Method for producing plant milk

Abstract

The purpose of the present invention is to provide a method for producing a plant milk which allows preparation of foamed milk having a sufficient amount of air bubbles. The purpose of the present invention is also to provide a method for producing plant milk in which phase separation and formation of insoluble matters are suppressed when mixed with coffee.　This method for producing plant milk comprises treating a raw material containing a plant protein with (I) protein glutaminase and (II) one or more enzymes selected from the group consisting of transglutaminase, pectinase, and glucose oxidase. The enzyme preparation for modifying plant milk contains the above (I) and (II).

Description

How plant-based milk is produced

[0001] The present invention relates to a method for producing plant-based milk and to an enzyme preparation for the modification of plant-based milk.

[0002] Foamed milk coffee (also known as caffè latte, cappuccino, latte macchiato, etc.) served at restaurants and the like is prepared by combining foamed milk (foamed milk) with coffee (or a mixture of coffee and liquid milk) to form a layer of foamed milk and a layer of coffee (or a mixture of coffee and liquid milk). In recent years, the market for dairy-free plant-based milk (e.g., oat milk) has expanded due to growing awareness of animal welfare and health consciousness. There is a demand for plant-based milk that can be used to prepare foamed milk. However, plant-based milks such as oat milk have poor foaming properties, making it difficult to produce foamed milk with a sufficient amount of foam. Furthermore, when plant-based milks such as oat milk are mixed with highly acidic coffee, such as light roast coffee, phase separation occurs or insoluble matter such as aggregates and precipitates is generated, resulting in a dark color of the mixture with coffee, and thus preventing the desired light brown color of the coffee beverage (e.g., the light brown color of a caffè latte made by mixing milk and coffee).

[0003] Patent Literature 1 discloses a method for producing a processed vegetable protein food or beverage material and / or a vegetable protein food or beverage product, comprising a step of treating the vegetable protein food or beverage material and / or vegetable protein food or beverage with a protein deamidase and at least one enzyme selected from the group consisting of lipase and cyclodextrin glucanotransferase. Patent Literature 2 discloses a method for producing oat milk using an alkaline protease and an emulsifier. Patent Literature 3 discloses a method for producing a liquid oat base or drink having an improved soluble oat protein content from an oat material containing starch and oat protein, characterized by solubilizing oat protein in an aqueous solvent, particularly water, by means of a protein deamidase and optionally decanting the product. However, it has not been previously known that the use of the specific enzymes described below in the present invention can produce vegetable milk capable of producing foamed milk with a sufficient amount of foam. Furthermore, it was not previously known that the use of the specific enzyme described below in the present invention makes it possible to produce plant-based milk that can solve the problems (phase separation, formation of insoluble matter) that arise when plant-based milk is mixed with highly acidic coffee.

[0004] WO2022 / 097675CN102349579AWO2014 / 123466

[0005] An object of the present invention is to provide a method for producing plant-based milk that can prepare foamed milk with a sufficient amount of bubbles. Another object of the present invention is to provide a method for producing plant-based milk that suppresses phase separation and the formation of insoluble matter when mixed with coffee (especially coffee with a high acidity) (i.e., improves mixing stability with coffee (especially coffee with a high acidity)).

[0006] The present inventors have conducted extensive research to solve the above problems and have found that, when producing plant-based milk (e.g., oat milk), it is possible to produce plant-based milk (e.g., oat milk) that can be used to prepare foamed milk with a sufficient amount of bubbles by allowing a specific enzyme of the present invention, described below, to act on a raw material containing plant protein (e.g., raw oats (e.g., whole oat flour)). The present inventors have also found that, when producing plant-based milk (e.g., oat milk), it is possible to produce plant-based milk that is inhibited from undergoing phase separation or producing insoluble matter when mixed with coffee (e.g., coffee with a high acidity) (i.e., has improved mixing stability with coffee (e.g., coffee with a high acidity)) by allowing a specific enzyme of the present invention, described below, to act on a raw material containing plant protein (e.g., raw oats (e.g., whole oat flour)). Based on these findings, the present inventors have conducted further research and have completed the present invention.

[0007] That is, the present invention provides the following: [1] A method for producing plant-based milk, which comprises treating a raw material containing a plant protein with the following (I) and (II): (I) protein glutaminase (II) one or more enzymes selected from the group consisting of transglutaminase, pectinase, and glucose oxidase [2] The method for producing plant-based milk according to [1] above, wherein (I) and (II) are selected from the group consisting of the following (1) to (6). (1) Protein glutaminase and transglutaminase (2) Protein glutaminase and pectinase (3) Protein glutaminase and glucose oxidase (4) Protein glutaminase, transglutaminase, and pectinase (5) Protein glutaminase, transglutaminase, and glucose oxidase (6) Protein glutaminase, transglutaminase, pectinase, and glucose oxidase [3] A method for producing plant-based milk according to [1] above, wherein (II) is transglutaminase and pectinase. [4] A method for producing plant-based milk according to [1] above, wherein (II) is transglutaminase and glucose oxidase. [5] A method for producing plant-based milk according to [1] above, wherein (II) is transglutaminase, pectinase, and glucose oxidase. [6] The manufacturing method according to any one of [1] to [5] above, wherein the raw material containing vegetable protein is raw oats and the vegetable milk is oat milk. [7] The manufacturing method according to any one of [1] to [6] above, wherein the vegetable milk is for use in foaming milk. [8] The manufacturing method according to any one of [1] to [7] above, wherein the vegetable milk is used to prepare a coffee beverage.

[0008] [9] An enzyme preparation for modifying plant-based milk, comprising the following (I) and (II): (I) protein glutaminase (II) one or more enzymes selected from the group consisting of transglutaminase, pectinase, and glucose oxidase

[10] The enzyme preparation according to [9] above, wherein (I) and (II) are selected from the group consisting of (1) to (6) below: (1) protein glutaminase and transglutaminase (2) protein glutaminase and pectinase (3) protein glutaminase and glucose oxidase (4) protein glutaminase, transglutaminase, and pectinase (5) protein glutaminase, transglutaminase, and glucose oxidase (6) protein glutaminase, transglutaminase, pectinase, and glucose oxidase

[11] The enzyme preparation according to [9] above, wherein (II) is transglutaminase and pectinase.

[12] The enzyme preparation according to [9] above, wherein (II) is transglutaminase and glucose oxidase.

[13] The enzyme preparation according to [9] above, wherein (II) is transglutaminase, pectinase, and glucose oxidase.

[14] The enzyme preparation according to any of [9] to

[13] above, wherein the plant-based milk is oat milk.

[15] The enzyme preparation according to any of [9] to

[14] above, wherein the plant-based milk is used for foaming milk.

[16] The enzyme preparation according to any of [9] to

[15] above, wherein the plant-based milk is used for preparing a coffee beverage.

[0009] According to the present invention, it is possible to produce plant-based milk (e.g., oat milk) that can be used to prepare foamed milk with a sufficient amount of bubbles. Furthermore, according to the present invention, it is possible to produce plant-based milk (e.g., oat milk) that is inhibited in phase separation and insoluble matter formation when mixed with coffee (especially highly acidic coffee) (i.e., that has improved mixing stability with coffee (especially highly acidic coffee)). Plant-based milk (e.g., oat milk) produced by the production method of the present invention can be used to prepare foamed milk with a sufficient amount of bubbles. Furthermore, when mixed with coffee (e.g., highly acidic coffee), it is inhibited in phase separation and insoluble matter formation, so that the white oat milk mixes uniformly with the coffee, resulting in a coffee beverage with a desired light brown color (e.g., a light brown like a caffè latte made by mixing milk and coffee).

[0010] FIG. 1 is a schematic diagram illustrating the method for evaluating the foaming properties of oat milk in Test Example 1.

[0011] The present invention will be described in detail below. The method for producing plant-based milk of the present invention comprises treating a raw material containing plant protein with the following (I) and (II): (I) protein glutaminase; and (II) one or more enzymes selected from the group consisting of transglutaminase, pectinase, and glucose oxidase.

[0012] Examples of the combination of enzymes (I) and (II) used in the production method of the present invention include the following combinations (1) to (6): (1) protein glutaminase and transglutaminase (2) protein glutaminase and pectinase (3) protein glutaminase and glucose oxidase (4) protein glutaminase, transglutaminase, and pectinase (5) protein glutaminase, transglutaminase, and glucose oxidase (6) protein glutaminase, transglutaminase, pectinase, and glucose oxidase

[0013] The protein glutaminase used in the present invention acts directly on amide groups in proteins to deamidate them without cleaving peptide bonds or cross-linking the proteins. There are no particular limitations on the type of protein glutaminase, so long as it has this effect. Protein glutaminase prepared from a culture medium of a microorganism that produces protein glutaminase can be used. Microorganisms used to prepare protein glutaminase are not particularly limited, but examples include microorganisms of the genera Chryseobacterium, Flavobacterium, and Empedobacter. For example, Chryseobacterium bacteria include Chryseobacterium proteolyticum, Flavobacterium bacteria include Flavobacterium aquatile, and Empedobacter brevis. Protein glutaminase can be prepared from a microbial culture solution by known protein separation and purification methods (such as centrifugation, UF concentration, salting out, and various types of chromatography using ion exchange resins). For example, the culture solution can be centrifuged to remove the bacterial cells, followed by a combination of salting out, chromatography, and the like to obtain the target enzyme. When recovering the enzyme from the bacterial cells, the bacterial cells can be disrupted, for example, by pressure treatment, ultrasonic treatment, or the like, and then separated and purified in the same manner as above to obtain the target enzyme. Alternatively, the bacterial cells may be recovered from the culture solution in advance by filtration, centrifugation, or the like, and then the above series of steps (disruption, separation, and purification of the bacterial cells) may be carried out. The enzyme may be powdered by a drying method such as freeze-drying or vacuum drying, and an appropriate excipient or drying aid may be used in this process. The protein glutaminase used in the present invention may be a commercially available product. Specific examples include protein glutaminases commercially available from Amano Enzyme Inc. under the trade names "Amano 500K Protein-Glutaminase" and "Protein-glutaminase "Amano" 500".

[0014] The activity unit of protein glutaminase used herein is measured and defined as follows. (1) 10 μl of an aqueous solution containing protein glutaminase is added to 100 μl of 176 mM phosphate buffer (pH 6.5) containing 30 mM Z-Gln-Gly, and the mixture is incubated at 37°C for 10 minutes. 100 μl of 12% TCA solution is then added to terminate the reaction. The enzyme is then diluted appropriately with 20 mM phosphate buffer (pH 6.0) to a concentration of 0.05 mg / ml. (2) After centrifugation (12,000 rpm, 4°C, 5 minutes), the supernatant is purified by NH (F-kit ammonia) (Roche). ３ The quantification is performed as follows. (3) To 100 μl of Reagent II solution (supplied with the F-kit), 10 μl of supernatant and 190 μl of 0.1 M triethanolamine buffer (pH 8.0) are added. After allowing to stand at room temperature for 5 minutes, 100 μl of the solution is used to measure the absorbance at 340 nm (E1). To the remaining 200 μl, 1.0 μl of Reagent III (glutamate dehydrogenase) is added, and after allowing to stand at room temperature for an additional 20 minutes, the absorbance at 340 nm (E2) of the remaining 200 μl is measured. The ammonia concentration in the reaction solution is determined from a calibration curve showing the relationship between ammonia concentration and the change in absorbance (340 nm), prepared using the ammonia standard solution supplied with the F-kit. (4) Protein concentration is measured using a protein assay CBB (Coomassie Brilliant Blue) solution (Nacalai Tesque) at a detection wavelength of 595 nm. BSA (manufactured by Pierce) is used as a standard. (5) Protein glutaminase activity is calculated using the following formula.

[0015]

[0016] In the production method of the present invention, the amount of protein glutaminase added is preferably 0.0001 to 10,000 U, more preferably 0.001 to 1,000 U, even more preferably 0.01 to 100 U, and particularly preferably 0.1 to 10 U in terms of enzyme activity per gram of protein contained in a raw material containing vegetable protein (for example, raw oats).

[0017] The transglutaminase used in the present invention is an enzyme that has the activity of catalyzing an acyl transfer reaction in which a glutamine residue in a protein or peptide serves as a donor and a lysine residue serves as an acceptor, and transglutaminases of various origins are known, such as those derived from mammals, fish, and microorganisms. The transglutaminase used in the present invention is not particularly limited in origin as long as it has the above-mentioned activity, and transglutaminases of any origin can be used, and recombinant enzymes can also be used. The transglutaminase used in the present invention may be a commercially available product, and as a specific example, microbial transglutaminases commercially available from Ajinomoto Co., Inc. under the trade name "Activa" TG can be used alone or in combination. In this specification, the enzymatic activity of transglutaminase is measured by reacting transglutaminase in a reaction system using benzyloxycarbonyl-L-glutamylglycine and hydroxylamine as substrates in a Tris buffer solution at 37°C and pH 6.0, forming an iron complex with the hydroxamic acid produced in the presence of trichloroacetic acid, measuring the absorbance at 525 nm, and determining the amount of hydroxamic acid using a calibration curve. One unit (1 U) of the enzyme is defined as the amount of enzyme that produces 1 μmole of hydroxamic acid per minute (see Japanese Patent Laid-Open No. 27471 / 1989).

[0018] When transglutaminase is used in the production method of the present invention, the amount of transglutaminase added is preferably 0.0001 to 10,000 U, more preferably 0.001 to 1,000 U, even more preferably 0.01 to 100 U, and particularly preferably 0.1 to 10 U in terms of enzymatic activity per gram of protein contained in a raw material containing vegetable protein (e.g., raw oats).

[0019] The pectinase used in the present invention is an enzyme that has the activity of catalyzing the hydrolysis of pectin (EC 3.2.1.15, etc.). This activity is also referred to as "pectinase activity." Specifically, pectinase activity may be the activity of catalyzing the hydrolysis of the α-1,4 glycosidic bond in the polygalacturonic acid chain that constitutes pectin. This "pectinase activity" also includes pectin lyase activity, which degrades polygalacturonic acid chains by β-elimination, pectin methylesterase activity, which demethylates the methyl ester groups of pectin, and protopectinase activity, which acts on water-insoluble protopectin to liberate water-soluble pectin. In this specification, the enzymatic activity of pectinase can be measured by the following procedure. Specifically, pectinase activity can be measured by incubating the enzyme with a substrate and measuring the enzyme-dependent degradation of the substrate. Degradation of the substrate can be measured, for example, using the generation of reducing ends (i.e., an increase in reducing power) as an indicator. The increase in reducing power can be measured, for example, by the dinitrosalicylic acid (DNS) method or the Somogyi-Nelson method. In the case of pectinase, the amount of enzyme that causes an increase in reducing power equivalent to 1 μmol of galacturonic acid per minute at 45°C and pH 4.5 when an enzymatic reaction is carried out using polygalacturonic acid as a substrate is defined as 1 U (unit). In the case of pectinase, an increase in reducing power equivalent to a certain amount of galacturonic acid may be interpreted as the production of that amount of galacturonic acid. The amount of galacturonic acid produced can be measured by known methods used for quantifying compounds, such as HPLC, LC / MS, GC / MS, and NMR. The pectinase used in the present invention may be a commercially available product; specifically, a pectinase commercially available from Nagase ChemteX Corporation under the trade name "Pectinase XP-534NEO" can be used.

[0020] When pectinase is used in the production method of the present invention, the amount of pectinase added is preferably 0.00001 to 100,000 U, more preferably 0.0001 to 10,000 U, even more preferably 0.001 to 1,000 U, and particularly preferably 0.01 to 100 U in terms of enzyme activity per gram of starch contained in a raw material containing vegetable protein (e.g., raw oats).

[0021] The glucose oxidase used in the present invention is an enzyme that catalyzes the reaction of glucose and oxygen as substrates to produce gluconolactone (gluconolactone is non-enzymatically hydrolyzed to gluconic acid) and hydrogen peroxide. The hydrogen peroxide produced by this reaction oxidizes SH groups in proteins, promoting the formation of SS bonds (disulfide bonds) and forming cross-linked structures within the proteins. Glucose oxidases of various origins, including those derived from microorganisms such as Aspergillus oryzae and plants, may be used, and their origin is not limited. Recombinant enzymes may also be used. A specific example of glucose oxidase is the microbial glucose oxidase commercially available from Shin-Nippon Chemical Industry Co., Ltd. under the trade name "Sumiteam PGO." In the present invention, the activity unit of glucose oxidase is defined as 1 U (unit)—the amount of enzyme that oxidizes 1 μmol of glucose per minute at 37°C and pH 7.0. In the present invention, the activity of glucose oxidase can be measured by the following method. Using glucose as a substrate, glucose oxidase is reacted in the presence of oxygen to generate hydrogen peroxide. Peroxidase is then reacted with the generated hydrogen peroxide in the presence of aminoantipyrine and phenol to generate a quinoneimine dye. The generated quinoneimine dye is measured at a wavelength of 500 nm. Specifically, the procedure is as follows: Glucose oxidase is dissolved in 0.1 mol / L phosphate buffer (potassium dihydrogen phosphate, adjusted to pH 7.0 with aqueous sodium hydroxide solution) with stirring, and the solution is diluted 50-fold with 0.1 mol / L phosphate buffer to obtain a GO solution. To an analytical cell, add 2.0 mL of a phenol-containing buffer solution (Milli-Q, 1.36 g of potassium dihydrogen phosphate, 3 mL of 5% phenol test solution, and 3 mL of 5% Triton X-100 solution, adjusted to pH 7.0 with aqueous sodium hydroxide solution to 100 mL), 500 μL of a 10% glucose solution, 500 μL of a 0.01% peroxidase solution (PO "amano" 3 (1250 U ± 250 U) used), and 100 μL of a 0.4% 4-aminoantipyrine solution in that order, mix by inversion, and keep at 37 ± 0.1°C for 10 minutes.100 μL of GO solution was placed in the analysis cell, and 11 points were automatically measured every 30 seconds for 5 minutes. The GO activity value was measured from the increment (slope) between 120 and 300 seconds. For the blank group, the value measured above was obtained by adding 0.1 mol / L phosphate buffer instead of the GO solution, and this value was subtracted from the GO test group. For oxidative / reductase enzymes other than glucose oxidase, the amount of enzyme required to oxidize or reduce 1 μmol of substrate per minute was defined as 1 U (unit).

[0022] When glucose oxidase is used in the production method of the present invention, the amount of glucose oxidase added is preferably 0.00001 to 100,000 U, more preferably 0.0001 to 10,000 U, even more preferably 0.001 to 1,000 U, and particularly preferably 0.001 to 100 U, or 0.01 to 100 U, in terms of enzyme activity per gram of starch contained in a raw material containing vegetable protein (for example, raw oats).

[0023] (1) Protein Glutaminase and Transglutaminase When protein glutaminase and transglutaminase are used in combination in the production method of the present invention, the weight ratio of the amounts added (protein glutaminase:transglutaminase) is, for example, 1:0.0001-10,000, preferably 1:0.001-1,000, more preferably 1:0.01-100, and even more preferably 1:0.1-10.

[0024] (2) Protein Glutaminase and Pectinase When protein glutaminase and pectinase are used in combination in the production method of the present invention, the weight ratio of the amounts added (protein glutaminase:pectinase) is, for example, 1:0.00001 to 10,000, preferably 1:0.0001 to 1,000, more preferably 1:0.001 to 100, and even more preferably 1:0.01 to 10.

[0025] (3) Protein Glutaminase and Glucose Oxidase When protein glutaminase and glucose oxidase are used in combination in the production method of the present invention, the weight ratio of the amounts added (protein glutaminase:glucose oxidase) is, for example, 0.00001 to 10,000, preferably 1:0.0001 to 1,000, more preferably 1:0.001 to 100, and even more preferably 1:0.01 to 10.

[0026] (4) Protein glutaminase, transglutaminase, and pectinase When protein glutaminase, transglutaminase, and pectinase are used in combination in the production method of the present invention, the weight ratio of the amounts added (protein glutaminase:transglutaminase:pectinase) is, for example, 1:0.0001-10000:0.00001-10000, preferably 1:0.001-1000:0.0001-1000, more preferably 1:0.01-100:0.001-100, and even more preferably 1:0.1-10:0.01-10.

[0027] (5) Protein glutaminase, transglutaminase, and glucose oxidase When protein glutaminase, transglutaminase, and glucose oxidase are used in combination in the production method of the present invention, the weight ratio of the amounts added (protein glutaminase:transglutaminase:glucose oxidase) is, for example, 1:0.0001-10000:0.000001-10000, preferably 1:0.001-1000:0.00001-1000, more preferably 1:0.01-100:0.0001-100, and even more preferably 1:0.1-10:0.001-10.

[0028] (6) Protein glutaminase, transglutaminase, pectinase, and glucose oxidase In the production method of the present invention, when protein glutaminase, transglutaminase, pectinase, and glucose oxidase are used in combination, the weight ratio of the amounts added (protein glutaminase: transglutaminase: pectinase: glucose oxidase) is, for example, 1:0.0001 to 10000:0.00001 to 10000:0.00001 to 10000, preferably 1:0.001 to 1000:0.0001 to 1000:0.0001 to 1000, more preferably 1:0.01 to 100:0.001 to 100:0.001 to 100, and even more preferably 1:0.1 to 10:0.01 to 10:0.01 to 10:0.01 to 10.

[0029] In the present invention, the vegetable protein-containing raw material to be treated with the enzyme can be a plant or a processed product thereof that is conventionally used as a raw material for vegetable milk. Examples include grains such as oats and rice, nuts and seeds such as almonds, cashews, and coconuts, and legumes such as soybeans and peas, as well as processed products thereof. The grains, nuts, and legumes can be whole grains, or can be ground, with the outer skin and germ removed. Ground products are preferred. Examples of processed grains, nuts, and legumes include vegetable milk (e.g., oat milk powder) produced by conventional manufacturing methods. In the present invention, commercially available vegetable protein-containing raw materials can also be used. Examples include whole oat flour (oat flour obtained by removing the husks and grinding oats) under the product name "Oat Flour" (Slow Food Kitchen) and "Oat Flour" (produced in Denmark by Sansho Co., Ltd.), and oat milk powder produced by conventional manufacturing methods under the product name "Oat Milk Powder GD-F" (Goudo Co., Ltd.).

[0030] In the present invention, oat milk can be produced by using a raw material derived from oats (e.g., whole oats, oats with the outer skin and germ removed, crushed oats, or processed oats) (referred to as "raw oats" in this specification) as the raw material containing vegetable protein to be treated with the enzyme.

[0031] The production method of the present invention can be carried out using the same raw materials and methods as ordinary plant-based milk, except that the milk is treated with the enzyme of the present invention.

[0032] For example, the production method of the present invention can produce the plant-based milk (e.g., oat milk) of the present invention by the following steps: (i) A raw material containing plant protein (e.g., raw oats) is mixed with water (if necessary, the raw material is pulverized in a mill or the like and then mixed with water, or the raw material is pulverized in a mill or the like together with water) to obtain a 1 to 30% suspension. (ii) To the resulting suspension, 0.01 to 100,000 U of α-amylase and the enzyme of the present invention are added per gram of starch contained in the raw material containing plant protein (e.g., raw oats), and the reaction is carried out at 20 to 80°C for 0.5 to 10 hours. (iii) After completion of the reaction in (ii), the mixture is heated at 90 to 100°C for 1 to 30 minutes to inactivate the enzyme. (iv) The mixture is subjected to solid-liquid separation by centrifugation or the like, and the liquid is recovered. The recovered liquid is stirred using a homogenizer while liquid oil (e.g., rapeseed oil) is added dropwise to the liquid. If necessary, the pH is adjusted to 6 to 10 using a pH adjuster to obtain the plant-based milk (for example, oat milk) of the present invention.

[0033] When multiple enzymes are added, they may be added in any order, either all at the same time or sequentially at staggered times.

[0034] The production method of the present invention can produce improved plant-based milk (e.g., oat milk). As used herein, "improved" refers to improved foaming properties and improved mixing stability with coffee (e.g., highly acidic coffee). As used herein, "improved foaming properties" refers to the plant-based milk produced by the production method of the present invention having improved foaming properties compared to plant-based milk produced without the enzyme of the present invention. The plant-based milk produced by the production method of the present invention also has improved foaming properties compared to plant-based milk produced using protein glutaminase, transglutaminase, pectinase, or glucose oxidase alone. As used herein, "improved mixing stability with coffee" refers to the plant-based milk produced by the production method of the present invention (referred to as "the plant-based milk of the present invention") being less susceptible to phase separation and insoluble matter formation when mixed with coffee compared to plant-based milk produced without the enzyme of the present invention (referred to as "control plant-based milk"). This also means that the color of the mixture of the plant-based milk of the present invention and coffee is lighter than the color of the mixture of the control plant-based milk and coffee. Conventional plant-based milks have had the problem of poor mixing stability, particularly with highly acidic coffee. However, the plant-based milk produced by the production method of the present invention has improved mixing stability with highly acidic coffee and is useful in that it can be used regardless of the acidity of the coffee. As used herein, "highly acidic coffee" refers to coffee with a pH of, for example, 4.0 to 5.5, 4.0 to 5.4, 4.0 to 5.3, 4.0 to 5.2, 4.0 to 5.1, or 4.0 to 5.0. The presence or absence of modification (improved foamability) can be evaluated in accordance with the evaluation of foamability in the test examples described below. The presence or absence of modification (improved mixing stability with coffee) can be evaluated in accordance with the sensory evaluation of the appearance (uniformity, color) of coffee containing oat milk in the test examples described below. The improved plant-based milk (e.g., oat milk) produced by the production method of the present invention has excellent foamability and is therefore useful as plant-based milk for preparing foamed milk (also referred to as "plant-based milk for foamed milk" in this specification).The improved plant-based milk (e.g., oat milk) produced by the production method of the present invention has excellent mixing stability with coffee, and is therefore useful as plant-based milk to be mixed with coffee to prepare a coffee beverage (also referred to herein as "plant-based milk for coffee beverages"). In the present invention, the "coffee beverage" may be any beverage containing a combination of coffee and plant-based milk, and includes, for example, foamed milk coffee (also known as caffè latte, cappuccino, latte macchiato, etc.) prepared to have a layer of foamed plant-based milk and a layer of coffee (or a mixture of coffee and liquid plant-based milk), and coffee with plant-based milk prepared by mixing liquid plant-based milk with coffee.

[0035] The present invention also relates to an enzyme preparation for modifying plant-based milk (hereinafter simply referred to as the enzyme preparation of the present invention), which contains the enzyme of the present invention. In the enzyme preparation of the present invention, the definition, amount, and method of addition (action time, action temperature, and method for terminating the enzymatic reaction) of each enzyme of the present invention, as well as examples of raw materials containing plant proteins to be treated with the enzyme, are the same as those in the production method of the present invention. The enzyme preparation of the present invention can be added to raw materials containing plant proteins (e.g., raw oats) and reacted in accordance with the method and amount of addition of the enzyme of the present invention described above in the production method of the present invention, to produce modified plant-based milk (e.g., oat milk).

[0036] The present invention will be described in more detail below based on examples and test examples, but the present invention is not limited to these.

[0037] In the following Examples and Comparative Examples, the enzymes shown in Table 1 were used.

[0038]

[0039] Test Example 1: Investigation of the Effect of Enzyme Addition on Improving Foaming Properties and Mixing Stability with Coffee (Production of Oat Milks in Examples 1 to 8 and Comparative Examples 2 to 5) Oat flour (trade name "Autoflower", Slow Food Kitchen) (protein content: 13%, starch content: 68%) was mixed with water to obtain a 6 w / w% oat flour suspension. To the resulting suspension, α-amylase (trade name "Spitase CP-40FG", units per g: 438,000 U, Nagase ChemteX Corporation) was added at 2.5 ppm relative to starch, and each enzyme shown in Tables 2-1 and 2-2 was added at the respective concentrations shown in Tables 2-1 and 2-2. The mixture was allowed to react at 60°C for 2 hours with stirring. After completion of the reaction, the mixture was heated at 95°C for 10 minutes and then cooled. The cooled suspension was centrifuged at 100 G for 1 minute, and the supernatant was recovered. Using a homogenizer (trade name "POLYTRON PT 3100", KINEMATICA), rapeseed oil (trade name "Sarasara (registered trademark) Canola Oil", J-Oil Mills) was added to the collected supernatant so that the concentration was 2.5%, and the mixture was stirred at 25,000 rpm for 4 minutes to obtain the oat milks of Examples 1 to 8 and Comparative Examples 2 to 5.

[0040] (Production of Oat Milk of Comparative Example 1) Oat milk of Comparative Example 1 was obtained in the same manner as in Examples 1 to 8 and Comparative Examples 2 to 5, except that the enzymes shown in Tables 2-1 and 2-2 were not added.

[0041] (Confirmation of Foaming Ability of Oat Milk) (Test Method) 30 g of the oat milk obtained in Examples 1 to 8 and Comparative Examples 1 to 5 was weighed and placed in a milk foamer (product name: Milk Cup Foamer MCF30W, UCC Ueshima Coffee Co., Ltd.) and foamed for 1 minute in hot mode to generate bubbles. Immediately after foaming was completed, the entire amount, including the foam and liquid oat milk, was added to a 100 ml Falcon tube containing 30 g of coffee, forming a foam layer of oat milk on top of a liquid layer consisting of a mixture of coffee and liquid oat milk. The height of the entire amount (i.e., from the bottom of the liquid layer to the top of the foam layer (Y in Figure 1)) and the height of the foam layer (i.e., from the boundary between the liquid layer and the foam layer to the top of the foam layer (X in Figure 1)) were measured, and the foam layer ratio was calculated according to the following formula. The foaming ability evaluation of each Example and Comparative Example is shown in Tables 3-1 and 3-2 as a relative value when the foam layer ratio of Comparative Example 1 is set to 100. The relative values ​​of the foam layer between each of Examples 1 to 8 and Comparative Examples 2 to 5 and Comparative Example 1 were obtained by preparing the oat milk of Comparative Example 1 (control) every time the tests were conducted for each of Examples 1 to 8 and Comparative Examples 2 to 5. (Formula for calculating foam layer ratio) Foam layer ratio = foam layer height (X) / total height (Y)

[0042] (Confirmation of Mixing Stability of Oat Milk with Coffee) (Coffee Production) 27 g of coffee powder (trade name "Juhla Mokka", ROBERT'S COFFEE) was weighed into a paper filter, and hot water was poured into it in 4 to 5 batches to produce coffee. The pH of the resulting coffee was approximately 4.8 to 5.0. (Preparation of Oat Milk and Coffee Mixture (Coffee with Oat Milk)) The produced coffee (approximately 70°C) was mixed with each of the oat milks (room temperature) from Examples 1 to 8 and Comparative Examples 1 to 5 in a weight ratio of 9:1 (coffee:oat milk), to produce each oat milk-containing coffee using the oat milk from Examples 1 to 8 and Comparative Examples 1 to 5. (Sensory Evaluation) Ten minutes after the oat milk-containing coffee was produced, a panel of three experts evaluated the appearance (uniformity, color brightness) of the oat milk-containing coffee according to the following sensory evaluation criteria. The evaluations of uniformity and color brightness were determined by discussion between the three experts. The results are shown in Tables 3-1 and 3-2.

[0043] [Sensory evaluation criteria (homogeneity)] Definition of homogeneity: The appearance of the liquid after mixing should be a homogeneous solution with no phase separation or precipitates or other insoluble matter. The appearance of the mixed liquid is evaluated according to A to C below. A: No precipitates or aggregates are observed, and it is a homogeneous solution with no phase separation. B: There is a gradual phase change (gradation), or the solution has no phase separation, but a smaller amount of aggregates than C is observed. C: Separation into multiple phases, or precipitates or aggregates are observed.

[0044] [Sensory evaluation criteria (color brightness)] Definition of color brightness: The mixture of oat milk and coffee must have a light brown color (a color similar to that of a cafe latte). Using the color of Comparative Example 1 (control) as the standard, the brightness of the color of the mixture is evaluated on a 5-point scale in increments of 0.5 points. Whenever Examples 1 to 8 and Comparative Examples 2 to 5 are tested, coffee containing oat milk is prepared using the oat milk of Comparative Example 1 (control) for comparison. 1 point: The color is the same as the control. 2 points: The color is slightly lighter than the control. 3 points: The brown color is lighter than the control. 4 points: The brown color is clearly lighter than the control. 5 points: The brown color is significantly lighter than the control.

[0045]

[0046]

[0047]

[0048]

[0049] Test Example 2: Investigation of the Effect of Enzyme Addition on Improving Foaming Properties and Mixing Stability with Coffee (Production of Oat Milks in Examples 9 to 23) Oat flour (trade name "Autoflower", Slow Food Kitchen) (protein content: 13%, starch content: 68%) was mixed with water to obtain a 6 w / w% oat flour suspension. To the resulting suspension, α-amylase (trade name "Spitase CP-40FG", units per g: 438,000 U, Nagase ChemteX Corporation) was added at 2.5 ppm relative to starch, and each enzyme shown in Tables 4-1 and 4-2 was added at the respective concentrations shown in Tables 4-1 and 4-2, and the mixture was allowed to react at 60°C for 2 hours with stirring. After completion of the reaction, the mixture was heated at 95°C for 10 minutes and then cooled. The cooled suspension was centrifuged at 100 G for 1 minute, and the supernatant was recovered. Using a homogenizer (trade name "POLYTRON PT 3100", KINEMATICA), rapeseed oil (trade name "Sarasara (registered trademark) Canola Oil", J-Oil Mills) was added to the collected supernatant so that the concentration was 2.5%, and the mixture was stirred at 25,000 rpm for 4 minutes to obtain the oat milks of Examples 9 to 23.

[0050] (Production of Oat Milk of Comparative Example 6) Oat milk of Comparative Example 6 was obtained in the same manner as in Examples 9 to 23, except that the enzymes shown in Tables 4-1 and 4-2 were not added.

[0051] (Confirmation of Foaming Ability of Oat Milk) (Test Method) 30 g of the oat milk obtained in Examples 9 to 23 was weighed and placed in a milk foamer (product name: Milk Cup Foamer MCF30W, UCC Ueshima Coffee Co., Ltd.) and foamed for 1 minute in hot mode to generate bubbles. Immediately after foaming was completed, the entire amount, including the foam and liquid oat milk, was added to a 100 ml Falcon tube containing 30 g of coffee, forming a foam layer of oat milk on top of a liquid layer consisting of a mixture of coffee and liquid oat milk. The height of the entire amount (i.e., from the bottom of the liquid layer to the top of the foam layer (Y in Figure 1)) and the height of the foam layer (i.e., from the boundary between the liquid layer and the foam layer to the top of the foam layer (X in Figure 1)) were measured, and the foam layer ratio was calculated according to the following formula. The foaming ability evaluation for each Example is shown in Tables 5-1 and 5-2 as a relative value when the foam layer ratio for Comparative Example 6 is set to 100. The relative values ​​of the foam layer between each of the above Examples and Comparative Example 6 were obtained by preparing the oat milk of Comparative Example 6 (control) every time the test for each Example was performed. (Formula for calculating the foam layer ratio) Foam layer ratio = foam layer height (X) / total height (Y)

[0052] (Confirmation of Mixing Stability of Oat Milk with Coffee) (Coffee Production) 27 g of coffee powder (trade name "Juhla Mokka", ROBERT'S COFFEE) was weighed into a paper filter, and hot water was poured into it in 4 to 5 batches to produce coffee. The pH of the resulting coffee was approximately 4.8 to 5.0. (Preparation of Oat Milk and Coffee Mixture (Coffee with Oat Milk)) The produced coffee (approximately 70°C) was mixed with each of the oat milks of Examples 9 to 23 (room temperature) in a weight ratio of 9:1 (coffee:oat milk), and each oat milk-containing coffee was produced using each of the oat milks of Examples 9 to 23. (Sensory Evaluation) Ten minutes after the production of the oat milk-containing coffees, a panel of three experts evaluated the appearance (uniformity, color brightness) of the oat milk-containing coffees according to the following sensory evaluation criteria. The evaluations of uniformity and color brightness were determined by discussion between the three experts. The results are shown in Tables 5-1 and 5-2.

[0053] [Sensory evaluation criteria (homogeneity)] Definition of homogeneity: The appearance of the liquid after mixing should be a homogeneous solution with no phase separation or precipitates or other insoluble matter. The appearance of the mixed liquid is evaluated according to A to C below. A: No precipitates or aggregates are observed, and it is a homogeneous solution with no phase separation. B: There is a gradual phase change (gradation), or the solution has no phase separation, but a smaller amount of aggregates than C is observed. C: Separation into multiple phases, or precipitates or aggregates are observed.

[0054] [Sensory evaluation criteria (color brightness)] Definition of color brightness: The mixture of oat milk and coffee must have a light brown color (a color similar to that of a cafe latte). Using the color of Comparative Example 6 (control) as the standard, the brightness of the color of the mixture is evaluated on a 5-point scale in increments of 0.5. Oat milk coffee is prepared using the oat milk of Comparative Example 6 (control) every time the test is performed for each of Examples 9 to 23, and a comparison is made. 1 point: The color is the same as the control. 2 points: The color is slightly lighter than the control. 3 points: The brown color is lighter than the control. 4 points: The brown color is clearly lighter than the control. 5 points: The brown color is significantly lighter than the control.

[0055]

[0056]

[0057]

[0058]

[0059] According to the present invention, a plant-based milk useful for foaming milk and / or coffee drinks can be provided.

[0060] This application is based on patent application No. 2024-057747 filed in Japan, the contents of which are incorporated in their entirety herein.

Claims

1. A method for producing plant-based milk, comprising treating a raw material containing plant protein with the following (I) and (II): (I) protein glutaminase, and (II) one or more enzymes selected from the group consisting of transglutaminase, pectinase, and glucose oxidase.

2. A method for producing plant-based milk according to claim 1, wherein (I) and (II) are selected from the group consisting of (1) to (6) below. (1) Protein glutaminase and transglutaminase (2) Protein glutaminase and pectinase (3) Protein glutaminase and glucose oxidase (4) Protein glutaminase, transglutaminase, and pectinase (5) Protein glutaminase, transglutaminase, and glucose oxidase (6) Protein glutaminase, transglutaminase, pectinase, and glucose oxidase 3. A method for producing plant-based milk according to claim 1, wherein (II) is transglutaminase and pectinase.

4. A method for producing plant-based milk according to claim 1, wherein (II) is transglutaminase and glucose oxidase.

5. The method for producing plant-based milk according to claim 1, wherein (II) is transglutaminase, pectinase, and glucose oxidase.

6. A manufacturing method according to any one of claims 1 to 5, wherein the raw material containing vegetable protein is raw oats, and the vegetable milk is oat milk.

7. The method according to any one of claims 1 to 5, wherein the plant-based milk is for use in foaming milk.

8. The method according to any one of claims 1 to 5, wherein the plant-based milk is used to prepare a coffee drink.

9. An enzyme preparation for modifying plant-based milk, comprising the following (I) and (II): (I) protein glutaminase; and (II) one or more enzymes selected from the group consisting of transglutaminase, pectinase, and glucose oxidase.

10. The enzyme preparation according to claim 9, wherein (I) and (II) are selected from the group consisting of (1) to (6) below: (1) protein glutaminase and transglutaminase (2) protein glutaminase and pectinase (3) protein glutaminase and glucose oxidase (4) protein glutaminase, transglutaminase, and pectinase (5) protein glutaminase, transglutaminase, and glucose oxidase (6) protein glutaminase, transglutaminase, pectinase, and glucose oxidase 11. The enzyme preparation according to claim 9, wherein (II) is transglutaminase and pectinase.

12. The enzyme preparation according to claim 9, wherein (II) is transglutaminase and glucose oxidase.

13. The enzyme preparation according to claim 9, wherein (II) is transglutaminase, pectinase, and glucose oxidase.

14. An enzyme preparation according to any one of claims 9 to 13, wherein the plant milk is oat milk.

15. An enzyme preparation according to any one of claims 9 to 13, wherein the plant-based milk is for foaming milk.

16. An enzyme preparation according to any one of claims 9 to 13, wherein the plant-based milk is used to prepare a coffee beverage.

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