Preventing condensation in nut milk

Abstract

The present invention aims to develop a method for effectively preventing nut milk from agglomerating. The present invention utilizes protein deamidase to treat nut milk, thereby improving its dispersibility and preventing agglomeration when added to liquid beverages or liquid foods.

Description

Technical Field

[0001] The present invention relates to nut milk. Specifically, the present invention relates to a nut milk with improved dispersibility (less likely to coagulate) 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 by reference. Background Art

[0002] Driven by allergy concerns, the rise in vegetarians, and religious reasons, plant-based soy proteins are gaining popularity as an alternative to animal-derived milk proteins, particularly milk. However, as soy becomes more prevalent, soy has also become a source of allergies, leading to a surge in the development of plant-based protein alternatives. In fact, proteins from cereals like peas, rice, and oats, as well as nut proteins from almonds, cashews, and peanuts, are increasingly being commercialized as soy alternatives in foods and beverages. This suggests a growing need and desire for a diverse range of plant-based protein alternatives to soy to mitigate allergies.

[0003] On the other hand, when replacing milk protein raw materials with plant-derived protein raw materials, direct substitution is sometimes not possible due to differences in protein type, functionality, or components that contribute to aroma and flavor. For example, nut milks such as almond milk and peanut milk are known to cause coagulation when added to acidic beverages such as coffee and black tea as a milk substitute. This coagulation does not typically occur with cow's milk and is a phenomenon unique to nut milks.

[0004] To the best of the present inventors' knowledge, there are no reports (in the literature, etc.) that clearly demonstrate the mechanism of protein aggregation caused by nut milk when added to acidic liquid foods, nor any countermeasures. While there have been repeated attempts by consumers to address this issue (e.g., mixing nut milk and coffee after minimizing the temperature difference, or slowly pouring coffee into nut milk), a fundamental solution has yet to be achieved.

[0005] On the other hand, it is known that milk protein dispersion stability becomes unstable under acidic conditions near its isoelectric point, easily leading to problems such as precipitation and aggregation in acidic milk beverages. To prevent this aggregation of milk proteins, polysaccharides such as pectin and carboxymethylcellulose are added (see, for example, Patent Documents 1 and 2). While the use of these dispersion stabilizers can potentially prevent aggregation of nut proteins, the use of additives is essential. Furthermore, the use of polysaccharides also carries the risk of increased viscosity depending on the amount added.

[0006] To prevent milk protein aggregation without adding additives, a coffee whitener treated with protein deamidase has been proposed (Patent Document 3). However, this coffee whitener contains an emulsifier and its use is limited to beverages such as coffee and black tea that use whitening agents.

[0007] Prior art literature

[0008] Patent Literature

[0009] Patent Document 1: International Publication No. 2012 / 176852 Pamphlet

[0010] Patent Document 2: Japanese Patent No. 3885194

[0011] Patent Document 3: International Publication No. 2011 / 108633 Pamphlet Summary of the Invention

[0012] Problems to be solved by the invention

[0013] The so-called coagulation phenomenon unique to nut milks reduces the value (utility value, commercial value, etc.) of nut milks, which are expected to see further growth in demand or expansion of their applications. Therefore, to increase the value of nut milks and promote their utilization and application, the present invention aims to create a method for effectively preventing coagulation of nut milks. In particular, it aims to provide a nut milk that is less susceptible to coagulation in liquid beverages (particularly acidic liquid beverages) or liquid foods (particularly acidic liquid foods), even without the addition of additives.

[0014] Technical solutions to solve problems

[0015] In view of the above technical problems, the present inventors have repeatedly conducted research and focused on protein deamidation, attempting to improve the dispersibility of nut milk by treating it with protein deamidase. However, there are no reports of using protein deamidase in nut milk.

[0016] First, regarding the addition of nut milk to coffee, a typical use of nut milk, we investigated whether treatment with protein deamidase could effectively prevent aggregation. Surprisingly, no protein aggregation occurred when almond milk was used after enzyme treatment. Based on this insight, detailed experiments envisioning various uses revealed that treatment with protein deamidase was highly effective in improving the general dispersibility of nut milk. In other words, we discovered a method for effectively preventing nut milk from agglomerating, and have succeeded in producing nut milk with improved dispersibility that is less prone to agglomeration even without the use of additives such as emulsifiers. Furthermore, this has yielded many useful insights into the use of nut milk in various beverages and foods. Based on these findings, we provide the following invention. Furthermore, as mentioned above, the use of protein deamidase has been proposed to improve the dispersibility of coffee whitener. However, coffee whitener is typically made from edible oils and fats as its primary raw material, and is homogenized using an emulsifier with excellent shear force, such as a high-pressure homogenizer, to which an emulsifier, and optionally milk components, thickeners, and flavorings, are added. Therefore, its raw materials, composition, and preparation methods are completely different from those of nut milk. Therefore, regardless of the effectiveness of the methods that are effective in improving the dispersibility of coffee whitener on nut milk, the possibility of their application is unpredictable.

[0017] [1] A nut milk obtained by treating with protein deamidase.

[0018] [2] The nut milk according to [1], wherein the nuts used as the raw material 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.

[0019] [3] The nut milk according to [1] or [2], wherein the nut protein concentration is 0.2% (w / v) to 10.0% (w / v).

[0020] [4] The nut milk according to any one of [1] to [3], wherein the dispersibility is improved by the above-mentioned treatment.

[0021] [5] The nut milk according to [4], wherein the protein does not aggregate when mixed with a weakly acidic to weakly alkaline liquid, and the pH of the mixed liquid is 5 or higher.

[0022] [6] The nut milk according to [5], wherein the pH of the liquid is 5 to 7.

[0023] [7] The nut milk according to [5], wherein the liquid is a beverage or liquid food selected from coffee, coffee beverages, tea, tea beverages, juice, juice beverages, sports drinks, nutritional supplement beverages, soups, curry, cocoa and chocolate beverages.

[0024] [8] The nut milk according to any one of [1] to [7], which does not contain an emulsifier or thickening polysaccharide for preventing aggregation.

[0025] [9] The nut milk according to any one of [1] to [8], wherein the protein deamidase is an enzyme derived from a microorganism of the genus Chryseobacterium.

[0026]

[10] The nut milk according to [9], wherein the Chryseobacterium microorganism is Chryseobacterium proteolyticum.

[0027]

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

[0028]

[12] The manufacturing method according to

[11] , comprising the following steps (1) and (2):

[0029] (1) Steps for preparing nut milk,

[0030] (2) A step of treating the nut milk prepared in (1) with protein deamidase.

[0031]

[13] The manufacturing method according to

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

[0032]

[14] The manufacturing method according to

[13] , further comprising the following step (3):

[0033] (3) Heat treatment step.

[0034]

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

[10] .

[0035]

[16] The beverage or liquid food according to

[15] , wherein the beverage or liquid food has a pH of 5 or above.

[0036]

[17] The beverage or liquid food according to

[15] , wherein the beverage or liquid food is a beverage or liquid food selected from coffee beverages, coffee creamers, tea beverages, fruit juice beverages, sports drinks, nutritional supplement beverages, soups, curry, cocoa beverages and chocolate beverages. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is a summary of experimental results (the relationship between protein concentration of nut milk and aggregation / aggregation prevention effect).

[0038] Figure 2This is a summary of experimental results (the relationship between liquid pH and aggregation / aggregation prevention effect).

[0039] Figure 3 This is a summary of the experimental results (the effect of preventing aggregation in various liquids). In addition, the results of Experiment 1 (preventing aggregation in coffee) are also recorded.

[0040] Figure 4 This is a summary of experimental results (anti-aggregation effects in nut milks other than almond milk).

[0041] Figure 5 This is a summary of experimental results (the relationship between liquid temperature and condensation / condensation prevention effect).

[0042] Figure 6 This is a summary of experimental results (study of enzyme treatment conditions (enzyme addition amount, reaction temperature, reaction time)). DETAILED DESCRIPTION

[0043] 1. Nut milk with improved dispersibility

[0044] A first aspect of the present invention relates to a nut milk (also known as a nut protein beverage) with improved dispersibility. The nut milk of the present invention is treated with a protein deamidase, and as a result of this treatment, its dispersibility is improved. The nut milk of the present invention exhibits excellent dispersibility and is therefore less likely to aggregate when added to beverages such as coffee and black tea, even without the use of additives for improving dispersibility (e.g., emulsifiers, thickening polysaccharides (pectin, carboxymethyl cellulose, etc.), salts). This characteristic allows it to be used in a variety of beverages or foods.

[0045] Nut milks, such as almond milk, are plant-based milks made from nuts. They are typically produced through processes such as crushing de-kerneled nuts, soaking / dissolving them in water, mixing / stirring them, filtering them, homogenizing them, and sterilizing them. The method for producing the nut milks used in the present invention is not particularly limited. Alternatively, nut milks from raw material manufacturers or commercially available sources can be purchased and used in the present invention.

[0046] The nut milk of the present invention can be obtained by treating nut milk with protein deamidase to improve its dispersibility. Hereinafter, for convenience of description, nut milk treated with protein deamidase will be referred to as "untreated nut milk."

[0047] The nuts used as raw materials for the unprocessed nut milk are not particularly limited, but examples of raw nuts include almonds, cashews, hazelnuts, pecans, macadamia nuts, pistachios, walnuts, Brazil nuts, peanuts, coconuts, chestnuts, sesame seeds, and pine nuts.

[0048] Unprocessed nut milk containing two or more types of nuts (for example, a combination of almonds and cashews or a combination of almonds and peanuts) may also be used.

[0049] The protein concentration in the untreated nut milk is not particularly limited, and 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 used. Furthermore, the protein concentration of nut milk after protein deamidase 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).

[0050] The protein deamidase used in the present invention has the following effect: directly act on the amide group of protein and deamidate without being accompanied by the cutting of peptide bonds and the cross-linking of protein. As long as it is an enzyme showing this effect, its kind, source, etc. are not particularly limited. As the example of protein deamidase, the disclosed protein deamidase derived from Chryseobacterium (Chryseobacterium) genus, Flavobacterium (Flavobacterium) genus, Empedobacter (Empedobacter) genus, Sphingobacterium (Sphingobacterium) genus, Aureobacterium (Aureobacterium) genus or class aroma fungi (Myroides) genus, commercially available protein glutaminase derived from Chryseobacterium genus, etc. can be enumerated. An enzyme derived from the genus Chryseobacterium is preferably used (a specific example is an enzyme derived from Chryseobacterium proteolyticus (for example, protein glutaminase "AMANO" 500, manufactured by Amano Enzyme Co., Ltd.)).

[0051] Protein deamidase can be produced from a culture broth of a microorganism that produces protein deamidase. The microorganism used to produce the protein deamidase is not particularly limited. Examples of enzyme-producing microorganisms include those belonging to the genera Chryseobacterium, Flavobacterium, Stibacillus, Sphingobacterium, Chrysobacterium, or Aromaticobacterium. Specific examples of microorganisms suitable for producing protein deamidase include Chryseobacterium sp. No. 9670, which belongs to the genus Chryseobacterium.

[0052] For example, protein deamidase can be obtained from the culture fluid or thalline of the above-mentioned microorganism. That is, if it is a secretory protein, it can be recovered from the culture fluid, if it is other than it, it can be recovered from the thalline. The method for preparing protein deamidase by culture fluid can use known protein separation, purification method (centrifugation, UF concentration, salting out, using various chromatography methods such as ion exchange resins, etc.). For example, the thalline can be removed by centrifugation of the culture fluid, and thereafter, the target enzyme is obtained by combining salting out, chromatography, etc. In the case of reclaiming the enzyme from the thalline, for example, after the thalline is crushed by pressure treatment, ultrasonic treatment, etc., the target enzyme is obtained by separating and refining as described above. In addition, it is also possible to carry out the above-mentioned series of operations (crushing, separation, and refining of the thalline) after the thalline is recovered in advance from the culture fluid by filtration, centrifugation, etc. The enzyme can be powdered by drying methods such as freeze drying and reduced pressure drying, and suitable excipients and drying aids can also be used at this moment.

[0053] In the present application, the activity of protein deamidase was measured using the following method.

[0054] (1) 0.1 ml of an aqueous solution containing a protein deamidase was added to 1 ml of a 0.2 M phosphate buffer (pH 6.5) containing 30 mM Z-Gln-Gly, and the mixture was incubated at 37°C for 10 minutes. Then, 1 ml of a 0.4 M TCA solution was added to stop the reaction. As a blank, a solution was prepared by adding 0.1 ml of an aqueous solution containing a protein deamidase to a solution prepared by adding 1 ml of a 0.2 M phosphate buffer (pH 6.5) containing 30 mM Z-Gln-Gly and 1 ml of a 0.4 M TCA solution, and incubating the solution at 37°C for 10 minutes.

[0055] (2) The amount of ammonia generated by the reaction was measured using Ammonia-test Wako (Wako Pure Chemical Industries, Ltd.) for the solution obtained in (1). The ammonia concentration in the reaction solution was determined from a calibration curve showing the relationship between ammonia concentration and absorbance (630 nm) prepared using an ammonia standard solution (ammonium chloride).

[0056] (3) The activity of protein deamidase was calculated by the following formula, taking the amount of enzyme that generates 1 μmol of ammonia in 1 minute as 1 unit.

[0057] Enzyme activity (U / mL) = ammonia concentration in reaction solution (mg / L) × (1 / 17.03) × (reaction solution volume / enzyme solution volume) × (1 / 10) × Df

[0058] (Wherein, 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)

[0059] The conditions for the treatment with protein deamidase are not particularly limited as long as they are effective in improving the dispersibility of nut milk. Optimal reaction conditions may be set by adjusting the reaction temperature, reaction time, and enzyme addition amount (enzyme concentration).

[0060] Without being limited to the above examples, 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. In addition, 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 substrate nut protein (g). In addition, as mentioned above, the protein concentration in the untreated nut milk is not particularly limited, and 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 deamidase.

[0061] Here, when setting the treatment conditions using protein deamidating enzyme, the following indicators (a) to (c) can be followed.

[0062] (a) When the reaction temperature is lowered, the reaction time is prolonged or the amount of enzyme added is increased (or both).

[0063] (b) When shortening the reaction time, the reaction temperature is increased (but not exceeding 70° C., preferably 60° C. or lower) or the amount of enzyme added is increased (or both).

[0064] (c) When the amount of enzyme added is reduced, the reaction temperature is increased (but not exceeding 70° C., preferably 60° C. or lower) or the reaction time is extended (or both).

[0065] More specific indicators for setting processing conditions are exemplified below.

[0066] When the reaction temperature is 5°C ≤ 15°C, the reaction time is set to more than 8 hours (preferably 24 hours or more), or the enzyme addition amount is set to 0.2 (U / g protein) or more (preferably 1 (U / g protein) or more).

[0067] When 15°C ≤ reaction temperature < 25°C, the reaction time is set to be longer than 7 hours, or the amount of enzyme added is set to be higher than 0.2 (U / g protein) (preferably 1 (U / g protein) or more).

[0068] When the reaction temperature is 25°C ≤ 40°C, the reaction time is set to more than 5 hours (preferably 7 hours or more), or the enzyme addition amount is set to 0.2 (U / g protein) or more (preferably 1 (U / g protein) or more).

[0069] When the reaction temperature is 40°C ≤ 50°C, the reaction time is preferably set to 3 hours or longer, or the amount of enzyme added is preferably set to 0.2 (U / g protein) or longer.

[0070] When 50 ≤ reaction temperature (the temperature is not more than 70°C, preferably 60°C or less), the reaction time is preferably set to 3 hours, or the enzyme addition amount is preferably set to 0.2 (U / g protein) or more.

[0071] As shown above, the nut milk of the present invention has excellent dispersibility and is less likely to cause protein aggregation. Typically, when mixed (added) with a weakly acidic (3 ≤ pH < 6) to weakly alkaline (8 ≤ pH < 11) liquid (where the pH of the mixed liquid is 5 or higher), protein aggregation does not occur. The pH of the mixed liquid in which 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) mixed with the nut milk of the present invention is not particularly limited. Examples thereof include coffee, coffee beverages, tea (including black tea, green tea, oolong tea, etc. Substances obtained by reducing extracts, substances obtained by reducing processed extracts (such as concentration, freeze-drying)), tea beverages (flavored tea, milk tea, tea beverages with added fruit juice, etc.), fruit juice, fruit juice beverages, sports drinks, nutritional supplement beverages (protein beverages, nursing nutritional beverages, etc.), soups (clear soups, stewed foods, mixed vegetables, beetroot soup, vegetable soup (such as tomato soup, corn soup, thick soup, pumpkin soup), miso juice), curry, cocoa, and chocolate beverages.

[0072] In a preferred embodiment of the present invention, taking advantage of its excellent dispersibility and resistance to protein aggregation, it does not contain emulsifiers (glycerol fatty acid esters, sucrose fatty acid esters, lecithin, saponins, etc.), thickening polysaccharides (pectin, carboxymethyl cellulose, etc.), salts (sea salt, calcium salts, phosphates, etc.) for preventing aggregation. In particular, it does not contain emulsifiers and thickening polysaccharides. In this way, according to the present invention, nut milk that meets consumer demand for products with few or no additives can be provided. In addition, in this preferred embodiment, the use of additives for other purposes (specifically, such as taste and flavor adjustment) is not hindered by the lack of the use of additives for prevention of aggregation.

[0073] 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 deamidase. Therefore, the nut milk of the present invention can be obtained typically by a production method comprising the following steps (1) and (2).

[0074] (1) Steps for preparing nut milk;

[0075] (2) A step of treating the nut milk prepared in (1) with protein deamidase.

[0076] Step (2), i.e., the treatment with protein deamidase, can be performed at any time before or after heat sterilization of nut milk. However, in order to simplify the manufacturing process, this step can be performed before heat sterilization of nut milk, and thereafter, a heat sterilization process that also inactivates protein deamidase is performed (in other words, step (2) can also be introduced into the manufacturing process of nut milk). Therefore, in a preferred embodiment, "(3) heat treatment step" is performed after step (2). The conditions of the heat treatment are not particularly limited as long as the protein deamidase can be inactivated and the nut milk can be sterilized. For example, the treatment is performed at a temperature of 70°C to 150°C for 1 second to 5 hours.

[0077] 2. Uses of Nut Milk

[0078] A second aspect of the present invention relates to uses of the nut milk of the present invention. The nut milk of the present invention exhibits excellent dispersibility and is less susceptible to protein aggregation. This property makes it suitable for use in a variety of beverages and liquid foods. Specifically, various beverages and liquid foods containing the nut milk of the present invention are provided.

[0079] As shown in the examples described below, detailed studies by the present inventors have revealed that: (i) treatment with a protein deamidase can expand the pH range where aggregation does not occur to the acidic side; and (ii) protein aggregation that occurs when nut milk is mixed with beverages, liquid foods, etc. depends on the pH of the beverage after the nut milk is mixed, and no protein aggregation occurs if the pH is 5 or above. In view of the above findings, the pH of the beverage or liquid food containing the nut milk of the present invention is preferably 5 or above. More specifically, the pH of the beverage or liquid food 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.

[0080] Examples of beverages or liquid foods include coffee drinks, coffee creamers (e.g., uses other than coffee, such as black tea, are also contemplated), tea drinks (flavored tea, milk tea, tea drinks with added juice, etc.), juice drinks, sports drinks, nutritional supplement drinks (protein drinks, nursing nutrition drinks, etc.), various soups, curry, cocoa drinks, and chocolate drinks. As can be seen from the above examples, the present invention is not limited to neutral beverages and liquid foods, and can also be used in weakly acidic beverages and liquid foods.

[0081] Nut milk, for example, is mixed with other raw materials in the middle of the manufacturing process of a beverage or liquid food. It is preferred to mix the nut milk in the final stage of the manufacturing process, i.e., after mixing with other raw materials and processing (at the stage of becoming the form / shape of the product). In addition, sterilization treatment, seasonings, preservatives, spices, antioxidants, etc. may be added for the purpose of adjusting the taste or maintaining the quality. On the other hand, mixing nut milk with a beverage or liquid food after the manufacturing process is completed (i.e., a substance in the form of a final product rather than an intermediate product) is also a preferred embodiment. In the case of this embodiment, the present invention can be applied without changing the manufacturing process of the beverage or liquid food.

[0082] Example

[0083] 1. Preventing Agglomeration in Coffee

[0084] To 100 mL of commercially available almond milk (manufactured by Rude, protein content 1.5%, raw materials: almonds, water) was added 1 U of protein glutaminase "AMANO" 500 (manufactured by Amano Enzyme, 500 U / g) per 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 at 95°C for 20 minutes, the mixture was cooled to 5°C to produce enzyme-treated almond milk.

[0085] Commercially available instant coffee was dissolved in hot water to prepare a 2% coffee solution. 20-30 mL of enzyme-treated almond milk (pH after addition of enzyme-treated almond milk was 5.7) was added to 150 mL of the coffee solution. No aggregation was observed. As a control, significant aggregation was observed when non-enzyme-treated almond milk was used. Furthermore, experiments were conducted under the same conditions using peanut milk instead of almond milk, yielding the same results (no aggregation occurred in the enzyme-treated peanut milk).

[0086] 2. Relationship between protein concentration of nut milk and aggregation / aggregation prevention effect

[0087] <Non-enzyme treatment>

[0088] (1) Method

[0089] Commercially available almond milk (manufactured by Rude, protein content 1.5%, raw materials: almonds, water) was diluted with tap water to a protein concentration of 0.1, 0.5, and 1.5% (w / v). The mixture was cooled to 5°C, and 5 mL of each was added to 50 mL of coffee solution heated to 90°C to confirm the presence of coagulation.

[0090] (2) Results

[0091] Agglomeration was observed in almond milk at any protein concentration ( Figure 1 ).

[0092] <Enzyme treatment>

[0093] (1) Method

[0094] To commercially available almond milk (manufactured by Rude, protein content 1.5%, raw materials: almonds, water), 1 U of protein glutaminase "AMANO" 500 (manufactured by Amano Enzyme, 500 U / g) was added per 1 g of protein in the almond milk, and the mixture was reacted at 50°C for 5 hours (deamidation reaction). The enzyme was heat-inactivated at 90°C for 15 minutes to produce 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 5 mL of each solution was added to 50 mL of coffee solution heated to 90°C to confirm the presence of aggregation.

[0095] (2) Results

[0096] No aggregation was observed in almond milk at any protein concentration ( Figure 1 ).

[0097] 3. Relationship between liquid pH and coagulation / coagulation prevention effect

[0098] (1) Method

[0099] After adjusting the pH with hydrochloric acid or sodium hydroxide, 15-20 mL of non-enzyme-treated almond milk or enzyme-treated almond milk (protein concentration 1.5% (w / v)) was added to hot water heated to 90°C, and coagulation was confirmed. Enzyme-treated almond milk was prepared using the method described in the experiment in 2. above.

[0100] (2) Results Figure 2 )

[0101] In the case of non-enzyme-treated almond milk, aggregation was observed in the mixed solution after addition at pH 2.5-7.0, while in the case of enzyme-treated almond milk, aggregation was observed in the mixed solution after addition at pH 2.7-4.8.

[0102] 4. Anti-agglomeration effect in various liquids

[0103] 4-1. Black tea

[0104] (1) Method

[0105] Black tea was prepared by pouring boiling water into a commercially available black tea bag (Twining, English Breakfast Tea) and extracting it for 2-3 minutes. The bag was then removed. Non-enzyme-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 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 using the method described in Experiment 2. above.

[0106] (2) Results Figure 3 )

[0107] In the case of non-enzyme-treated almond milk, a little coagulation was observed. As a control, no coagulation was observed in the enzyme-treated almond milk.

[0108] 4-2. Lemon tea

[0109] (1) Method

[0110] Black tea was prepared by pouring boiling water into a commercially available black tea bag (Twining, English Breakfast Tea) and extracting it for 2-3 minutes. The bag was then removed. Lemon juice was added to the tea to adjust the pH. Then, either non-enzymatically treated almond milk or enzymatically treated almond milk (protein concentration 1.5% (w / v)) was added to the tea and the presence of aggregation was checked. The temperature of the tea immediately before the addition of the almond milk was 70°C. The enzymatically treated almond milk was prepared using the method described in Experiment 2. above.

[0111] (2) Results Figure 3 )

[0112] When the pH before adding almond milk was 3.5, aggregation was observed in both non-enzyme-treated and enzyme-treated almond milks. Furthermore, the pH of the black tea after adding non-enzyme-treated almond milk was 3.9, while the pH of the black tea after adding enzyme-treated almond milk was 4.1.

[0113] On the other hand, when the pH before adding almond milk was 4.0, coagulation was observed in the non-enzyme-treated almond milk, but no coagulation was observed in the enzyme-treated almond milk. Furthermore, the pH of the black tea after adding non-enzyme-treated almonds was 4.9, while the pH of the black tea after adding enzyme-treated almond milk was 5.0.

[0114] 4-3. Decaffeinated coffee

[0115] (1) Method

[0116] Boiling hot water was poured into commercially available decaffeinated coffee powder (Nescafe Gold, manufactured by Nestlé) to dissolve it thoroughly, preparing a decaffeinated coffee solution. Non-enzymatically treated almond milk or enzyme-treated almond milk (protein concentration 1.5% (w / v)) was then added to the solution and the presence of aggregation was checked. The decaffeinated coffee solution immediately before the addition of the almond milk was at 80°C and had a pH of 5.3. The pH of the decaffeinated coffee solution after the addition of the almond milk was 5.8. The enzyme-treated almond milk was prepared using the method described in Experiment 2. above.

[0117] (2) Results Figure 3 )

[0118] Agglomeration was observed in non-enzyme treated almond milk, but not in enzyme treated almond milk.

[0119] 4-4. Tomato soup

[0120] (1) Method

[0121] A prescribed amount of boiling water was poured into commercially available chicken bouillon granules (Unilever, Knorr Chicken Cube) to completely dissolve the bouillon granules. After preparing the broth, commercially available tomato paste was added. After adjusting the pH of the tomato broth by varying the amount of tomato paste, either non-enzyme-treated almond milk or enzyme-treated almond milk (protein concentration 1.5% (w / v)) was added and the presence of coagulation was checked. The tomato broth was held at 80°C immediately before the addition of the almond milk. The enzyme-treated almond milk was prepared using the method described in Experiment 2. above.

[0122] (2) Results Figure 3 )

[0123] When the pH before adding almond milk was 5.0, coagulation was observed in the non-enzyme-treated almond milk, but no coagulation was observed in the enzyme-treated almond milk. Furthermore, the pH of the tomato soup after adding the non-enzyme-treated almond milk and the tomato soup after adding the enzyme-treated almond milk were both 5.4.

[0124] When the pH before adding almond milk was 4.0, aggregation was observed in both non-enzyme-treated and enzyme-treated almond milk. Furthermore, the pH of the tomato soup after adding non-enzyme-treated almond milk and the tomato soup after adding enzyme-treated almond milk was 4.0. Furthermore, it is believed that the strong buffering capacity of the citric acid added to the pH adjuster for tomato paste prevented the pH from changing even after the addition of almond milk, and aggregation occurred.

[0125] 5. Anti-agglomeration effect in nut milks other than almond milk

[0126] (1) Method

[0127] Commercially available peanut milk (Rude, protein content 2.0%, raw materials: peanuts, water), cashew milk (PLENISH, protein content 0.9%, raw materials: water, cashews, salt), pistachio milk (Borna Food, protein content 1.0%), and hazelnut milk (Plenish, protein content 0.6%) were added with 1U of protein glutaminase "AMANO" 500 (500U / g, Amano Enzyme Co., Ltd.) per 1g of nut protein and reacted at 50°C for 5 hours (deamidation reaction). After the enzyme reaction, the mixture was immediately inactivated by treating at 90°C for 15 minutes, cooled in running water, and then cooled to 5°C in a refrigerator. Then, 5mL of each solution was added to 50mL of a coffee solution heated to 90°C to check for aggregation.

[0128] (2) Results Figure 4 )

[0129] Aggregation was observed in all of the peanut, cashew, pistachio, and hazelnut milks without enzyme treatment, but no agglomeration was observed with enzyme treatment. This result suggests that similar effects can be achieved with enzyme treatment in nut milks other than almond milk.

[0130] 6. Relationship between liquid temperature and coagulation / coagulation prevention effect

[0131] <Change the coffee temperature (almond milk is kept constant at 5°C)>

[0132] (1) Method

[0133] 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 coagulation was checked.

[0134] (2) Results Figure 5 )

[0135] In the non-enzyme-treated almond milk, aggregation was observed at coffee temperatures above 60°C, and the amount of aggregation increased with increasing temperature. On the other hand, in the enzyme-treated almond milk, an anti-aggregation effect was observed (coffee temperatures of 60°C and 90°C).

[0136] Changing the coffee temperature (almond milk is kept constant at 90°C)

[0137] (1) Method

[0138] 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 coagulation was checked.

[0139] (2) Results Figure 5 )

[0140] Even when 90°C non-enzymatically treated almond milk was added to 90°C coffee, aggregation was observed. Furthermore, when 90°C non-enzymatically treated almond milk was added to 50°C coffee, aggregation was also observed, but no aggregation was observed when 90°C non-enzymatically treated almond milk was added to 40°C coffee. When 90°C non-enzymatically treated almond milk was added to 50°C coffee, it is believed that the coffee temperature temporarily became high, causing aggregation. Furthermore, it is believed that high temperatures after mixing (50°C or above) promote aggregation.

[0141] In case of addition of enzyme treated almond milk (coffee at 40°C or 90°C), no coagulation was observed.

[0142] 7. Study on enzyme treatment conditions (enzyme addition amount, reaction temperature, reaction time)

[0143] (1) Method

[0144] Commercially available almond milk (manufactured by Rude, protein content 1.5%, raw materials: almonds, water) was added with 0.2 U, 1 U, or 5 U of protein glutaminase "AMANO" per 1 g of protein in the almond milk. The reaction was allowed to proceed at a specified temperature (5°C, 15°C, 25°C, 40°C, or 50°C) for 3-24 hours (deamidation reaction). Following the enzymatic reaction, the solution was immediately inactivated by heating at 90°C for 15 minutes. The solution was then cooled in running water and then to 5°C in a refrigerator. Then, 5 mL of each solution was added to 50 mL of coffee solution heated to 90°C to confirm the presence of aggregation.

[0145] (2) Results Figure 6 )

[0146] It is known that the effect varies according to enzyme addition, reaction temperature, and reaction time, but coagulation can be prevented by adjusting the above-mentioned conditions. Specifically, if the enzyme addition is increased or the reaction time (or both) is prolonged when the reaction temperature is low, the desired effect can be obtained. For example, even if the reaction temperature is 5°C, when the enzyme addition is more than 1U or when the reaction is long, coagulation can be effectively prevented. On the other hand, if the reaction time is short, the reaction temperature is raised or the enzyme addition is increased (or both), the desired effect can be obtained. For example, even if the reaction time is 3 hours, when the reaction temperature is set to more than 40°C or when the enzyme addition is set to more than 1U, the coagulation prevention effect can be obtained. In addition, if the reaction temperature is raised or the reaction time (or both) is prolonged, the enzyme addition can be reduced. For example, if the reaction temperature is set to more than 25°C or when the reaction time is long, the enzyme addition can be set to below 0.2U.

[0147] Conclusion

[0148] The same anti-aggregation effect was observed regardless of the nut protein concentration within the range of 0.1% to 1.5% (w / v). This indicates that enzymatic treatment with protein deamidase is effective in preventing aggregation in nut milks with various protein concentrations, demonstrating its high versatility.

[0149] While it depends on the type of liquid with which the nut milk is mixed, a trend is that without enzymatic treatment using protein deamidase, aggregation occurs when the pH reaches 7 or below after mixing with the nut milk. However, with enzymatic treatment, the lower limit for aggregation can be extended to pH 5. If the pH of the liquid after mixing with nut milk is 5 or above, it is possible to use it in acidic liquid foods such as sour dairy soups, in addition to beverages like coffee and black tea. Furthermore, if the pH of the liquid after mixing with milk is 5 or above, lemon milk tea, which is difficult to prepare even with cow's milk, can be prepared, making it applicable to various beverages and liquid foods using sour fruits.

[0150] First, the pH of the liquid used to mix the nut milk has a significant impact on coagulation. Second, the higher the temperature of the liquid, the easier it is to coagulate.

[0151] The effect varies depending on the amount of enzyme added (enzyme concentration), reaction temperature, and reaction time.

[0152] The same effect was observed not only in almond milk but also in peanut milk, cashew milk, pistachio milk, and hazelnut milk. Therefore, it is believed that enzymatic treatment with protein deamidase is effective in preventing the aggregation of nut milks in general.

[0153] Industrial applicability

[0154] The present invention provides a nut milk that exhibits excellent dispersibility even without the use of additives such as emulsifiers. This high dispersibility enhances the value of the nut milk itself and beverages and liquid foods containing it. Furthermore, it enables the provision of new beverages and liquid foods that were previously unattainable.

[0155] The nut milk provided by the present invention is not limited to existing uses and is expected to be used or applied in a variety of applications (particularly acidic beverages and acidic liquid foods). The absence of additives such as emulsifiers is a significant advantage of the present invention. Furthermore, even when the nut milk is added to coffee as a substitute for milk, soy milk, etc., no special procedures are required to prevent coagulation, thereby improving consumer convenience.

[0156] The embodiments and examples of the invention described above do not limit the present invention in any way. Various modifications are encompassed by the present invention within the scope of the claims and readily conceivable by those skilled in the art. The contents of papers, published patent publications, patent publications, and the like explicitly mentioned in this specification are incorporated by reference in their entirety.

Claims

1. Use of nut milk obtained by treatment with a protein deamidase, and mixing it with a beverage or liquid food to produce a nut milk-containing beverage or nut milk-containing liquid food, wherein the nut milk-containing beverage or nut milk-containing liquid food has a pH of 5 to 7 and does not cause protein aggregation.

2. The use according to claim 1, wherein The raw nuts of 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.

3. The use according to claim 1 or 2, wherein The nut protein concentration of the nut milk is 0.2% w / v to 10.0% w / v.

4. The use according to claim 1 or 2, wherein The dispersibility of the nut milk in beverages or liquid foods is improved by the treatment.

5. The use according to claim 1 or 2, wherein The beverage or liquid food is selected from the group consisting of coffee, coffee beverages, tea, tea beverages, juice, juice beverages, sports drinks, nutritional supplement beverages, soups, curry, cocoa and chocolate beverages.

6. The use according to claim 1 or 2, wherein The nut milk does not contain emulsifiers and thickening polysaccharides to prevent coagulation.

7. The use according to claim 1 or 2, wherein The protein deamidase is an enzyme derived from a microorganism of the genus Chryseobacterium.

8. The use according to claim 7, wherein The microorganism of the genus Chryseobacterium is Chryseobacterium utilis.

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