Prevention of coagulation of vegetable milk
Patent Information
- Authority / Receiving Office
- JP · JP
- Patent Type
- Applications
- Current Assignee / Owner
- AMANO ENZYME INC
- Filing Date
- 2024-12-27
- Publication Date
- 2026-04-22
Abstract
Description
[Technical field]
[0001] The present invention relates to plant-based milk. More specifically, the present invention relates to plant-based milk with improved dispersibility (less prone to clumping) and its uses. This application claims priority based on Japanese Patent Application No. 2019-029904 filed on February 21, 2019, and Japanese Patent Application No. 2019-077841 filed on April 16, 2019, 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 coagulate when added to high-temperature acidic beverages such as coffee and tea as a substitute for cow's milk. Normally, this type of coagulation does not occur in cow's milk, and is a phenomenon unique to plant-based milks such as nut milk.
[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 high-temperature 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 plant-based milk such as nut milk, reduces the value (utility value, commercial value, etc.) of plant-based 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 plant-based milk and promote its use and application, the present invention aims to create an effective means for preventing aggregation of plant-based milk, and in particular to provide plant-based milk that is less likely to aggregate when used for high-temperature liquid beverages (particularly acidic liquid beverages) and high-temperature liquid foods (particularly acidic liquid foods) without the addition of additives. [Means for solving the problem]
[0009] In light of the above problems and through repeated investigations, the present inventors have focused on protein deamidation and attempted to improve the dispersibility of vegetable milk when added to high-temperature beverages or liquid foods by treating the same with protein deamidating enzymes. However, there have been no reports to date of the use of vegetable milk treated with protein deamidating enzymes in high-temperature beverages or liquid foods.
[0010] First, in regard to the addition of nut milk to hot coffee, which is one of the typical uses of nut milk, we investigated whether treatment with protein deamidase is 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 as a result, 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 nut milk with improved dispersibility and less aggregation when added to high-temperature beverages or liquid foods without the use of additives such as emulsifiers was successfully prepared. In addition, many findings that are useful for using nut milk in various beverages and foods were obtained. In addition, it was revealed that treatment with protein deamidase is effective in preventing aggregation when added to high-temperature beverages or liquid foods in plant-based milks other than nut milk, such as soy milk, oat milk, pea milk, and hemp milk, when added to high-temperature beverages or liquid 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 having excellent shearing force, such as a high-pressure homogenizer, and is completely different from vegetable 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 vegetable milk. [1] Plant-based milk that has been treated with protein deamidating enzyme to prepare a high-temperature plant-based milk-containing liquid food or beverage. [2] The plant-based milk described in [1], wherein the plant-based milk is nut milk, soy milk, pea milk, oat milk or hemp milk. [3] The plant-based milk described in [2], wherein the nuts used to make the nut milk 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. [4] The plant-based milk according to any one of [1] to [3], wherein the raw material plant protein concentration is 0.2% (w / v) to 10.0% (w / v). [5] The plant-based milk described in any one of [1] to [4], whose dispersibility is improved by the treatment. [6] The plant-based milk described in [5], which does not cause protein aggregation when mixed with a weakly acidic to weakly alkaline liquid food or beverage (provided that the pH of the mixed liquid is 5 or higher). [7] The plant-based milk described in [6], wherein the liquid food or beverage has a pH of 5 to 7. [8] The plant-based milk described in [6], wherein the liquid food or beverage is a raw material, intermediate product or final product of a beverage or liquid food selected from the group consisting of coffee, coffee beverages, tea, tea beverages, fruit juice, fruit juice beverages, sports drinks, nutritional supplements, soup, curry, cocoa and chocolate beverages. [9] A plant-based milk described in any one of [1] to [8], which does not contain emulsifiers or thickening polysaccharides to prevent aggregation.
[10] The plant-based milk described in any one of [1] to [9], wherein the protein deamidase is an enzyme derived from a microorganism of the genus Chryseobacterium.
[11] The plant-based milk described in
[10] , wherein the Chryseobacterium microorganism is Chryseobacterium proteolyticum.
[12] A method for producing vegetable milk with improved dispersibility, comprising treating vegetable milk with protein deamidating enzyme.
[13] The method according to
[12] , comprising the following steps (1) and (2): (1) preparing plant-based milk; (2) A step of treating the plant-based milk prepared in (1) with protein deamidating enzyme.
[14] The method of manufacturing according to
[13] , wherein the plant-based milk in step (1) is plant-based milk before heat sterilization.
[15] The method according to
[14] , further comprising the step (3): (3) A step of heat treatment.
[16] A liquid food or beverage containing the plant-based milk described in any one of [1] to
[11] .
[17] The liquid food or beverage described in
[16] , which has a pH of 5 or higher.
[18] The liquid food or drink according to
[16] , which is a beverage or liquid food selected from the group consisting of coffee beverages, coffee whiteners, tea beverages, fruit juice beverages, sports drinks, nutritional supplements, soups, curries, cocoa beverages and chocolate beverages.
[19] A method for producing a liquid food or beverage, comprising mixing vegetable milk treated with protein deamidating enzyme under high temperature conditions with a raw material, intermediate product or final product of the liquid food or beverage.
[20] The method according to
[19] , comprising the following steps (1) and (2): (1) providing a plant-based milk treated with protein deamidating enzyme; (2) A step of mixing the plant-based milk prepared in (1) with a liquid food or beverage raw material, intermediate product, or final product under high temperature conditions. [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)). [Figure 7] Summary of experimental results (anti-coagulation effect in soy milk). [Figure 8] Summary of experimental results (anti-coagulation effect in plant-based milk). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0012] 1. Plant-based milk with improved dispersibility A first aspect of the present invention relates to a plant-based milk (also called a plant protein-containing beverage) with improved dispersibility when added to a hot liquid food or beverage (beverage or liquid food). The term "liquid food or beverage" includes not only final products but also those used as ingredients for other foods and beverages, or intermediate products. The plant-based milk of the present invention is treated with protein deamidating enzyme, and as a result of the treatment, its dispersibility is improved. Since the plant-based milk of the present invention exhibits excellent dispersibility when added to a hot liquid food or beverage, it is unlikely to aggregate when added to a hot beverage such as coffee or tea, even without the use of additives for increasing dispersibility (e.g., emulsifiers, thickening polysaccharides (pectin, carboxymethylcellulose, etc.), salts). This characteristic allows it to be used in a variety of beverages and foods.
[0013] The vegetable milk is, for example, milk made from various nuts, soybeans, oats, peas, hemp, lupine beans, broad beans, chickpeas, barley, wheat, rice, barnyard millet, millet, canary seeds, teff, keanu, or flaxseed. Nut milk (also called nut protein-containing beverage), typified by almond milk, is a vegetable milk made from nuts, and is generally prepared by processes such as crushing, soaking / dissolving, mixing / stirring, filtering, homogenizing, and sterilization of de-shelled nuts. The preparation method of the nut milk used in the present invention is not particularly limited. The nuts used as the raw material for the nut milk are not particularly limited. 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. In addition, vegetable milk provided by a raw material manufacturer or purchased commercially may be used in the present invention.
[0014] The plant-based milk of the present invention can be obtained by treating plant-based milk with protein deamidating enzyme to improve its dispersibility. Hereinafter, for convenience of explanation, the plant-based milk to be subjected to the treatment with protein deamidating enzyme is referred to as "untreated plant-based milk".
[0015] Unprocessed plant-based milk made from a combination of two or more types of raw plant material (for example, a combination of almonds and cashew nuts or a combination of almonds and peanuts) can also be used.
[0016] The protein concentration in the untreated plant milk (raw material plant protein concentration) is not particularly limited, but untreated plant 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. The protein concentration of the plant 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), and more preferably 0.2% (w / v) to 5.0% (w / v).
[0017] 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.
[0018] 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.
[0019] 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.
[0020] 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.)
[0021] As long as it is effective in improving the dispersibility of plant-based milk, the conditions for the treatment with protein deamidating enzyme are not particularly limited, and optimal reaction conditions can be set by adjusting the reaction temperature, reaction time, and amount of enzyme added (enzyme concentration).
[0022] 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. Furthermore, 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" refers to the number of units per g of substrate plant protein. As mentioned above, the protein concentration in the untreated plant-based milk is not particularly limited, but untreated plant-based 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.
[0023] 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.
[0024] 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.
[0025] As described above, the plant-based milk of the present invention has excellent dispersibility when added to a liquid food or drink at a high temperature, and is less likely to cause protein aggregation. The high temperature in the present invention is not particularly limited as long as it is high enough to cause protein aggregation in the plant-based milk by heat, but is, for example, 50°C or higher, preferably 60°C or higher, more preferably 70°C or higher, more preferably 80°C or higher, and most preferably 90°C or higher. The upper limit of the high temperature is, for example, 100°C. The temperature of the plant-based milk is not particularly limited, but it is preferable that the temperature of the liquid food or drink after mixing with the plant-based milk is the above-mentioned high temperature condition (i.e., for example, 50°C or higher). Typically, when mixed (added) to a liquid food or drink that is weakly acidic (3≦pH<6) to weakly alkaline (8≦pH<11) (provided that the pH of the mixed liquid is 5 or higher), protein aggregation does not occur. The pH of the liquid food or drink after mixing at which protein aggregation does not occur is, for example, 5 to 10, preferably 5 to 9, and more preferably 5 to 7. The liquid food and beverages (beverages, liquid foods) with which the plant-based milk of the present invention is mixed are not particularly limited, and examples include coffee, coffee beverages, tea (black tea, green tea, oolong tea, etc., including those obtained by reducing the extract and those obtained by reducing the extract after processing (e.g. concentrating, freeze-drying)), tea beverages (flavored tea, milk tea, tea beverages containing fruit juice, etc.), fruit juice, fruit juice beverages, sports drinks, nutritional supplements (protein drinks, nutritional drinks for nursing care, etc.), soups (bouillon-based soups, stews, chowder, borsch, vegetable soups (e.g. tomato soup, corn soup, potage, pumpkin soup), miso soup), curry, cocoa, and chocolate beverages.
[0026] In a preferred embodiment of the present invention, taking advantage of the characteristics of excellent dispersibility and resistance to protein aggregation, the product does not contain emulsifiers (glycerin fatty acid esters, sucrose fatty acid esters, lecithin, saponin, etc.), thickening polysaccharides (pectin, carboxymethylcellulose, etc.), salts (seasalt, calcium salts, phosphates, etc.) or the like for preventing aggregation. In particular, the product does not contain emulsifiers or thickening polysaccharides. In this way, the present invention provides a plant-based milk that meets consumer needs 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.
[0027] As is apparent from the above description, the plant-based milk of the present invention can be produced by treating untreated plant-based milk with protein deamidating enzyme. Thus, the plant-based milk of the present invention can typically be obtained by a production method including the following steps (1) and (2). (1) preparing plant-based milk; (2) A step of treating the plant-based milk prepared in (1) with protein deamidating enzyme.
[0028] Step (2), i.e., the treatment with protein deamidase, may be carried out either before or after the heat sterilization of the plant-based milk. However, in order to simplify the production process, it is preferable to carry out this step before the heat sterilization of the plant-based milk, and then to carry out a heat sterilization step that also inactivates the protein deamidase (in other words, step (2) may be incorporated into the production process of the plant-based milk). Therefore, in a preferred embodiment, "(3) a heat treatment step" is carried out after step (2). The conditions for the heat treatment are not particularly limited as long as they allow the inactivation of protein deamidase and the sterilization of the plant-based milk. For example, the treatment is carried out at a temperature of 70°C to 150°C for 1 second to 5 hours.
[0029] 2. Uses of plant-based milk The second aspect of the present invention relates to uses of the plant-based milk of the present invention. The plant-based milk of the present invention has excellent dispersibility when mixed with liquid food and beverages under high temperature conditions, and is less likely to cause protein aggregation. That is, the plant-based milk of the present invention is used to prepare high-temperature liquid food and beverages containing plant-based milk. Due to this characteristic, it is suitable for use in various beverages and liquid foods. That is, various beverages and liquid foods containing the plant-based milk of the present invention are provided.
[0030] 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 that occurs when vegetable milk is mixed with high-temperature beverages, liquid foods, etc. depends on the pH of the beverage after mixing with vegetable milk, and protein aggregation does not occur at a pH of 5 or higher. In view of this finding, the pH of liquid foods and beverages containing the vegetable milk of the present invention is preferably 5 or higher. More specifically, the pH of liquid foods and beverages containing the vegetable milk of the present invention is preferably 5 to 9, more preferably 5 to 8, and even more preferably 5 to 7.5.
[0031] Examples of liquid foods and drinks (drinks or liquid foods) include coffee drinks, coffee whitener (usage for other things than coffee, such as black tea, is also envisioned), tea drinks (flavored tea, milk tea, tea drinks containing fruit juice, etc.), fruit juice drinks, sports drinks, nutritional supplement drinks (protein drinks, nutritional drinks for nursing care, etc.), various soups, curries, cocoa drinks, and chocolate drinks. As can be seen from these examples, the present invention can be used not only for neutral drinks and liquid foods, but also for weakly acidic drinks and liquid foods.
[0032] For example, the vegetable milk is mixed with other raw materials during the manufacturing process of the liquid food or beverage. Preferably, the vegetable milk is mixed at the final stage of the manufacturing process, i.e., after the other raw materials have been mixed and processed (the stage at which the product has taken on the form / shape). However, sterilization treatment and the addition of seasonings, preservatives, flavorings, antioxidants, etc. for the purpose of adjusting the taste and maintaining the quality may be performed thereafter. On the other hand, it is also a preferred embodiment to mix the vegetable milk with the liquid food or beverage after the manufacturing process has been completed (i.e., in the form of a final product rather than an intermediate product). In this embodiment, the present invention can be applied without changing the manufacturing process of the liquid food or beverage.
[0033] As is apparent from the above explanation, the vegetable milk-containing liquid food or drink of the present invention can be produced by mixing vegetable milk treated with protein deamidating enzyme with a raw material, intermediate product, or final product of the liquid food or drink under high temperature conditions. Thus, the liquid food or drink of the present invention can typically be obtained by a production method including the following steps (1) and (2). (1) preparing a plant-based milk treated with a protein deamidating enzyme; (2) Mixing the plant-based milk prepared in (1) with a liquid food or beverage raw material, intermediate product, or final product under high temperature conditions. 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.
[0062] 8. Preventing clumping in soy milk Soy milk, which has a unique flavor and nutrition, is widely used not only as a substitute for cow's milk, but also as an ingredient or additive in various foods and beverages. Soy milk with improved dispersibility is expected to improve the quality of existing applications and also to be used in new applications. Therefore, we investigated whether treatment with protein deamidase is effective in preventing the aggregation of soy milk.
[0063] (1) Method Protein glutaminase "Amano" 500 (Amano Enzyme, 500U / g) was added at 5U or 15U per 1g of soy protein to commercially available soy milk (manufactured by Sojasun, product name "SOJA NATURE SANS SUCRE", protein content 3.6% (w / w), raw materials: soybeans, water) and reacted at 50°C for 5 hours (deamidation reaction). After the enzyme reaction, the enzyme was immediately inactivated by treatment at 90°C for 15 minutes, cooled in running water, and then cooled to 5°C in a refrigerator. 15mL of each was added to 150mL of coffee solution heated to 90°C to check for the presence or absence of aggregation.
[0064] (2) Results (Figure 7) Aggregation was observed without enzyme treatment, but no aggregation was observed with enzyme treatment. This result indicates that the aggregation prevention effect can be obtained by enzyme treatment even in soy milk.
[0065] Treatment with protein deamidase was also effective in preventing aggregation in soy milk. Thus, similar to nut milk, treatment with protein deamidase can prepare soy milk with improved dispersibility (i.e., less prone to aggregation). Soy milk with improved dispersibility can be used for applications where untreated soy milk cannot be used (or is not suitable for use) due to aggregation. In light of the above experimental results, the conditions for treatment with protein deamidase can be the same as those for nut milk.
[0066] 9. Prevents clumping in other plant-based milks In addition to soy milk, various plant-based milks with unique flavors and nutrients are widely used not only as substitutes for cow's milk, but also as ingredients and additives in various foods and beverages. Plant-based milks with improved dispersibility are expected to improve the quality of existing applications and also to be used in new applications. Therefore, we investigated whether treatment with protein deamidase is effective in preventing aggregation of various plant-based milks.
[0067] (1) Method Protein glutaminase "Amano" 500 (Amano Enzyme, 500U / g) was added at 1U or 5U per 1g of protein to commercially available pea milk (Mihgtysociety, protein content 3.2% (w / w)), oat milk (liquatsvegetals, protein content 1.4% (w / w)), and hemp milk (Ecomil, protein content 1.0% (w / w)), 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. 15mL of each was added to 150mL of coffee solution heated to 90°C to check for the presence or absence of aggregation.
[0068] (2) Results (Figure 8) Aggregation was observed without enzyme treatment, but no aggregation was observed with enzyme treatment. This result indicates that the anti-aggregation effect can be achieved with various plant-based milks by enzyme treatment.
[0069] Treatment with protein deamidase was also effective in preventing aggregation in various plant-based milks. Thus, similar to nut milk, various plant-based milks with improved dispersibility (i.e., less prone to aggregation) can be prepared by treatment with protein deamidase. Various plant-based milks with improved dispersibility can be used for applications where untreated plant-based milks cannot be used (or are not suitable for use) due to aggregation. In light of the above experimental results, the conditions for treatment with protein deamidase can be the same as those for nut milk. [Industrial Applicability]
[0070] The present invention provides a plant-based milk that has excellent dispersibility when added to high-temperature liquid food and beverages (beverages or liquid foods) without the use of additives such as emulsifiers. High dispersibility enhances the value of the plant-based milk itself and liquid food and beverages that use the plant-based milk. It also makes it possible to provide new liquid food and beverages that have not been possible in the past.
[0071] The plant-based 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 significant advantage of the present invention. In addition, even when plant-based milk is added to high-temperature coffee as a substitute for cow's milk, no special operation is required to prevent clumping, improving convenience for consumers.
[0072] 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. A plant-based milk used as an additive to liquid food and beverages at temperatures of 60°C or higher, The aforementioned liquid food and beverage has a pH of 5 to 10 after the addition of plant-based milk. The aforementioned plant-based milk is a plant-based milk that has been treated with protein glutaminase.
2. The plant-based milk according to claim 1, wherein the plant-based milk is nut milk, soy milk, pea milk, oat milk, or hemp milk.
3. The plant-based milk according to claim 2, wherein the nuts used as raw materials for the nut milk are one or more nuts selected from almonds, cashews, hazelnuts, pecans, macadamia nuts, pistachios, walnuts, Brazil nuts, peanuts, coconuts, chestnuts, sesame seeds, and pine nuts.
4. The plant-based milk according to any one of claims 1 to 3, wherein the raw material plant protein concentration is 0.2% (w / v) to 10.0% (w / v).
5. The plant-based milk according to any one of claims 1 to 4, wherein the dispersibility is improved by the above-mentioned process.
6. The plant-based milk according to claim 5, wherein protein aggregation does not occur when mixed with a weakly acidic to weakly alkaline liquid food or beverage (provided the pH of the mixture is 5 or higher).
7. The plant-based milk according to claim 6, wherein the pH of the liquid food or beverage is 5 to 7.
8. The plant-based milk according to any one of claims 1 to 7, wherein the liquid food or beverage is selected from the group consisting of coffee, coffee beverages, tea, tea beverages, fruit juice, fruit juice beverages, sports drinks, nutritional supplements, soups, curries, cocoa, and chocolate beverages, and is an ingredient, intermediate product, or final product of a beverage or liquid food.
9. A plant-based milk according to any one of claims 1 to 8, which does not contain emulsifiers and thickening polysaccharides for preventing coagulation.
10. The plant-based milk according to any one of claims 1 to 9, wherein the protein glutaminase is an enzyme derived from a microorganism of the genus Chryseobacterium.
11. The plant-based milk according to claim 10, wherein the microorganism of the genus Chryseobacterium is Chryseobacterium proteoricum.
12. A liquid food or beverage containing plant-based milk, obtained by adding the plant-based milk described in any one of claims 1 to 11 to a liquid food or beverage at a temperature of 60°C or higher, and having a pH of 5 to 10.
13. The liquid food or beverage according to claim 12, wherein the pH of the plant-based milk-containing liquid food or beverage is 5 to 9.
14. The liquid food or beverage according to claim 12 or 13, 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.
15. A method for producing a liquid food containing plant milk, comprising mixing plant-based milk treated with protein glutaminase with a raw material, intermediate product, or final product of a liquid food or beverage at a temperature of 60°C or higher to prepare a liquid food or beverage containing plant milk with a pH of 5 to 10.
16. The manufacturing method according to claim 15, comprising the following steps (1) and (2): (1) Steps to prepare plant-based milk treated with protein glutaminase, (2) A step of mixing the plant-based milk prepared in (1) with a raw material, intermediate product, or final product of a liquid food or beverage at 60°C or higher to obtain a liquid food or beverage containing plant-based milk with a pH of 5 to 10.