Prevention of aggregation in nut milk

Abstract

To provide effective means for preventing the aggregation in a nut milk.SOLUTION: The present invention provides a nut milk for addition to beverages or liquid foods, where the beverages or liquid food exhibit a pH of 5 to 7 after the addition of the nut milk, and where the nut milk has been treated with a protein deamidase. Preferably, the raw material nut is one or two or more nuts selected from almonds, cashew nuts, hazelnuts, pecan nuts, macadamia nuts, pistachios, walnuts, Brazil nuts, peanuts, coconuts, chestnuts, sesame, and pine nuts.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to nut milk. More specifically, the present invention relates to nut milk with improved dispersibility (hard to aggregate) and its uses. This application claims priority based on Japanese Patent Application No. 2019-029904 filed on February 21, 2019, the entire contents of which are incorporated by reference. [Background technology]

[0002] Due to allergy issues, an increase in vegetarians, and religious reasons, soybean-derived proteins, which are of plant origin, have become popular as an alternative ingredient to food and beverages that use animal-derived dairy protein sources such as milk. However, as soybeans have become more popular, it has become clear that soybeans also cause allergies, and in recent years, there has been active development of plant-derived protein ingredients to replace soybeans. In fact, grain-derived proteins such as peas, rice, and oats, as well as nut proteins such as almonds, cashews, and peanuts, are being commercialized one after another as food and beverage products to replace soybeans, and there is a high need and demand for diversification of plant-derived protein ingredients to replace soybeans in order to avoid allergies.

[0003] On the other hand, when replacing dairy protein ingredients with plant-derived protein ingredients, there are cases where they cannot be substituted as they are due to differences in the type and functionality of the protein, or the components that make up the aroma and taste. For example, it is known that nut milks such as almond milk and peanut milk will clump when added to acidic beverages such as coffee and tea as a substitute for cow's milk. This type of clumping does not usually occur with cow's milk, and is a phenomenon unique to nut milks.

[0004] As far as the inventors know, there are no reports (literature, etc.) that clearly show the mechanism of protein aggregation that occurs when nut milk is added to acidic liquid foods or countermeasures against it. Although there are trial and error among consumers to find countermeasures (for example, mixing nut milk and coffee after reducing the temperature difference, or slowly pouring coffee into nut milk, etc.), the problem has not yet been fundamentally solved.

[0005] On the other hand, it is known that the dispersion stability of milk proteins becomes unstable under acidic conditions near the isoelectric point, and acidic milk beverages are prone to precipitation and aggregation. In order to prevent the aggregation of milk proteins, polysaccharides such as pectin and carboxymethylcellulose are added (see, for example, Patent Documents 1 and 2). Although the use of such dispersion stabilizers may also prevent the aggregation of nut proteins, the use of additives is essential. In addition, when polysaccharides are used, there is a possibility that side effects such as increased viscosity may occur depending on the amount added.

[0006] Regarding the prevention of milk protein aggregation without adding additives, a coffee whitener treated with protein deamidation enzyme has been proposed (Patent Document 3). However, this coffee whitener is a product containing an emulsifier, and its use is limited to products in which whiteners are used, 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 Brochure Summary of the Invention [Problem to be solved by the invention]

[0008] The aggregation phenomenon, which can be said to be unique to nut milk, reduces the value (use value, commercial value, etc.) of nut milk, which is expected to see further growth in demand and expansion of uses in the future. Therefore, in order to increase the value of nut milk and promote its use or application, the present invention aims to create an effective means for preventing aggregation of nut milk, and in particular, to provide nut milk that is less likely to aggregate when used in liquid beverages (particularly acidic liquid beverages) and liquid foods (particularly acidic liquid foods) without adding additives. [Means for solving the problem]

[0009] In light of the above problems, the present inventors have been studying the issue and have focused on protein deamidation, and have attempted to improve the dispersibility of nut milk by treating it with a protein deamidating enzyme. However, there have been no reports of the use of protein deamidating enzyme in nut milk.

[0010] First, in regard to the addition of nut milk to coffee, which is one of the typical uses, it was investigated whether treatment with protein deamidase was effective in preventing aggregation. Surprisingly, when almond milk after enzyme treatment was used, protein aggregation did not occur. Based on this knowledge, detailed experiments were carried out assuming various uses, and it was revealed that treatment with protein deamidase is extremely effective in improving the dispersibility of nut milk in general. In other words, an effective means for preventing aggregation of nut milk was found, and a nut milk with improved dispersibility and less tendency to aggregate even without the use of additives such as emulsifiers was successfully prepared. In addition, many useful findings were obtained when using nut milk in various beverages and foods. Based on these results, the following invention is provided. As mentioned above, the use of protein deamidating enzyme has been proposed to improve the dispersibility of coffee whitener, but coffee whitener is generally prepared by homogenizing an emulsion containing edible oils and fats as the main raw material, an emulsifier, and, if necessary, milk components, thickeners, flavorings, etc., in an emulsifier with excellent shear force such as a high-pressure homogenizer, and is completely different from nut milk in terms of raw materials, composition, preparation method, etc. Therefore, it is impossible to predict the effectiveness of a means for improving the dispersibility of coffee whitener, let alone the possibility of its application to nut milk. [1] Nut milk that has been treated with protein deamidating enzymes. [2] The nut milk according to [1], wherein the raw nuts are one or more nuts selected from almonds, cashew nuts, hazelnuts, pecan nuts, macadamia nuts, pistachios, walnuts, Brazil nuts, peanuts, coconuts, chestnuts, sesame seeds and pine nuts. [3] The nut milk according to [1] or [2], having a nut protein concentration of 0.2% (w / v) to 10.0% (w / v). [4] The nut milk according to any one of [1] to [3], wherein the dispersibility is improved by the treatment. [5] The nut milk described in [4], which does not cause protein aggregation when mixed with a weakly acidic to weakly alkaline liquid (provided that the pH of the mixed liquid is 5 or higher). [6] The nut milk described in [5], wherein the pH of the liquid is 5 to 7. [7] The nut milk described in [5], wherein the liquid is a beverage or liquid food selected from the group consisting of coffee, coffee drinks, tea, tea drinks, fruit juice, fruit juice drinks, sports drinks, nutritional supplements, soup, curry, cocoa and chocolate drinks. [8] A nut milk according to any one of [1] to [7], which does not contain emulsifiers or thickening polysaccharides to prevent aggregation. [9] The nut milk described in any one of [1] to [8], wherein the protein deamidase is an enzyme derived from a microorganism of the genus Chryseobacterium.

[10] The nut milk described in [9], wherein the Chryseobacterium microorganism is Chryseobacterium proteolyticum.

[11] A method for producing nut milk with improved dispersibility, comprising treating nut milk with protein deamidating enzyme.

[12] The method according to

[11] , comprising the following steps (1) and (2): (1) Prepare nut milk; (2) A step of treating the nut milk prepared in (1) with protein deamidating enzyme.

[13] The method of producing the nut milk described in

[12] , wherein the nut milk in step (1) is a nut milk before heat pasteurization.

[14] The method according to

[13] , further comprising the step (3): (3) A step of heat treatment.

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

[10] .

[16] A beverage or liquid food according to

[15] , which has a pH of 5 or higher.

[17] The beverage or liquid food according to

[15] , which is a beverage or liquid food selected from the group consisting of coffee beverages, coffee whiteners, tea beverages, fruit juice beverages, sports beverages, nutritional supplements, soups, curries, cocoa beverages and chocolate beverages. [Brief description of the drawings]

[0011] [Figure 1] Summary of experimental results (relationship between protein concentration of nut milk and aggregation / anti-aggregation effect). [Diagram 2] Summary of experimental results (relationship between liquid pH and flocculation / anti-flocculation effect). [Diagram 3] Summary of experimental results (anti-agglomeration effect in various liquids). The results of Experiment 1 (anti-agglomeration effect in coffee) are also shown. [Figure 4] Summary of experimental results (anti-coagulation effect in nut milks other than almond milk). [Diagram 5] Summary of experimental results (relationship between liquid temperature and aggregation / anti-agglomeration effect). [Figure 6] Summary of experimental results (examination of enzyme treatment conditions (amount of enzyme added, reaction temperature, reaction time)). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] 1. Nut milk with improved dispersibility The first aspect of the present invention relates to a nut milk (also called a nut protein-containing beverage) with improved dispersibility. The nut milk of the present invention is treated with protein deamidating enzyme, and as a result of the treatment, its dispersibility is improved. Since the nut milk of the present invention exhibits excellent dispersibility, it is difficult to aggregate when added to beverages such as coffee and tea, even without the use of additives for improving dispersibility (e.g., emulsifiers, thickening polysaccharides (e.g., pectin, carboxymethylcellulose, etc.), salts). This characteristic allows it to be used in various beverages and foods.

[0013] Nut milk, such as almond milk, is a vegetable milk made from nuts, and is generally prepared by crushing de-shelled nuts, soaking / dissolving, mixing / stirring, filtering, homogenizing, sterilizing, etc. The method for preparing the nut milk used in the present invention is not particularly limited. In addition, nut milk provided by a raw material manufacturer or commercially available nut milk may be purchased and used in the present invention.

[0014] The nut milk of the present invention can be obtained by treating the nut milk with a protein deamidating enzyme to improve its dispersibility. Hereinafter, for the sake of convenience, the nut milk to be treated with the protein deamidating enzyme is referred to as "untreated nut milk".

[0015] The nuts used as raw materials for the unprocessed nut milk are not particularly limited, and examples of raw nuts include almonds, cashew nuts, hazelnuts, pecan nuts, macadamia nuts, pistachios, walnuts, Brazil nuts, peanuts, coconuts, chestnuts, sesame seeds, and pine nuts.

[0016] Unprocessed nut milks that combine two or more nuts (e.g., almonds and cashews or almonds and peanuts) can also be used.

[0017] The protein concentration in the untreated nut milk is not particularly limited, but the protein concentration is, for example, 0.2% (w / v) to 10.0% (w / v), preferably 0.2% (w / v) to 8.0% (w / v), more preferably 0.2% (w / v) to 5.0% (w / v). The protein concentration of the nut milk after the 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), more preferably 0.2% (w / v) to 5.0% (w / v).

[0018] The protein deamidase used in the present invention has an action of directly acting on amide groups of a protein to deamidate without cleavage of peptide bonds or crosslinking of the protein. The type, origin, etc. of the enzyme are not particularly limited as long as it exhibits the above action. Examples of protein deamidase include protein deamidases derived from the genus Chryseobacterium, Flavobacterium, Empedobacter, Sphingobacterium, Aureobacterium, or Myroides, which are disclosed in JP-A-2000-50887, JP-A-2001-218590, WO2006 / 075772, etc., and commercially available protein glutaminases derived from the genus Chryseobacterium. Preferably, an enzyme derived from the genus Chryseobacterium (specifically, an enzyme derived from Chryseobacterium proteolyticum (for example, Protein Glutaminase "Amano" 500, manufactured by Amano Enzyme Inc.)) is used.

[0019] The protein deamidase may be prepared from a culture medium of a microorganism that produces protein deamidase. The microorganism used for preparing protein deamidase is not particularly limited, and may be, for example, a microorganism that produces the enzyme and belongs to the genus Chryseobacterium, Flavobacterium, Empedobacter, Sphingobacterium, Aureobacterium, or Myroides. A specific example of a microorganism suitable for preparing protein deamidase is Chryseobacterium sp. No. 9670, which belongs to the genus Chryseobacterium.

[0020] For example, protein deamidase can be obtained from the culture solution or cells of the above-mentioned microorganism. That is, if it is a secretory protein, it can be recovered from the culture solution, and if it is not, it can be recovered from the cells. A known protein separation and purification method (centrifugation, UF concentration, salting out, various chromatographies using ion exchange resins, etc.) can be used to prepare protein deamidase from the culture solution. For example, the culture solution can be centrifuged to remove the cells, and then the target enzyme can be obtained by combining salting out, chromatography, etc. When the enzyme is recovered from the cells, the target enzyme can be obtained by, for example, crushing the cells by pressure treatment, ultrasonic treatment, etc., and then separating and purifying the cells in the same manner as above. Note that the above series of steps (cell disruption, separation, purification) may be performed after the cells are recovered from the culture solution in advance by filtration, centrifugation, etc. The enzyme may be powdered by a drying method such as freeze-drying or vacuum drying, and in that case, an appropriate excipient or drying aid may be used.

[0021] In the present application, the activity of protein deamidase is measured by the following method. (1) 0.1 ml of an aqueous solution containing protein deamidase is added to 1 ml of 0.2 M phosphate buffer (pH 6.5) containing 30 mM Z-Gln-Gly, and the mixture is incubated at 37°C for 10 minutes, after which 1 ml of 0.4 M TCA solution is added to stop the reaction. As a blank, 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 are added, and 0.1 ml of an aqueous solution containing protein deamidase is added to the mixture, and the mixture is incubated at 37°C for 10 minutes. (2) Using the solution obtained in (1), measure the amount of ammonia produced by the reaction using an Ammonia Test Wako (Wako Pure Chemical Industries, Ltd.). The ammonia concentration in the reaction solution is calculated from a calibration curve showing the relationship between ammonia concentration and absorbance (630 nm) created using an ammonia standard solution (ammonium chloride). (3) Protein deamidase activity is calculated by the following formula, where 1 unit is the amount of enzyme that produces 1 μmol of ammonia per minute. 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, Df is the dilution ratio of the enzyme solution, and 17.03 is the molecular weight of ammonia.)

[0022] As long as it is effective in improving the dispersibility of nut milk, the conditions for the treatment with protein deamidating enzyme are not particularly limited, and the reaction temperature, reaction time, and amount of enzyme added (enzyme concentration) can be adjusted to set optimal reaction conditions.

[0023] Although not limited to this example, the reaction temperature may be set within the range of, for example, 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 may be set within the range of, for example, 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. In addition, the amount of enzyme added may be set within the range of, for example, 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" is the number of units per substrate nut protein (g). As mentioned above, the protein concentration in the untreated nut milk is not particularly limited, but untreated nut milk having 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 deamidating enzyme.

[0024] Here, when setting the conditions for the treatment with protein deamidase, the following guidelines (a) to (c) should be observed. (a) If the reaction temperature is lowered, the reaction time or the amount of enzyme added should be increased (or both). (b) To shorten the reaction time, the reaction temperature is increased (but not exceeding 70°C, preferably not exceeding 60°C) and / or the amount of enzyme added is increased. (c) When the amount of enzyme added is reduced, the reaction temperature is increased (but not exceeding 70°C, preferably not exceeding 60°C) and / or the reaction time is increased.

[0025] More specific indexes for setting the processing conditions are given below. When the reaction temperature is 5°C or less and <15°C, the reaction time is set to more than 8 hours (preferably 24 hours or more) or the amount of enzyme added is set to 0.2 (U / g protein) or more (preferably 1 (U / g protein) or more). When the reaction temperature is 15°C or less 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 1 (U / g protein) or more). When the reaction temperature is 25°C or less and <40°C, the reaction time is set to more than 5 hours (preferably 7 hours or more) or the amount of enzyme added is set to 0.2 (U / g protein) or more (preferably 1 (U / g protein) or more). When the reaction temperature is 40° C. or less and <50° C., the reaction time is preferably 3 hours or more, or the amount of enzyme added is preferably 0.2 (U / g protein) or more. When the reaction temperature is 50 or less (however, the temperature does not exceed 70°C, and preferably is 60°C or less), the reaction time is preferably 3 hours or more, 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 has excellent dispersibility and is less likely to cause protein aggregation. Typically, when mixed (added) to a weakly acidic (3≦pH<6) to weakly alkaline (8≦pH<11) liquid (provided that the pH of the mixed liquid is 5 or higher), protein aggregation does not occur. The pH of the liquid after mixing where protein aggregation does not occur is, for example, 5 to 10, preferably 5 to 9, and more preferably 5 to 7. The liquid (beverage, liquid food) with which the nut milk of the present invention is mixed is 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., concentrating or freeze-drying)), tea beverages (flavored tea, milk tea, tea beverages containing fruit juice, etc.), fruit juice, fruit juice beverages, sports drinks, nutritional supplement beverages (protein beverages, nutritional beverages for nursing care, etc.), soup (bouillon-based soup, stew, chowder, borsch, vegetable soup (e.g., tomato soup, corn soup, potage, pumpkin soup), miso soup), curry, cocoa, and chocolate beverages.

[0027] In a preferred embodiment of the present invention, the characteristics of excellent dispersibility and low protein aggregation are utilized, and emulsifiers (glycerin fatty acid esters, sucrose fatty acid esters, lecithin, saponin, etc.), thickening polysaccharides (pectin, carboxymethylcellulose, etc.), salts (salt (seasalt), calcium salts, phosphates, etc.), etc., for preventing aggregation are not contained. In particular, emulsifiers and thickening polysaccharides are not contained. Thus, according to the present invention, a nut milk is provided that meets the needs of consumers for products with few or no additives. Note that even in this preferred embodiment, the use of additives for other purposes (specifically, for example, adjusting taste and flavor) other than for preventing aggregation is not precluded.

[0028] As can be seen from the above description, the nut milk of the present invention can be produced by treating untreated nut milk with protein deamidating enzyme. Therefore, the nut milk of the present invention can be typically obtained by a production method including the following steps (1) and (2). (1) Prepare nut milk; (2) A step of treating the nut milk prepared in (1) with protein deamidating enzyme.

[0029] Step (2), i.e., the treatment with protein deamidating enzyme, may be carried out either before or after the heat sterilization of the nut milk. However, in order to simplify the production process, it is preferable to carry out this step before the heat sterilization of the nut milk, and then to carry out a heat sterilization step that also inactivates the protein deamidating enzyme (in other words, step (2) may be incorporated into the production process of the nut milk). Therefore, in a preferred embodiment, "(3) a heat treatment step" is carried out after step (2). The conditions of the heat treatment are not particularly limited as long as the protein deamidating enzyme can be inactivated and the nut milk can be sterilized. For example, the treatment is carried out at a temperature of 70°C to 150°C for 1 second to 5 hours.

[0030] 2. Uses of nut milk The second aspect of the present invention relates to the use of the nut milk of the present invention. The nut milk of the present invention has excellent dispersibility and is less likely to cause protein aggregation. Due to this characteristic, it is suitable for use in various beverages and liquid foods. In other words, various beverages and various liquid foods containing the nut milk of the present invention are provided.

[0031] As shown in the examples below, detailed studies by the present inventors have revealed that (i) the pH range in which aggregation does not occur can be expanded to the acidic side by treatment with protein deamidase, and (ii) protein aggregation occurring when nut milk is mixed with beverages, liquid foods, etc. depends on the pH of the beverages, etc. after mixing with nut milk, and protein aggregation does not occur if the pH is 5 or higher. In view of this finding, 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 whiteners (usage for other than coffee, such as black tea, is also envisioned), tea beverages (flavored tea, milk tea, tea beverages containing fruit juice, etc.), fruit juice beverages, sports drinks, nutritional supplement beverages (protein beverages, nutritional beverages for nursing care, etc.), various soups, curries, cocoa beverages, and chocolate beverages. 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.

[0033] Nut milk is mixed with other raw materials during the manufacturing process of beverages or liquid foods, for example. Preferably, nut milk is mixed at the final stage of the manufacturing process, that is, after other raw materials are mixed and processed (the stage in which the product is in the form / shape). However, sterilization treatment, addition of seasonings, preservatives, flavorings, antioxidants, etc. for the purpose of adjusting taste and maintaining quality, etc. may be performed thereafter. On the other hand, it is also a preferred embodiment to mix nut milk with beverages or liquid foods after the manufacturing process is completed (i.e., in the form of a final product, not an intermediate product). In this embodiment, the present invention can be applied without changing the manufacturing process of beverages or liquid foods. EXAMPLES

[0034] 1. Preventing clumping in coffee Protein glutaminase "Amano" 500 (Amano Enzyme, 500U / g) was added to 100mL of commercially available almond milk (Rude, protein content 1.5%, ingredients: almonds, water) at 1U per 1g of protein in the almond milk and reacted at 50℃ for 5 hours (deamidation reaction). After treating at 95℃ for 20 minutes to heat inactivate the enzyme, the mixture was cooled to 5℃ to obtain enzyme-treated almond milk.

[0035] Commercially available instant coffee was dissolved in hot water to prepare a coffee solution (2%). When 20-30 mL of enzyme-treated almond milk was added to 150 mL of the coffee solution (pH after adding enzyme-treated almond milk was 5.7), no aggregation was observed. In contrast, when non-enzyme-treated almond milk was used, obvious aggregation was observed. Furthermore, when an experiment was conducted under the same conditions using peanut milk instead of almond milk, the same results were obtained (no aggregation occurred with enzyme-treated peanut milk).

[0036] 2. Relationship between protein concentration of nut milk and aggregation / anti-aggregation effect <No enzyme treatment> (1) Method Commercially available almond milk (Rude, protein content 1.5%, ingredients: almonds, water) was diluted with tap water to protein concentrations of 0.1, 0.5, and 1.5% (w / v), then cooled to 5°C and 5 mL of each was added to 50 mL of coffee solution heated to 90°C to check for the presence or absence of aggregation.

[0037] (2) Results Aggregation was observed in almond milk at all protein concentrations (Figure 1).

[0038] <Enzyme treatment included> (1) Method Protein glutaminase "Amano" 500 (Amano Enzyme, 500U / g) was added to commercially available almond milk (Rude, protein content 1.5%, raw materials: almonds, water) at 1U per 1g of protein in the almond milk and reacted at 50°C for 5 hours (deamidation reaction). The enzyme was heat-inactivated by treating at 90°C for 15 minutes to obtain enzyme-treated almond milk. The enzyme-treated almond milk was diluted with tap water to protein concentrations of 0.1, 0.5, 0.75, 1.0, and 1.5% (w / v), cooled to 5°C, and 5mL of each was added to 50mL of coffee solution heated to 90°C to check for the presence or absence of aggregation.

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

[0040] 3. Relationship between liquid pH and flocculation / anti-flocculation 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 hot water heated to 90°C, and aggregation was confirmed. The enzyme-treated almond milk was prepared by the method described in the experiment in 2 above.

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

[0042] 4. Anti-aggregation effect in various liquids 4-1. Black tea (1) Method Boiling water was poured into a commercially available black tea tea bag (Twinings, English Breakfast), and after 2-3 minutes of extraction, the tea bag was removed to prepare black tea. Non-enzyme-treated almond milk or enzyme-treated almond milk (protein concentration 1.5% (w / v)) was added to this black tea, and the presence or absence of aggregation was confirmed. The black tea immediately before the addition of the almond milk was at 80°C and had a pH of 5.2. The pH of the black tea after the addition of the almond milk was 5.9. The enzyme-treated almond milk was prepared by the method described in the experiment in 2 above.

[0043] (2) Results (Figure 3) A small amount of aggregation was observed in the non-enzyme treated almond milk, in contrast to no aggregation observed in the enzyme treated almond milk.

[0044] 4-2. Lemon tea (1) Method Boiling water was poured into a commercially available black tea tea bag (Twinings, English Breakfast), and after 2-3 minutes of extraction, the tea bag was removed to prepare black tea. Lemon juice was added to this black 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 the presence or absence of aggregation. The black tea was at 70°C just before the almond milk was added. The enzyme-treated almond milk was prepared by the method described in the experiment in 2 above.

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

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

[0047] 4-3. Decaf (1) Method A decaffeinated coffee solution was prepared by pouring boiling water into commercially available decaffeinated coffee powder (Nestle, Nescafe Gold) and dissolving it thoroughly. Non-enzyme-treated almond milk or enzyme-treated almond milk (protein concentration 1.5% (w / v)) was added to this, and the presence or absence of aggregation was confirmed. The decaffeinated coffee solution immediately before the addition of almond milk was 80°C and had a pH of 5.3. The pH of the decaffeinated coffee solution after the addition of almond milk was 5.8. The enzyme-treated almond milk was prepared by the method described in the experiment in 2 above.

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

[0049] 4-4. Tomato soup (1) Method A specified amount of boiling water was poured into commercially available chicken soup stock (Knorr Chicken Cube, manufactured by Unilever) to completely dissolve the stock, and chicken soup was prepared, followed by the addition of commercially available tomato puree. 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 aggregation was confirmed. The tomato soup was at 80°C just before the addition of the almond milk. The enzyme-treated almond milk was prepared by the method described in the experiment in 2 above.

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

[0051] When the pH before adding almond milk was 4.0, aggregation 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 the pH of the tomato soup after adding enzyme-treated almond milk were both 4.0. It is considered that the pH did not change even when almond milk was added because of the strong buffering capacity of the pH adjuster citric acid added to the tomato puree, and thus aggregation occurred.

[0052] 5. Anti-clumping effect in nut milks other than almond milk (1) Method Protein glutaminase "Amano" 500 (Amano Enzyme, 500U / g) was added at 1U per 1g of nut protein to commercially available peanut milk (Rude, protein content 2.0%, raw materials: peanuts, water), commercially available cashew nut milk (PLENISH, protein content 0.9%, raw materials: water, cashew nuts, salt), pistachio milk (Borna Food, protein content 1.0%), and hazelnut milk (Plenish, protein content 0.6%), and reacted at 50°C for 5 hours (deamidation reaction). After the enzyme reaction, the enzyme was immediately inactivated by treating at 90°C for 15 minutes, cooled in running water, and then cooled to 5°C in a refrigerator. 5mL of each was added to 50mL of coffee solution heated to 90°C to check for the presence or absence of aggregation.

[0053] (2) Results (Figure 4) Peanut milk, cashew nut milk, pistachio milk, and hazelnut milk all showed aggregation without enzyme treatment, but did not show aggregation after enzyme treatment. This result indicates that the same effect of enzyme treatment can be obtained for nut milks other than almond milk.

[0054] 6. Relationship between liquid temperature and aggregation / anti-agglomeration effect <Vary the temperature of the coffee (almond milk is kept at a constant 5℃)> (1) Method 5 mL of non-enzyme-treated almond milk or enzyme-treated almond milk cooled to 5°C was added to 50 mL of coffee adjusted to each temperature, and the presence or absence of aggregation was confirmed.

[0055] (2) Results (Fig. 5) In the case of non-enzyme-treated almond milk, aggregation was observed when the coffee temperature was 60°C or higher, and the amount of aggregation increased as the temperature increased. On the other hand, the enzyme-treated almond milk showed an anti-aggregation effect (coffee temperatures of 60°C and 90°C).

[0056] <Vary the temperature of the coffee (almond milk is kept at 90℃)> (1) Method 5 mL of non-enzyme-treated almond milk or enzyme-treated almond milk heated to 90°C was added to 50 mL of coffee adjusted to each temperature, and the presence or absence of aggregation was confirmed.

[0057] (2) Results (Fig. 5) Aggregation was observed when non-enzyme-treated almond milk at 90°C was added to coffee at 90°C. Aggregation was also observed when non-enzyme-treated almond milk at 90°C was added to coffee at 50°C, but no aggregation was observed when non-enzyme-treated almond milk at 90°C was added to coffee at 40°C. It is thought that when non-enzyme-treated almond milk at 90°C was added to coffee at 50°C, the temperature of the coffee temporarily rose, causing aggregation. It is also thought that a higher temperature after mixing (50°C or higher) makes the coffee more likely to aggregate.

[0058] No aggregation was observed when enzyme-treated almond milk was added (40°C or 90°C coffee).

[0059] 7. Examination of enzyme treatment conditions (enzyme addition amount, reaction temperature, reaction time) (1) Method Protein glutaminase "Amano" was added to commercially available almond milk (Rude, protein content 1.5%, raw materials: almonds, water) at 0.2 U, 1 U or 5 U per 1 g 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 (deamidation reaction). After the enzyme reaction, the enzyme was quickly inactivated by treatment at 90°C for 15 minutes, cooled in running water, and then cooled to 5°C in a refrigerator. 5 mL of each was added to 50 mL of coffee solution heated to 90°C, and the presence or absence of aggregation was confirmed.

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

[0061] <Summary> The same anti-aggregation effect was observed regardless of the nut protein concentration in the range of 0.1-1.5% (w / v). In other words, it was shown that enzyme treatment with protein deamidase is effective in preventing aggregation in nut milks with various protein concentrations and is highly versatile. Although it depends on the type of liquid to be mixed with the nut milk, the tendency is that without enzymatic treatment with protein deamidase, the nut milk will aggregate when the pH drops below 7 after mixing with nut milk, but 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 for beverages such as coffee and tea, but also for acidic liquid foods such as sour milk soup. In addition, if the pH of the liquid after mixing with milk is 5 or higher, milk lemon tea, which is difficult to prepare even with milk, can be prepared, so it can also be applied to various beverages and liquid foods using sour fruits. Basically, the pH of the liquid you mix the nut milk with has a big effect on clumping, followed by the higher the temperature of the liquid, the more likely it is to clump. The effect varies depending on the amount of enzyme added (enzyme concentration), reaction temperature, and reaction time. The same effect was observed not only in almond milk, but also in peanut milk, cashew nut milk, pistachio milk, and hazelnut milk. Therefore, it is considered that enzyme treatment with protein deamidase is effective in preventing aggregation in nut milk in general. [Industrial Applicability]

[0062] The present invention provides a nut milk that has excellent dispersibility without the use of additives such as emulsifiers. High dispersibility enhances the value of the nut milk itself and beverages and liquid foods that use the nut milk. It also makes it possible to provide new beverages and liquid foods that could not be realized in the past.

[0063] The nut milk provided by the present invention is not limited to existing uses, but is expected to be used or applied to various uses (especially acidic beverages and acidic liquid foods). The fact that additives such as emulsifiers can be eliminated is a very big advantage of the present invention. In addition, even when the nut milk is added to coffee, etc. as a substitute for cow's milk or soy milk, special operations to prevent clumping are not required, which improves convenience for consumers.

[0064] The present invention is not limited to the above-mentioned embodiment and examples. Various modifications within the scope of the claims and within the scope that can be easily conceived by a person skilled in the art are also included in the present invention. The contents of papers, published patent publications, patent publications, etc. specified in this specification are all incorporated by reference.

Claims

1. Nut milk used as an additive to beverages or liquid foods, The aforementioned beverage or liquid food has a pH of 5 to 7 after the addition of nut milk. The aforementioned nut milk is nut milk that has been treated with protein glutaminase.

2. The nut milk according to claim 1, wherein the raw material nuts 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 the nut protein concentration is 0.2% (w / v) to 10.0% (w / v).

4. The nut milk according to any one of claims 1 to 3, wherein the dispersibility is improved by the above-mentioned process.

5. The nut milk according to any one of claims 1 to 4, wherein the beverage or liquid food 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.

6. The nut milk according to any one of claims 1 to 5, which does not contain emulsifiers and thickening polysaccharides for preventing aggregation.

7. The nut milk according to any one of claims 1 to 6, wherein the protein glutaminase is an enzyme derived from a microorganism of the genus Chryseobacterium.

8. The nut milk according to claim 7, wherein the microorganism of the genus Chryseobacterium is Chryseobacterium proteoricum.

9. A nut milk-containing beverage or liquid food obtained by adding the nut milk described in any one of claims 1 to 8 to a beverage or liquid food, wherein the pH is 5 to 7.

10. The beverage or liquid food according to claim 9, 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.

11. Nut milk treated with protein glutaminase.