Method for producing malt fermented liquid

Abstract

The present invention provides: a method for producing a fermented malt liquid that has a step for inoculating a Kluyveromyces marxianus yeast into a liquid fermentation material containing a saccharified malt and carrying out aerated fermentation; and a method for producing a fermented malt beverage that uses, as a raw material, the fermented malt liquid obtained by the method for producing a fermented malt liquid. These methods provide a fermented malt liquid having a low ethanol density and a fruity aroma, and a fermented malt beverage obtained from the fermented malt liquid.

Description

[Technical Field]

[0001] The present invention relates to a method for producing a fermented liquid having a low ethanol concentration and a fruity aroma, using malt saccharified products as a fermentation raw material, and a method for producing a beverage containing the fermented liquid produced by this method as a raw material. This application claims priority based on Japanese Patent Application No. 2020-104615, filed in Japan on June 17, 2020, and the contents of that application are incorporated herein by reference. [Background technology]

[0002] In recent years, a wide variety of alcoholic beverages have been launched to meet the diverse tastes of consumers. For example, there is a growing demand for alcoholic beverages with relatively low alcohol content because they are easier to drink. On the other hand, in the case of fermented alcoholic beverages, fermentation produces various aromatic components in addition to ethanol by yeast, contributing to the flavor of the beverage.

[0003] In fermented alcoholic beverages, the various aroma components produced during fermentation depend on the type of yeast used. For example, the most widely used yeast in beer and wine brewing is of the genus Saccharomyces, but in recent years, it has been possible to produce beers with different flavors from conventional beers by using yeasts other than those of the genus Saccharomyces. For example, a method is known for producing beer with 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 (Patent Document 1).

[0004] On the other hand, there is also a thriving development of food and beverages that utilize yeast fermentation, in addition to alcoholic beverages. For example, by fermenting rice flour saccharification liquid, a rice flour saccharification fermented beverage with excellent digestibility and absorption can be obtained. By using a specific strain of Kluyveromyces marxianus, a yeast isolated from kefir, as the yeast used in this process, a rice flour saccharification fermented beverage that is fruity and makes the most of the sweetness of the rice flour saccharification liquid itself can be obtained (Non-Patent Literature 1). Kluyveromyces marxianus has the ability to produce ethyl acetate from lactose only under aeration conditions (Non-Patent Literature 2). [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] International Publication No. 2014 / 202564 [Non-patent literature]

[0006] [Non-Patent Document 1] Mihoko Tominaga et al., "Fermentation of rice flour saccharification liquid by yeast isolated from kefir, Kluyveromyces marxianus HU-1," Journal of the Japan Society for Bioscience, Biotechnology, and Agrochemistry, 1998, Vol. 72, No. 4, pp. 481-488. [Non-Patent Document 2] Urit et.al.,Engineering in Life Sciences,2013, vol.13(3) p.247-260. [Overview of the project] [Problems that the invention aims to solve]

[0007] In the production of fermented alcoholic beverages, suppressing the degree of fermentation to keep the ethanol concentration low can also reduce the content of aromatic components and may result in unpleasant odors due to insufficient fermentation. The present invention aims to provide a method for producing a malt ferment liquid having a low ethanol concentration and a fruity aroma, a method for producing a malt ferment beverage using the malt ferment liquid produced by the said method, and a malt ferment beverage produced by the said method. [Means for solving the problem]

[0008] The inventors of the present invention conducted intensive research to solve the above problems and, as a result, discovered that by using the yeast species Cluyveromyces marxianus, which had not been conventionally used for fermentation of fermentation raw materials containing malt saccharified products, and performing aerated fermentation, ethanol production is suppressed, and instead, the production of aroma components such as esters is promoted, thereby enabling the production of a malt fermentation liquid with a fruity aroma and a low ethanol concentration, thus completing the present invention.

[0009] The method for producing malt fermented liquid and the method for producing malt fermented beverage according to the present invention are as follows [1] to [8]. [1] A fermentation liquid containing malt saccharified products is inoculated with the yeast species Cluyveromyces marxianus and fermented at 0-28°C. The dissolved oxygen concentration in the fermentation liquid is maintained at 1 ppm (mg / L) or higher. It has a process of aeration and fermentation, The Brix of the aforementioned fermentation raw material liquid is 4° or higher, and the ethanol concentration is less than 1.0% (vol / vol). the law of nature, The fermentation time for the aforementioned aeration fermentation is within 3 days. A method for producing malt fermentation liquid. [2] The method for producing the malt fermented liquid according to [1], wherein the Brix of the fermented raw material liquid is 4° to 20°. [3] A method for producing a malt ferment liquor according to [1] or [2], wherein the propyl acetate concentration is 0.1 ppm (mg / L) or higher. [4] A method for producing a malt ferment liquor with an ethanol concentration of 0.5% (vol / vol) or less, according to any of the methods described in [1] to [3] above. [5] A method for producing a malt ferment liquor with an ethyl acetate concentration of 50 ppm (mg / L) or more, according to any of the above [1] to [4]. [6] A method for producing a malt fermentation beverage, which comprises producing a malt fermentation broth by the method for producing a malt fermentation broth according to any one of [1] to [5] above, and using the obtained malt fermentation broth as a raw material to produce a malt fermentation beverage. [7] The method for producing a malt fermentation beverage according to [6] above, which comprises a step of diluting the malt fermentation broth. [8] The method for producing a malt fermentation beverage according to [6] or [7] above, wherein the malt fermentation beverage is a beer-like foaming beverage 。 [Advantages of the Invention]

[0010] In the method for producing a malt fermentation broth according to the present invention, aeration fermentation is carried out using a yeast of the Kluyveromyces marxianus species, which has a low ethanol-producing ability and a high ability to produce esters such as ethyl acetate, isoamyl acetate, β-phenethyl acetate, propyl acetate, etc., and alcohols having 3 or more carbon atoms such as isoamyl alcohol and β-phenethyl alcohol. Therefore, the method for producing a malt fermentation broth according to the present invention can provide a malt fermentation broth having a low ethanol concentration and a fruity aroma. Further, by using the malt fermentation broth, a malt fermentation beverage having a low ethanol concentration and a fruity aroma can be provided. [Brief Description of the Drawings]

[0011] [Figure 1] It is a diagram showing the results of a sensory test of "intensity of wort odor" conducted in Example 5. [Figure 2] [[ID=​​​​​​​​​​​​​In the present invention and this specification, "malt-fermented beverage" means a beverage produced using malt or a processed product thereof as a raw material and manufactured through a fermentation process using yeast. The malt-fermented beverage may be an alcoholic beverage, or it may be a so-called non-alcoholic beverage or low-alcohol beverage with an ethanol concentration of less than 1.0% (vol / vol). Furthermore, the ethanol concentration of the malt-fermented beverage is preferably less than 0.5% (vol / vol), more preferably less than 0.1% (vol / vol), and even more preferably less than 0.05% (vol / vol).

[0013] Examples of malt-fermented beverages include beer-like sparkling beverages. In the present invention and this specification, "beer-like sparkling beverage" means a sparkling beverage that has a flavor, taste, and texture equivalent to or similar to beer, and that has beer-like characteristics (a taste that evokes beer in terms of aroma and flavor). Specifically, examples of malt-fermented beer-like sparkling beverages include beer, sparkling alcoholic beverages made from malt, etc. In addition, liqueurs obtained by mixing malt fermentation liquid with ethanol-containing distillate may also be used. Ethanol-containing distillate is a solution containing ethanol obtained by a distillation operation, and may be, for example, raw material alcohol (ethanol), or distilled spirits such as whiskey, brandy, vodka, rum, tequila, gin, shochu, etc.

[0014] The present invention relates to a method for producing malt fermented liquid, which includes the step of inoculating a fermentation raw material liquid containing malt saccharified products with Kluiveromyces marxianus yeast and carrying out aeration fermentation. Kluiveromyces marxianus yeast has a lower ethanol production capacity during aeration fermentation than Saccharomyces yeast, but instead has a high capacity to produce aroma components such as esters and alcohols with 3 or more carbon atoms. Therefore, by using Kluiveromyces marxianus yeast, a fermented liquid with a low ethanol concentration and rich in esters such as ethyl acetate, isoamyl acetate, β-phenethyl acetate, and propyl acetate can be obtained.

[0015] The yeast used in this invention is not particularly limited as long as it is of the species Cluyveromyces marxianus. However, from the standpoint of safety when applying the malt fermentation liquid produced to food and beverages, it is preferable to use Cluyveromyces marxianus yeast isolated from food and beverages, their ingredients, or edible plants and animals. These Cluyveromyces marxianus yeasts can be appropriately selected and used from strains of Cluyveromyces marxianus available from strain preservation institutions or distributors such as the National Biotechnology Center (NBRC) of the National Institute of Technology and Evaluation and the National Collection of Yeast Culture (NCYC).

[0016] In the present invention, the fermentation liquid obtained by inoculating and fermenting with Cluyveromyces marxianus yeast is not particularly limited as long as it contains malt saccharified products and contains a carbon source or nitrogen source that Cluyveromyces marxianus yeast can utilize. For example, it may consist only of malt saccharified products, or it may contain a carbon source or nitrogen source other than malt saccharified products.

[0017] In the present invention and this specification, "malt saccharified product" refers to a product obtained by saccharifying the sugars in malt with various enzymes. The enzymes used in the saccharification process may be enzymes naturally present in malt, or enzymes derived from sources other than malt. For example, by adding water to malt or its crushed product and holding it at 50-60°C, saccharified product can be obtained by amylase derived from malt. In this case, saccharifying enzymes derived from sources other than malt may be added, or enzymes other than saccharifying enzymes, such as proteolytic enzymes, may be used in combination.

[0018] In the present invention, as the saccharified malt, for example, wort or malt extract can be used. Malt extract is a concentrated form of wort and mainly consists of maltose. Malt extract can be used as a beverage by diluting it with carbonated water, or as a raw material for food and beverages. Concentration of wort can be carried out by conventional methods. Vacuum concentration is preferred because it minimizes the impact on the components in the wort, but it can also be concentrated by heating to remove the solvent.

[0019] Wort is prepared by heating a mixture containing malt and raw water to saccharify the starch in the malt. It is preferable to use malt milled, which is obtained by grinding the malt. The grinding of the malt can be carried out by conventional methods. The malt milled may have undergone processing that is normally done before and after the grinding process.

[0020] The mixture containing malt and raw water may also contain fermentation raw materials other than malt. These fermentation raw materials may be grain raw materials or carbohydrate raw materials. Examples of grain raw materials other than malt include barley and wheat, rice, corn, soybeans and other legumes, and potatoes. The grain raw materials can also be used as grain grinds, grain syrups, grain extracts, etc. The grain grinds may be corn starch, corn grits, etc., which have undergone processing that is normally done before and after grinding. Examples of carbohydrate raw materials include sugars such as liquid sugar.

[0021] The wort may be used as a fermentation raw material as is, but it is preferable to boil it before inoculating it with yeast. This boiling process sterilizes the wort and allows for safer fermentation. When boiling, it is preferable to remove insoluble solids by filtration or other means before boiling. The filtration process is not particularly limited and can be appropriately selected from solid-liquid separation processes commonly used in the manufacturing process of food and beverages, such as diatomaceous earth filtration or filter filtration. Alternatively, instead of the filtrate of this sugar solution, a mixture of malt extract and warm water may be used and boiled. The boiling method and conditions can be determined as appropriate.

[0022] By adding herbs and other ingredients as appropriate before or during boiling, a malt fermentation liquid with a desired flavor can be produced. Hops, in particular, are preferably added before or during boiling. Boiling in the presence of hops allows for efficient extraction of the hop's flavor and aroma components. The amount of hops added, the method of addition (e.g., adding in several stages), and the boiling conditions can be determined as appropriate.

[0023] Before inoculating the boiled wort with yeast, it is preferable to remove any residue, such as proteins, that has settled. This residue can be removed by any solid-liquid separation method, but generally, a tank called a whirlpool is used to remove the sediment. The temperature of the boiling liquid at this time should be 15°C or higher, and is generally set to around 50-80°C. The clear wort (filtrate) after the residue has been removed is cooled to an appropriate fermentation temperature using a plate cooler or the like. This residue-free wort becomes the raw material for fermentation.

[0024] The cooled fermentation raw material liquid is inoculated with Cluyveromyces marxianus yeast, and fermentation is carried out. The cooled fermentation raw material liquid may be used for fermentation as is, or it may be used for fermentation after being adjusted to the desired extract concentration. In the present invention, the fermentation raw material liquid inoculated with Cluyveromyces marxianus yeast preferably has a Brix of 4° to 20°, more preferably 7° to 18°, even more preferably 9° to 16°, and still more preferably 10° to 14°.

[0025] In this invention and in this specification, the Brix value of the fermentation raw material liquid can be measured by conventional methods using a measuring device such as a refractometer (RX-5000α, manufactured by Atago Corporation).

[0026] Malt ferment is obtained by aerated fermentation of a fermentation raw material liquid inoculated with Cluyveromyces marxianus yeast. When Cluyveromyces marxianus yeast is fermented under aerobic conditions, ethanol production is further suppressed, and the production of esters and alcohols with 3 or more carbon atoms is increased, resulting in a malt ferment with superior aroma and a lower ethanol concentration.

[0027] Aerated fermentation is a fermentation method carried out under aerobic conditions in which oxygen or air can be supplied to the culture medium, and can be performed by conventional methods. For example, shaking culture, stirring culture, and aerated stirring culture can be used. The stirring conditions and the amount of air or oxygen gas supplied should be as long as they allow the culture environment to be kept aerobic, and can be appropriately selected depending on the culture apparatus, viscosity of the culture medium, etc. For example, when performing large-scale culture, such as in culture using a jar fermenter, the amount of dissolved oxygen in the fermentation liquid can be monitored while culturing and fermenting, and in this case, air or oxygen gas can be continuously or intermittently supplied so that the amount of dissolved oxygen in the fermentation liquid is above a predetermined amount (e.g., 1 ppm (mg / L)).

[0028] The fermentation conditions in the method for producing malt fermented liquid according to the present invention can be appropriately selected from among the general yeast fermentation conditions that can maintain an aerobic culture environment during fermentation, and can be modified as necessary. For example, the fermentation temperature is 0 to 35°C, preferably 12 to 28°C, and more preferably 15 to 25°C. By carrying out fermentation within the above temperature range, the production of esters and alcohols having 3 or more carbon atoms by the Cluyveromyces marxianus yeast can be carried out efficiently. The fermentation time is not particularly limited, but is preferably within 7 days, more preferably within 4 days, and even more preferably within 3 days.

[0029] The malt ferment liquor produced in this way has a low ethanol concentration and a high content of aromatic compounds. These aromatic compounds include ethyl acetate, isoamyl acetate, isoamyl alcohol, β-phenethyl acetate (1-phenylethyl acetate), β-phenethyl alcohol (2-phenylethanol), and propyl acetate. These are the components that cause fruity aromas, and because of their high content, malt ferment liquor obtained by aeration fermentation with Cluyveromyces marxianus yeast has an excellent fruity aroma.

[0030] The ethanol concentration of the malt ferment liquor obtained by the present invention is not particularly limited. For example, when a fermentation raw material liquid with a Brix of 4° to 20° is subjected to aeration fermentation, the ethanol concentration of the produced malt ferment liquor is less than 1.0% (vol / vol), preferably 0.5% (vol / vol) or less, more preferably 0.25% (vol / vol) or less, and even more preferably 0.15% (vol / vol) or less.

[0031] In this invention and specification, the ethanol concentration of malt fermented liquid and beverages can be measured by methods such as the enzymatic method using the multi-functional biosensor BF-5 (manufactured by Oji Instruments Co., Ltd.), the distillation-density (specific gravity) method, the gas chromatography (GC) analysis method, and the oxidation method.

[0032] The malt ferment liquor obtained by the present invention preferably contains propyl acetate above the detection limit. Propyl acetate is hardly detectable in ferment liquors fermented using Saccharomyces or Pichia yeasts, which are conventionally used in fermentation, and is an ester produced by aeration fermentation using Cluyveromyces marxianus yeast. The concentration of propyl acetate in the malt ferment liquor obtained by the present invention is not particularly limited. For example, when a fermentation raw material liquid with a Brix of 4° to 20° is aeration fermented, the propyl acetate concentration of the produced malt ferment liquor is preferably 0.1 ppm (mg / L) or higher, more preferably 0.4 ppm (mg / L) or higher, and even more preferably 0.7 ppm (mg / L) or higher. Furthermore, when a fermentation raw material liquid with a Brix of 4° to 20° is subjected to aeration fermentation, the propyl acetate concentration of the produced malt ferment liquor is preferably 20.0 ppm (mg / L) or less, more preferably 15.0 ppm (mg / L) or less, and even more preferably 10.0 ppm (mg / L) or less.

[0033] The Cluyveromyces marxianus yeast also exhibits excellent ethyl acetate production ability. The ethyl acetate concentration of the malt ferment broth obtained by the present invention is not particularly limited. For example, when a fermentation raw material with a Brix of 4° to 20° is subjected to aeration fermentation, the ethyl acetate concentration of the produced malt ferment broth is preferably 50 ppm (mg / L) or higher, more preferably 100 ppm (mg / L) or higher, and even more preferably 150 ppm (mg / L) or higher. Furthermore, when a fermentation raw material with a Brix of 4° to 20° is subjected to aeration fermentation, the ethyl acetate concentration of the produced malt ferment broth is preferably 500.0 ppm (mg / L) or lower, more preferably 450.0 ppm (mg / L) or lower, and even more preferably 400.0 ppm (mg / L) or lower.

[0034] The malt ferment liquor obtained by the present invention contains a large amount of isoamyl acetate and isoamyl alcohol. The concentrations of isoamyl acetate and isoamyl alcohol in the malt ferment liquor obtained by the present invention are not particularly limited. For example, when a fermentation raw material liquid with a Brix of 4° to 20° is subjected to aeration fermentation, the total concentration of isoamyl acetate and isoamyl alcohol in the produced malt ferment liquor is preferably 15 ppm (mg / L) or more, and more preferably 30 ppm (mg / L) or more.

[0035] The malt ferment liquor obtained by the present invention contains a large amount of β-phenethyl acetate and β-phenethyl alcohol. The concentrations of β-phenethyl acetate and β-phenethyl alcohol in the malt ferment liquor obtained by the present invention are not particularly limited. For example, when a fermentation raw material liquid with a Brix of 4° to 20° is subjected to aeration fermentation, the total concentration of β-phenethyl acetate and β-phenethyl alcohol in the produced malt ferment liquor is preferably 10 ppm (mg / L) or more, and more preferably 15 ppm (mg / L) or more.

[0036] The amounts of esters such as ethyl acetate, isoamyl acetate, β-phenethyl acetate, and propyl acetate, as well as alcohols with 3 or more carbon atoms such as isoamyl alcohol and β-phenethyl alcohol, in malt fermented liquid or malt fermented beverages can be determined, for example, from the peak area of ​​a chromatograph obtained by GC analysis. The quantitative method from the peak area is not particularly limited, but examples include the area percentage method, the internal standard method, the standard addition method, and the absolute calibration curve method. Specifically, it can be determined by the method described in the examples below.

[0037] The malt fermentation liquid obtained in this invention can be used as a raw material for food and beverages as is. The food and beverages produced using this malt fermentation liquid as a raw material are not particularly limited. As food and beverages produced using the malt fermentation liquid obtained in this 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.

[0038] For example, the obtained malt fermentation liquid can be subjected to solid-liquid separation to remove yeast and other contaminants, thereby obtaining a malt fermented beverage. The solid-liquid separation treatment can be any method capable of removing yeast and other contaminants, such as centrifugal separation, diatomaceous earth filtration, or filter filtration using a filter with an average pore size of approximately 0.4 to 0.5 μm, and these treatments may be combined. Hops, hop extract, flavorings, etc., may also be added to the obtained malt fermentation liquid to adjust the flavor of the fermented malt beverage. Flavor adjustment may be performed before or after the solid-liquid separation treatment. The malt fermentation liquid after solid-liquid separation may also be stored and aged under low-temperature conditions of approximately 0°C. Further treatments such as pH adjustment and heat treatment may be performed according to conventional methods to produce the malt fermented beverage.

[0039] The obtained malt fermented liquid can be diluted as appropriate to produce a malt fermented beverage with extract concentration and ethanol concentration 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 for example, it can be diluted 2 to 10 times, with 2 to 5 times dilution being more preferable, and 2 to 3 times dilution being even more preferable. The step of diluting the malt fermented liquid may be performed after the fermentation process is completed, after the removal of yeast, etc., or on the malt fermented liquid after maturation.

[0040] The concentrations of ethanol, various esters, and alcohols with 3 or more carbon atoms in a malt fermented beverage obtained using the malt fermented liquid obtained in the present invention as a raw material are not particularly limited. By using the malt fermented liquid obtained in the present invention as a raw material, a malt fermented beverage with a low ethanol concentration and a fruity aroma can be produced. Examples of such malt fermented beverages include a malt fermented beer-like sparkling beverage having a propyl acetate concentration of 0.1 ppm (mg / L) or more and an ethanol concentration of 0.5% (vol / vol) or less. For such malt fermented beer-like sparkling beverages, the propyl acetate concentration is preferably 0.2 ppm (mg / L) or more, more preferably 0.3 ppm (mg / L) or more. For such malt fermented beer-like sparkling beverages, the propyl acetate concentration is preferably 20.0 ppm (mg / L) or less, more preferably 15.0 ppm (mg / L) or less, and even more preferably 10.0 ppm (mg / L) or less. For the malt-fermented beer-like sparkling beverage, the ethanol concentration is preferably 0.25% (vol / vol) or less, more preferably 0.1% (vol / vol) or less, and even more preferably 0.05% (vol / vol) or less. [Examples]

[0041] 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.

[0042] <Analysis of aroma components such as ethyl acetate> In subsequent experiments, unless otherwise specified, the concentrations of ethyl acetate, isoamyl acetate, isoamyl alcohol, β-phenethyl acetate, and β-phenethyl alcohol in the fermentation raw material liquid, malt fermentation liquid, or beverage were measured by GC analysis using hexyl alcohol and hexyl acetate as internal standards after extraction with carbon disulfide.

[0043] Specifically, first, 30 g of the sample was taken into a container, 6 g of ammonium sulfate and 3 mL of carbon disulfide were added, and 150 μL of a mixture of 500 ppm hexyl alcohol and 20 ppm hexyl acetate was added as an internal standard. The mixture was then shaken and extracted for 10 minutes. After that, the mixture was centrifuged at 3,000 rpm for 10 minutes, and the solvent layer was collected and subjected to GC analysis. For the GC analysis, 2 μL of the collected solvent layer was injected into a column (DB-FFAP, 30 m × 0.25 mm ID; 0.25 μm F.T., Agilent) using the split injection method, held at 40°C for 2 minutes, and then heated to 230°C at a rate of 7°C / min and held for 10 minutes. The concentrations of each component in the analytical sample were calculated by creating calibration curves for each component, using hexyl alcohol as the internal standard for ethyl acetate, isoamyl acetate, isoamyl alcohol, and β-phenethyl alcohol, and hexyl acetate as the internal standard for β-phenethyl acetate.

[0044] <Analysis of propyl acetate> In Examples 1 and 2, propyl acetate in the malt ferment broth or beverage was measured by SPME-GC / MS (Solid Phase Micro Extraction - Gas Chromatography / Mass Spectrometry). Specifically, adsorbed components were subjected to GC / MS analysis. First, 5 g of the sample was taken into a 20 mL vial, mixed, and then the lid was loosened to release the gas. The vial was heated at 60°C for 30 minutes while being stirred, and then the gas phase components in the headspace were adsorbed onto SPME (DVB / CAR / PDMS (SUPELCO), film thickness 50 / 30 μm, length 1 cm) and subjected to GC / MS analysis (splitless method, Agilent DB-WAX column, 60 m × 0.250 mm × 0.25 μm). The GC / MS analysis was performed by holding at 38°C for 10 minutes, then raising the temperature to 230°C at a rate of 3°C / min, and then holding for 23 minutes. The propyl acetate concentration in the sample was calculated using the standard addition method.

[0045] In Examples 3 and 4, propyl acetate in malt ferment broth or beverages was measured by headspace gas chromatography-mass spectrometry (HS-GC / MS). Specifically, 1 ml of sample and 3 g of sodium chloride were added to 9 ml of ultrapure water, and the propyl acetate in the sample was measured by HS-GC / MS. The concentration of propyl acetate in the sample was quantified using an absolute calibration curve. Specifically, a calibration curve was first created using standard substances from the amount of component (μg) and peak area value. Using this calibration curve, the propyl acetate content (μg) was determined from the peak area value of propyl acetate measured by HS-GC / MS, and the concentration of propyl acetate in the sample (μg / mL) was quantified by dividing this value by the volume of the sample (mL).

[0046] Here, we confirmed that the concentration of propyl acetate in the sample, quantified by the standard addition method after measuring propyl acetate in the sample using the SPME-GC / MS method, and the concentration of propyl acetate in the sample, quantified by the absolute calibration curve method after measuring propyl acetate in the sample using the HS-GC / MS method, were in general agreement.

[0047] <Ethanol concentration measurement> In subsequent experiments, unless otherwise specified, the ethanol concentration in the malt ferment broth or beverage was measured by the following method. The ethanol concentration in the sample was diluted with distilled water to 0.5-2.0% (vol / vol), and the solution was measured using a multi-functional biosensor (BF-5, manufactured by Oji Instruments Co., Ltd.). The obtained measurement value was multiplied by the dilution factor to determine the ethanol concentration (%(vol / vol)) in the sample.

[0048] The multifunctional biosensor is a device that electrochemically detects hydrogen peroxide (H2O2) generated by oxidation with alcohol oxidase, and can measure the total concentration of methanol and ethanol. Since yeast does not biosynthesize methanol, the malt fermentation broth contains only a trace amount of methanol within the error range. Therefore, the measured value by the multifunctional biosensor is the measured value of the ethanol amount in the malt fermentation broth sample.

[0049] In the case of a sample with an ethanol concentration of less than 0.5%, using the multifunctional biosensor, for a solution obtained by diluting the sample so that the alcohol concentration becomes 0.01 to 0.04% (vol / vol), the ethanol concentration was measured by a highly sensitive measurement method using a buffer solution (SL04-0015, manufactured by Oji Scientific Instruments Co., Ltd.) without sodium azide.

[0050] <Brix Measurement> In the subsequent experiments, unless otherwise specified, the Brix of the fermentation raw material liquid, malt fermentation broth, or beverage was measured using a digital refractometer (RX-5000α, manufactured by Atago Co., Ltd.).

[0051] <pH Measurement> In the subsequent experiments, unless otherwise specified, the pH of the fermentation raw material liquid, malt fermentation broth, or beverage was measured using a pH meter (LAQUAtwin-pH-33, manufactured by Horiba, Ltd.).

[0052] <Oligosaccharide Measurement> In the subsequent experiments, unless otherwise specified, the oligosaccharides in the fermentation raw material liquid, malt fermentation broth, or beverage were analyzed by HPLC (high performance liquid chromatography) (Prominence (registered trademark) (manufactured by Shimadzu Corporation); RI detector: RID-20A (manufactured by Shimadzu Corporation); hydrocarbon analysis column: Aminex HPX-42A carbohydorate Column (manufactured by BIO-RAD)). The HPLC analysis was performed at a column temperature of 80°C using degassed ultrapure water as the mobile phase at a flow rate of 0.5 mL / min.

[0053] [Example 1] Six strains of Cluyveromyces marxianus yeast were used to ferment aerated wort or diluted malt extract, and the composition of the resulting fermented liquid was examined. The Cluyveromyces marxianus yeast strains used were NBRC260 (isolated from air), NBRC272 (isolated from miso), NBRC277, NBRC482 (isolated from the mash of Chinese liquor (kaoliangchiu)), NBRC483 (isolated from Korean yeast cake), and NBRC690 (all strains owned by the Biotechnology Center of the National Institute of Technology and Evaluation).

[0054] <Preparation of fermentation raw material liquid (a)> Wort (a) was produced using a 200L scale brewing facility. 40 kg of crushed malt and starchy adjuncts, which are the raw materials for fermentation, and 160 L of raw water were added to the mashing tank, and the mixture in the mashing tank was heated according to conventional methods to saccharify it. Next, the resulting saccharified liquid was filtered, and hops were added to the filtrate, which was then boiled. After boiling, the filtrate was transferred to a sedimentation tank to separate and remove the precipitate, and then cooled to obtain wort (a). The analytical values ​​of the obtained wort (a) were as follows. This wort (a) was diluted with water to a Brix of 11° to obtain the fermentation raw material liquid (a).

[0055] [Table 1]

[0056] <Preparation of fermentation raw material liquids (b) to (d)> The three types of malt extracts listed in Table 2 were diluted with water to a Brix of 11°, mixed, and then autoclaved to obtain the fermentation raw material liquids (b) to (d), respectively.

[0057] [Table 2]

[0058] <100mL stirring fermentation> Six strains of Cluyveromyces marxianus yeast were pre-cultured in fermentation liquid (a) at an incubator rotation speed of 160 rpm and 25°C. Next, 100 mL of the fermentation liquid listed in Table 3 was placed in a 500 mL baffled Erlenmeyer flask, and the pre-cultured yeast strains were added to it, with a bacterial count of 2.0 × 10⁶ per mL of wort. 5 The yeast cells were added in such a manner that the total number of cells was 1.3 to 3.0 × 10¹⁶. This Erlenmeyer flask was incubated in a 160 rpm incubator at 20°C for 24 to 28 hours, and the number of yeast cells was 1.3 to 3.0 × 10¹⁶ per 1 mL of fermentation liquid. 8 After the individual components were separated, fermentation was terminated to obtain a fermented liquid. For fermented raw material liquids (b) to (d), one strain of Cluyveromyces marxianus yeast was used to obtain a fermented liquid in the same manner as when using fermented raw material liquid (a).

[0059] <300mL static fermentation> A 300 mL Brix 11° fermentation raw material liquid (a) is placed in a 500 mL sterile disposable container (Corning), and the yeast count is 2.0 × 10¹⁶ per mL of wort. 7 The ingredients were added in such a manner that they formed into individual particles, and the mixture was allowed to ferment at 20°C for 3 days to obtain the fermentation liquid.

[0060] <Analysis of various components> After fermentation was complete, the fermentation liquid was centrifuged, the supernatant was collected, and the yeast was removed by filtering through a 0.2 μm filter. The ethanol concentration and the concentration of each aroma component were quantified for each fermentation liquid after yeast removal using the method described above. The results are shown in Table 3.

[0061] <Sensory evaluation of malt-fermented beverages> After removing the yeast, the fermented liquid was diluted three times with carbonated water, and then hop flavoring was added to prepare a malt-fermented beverage. The resulting malt-fermented beverage was then subjected to a sensory evaluation by five panelists.

[0062] [Table 3]

[0063] In the 300 mL static fermentation, the ethanol concentration of the resulting fermented liquid was 0.68% (vol / vol), whereas in the 100 mL stirred fermentation, the ethanol concentration of the resulting fermented liquid was consistently below 0.15% (vol / vol). Furthermore, the fermented liquids obtained from the 100 mL stirred fermentation were all significantly higher in ethyl acetate concentration, the combined concentration of isoamyl acetate and isoamyl alcohol, and the combined concentration of β-phenethyl acetate and β-phenethyl alcohol compared to those obtained from the 300 mL static fermentation. In particular, the ethyl acetate concentration was 7.3 ppm in the fermented liquid obtained from the 300 mL static fermentation, while it was consistently above 100 ppm in all fermented liquids obtained from the 100 mL stirred fermentation. In addition, propyl acetate, which was not detected at all in the fermented liquid obtained from the 300 mL static fermentation, was detected at 0.72–2.64 ppm in the fermented liquid obtained from the 100 mL stirred fermentation.

[0064] In the sensory evaluation, all five panelists detected a fruity aroma reminiscent of pears in all fermentation liquids obtained from 100 mL stirred fermentation, but this aroma was not detected in the fermentation liquid obtained from 300 mL static fermentation. Propyl acetate is one of the representative components of pear aroma.

[0065] In other words, regardless of the strain of Cluyveromyces marxianus yeast used or the fermentation liquid, stirring fermentation resulted in less ethanol production and more production of aroma components such as ethyl acetate compared to static fermentation, particularly propyl acetate, which is an aroma component of pear. These results indicate that aeration fermentation using Cluyveromyces marxianus yeast yields a malt ferment liquid with a low ethanol concentration and a strong fruity aroma, and that using this malt ferment liquid yields a malt fermented beverage with a low ethanol concentration and a strong fruity aroma.

[0066] [Example 2] Fermentation was carried out using various methods with Cluyveromyces marxianus yeast and Pichia cluiberi yeast, and the composition of the resulting fermented liquid was examined. As for Cluyveromyces marxianus yeast, strain NBRC483, which was also used in Example 1, was used.

[0067] <Jar Fermentation Test> 600 mL of the fermentation raw material liquid (a) used in Example 1 was placed in a 2 L jar fermenter (manufactured by Marubishi Bioengin Co., Ltd.), and an antifoaming agent (KM-72GS, manufactured by Shin-Etsu Chemical Co., Ltd.) was added to a final concentration of 0.165 g / L. Subsequently, the number of yeast cells in the jar fermenter was 2.0 × 10⁶ per 1 mL of fermentation raw material liquid. 5 Fermentation was initiated by adding the yeast to a certain concentration. Fermentation was started at 20°C, 240 rpm, and 0.5 vvm. The dissolved oxygen concentration was monitored, and the stirring speed was increased to maintain the oxygen supply so that the dissolved oxygen concentration, which decreases with yeast growth, remains above 1 ppm (mg / L). In the case of the NBRC483 strain, the number of yeast cells was approximately 1.8 × 10 per 1 mL of fermentation liquid. 8 Fermentation was completed at one point, and the fermented liquid was obtained. In the case of Pichia kruiberi yeast, the number of yeast cells was approximately 2.0 × 10 per 1 mL of fermented liquid. 8 The fermentation process was completed, and a fermentation liquid was obtained.

[0068] <Fermentation Tank Fermentation Test> 60 L of the fermentation raw material liquid (a), which was also used in Example 1, was placed in a 100 L fermentation tank, and the defoaming agent KM-72GS was added to a final concentration of 0.165 g / L. Next, the number of yeast cells in the fermentation tank was increased to 2.0 × 10⁶ per 1 mL of fermentation raw material liquid. 5 The yeast was added to a concentration of 1.8 × 10¹⁴ cells and fermented. During fermentation, the liquid was pumped from the sampling valve port of the fermentation tank to the aeration line, passed through a static mixer and a dissolved oxygen meter, and returned to the bottom of the tank for circulation. To prevent oxygen deficiency during yeast growth, the dissolved oxygen level was monitored, and intermittent aeration was performed to maintain a dissolved oxygen concentration of 1 ppm (mg / L) or higher. In the case of the NBRC483 strain, the number of yeast cells was approximately 1.8 × 10¹⁴ cells per 1 mL of fermentation liquid. 8Fermentation was terminated after [X] cells, and a fermentation broth was obtained. In the case of Pichia kudriavzevii yeast, the number of yeast cells was 3.0×10 per 1 mL of the fermentation broth 7 Fermentation was terminated after [X] cells, and a fermentation broth was obtained.

[0069] <Static Fermentation Test> 7 L of the fermentation raw material liquid (a) used in Example 1 was placed in a sterilized container (10 L capacity Steritainer, manufactured by Sekisui Plastics Co., Ltd.). Next, the number of yeast cells was adjusted to about 1.0×10 per 1 mL of the fermentation raw material liquid and added to the container, and static fermentation was carried out at 20 °C for 6 days to obtain a fermentation broth. The number of bacteria at the end of fermentation was 1.4×10 per 1 mL of the fermentation broth for the NBRC483 strain and 6×10 for Pichia kudriavzevii yeast 7 cells, and for Pichia kudriavzevii yeast it was 6×10 8 cells. 7

[0070] <Analysis of Various Components> In the same manner as in Example 1, the ethanol concentration and the concentrations of each aroma component of the malt fermentation broth obtained after fermentation were quantified by the above method. The results are shown in Table 4.

[0071]

Table 4

[0072] ​In jar fermentation tests and fermentation tank fermentation tests using strain NBRC483, the ethanol concentration and contained aroma components showed trends very similar to those of the 100 mL stirred fermentation in Example 1. Specifically, in all test samples, the ethanol concentration was 0.15% (vol / vol) or less, the ethyl acetate concentration was 60 ppm (mg / L) or higher, and the propyl acetate concentration was 0.15 ppm (mg / L) or higher. Other aroma components also tended to be high. On the other hand, in static fermentation tests using strain NBRC483 and static fermentation tests and aeration fermentation tests using Pichia kruiberi yeast, propyl acetate was detected at a maximum of only 0.02 ppm (mg / L), and ethyl acetate was detected at a maximum of 35.4 ppm (mg / L) (static test of Pichia kruiberi yeast). Furthermore, in the static fermentation test, the ethanol concentration of the obtained fermentation liquid was 0.81% (vol / vol) (NBRC483 strain) and 0.52% (vol / vol) (Pichia kruiberi yeast), which was higher than that in the aerated fermentation test.

[0073] [Example 3] Stirring fermentation experiment using Cluyveromyces marxianus yeast and other bacterial species Using the same initial bacterial count, container, incubator rotation speed, and temperature as in Example 1's <100mL stirred fermentation>, the fermentation raw material liquid (a) was used, and the test strains were increased and tested with aeration stirring. The yeasts used were NBRC483, a Cluyveromyces marxianus species yeast, and NBRC10005, a type strain of Cluyveromyces marxianus species yeast, which were used in Example 1. In addition, two strains of Pichia kruiberi yeast, one strain of Torulaspora delbrueckii yeast, one strain of Saccharomycodes ludwigii yeast, one strain of Saccharomyces cerevisiae yeast, and one strain of Saccharomyces pastorianus yeast, all commonly used in non-alcoholic beer production, were used as control strains in the experiment. In Table 5, the type strain of each yeast species is indicated as TS next to the species name. The fermentation time was set to 20-28 hours, and the bacterial count concentration of each yeast was measured by turbidity (OD). 660 After fermentation was allowed to reach 4.0-7.0, the fermentation was stopped and each fermented liquid was obtained. Saccharomyces ludwigii showed aggregation of the fungal cells and turbidity (OD 660 Because it was difficult to measure the ethanol concentration, the culture was terminated at the time of the longest culture time among all strains. The ethanol concentration and the concentration of each aroma component in each fermentation liquid are shown in Table 5.

[0074] [Table 5]

[0075] The test results showed that the ethanol concentration was less than 0.5% in all test groups. The type strain NBRC10005 of the Cluyveromyces marxianus yeast, while showing a tendency towards lower concentrations than other Cluyveromyces marxianus yeast strains, still met the requirements of ethyl acetate at 50 ppm or higher and propyl acetate at 0.1 ppm or higher. In contrast, the control strain had a maximum ethyl acetate concentration of only about 3 ppm, and no samples showed detectable levels of propyl acetate.

[0076] [Example 4] Fermentation tests using Cluyveromyces marxianus yeast at various Brix levels of fermentation liquids NBRC483 strain was pre-cultured in an incubator at 160 rpm and 25°C. Malt extract Maltax 10 (Senson) was diluted with water to create fermentation raw material solutions with Brix levels of 4°, 7°, 9°, 12°, 16°, 18°, and 20°. 100 mL of each solution was placed in 500 mL baffled Erlenmeyer flasks. The pre-cultured yeast strains were then added to these solutions, with a bacterial count of 2.0 × 10⁴ per mL of wort. 5 The yeast cells were added in such a manner that the number of cells was approximately 1.8 × 10⁶ per ml. This Erlenmeyer flask was incubated in a 160 rpm incubator at 20°C for 26-30 hours with aeration and stirring, and the number of yeast cells was approximately 1.8 × 10⁶ per ml. 8 Fermentation was completed at one stage, and a fermentation liquid was obtained. When using fermentation liquids with Brix 18° and 20°, yeast growth took a long time, and even after 30 hours of incubation, the bacterial count was only 1.0 × 10⁶. 8 The number of samples was only about [number]. The test results for each Brix level of the fermentation raw material liquid are shown in Table 6. All test groups met the requirements of 50 ppm or more of ethyl acetate and 0.1 ppm or more of propyl acetate.

[0077] [Table 6]

[0078] [Example 5] Sensory evaluation The fermented liquid obtained in Example 1 (100 ml stirred fermentation) and the fermented liquid obtained in Example 3 were diluted with carbonated water using the fermentation raw material liquid to a Brix of 5° (2.2 times dilution), and hop flavoring was added to prepare each beverage. Each of the resulting beverages was subjected to a sensory evaluation using a scoring method by five experienced panelists. For "strength of wort aroma," water was evaluated as 1 point and the diluted fermentation raw material liquid (a) as 7 points. However, for beverages using fermentation raw material liquids (b), (c), and (d), "strength of wort aroma" was not evaluated because the type of fermentation raw material liquid was different. For "pear-like aroma," "fruity aroma," and "deliciousness," the wort fermentation liquid using Saccharomyces pastorianus (NBRC11024), a representative beer yeast, was evaluated as 4 points for each beverage. The sensory evaluation was conducted randomly, with the details such as the name of each beverage concealed, and the order of evaluation differed among the panelists. For "strength of wort aroma," "pear-like aroma," and "fruity aroma," aroma was evaluated, while for "deliciousness," taste was evaluated. The results for each evaluation are shown in Tables 7-8 and Figures 1-3. Since the strength of wort aroma was similarly low in all beverages, it can be assumed that each fermentation liquid progressed to a similar degree sensorially. In addition, beverages using aerated fermentation liquid of Cluyveromyces marxianus yeast showed significantly stronger "pear-like aroma" and "fruity aroma" compared to the control. Student's t-test was used for statistical analysis (*: p<0.05, **: p<0.01). This characteristic was similarly confirmed even when different types of wort were used. On the other hand, beverages using other strains of yeast, or beverages using static fermentation liquid of Cluyveromyces marxianus yeast, did not show significantly higher values ​​for these items compared to the control. From the above, it can be said that the aerated fermentation liquid produced by the Cluyveromyces marxianus yeast is characterized by a strong, sensual "pear-like aroma" and "fruity aroma."

[0079] [Table 7]

[0080] [Table 8]

[0081] [Example 6] To investigate the relationship between "pear-like aroma" and "deliciousness," we examined the correlation between the two. Spearman's rank correlation coefficient was used for significance testing. The results showed a significant positive correlation (ρ=0.574, p=0.000) between deliciousness and pear-like aroma (Figure 4). It was found that the pear-like aroma of the fermented liquid contributed to the overall deliciousness of the fermented liquid. [Industrial applicability]

[0082] The method for producing malt fermented liquid according to the present invention uses a yeast of the species Cluyveromyces marxianus, which has low ethanol production capacity and high capacity to produce esters such as ethyl acetate, isoamyl acetate, β-phenethyl acetate, and propyl acetate, as well as alcohols with 3 or more carbon atoms such as isoamyl alcohol and β-phenethyl alcohol, and is used for aeration fermentation. Therefore, the method for producing malt fermented liquid according to the present invention can provide a malt fermented liquid with a low ethanol concentration and a fruity aroma. Furthermore, by using this malt fermented liquid, a malt fermented beverage with a low ethanol concentration and a fruity aroma can be provided.

Claims

1. The process involves inoculating a fermentation raw material liquid containing malt saccharified products with Cluyveromyces marxianus yeast, and carrying out aeration fermentation at 0-28°C while maintaining a dissolved oxygen concentration of 1 ppm (mg / L) or higher in the fermentation liquid. The Brix of the fermentation raw material liquid is 4° or higher, and the ethanol concentration is less than 1.0% (vol / vol), A method for producing malt fermented liquid, wherein the fermentation time for the aeration fermentation is three days or less.

2. The method for producing malt fermented liquid according to claim 1, wherein the Brix of the fermented raw material liquid is 4° to 20°.

3. A method for producing a malt ferment liquor according to claim 1 or 2, wherein the propyl acetate concentration is 0.1 ppm (mg / L) or higher.

4. A method for producing a malt ferment liquor according to any one of claims 1 to 3, wherein the ethanol concentration is 0.5% (vol / vol) or less.

5. A method for producing a malt ferment liquor according to any one of claims 1 to 4, wherein the ethyl acetate concentration is 50 ppm (mg / L) or higher.

6. A method for producing a malt fermented beverage, comprising producing a malt fermented liquid by the method for producing a malt fermented liquid described in any one of claims 1 to 5, and producing a malt fermented beverage using the obtained malt fermented liquid as a raw material.

7. A method for producing a malt fermented beverage according to claim 6, comprising the step of diluting the malt fermented liquid.

8. The method for producing a malt-fermented beverage according to claim 6 or 7, wherein the malt-fermented beverage is a beer-like sparkling beverage.

Images