Preventing coagulation of plant-based milk

Abstract

The objective of this study is to develop an effective method for preventing the aggregation of plant-based milk under high-temperature conditions. By treating plant-based milk with protein deamidating enzyme, the dispersibility under high-temperature conditions is improved, and aggregation is prevented when added to hot liquid beverages or liquid foods.

Description

Technical Field

[0001] The present invention relates to plant milk. Specifically, it relates to plant milk with improved dispersibility (difficult to aggregate) and its uses, etc. This application claims priority based on Japanese Patent Application No. 2019-029904 filed on February 21, 2019, and Japanese Patent Application No. 2019-077841 filed on April 16, 2019, and the entire contents of the patent applications are incorporated herein by reference.

Background Art

[0002] Due to allergy problems, the increasing number of vegetarians, religious reasons, etc., as an alternative raw material for food and beverages using milk protein sources derived from animals, such as milk, plant-derived proteins from soybeans have become widespread. However, as the spread has progressed, it has been found that soybeans can also cause allergies, and in recent years, the development of plant-derived protein raw materials as alternatives to soybeans has been active. In fact, proteins derived from grains such as peas, rice, and oats, and nut proteins such as almonds, cashew nuts, and peanuts have been successively commercialized as foods and beverages as alternatives to soybeans, and it can be said that the need and desire for diversification of other plant-derived protein raw materials as alternatives to soybeans for allergy avoidance are high.

[0003] On the other hand, when replacing the milk protein raw material with a plant-derived protein raw material, there are scattered cases where it cannot be directly replaced because the types and functionalities of the proteins, or the components that make up the aroma and taste are different. For example, it is known that nut milks such as almond milk and peanut milk aggregate when added to high-temperature acidic beverages such as coffee and black tea as an alternative to milk. Usually, such aggregation does not occur in milk, and it can be said to be a phenomenon peculiar to plant milks such as nut milk.

[0004] To the best of our knowledge, there are no reports (literature, etc.) that clearly explain the mechanism of protein aggregation that occurs when nut milk is added to high-temperature acidic liquid foods, or how to deal with it. Although there has been some trial and error among consumers regarding countermeasures (for example, mixing nut milk and coffee after reducing the temperature difference, or pouring coffee into nut milk slowly), a fundamental solution has yet to be found.

[0005] On the other hand, milk proteins exhibit unstable dispersion stability under acidic conditions near their isoelectric point, and it is known that acidic milk beverages are prone to precipitation and aggregation. To prevent this aggregation of milk proteins, polysaccharides such as pectin and carboxymethylcellulose are added (see, for example, Patent Documents 1 and 2). While it may be possible to prevent the aggregation of nut proteins by using such dispersion stabilizers, the use of additives is essential. Furthermore, when polysaccharides are used, there is a possibility of side effects such as increased viscosity depending on the amount added.

[0006] Regarding preventing the aggregation of milk proteins without adding additives, a coffee whitener treated with protein deamidation enzymes has been proposed (Patent Document 3). However, this coffee whitener is a product containing emulsifiers, and its use is limited to products that use whitener, such as coffee and tea. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] International Publication No. 2012 / 176852 brochure [Patent Document 2] Patent No. 3885194 [Patent Document 3] International Publication No. 2011 / 108633 Pamphlet [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] The coagulation phenomenon, which is characteristic of plant-based milks such as nut milk, reduces the value (utility value, commercial value, etc.) of plant-based milks, which are expected to see further increases in demand and expanded applications in the future. Therefore, in order to enhance the value of plant-based milks and promote their use and application, the present invention aims to create an effective means for preventing coagulation of plant-based milks, and in particular to provide plant-based milks that are less prone to coagulation in high-temperature liquid beverages (especially acidic liquid beverages) and high-temperature liquid foods (especially acidic liquid foods) without the addition of additives. [Means for solving the problem]

[0009] In light of the above issues, the inventors focused on protein deamidation and attempted to improve the dispersibility of plant-based milk when added to high-temperature beverages and liquid foods by treating it with a protein deamidation enzyme. It should be noted that there have been no previous reports of using plant-based milk treated with a protein deamidation enzyme in high-temperature beverages or liquid foods.

[0010] First, regarding its addition to hot coffee, a typical use for nut milk, we investigated whether treatment with a protein deamidation enzyme was effective in preventing aggregation. Surprisingly, no protein aggregation occurred when using almond milk treated with the enzyme. Based on this finding, we conducted detailed experiments considering various applications, and the results revealed that treatment with a protein deamidation enzyme is extremely effective in improving the dispersibility of nut milk in general. In other words, we found an effective means of preventing aggregation of nut milk, improving its dispersibility and successfully preparing nut milk that is less likely to aggregate when added to high-temperature beverages and liquid foods without the use of emulsifiers or other additives. Furthermore, this research provided many useful insights for using nut milk in various beverages and foods. In addition, it was revealed that treatment with a protein deamidation enzyme is effective in preventing aggregation when added to high-temperature beverages and liquid foods for other plant-based milks such as soy milk, oat milk, pea milk, and hemp milk. Based on these results, the following invention is provided. As mentioned above, the use of protein deamidation enzymes to improve the dispersibility of coffee whitener has been proposed. However, coffee whitener is generally prepared by homogenizing an emulsified liquid, which is made primarily from edible oils and fats, with emulsifiers, and optionally milk components, thickeners, and flavorings, using an emulsifier with excellent shearing power, such as a high-pressure homogenizer. Its raw materials, composition, and preparation method are completely different from those of plant-based milk. Therefore, it is impossible to predict the effectiveness of methods effective in improving the dispersibility of coffee whitener on plant-based milk, let alone the possibility of their application. [1] Vegetable milk treated with a protein deamidation enzyme for preparing high-temperature vegetable milk-containing liquid food and beverages. [2] The plant-based milk according to [1], wherein the plant-based milk is nut milk, soy milk, pea milk, oat milk, or hemp milk. [3] The vegetable milk according to [2], wherein the nuts used as raw materials for the nut milk are one or more nuts selected from almonds, cashews, hazelnuts, pecans, macadamia nuts, pistachios, walnuts, Brazil nuts, peanuts, coconuts, chestnuts, sesame seeds, and pine nuts. [4] A plant-based milk according to any one of items [1] to [3], wherein the raw material plant protein concentration is 0.2% (w / v) to 10.0% (w / v). [5] The plant-based milk according to any one of [1] to [4], wherein the dispersibility is improved by the above treatment. [6] A plant-based milk as described in [5] that does not cause protein aggregation when mixed with weakly acidic to weakly alkaline liquid food or beverage (provided the pH of the mixture is 5 or higher). [7] The plant-based milk according to [6], wherein the pH of the liquid food or beverage is 5 to 7. [8] The plant milk according to [6], wherein the liquid food or beverage is selected from the group consisting of coffee, coffee beverages, tea, tea beverages, fruit juice, fruit juice beverages, sports drinks, nutritional supplements, soups, curries, cocoa, and chocolate beverages, and is an ingredient, intermediate product, or final product of a beverage or liquid food. [9] A plant-based milk according to any one of items [1] to [8], which does not contain emulsifiers or thickening polysaccharides for preventing coagulation.

[10] A plant-based milk as described in any one of the following [1] to [9], wherein the protein deamide enzyme is derived from a microorganism of the genus Chryseobacterium.

[11] The plant milk described in

[10] , wherein the microorganism of the genus Chryseobacterium is Chryseobacterium proteoricum.

[12] A method for producing plant-based milk with improved dispersibility, characterized by treating plant-based milk with a protein deamidation enzyme.

[13] The manufacturing method according to

[12] , comprising the following steps (1) and (2): (1) Steps to prepare plant-based milk, (2) A step in which the plant-based milk prepared in (1) is treated with a protein deamidation enzyme.

[14] The manufacturing method according to

[13] , wherein the plant milk in step (1) is plant milk before heat sterilization.

[15] The manufacturing method described in

[14] further includes the following step (3): (3) The step of heat treatment. A liquid food or drink containing the plant milk according to any one of

[16] [1] to

[11] .

[17] The liquid food or drink according to

[16] , which is a liquid food or drink having a pH of 5 or more.

[18] The liquid food or drink according to

[16] , which is a drink or liquid food selected from the group consisting of coffee beverages, coffee creamers, tea beverages, fruit juice beverages, sports beverages, nutritional supplements, soups, curries, cocoa beverages and chocolate beverages.

[19] A method for producing a liquid food or drink, which comprises mixing a plant milk treated with a protein deamidase with a raw material, intermediate product or final product of the liquid food or drink under high temperature conditions.

[20] The production method according to

[19] , comprising the following steps (1) and (2): (1) A step of preparing a plant milk treated with a protein deamidase. (2) A step of mixing the plant milk prepared in (1) with a raw material, intermediate product or final product of the liquid food or drink under high temperature conditions.

Brief Description of Drawings

[0011] [Figure 1] Summary of experimental results (relationship between protein concentration of nut milk and aggregation / aggregation prevention effect). [Figure 2] Summary of experimental results (relationship between pH of liquid and aggregation / aggregation prevention effect). [Figure 3] Summary of experimental results (aggregation prevention effect in various liquids). The results of Experiment 1 (aggregation prevention in coffee) are also included. [Figure 4] Summary of experimental results (aggregation prevention effect with nut milk other than almond milk). [Figure 5] Summary of experimental results (relationship between temperature of liquid and aggregation / aggregation prevention effect). [Figure 6] Summary of experimental results (examination of enzyme treatment conditions (enzyme addition amount, reaction temperature, reaction time)). [Figure 7] Summary of experimental results (aggregation prevention effect in soy milk). [Figure 8] Summary of experimental results (aggregation prevention effect in plant milk).

Mode for Carrying Out the Invention

[0012] 1. Plant milk with improved dispersibility The first aspect of the present invention relates to a plant milk (also called a plant protein-containing beverage) having improved dispersibility when added to a high-temperature liquid food or drink (beverage or liquid food). The "liquid food or drink" includes not only the final product but also those used as raw materials for other foods, beverages, etc., or intermediate products. The plant milk of the present invention has been treated with a protein deamidase, and as a result of this treatment, its dispersibility has been improved. Since the plant milk of the present invention exhibits excellent dispersibility when added to a high-temperature liquid food or drink, it is difficult to aggregate when added to a high-temperature beverage such as coffee or tea, for example, without using additives (for example, emulsifiers, thickening polysaccharides (pectins, carboxymethylcellulose, etc.), salts) to enhance dispersibility. This property enables it to be used in various beverages and foods.

[0013] Plant milk is, for example, milk made from various nuts, soybeans, oats, peas, hemp, lupin beans, broad beans, chickpeas, barley, wheat, rice, millet, foxtail millet, canary seed, teff, quinoa, or flax seeds. Nut milk represented by almond milk (also called a nut protein-containing beverage) is a plant milk made from nuts. Generally, it is prepared by processes such as crushing of shelled nuts, soaking / dissolving, mixing / stirring, filtering, homogenization, and sterilization. The method for preparing the nut milk used in the present invention is not particularly limited. The nuts used as the raw material for nut milk are not particularly limited. Examples of the raw nuts include almonds, cashew nuts, hazelnuts, pecans, macadamia nuts, pistachios, walnuts, Brazil nuts, peanuts, coconuts, chestnuts, sesame seeds, and pine nuts. Also, as the plant milk, it may be purchased from a raw material manufacturer or commercially available plant milk and used in the present invention.

[0014] The plant-based milk of the present invention is obtained by treating plant-based milk with a protein deamidation enzyme to improve its dispersibility. For the sake of convenience, the plant-based milk subjected to treatment with the protein deamidation enzyme will be referred to as "untreated plant-based milk" below.

[0015] Unprocessed plant-based milk can also be used, which is made using two or more types of raw plant materials (for example, a combination of almonds and cashews, or almonds and peanuts).

[0016] The protein concentration in untreated plant milk (raw material protein concentration) is not particularly limited, but untreated plant milk with a protein concentration of, for example, 0.2%(w / v) to 10.0%(w / v), preferably 0.2%(w / v) to 8.0%(w / v), and more preferably 0.2%(w / v) to 5.0%(w / v) is used. Similarly, the protein concentration of plant milk after protein deamidation enzyme treatment is also, for example, 0.2%(w / v) to 10.0%(w / v), preferably 0.2%(w / v) to 8.0%(w / v), and more preferably 0.2%(w / v) to 5.0%(w / v).

[0017] The protein deamide enzyme used in the present invention has the effect of deamidering by directly acting on the amide group of a protein without cleaving peptide bonds or crosslinking proteins. The type and origin of the enzyme are not particularly limited as long as it exhibits this effect. Examples of protein deamide enzymes include protein deamide enzymes from the genera Chryseobacterium, Flavobacterium, Empedobacter, Sphingobacterium, Aureobacterium, or Myroides disclosed in Japanese Patent Publication No. 2000-50887, Japanese Patent Publication No. 2001-218590, WO2006 / 075772, etc., as well as commercially available protein glutaminase derived from the genus Chryseobacterium. Preferably, an enzyme derived from the genus Chryseobacterium is used (a specific example being an enzyme derived from Chryseobacterium proteoricum (for example, protein glutaminase "Amano" 500, manufactured by Amano Enzyme Co., Ltd.)).

[0018] The protein deamide enzyme can be prepared from a culture medium of a microorganism that produces the protein deamide enzyme. The microorganism used to prepare the protein deamide enzyme is not particularly limited, but any microorganism that produces the enzyme, such as those belonging to the genera Chryseobacterium, Flavobacterium, Empedobacter, Sphingobacterium, Aureobacterium, or Myroides, can be used. A specific example of a microorganism suitable for preparing the protein deamide enzyme is Chryseobacterium sp. No. 9670, which belongs to the genus Chryseobacterium.

[0019] For example, protein deamidase can be obtained from the culture medium or cells of the above-mentioned microorganisms. That is, secreted proteins can be recovered from the culture medium, and other proteins can be recovered from the cells. Methods for preparing protein deamidase from the culture medium include known protein separation and purification methods (centrifugation, UF concentration, salting out, various chromatography methods using ion exchange resins, etc.). For example, the culture medium can be centrifuged to remove the cells, and then the desired enzyme can be obtained by combining salting out, chromatography, etc. When recovering the enzyme from the cells, for example, the cells can be crushed by pressurization, sonication, etc., and then the desired enzyme can be obtained by separation and purification in the same manner as above. Alternatively, the cells can be recovered from the culture medium beforehand by filtration, centrifugation, etc., before performing the above series of steps (crushing, separation, and purification of cells). The enzyme may be powdered by drying methods such as freeze-drying or vacuum drying, and appropriate excipients and drying aids may be used at that time.

[0020] In this application, the activity of the protein deamidase is measured by the following method. (1) Add 0.1 ml of aqueous solution containing protein deamidase to 1 ml of 0.2 M phosphate buffer (pH 6.5) containing 30 mM Z-Gln-Gly, incubate at 37°C for 10 minutes, then add 1 ml of 0.4 M TCA solution to stop the reaction. Prepare a blank by adding 0.1 ml of aqueous solution containing protein deamidase to 1 ml of 0.2 M phosphate buffer (pH 6.5) containing 30 mM Z-Gln-Gly and 1 ml of 0.4 M TCA solution, and incubate at 37°C for 10 minutes. (2) The amount of ammonia produced by the reaction is measured using the ammonia test Wako (Wako Pure Chemical Industries) on the solution obtained in (1). The ammonia concentration in the reaction solution is determined from a calibration curve that shows the relationship between ammonia concentration and absorbance (630 nm) prepared using ammonia standard solution (ammonium chloride). (3) The activity of the protein deamidase is calculated using the following formula, with the amount of enzyme that produces 1 μmol of ammonia per minute being defined as 1 unit. Enzyme activity (U / mL) = Ammonia concentration in reaction solution (mg / L) × (1 / 17.03) × (Volume of reaction solution / Volume of enzyme solution) × (1 / 10) × Df (In the formula, the volume of the reaction solution is 2.1, the volume of the enzyme solution is 0.1, and Df is the dilution ratio of the enzyme solution. Also, 17.03 is the molecular weight of ammonia.)

[0021] As long as it is effective in improving the dispersibility of plant-based milk, the conditions for treatment with protein deamidation enzymes are not particularly limited; the optimal reaction conditions can be set by adjusting the reaction temperature, reaction time, and enzyme addition amount (enzyme concentration).

[0022] While not limited to this example, the reaction temperature can be set, for example, within the range of 2°C to 70°C, preferably within the range of 5°C to 60°C, and more preferably within the range of 15°C to 50°C. Similarly, the reaction time can be set, for example, within the range of 10 minutes to 7 days, preferably within the range of 30 minutes to 3 days, and more preferably within the range of 1 hour to 1 day. The amount of enzyme added can be set, for example, within the range of 0.01 (U / g protein) to 500 (U / g protein), preferably within the range of 0.02 (U / g protein) to 50 (U / g protein), and more preferably within the range of 0.2 (U / g protein) to 5 (U / g protein). Here, "U / g protein" refers to the number of units per (g) of substrate plant protein. As mentioned above, the protein concentration in untreated plant milk is not particularly limited, but untreated plant milk with a protein concentration of, for example, 0.2%(w / v) to 10.0%(w / v), preferably 0.2%(w / v) to 8.0%(w / v), and more preferably 0.2%(w / v) to 5.0%(w / v) is subjected to treatment with protein deamide enzymes.

[0023] When setting the processing conditions using protein deamidation enzymes, it is recommended to follow the following indicators (a) to (c). (a) If the reaction temperature is lowered, increase the reaction time or increase the amount of enzyme added (or both). (b) To shorten the reaction time, increase the reaction temperature (but not exceed 70°C, preferably 60°C or lower) or increase the amount of enzyme added (or both). (c) If the amount of enzyme added is reduced, the reaction temperature should be increased (but not exceeding 70°C, preferably 60°C or lower) or the reaction time should be increased (or both).

[0024] The following are examples of more specific indicators to consider when setting processing conditions. If the reaction temperature is 5°C or less than 15°C, the reaction time should be longer than 8 hours (preferably 24 hours or more), or the amount of enzyme added should be 0.2 U / g protein or more (preferably 1 U / g protein or more). If the reaction temperature is 15°C or less than 25°C, the reaction time should be longer than 7 hours, or the amount of enzyme added should be greater than 0.2 U / g protein (preferably 1 U / g protein or more). If the reaction temperature is 25°C or less than 40°C, the reaction time should be longer than 5 hours (preferably 7 hours or more), or the amount of enzyme added should be 0.2 U / g protein or more (preferably 1 U / g protein or more). If the reaction temperature is 40°C or less than 50°C, the reaction time should preferably be 3 hours or more, or the amount of enzyme added should preferably be 0.2 U / g protein or more. If the reaction temperature is 50 or less (however, the temperature should not exceed 70°C, preferably 60°C or lower), the reaction time should preferably be 3 hours or more, or the amount of enzyme added should preferably be 0.2 (U / g protein) or more.

[0025] As described above, the plant-based milk of the present invention exhibits excellent dispersibility when added to high-temperature liquid food and beverages and is less prone to protein aggregation. In the present invention, high temperature is not particularly limited as long as it is high enough to cause protein aggregation of the plant-based milk due to heat, but for example, it is 50°C or higher, preferably 60°C or higher, more preferably 70°C or higher, more preferably 80°C or higher, and most preferably 90°C or higher. The upper limit of high temperature is, for example, 100°C. The temperature of the plant-based milk is not particularly limited, but it is preferable that the temperature of the liquid food and beverage after mixing with the plant-based milk is within the above high temperature conditions (i.e., for example, 50°C or higher). Typically, protein aggregation does not occur when mixed (added) to a weakly acidic (3≦pH<6) to weakly alkaline (8≦pH<11) liquid food and beverage (provided that the pH of the mixture is 5 or higher). The pH of the liquid food and beverage after mixing that does not cause protein aggregation is, for example, 5 to 10, preferably 5 to 9, and more preferably 5 to 7. The liquid food and beverages (drinks, liquid foods) to which the plant-based milk of the present invention is mixed are not particularly limited, and examples include coffee, coffee beverages, tea (black tea, green tea, oolong tea, etc., including those obtained by reducing the extract and those obtained by reducing the extract after processing (e.g., concentration, freeze-drying)), tea beverages (flavored tea, milk tea, fruit juice tea beverages, etc.), fruit juice, fruit juice beverages, sports drinks, nutritional supplements (protein drinks, nursing nutrition drinks, etc.), soups (bouillon-based soups, stews, chowders, borscht, vegetable soups (e.g., tomato soup, corn soup, potage, pumpkin soup), miso soup), curry, cocoa, and chocolate beverages.

[0026] In one preferred embodiment of the present invention, taking advantage of its excellent dispersibility and resistance to protein aggregation, it does not contain emulsifiers (glycerin fatty acid esters, sucrose fatty acid esters, lecithin, saponins, etc.), thickening polysaccharides (pectin, carboxymethylcellulose, etc.), salts (sea salt, calcium salts, phosphates, etc.) used to prevent aggregation. In particular, it does not contain emulsifiers or thickening polysaccharides. Thus, the present invention provides a plant-based milk that meets consumer needs for products with few or no additives. It should be noted that even in this preferred embodiment, the use of additives for purposes other than preventing aggregation (specifically, for example, for adjusting taste and flavor) is not prohibited.

[0027] As can be seen from the above explanation, the plant-based milk of the present invention can be produced by treating untreated plant-based milk with a protein deamidation enzyme. Therefore, the plant-based milk of the present invention can typically be obtained by a production method comprising the following steps (1) and (2). (1) Steps to prepare plant-based milk, (2) A step in which the plant-based milk prepared in (1) is treated with a protein deamidation enzyme.

[0028] Step (2), i.e., treatment with a protein deamidase enzyme, may be performed either before or after pasteurization of the plant-based milk. However, for the sake of simplifying the manufacturing process, it is preferable to perform this step before pasteurization of the plant-based milk, followed by a pasteurization step that also inactivates the protein deamidase enzyme (in other words, step (2) may be incorporated into the plant-based milk manufacturing process). Therefore, in a preferred embodiment, step (3) "heat treatment step" is performed after step (2). The conditions for the heat treatment are not particularly limited as long as they allow for the inactivation of the protein deamidase enzyme and the sterilization of the plant-based milk. For example, the treatment may be performed at a temperature of 70°C to 150°C for 1 second to 5 hours.

[0029] 2. Uses of plant-based milk A second aspect of the present invention relates to the uses of the plant-based milk of the present invention. The plant-based milk of the present invention exhibits excellent dispersibility when mixed with liquid food and beverages under high-temperature conditions, and is less prone to protein aggregation. That is, the plant-based milk of the present invention can be used in the preparation of high-temperature liquid food and beverages containing plant-based milk. Due to this characteristic, it is suitable for use in various beverages and liquid foods. In other words, various beverages and liquid foods containing the plant-based milk of the present invention are provided.

[0030] As shown in the examples described below, detailed studies by the inventors have revealed that (i) the pH range in which aggregation does not occur can be extended to the acidic side by treatment with a protein deamidation enzyme, and (ii) protein aggregation that occurs when plant-based milk is mixed with high-temperature beverages or liquid foods depends on the pH of the beverage after mixing with plant-based milk, and does not occur if the pH is 5 or higher. In view of this finding, it is preferable that the pH of the liquid food or beverage containing the plant-based milk of the present invention is 5 or higher. More specifically, the pH of the liquid food or beverage containing the plant-based milk of the present invention is preferably 5 to 9, more preferably 5 to 8, and even more preferably 5 to 7.5.

[0031] Examples of liquid food and beverages (beverages or liquid foods) include coffee beverages, coffee whiteners (which can also be used with tea, etc.), tea beverages (flavored teas, milk teas, fruit juice teas, etc.), fruit juices, sports drinks, nutritional supplements (protein drinks, nutritional drinks for the elderly, etc.), various soups, curries, cocoa drinks, and chocolate drinks. As can be seen from these examples, the present invention can be used not only for neutral beverages and liquid foods, but also for weakly acidic beverages and liquid foods.

[0032] Plant-based milk is mixed with other raw materials, for example, during the manufacturing process of liquid food and beverages. Preferably, plant-based milk is mixed in at the final stage of the manufacturing process, i.e., after the other raw materials have been mixed and processed (at the stage where it takes the form / shape of a product). However, sterilization, or the addition of seasonings, preservatives, flavorings, antioxidants, etc., for the purpose of adjusting the taste or maintaining quality may be carried out afterward. On the other hand, it is also a preferred embodiment to mix plant-based milk into liquid food and beverages after the manufacturing process is completed (i.e., in the form of a final product rather than an intermediate product). In this embodiment, the present invention can be applied without changing the manufacturing process of liquid food and beverages.

[0033] As can be seen from the above description, the plant-based milk-containing liquid food and beverage of the present invention can be produced by mixing plant-based milk treated with protein deamidation enzymes with raw materials, intermediate products, or final products of liquid food and beverages under high temperature conditions. Accordingly, the liquid food and beverage of the present invention can typically be obtained by a production method comprising the following steps (1) and (2). (1) Step to prepare plant-based milk treated with protein deamidation enzyme (2) A step of mixing the plant-based milk prepared in (1) with the raw material, intermediate product, or final product of a liquid food or beverage under high temperature conditions. [Examples]

[0034] 1. Preventing coagulation in coffee To 100 mL of commercially available almond milk (Rude brand, protein content 1.5%, ingredients: almonds, water), protein glutaminase "Amano" 500 (Amano Enzyme Co., Ltd., 500 U / g) was added at a rate of 1 U per gram of protein in the almond milk, and the mixture was reacted at 50°C for 5 hours (deamide reaction). The enzyme was then inactivated by heat treatment at 95°C for 20 minutes, and the mixture was cooled to 5°C to obtain enzyme-treated almond milk.

[0035] Commercial instant coffee was dissolved in hot water to prepare a 2% coffee solution. When 20-30 mL of enzyme-treated almond milk was added to 150 mL of the coffee solution (the pH after adding the enzyme-treated almond milk was 5.7), no coagulation was observed. In contrast, when unenzyme-treated almond milk was used, clear coagulation was observed. Furthermore, when peanut milk was used instead of almond milk under the same conditions, similar results were obtained (no coagulation occurred with enzyme-treated peanut milk).

[0036] 2. Relationship between protein concentration in nut milk and its coagulation / coagulation prevention effect <No enzyme treatment> (1) Method Commercially available almond milk (Rude brand, 1.5% protein content, ingredients: almonds, water) was diluted with tap water to achieve protein concentrations of 0.1%, 0.5%, and 1.5% (w / v). After cooling to 5°C, 5 mL of each solution was added to 50 mL of coffee solution heated to 90°C, and the presence or absence of coagulation was checked.

[0037] (2) Results Almond milk at all protein concentrations showed aggregation (Figure 1).

[0038] <Enzyme-treated> (1) Method Commercially available almond milk (manufactured by Rude, protein content 1.5%, ingredients: almonds, water) was mixed with protein glutaminase "Amano" 500 (manufactured by Amano Enzyme Co., Ltd., 500 U / g), with 1 U added per gram of protein in the almond milk. The mixture was reacted at 50°C for 5 hours (deamide reaction). The enzyme was then inactivated by heat treatment at 90°C for 15 minutes to obtain enzyme-treated almond milk. The enzyme-treated almond milk was diluted with tap water to achieve protein concentrations of 0.1%, 0.5%, 0.75%, 1.0%, and 1.5% (w / v). After cooling to 5°C, 5 mL of each solution was added to 50 mL of coffee solution heated to 90°C, and the presence or absence of coagulation was checked.

[0039] (2) Results No coagulation was observed in any of the almond milk samples at any of the protein concentrations (Figure 1).

[0040] 3. Relationship between liquid pH and flocculation / flocculation prevention effect (1) Method After adjusting the pH with hydrochloric acid or sodium hydroxide, 15-20 mL of non-enzyme-treated or enzyme-treated almond milk (protein concentration 1.5% (w / v)) was added to water heated to 90°C, and coagulation was observed. The enzyme-treated almond milk was prepared using the method described in experiment 2 above.

[0041] (2) Results (Figure 2) In the case of non-enzyme-treated almond milk, flocculation was observed in the mixed solution at a pH of 2.5-7.0 after addition. On the other hand, in the case of enzyme-treated almond milk, flocculation was observed in the mixed solution at a pH of 2.7-4.8 after addition.

[0042] 4. Anti-coagulation effect in various liquids 4-1. Black Tea (1) Method A commercially available tea bag (Twinings English Breakfast) was placed in boiling water and steeped for 2-3 minutes. After steeping, the tea bag was removed to prepare the tea. Non-enzyme-treated almond milk or enzyme-treated almond milk (protein concentration 1.5% (w / v)) was added to this tea, and the presence or absence of coagulation was checked. The tea immediately before adding the almond milk was at 80°C and pH 5.2. The pH of the tea after adding the almond milk was 5.9. The enzyme-treated almond milk was prepared using the method described in experiment 2 above.

[0043] (2) Results (Figure 3) In the case of non-enzyme-treated almond milk, slight coagulation was observed. In contrast, no coagulation was observed in enzyme-treated almond milk.

[0044] 4-2. Lemon Tea (1) Method A commercially available tea bag (Twinings English Breakfast) was placed in boiling water and steeped for 2-3 minutes. After steeping, the tea bag was removed to prepare the tea. Lemon juice was added to this tea to adjust the pH, and then non-enzyme-treated almond milk or enzyme-treated almond milk (protein concentration 1.5% (w / v)) was added to check for coagulation. The tea temperature immediately before adding the almond milk was 70°C. The enzyme-treated almond milk was prepared using the method described in experiment 2 above.

[0045] (2) Results (Figure 3) When the pH before adding almond milk was 3.5, coagulation was observed in both non-enzyme-treated and enzyme-treated almond milk. Furthermore, the pH of the tea after adding non-enzyme-treated almond milk was 3.9, and the pH of the tea after adding enzyme-treated almond milk was 4.1.

[0046] On the other hand, when the pH before adding almond milk was 4.0, coagulation was observed in non-enzyme-treated almond milk, but not in enzyme-treated almond milk. Furthermore, the pH of the tea after adding non-enzyme-treated almond milk was 4.9, and the pH of the tea after adding enzyme-treated almond milk was 5.0.

[0047] 4-3. Decaf (1) Method A decaffeinated coffee solution was prepared by pouring boiling water over commercially available decaffeinated coffee powder (Nestlé, Nescafé Gold) and stirring thoroughly. Non-enzyme-treated almond milk or enzyme-treated almond milk (protein concentration 1.5% (w / v)) was added to this solution, and the presence or absence of coagulation was checked. The decaffeinated coffee solution immediately before adding the almond milk was at 80°C and pH 5.3. The pH of the decaffeinated coffee solution after adding the almond milk was 5.8. The enzyme-treated almond milk was prepared using the method described in experiment 2 above.

[0048] (2) Results (Figure 3) Coagulation was observed in non-enzyme-treated almond milk, but not in enzyme-treated almond milk.

[0049] 4-4. Tomato Soup (1) Method Commercially available chicken soup stock (Unilever, Knorr Chicken Cubes) was dissolved in a specified amount of boiling water to prepare chicken soup, and then commercially available tomato puree was added. After adjusting the pH of the tomato soup by increasing or decreasing the amount of puree added, non-enzyme-treated almond milk or enzyme-treated almond milk (protein concentration 1.5% (w / v)) was added, and the presence or absence of coagulation was checked. The temperature of the tomato soup immediately before adding the almond milk was 80°C. The enzyme-treated almond milk was prepared using the method described in experiment 2 above.

[0050] (2) Results (Figure 3) When the pH before adding almond milk was 5.0, coagulation was observed with non-enzyme-treated almond milk, but not with enzyme-treated almond milk. The pH of the tomato soup after adding non-enzyme-treated almond milk and after adding enzyme-treated almond milk were both 5.4.

[0051] When the pH before adding almond milk was 4.0, coagulation was observed in both non-enzyme-treated and enzyme-treated almond milk. The pH of the tomato soup after adding non-enzyme-treated almond milk and after adding enzyme-treated almond milk was both 4.0. It is thought that the strong buffering capacity of the citric acid pH adjuster added to the tomato puree prevented the pH from changing when almond milk was added, resulting in coagulation.

[0052] 5. Anticoagulation effect of nut milks other than almond milk. (1) Method Commercially available peanut milk (Rude, 2.0% protein content, ingredients: peanuts, water), commercially available cashew milk (PLENISH, 0.9% protein content, ingredients: water, cashews, salt), pistachio milk (Borna Food, 1.0% protein content), and hazelnut milk (Plenish, 0.6% protein content) were mixed with protein glutaminase "Amano" 500 (Amano Enzyme Co., Ltd., 500U / g), with 1 unit added per gram of nut protein. The mixture was reacted at 50°C for 5 hours (deamide reaction). After the enzymatic reaction, the enzyme was quickly deactivated by treating at 90°C for 15 minutes. After cooling in running water, the mixture was cooled to 5°C in a refrigerator. 5 mL of each mixture was then added to 50 mL of coffee solution heated to 90°C, and the presence or absence of coagulation was checked.

[0053] (2) Results (Figure 4) Coagulation was observed in peanut milk, cashew milk, pistachio milk, and hazelnut milk without enzyme treatment, but not after enzyme treatment. This result indicates that similar effects can be obtained from enzyme treatment of nut milks other than almond milk.

[0054] 6. Relationship between liquid temperature and coagulation / coagulation prevention effect <Coffee temperature is varied (almond milk is kept constant at 5°C)> (1) Method 50 mL of coffee, adjusted to each temperature, was mixed with 5 mL of either non-enzyme-treated or enzyme-treated almond milk cooled to 5°C, and the presence or absence of coagulation was checked.

[0055] (2) Results (Figure 5) In non-enzyme-treated almond milk, coagulation was observed at coffee temperatures above 60°C, and the amount of coagulation increased with increasing temperature. On the other hand, enzyme-treated almond milk showed an anti-coagulation effect (at coffee temperatures of 60°C and 90°C).

[0056] <Fluctuate the coffee temperature (almond milk remains constant at 90°C)> (1) Method 50 mL of coffee adjusted to each temperature was mixed with 5 mL of non-enzyme-treated or enzyme-treated almond milk heated to 90°C, and the presence or absence of coagulation was checked.

[0057] (2) Results (Figure 5) Coagulation was observed when 90°C unenzyme-treated almond milk was added to 90°C coffee. Coagulation was also observed when 90°C unenzyme-treated almond milk was added to 50°C coffee, but not when 90°C unenzyme-treated almond milk was added to 40°C coffee. It is thought that when 90°C unenzyme-treated almond milk was added to 50°C coffee, the coffee temperature temporarily rose, causing coagulation. Furthermore, it is thought that coagulation is more likely to occur at higher temperatures after mixing (above 50°C).

[0058] When enzyme-treated almond milk was added (to coffee at 40°C or 90°C), no coagulation was observed.

[0059] 7. Examination of enzyme treatment conditions (amount of enzyme added, reaction temperature, reaction time) (1) Method Commercial almond milk (manufactured by Rude, protein content 1.5%, ingredients: almonds, water) was mixed with protein glutaminase "Amano" at a concentration of 0.2U, 1U, or 5U per gram of protein in the almond milk, and reacted at a specified temperature (5°C, 15°C, 25°C, 40°C, or 50°C) for 3-24 hours (deamide reaction). After the enzymatic reaction, the enzyme was quickly deactivated by treating it at 90°C for 15 minutes, cooled under running water, and then chilled to 5°C in a refrigerator. 5 mL of each solution was then added to 50 mL of coffee solution heated to 90°C, and the presence or absence of coagulation was checked.

[0060] (2) Results (Figure 6) The effect varies depending on the amount of enzyme added, the reaction temperature, and the reaction time, but it can be seen that aggregation can be prevented by adjusting these conditions. Specifically, if the reaction temperature is low, the desired effect can be obtained by increasing the amount of enzyme added or increasing the reaction time (or both). For example, even at a reaction temperature of 5°C, aggregation can be effectively prevented if the amount of enzyme added is 1 U or more, or if the reaction time is long. On the other hand, if the reaction time is short, the desired effect can be obtained by increasing the reaction temperature or increasing the amount of enzyme added (or both). For example, even with a reaction time of 3 hours, aggregation prevention can be achieved if the reaction temperature is set to 40°C or higher or if the amount of enzyme added is 1 U or more. Also, the amount of enzyme added can be reduced by increasing the reaction temperature or increasing the reaction time (or both). For example, if the reaction temperature is set to 25°C or higher, or the reaction time is long, the amount of enzyme added can be reduced to 0.2 U or less.

[0061] <Summary> • In the range of nut protein concentrations from 0.1% to 1.5% (w / v), a similar anti-aggregation effect was observed regardless of the concentration. In other words, enzymatic treatment with protein deamidation enzymes is effective in preventing the aggregation of nut milk at various protein concentrations, demonstrating its high versatility. • Depending on the type of liquid mixed with the nut milk, the general trend is that without enzymatic treatment with a protein deamidation enzyme, the mixture will aggregate if the pH drops below 7 after mixing with the nut milk. However, with enzymatic treatment, the lower limit of aggregation can be extended to pH 5. It was shown that if the pH of the liquid after mixing with nut milk is 5 or higher, it can be used not only in beverages such as coffee and tea, but also in acidic liquid foods such as sour milk soups. Furthermore, if the pH of the liquid after mixing with milk is 5 or higher, it was possible to prepare milk lemon tea, which is difficult to do even with cow's milk, suggesting that it can be applied to various beverages and liquid foods using sour fruits. Basically, the pH of the liquid mixed with the nut milk greatly affects coagulation, followed by the fact that higher liquid temperatures make coagulation more likely. The effect varies depending on the amount of enzyme added (enzyme concentration), reaction temperature, and reaction time. The same effect was observed not only with almond milk, but also with peanut milk, cashew milk, pistachio milk, and hazelnut milk. Therefore, it is considered that enzymatic treatment with protein deamidation enzymes is effective in preventing coagulation of nut milks in general.

[0062] 8. Preventing coagulation in soy milk Soy milk, with its unique flavor and nutritional value, is widely used not only as a substitute for cow's milk but also as an ingredient and additive in various foods and beverages. Improved dispersibility of soy milk offers the potential for not only enhanced quality in existing applications but also new uses. Therefore, we investigated whether treatment with protein deamidation enzymes is effective in preventing soy milk aggregation.

[0063] (1) Method Commercially available soy milk (Sojasun Co., Ltd., product name "SOJA NATURE SANS SUCRE", protein content 3.6% (w / w), ingredients: soybeans, water) was mixed with protein glutaminase "Amano" 500 (Amano Enzyme Co., Ltd., 500U / g), with 5U or 15U added per gram of soy protein, and reacted at 50°C for 5 hours (deamide reaction). After the enzymatic reaction, the enzyme was quickly deactivated by treating at 90°C for 15 minutes, cooled under running water, and then chilled to 5°C in a refrigerator. 15mL of each mixture was then added to 150mL of coffee solution heated to 90°C, and the presence or absence of coagulation was checked.

[0064] (2) Results (Figure 7) Aggregation was observed without enzyme treatment, but not with enzyme treatment. This result indicates that enzyme treatment can also prevent aggregation in soy milk.

[0065] Treatment with protein deamidase enzymes was also effective in preventing coagulation in soy milk. Therefore, similar to nut milk, soy milk with improved dispersibility (i.e., less prone to coagulation) can be prepared by treatment with protein deamidase enzymes. Soy milk with improved dispersibility can be used for applications where untreated soy milk cannot be used (or is unsuitable for use) due to coagulation. Based on the above experimental results, the conditions for treatment with protein deamidase enzymes can be the same as those used for nut milk.

[0066] 9. Prevention of coagulation in other plant-based milks In addition to soy milk, various plant-based milks with unique flavors and nutritional value are widely used not only as substitutes for cow's milk but also as ingredients and additives in a variety of foods and beverages. Plant-based milks with improved dispersibility offer the potential for improved quality in existing applications as well as new applications. Therefore, we investigated whether treatment with protein deamidation enzymes is effective in preventing the aggregation of various plant-based milks.

[0067] (1) Method Commercially available pea milk (Mihgty Society, protein content 3.2% (w / w)), oat milk (Liquats Vegetals, protein content 1.4% (w / w)), and hemp milk (Ecomil, protein content 1.0% (w / w)) were mixed with protein glutaminase "Amano" 500 (Amano Enzyme Co., Ltd., 500 U / g), with 1 U or 5 U per gram of protein added, and the mixture was reacted at 50°C for 5 hours (deamide reaction). After the enzymatic reaction, the enzyme was quickly deactivated by treating it at 90°C for 15 minutes, cooled under running water, and then chilled to 5°C in a refrigerator. 15 mL of each mixture was then added to 150 mL of coffee solution heated to 90°C, and the presence or absence of coagulation was checked.

[0068] (2) Results (Figure 8) Agglutination was observed without enzyme treatment, but not with enzyme treatment. This result indicates that enzyme treatment can prevent agglutination in various types of plant-based milks.

[0069] Treatment with protein deamide enzymes was also effective in preventing coagulation in various plant-based milks. Therefore, similar to nut milk, treatment with protein deamide enzymes can be used to prepare various plant-based milks with improved dispersibility (i.e., less prone to coagulation). These plant-based milks with improved dispersibility can be used in applications where untreated plant-based milks cannot be used (or are unsuitable for use) due to coagulation. Based on the above experimental results, the conditions for treatment with protein deamide enzymes can be the same as those used for nut milk. [Industrial applicability]

[0070] This invention provides a plant-based milk that exhibits excellent dispersibility when added to high-temperature liquid food or beverages (beverages or liquid foods) without the use of additives such as emulsifiers. High dispersibility enhances the value of both the plant-based milk itself and the liquid food or beverages using it. Furthermore, it makes it possible to provide novel liquid food or beverages that were previously unattainable.

[0071] The plant-based milk provided by this invention is expected to be used or applied in a variety of applications (particularly in acidic beverages and acidic liquid foods), not limited to existing uses. The fact that it eliminates the need for additives such as emulsifiers is a major advantage of this invention. Furthermore, when the plant-based milk is added to high-temperature coffee or other beverages as a substitute for cow's milk, special operations to prevent coagulation are unnecessary, thus improving convenience for consumers.

[0072] This invention is not limited in any way to the descriptions of embodiments and examples of the invention described above. Various modifications are also included in this invention, provided that they do not depart from the scope of the claims and are easily conceivable by those skilled in the art. The contents of papers, published patent gazettes, and other documents explicitly mentioned herein shall be cited in their entirety.

Claims

1. A plant-based milk used as an additive to liquid food and beverages at temperatures of 60°C or higher, The aforementioned liquid food and beverage has a pH of 5 to 7 after the addition of plant-based milk. The aforementioned plant-based milk is a plant-based milk that has been treated with a protein deamidation enzyme.

2. The plant-based milk according to claim 1, wherein the plant-based milk is nut milk, soy milk, pea milk, oat milk, or hemp milk.

3. The plant-based milk according to claim 2, wherein the nuts used as raw materials for the nut milk are one or more nuts selected from almonds, cashews, hazelnuts, pecans, macadamia nuts, pistachios, walnuts, Brazil nuts, peanuts, coconuts, chestnuts, sesame seeds, and pine nuts.

4. The plant-based milk according to any one of claims 1 to 3, wherein the raw material plant protein concentration is 0.2% (w / v) to 10.0% (w / v).

5. The plant-based milk according to any one of claims 1 to 4, wherein the dispersibility is improved by the above-mentioned process.

6. The plant-based milk according to claim 5, wherein protein aggregation does not occur when mixed with a weakly acidic to weakly alkaline liquid food or beverage (provided the pH of the mixture is 5 or higher).

7. The plant-based milk according to claim 6, wherein the pH of the liquid food or beverage is 5 to 7.

8. The plant-based milk according to any one of claims 1 to 7, wherein the liquid food or beverage is selected from the group consisting of coffee, coffee beverages, tea, tea beverages, fruit juice, fruit juice beverages, sports drinks, nutritional supplements, soups, curries, cocoa, and chocolate beverages, and is an ingredient, intermediate product, or final product of a beverage or liquid food.

9. A plant-based milk according to any one of claims 1 to 8, which does not contain emulsifiers and thickening polysaccharides for preventing coagulation.

10. The plant-based milk according to any one of claims 1 to 9, wherein the protein deamide enzyme is an enzyme derived from a microorganism of the genus Chryseobacterium.

11. The plant-based milk according to claim 10, wherein the microorganism of the genus Chryseobacterium is Chryseobacterium proteoricum.

12. A liquid food or beverage containing plant-based milk, obtained by adding the plant-based milk described in any one of claims 1 to 11 to a liquid food or beverage at a temperature of 60°C or higher, and having a pH of 5 to 7.

13. The liquid food or beverage according to claim 12, which is a beverage or liquid food selected from the group consisting of coffee beverages, coffee whiteners, tea beverages, fruit juices, sports drinks, nutritional supplements, soups, curries, cocoa beverages, and chocolate beverages.

14. A method for producing a liquid food product containing plant milk, comprising mixing plant-based milk treated with a protein deamidation enzyme with a raw material, intermediate product, or final product of a liquid food product at a temperature of 60°C or higher to prepare a liquid food product containing plant milk with a pH of 5 to 7.

15. The manufacturing method according to claim 14, comprising the following steps (1) and (2): (1) A step of preparing plant-based milk treated with a protein deamidation enzyme, (2) A step of mixing the plant-based milk prepared in (1) with a raw material, intermediate product, or final product of a liquid food or beverage at 60°C or higher to obtain a liquid food or beverage containing plant-based milk with a pH of 5 to 7.

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