methional was obtained.

[0099] [Example 5] Fermentation raw material liquid containing RAG － The phenotype Kluyveromyces lactis was inoculated and the fermentation time was varied, and the bacterial concentration, ethanol concentration, and methional concentration of the resulting fermentation broth were examined. － The Kluyveromyces lactis NBRC648 strain was used as the phenotype Kluyveromyces lactis, and wort A was used as the fermentation raw material liquid.

[0100] A 200 mL Erlenmeyer flask was charged with 160 mL of wort, and the yeast was pre-cultured in the same manner as in Example 1, except that the number of days of culture and the initial cell count were as shown in Table 11, so that the initial cell count was 1.0 × 10 per mL of wort. ６ The wort in a conical flask was inoculated with the inoculated yeast so that the number of bacteria reached 100. The inoculated yeast was then cultured in an incubator at 60 rpm at 18°C ​​under normal pressure for fermentation. The bacterial concentration (achieved bacterial count), ethanol concentration, and methional concentration of the resulting fermented broth were quantified. The results are shown in Table 11.

[0101]

[0102] In Table 11, the column with a culture time of 0 hours shows the measurement results of the fermentation raw material liquid (wort) before inoculation with yeast. As shown in Table 11, the longer the culture time, the higher the methional reduction rate. When the contact time between yeast and wort was 4 hours or more, a malt fermentation liquid was obtained in which the ethanol concentration per Brix 5.0 mass% was less than 0.05 v / v%, methional was reduced by 85 mass% or more compared to the wort (fermentation raw material liquid), and the methional concentration per Brix 5.0 mass% was 3 ppb or less.

[0103] [Example 6] Fermentation raw material liquid containing RAG － The oxygen supply conditions during inoculation and fermentation of phenotype Kluyveromyces lactis were varied, and the bacterial concentration, ethanol concentration, and methional concentration of the resulting fermentation broth were examined. － The Kluyveromyces lactis NBRC648 strain was used as the phenotype Kluyveromyces lactis, and wort A was used as the fermentation raw material liquid.

[0104] (1) Fermentation under oxygen supply condition 2 (Reference Example 1) 400 mL of wort was placed in a 500 mL jar fermenter, and yeast was added at an initial cell count of 2.0 × 10 per mL of wort. ５ After inoculation to obtain a total of 1000 yeast cells, the pre-cultured yeast was inoculated into the wort and fermented in the same manner as in Example 1, except that a 0.2 μm filter was attached to the exhaust port to prevent active air supply but allow air in and out, the stirring blade was rotated at 50 rpm, and fermentation was carried out at 18°C ​​and atmospheric pressure for 1 day. The bacterial concentration (achieved bacterial count), ethanol concentration, and methional concentration of the resulting fermentation broth were quantified. The results are shown in Table 12.

[0105] (2) Fermentation under oxygen supply condition 4 (Reference Example 1) 400 mL of wort was placed in a 500 mL jar fermenter, and yeast was added at an initial cell count of 2.0 × 10 per mL of wort. ５ The pre-cultured yeast was inoculated into the wort and fermented in the same manner as in (1) above, except that a 0.2 μm diameter filter was attached to the exhaust port, nitrogen gas was blown in, and the stirring blade was rotated at 50 rpm to stir. The fermentation was carried out at 18°C ​​and atmospheric pressure for 1 day. The bacterial concentration (achieved bacterial count), ethanol concentration, and methional concentration of the resulting fermentation broth were quantified. The results are shown in Table 12.

[0106]

[0107] In Table 12, the column "oxygenation condition" "2" shows the results when fermentation was performed under conditions where air was not actively supplied but air was allowed to enter and exit (oxygenation condition 2 of Reference Example 1), and the column "4" shows the results when fermentation was performed under conditions where nitrogen gas was blown in (oxygenation condition 4 of Reference Example 1). In fermentation under oxygenation condition 2, which is an anaerobic environment, the ethanol concentration per Brix 5.0 mass% was less than 0.05 v / v%, and compared to the wort (fermentation raw material liquid), methional was reduced by 85 mass% or more, and a malt fermentation liquid having a methional concentration per Brix 5.0 mass% or less of 3 ppb was obtained. On the other hand, in fermentation under oxygenation condition 4, which is a completely anaerobic environment, the methional reduction rate was only about 30%, and fermentation was insufficient.

[0108] Next, fermentation was carried out under oxygen supply condition 4 in the same manner as in (2) above, except that the initial cell count was set to the amount shown in Table 13. The cell concentration (achieved cell count), ethanol concentration, and methional concentration of the resulting fermentation broth were quantified. The results are shown in Table 13.

[0109]

[0110] As shown in Table 13, the inoculation number (initial number of bacteria) was 3 × 10 ６ When the cell count exceeds 3 × 10 cells / mL, even in a completely anaerobic environment, the number of bacteria reached is 3 × 10 ６ The malt fermentation broth had an ethanol concentration of more than 0.05 v / v% per Brix 5.0% by mass, a methional concentration of 85% by mass or more reduced compared to the wort (fermentation raw material broth), and a methional concentration of 3 ppb or less per Brix 5.0% by mass. These results demonstrate that even in a completely anaerobic environment where yeast growth is suppressed during fermentation, a malt fermentation broth with the desired low alcohol concentration and low methional concentration can be obtained if the initial cell count is sufficiently high.

[0111] [Example 7] Fermentation raw material liquid containing RAG － The fermentation temperature during inoculation with phenotypic Kluyveromyces lactis and fermentation was varied, and the bacterial concentration, ethanol concentration, and methional concentration of the resulting fermentation broth were examined. Wort A was used as the fermentation raw material liquid.

[0112] RAG － The phenotype Kluyveromyces lactis strain NBRC648 was used, and 160 mL of wort was placed in a 200 mL Erlenmeyer flask. The wort was inoculated with the pre-cultured yeast and fermented by shaking culture at 60 rpm in an incubator at 18°C ​​and atmospheric pressure in the same manner as in Example 1, except that the fermentation temperature and initial cell count were set as shown in Table 14. The cell concentration (achieved cell count), ethanol concentration, and methional concentration of the resulting fermentation broth were quantified. The results are shown in Table 14.

[0113]

[0114] As shown in Table 14, even when the fermentation temperature was low and yeast barely grew during the fermentation period, the number of bacteria reached was 3 x 10 ６ When the concentration exceeded 100 cells / mL, the methional reduction rate could be 90% or more, and a malt fermentation liquid could be prepared in which the ethanol concentration per Brix 5.0 mass% was less than 0.05 v / v% and the ethanol concentration per Brix 5.0 mass% was less than 0.05 v / v%.

[0115] [Example 8] The fermentation temperature was set at 1°C, and the bacterial concentration, ethanol concentration, and methional concentration of the obtained fermented liquid were examined. Wort A was used as the fermentation raw material liquid. RAG yeast was used. － Kluyveromyces lactis NBRC648 strain and Kluyveromyces lactis R33 strain, which are phenotypically Kluyveromyces lactis, and Saccharomyces ludwigii NBRC798TS strain were used.

[0116] 240 mL of wort was placed in a 300 mL Erlenmeyer flask, and the pre-cultured yeast was inoculated into the wort. Fermentation was carried out by shaking culture at 60 rpm in an incubator at 18°C ​​and atmospheric pressure in the same manner as in Example 1, except that the fermentation temperature, yeast species, and initial cell count were as shown in Table 15. The cell concentration (achieved cell count), ethanol concentration, and methional concentration of the resulting fermentation broth were quantified. The results are shown in Table 15.

[0117] Each of the fermented broths was diluted 2.56 times to prepare a fermented malt beer-like sparkling beverage, and the taste of each beverage was evaluated by five experienced panelists. The results are shown in Table 16.

[0118]

[0119]

[0120] As shown in Table 15, the methional reduction rate was 85% or more in the fermented liquid obtained using any of the yeasts, and the fermented liquid was sufficiently fermented. －The fermentation broths obtained using the Kluyveromyces lactis NBRC648 strain and the Kluyveromyces lactis R33 strain, which are phenotypes of Kluyveromyces lactis, also had low ethanol production and were characterized by being mild, but refreshing, light, and easy to drink compared to the fermentation broth obtained using the Saccharomyces ludwigii NBRC798TS strain (Table 16).

[0121] [Example 9] Pilot scale RAG － Fermentation was carried out using the phenotype Kluyveromyces lactis, and the bacterial concentration, ethanol concentration, and methional concentration of the resulting fermentation liquid were examined. Wort D was used as the fermentation raw material liquid. RAG yeast was used. － The Kluyveromyces lactis NBRC648 strain and the Kluyveromyces lactis R86 strain, which are phenotypically Kluyveromyces lactis, were used.

[0122] <Fermentation in Fermentation Tank> 100 L of wort D (Brix 9.0%) was placed in a 200 L fermentation tank, and yeast was added to the fermentation tank so that the initial cell count was the amount shown in Table 17. The fermentation tank was then pressurized to 0.026 MPa, and fermentation was carried out at 18°C ​​for 3 days under conditions in which air was supplied at 0.67 NL / min per 100 L of fermentation liquid (oxygen supply condition 3 in Reference Example 1). The bacterial concentration (achieved cell count), ethanol concentration, and methional concentration of the resulting fermentation liquid were quantified. The results are shown in Table 17.

[0123]

[0124] As shown in Table 17, even at the pilot scale, RAG － It was confirmed that fermentation using the phenotype Kluyveromyces lactis under appropriate oxygen-providing conditions can produce a malt fermentation liquid having an ethanol concentration of less than 0.05 v / v% per 5.0% Brix by mass, in which methional has been reduced by 85% by mass or more compared to the wort (fermentation raw material liquid), and in which the methional concentration per 5.0% Brix by mass is 3 ppb or less.

Claims

1. A method for producing a malt fermentation liquid by inoculating a yeast into a fermentation raw material liquid containing a saccharified product of malt and fermenting the liquid, the method comprising the steps of: － A method for producing a malt fermentation liquor, comprising: a phenotype of Kluyveromyces lactis; and the malt fermentation liquor has an ethanol concentration of less than 0.05 v / v% per Brix 5.0 mass%.

2. A method for producing a malt fermentation liquor according to claim 1, wherein the malt fermentation liquor has a methional concentration of 3.0 ppb or less per Brix 5.0 mass%.

3. A method for producing a malt fermentation liquor as described in claim 1, wherein the methional concentration of the malt fermentation liquor is 15 mass% or less of the methional concentration of the fermentation raw material liquid.

4. The bacterial concentration of the malt fermentation liquid at the end of fermentation is 3.0 x 10 ６ The method for producing a malt fermentation liquor according to claim 1, wherein the malt fermentation liquor concentration is 10 ... cells / mL or more.

5. The bacterial concentration of the malt fermentation liquid at the start of fermentation is 2.0 x 10 ４ The method for producing a malt fermentation liquor according to claim 1, wherein the malt fermentation liquor concentration is 10 ... cells / mL or more.

6. The method for producing a malt fermentation liquid according to claim 1, wherein the fermentation is carried out under oxygen-providing conditions, and the oxygen-providing conditions are conditions in which the time required to increase the dissolved oxygen concentration of pure water from 6.0 ppm to 8.0 ppm at 18°C ​​and normal pressure is more than 0 hours and not more than 5 hours.

7. The method for producing a malt fermentation liquid according to claim 1, wherein the fermentation is carried out under conditions in which no oxygen is added.

8. The method for producing a malt fermentation liquid according to claim 1, wherein the fermentation is carried out at a temperature of 1°C or higher.

9. The method for producing a malt fermentation liquid according to claim 1, wherein the fermentation is carried out for 4 hours or more.

10. The method for producing a malt fermentation liquid according to claim 1, wherein the fermentation is carried out for 24 hours or more.

11. A method for producing a fermented malt beverage, comprising producing a malt fermentation liquid by the method for producing a malt fermentation liquid according to any one of claims 1 to 10, and then producing a fermented malt beverage using the resulting malt fermentation liquid as a raw material.

12. The method for producing the fermented malt beverage according to claim 11, further comprising a step of diluting the malt fermentation liquid.

13. A method for producing a fermented malt beverage according to claim 11, comprising producing a fermented malt beverage containing 3 to 7 mass% of original wort extract.

14. The method for producing a fermented malt beverage according to claim 11, wherein the fermented malt beverage is a beer-like sparkling beverage.

15. A fermented malt beverage having an ethanol concentration of less than 0.05 v / v% per 5.0 mass% Brix and a methional concentration of 3.0 ppb or less per 5.0 mass% Brix.

16. The malt fermented beverage according to claim 15, which is a beer-like sparkling beverage.

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