NL2028706B1

NL2028706B1 - Method of producing fermented malt-based liquid

Info

Publication number: NL2028706B1
Application number: NL2028706A
Authority: NL
Inventors: Miyoshi Miho; Ikeda Yuka; Onishi Makiko; Kanda Tomomasa; Sunagawa Tadahiro
Original assignee: Asahi Group Holdings Ltd
Priority date: 2021-07-12
Filing date: 2021-07-12
Publication date: 2023-01-18
Also published as: WO2023286789A1; AU2022310480A1

Abstract

A method of producing a fermented malt-based liquid having a low ethanol concentration and a pineapple-like fruity flavor, and a fermented malt beverage produced from the fermented malt-based liquid obtained by the production method are provided. That is, the present invention relates to a method of producing a fermented malt-based liquid, including a step of inoculating a yeast of the genus Cyberlindnera into a fermentation raW material liquid containing a malt saccharified product and performing aerated fermentation on the fermentation raW material liquid, and a method of producing a fermented malt beverage, in Which a fermented malt beverage is produced using the fermented malt-based liquid obtained by the method of producing a fermented malt-based liquid as a raW material.

Description

METHOD OF PRODUCING FERMENTED MALT-BASED LIQUID

BACKGROUND OF THE INVENTION Field of the Invention

[0001] The present invention relates to a method of producing a fermented malt-based liquid obtained by using a malt saccharified product as a fermentation raw material and having a low ethanol concentration and a pineapple-like fruity flavor, and a method of producing a beverage containing the fermented liquid produced by the production method as a raw material.

Description of Related Art

[0002] In recent years, with the diversification of consumer’s preferences in alcoholic beverages, a wide variety of products have been put on the market. For example, there is an increasing demand for alcoholic beverages with a relatively low alcohol concentration because such beverages are easier to drink. Meanwhile, in a case of fermented alcoholic beverages, ethanol and various flavor components are produced by yeasts through fermentation, and the fermentation contributes to the flavor of the beverages.

[0003] In such fermented alcoholic beverages, the various flavor components produced during fermentation depend on the kind of yeasts used in the fermentation. For example, the yeast most widely used for brewing beer and wine is the genus Saccharomyces, but in recent years, beer with a flavor different from that of beer or the like known in the art has also been produced by using yeasts other than the genus Saccharomyces.

[0004] For example, a method of producing beer with a high content of isoamyl acetate (IAAT) and a high content of ethyl acetate by fermenting wort under aerobic conditions using yeasts of the genus Pichia such as Pichia kluyveri is known (PCT International Publication No. W02014/202564). Further, it has been reported that yeasts of the genus Cyberlindnera and wort can be brought into contact with each other and left to stand still and ferment to obtain a fermented malt-based liquid, and the obtained fermented malt-based liquid contains a high content of esters such as isoamyl acetate and ethyl acetate and has a low ethanol concentration (Belluta, et al., Journal of the American Society of Brewing Chemists, 2019, DOI:

10.1080/03610470.2019.1569452, and Belluta, et al., Fermentation, 2019, DOI:

10.339()/ fermentation 5040103).

[0005] Further, the esters are flavor components mainly produced in a fermentation step carried out using yeasts, and the ester flavor is a kind of characteristic flavor of a brewed liquid such as wine, sake, or beer. The esters have multiple fruity flavor components. Isoamyl acetate mainly has a banana-like flavor, ethyl caproate has an apple-like flavor, isoamyl propionate has a pineapple-like flavor, and B-phenethyl acetate has a raspberry-like flavor. Fresh fruits contain a high content of esters, and the content of esters decreases as the freshness decreases. For example, the proportion of ester with respect to isobutanol is high in strawberries immediately after harvest, whereas the proportion thereof is reversed in strawberries that have become rotten due to storage for a long time (Kim et al., Sensors, 2013, vol. 13, pp. 7939 to 7978). Further, isobutanol is a flavor component that smells like an oil-based marker. Citation List Patent Literature

[0006] [PTL 1] International Patent Publication No. 2014/202564 Non Patent Literature

[0007] [NPL 1} Belluta, et al., Journal of the American Society of Brewing Chemists, 2019, DOI:

10.1080/03610470.2019.1569452 [NPL 2] Belluta, et al., Fermentation, 2019, DOI: 10.3390/fermentation5040103 [NPL 3} Kim et al., Sensors, 2013, vol.13, p.7939-7978.

SUMMARY OF THE INVENTION

[0008] In the production of fermented alcoholic beverages, in a case where the degree of fermentation is suppressed in order to suppress the ethanol concentration to be low, the content of flavor components may also be decreased, or an unpleasant odor due to fermentation failure may OCCUT. An object of the present invention is to provide a method of producing a fermented malt- based liquid having a low ethanol concentration and a fruity flavor, a method of producing a fermented malt beverage using the fermented malt-based liquid produced by the production method, and a fermented malt beverage produced by the production method.

[0009] As a result of intensive research conducted by the present inventors in order to solve the above-described problems, it was found that a fermented malt-based liquid having a fruity flavor and a low ethanol concentration can be produced by performing aerated fermentation using yeasts of the genus Cyberlindnera which have not been used for fermenting a fermentation raw material liquid containing a malt saccharified product in the related art so that the production of ethanol is suppressed and the production of flavor components such as esters is promoted, thereby completing the present invention.

[0010] The method of producing a fermented malt-based liquid, the method of producing a fermented malt beverage, and the fermented malt beverage according to the present invention are as follows.

[1] A method of producing a fermented malt-based liquid, including: a step of inoculating a yeast of the genus Cyberlindnera into a fermentation raw material liquid containing a malt saccharified product and performing aerated fermentation on the fermentation raw material liquid.

[2] The method of producing a fermented malt-based liquid according to [1], in which a fermented malt-based liquid containing less than 0.2% (vol/vol) of ethanol is produced.

[3] The method of producing a fermented malt-based liquid according to [1] or [2], in which a fermented malt-based liquid in which a concentration of isoamyl propionate is 1.4 pg/L or greater per Brix and a ratio of a total concentration of isoamyl acetate and B-phenethyl acetate to a concentration of isobutanol [([concentration of isoamyl acetate (img/L)] + [concentration of [3- phenethyl acetate {mg/L)})/| concentration of isobutanol (mg/L)}] is 5.1 or greater is produced.

[4] The method of producing a fermented malt-based liquid according to any one of [1] to

[3], in which the fermentation raw material liquid has a Brix of 10% to 20% and a pH of 3 to 6.

[5] The method of producing a fermented malt-based liquid according to any one of [1] to

[4], in which the aerated fermentation is performed at 6°C to 28°C under a condition that a concentration of dissolved oxygen in the fermented liquid is 1 ppm or greater.

[6] The method of producing a fermented malt-based liquid according to any one of [1] to

[5], in which 1.0 x 10% to 2.0 x 107 yeasts per | mL are inoculated into the fermentation raw material liquid.

[7] The method of producing a fermented malt-based liquid according to any one of [1] to

[6], in which the yeast is one or more selected from the group consisting of Cyberlindnera jadinii,

Cyberlindnera mrakii, Cyberlindnera fabianii, Cyberlindnera saturnus, and Cvberlindnera subsufficiens.

[8] A method of producing a fermented malt beverage, in which a fermented malt beverage is produced using a fermented malt-based liquid obtained by the method of producing a fermented malt-based liquid according to any one of [1] to [7] as a raw material.

[9] The method of producing a fermented malt beverage according to [8], including: a step of diluting the fermented malt-based liquid.

[10] The method of producing a fermented malt beverage according to [8} or [9], in which the fermented malt beverage is a beer-like sparkling beverage.

11} A fermented malt beverage comprising a fermented malt-based liquid obtained by the method of producing a fermented malt-based liquid according to any one of [1] to [7], in which the concentration of isoamyl propionate is 1.4 pg/L or greater per Brix, a ratio of a total concentration of isoamyl acetate and 3-phenethyl acetate to a concentration of isobutanol [([concentration of 1soamyl acetate (mg/L)] + [concentration of f3-phenethyl acetate (mg/L )])/[concentration of isobutanol (mg/L )]] is 5.1 or greater, and the concentration of ethanol is 0.5% (vol/vol) or less.

[12] The fermented malt beverage according to [11], in which the fermented malt beverage is a beer-like sparkling beverage.

[0011] In the method of producing a fermented malt-based liquid according to the present invention, aerated fermentation is performed using yeasts of the genus Cyberlindnera, which have a low ethanol-producing ability and a high ability to produce esters such as isoamyl propionate. Therefore, according to the method of producing a fermented malt-based liquid of the present invention, it is possible to provide a fermented malt-based liquid having a low ethanol concentration and a pineapple-like fruity flavor. Further, it is possible to provide a fermented malt beverage having a low ethanol concentration and a pineapple-like fruity flavor using the fermented malt-based liquid.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIG. 1 is a diagram showing a correlation between sensory evaluation of (e) deliciousness and sensory evaluation of (b) pineapple-like flavor (deliciousness x pineapple-like flavor) based on the sensory evaluation results obtained in Examples 1 and 2.

FIG. 2 is a diagram showing a correlation between sensory evaluation of (e} deliciousness and sensory evaluation of (d) intensity of miscellaneous odor (deliciousness X intensity of miscellaneous odor) based on the sensory evaluation results obtained in Examples 1 and 2.

5 DETAILED DESCRIPTION OF THE INVENTION

[0013] In the present invention and the specification of the present application, a “fermented malt beverage” indicates a beverage produced by performing a fermentation step with one or more yeasts using malt or a processed product thereof as a raw material. The fermented malt beverage may be an alcoholic beverage or may be a so-called non-alcoholic beverage or a low-alcoholic beverage having an ethanol concentration of less than 1.0% (vol/vol). Further, the concentration of ethanol in the fermented malt beverage is preferably less than 0.5% (vol/vol), more preferably less than 0.1% (vol/vol), and still more preferably less than 0.05% (vol/vol).

[0014] Examples of the fermented malt beverage include beer-like sparkling beverages. In the present invention and the specification of the present application. “beer-like sparkling beverage™ indicates a sparkling beverage which has a savor, a taste, and a texture equivalent to or similar to those of beer and has a beer-like taste (a taste reminiscent of beer in terms of flavor). Specific examples of the fermented malt beer-like sparkling beverage include beer and sparkling liquid obtained by using malt as a raw material. Further, liqueurs obtained by mixing a fermented malt- based liquid with an ethanol-containing distillate may also be used. The ethanol-containing distillate is a solution containing ethanol obtained by performing a distillation operation and may be, for example, alcohol (ethanol) for a raw material, and examples thereof include distilled liquids (spirits) such as whiskey, brandy, vodka, rum, tequila, gin, and shochu.

[0015] The method of producing a fermented malt-based liquid according to the present invention includes a step of inoculating one or more yeasts of the genus Cyberlindnera into a fermentation raw material liquid containing a malt saccharified product and performing aerated fermentation on the fermentation raw material liquid. The yeasts of the genus Cyberlindnera have a lower ethanol- producing ability during aerated fermentation than that of the yeasts of the genus Saccharomyces, but have a higher ability to produce flavor components such as esters. Therefore, a fermented liquid that has a low ethanol concentration and is rich in esters such as isoamyl propionate, isoamyl acetate, and [-phenethyl acetate is obtained by using the yeasts of the genus Cyberlindnera.

[0016]

The one or more yeasts used in the present invention are not particularly limited as long as yeasts of the genus Cyberlindnera are used. From the viewpoint of safety in a case where a produced fermented malt-based liquid is applied to food and drink, it is preferable that the yeasts be yeasts of the genus Cyberlindnera separated from food and drink, materials thereof, and edible animals and plants. Examples of the yeasts of the genus Cyberlindnera include yeasts belonging to Cyberlindnera jadinii, Cyberlindnera mrakii, Cyberlindnera fabianii, Cyberlindnera saturnus, and Cyberlindnera subsufficiens. These yeasts of the genus Cyberlindnera can be appropriately selected from strains of yeasts of the genus Cyberlindnera available from yeast strain storage organizations such as the National Institute of Technology and Evaluation, Biotechnology Resource Center (NBRC), National Collection of Yeast Cultures (NCYC), and distributors and then used. In the present invention, the yeasts of the genus Cyberlindnera to be inoculated into the fermentation raw material liquid may be used alone or two or more kinds thereof. Further, a combination of the yeasts of the genus Cyberlindnera and other yeasts may be inoculated into the fermentation raw material liquid.

[0017] In the present invention, the fermentation raw material liquid to be fermented by being inoculated with one or more yeasts of the genus Cyberlindnera is not particularly limited as long as the fermentation raw material liquid contains a malt saccharified product and has a carbon source or a nitrogen source that can be assimilated with the yeasts of the genus Cvberlindnera. For example, the fermentation raw material liquid may be formed of only a malt saccharified product or may have a carbon source or a nitrogen source other than the malt saccharified product.

[0018] In the present invention and the specification of the present application, the “malt saccharified product” is obtained by performing a saccharification treatment on saccharides in malt with various enzymes. The enzyme used for the saccharification treatment may be an enzyme originally contained in malt or an enzyme derived from a source other than malt. For example. a saccharified product is obtained by amylase derived from malt by adding water to malt or a ground malt and holding the temperature at 50°C to 60°C. Here, a saccharifying enzyme derived from a source other than malt may be further added thereto, or an enzyme other than the saccharifying enzyme such as a proteolytic enzyme may be used in combination with the amylase derived from malt.

[0019] In the present invention, for example, wort or a malt extract can be used as the malt saccharified product. Malt extract is obtained by concentrating wort and contains maltose as a main component. The malt extract is used as a beverage by being diluted with carbonated water or as a raw material for food and drink. The wort can be concentrated by a method known in the art. It is preferable that the wort be concentrated under reduced pressure because the unintentional change on the components in the wort is suppressed, but the wort can also be concentrated by being heated to remove the solvent.

[0020] Wort is prepared by preparing a mixture containing malt and water used as a raw material, heating the mixture, and saccharifying the starchy substance of the malt. It is preferable that the malt be used as a malt ground product obtained by performing a grinding treatment. The grinding treatment can be performed on malt according to a method know in the art. The malt ground product may be obtained by performing a treatment that is typically carried out before and after the grinding treatment.

[0021] 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 saccharine raw material. Examples of grain raw materials other than malt include wheat varieties such as barley and wheat, rice, corn, beans such as soybean, and potatoes. The grain raw material can also be used as a grain ground product, a grain syrup, a grain extract, or the like. The grain ground product may be subjected to a treatment that is typically carried out before and after the grinding treatment, such as com starch or corn grits. Examples of the saccharine raw material include saccharides such as liquid sugar.

[0022] The wort may be used as a fermentation raw material liquid as it is, but it is preferable that the wort be subjected to a boiling treatment before inoculation with yeasts. By performing a boiling treatment, the fermentation raw material liquid can be sterilized and is fermented more safely. In a case where a boiling treatment is performed, it is preferable that an insoluble solid content be removed from the wort 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 typically performed in the step of producing food and drink such as diatomaceous earth filtration or filter filtration. Further, warm water may be added to malt extract and boiled for use in place of the filtrate of this saccharified solution (wort). The boiling method and the conditions thereof can be appropriately determined.

[0023]

A fermented malt-based liquid having a desired flavor can be produced by appropriately adding fragrant grass or the like before or during the boiling treatment. In particular, it is preferable that hops be added before or during the boiling treatment. By performing the boiling treatment in the presence of hops, the savor and flavor components of hops can be efficiently extracted. The amount of hops to be added, the addition form {for example, several separate additions), and the boiling conditions can be appropriately determined.

[0024] It is preferable that lees such as proteins generated by precipitation be removed from the boiled wort before inoculation with yeasts. The lees may be removed by any solid-liquid separation treatment, but the precipitate is typically removed using a tank called a whirlpool. Here, the temperature of the boiled wort may be 15°C or higher and typically in a range of 50°C to 80°C. The clarified wort (filtrate) after removal of the lees is cooled to an appropriate fermentation temperature with a plate cooler or the like. The wort after removal of the lees is the fermentation raw material Hquid.

[0025] The cooled fermentation raw material liquid is inoculated with yeasts of the genus Cyberlindnera and fermented. The cooled fermentation raw material liquid may be provided for fermentation as it is or may be provided for fermentation after being adjusted to have the desired extract concentration (Brix of the fermentation raw material liquid to be inoculated with the yeasts) described below. The amount of the yeasts of the genus Cyberlindnera to be added to the fermentation raw material liquid is not particularly limited, but is preferably in a range of 1.0 x 10% to 2.0 x 107 per 1 mL of the fermentation raw material liquid and, for example, the amount thereof can be set to approximately 2 x 10° (2 x 105 cells/mL) per 1 mL of the fermentation raw material liquid. In the present invention, the Brix of the fermentation raw material liquid to be inoculated with the yeasts of the genus Cyberlindnera is preferably in a range of 10% to 20%, more preferably in a range of 11% to 18%, and still more preferably in a range of 12% to 16%. Further, in the present invention, the pH of the fermentation raw material liquid inoculated with the yeasts of the genus Cyberlindnera is preferably in a range of 3 to 6 and more preferably in a range of 3.5 10 5.5.

[0026] Further, in the present invention and the specification of the present application, the Brix value of the fermentation raw material liquid can be measured by a method known in the art using a measuring device such as a refractometer (RX-50000, manufactured by Atago Co. Ltd.).

[0027]

A fermented malt-based liquid is obtained by performing aerated fermentation on the fermentation raw material liquid inoculated with the one or more yeasts of the genus Cyberlindnera. By inoculating the yeasts of the genus Cyberlindnera into the fermentation raw material liquid containing wort and carrying out fermentation under aerobic conditions, a fermented malt-based liquid is obtained in which the production of ethanol is further suppressed, the amount of esters produced is further increased, the flavor is excellent, and the ethanol concentration is low. Further, in the present invention, the amount of isobutanol produced is low and the wort odor and the like are suppressed by performing aerated fermentation using the one or more yeasts of the genus Cyberlindnera, and thus a refreshing fermented malt-based liquid with less miscellaneous odor is obtained. Further, in the present invention and the specification of the present application, the miscellaneous odor indicates an odor of stuffy socks after wearing, wine flavor, wort odor, thinner odor, or the like.

[0028] The aerated fermentation is fermentation performed under aerobic conditions in which oxygen or air can be supplied into the medium, and can be performed by a method known in the art. Examples of the method include a shaking culture method, a stirring culture method, and an aeration stirring culture method. Further, the stirring conditions and the aeration amount of air and oxygen gas may be conditions set such that the culture environment can be maintained aerobic and may be appropriately selected depending on the culture device, the viscosity of the medium, and the like. For example, in a case of mass culture such as culture using a jar fermenter, the culture and fermentation can be made while the amount of dissolved oxygen in the fermented Hquid is monitored, and air or oxygen gas can be continuously or intermittently aerated such that the amount of dissolved oxygen in the fermented liquid reaches a predetermined amount (for example, I ppm (mg/L) or greater.

[0029] The fermentation conditions for the method of producing a fermented malt-based liquid according to the present invention are appropriately selected from the conditions that enable the culture environment during the fermentation to be maintained aerobic among the typical fermentation conditions for yeasts and can be modified as necessary. For example, the fermentation temperature is set to be in a range of 0°C to 35°C, preferably in a range of 6°C to 28°C, more preferably in a range of 8°C to 25°C, and still more preferably in a range of 10°C to 20°C. By performing fermentation in the above-described temperature range, the production of esters by the yeast of the genus Cyberlindnera is efficiently carried out. The fermentation time is not particularly limited, but is preferably 7 days or shorter, more preferably 4 days or shorter, and still more preferably 3 days or shorter. For example, the time at which the growth of a predetermined amount of yeasts is achieved can be set as the end of fermentation. As the standard for the end of fermentation, the time at which ODeso of the fermented liquid reaches 1.0 to 6.5, the time at which the yeasts grow to an amount of approximately 2.0 x 107 cells to 2.5 x 10° cells (2.0 x 107 t0 2.5 x 10° cells/mL) per 1 mL of the fermented liquid, and the time at which the yeasts grow to an amount of approximately 6.5 x 107 cells to 1.1x 10% cells (6.5 x 107 to 1.1 x 165 cells/mL) per 1 mL of the fermented liquid are exemplary examples.

[0030] The fermented malt-based liquid produced in the above-described manner has a low ethanol concentration and a high content of flavor components as described below. Examples of the flavor components include isoamyl propionate. isoamyl acetate, and B-phenethyl acetate (1-phenylethyl acetate). These components are the causative components of the fruity flavor, and since the content of these components is high, the fermented malt-based liquid obtained by aerated fermentation of the yeasts of the genus Cyberlindnera has an excellent fruity flavor.

[0031] In particular, the amount of isoamyl propionate produced by other yeasts is extremely small, but a fermented malt-based liquid in which the concentration of isoamyl propionate is 1.4 pg/L or greater per Brix and the pineapple-like fruity flavor is excellent is obtained by aerated fermentation of the yeasts of the genus Cyberlindnera. The concentration of isoamyl propionate per Brix can be calculated by dividing the quantitative value (ug/L) of isoamyl propionate by the Brix concentration of the fermentation raw material liquid. The concentration of isoamyl propionate in the fermented malt-based liquid obtained by the present invention per Brix is preferably in a range of 1.4 to 30 pg/L, more preferably in a range of 1.4 to 20 pg/L, and still more preferably in a range of 1.4to 15 pg/L.

[0032] The concentration of isoamyl acetate in the fermented malt-based liquid obtained by the present invention per Brix is preferably 0.1 mg/L or greater, more preferably in a range of 0.1 to

2.5 mg/L, still more preferably in a range of 0.1 to 2.0 mg/L, and even still more preferably in a range of 0.3 to 2.0 mg/L. The concentration of B-phenethyl acetate in the fermented malt-based liquid obtained by the present invention per Brix is preferably 0.05 mg/L or greater, more preferably in a range of 0.05 to 1.5 mg/L, and still more preferably in a range of 0.05 to 1.0 mg/L. Further, the concentration of isobutanol in the fermented malt-based liquid obtained by the present invention per Brix is preferably 0.5 mg/L or less. The concentration of isoamyl acetate, p- phenethyl acetate and isobutanol per Brix can be calculated by dividing the quantitative value

(mg/L) of isoamyl acetate, [3-phenethyl acetate and isobutanol by the Brix concentration of the fermentation raw material liquid.

[0033] In a case where the fermentation raw material liquid containing wort is aerated and fermented with the one or more yeasts of the genus Cyberlindnera, the amount of esters produced is large, while the amount of isobutanol produced is small. Therefore, the fermented malt-based liquid obtained by the present invention can have a fresh fruity flavor of freshly harvested fruit or the like, which is preferable. The ratio of the total concentration of the isoamyl acetate and f3- phenethyl acetate to the concentration of isobutanol [{[concentration of isoamyl acetate (mg/L )] + [concentration of B-phenethyl acetate (mg/L)})/[concentration of isobutanol (mg/L)]] (hereinafter, also referred to as the “(1IAmAc + bPEAc)/IBA ratio”) of the fermented malt-based liquid obtained by the present invention is preferably 5.1 or greater, more preferably in a range of 5.1 to 400, still more preferably in a range of 5.1 to 300, and even still more preferably in a range of 5.1 to 200.

[0034] The concentration of ethanol in the fermented malt-based liquid obtained by the present invention is not particularly limited. For example, in a case where the fermentation raw material liquid having a Brix of 4% to 20% is aerated and fermented, the concentration of ethanol in the produced fermented malt-based liquid is less than 0.5% (vol/vol), preferably 0.2% (vol/vol) or less, still more preferably 0.1% (vol/vol) or less, and even still more preferably less than 0.05% (vol/vol).

[9035] Further, in the present invention and the specification of the present application, the concentration of ethanol in the fermented malt-based liquid or the beverage can be measured by an enzyme method using a multifunctional biosensor BF-5 (manufactured by Oji Scientific Instruments Co., Ltd.), a distillation-density (specific gravity) method, or a gas chromatography (GC) analysis method, an oxidation method, or the like.

[0036] The content of esters such as isoamyl propionate, isoamyl acetate, and B-phenethyl acetate and isobutanol in the fermented malt-based liquid or the fermented malt beverage can be acquired from, for example, the peak area of the chromatograph obtained by GC analysis. The quantification method based on the peak area is not particularly limited, and examples thereof include an area percentage method, an internal standard method, a standard addition method, and an absolute calibration curve method. Specifically, the content thereof can be acquired by the method described in the following examples.

[0037] The fermented malt-based liquid obtained in the present invention can be used as a raw material for food and drink as it is. The food and drink produced from using the fermented malt- based liquid as a raw material is not particularly limited. As the food and drink produced using the fermented malt-based liquid obtained in the present invention as a raw material, a beverage is preferable, a fermented malt beverage is more preferable, and a fermented malt beer-like sparkling beverage is still more preferable.

[0038] For example, a fermented malt beverage can be obtained by performing a solid-liquid separation treatment on the obtained fermented malt-based liquid to remove the yeasts and the like. The solid-liquid separation treatment may be a method that enables the yeasts and the like to be removed, and examples thereof include a centrifugal separation treatment, a diatomaceous earth filtration treatment, and a filter filtration treatment using a filter having an average pore diameter of approximately 0.4 to 0.5 um, and these treatments may be used in combination. Further, the flavor of the fermented malt beverage may be adjusted by adding hops, hop extracts, flavors, or the like to the obtained fermented malt-based liquid. The flavor may be adjusted before the solid-liquid separation treatment or after the solid-liguid separation treatment. Further, the fermented malt- based liquid after the solid-liquid separation treatment may be stored and aged under a low temperature condition of approximately 0°C. In order to produce a fermented malt beverage, for example, a pH adjustment treatment, a heat treatment, or the like may be further carried out according to a method known in the art.

[0039] A fermented malt beverage in which the extract concentration and the ethanol concentration are each in a desired range can also be produced by appropriately diluting the obtained fermented malt-based liquid. The liquid used for the dilution is not particularly limited, and examples thereof include water and carbonated water. The dilution rate is not particularly limited, and the fermented malt-based liquid can be diluted, for example, 2 to 10 times, more preferably 2 to 6 times, and still more preferably 4 to 6 times. The step of diluting the fermented malt-based liquid may be performed after the end of the fermentation step, after the removal of the yeasts and the like, or on the fermented malt-based liquid after the aging.

[0040] The concentrations of ethanol, various esters, and isobutanol in the fermented malt beverage obtained using the fermented malt-based liquid obtained in the present invention as the raw material are not particularly limited. A fermented malt beverage having a low ethanol concentration and a pineapple-like fruity flavor can be produced by using the fermented malt-based liquid obtained in the present invention as a raw material. Further, since the fermented malt-based liquid obtained in the present invention is used as a raw material, the content of esters is large, while the content of isobutanol is small. Therefore, a fermented malt beverage having a fresh fruity flavor of freshly harvested fruit or the like can be produced. As the fermented malt beverage, for example, a fermented malt beverage in which the concentration of isoamyl propionate is 1.4 ug/L or greater per Brix, the GAmAc + bPEAc)/IBA ratio is 5.1 or greater, and the concentration of ethanol is less than 0.5% (vol/vol) is preferable, and a fermented malt beer- like sparkling beverage in which the concentration of isoamyl propionate is 1.4 pg/L or greater per Brix, the (iAmAc + bPEAc)/IBA ratio is 5.1 or greater, and the concentration of ethanol is less than 0.05% (vol/vol) is particularly preferable. As the fermented malt beer-like sparkling beverage, a fermented malt beer-like sparkling beverage in which the concentration of isoamyl propionate is in a range of 1.4 to 30 ug/L per Brix, the (1IAmAc + bPEAc)/IBA ratio is in a range of

5.1 to 400, and the concentration of ethanol is less than 0.5% (vol/vol) is more preferable, and a fermented malt beer-like sparkling beverage in which the concentration of isoamyl propionate is in a range of 1.4 to 20 pg/L per Brix, the (iAmAc + bPEAc)/IBA ratio is in a range of 5.1 to 300, and the concentration of ethanol is less than 0.05% (vol/vol) is still more preferable. Examples

[0041] Hereinafter, the present invention will be described in more detail based on the examples and the reference examples, but the present invention is not limited to the following examples.

[0042] <Measurement of concentration of ethanol> In the following experiments, the concentration of ethanol in a fermented malt-based liquid or a fermented malt beverage was measured by the following method unless otherwise specified. A solution obtained by diluting a sample with distilled water such that the concentration of ethanol reached 0.5 to 2.0% (vol/vol) was measured by a multifunctional biosensor (BF-5, manufactared by Oji Scientific Instruments Co., Ltd.). The value obtained by multiplying the obtained measured value by the dilution rate was set as the concentration (% (vol/vol) of ethanol in the sample.

[0043] Further, the multifunctional biosensor is a device that electrochemically detects hydrogen peroxide (H20:) generated by oxidation by alcohol oxidase and can measure the total concentration of methanol and ethanol. Since yeasts do not biosynthesize methanol, the fermented malt-based liquid contains only a trace amount of methanol, which can be said to be within the range of error. Therefore, the value measured by the multifunctional biosensor is a measured value of the amount of ethanol in the fermented malt-based liquid sample.

[0044] In a case of a sample having an ethanol concentration of less than (0.5% (vol/vol), the measurement of the concentration of ethanol was performed on a solution obtained by diluting the sample such that the alcohol concentration reached 0.01% to 0.04% (vol/vol) using the above- described multifunctional biosensor according to a high-sensitivity measurement method using a buffer solution free of sodium azide (SL04-0015, manufactured by Oji Scientific Instruments Co., Lid).

[0045] <Analysis of isobutanol, isoamyl acetate, and B-phenethyl acetate> In the following experiments, the concentrations of isobutanol, isoamyl acetate, and [3- phenethyl acetate in the fermentation raw material liquid, the fermented malt-based liquid, or the fermented malt beverage were quantified by performing extraction with carbon disulfide and analyzing the extracts with FID-GC (hydrogen flame ionization detector-gas chromatography) using hexyl alcohol and hexyl acetate as internal standards, unless otherwise specified. Specifically, first, 30 g of the sample was collected in a container, 6 g of ammonium sulfate and 3 mL of carbon disulfide were added thereto, 150 pL of a mixed solution containing 500 mg/L of hexyl alcohol and 20 mg/L of hexyl acetate was added thereto as an internal standard substance, and the resulting solution was extracted by shaking extraction for 10 minutes. Thereafter, the centrifugal separation treatment was performed at 3000 rpm for 10 minutes so that the solvent layer was recovered. The recovered solvent layer was used as an analysis sample and subjected to GC analysis.

[0046] The GC analysis was performed by injecting 2 uL of the analysis sample into a column (DB- FFAP, 30 m = 0.25 mm ID: 0.25 um EF. T., manufactured by Agilent Technologies, Inc.) at an injection temperature of 250°C using a split injection method, and the analysis sample was held at 40°C for 2 minutes, heated to 230°C at 7°C/min, and held for 10 minutes. The concentration of each component in the analysis sample was quantified by creating a calibration curve for each component based on the ratio between the peak area of each component and the peak area of the internal standard using hexyl alcohol as an internal standard in a case of isoamyl acetate and isobutanol and using hexyl acetate as an internal standard in a case of 3-phenethyl acetate. This quantified value indicates a value rounded off to the first decimal place. In a case where the quantified value of isobutanol is 0.0, the (iIAmAc + bPEAc)/IBA ratio was acquired as a maximum value of 0.049.

[0047] <Analysis of isoamyl propionate> In the following experiments, the concentration of isoamyl propionate in the fermentation raw material liquid, the fermented malt-based liquid, or the fermented malt beverage was analyzed using GCMS-TQ8040NX (Shimadzu Corporation) unless otherwise specified. Specifically, first, 5S mL of a sample was collected in a container and incubated at 60°C for 15 minutes, and the gas phase was adsorbed at 60°C for 30 minutes using SPME Arrow (1.10 mnvDivinyl benzene/Carbon WR/PDMS) (manufactured by Shimadzu Corporation). The sample was splitlessly injected at an injection temperature of 250°C, and a DB-WAX column (60 m x

0.250 mm x 0.25 un, manufactured by Agilent Technologies, Inc.) was used for separation. The oven temperature was programmed to rise from 40°C (held for 3 minutes) to 230°C at a rate of 5°C/min and to be held for 15 minutes after reaching 230°C. The concentration of components in the analysis sample was quantified from the calibration curve created according to the absolute calibration curve method after the measurement in an MRM mode (m/z75 > 57). The numerical value indicates a value rounded off to the first decimal place. The concentration of isoamy! propionate per Brix was calculated by dividing the obtained numerical value by the Brix of the fermentation raw material liquid.

[0048] <Brix measurement> In the following experiments, the Brix of the fermentation raw material liquid, the fermented malt-based liquid, or the fermented malt beverage was measured using a digital refractometer (RX- 50000, manufactured by Atago Co. Ltd.) unless otherwise specified.

[0049] <pH measurement> In the following experiments, the pH of the fermentation raw material liquid, the fermented malt-based liquid, or the fermented malt beverage was measured using a pH-measuring device (LAQUAtwin-pH-33, manufactured by Horiba, Ltd.) unless otherwise specified.

[0050] <Preparation of wort A> Wort A was produced using a 200 L scale charging facility. First, 40 kg of ground malt and starch auxiliary raw materials serving as fermentation raw materials and 160 L of raw material water were pat into a charging tank. Next, the mixture in the charging tank was heated and saccharified according to a method known in the art, and the obtained saccharified solution was filtered. Hops were added to the obtained filtrate, and the filtrate was boiled. The boiled filtrate was transferred to a settling tank to separate and remove the precipitate, and the resultant was cooled, thereby obtaining wort A. The analysis values of the obtained wort A are as listed in Table

1.

[0051] [Table 1] Brix

0.41%

[0052] <Preparation of wort B and wort C> The commercially available malt extracts listed in Table 2 were diluted with water such that the Brix reached 12.8% and mixed to obtain each of wort B and wort C.

[0053] [Table 2]

[0054] [Example 1] Wort was aerated and fermented in a flask using various yeasts of the genus Cyberlindnera, and the composition of the obtained fermented liquid was examined.

[0055] <Yeasts> As the yeasts of the genus Cyberlindnera, 7 strains of Cyberlindnera jadinii (NBRC987 strain (type strain), NBRC619 strain, NBRC 1086 strain, NBRC10707 strain, NBRC10708 strain, NBRC112339 strain, and NCYC193 strain), 4 strains of Cyberlindnera mrakii (NBRC897 stram {type strain), NBRC895 strain, NBRC896 strain, and NC YC2328 strain), 4 strains of Cyberlindnera fabianii (NBRC1370 strain (type strain), NBRC 1253 strain, NBRC1254 strain, and

NCYC2662), 5 strains of Cyberlindnera saturnus (NBRC10697 strain {type strain), NBRC810 strain, NBRC941 strain, NBRC11021 strain, and NBRC 100360 strain), and 3 strains of Cyberlindnera subsufficiens (NBRC1466 strain (type strain), NBRC1467 strain, and NCYC2314 strain) were used. For comparison, Pichia kluyveri NBRC1165 strain (type strain), Torulaspora delbrueckii NBRC1083 strain, Saccharomycodes ludwigii NBRC798 strain (type strain), Saccharomyces pastorianus NBRC11024 strain (type strain), Saccharomyces cerevisiae NBRC104081 strain (type strain), and Frootzen were used.

[0056] <100 mL flask aeration stirring fermentation> The wort A was used as the fermentation raw material liquid. First, each yeast cultured overnight in wort A was diluted with water such that the Brix reached 5% and washed once with water and added to each of 100 mL wort A (Brix of 12.8%) placed in a 500 mL Erlenmeyer flask with a baffle such that the number of yeasts reached 2.0 x 10° per 1 mL of wort. Next, the wort inoculated with the yeasts was aerated and stirred at an incubator rotation speed of 160 rpm at 20°C, and fermented until the concentration of the yeasts reached ODgso of 4.0 to 6.5. Thereafter, the concentration of ethanol and the concentration of various flavor components in the obtained fermented liquid were quantified. The results thereof are listed in Table 3. In the table, “Cj” represents Cyberlindnera jadinii, “Cm” represents Cyberlindnera Mrakii, “Cf” represents Cyberlindnera fabianii, “Csa” represents Cyberlindnera saturnus, “Csu” represents Cyberlindnera subsufficiens, “Pk” represents Pichia kluyveri, “Tb” represents Torulaspora delbrueckii, “SI” represents Saccharomycodes ludwigii, “Sp” represents Saccharomyces pastorianus, and “Sc” represents Saccharomyces cerevisiae. In addition, “isoamyl propionate/Brix” indicates the concentration of isoamyl propionate in the fermented liquid per Brix.

[0057] [Table 3]

east Stain acetate acetate propionate bPEACYIBA | propionate/Brix Co [we [| |e a] wee | os | ow | a |e ww

[0058]

In the fermented liquid for which Cyberlindnera saturnus was used, ODsgo stayed at around

3. In the fermented liquid for which Saccharomycodes ludwigii was used, the yeast concentration did not reach ODseo of 4.0 due to the exhibited aggregating properties of the yeast. Therefore, the culture of these two fermented liquids was ended according to the longest culture time among all the strains. As a result, in the genus Cyberlindnera, the concentration of ethanol was less than

0.05% (vol/vol) per Brix of 12.8%, the concentration of isoamyl propionate was 1.4 ug/L or greater per Brix, and the (iAmAc + bPEAc)/IBA ratio was 5.1 or greater.

[0059] <Sensory evaluation> The sensory evaluation was carried out by 5 skilled panelists using a liquid obtained by diluting each fermented liquid to 2.56 times as an evaluation sample based on the semantic differential (SD) method of 1 to 7 points. The evaluation items are (a) intensity of wort odor (index of fermentation degree), (b) pineapple-like flavor, (c) fruity flavor, (d) intensity of miscellaneous odor, and (e) deliciousness. The items (a) to (d) were evaluated based on the flavor, and the item ({e) was evaluated based on the taste. The amount of each evaluation sample was set to 20 mL, and the order of evaluation was randomized so as to differ depending on the panelists.

[0060] In regard to the item (a), water was used for 1 point and the wort A was used for 7 points as a sample.

In regard to the items (b) to {e), scoring was performed using Pichia kluyveri as a control (4 points). In regard to the item (d) miscellaneous odor, an odor of stuffy socks after wearing, wine flavor, wort odor, and thinner odor were evaluated, and it was evaluated that miscellaneous odor was present in a case where the intensity of any of these odors was higher than that of the control.

[0061] [Table 4]

Eee Yeast Strain EE po] SE oe) SE re Ere poe value of |{pvalue{ valucof |pvaluc{ valuecof |[pvalue| valucof | pvalue | valucof Ip value

SCOICS SCOICS SCOICS SCOICS SCOICS SC | 0 I EO EE ET EE EE ET ¢ Coens foro fof ofp fe

[0062] The evaluation results are listed in Table 4. As shown by the analysis values in Table 3, it was clarified that the fermented liquid of the yeasts of the genus Cvberlindnera had a significantly strong pineapple-like flavor and a low intensity of miscellaneous odor as compared with the fermented liquid of the yeasts of the comparative control (Table 4). The significance test between the control and the evaluation sample was carried out using the Mann-Whitney U test,

[0063] [Example 2] Various yeasts of the genus Cyberlindnera were inoculated into the wort B and the wort C per Brix of 12.8% and aerated and fermented in a flask, and the composition of the obtained fermented liquid was examined. As the yeasts, 2 strains of Cyberlindnera jadinii (NBRC987 strain (type strain) and NBRC1 12339 strain), Cyberlindnera Mrakii NBRC897 strain (type strain), Cyberlindnera fabianii NBRC1370 strain (type strain), and Pichia kluyveri NBRC1165 strain {type strain) were used.

[0064] <100 mL flask aeration stirring fermentation>

The wort B or wort C was used as the fermentation raw material liquid. First, each yeast cultured overnight in wort A was diluted with water such that the Brix reached 5% and was washed once with water and added to 100 mL wort B or wort C (Brix of 12.8%) placed in a 500 mL Erlenmeyer flask with a baffle such that the number of yeasts reached 2.0 x 10° per 1 mL of wort.

Next, the wort inoculated with the yeasts was aerated and stirred at an incubator rotation speed of 160 rpm at 20°C and fermented until the number of yeasts reached 1.0 to 2.5 x 10° per 1 mL of wort. Thereafter, the concentration of ethanol and the concentration of various flavor components in the obtained fermented liquid were quantified. The results thereof are listed in Table 5.

[0065] [Table 5] Yeast Strain acetate acetate propionate bPEACYIBA | propionate/Brix on wo we |e ww Yeast Strain acetate acetate propionate bPEACYIBA | propionate/Brix EE [on [ost [at I | :

[0066] As a result, as in the case where the wort A of Example 1 was fermented, in the fermented liquid for which the yeasts of the genus Cyberlindnera were used, the concentration of ethanol was less than 0.05% (vol/vol) per Brix of 12.8%, the concentration of isoamyl propionate was 1.4 pg/L or greater per Brix, and the (iAmAc + bPEAc)/IBA ratio was 5.1 or greater.

[0067] <Sensory evaluation>

Each of the obtained fermented liquids was subjected to sensory evaluation in the same manner as in Example 1. However, in regard to the item (a), water was used for 1 point and the wort B or the wort C was used for 7 points as a sample.

The evaluation results are listed in Table 6. As a result, it was clarified that the fermented liquid for which the yeasts of the genus Cyberlindnera were used had a strong pineapple-like flavor and a low intensity of miscellaneous odor as compared with the comparative control (Table 6). The significance test between the control and the evaluation sample was carried out using the Mann-Whitney U test.

[0068] [Table 6] eT pI odor flavor miscellaneous odor Yeast Stain Average Average Average Average Average

SCOICS SCOICS SCOICS SCOICS SCOICS a a | [ow | Lew | ee [Le | hes Io LL Fermented liquid for which wort C having Brix of 12.8% was used

EE fone jo DD DLD

[0069] [Reference Example 1] The correlation between sensory evaluation of (e) deliciousness and sensory evaluation of (b) pineapple-like flavor (deliciousness x pineapple-like flavor) and the correlation between sensory evaluation of (e) deliciousness and sensory evaluation of (d) intensity of miscellaneous odor (deliciousness x intensity of miscellaneous odor) were confirmed using the sensory evaluation data obtained in [Example 1] and [Example 2]. The significance test was carried out using the Spearman’s rank correlation coefficient in order to calculate the rank correlation coefficient. The results thereof are shown in FIGS. | and 2.

[0070] As a result, it was confirmed that (e) deliciousness and (b) pineapple-like flavor have a significant positive correlation (p = 0.693, p = 0.000) (FIG. 1). Further, it was also confirmed that (e) deliciousness and (d) intensity of miscellaneous odor have a significant negative correlation (p = -0.640, p = 0.000) (FIG. 2). As shown in these results, it was clarified that the deliciousness increases as the pineapple-like flavor strengthens or the miscellaneous odor weakens.

[0071] [Example 3] Various yeasts of the genus Cyberlindnera were inoculated into the wort B with a Brix of 10% or 20% and aerated and fermented in a flask, and the composition of the obtained fermented liquid was examined. As the yeasts, 2 strains of Cyberlindnera jadinii (NBRC987 strain (type strain) and NBRC112339 strain), Cyberlindnera Mrakii NBRC897 strain (type strain), and Cyberlindnera fabianii NBRC1370 strain (type strain) were used.

[0072] <100 mL flask aeration stirring fermentation> As the fermentation raw material liquid, a malt extract “MALT extract (Non-diastatic)” (manufactured by Coopers) listed in Table 2 was used by being diluted with water such that the Brix reached 10% or 20%. Each yeast cultured overnight in wort A was diluted with water such that the Brix reached 5% and was washed once with water and added to 100 mL of the fermentation raw material liquid (Brix of 10% or 20%) placed in a 500 mL Erlenmeyer flask with a baffle such that the number of yeasts reached 2.0 x 10° per I mL of wort. The wort was aerated and stirred at an incubator rotation speed of 160 rpm at 20°C and fermented until the number of yeasts reached 1.0 to 2.5 x 103 per 1 mL of wort, and the concentration of ethanol and the concentration of the flavor components in the fermented liquid were quantified. The results thereof are listed in Table 7.

[0073] [Table 7]

‘ermented liquid for which fermentation raw material liquid having Brix of 10% was used Yeast Stain acetate acetate propionate | bPEAcYIBA | propionate/Brix Fermented figuid for which fermentation raw material liquid having Brix of 20% was used f-phenethyl (iAmAc + Isoamyl Yeast Stain acetate acetate propionate bPEACYIBA | propionate/Brix

[0074] As a result, as in the case where the wort B having a Brix of 12.8% was used as the fermentation raw material liquid, in the fermented liquid obtained using the wort B having a Brix of 10% or 20% as the fermentation raw material liquid in the yeasts of the genus Cyberlindnera, the concentration of ethanol was less than 0.05% (vol/vol), the concentration of isoamyl propionate was 1.4 ng/L or greater per Brix, and the (AmAc + bPEACc)/IBA ratio was 5.1 or greater.

[0075] [Example 4] It was investigated whether the composition of the fermented liquid obtained by inoculating the yeasts of the genus Cyberlindnera into the wort A and performing aerated fermentation in a flask was changed depending on the number of yeasts at the end of fermentation. As the yeast, Cyvberlindnera jadinii NBRC112339 strain was used.

[0076] <100 mL flask aeration stirring fermentation> The wort A was used as the fermentation raw material liquid. First, the yeasts cultured overnight in the wort A were diluted with water such that the Brix reached 5% and washed once with water and added to 100 mL wort A (Brix of 12.8%) placed in a 500 ml. Erlenmeyer flask with a baffle such that the number of yeasts reached 2.0 x 10° per 1 mL of wort. Next, the wort inoculated with the yeasts was aerated and stirred at an incubator rotation speed of 160 rpm at

20°C and fermented until the number of yeasts reached (a) 6.5 x 107, (b) 8.6 x 107, and (©) 1.1 x 10% per 1 mL of wort. Thereafter, the concentration of ethanol and the concentration of various flavor components in the obtained fermented liquid were quantified. The results thereof are listed in Table 8.

[0077] [Table 8] Isoamyl f-phenethyl Isoamyl (iAmAc + Isoamyl condition cov | met | ower | ome | owt | | wr

[0078] The tendency that the concentration of isoamyl acetate, the concentration of B-phenethyl acetate, and the concentration of isoamyl propionate in the obtained fermented liquid increased as the number of yeasts at the end of the fermentation increased was observed. The concentration of isobutanol was also high depending on the amount of yeasts inoculated into the fermentation raw material liquid, but was not affected as much as the esters.

[0079] [Example 5] The wort A was inoculated with the yeasts of the genus Cyberlindnera and aerated and fermented in a jar fermenter, and. the composition of the obtained fermented liquid was examined. As the yeast, Cyberlindnera jadinii NBRC112339 strain was used.

[9080] <Jar fermenter fermentation> 600 mL of the wort A (Brix of 12.8%) was placed in a 2 L jar fermenter (manufactured by B. E. Maruhishi Co., Ltd.), and an antifoaming agent (KM-72GS, manufactured by Shin-Etsu Chemical Co., Ltd.) was added thereto such that the final concentration thereof reached 0.165 g/L. Next, fermentation was performed in the jar fermenter under any of three conditions that (a) yeasts were added such that the number of yeasts reached 2.0 x 10" per 1 mL of the wort and the wort was fermented at a temperature of 20°C, (b) yeasts were added such that the number of yeasts reached

2.0 x 10° per 1 mL of the wort and the wort was fermented at a temperature of 20°C, or (¢) yeasts were added such that the number of yeasts reached 2.0 x 10° per 1 mL of the wort and the wort was fermented at a temperature of 17°C. The aeration during fermentation was started under conditions of 240 rpm and 0.5 vvm, the concentration of dissolved oxygen was monitored, and the oxygen supply amount was maintained by increasing the stirring rate such that the concentration of dissolved oxygen that was decreased with yeast growth was held at | ppm or greater. After the fermentation was carried out until the number of yeasts was approximately 1.0 x 10% per 1 mL of the fermented liquid, the concentration of ethanol and the concentration of the flavor components in the fermented liquid were quantified in the same manner as in Example |. The results thereof are listed in Table 9.

[0081] [Table 9] condition a | wer | ome | owe | owt | | wr

[0082] As a result, similar to Example 1 and the like in which the fermentation was carried out by aeration stirring culture in a flask, the concentration of ethanol in the obtained fermented liquid was less than 0.05% (vol/vol) per Brix of 12.8%, the concentration of isoamyl propionate in the obtained fermented liquid was 1.4 pg/L or greater per Brix, and the GAmAc + bPEAc)/IBA ratio was 5.1 or greater under all the conditions (a) to {(c).

[0083] [Example 6] The wort A was inoculated with the yeasts of the genus Cyberlindnera and aerated and fermented in a 100 L fermentation tank, and the composition of the obtained fermented liquid was examined. As the yeast, Cyberlindnera jadinii NBRC112339 strain was used.

[0084] <Fermentation tank fermentation> 60 L of the wort A (Brix of 12.8%) was placed in a 100 L fermentation tank. Next, the fermentation was carried out by adding the yeasts to the fermentation tank such that the number of yeasts reached 2.0 x 10° per 1 mL of the wort. During the fermentation, the liquid was allowed to flow from a sampling valve port of the fermentation tank to an aeration line using a pump, passed through a dissolved oxygen meter from a static mixer, and returned to the bottom of the tank for circulation. While the amount of dissolved oxygen was monitored so as not to cause oxygen deficiency during yeast growth, aeration was performed intermittently such that the concentration of dissolved oxygen reached 1 ppm or greater. The fermentation was ended before and after the number of yeasts reached 2.0 x 107 per 1 mL of the fermented liquid. After the end of the fermentation, the fermented liquid contained less than 0.05% (vol/vol) of ethanol per Brix of

12.8% and had a pineapple-like fruitiness and a refreshing feeling with little miscellaneous odor.

[0085] [Reference Example 2] The wort A was inoculated with the yeasts of the genus Cyberlindnera and allowed to stand in a flask for fermentation, and the composition of the obtained fermented liquid was examined. As the yeasts, 2 strains of Cyberlindnera jadinii (NBRC987 strain (type strain) and NBRC112339 strain), Cyberlindnera Mrakii NBRC897 strain (type strain), Cyberlindnera fabianii NBRC1370 strain (type strain), Cvberlindnera saturnus NBRC 10697 strain (type strain), and Cyberlindnera subsufficiens NBRC1466 strain {type strain) were used.

[0086] <Standing fermentation> Each yeast cultured overnight in the wort A was diluted with water such that the Brix reached 5% and was washed once with water and added to 170 mL wort A (Brix of 12.8%) placed ina 200 mL Erlenmeyer flask with a baffle such that the number of yeasts reached 2.0 x 107 per 1 mL of wort, and standing fermentation was carried out at 20°C for 3 days. After the end of the fermentation, the concentration of ethanol and the concentration of the flavor components in the fermented liquid were quantified in the same manner as in Example 1. The results thereof are listed in Table 10.

[0087] [Table 10]

“‘ermented liquid for which wort B having Brix of 10% was used Yeast Stain acetate acetate propionate bPEAC)IBA | propionate/Brix

[0088] In the fermented liquid subjected to standing fermentation, the concentration of ethanol was

0.25% (vol/vol) or greater regardless of which yeasts of the genus Cyberlindnera were used, which was greater than the case where aerated fermentation was performed. Further, as reported in Belluta, et al., Journal of the American Society of Brewing Chemists, 2019, DOI:

10.1080/03610470.2019.1569452 and the like, all the concentrations of isoamyvl acetate, B- phenethyl acetate, and isoamy! propionate in the fermented liquid subjected to standing fermentation were greater than the case where aeration fermentation was performed using the yeasts of the genus Saccharomyces, but the concentrations thereof were clearly lower than the case where aeration culture was performed using the yeasts of the genus Cvberlindnera.

Claims

CONCLUSIONS

A method of producing a malt-based fermented liquor, comprising: a step of inoculating a yeast of the genus Cyberlindnera into fermentation stock liquor comprising a malt saccharified product and performing aerated fermentation on the fermentation stock liquor .

The method for producing a malt-based fermented liquid according to claim 1, wherein a malt-based fermented liquid comprising less than 0.2% (vol/vol) ethanol is produced.

The method for producing a malt-based fermented liquid according to claim 1 or 2, wherein a malt-based fermented liquid is produced in which the concentration of isoamyl propionate is 1.4 pg/L or higher per Brix and the ratio of the total concentration of 1soamyl acetate and [B-phenethyl acetate relative to the concentration of isobutanol [([concentration of isoamyl acetate (mg/L)] + [concentration of B-phenethyl acetate (mg/L )]) / [concentration of isobutanol (mg/L )]] 5.1 or higher.

A process for producing a malt-based fermented liquor according to any one of claims 1 to 3, wherein the fermentation stock liquor has a Brix of 10% to 20% and a pH of 3 to 6.

The method for producing a malt-based fermented liquor according to any one of claims 1 to 4, wherein the aerated fermentation is carried out at 6°C to 28°C under the condition that the dissolved oxygen concentration in the fermented liquor is 1 ppm or higher.

A process for producing a malt-based fermented liquor according to any one of claims 1 to 5, wherein 1.0 = 10 7 to 2.0 x 10 7 yeasts per | ml in the fermentation stock liquid.

A process for producing a malt-based fermented liquor according to any one of claims 1 to 6, wherein the yeast comprises one or more species selected from the group consisting of Cyberfindnera Jadinii, Cyberlindnera murakii, Cyberlindnera fabianii, Cyberlindnera saturnus, and Cyberlindnera subsaffinics.

A method for producing a fermented malt beverage, wherein a fermented malt beverage is produced by using a fermented malt-based liquid obtained by the method for producing a fermented malt-based liquid according to any one of claims 1 to 7 as raw material.

A method of producing a fermented malt beverage according to claim 8, comprising a step of diluting the fermented malt-based liquid.

A method of producing a fermented malt beverage according to claim 8 or 9, wherein the fermented malt beverage is a beer-like sparkling beverage.

A fermented malt beverage comprising a malt-based fermented liquid obtained by the process for producing a malt-based fermented liquid according to any one of claims 1 to 7, wherein the concentration of isoamyl propionate is 1.4 µg/L or more per Brix, the ratio of the total concentration of isoamyl acetate and β-phenethyl acetate to the concentration of isobutanol [{[concentration of isoamyl acetate (mg/L)] + [concentration of [3-phenethyl acetate (mg/L)]) / [concentration of isobutanol (mg/L) )]|] is 5.1 or more, and the concentration of ethanol is 0.5% (vol/vol) or less.

The fermented malt beverage of claim 11, wherein the fermented malt beverage is a beer-like sparkling beverage.