Prevention of agglomeration of nut milk

Abstract

The invention relates to prevention of agglomeration of nut milk. The present invention addresses the problem of developing a method for effectively preventing agglomeration of nut milk. According to the present invention, nut milk is treated with a protein deamidase, thereby improving the dispersibility thereof and preventing agglomeration when added to a liquid beverage, a liquid food or the like. The nut milk according to the present invention is characterized by being obtained by performing a treatment using a protein deamidase.

Description

This application is a divisional application of Chinese invention patent application No. 202080015431.9, filed on February 19, 2020, entitled "Prevention of Coagulation of Nut Milk". Technical Field

[0001] This invention relates to a nut milk. More specifically, this invention relates to a nut milk with improved dispersibility (less prone to coagulation) and its uses, etc. This application claims priority based on Japanese Patent Application No. 2019-029904, filed on February 21, 2019, the entire contents of which are incorporated herein by reference. Background Technology

[0002] Against the backdrop of allergy issues, the rise of vegetarianism, and religious reasons, plant-based proteins, particularly those derived from soybeans, have become increasingly popular as alternatives to animal-derived milk proteins, such as those found in milk. However, along with this growing popularity, soybeans have also become a source of allergies. In recent years, the development of plant-based protein alternatives to soybeans has flourished. In fact, grain proteins derived from peas, rice, and oats, and nut proteins from almonds, cashews, and peanuts, are gradually being commercialized as soybean substitutes in food and beverages. It can be said that, to avoid allergies, the need and expectation for diversification of plant-based protein alternatives to soybeans is growing.

[0003] On the other hand, when milk protein sources are replaced with plant-derived proteins, the types, functions, and aroma and flavor components of the proteins differ, sometimes making direct substitution impossible. For example, it is known that coagulation occurs when almond milk or peanut milk (nut milk) is added to acidic beverages such as coffee or black tea as a substitute for cow's milk. This coagulation typically does not occur with cow's milk and is a phenomenon unique to nut milk.

[0004] To the best of our knowledge, there are no reports (in the literature, etc.) clearly demonstrating the mechanism of protein coagulation caused by adding nut milk to acidic liquid foods. Although repeated experiments by consumers have been conducted (e.g., mixing nut milk and coffee after reducing the temperature difference, or slowly pouring coffee into nut milk), a fundamental solution has not yet been achieved.

[0005] On the other hand, it is known that the dispersion stability of milk proteins becomes unstable under acidic conditions near their isoelectric point, easily leading to problems such as precipitation and aggregation in acidic milk beverages. To prevent the aggregation of these milk proteins, polysaccharides such as pectin and carboxymethyl cellulose are added (see, for example, Patent Documents 1 and 2). Although it is possible to prevent the aggregation of nut proteins by using the aforementioned dispersion stabilizers, the use of additives is indispensable. Furthermore, when polysaccharides are used, there is a possibility of increased viscosity due to the amount added.

[0006] Regarding preventing the coagulation of milk proteins without adding additives, a coffee creamer obtained by treating proteins with deamidase was proposed (Patent Document 3). However, this coffee creamer becomes a product containing emulsifiers, and its use is limited to beverages such as coffee and black tea that use whitening agents. Existing technical documents Patent documents

[0007] Patent Document 1: International Publication No. 2012 / 176852 Patent Document 2: Japanese Patent No. 3885194 Patent Document 3: International Publication No. 2011 / 108633 Summary of the Invention The problem that the invention aims to solve

[0008] The inherent coagulation phenomenon in nut milk reduces its value (utility value, commercial value, etc.) in anticipation of increased demand or wider applications. Therefore, to enhance the value of nut milk and promote its utilization and application, the objective of this invention is to create a method for effectively preventing coagulation in nut milk, and in particular, to provide a nut milk that is not prone to coagulation in liquid beverages (especially acidic liquid beverages) and liquid foods (especially acidic liquid foods), even without the addition of additives. Technical solutions for solving the problem

[0009] In light of the aforementioned technical problems and repeated research efforts, the inventors focused on the deamidation of proteins and attempted to improve the dispersibility of nut milk by treating it with protein deamidases. Furthermore, no examples of using protein deamidases in nut milk have been reported to date.

[0010] First, regarding the addition of nut milk to coffee, a typical use of nut milk, we investigated whether treatment with protein deamidase was effective in preventing coagulation. Surprisingly, no protein coagulation occurred when using almond milk treated with the enzyme. Based on this insight, detailed experiments were conducted for various envisioned applications, and the results showed that treatment with protein deamidase was very effective in improving the dispersibility of nut milk. In other words, a method to effectively prevent coagulation of nut milk was discovered, and success was achieved in the preparation of nut milk with improved dispersibility that is less prone to coagulation even without the use of emulsifiers or other additives. Furthermore, many beneficial insights were gained when using nut milk in various beverages and foods. Based on the above results, the following invention is provided. In addition, as mentioned above, the use of protein deamidase in improving the dispersibility of coffee creamer was proposed. However, coffee creamer is usually prepared by homogenizing an emulsion containing emulsifiers, milk components, thickeners, flavorings, etc., using an emulsifier with excellent shear force, such as a high-pressure homogenizer, with edible oils as the main raw material. Compared with nut milk, its raw materials, composition, and preparation method are completely different. Therefore, regardless of the effectiveness of methods that improve the dispersibility of coffee creamers for nut milk, the possibility of their application is unpredictable. [1] A nut milk obtained by processing with protein deamidase. [2] According to the nut milk described in [1], the nuts used as raw materials are one or more of the following: almonds, cashews, hazelnuts, pecans, macadamia nuts, pistachios, walnuts, Brazil nuts, peanuts, coconuts, chestnuts, sesame seeds and pine nuts. [3] According to the nut milk described in [1] or [2], the nut protein concentration is 0.2% (w / v) to 10.0% (w / v). [4] The nut milk described in any of [1] to [3], wherein the dispersibility is improved by the above treatment. [5] According to the nut milk described in [4], when mixed with a weakly acidic to weakly alkaline liquid, no protein coagulation occurs, wherein the pH of the mixture is 5 or higher. [6] According to the nut milk described in [5], the pH of the liquid is 5 to 7. [7] According to the nut milk described in [5], the liquid is a beverage or liquid food selected from coffee, coffee beverage, tea, tea beverage, fruit juice, fruit juice beverage, sports drink, nutritional supplement beverage, soup, curry, cocoa and chocolate beverage. [8] The nut milk described in any of [1] to [7] does not contain emulsifiers and thickening polysaccharides used to prevent coagulation. [9] According to any one of [1] to [8], the nut milk contains a protein deamidase that is an enzyme derived from a microorganism of the genus *Chlorella*.

[10] According to the nut milk described in [9], the genus Chryseobacterium is Chryseobacterium proteolyticum.

[11] A method for manufacturing nut milk with improved dispersibility, characterized in that the nut milk is treated with a protein deamidase.

[12] The manufacturing method described in

[11] includes the following steps (1) and (2): (1) Steps for preparing nut milk (2) The steps of treating the nut milk prepared in (1) with protein deamidase.

[13] According to the manufacturing method described in

[12] , the nut milk in step (1) is nut milk before heating and sterilization.

[14] The manufacturing method described in

[13] further includes the following step (3): (3) Heat treatment steps.

[15] A beverage or liquid food containing nut milk as described in any one of [1] to

[10] .

[16] The beverage or liquid food described in

[15] is a beverage or liquid food with a pH of 5 or higher.

[17] The beverage or liquid food described in

[15] is selected from coffee beverages, coffee creamer, tea beverages, fruit juice beverages, sports drinks, nutritional supplement beverages, soups, curries, cocoa beverages and chocolate beverages. Attached Figure Description

[0011] Figure 1 This is a summary of the experimental results (the relationship between protein concentration in nut milk and coagulation / coagulation prevention effect). Figure 2 This is a summary of experimental results (the relationship between the pH of the liquid and the effect of coagulation / coagulation prevention). Figure 3 This is a summary of the experimental results (the effects of preventing condensation in various liquids). The results of Experiment 1 (prevention of condensation in coffee) are also recorded. Figure 4 This is a summary of the experimental results (the effect of preventing coagulation in nut milk other than almond milk). Figure 5 It is a summary of experimental results (the relationship between liquid temperature and coagulation / coagulation prevention effect). Figure 6 It is a summary of experimental results (studies on enzyme treatment conditions (enzyme addition amount, reaction temperature, reaction time)). Detailed Implementation

[0012] 1. Nut milk with improved dispersibility The first aspect of this invention relates to a nut milk (also known as a nut protein beverage) with improved dispersibility. The nut milk of this invention is treated with a protein deamidase, resulting in improved dispersibility. The nut milk of this invention exhibits excellent dispersibility, and therefore, even without the use of additives for improving dispersibility (e.g., emulsifiers, thickening polysaccharides (pectin, carboxymethyl cellulose, etc.), salts), it is not prone to coagulation when added to beverages such as coffee or black tea. This property allows it to be used in a wide variety of beverages or foods.

[0013] Nut milk, exemplified by almond milk, is a plant-based milk made from nuts. It is typically prepared through processes such as crushing, soaking / dissolving, mixing / stirring, filtering, homogenizing, and sterilizing de-shelled nuts. There are no particular limitations on the preparation method of the nut milk used in this invention. Alternatively, nut milk provided by the raw material manufacturer or commercially available can be purchased and used in this invention.

[0014] The nut milk of the present invention can be obtained by treating nut milk with a protein deamidase to improve its dispersibility. Hereinafter, for ease of explanation, the nut milk treated with the protein deamidase will be referred to as "untreated nut milk".

[0015] There are no particular restrictions on the nuts that can be used as ingredients in unprocessed nut milk. Examples of nuts that can be used as ingredients include almonds, cashews, hazelnuts, pecans, macadamia nuts, pistachios, walnuts, Brazil nuts, peanuts, coconuts, chestnuts, sesame seeds, and pine nuts.

[0016] Unprocessed nut milk can also be used, which combines two or more types of nuts (e.g., almonds and cashews or almonds and peanuts).

[0017] There are no particular limitations on the protein concentration in untreated nut milk; 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) of untreated nut milk is used. Similarly, the protein concentration in nut milk treated with protein deamidase 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).

[0018] The protein deamidase used in this invention has the following function: it acts directly on the amide group of a protein to perform deamidation without the cleavage of peptide bonds or cross-linking of the protein. There are no particular limitations on the type or source of any enzyme exhibiting this function. Examples of protein deamidases include those derived from the genera *Chryseobacterium*, *Flavobacterium*, *Empedobacter*, *Sphingobacterium*, *Aureobacterium*, or *Myroides*, as disclosed in Japanese Patent Application Publication Nos. 2000-50887, 2001-218590, and WO2006 / 075772, as well as commercially available protein glutaminases derived from the genus *Chryseobacterium*. Enzymes derived from the genus *Chlorella* are preferred (specifically, enzymes derived from *Chlorella pyrenoidosa* (e.g., protein glutaminase "AMANO" 500 manufactured by Amano Enzyme Co., Ltd.)).

[0019] Protein deamidases can be prepared using substances derived from the culture medium of microorganisms that produce protein deamidases. There are no particular limitations on the microorganisms used for the preparation of protein deamidases; for example, microorganisms belonging to the genera *Chrysobacterium*, *Flavobacterium*, *Sphingomonas*, *Chrysobacterium*, or *Aromatica* can be used. As a specific example of a suitable microorganism for the preparation of protein deamidases, *Chrysobacterium* sp. No. 9670 belongs to the genus *Chrysobacterium*.

[0020] For example, protein deamidases can be obtained from the culture medium or cells of the aforementioned microorganisms. That is, if it is a secreted protein, it can be recovered from the culture medium; if it is not, it can be recovered from the cell. Methods for preparing protein deamidases from the culture medium can utilize known protein separation and purification methods (centrifugation, UF concentration, salting out, various chromatographic methods using ion exchange resins, etc.). For example, the culture medium can be centrifuged to remove the cells, followed by a combination of salting out, chromatography, etc., to obtain the target enzyme. In the case of recovering the enzyme from the cell, the cells can be broken up by pressure treatment, ultrasonic treatment, etc., and then separated and purified in the same manner as described above to obtain the target enzyme. Alternatively, the series of steps described above (crushing, separation, and purification of the cells) can be performed after the cells have been recovered from the culture medium beforehand by filtration, centrifugation, etc. The enzyme can be powdered by freezing-drying, vacuum drying, etc., in which case appropriate excipients and drying aids can also be used.

[0021] In this application, the activity of protein deamidases was determined using the following method. (1) Add 0.1 ml of an 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 min, and then add 1 ml of 0.4 M TCA solution to stop the reaction. As a blank, prepare a solution by adding 0.1 ml of an aqueous solution containing protein deamidase to a solution obtained by adding 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 min. (2) The amount of ammonia produced by the reaction was determined using Ammonia-test Wako for the solution obtained in (1). The ammonia concentration in the reaction solution was determined from a standard curve showing the relationship between ammonia concentration and absorbance (630 nm) prepared using ammonia standard solution (ammonium chloride). (3) The activity of protein deamidase is: the amount of enzyme that generates 1 μmol of ammonia in 1 minute is taken as 1 unit, and is calculated by the following mathematical formula. Enzyme activity (U / mL) = Ammonia concentration in reaction solution (mg / L) × (1 / 17.03) × (Reaction solution volume / Enzyme solution volume) × (1 / 10) × Df (In the formula, the reaction solution volume is 2.1, the enzyme solution volume is 0.1, and Df is the dilution factor of the enzyme solution. In addition, 17.03 is the molecular weight of ammonia.)

[0022] As long as it effectively improves the dispersibility of nut milk, there are no particular restrictions on the conditions for using protein deamidase. Simply adjust the reaction temperature, reaction time, and enzyme addition amount (enzyme concentration) to set the optimal reaction conditions.

[0023] Not limited to the examples above, the reaction temperature can be set within, for example, a range of 2°C to 70°C, preferably within a range of 5°C to 60°C, and more preferably within a range of 15°C to 50°C. Similarly, the reaction time can be set within, for example, a range of 10 minutes to 7 days, preferably within a range of 30 minutes to 3 days, and more preferably within a range of 1 hour to 1 day. Furthermore, the amount of enzyme added can be set within, for example, a range of 0.01 (U / g protein) to 500 (U / g protein), preferably within a range of 0.02 (U / g protein) to 50 (U / g protein), and more preferably within a range of 0.2 (U / g protein) to 5 (U / g protein). Here, "U / g protein" refers to the number of units of protein (g) per substrate nut. Furthermore, as mentioned above, there is no particular limitation on the protein concentration in untreated nut milk, and untreated nut 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 for treatment with protein deamidase.

[0024] Here, when setting the treatment conditions using protein deamidase, the following indicators (a) to (c) can be followed. (a) While lowering the reaction temperature, extend the reaction time or increase the amount of enzyme added (or both). (b) In the case of shortening the reaction time, increase the reaction temperature (wherein, set to a temperature not exceeding 70°C, preferably set to a temperature below 60°C) or increase the amount of enzyme added (or both). (c) While reducing the amount of enzyme added, increase the reaction temperature (wherein, set to a temperature not exceeding 70°C, preferably set to a temperature below 60°C) or extend the reaction time (or both).

[0025] The following examples illustrate more specific metrics when setting processing conditions. When the reaction temperature is between 5°C and 15°C, the reaction time is set to more than 8 hours (preferably more than 24 hours), or the amount of enzyme added is set to more than 0.2 (U / g protein) (preferably more than 1 (U / g protein)). When the reaction temperature is between 15°C and 25°C, the reaction time is set to more than 7 hours, or the amount of enzyme added is set to more than 0.2 (U / g protein) (preferably more than 1 (U / g protein)). When the reaction temperature is between 25°C and 40°C, the reaction time is set to more than 5 hours (preferably more than 7 hours), or the amount of enzyme added is set to more than 0.2 (U / g protein) (preferably more than 1 (U / g protein)). When the reaction temperature is between 40℃ and 50℃, it is preferable to set the reaction time to 3 hours or more, or preferably to set the enzyme addition amount to 0.2 (U / g protein) or more. When the reaction temperature is 50 ≤ (wherein, the temperature is set to not more than 70°C, preferably below 60°C), the reaction time is preferably set to 3 hours, or the amount of enzyme added is preferably 0.2 (U / g protein) or more.

[0026] As described above, the nut milk of the present invention exhibits excellent dispersibility and is less prone to protein aggregation. Typically, when mixed (added) with a weakly acidic (3 ≤ pH < 6) to a weakly alkaline (8 ≤ pH < 11) liquid (wherein the pH of the mixture is 5 or higher), protein aggregation does not occur. The pH of the mixed liquid that does not generate protein aggregation is, for example, 5 to 10, preferably 5 to 9, and more preferably 5 to 7. There are no particular limitations on the liquids (beverages, liquid foods) used in the mixing of the nut milk of this invention. Examples include coffee, coffee beverages, tea (including black tea, green tea, oolong tea, etc.; substances obtained from reductive extracts, substances obtained from the reduction of processed extracts (e.g., concentration, freeze-drying), tea beverages (flavored tea, milk tea, tea beverages with added fruit juice, etc.), fruit juice, fruit juice beverages, sports drinks, nutritional supplements (protein drinks, nutritional drinks, etc.), soups (clear soups, stews, mixed vegetables, borscht, vegetable soups (e.g., tomato soup, corn soup, thick soup, pumpkin soup), miso soup), curry, cocoa, and chocolate beverages.

[0027] In a preferred embodiment of the invention, taking advantage of its excellent dispersibility and resistance to protein aggregation, it is free of emulsifiers (glycerol fatty acid esters, sucrose fatty acid esters, lecithin, saponins, etc.) used to prevent aggregation, thickening polysaccharides (pectin, carboxymethyl cellulose, etc.), and salts (sea salt, calcium salts, phosphates, etc.). In particular, it is free of emulsifiers and thickening polysaccharides. Thus, according to the invention, nut milk that meets consumer demand for products with few or no additives can be provided. Furthermore, in this preferred embodiment, the absence of additives for preventing aggregation does not preclude the use of additives for other purposes (specifically, such as flavor and taste adjustments).

[0028] As can be seen from the above description, the nut milk of the present invention can be manufactured by treating untreated nut milk with a protein deamidase. Therefore, the nut milk of the present invention can typically be obtained by a manufacturing method including the following steps (1) and (2). (1) Steps for preparing nut milk; (2) The steps of treating the nut milk prepared in (1) with protein deamidase.

[0029] Step (2), namely the treatment using protein deamidase, can be performed at any time before or after the heat sterilization of the nut milk. However, to simplify the manufacturing process, this step can be performed before the heat sterilization of the nut milk, followed by a heat sterilization process that also inactivates the protein deamidase (in other words, step (2) can also be incorporated into the manufacturing process of the nut milk). Therefore, in a preferred embodiment, the "(3) heat treatment step" is performed after step (2). The conditions for the heat treatment are not particularly limited as long as they enable the inactivation of the protein deamidase and the sterilization of the nut milk. For example, treatment can be performed at a temperature of 70°C to 150°C for 1 second to 5 hours.

[0030] 2. Uses of nut milk A second aspect of the invention relates to the use of the nut milk of the present invention. The nut milk of the present invention exhibits excellent dispersibility and is less prone to protein aggregation. This characteristic makes it suitable for use in a wide variety of beverages or liquid foods. Specifically, various beverages and liquid foods formulated with the nut milk of the present invention are provided.

[0031] As described in the embodiments below, based on detailed research by the inventors, it has been found that: (i) treatment with protein deamidase can extend the pH region where coagulation does not occur to the acidic side; (ii) protein coagulation that occurs when nut milk is mixed with beverages, liquid foods, etc., depends on the pH of the beverage, etc., after mixing with the nut milk; if the pH is 5 or higher, protein coagulation does not occur. In view of the above, the pH of beverages or liquid foods containing the nut milk of the present invention is preferably 5 or higher. More specifically, the pH of beverages or liquid foods containing the nut milk of the present invention is preferably 5 to 9, more preferably 5 to 8, and even more preferably 5 to 7.5.

[0032] Examples of beverages or liquid foods include: coffee beverages, coffee creamers (for example, their use in addition to coffee such as black tea), tea beverages (flavored tea, milk tea, tea beverages with added fruit juice, etc.), fruit juice beverages, sports drinks, nutritional supplement beverages (protein drinks, nutritional drinks, etc.), various soups, curries, cocoa beverages, and chocolate beverages. As can be seen from the above examples, this invention is not limited to neutral beverages and liquid foods; it can also be used in weakly acidic beverages and liquid foods.

[0033] Nut milk, for example, is mixed with other raw materials during the manufacturing process of beverages or liquid foods. Preferably, it is mixed with other raw materials at the final stage of the manufacturing process, after processing (to achieve the form / shape of the finished product). Subsequently, sterilization, and the addition of seasonings, preservatives, flavorings, antioxidants, etc., for purposes such as flavor adjustment or quality preservation, may also be performed. Alternatively, mixing nut milk into the beverage or liquid food (i.e., a substance in the form of a final product, not an intermediate product) after the manufacturing process is completed is also a preferred method. In this case, the present invention can be applied without changing the manufacturing process of the beverage or liquid food. Example

[0034] 1. The anti-coagulation agents in coffee 1 unit of protein glutaminase "AMANO" 500 (manufactured by Amano Enzyme Co., Ltd., 500 U / g) was added to 100 mL of commercially available almond milk (manufactured by Rude Co., Ltd., protein content 1.5%, raw materials: almonds, water) for every 1 g of protein in the almond milk. The mixture was reacted at 50°C for 5 hours (deamidation reaction). After heat inactivation of the enzyme by treating at 95°C for 20 minutes, the mixture was cooled to 5°C to produce enzyme-treated almond milk.

[0035] Commercially available instant coffee was dissolved in hot water to prepare a 2% coffee solution. 20-30 mL of enzyme-treated almond milk (pH 5.7 after adding enzyme-treated almond milk) was added to 150 mL of the coffee solution; no coagulation was observed. As a control, significant coagulation was observed when using non-enzyme-treated almond milk. Furthermore, peanut milk was used instead of almond milk, and the experiment was conducted under the same conditions, yielding the same results (no coagulation occurred in enzyme-treated peanut milk).

[0036] 2. The relationship between protein concentration in nut milk and coagulation / coagulation prevention effect. <Enzyme-free treatment> (1) Method Dilute commercially available almond milk (manufactured by Rude Company, protein content 1.5%, raw materials: almonds, water) with tap water to make protein concentrations of 0.1%, 0.5%, and 1.5% (w / v). Cool to 5°C and add 5 mL of each to 50 mL of coffee solution heated to 90°C to check for coagulation.

[0037] (2) Results Aggregation was confirmed in almond milk of any protein concentration. Figure 1 ).

[0038] <Enzyme treatment> (1) Method 1 U of protein glutaminase "AMANO" 500 (manufactured by Amano Enzyme Co., Ltd., 500 U / g) was added to commercially available almond milk (manufactured by Rude Co., Ltd., protein content 1.5%, raw materials: almonds, water) per 1g of protein in the almond milk. The mixture was reacted at 50°C for 5 hours (deamidation reaction). The enzyme was then thermally inactivated by treating at 90°C for 15 minutes, resulting in 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 of coagulation was checked.

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

[0040] 3. The relationship between the pH of a liquid and its coagulation / coagulation prevention effect. (1) Method After adjusting the pH with hydrochloric acid or sodium hydroxide, add 15-20 mL of non-enzyme-treated almond milk or enzyme-treated almond milk (protein concentration 1.5% (w / v)) to hot water heated to 90°C, and confirm coagulation. Alternatively, enzyme-treated almond milk can be prepared using the method described in section 2 above.

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

[0042] 4. Anti-coagulation effect in various liquids 4-1. Black tea (1) Method Boiling water was poured into commercially available black tea bags (manufactured by Twining, British Breakfast Tea), and steeped for 2-3 minutes. The tea bags were then removed to prepare black tea. Non-enzymatically treated almond milk or enzyme-treated almond milk (protein concentration 1.5% (w / v)) was added to the black tea to check for coagulation. The black tea before adding the almond milk was at 80°C and pH 5.2. The pH of the black tea after adding the almond milk was 5.9. Furthermore, the enzyme-treated almond milk was prepared using the method described in section 2 above.

[0043] (2) Results Figure 3 ) A small amount of coagulation was observed in the non-enzymatically treated almond milk. As a control, no coagulation was observed in the enzymatically treated almond milk.

[0044] 4-2. Lemon tea (1) Method Boiling water was poured into commercially available black tea bags (manufactured by Twining, British Breakfast Tea), and steeped for 2-3 minutes. The tea bags were then removed to prepare black tea. Lemon juice was added to the black tea to adjust the pH. Then, non-enzyme-treated almond milk or enzyme-treated almond milk (protein concentration 1.5% (w / v)) was added to check for coagulation. The black tea was kept at 70°C just before adding the almond milk. Furthermore, the enzyme-treated almond milk was prepared using the method described in section 2 above.

[0045] (2) Results Figure 3 ) Coagulation was observed in both non-enzyme-treated and enzyme-treated almond milk at a pH of 3.5 before the addition of almond milk. Furthermore, the pH of black tea with non-enzyme-treated almond milk was 3.9, while the pH of black tea with enzyme-treated almond milk was 4.1.

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

[0047] 4-3. Decaffeinated coffee (1) Method A decaffeinated coffee solution was prepared by dissolving commercially available decaffeinated coffee powder (Nescafe Gold, manufactured by Nestlé) in boiling water. Non-enzymatically treated almond milk or enzyme-treated almond milk (1.5% protein concentration (w / v)) was then added to check for coagulation. The decaffeinated coffee solution was at 80°C and pH 5.3 just before adding the almond milk. 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 section 2 above.

[0048] (2) Results Figure 3 ) Coagulation was observed in non-enzyme-treated almond milk, but no coagulation was observed in enzyme-treated almond milk.

[0049] 4-4. Tomato Soup (1) Method Add a specified amount of boiling water to commercially available chicken bouillon cubes (manufactured by Unilever, Knorr Chicken Cube) to completely dissolve the cubes and prepare the chicken broth. Then, add commercially available tomato paste. Adjust the pH of the tomato broth by increasing or decreasing the amount of tomato paste added. Next, add either non-enzymatically treated almond milk or enzyme-treated almond milk (protein concentration 1.5% (w / v)) and check for coagulation. The tomato broth should be at 80°C just before adding the almond milk. Furthermore, the enzyme-treated almond milk was prepared using the method described in section 2 above.

[0050] (2) Results Figure 3 ) At a pH of 5.0 before the addition of almond milk, coagulation was observed in the non-enzyme-treated almond milk, but not in the enzyme-treated almond milk. Furthermore, the pH of the tomato soup after adding both non-enzyme-treated and enzyme-treated almond milk was 5.4.

[0051] Coagulation was observed in both non-enzyme-treated and enzyme-treated almond milk at a pH of 4.0 before the addition of almond milk. Furthermore, the pH of tomato soup after adding non-enzyme-treated almond milk and tomato soup after adding enzyme-treated almond milk were both 4.0. It is further believed that the citric acid added to the tomato paste, a pH adjuster, has a strong buffering capacity, so even with the addition of almond milk, the pH did not change, and coagulation occurred.

[0052] 5. Anti-coagulation effect in nut milk other than almond milk (1) Method Commercially available peanut milk (manufactured by Rude Corporation, 2.0% protein content, raw materials: peanuts, water), cashew milk (manufactured by PLENISH Corporation, 0.9% protein content, raw materials: water, cashews, salt), pistachio milk (manufactured by Borna Foods Corporation, 1.0% protein content), and hazelnut milk (manufactured by Plenish Corporation, 0.6% protein content) were treated with 1U of protein glutaminase "AMANO" 500 (manufactured by Amano Enzyme Corporation, 500U / g) per 1g of nut protein, and reacted at 50°C for 5 hours (deamidation reaction). After the enzyme reaction, the milk was rapidly inactivated by treating it at 90°C for 15 minutes, cooled in running water, and then cooled to 5°C in a refrigerator. Subsequently, 5mL of each milk was added to 50mL of coffee solution heated to 90°C to check for coagulation.

[0053] (2) Results Figure 4 ) Coagulation was observed in all types of milk—peanut milk, cashew milk, pistachio milk, and hazelnut milk—without enzyme treatment, but not with enzyme treatment. This indicates that the same effect of enzyme treatment can be achieved in nut milks other than almond milk.

[0054] 6. The relationship between liquid temperature and coagulation / coagulation prevention effect <Change the coffee temperature (almond milk is kept constant at 5℃)> (1) Method Add 5 mL of non-enzyme-treated almond milk or enzyme-treated almond milk cooled to 5°C to 50 mL of coffee at each temperature, and check for coagulation.

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

[0056] <Change the coffee temperature (almond milk is kept constant at 90℃)> (1) Method Add 5 mL of non-enzyme-treated almond milk or enzyme-treated almond milk heated to 90°C to 50 mL of coffee at each temperature, and check for coagulation.

[0057] (2) Results Figure 5 ) Even when non-enzymatically treated almond milk at 90°C is added to coffee at 90°C, coagulation is observed. Furthermore, coagulation is also observed when non-enzymatically treated almond milk at 90°C is added to coffee at 50°C, but no coagulation is observed when it is added to coffee at 40°C. The addition of non-enzymatically treated almond milk at 90°C to coffee at 50°C is believed to cause coagulation due to the coffee's temporary high temperature. It is also believed that higher temperatures after mixing (above 50°C) tend to induce coagulation.

[0058] No coagulation was observed when almond milk was treated with added enzymes (coffee at 40°C or 90°C).

[0059] 7. Study on enzyme treatment conditions (enzyme dosage, reaction temperature, reaction time) (1) Method Add 0.2U, 1U, or 5U of protein glutaminase "AMANO" to commercially available almond milk (manufactured by Rude, protein content 1.5%, raw materials: almonds, water) relative to 1g of protein in the milk. React at specified temperatures (5℃, 15℃, 25℃, 40℃, or 50℃) for 3-24 hours (deamidation reaction). After the enzyme reaction, rapidly inactivate the enzyme by treating at 90℃ for 15 minutes. After cooling in running water, cool to 5℃ in a refrigerator. Then, add 5mL of each solution to 50mL of coffee solution heated to 90℃ and check for coagulation.

[0060] (2) Results Figure 6 ) It is known that the effect varies depending on the amount of enzyme added, the reaction temperature, and the reaction time, but aggregation can be prevented by adjusting these conditions. Specifically, increasing the amount of enzyme added or extending the reaction time (or both) at a low reaction temperature yields the desired effect. For example, even at a reaction temperature of 5°C, aggregation can be effectively prevented by adding 1 U or more of enzyme or by a longer reaction time. On the other hand, increasing the reaction temperature or increasing the amount of enzyme added (or both) at a short reaction time yields the desired effect. For example, even at a reaction time of 3 hours, aggregation prevention is achieved by setting the reaction temperature to 40°C or higher or adding 1 U or more of enzyme. Furthermore, increasing the reaction temperature or extending the reaction time (or both) allows for a reduction in the amount of enzyme added. For example, setting the reaction temperature to 25°C or higher or setting the reaction time to a longer time allows for an enzyme addition of 0.2 U or less.

[0061] <Conclusion> • A uniform anti-coagulation effect was observed across a nut protein concentration range of 0.1–1.5% (w / v). This demonstrates that enzymatic treatment with protein deamidases is effective and versatile in preventing coagulation in nut milk with a wide variety of protein concentrations. While it depends on the type of liquid used to mix the nut milk, a general trend is observed: without enzymatic treatment using protein deamidases, coagulation occurs below pH 7 after mixing with the nut milk. However, with enzymatic treatment, the lower limit of coagulation can be extended to pH 5. If the pH of the liquid after mixing the nut milk is above 5, it shows that it can be used not only in beverages such as coffee and black tea, but also in acidic liquid foods such as sour dairy soups. Furthermore, if the pH of the liquid after mixing with milk is above 5, lemon milk tea, which is difficult to prepare even with cow's milk, can be made. Therefore, it can also be applied to various beverages or liquid foods using sour fruits. First, the pH of the liquid in mixed nut milk has a significant impact on coagulation. Second, the higher the temperature of the liquid, the easier it is to coagulate. • The effect varies depending on the amount of enzyme added (enzyme concentration), reaction temperature, and reaction time. • This effect was not limited to almond milk; it was also observed in peanut milk, cashew milk, pistachio milk, and hazelnut milk. Therefore, it is believed that enzymatic treatment with protein deamidases is effective in preventing coagulation in nut milk. Industrial availability

[0062] This invention provides a nut milk that exhibits excellent dispersibility even without the use of emulsifiers or other additives. This high dispersibility enhances the value of the nut milk itself and beverages and liquid foods using it. Furthermore, it enables the creation of novel beverages and liquid foods that were previously unattainable.

[0063] The nut milk provided by this invention is not limited to its existing uses and is expected to be utilized or applied in a variety of applications (especially acidic beverages and acidic liquid foods). A significant advantage of this invention is that it eliminates the need for additives such as emulsifiers. Furthermore, even when the nut milk is added to coffee or similar products as a substitute for milk or soy milk, no special procedures to prevent coagulation are required, thus improving consumer convenience.

[0064] The description of the embodiments and examples described above does not limit the present invention in any way. Various modifications are included in the present invention without departing from the scope readily conceived by those skilled in the art. The contents of all papers, published patent gazettes, and patent gazettes explicitly stated in this specification are incorporated herein by reference.

Claims

1. A nut-based milk, characterized in that, It is obtained by processing with protein deamidase.

2. The nut milk according to claim 1, wherein, The raw materials 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.

3. The nut milk according to claim 1 or 2, wherein, Nut protein concentration ranges from 0.2% w / v to 10.0% w / v.

4. The nut milk according to claim 1 or 2, wherein, The dispersion is improved through the aforementioned treatment.

5. The nut milk according to claim 4, wherein, No protein aggregation occurs when mixed with weakly acidic to weakly alkaline liquids, and the pH of the mixture is above 5.

6. The nut milk according to claim 5, wherein, The pH of the liquid is 5 to 7.

7. The nut milk according to claim 5, wherein, The liquid is selected from beverages or liquid foods such as coffee, coffee drinks, tea, tea drinks, fruit juice, fruit juice drinks, sports drinks, nutritional supplement drinks, soup, curry, cocoa and chocolate drinks.

8. The nut milk according to claim 1 or 2, wherein, It does not contain emulsifiers or thickening polysaccharides used to prevent coagulation.

9. The nut milk according to claim 1 or 2, wherein, The protein deamidase is an enzyme derived from the genus *Chlorella*.

10. The nut milk according to claim 9, wherein, The *Aureobacterium* species mentioned is *Aureobacterium utilis*.

11. A method for manufacturing nut milk with improved dispersibility, characterized in that, In the manufacturing method, nut milk is treated with protein deamidase.

12. The manufacturing method according to claim 11, wherein, The manufacturing method includes the following steps (1) and (2): (1) Steps for preparing nut milk; (2) The steps of treating the nut milk prepared in (1) with protein deamidase.

13. The manufacturing method according to claim 12, wherein, The nut milk in step (1) is the nut milk before it is heated and sterilized.

14. The manufacturing method according to claim 13, wherein, It also includes the following step (3): (3) Heat treatment steps.

15. A beverage or liquid food, characterized in that, Combined with the nut milk as described in claim 1 or 2.

16. The beverage or liquid food according to claim 15, wherein, The beverage or liquid food mentioned is a beverage or liquid food with a pH of 5 or higher.

17. The beverage or liquid food according to claim 15, wherein, The beverage or liquid food is selected from coffee beverages, coffee creamer, tea beverages, fruit juice beverages, sports drinks, nutritional supplement beverages, soups, curries, cocoa beverages, and chocolate beverages.

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