Modified legume proteins with improved colloidal stability

Abstract

This invention relates to a novel method for obtaining aqueous dispersions of modified legume proteins with improved colloidal stability using protein deamidases.

Description

[0001] References to sequence lists This application contains a sequence list in a computer-readable form, which is incorporated herein by reference. Technical Field

[0002] This invention relates to the use of protein deamidases and heat treatment in a method for obtaining an aqueous dispersion of modified legume protein with improved colloidal stability. Background Technology

[0003] In recent years, there has been an increase in the number of people pursuing vegan, vegetarian, or non-dairy diets for health and other reasons. Furthermore, animal-derived foods (especially dairy cows) are increasingly attracting attention due to their high environmental costs. These factors are driving a growing demand for dairy alternatives—plant-based foods (including milk, non-dairy creamer, cheese, yogurt, and ice cream)—that replace traditionally milk-based foods.

[0004] Legumes are considered a valuable and low-cost source of high-quality protein products, such as concentrates and isolates. Industrial-scale production typically uses soybeans as a protein source, while pea protein (such as pea protein isolates) can be seen as a promising alternative to soybean-derived protein products.

[0005] Plant proteins (including legume proteins) typically have lower solubility and poorer functional properties, and may require extensive modification to achieve industrially viable products that meet consumer demands in terms of taste, texture, and appearance. Such extensive modification can pose significant challenges for manufacturers of plant proteins and plant-derived foods, especially when it comes to obtaining plant-based foods that meet consumer clean label requirements while maintaining low manufacturing costs.

[0006] Deamidation is known to improve the solubility of plant proteins, thereby enhancing their functional properties, including foaming activity, foaming stability, emulsifying activity, and emulsifying stability. Deamidation can be chemical or enzymatic; in the latter case, protein deamidases, in particular, can provide an effective means of modifying plant proteins without impairing the sensory or technical functional properties of the modified proteins.

[0007] EP 4 201 216 A1 describes the treatment of plant milk with protein deamidases to prevent coagulation when added to hot liquid beverages, hot liquid foods, etc. Therefore, EP 4 201 216 A1 relates to preventing aggregation of plant milk when mixed with heated liquid foods or beverages (such as coffee). EP 4 201 216 A1 does not describe the use of protein deamidases and heat treatment for improving the colloidal stability of aqueous dispersions of modified legume proteins.

[0008] US 2023 / 0345970 A1 describes deamidated pea protein isolates, methods for preparing deamidated pea protein isolates, and the use of deamidated pea protein isolates in food and beverage compositions. US 2023 / 0345970 A1 does not relate to the use of protein deamidases and heat treatment for improving the colloidal stability of aqueous dispersions of modified legume proteins.

[0009] In view of the above, the object of the present invention is to discover an improved method for obtaining an aqueous dispersion of modified legume protein with improved colloidal stability. Summary of the Invention

[0010] The inventors of this invention unexpectedly discovered that by treating an aqueous solution of legume protein isolate with a protein deamidase, followed by heat treatment, a modified aqueous dispersion of legume protein is obtained. This dispersion exhibits improved colloidal stability, dispersibility, and reduced risk of flocculation, even during storage. This results in a stable, sensorily pleasing modified aqueous dispersion of legume protein with the added benefit of being particularly suitable for mixing with other beverages, such as acidic beverages. Therefore, a modified aqueous dispersion of legume protein with improved solubility and mouthfeel, as well as improved beverage stability, can be obtained.

[0011] Therefore, the modified legume protein claimed in this paper not only meets consumers' demand for satisfactory sensory properties, but also achieves storage and beverage stability.

[0012] Therefore, the present invention provides a method for obtaining an aqueous dispersion of modified legume protein, the method comprising the following steps: (a) Provide an aqueous solution of legume protein isolate with a protein content in the range of 1%-20% (w / w); (b) Treating the aqueous solution from step (a) with a protein deamidase to obtain enzymatically deamidated legume protein; and (c) The enzymatically deamidated legume protein from step (b) is subjected to heat treatment to obtain an aqueous dispersion of the modified legume protein. In steps (b) and (c), the pH is in the range of pH 6-8, and the legume protein isolate is derived from or obtained from peas, soybeans, broad beans, lentils, or any combination thereof.

[0013] By using the methods claimed and disclosed herein, an aqueous dispersion of modified legume protein was obtained, which, after being stored at a temperature of at least 4°C for 15 minutes, exhibited improved colloidal stability compared to an aqueous dispersion of modified legume protein prepared using a similar method but without the use of protein deamidase and heat treatment.

[0014] In the context of this invention, it is necessary to distinguish between colloidal stability and coffee stability, as these two terms refer to different aspects of beverage formulation and preservation. Colloidal stability refers to the ability of a colloidal system (such as an emulsified beverage or suspension) to remain uniformly dispersed over time without separation of its constituent phases. The method of this invention produces an aqueous dispersion of modified legume protein with improved colloidal stability. This means that even after prolonged storage, the aqueous dispersion remains uniformly mixed, and the protein particles do not settle or aggregate. The improvement in colloidal stability is achieved through enzymatic deamidation and subsequent heat treatment, which ensures that the protein particles remain well dispersed in solution. On the other hand, coffee stability specifically relates to the ability of an aqueous dispersion of legume protein to remain stable when mixed with other beverages, such as coffee or tea, preferably hot coffee or tea. Unlike colloidal stability, coffee stability focuses on maintaining uniformity and preventing separation or coagulation when the aqueous dispersion of legume protein is combined with acidic beverages.

[0015] Preferably, the enzymatic treatment with protein deamidase is carried out at a temperature ranging from 50°C to 70°C for 15-90 minutes. In the examples, the treatment with protein deamidase is carried out at a temperature ranging from 60°C to 70°C for 30-60 minutes. (See Example 3 and...) Figure 3 and Figure 4 As shown, the method disclosed herein for obtaining aqueous dispersions of legume proteins with improved colloidal stability can be carried out with shorter enzyme incubation times and higher enzyme incubation temperatures. These improvements translate to shorter downtime, reduced energy consumption, and the ability to produce high-quality products more efficiently.

[0016] The modified legume protein obtained according to the method of the present invention exhibits improved colloidal stability, which is achieved without the need for the addition of emulsifiers and / or stabilizers and without adjusting the pH outside the pH range of 6-8. Therefore, a modified legume protein is obtained that requires minimal processing and can be labeled as a clean label product. Thus, in some preferred embodiments, emulsifiers and / or stabilizers are absent in any of steps (a), (b), and / or (c).

[0017] Using the method of the present invention, an aqueous dispersion of modified legume protein can be obtained, which exhibits improved immediate and long-term colloidal stability, as well as improved stability when mixed with acidic beverages. Therefore, the present invention further relates to an aqueous dispersion of modified legume protein in the pH range of 6-8, wherein the modified legume protein is obtained by treatment with a protein deamidase followed by a heating step, and wherein the legume protein is derived from or obtained from legume protein isolates of peas, soybeans, broad beans, lentils, or any combination thereof.

[0018] In particular, the inventors of this invention have discovered that the modified legume protein aqueous dispersion according to the invention exhibits anti-flocculation properties immediately after its preparation, as well as after storage under refrigeration and at room temperature for several days or weeks. Therefore, it is possible to obtain a modified legume protein aqueous dispersion possessing functional properties required and desired by consumers, including creaminess, mouthfeel, and stability.

[0019] Therefore, modified legume protein aqueous dispersions with functional properties that consumers need and desire, including creaminess, mouthfeel, and stability, can be obtained. Thus, the modified legume protein aqueous dispersions disclosed herein and obtained using the methods of the present invention not only possess satisfactory sensory properties but can also be used to obtain healthier, clean-label dairy alternatives that are high in protein and low in added fat and sugar.

[0020] This invention also provides the use of protein deamidases in the production of aqueous dispersions of modified legume proteins to improve colloidal stability.

[0021] The present invention also provides the use of protein deamidase and heat treatment in the production of aqueous dispersions of modified legume proteins to improve colloidal stability. Attached Figure Description

[0022] Figure 1 The colloidal stability of legume-based suspensions obtained using the methods disclosed herein, with or without the addition of protein deamidases, is demonstrated.

[0023] Figure 2 The colloidal stability of legume-based suspensions prepared with or without heat treatment steps was demonstrated.

[0024] Figure 3 The colloidal stability of legume-based suspensions obtained with or without protein deamidases and / or heat treatment was demonstrated.

[0025] Figure 4The colloidal stability of legume-based suspensions obtained with or without protein deamidases and / or heat treatment was demonstrated.

[0026] sequence SEQ ID NO: 1: Derived from Visceral Chlorella ( Chryseobacterium viscerum Protein deamidase (strain formerly known as Chrysobacterium) Chryseobacterium (Species-62563), which has a mature polypeptide sequence as shown in SEQ ID NO: 2.

[0027] SEQ ID NO: 2: Mature polypeptide sequence of a protein deamidase derived from species-62563 of the genus *Aureobacterium*.

[0028] SEQ ID NO: 3: Derived from *Cyclophorus pyogenes* ( Chryseobacterium proteolyticum The protein deamidase has a mature polypeptide sequence as shown in SEQ ID NO: 4.

[0029] SEQ ID NO: 4: Mature polypeptide sequence of protein deamidase derived from Chrysobacterium utilis.

[0030] SEQ ID NO: 5: Derived from *Aureobacillus brevis* ( Chryseobacterium gambrini The protein deamidase has a mature polypeptide sequence as shown in SEQ ID NO: 6.

[0031] SEQ ID NO: 6: Mature polypeptide sequence of a protein deamidase derived from *Cyclophorus brevis*.

[0032] SEQ ID NO: 7: Derived from Culex pipiens lucida ( Chryseobacterium culicis The protein deamidase has a mature polypeptide sequence as shown in SEQ ID NO: 8.

[0033] SEQ ID NO: 8: Mature polypeptide sequence of protein deamidase derived from Culex pipiens.

[0034] SEQ ID NO: 9: Originating from sewage-borne *Cyclocarya paliurus* ( Chryseobacterium defluvii The protein deamidase has a mature polypeptide sequence as shown in SEQ ID NO: 10.

[0035] SEQ ID NO: 10: Mature polypeptide sequence of protein deamidase derived from *Cryptospirobacter septicemia*. Detailed Implementation

[0036] Based on this detailed description, the following definitions apply. Note that the singular forms “a / an” and “the” include plural indicators unless the context explicitly indicates otherwise.

[0037] This document uses the term "about" to refer to a value or parameter, including aspects of that value or parameter itself. For example, a description of "about X" includes aspect "X". When used in conjunction with a measurement, "about" includes at least a range covering the uncertainty associated with the method of measuring that particular value, and may include a range of two standard deviations positive or negative around the said value.

[0038] Unless otherwise defined or explicitly indicated by the context, all percentages are weight percentages (percentage w / w or "% (w / w)").

[0039] The accompanying drawings are for illustrating certain convenient embodiments of the invention and should not be construed as limiting the invention.

[0040] Furthermore, it will be understood that, unless otherwise stated, the embodiments described in connection with one of the aspects described herein can also be applied to the other aspects.

[0041] Unless otherwise defined or explicitly indicated by the context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0042] This invention is based on the unexpected and inventive discovery that, even after storage or mixing with acidic beverages, aqueous dispersions of modified legume proteins can be obtained by treating legume protein isolates with a protein deamidase followed by a heat treatment step. These dispersions exhibit improved colloidal stability, dispersibility, and a reduced risk of flocculation. The result is stable, for example, shelf-stable, and sensorily satisfactory modified legume proteins. When provided as aqueous dispersions of modified legume proteins, these proteins offer the added benefit of being particularly suitable for mixing with other beverages, such as acidic beverages.

[0043] The unexpected findings reported in this article are illustrated in Examples 2 and 3, and at least Figure 1-4 As explained herein, and based on these general observations, the inventors of the present invention anticipate that the same benefits can be obtained from any suitable legume protein isolate derived from or obtained from peas, soybeans, broad beans, lentils, or any combination thereof using the methods of the present invention.

[0044] In a first aspect, the present invention relates to a method for obtaining an aqueous dispersion of modified legume protein, the method comprising the steps of: (a) Provide an aqueous solution of legume protein isolate with a protein content in the range of 1%-20% (w / w); (b) Treating the aqueous solution from step (a) with a protein deamidase to obtain enzymatically deamidated legume protein; and (c) The enzymatically deamidated legume protein from step (b) is subjected to heat treatment to obtain an aqueous dispersion of the modified legume protein. In steps (b) and (c), the pH is in the range of pH 6-8, and the legume protein isolate is derived from or obtained from peas, soybeans, broad beans, lentils, or any combination thereof.

[0045] The term "modified legume protein" refers to legume protein that has been treated with a protein deamidase and subjected to heat treatment. Therefore, the term "enzymatically deamidated legume protein" refers to legume protein treated with a protein deamidase for deamidation. Those skilled in the art will know suitable analytical methods for determining whether enzymatic deamidation has occurred in legume proteins. One such method is illustrated in Example 1 by measuring the free ammonium content (NH4). Modified legume proteins can be ingested by humans or animals, preferably by humans. The modified legume proteins claimed herein are plant-based.

[0046] Modified legume proteins can exist in aqueous dispersions and can be referred to as aqueous dispersions of modified legume proteins. The term "aqueous dispersion" has its general meaning well known and understood by those skilled in the art. It refers to a liquid system in which very small solid particles are uniformly dispersed in an aqueous solution, such as water.

[0047] The modified legume protein aqueous dispersion claimed herein comprises at least one enzymatically deamidated legume protein and may be combined with or without other food ingredients to produce the modified legume protein aqueous dispersion. In some embodiments, the modified legume protein aqueous dispersion is intended for use as a beverage, such as a dairy alternative milk or drink. As used herein, the terms “drink,” “milk,” and “beverage” are used interchangeably and, unless otherwise stated, have the same meaning.

[0048] The modified legume proteins obtained according to the present invention exhibit improved colloidal stability and improved acid and / or thermal stability, and can be incorporated into a wide variety of foods. For example, the modified legume proteins can be used directly as food and have certain commercial value.

[0049] In the context of this invention, the term "colloidal stability" refers to the ability of particles in a liquid (e.g., an aqueous solution) to remain stable in a dispersion system. The ability of modified legume proteins to remain stable in a dispersion system without forming aggregates or clusters that, for example, cause changes in particle size within the dispersion system, signifies colloidal stability. Furthermore, the modified legume proteins of this invention exhibit improved anti-sedimentation properties, i.e., resistance to particle sedimentation. The colloidal stability and sedimentation of the modified legume proteins can be determined by visually inspecting the aqueous dispersion of the modified legume proteins. Visual inspection can be performed immediately as well as after long-term storage, for example, by placing the aqueous dispersion of the modified legume proteins in a transparent beaker, allowing for visual inspection both immediately after pouring the liquid and after storage. Alternatively, or in addition to visual inspection, colloidal stability and sedimentation can also be assessed based on particle size analysis (e.g., particle size distribution in an aqueous suspension). For example, visual inspection can be performed after incubating an aqueous suspension of the modified protein at room temperature or under refrigeration for 5–60 minutes.

[0050] Without wishing to be bound by any particular theory, it is believed that the colloidal stability of the modified legume protein obtained according to the methods disclosed herein is achieved by treating the legume protein isolate with a protein deamidase. Therefore, in some embodiments, the modified legume protein exhibits improved colloidal stability compared to modified legume proteins prepared using similar methods in which a protein deamidase is not added to an aqueous solution of the legume protein isolate.

[0051] In particular, after being stored at a temperature of at least 4°C for 15 minutes, the modified legume protein aqueous dispersion exhibits improved colloidal stability compared to an aqueous dispersion of modified legume protein prepared using a similar method in which protein deamidase is not added to an aqueous solution of the legume protein isolate.

[0052] Specifically, after storage at at least 4°C for 15 minutes, the modified legume protein aqueous dispersion exhibited improved colloidal stability compared to aqueous dispersions prepared using a similar method but without the heat treatment step following protein deamidase treatment. This is as demonstrated at least by Example 3 and... Figure 3 and 4 The results shown confirm that the use of protein deamidase followed by a heat treatment step results in excellent colloidal stability in the aqueous dispersion of legume proteins.

[0053] The modified legume protein of the present invention, such as the modified legume protein aqueous dispersion, is storable and can be stored under refrigerated conditions or at room temperature.

[0054] In the context of this invention, room temperature conditions include temperatures of about 15°C to 25°C (e.g., about 18°C ​​to 22°C), and low temperature and / or refrigeration conditions mean temperatures of about 2°C to 5°C (e.g., about 4°C).

[0055] The modified legume protein, and particularly its aqueous dispersion, exhibits improved beverage stability. Without wishing to be bound by any particular theory, the improved colloidal stability, and especially the improved beverage stability, reported herein is not solely a result of the improved solubility of the protein in the dispersion system. That is, both the modified legume protein and its aqueous dispersion possess improved protein solubility in liquid dispersion systems and further improved stability in liquids. This is observed, for example, when the aqueous dispersion of the modified legume protein is mixed with an acidic, optionally geothermal, beverage (such as coffee or tea), where no visible flocculation or sedimentation is observed during mixing.

[0056] Therefore, in some embodiments, the modified legume protein aqueous dispersion exhibits improved stability when mixed with acidic beverages. Furthermore, in some embodiments, the modified legume protein aqueous dispersion exhibits improved stability when mixed with hot acidic beverages.

[0057] In some embodiments, the modified legume protein aqueous dispersion is suitable for mixing with a hot, acidic beverage (e.g., a coffee or tea beverage). In the context of this invention, a hot, acidic beverage means a beverage having a temperature of at least 85°C when mixed with the modified legume protein aqueous dispersion. In some embodiments, the hot, acidic beverage has a temperature of at least 85°C, such as at least 87°C, at least 90°C, at least 92°C, or at least 95°C, prior to mixing with the modified legume protein aqueous dispersion. In some embodiments, the modified legume protein aqueous dispersion exhibits improved stability, such as reduced flocculation levels, when mixed with a hot, acidic beverage compared to a modified legume protein aqueous dispersion prepared using a similar method in which a protein deamidase is not added to an aqueous solution of the legume protein isolate.

[0058] The aqueous dispersions of the modified legume proteins are stable over time, meaning the aqueous dispersions are stable and the proteins and other components contained therein do not precipitate. Stability can be observed immediately after obtaining the aqueous dispersions of the modified proteins, and can be visually observed at 5, 10, 15, or even 30 minutes after obtaining the aqueous dispersions of the modified proteins.

[0059] The modified legume protein aqueous dispersions are stable over extended periods of time, namely, the aqueous dispersions are stable for 7 days, 14 days, 21 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, and 12 months (e.g., 4 months or 8 months) under refrigerated and room temperature conditions, and the proteins and other components contained therein do not precipitate.

[0060] Preferably, the legume protein isolate has a protein content of at least 80% (w / w, on a dry matter basis).

[0061] Preferably, the legume protein isolate has a protein content of up to 95% (w / w, on a dry matter basis).

[0062] In a preferred embodiment, the legume protein isolate has a protein content of about 82% (w / w, on a dry matter basis).

[0063] Preferably, the modified legume protein has a lipid content of at least 0% (w / w, on a dry matter basis).

[0064] Preferably, the modified legume protein has a lipid content of up to 10% (w / w, on a dry matter basis).

[0065] In a preferred embodiment, the modified legume protein has a lipid content of about 5% (w / w, on a dry matter basis).

[0066] In some embodiments, additional food ingredients are added to the modified legume protein or an aqueous dispersion of the modified legume protein. The additional food ingredients can be any food ingredient that a person skilled in the art would find useful. The additional food ingredients can be solid or liquid. The additional food ingredients may or may not be plant-based. In some embodiments, the additional food ingredient is water.

[0067] Modified legume proteins or modified legume protein aqueous dispersions can be fortified with plant-based dairy alternative powders (such as soy milk powder, or concentrated or isolated proteins, such as soy protein isolates, soy protein concentrates, pea protein isolates, or pea protein concentrates). In some embodiments, the modified legume protein aqueous dispersion is a pea-based beverage fortified with soy or pea protein to a protein level of 1%-20% (w / w).

[0068] Additional food ingredients that may be added to modified legume proteins or to aqueous dispersions of modified legume proteins include, but are not limited to, lipids (e.g., oils, especially vegetable oils), sugars (e.g., sucrose), proteins, synthetic amino acids in various forms, dietary fiber, salt, minerals, flavorings, vitamins, and any combination thereof.

[0069] In some embodiments, lipids are added to an aqueous solution of legume protein isolates and / or an aqueous dispersion of modified legume proteins and / or modified legume proteins. The lipids can be vegetable oils or mixtures of vegetable oils. Lipids can be selected from rapeseed oil, flaxseed oil, safflower oil, soybean oil, olive oil, sunflower oil, palm oil, and combinations thereof. In this embodiment, the lipid is soybean oil. The selection of a suitable lipid can be based on the type of food desired.

[0070] In some embodiments, sugar is optionally added together with lipids to an aqueous solution of legume protein isolate and / or an aqueous dispersion of modified legume protein and / or modified legume protein. In embodiments, the sugar is sucrose. In a preferred embodiment, sucrose and soybean oil are added to the aqueous solution of legume protein isolate.

[0071] In some embodiments, salt is added to an aqueous solution of legume protein isolate and / or an aqueous dispersion of modified legume protein. The salt may be sodium chloride, dicalcium carbonate, dicalcium phosphate, tricalcium phosphate, calcium carbonate, or any combination thereof.

[0072] In some embodiments, vitamins and / or minerals are added to an aqueous solution of legume protein isolate and / or an aqueous dispersion of modified legume protein and / or modified legume protein. Vitamins may be vitamin A, vitamin C, vitamin D, vitamin E, vitamin B12, thiamine (vitamin B1), riboflavin (vitamin B2), niacin (vitamin B3), vitamin B6, vitamin K, folic acid (vitamin B9), and mixtures thereof. Minerals may be calcium, phosphorus, magnesium, sodium, potassium, chloride, iron, zinc, iodine, selenium, copper, and mixtures thereof.

[0073] The modified legume proteins and their aqueous dispersions obtained according to the method claimed herein do not require the addition of emulsifiers and / or stabilizers to achieve the claimed properties. In particular, using the method of the present invention, modified legume proteins with improved stability, particularly improved colloidal stability, are obtained without the need to add emulsifiers and / or stabilizers during or in any step of the production of the final product.

[0074] The modified legume protein aqueous dispersion does not flocculate or precipitate even after prolonged storage, such as after several weeks or months. Therefore, in preferred embodiments, the modified legume protein and / or the modified legume protein aqueous dispersion are substantially free of added emulsifiers and / or stabilizers. Furthermore, in preferred embodiments, stabilizers and / or emulsifiers are not present in any step of the method claimed herein.

[0075] As used herein, the term "substantially free" is used to describe compositions or products containing only trace or negligible amounts of a particular substance or component. It indicates that the presence of the specified substance or component is minimal and does not affect the overall characteristics or function of the invention. In embodiments, "substantially free" means 0% (w / w) or 0% (w / v).

[0076] As used herein, the terms “emulsifier” and “stabilizer” mean added emulsifiers and stabilizers that are not naturally present in the materials used to prepare modified legume proteins. Examples of such emulsifiers and stabilizers include, but are not limited to, thickeners (such as carboxymethyl cellulose, gellan gum, hydroxypropyl starch, and agar) and emulsifiers (such as monoglycerides and diglycerides).

[0077] In the context of this invention, legume proteins are derived from or obtained from peas, soybeans, broad beans, lentils, or any combination thereof.

[0078] Soybeans, peas, broad beans, and lentils all belong to the legume family (Fabaceae) and are pods or fruits of the legume family (Fabaceae). Fabaceae Based on protein content, legume protein products can be classified into three main categories: powder, concentrate, and isolate, with the highest protein content found in isolate, followed by concentrate and lastly powder.

[0079] In the context of this invention, legume proteins are derived from or obtained from legume protein isolates. The term "legume protein isolate" refers to a composition in which the protein content is increased compared to the pre-processed state (i.e., the plant protein raw material) due to protein extraction or concentration. Typically, such an isolate refers to a composition in which the protein content is 80% (w / w) or higher (e.g., ranging from 80% to 99% (w / w)). For some commercial soybean protein isolates or pea protein isolates, the protein content is about 82% (w / w) or higher. Some purified soybean protein isolates or pea protein isolates can achieve a protein content of 90% (w / w) or higher.

[0080] The legume protein isolate used in the method of this invention can be a commercially available isolate or can be prepared using methods known to those skilled in the art. The legume protein isolate can be a spray-dried or freeze-dried legume protein isolate.

[0081] In the method of this invention, the legume protein is derived from or obtained from legume protein isolates, such as pea protein isolates, soybean protein isolates, broad bean protein isolates, lentil protein isolates, or any combination thereof. In a preferred embodiment, the legume protein is derived from or obtained from pea protein isolates, soybean protein isolates, or combinations thereof.

[0082] In the method of this invention, the legume protein isolate is provided as an aqueous solution of the legume protein isolate, the protein content of which is in the range of 1%-20% (w / w). The term "aqueous solution of legume protein isolate" means, for example, an aqueous liquid in which the legume protein isolate in powder form is dissolved, such as water. The aqueous solution of the protein isolate may or may not have a uniform distribution of the legume protein isolate in the aqueous solution.

[0083] In a preferred embodiment, the aqueous solution of the legume protein isolate has a protein content in the range of 3%-20% (w / w). In another embodiment, the aqueous solution of the legume protein isolate has a protein content of about 3% (w / w), about 4% (w / w), about 5% (w / w), about 6% (w / w), about 7% (w / w), about 8% (w / w), about 9% (w / w), about 11% (w / w), about 13% (w / w), about 15% (w / w), about 17% (w / w), or about 20% (w / w). In some embodiments, the aqueous solution of the legume protein isolate has a protein content in the range of 5%-15% (w / w).

[0084] In some embodiments, the aqueous solution of the legume protein isolate has a lipid content in the range of 0%-2% (w / w), such as 0%-1% (w / w). In some embodiments, the aqueous solution of the legume protein isolate has a lipid content of about 0.5% (w / w) or about 1% (w / w).

[0085] The protein deamidase used to treat the aqueous solution of legume protein isolate is maintained at a temperature within the range of 20°C to 80°C, such that the legume protein isolate is enzymatically deamidated by the protein deamidase to produce enzymatically deamidated legume protein. In some embodiments, the aqueous solution of legume protein isolate is maintained at a temperature between 25°C-40°C, 30°C-45°C, 35°C-50°C, 40°C-55°C, 50°C-60°C, 50°C-65°C, 55°C-70°C, or 55°C-65°C. In other embodiments, the temperature of the aqueous solution of legume protein isolate is maintained at about 20°C, 25°C, about 30°C, about 35°C, about 40°C, about 45°C, about 50°C, about 55°C, about 60°C, about 65°C, or about 70°C.

[0086] In one embodiment, the treatment with protein deamidase was performed at a temperature in the range of 50°C to 70°C, for example, at a temperature in the range of 55°C to 70°C. In another embodiment, the treatment with protein deamidase was performed at a temperature in the range of 60°C to 70°C, for example, at a temperature in the range of 60°C to 65°C.

[0087] In some embodiments, an aqueous solution of legume protein isolate is held with an added protein deamidase at 20°C–80°C for at least 10 minutes to allow enzymatic deamidation of the legume protein. In some embodiments, the aqueous solution of legume protein isolate is held for about 10, about 15, about 20, about 25, about 30, about 60, about 120, about 180, or about 240 minutes to allow enzymatic deamidation of the legume protein isolate. In some embodiments, the aqueous solution of legume protein isolate is held for at least about 10, 30, 60, or 90 minutes. In some embodiments, the aqueous solution of legume protein isolate is held for 30 minutes. In some embodiments, the aqueous solution of legume protein isolate is held for 60 minutes.

[0088] Preferably, the treatment with protein deamidase is carried out at a temperature ranging from 50°C to 70°C for 15-90 minutes. In the examples, the treatment with protein deamidase is carried out at a temperature ranging from 60°C to 70°C for 30-60 minutes.

[0089] In the method of this invention, the pH is in the range of pH 6-8. For example, in the method disclosed herein, the pH can be about 6.2, 6.5, 6.7, 7, 7.2, 7.5, or 7.7. In the context of this invention, the pH can be measured on any of the following: an aqueous solution of legume protein isolate, an aqueous solution of enzymatically deamidated legume protein, and an aqueous dispersion of modified legume protein.

[0090] According to the method disclosed herein, it is not necessary to adjust the pH to below 6 during any step in obtaining the modified legume protein or an aqueous dispersion of the modified legume protein. Therefore, a simpler production method is achieved that does not affect the final properties of the modified legume protein or the aqueous dispersion of the modified legume protein, particularly its colloidal stability.

[0091] Other parameters (such as temperature range and length of enzymatic treatment) will vary depending on the legume protein, the additional enzymes used, and the desired end product. Technicians will know how to determine appropriate additional process parameters based on, for example, the legume protein used, the end product, and the enzymes.

[0092] Following enzymatic deamidation of legume proteins, heat treatment is performed. Heat treatment also inactivates the protein deamidase. In some embodiments, the heat treatment is carried out at 80°C–125°C for 5–30 minutes. For example, the heat treatment is carried out at 85°C–121°C for 10–20 minutes. In some embodiments, the heat treatment is carried out at 90°C for 5, 10, 15, 20, 25, or 30 minutes. In some preferred embodiments, the heat treatment is carried out at 90°C for 10 minutes. In some embodiments, the heat treatment is carried out at 85°C for 5, 10, 15, 20, 25, or 30 minutes. In some preferred embodiments, the heat treatment is carried out at 85°C for 15 minutes. In some embodiments, the heat treatment is carried out at 115°C for 2, 5, 10, 15, or 20 minutes. In some embodiments, the heat treatment is carried out at 120°C for 2, 5, 10, 15, or 20 minutes.

[0093] In some embodiments, the heat treatment further includes ultra-high temperature (UHT) treatment lasting for 1-120 seconds at a temperature in the range of 126°C-150°C, such as lasting for 1-90 seconds at a temperature in the range of 130°C-145°C.

[0094] In some embodiments, the heat treatment and inactivation of protein deamidases are ultra-high temperature (UHT) treatments. UHT treatment can be direct or indirect. In some embodiments, the UHT treatment is performed at a temperature in the range of 121°C-150°C for 1-120 seconds. In some embodiments, the UHT treatment is performed at a temperature in the range of 130°C-145°C for 1-90 seconds. In another embodiment, the UHT treatment is performed at 135°C-154°C for 1-10 seconds. In another embodiment, the UHT treatment is performed at 140°C-150°C for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 seconds. In another embodiment, the UHT treatment is performed at 140°C-145°C for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 seconds. In some embodiments, the UHT treatment is performed at 143°C for 1, 2, 3, 4, 5, 6, 7, or 8 seconds.

[0095] In some embodiments, the heat treatment is performed in two steps, including a first step of heat treatment at a temperature in the range of 80°C-125°C for 5-30 minutes, such as at a temperature in the range of 85°C-121°C for 10-20 minutes, followed by UHT treatment at a temperature in the range of 126°C-150°C for 1-120 seconds, such as at a temperature in the range of 130°C-145°C for 1-90 seconds.

[0096] After heat treatment, the aqueous dispersion of the modified legume protein can be cooled. The aqueous dispersion can be separated into a solid stream and a liquid stream, for example, by centrifugation. Centrifugation can occur in a sedimentation centrifuge. After centrifugation, the liquid stream can be harvested or collected and used as the aqueous dispersion of the modified legume protein. The liquid stream may still contain some solids. In some embodiments, the liquid stream contains 1%-80% solids. In other embodiments, the liquid stream contains 1%-10%, 5%-20%, 10%-25%, 20%-35%, 25%-40%, 30%-45%, 35%-50%, 40%-55%, 45%-60%, 50%-65%, 55%-70%, 60%-75%, or 65%-80% solids. In some embodiments, the liquid stream contains 10%-15% solids.

[0097] In some embodiments, the modified legume protein aqueous dispersion does not separate into solid and liquid streams, i.e., the modified legume protein aqueous dispersion is not subjected to a centrifugation step. In such embodiments, the modified legume protein aqueous dispersion is used directly, for example, to obtain dairy alternative foods, such as dairy alternative beverages. The modified legume protein aqueous dispersion obtained according to the method of the invention has improved dispersibility and colloidal stability, regardless of whether a centrifugation step is performed.

[0098] In some embodiments, the liquid stream is used directly as an aqueous dispersion of modified legume protein. Additional food ingredients can be added to the liquid stream to produce the modified legume protein aqueous dispersion. For example, legume protein-derived liquid streams can be formulated using, for instance, sodium chloride (NaCl), oil, sugar, and flavorings. It may be homogenized. It may be UHT or ESL treated and aseptically packaged.

[0099] In some embodiments, the modified legume protein aqueous dispersion is a dairy alternative beverage. Examples of dairy alternative beverages include soy beverages, pea beverages, fava bean beverages, lentil beverages, and beverages containing any combination thereof. In embodiments, the plant-based beverage is a soy beverage, pea beverage, or a beverage containing a combination thereof. In some embodiments, the dairy alternative beverage is prepared from soy protein isolate or pea protein isolate.

[0100] Aqueous dispersions of modified proteins are suitable for a wide range of applications, including but not limited to use as dairy alternative beverages, such as dairy alternative “milk”; for mixing with other types of beverages, such as for mixing with acidic beverages (e.g., coffee or tea) to obtain ready-to-drink (RTD) acidic beverages; and as protein drinks.

[0101] In some embodiments, the aqueous dispersion of the modified legume protein is a canned or packaged beverage. The aqueous dispersion of the modified legume protein can be contained and stored in any type of can or packaging material deemed suitable by those skilled in the art. In some embodiments, the canned or packaged beverage containing the aqueous dispersion of the modified legume protein is subjected to can sterilization. For example, can sterilization may be performed at 80°C-125°C for 5-30 minutes, preferably at 110°C-121°C for 10-20 minutes.

[0102] Aqueous solutions of legume protein isolates can be further processed, for example, by treatment with additional enzymes, including, for example, additional hydrolases. These additional enzymes include, but are not limited to, one or more hydrolases selected from the group consisting of: pectinase, hemicellulase, xylanase, β-glucanase, mannanase, glucanase, glucosylamylase, isoamylase, α-amylase, β-amylase, and mixtures thereof.

[0103] The enzymes used in the methods of this invention can be added to an aqueous dispersion containing legume protein isolates in any suitable form (e.g., as a liquid, particularly a stabilized liquid), or they can be added as a substantially dry powder or granules. For example, granules can be produced as disclosed in U.S. Patent Nos. 4,106,991 and 4,661,452. Liquid enzyme preparations can be stabilized, for example, by adding sugars or sugar alcohols or lactic acid according to established procedures. Other enzyme stabilizers are well known in the art.

[0104] In another aspect, the present invention relates to an aqueous dispersion of modified legume protein in the pH range of 6-8, wherein the modification is obtained by enzymatic deamidation with a protein deamidase followed by a heat treatment step, and wherein the legume protein is derived from or obtained from legume protein isolates of peas, soybeans, broad beans, lentils or any combination thereof, preferably legume protein isolates of peas, soybeans or combinations thereof.

[0105] In another aspect, the present invention relates to the use of protein deamidase in the production of aqueous dispersions of modified legume proteins in the pH range of 6-8 for improving colloidal stability, wherein the modified legume protein is derived from or obtained from legume protein isolates of peas, soybeans, broad beans, lentils or any combination thereof.

[0106] In another aspect, the present invention relates to the use of protein deamidase and heat treatment in the production of aqueous dispersions of modified legume proteins in the pH range of 6-8 for improving colloidal stability, wherein the modified legume protein is derived from or obtained from legume protein isolates of peas, soybeans, broad beans, lentils or any combination thereof.

[0107] Protein deamidase In the method of the present invention, legume protein isolates are treated with protein deamidases to obtain modified aqueous dispersions of legume proteins.

[0108] In this invention, protein deamidase refers to an enzyme that acts directly on the amide groups of the amino acid side chains that make up a protein to cause deamidation and release ammonia without cleaving the peptide bonds of the protein or cross-linked proteins.

[0109] The term "deamidase" refers to the activity of protein-glutamine glutaminase (also known as glutamylpeptide glutaminase), as described in EC 3.5.1.44, which catalyzes the hydrolysis of γ-amides of glutamines substituted at the carboxyl position or both the α-amino and carboxyl positions (e.g., L-glutamylglycine and L-phenylalanyl-L-glutamylglycine). Therefore, deamidases can deamidate glutamine residues in proteins to glutamate residues, and deamidases are also referred to as protein glutamine deamidases. Deamidases include Cys-His-Asp catalytic triads (e.g., Cys-156, His-197, and Asp-217, as described in Hashizume et al., "Crystal structures of protein glutaminase and its proforms converted into enzyme-substrate complex"). Journal of Biological Chemistry (See [Journal of Biochemistry], Volume 286, Issue 44, pp. 38691–38702) and belongs to InterPro entry IPR041325.

[0110] Deamidases may also include protein asparaginases, which act directly on the amide group of the side chain of asparagine residues in a protein to release ammonia and thus convert the asparagine residues to aspartic acid residues. In this invention, either protein glutaminase or protein asparaginase, or a combination of both, can be used as the protein deamidase. An example of a protein deamidase used in this invention is protein glutaminase.

[0111] The protein deamidases used in the methods of this invention can be obtained from microorganisms of any genus. For the purposes of this invention, the term "obtained from," as used herein in conjunction with a given source, should mean that the polypeptide encoded by the polynucleotide is produced by that source or by a strain that has inserted a polynucleotide from that source. In examples, polypeptides obtained from a given source are secreted extracellularly.

[0112] Protein deamidases can be obtained from microorganisms using any suitable technique. For example, an enzyme preparation can be obtained by fermenting a suitable microorganism and subsequently isolating the protein deamidase preparation from the resulting fermentation broth or microorganism using methods known in the art. Protein deamidases can also be obtained using recombinant DNA technology. Such methods typically involve culturing host cells transformed with a recombinant DNA vector containing a DNA sequence encoding the protein deamidase, and operatively linking the DNA sequence to a suitable expression signal such that the enzyme can be expressed in a culture medium under conditions allowing enzyme expression, and recovering the enzyme from the culture. The DNA sequence can also be incorporated into the genome of a host cell. The DNA sequence can be genomic, cDNA, or synthetically derived, or any combination thereof, and can be isolated or synthesized according to methods known in the art.

[0113] Protein deamidases can be purified. As used herein, the term "purified" includes protein deamidase proteins that are substantially free of insoluble components from the producing organism. The term "purified" also includes protein deamidase proteins that are substantially free of insoluble components from the native organism from which the enzyme was obtained. Preferably, the enzyme can also be separated from some soluble components of the source organism and the culture medium. More preferably, separation can be performed by one or more unit operations: filtration, precipitation, or chromatography.

[0114] There are no particular limitations on the type or source of the protein deamidase used in this invention. Examples of protein deamidases include those derived from the genera *Chlorella* and *Flavobacterium*. Flavobacterium genus), Stenotrophomonas ( Empedobacter genus), Sphingosine Bacillus ( Sphingobacterium genus), Chlorella ( Aureobacterium genus) or genus of aromatic fungi ( Myroides Protein deamidases (genus).

[0115] Protein deamidases can originate from any of the sources mentioned herein. In the context of this invention, the term "originating from" means that the enzyme can be isolated from the organism in which it naturally exists, i.e., the amino acid sequence of the protein deamidase is identical to that of the natural polypeptide. The term "originating from" also means that the enzyme can be recombinantly produced in a host organism, and the recombinant enzyme has an amino acid sequence identical to that of the natural enzyme, or has a modified amino acid sequence (e.g., with one or more amino acids that are deleted, inserted, and / or substituted), i.e., the recombinant enzyme is a mutant of the natural amino acid sequence. The meaning of "natural enzyme" includes natural variants. Furthermore, the term "originating from" includes enzymes synthesized, for example, through peptide synthesis. The term "originating from" also includes enzymes that have been modified in vivo or in vitro, for example, through glycosylation, phosphorylation, etc. For recombinant enzymes, the term "originating from" refers to the identity of the enzyme, not the identity of the host organism from which the recombinant enzyme is produced.

[0116] In some embodiments, protein deamidases may be derived from the genus *Chlorella*, such as species of *Chlorella*. 62563 Beer God Golden Bacterium ( C. gambrini Culex pipiens ( ), C. culicis ), wastewater vulcanii ( C. defluvii ) or pyruvum valerate ( C. proteolyticum In some embodiments, the deamidase in the method of the present invention is derived from or obtained from species of the genus *Chlorella*. -62563 .

[0117] EP1839491 discloses the presence of Corynebacterium glutamicum ( Corynebacterium glutamicum The clone of a protein glutaminase from *Chlorella vulgaris* expressed in the sample. Deamidases are also commercially available, for example, protein glutaminases derived from *Chlorella*, such as "Amano PG500" (manufactured by Amano Enzyme Products Co., Ltd.).

[0118] For example, protein deamidases can be obtained from the culture medium of the aforementioned microorganisms.

[0119] Protein deamidases are produced by microbial cells in an inactive proform containing a propeptide domain tightly bound to the deamidase domain. This proform is expressed as a fusion protein with reduced deamidase activity to protect the viability of the host cell. Essentially, the fusion protein is post-processed to remove the propeptide and release the active deamidase outside the host cell. However, in recombinant expression systems, the fusion protein is secreted outside the host cell as an inactive proform containing the propeptide. The propeptide can then be enzymatically cleaved to separate it from the mature deamidase. The protein deamidase of the methods and compositions of the present invention is a mature deamidase in which the propeptide has been removed. In some embodiments, the propeptide is enzymatically cleaved by an endopeptidase. In some embodiments, the propeptide may still be present in the composition containing the mature deamidase.

[0120] The recombinant mature protein deamidases used in the method of this invention comprise polypeptides of SEQ ID NO: 2, 4, 6, 8, and 10. Each mature protein deamidase is derived from a proto-deamidase polypeptide comprising polypeptides of SEQ ID NO: 1, 3, 5, 7, and 9, respectively. The proto-deamidase polypeptide contains a propeptide fused to the N-terminus of a deamidase identical to that of the polypeptides of SEQ ID NO: 2, 4, 6, 8, or 10. The propeptide can be enzymatically cleaved from the proto-deamidase polypeptide to release the mature deamidase. A naturally occurring propeptide sequence is provided in the proto-deamidase polypeptide.

[0121] The methods and compositions of the present invention comprise a mature deamidase and optionally a second polypeptide derived from the propeptide of the deamidase. The second polypeptides described herein are mutant variants of naturally occurring propeptides. These variant propeptide sequences have been found to bind weakly to their corresponding deamidases, making them more readily enzymatically cleaved after recombinant expression and secretion from host cells. The polypeptides of SEQ ID NO: 1 to 10 are derived from species of the genus *Chlorella* and are described in the applicant's PCT application, publication number WO2023 / 170177 A1 (incorporated herein by reference).

[0122] After expressing the original polypeptide in a recombinant expression system, the propeptide is cleaved using a site-specific endopeptidase, leaving an active mature deamidase. In some embodiments, the cleaved propeptide is not purified from the mature deamidase. Therefore, the propeptide can be present in a composition containing a mature deamidase.

[0123] According to a preferred embodiment, the protein deamidase used in the method of the present invention is derived from or obtained from species of the genus *Chlorella*, such as *Chlorella utilis* or *Chlorella viviparus*.

[0124] In the context of this invention, the term "mature polypeptide" means a polypeptide in its mature form after N-terminal processing (e.g., removal of a signal peptide). A "signal peptide" is an amino acid sequence attached to the N-terminal portion of a protein that facilitates its secretion outside the cell. The mature form of an extracellular protein lacks a signal peptide, which is cleaved during the secretion process.

[0125] In the examples, the deamidase is selected from polypeptides having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with SEQ ID NO: 2.

[0126] In the examples, the deamidase is selected from polypeptides having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with SEQ ID NO: 4.

[0127] In the examples, the deamidase is selected from polypeptides having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with SEQ ID NO: 6.

[0128] In the examples, the deamidase is selected from polypeptides having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with SEQ ID NO: 8.

[0129] In the examples, the deamidase is selected from polypeptides having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with SEQ ID NO: 10.

[0130] In the examples, the deamidase is selected from polypeptides having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with the mature polypeptide of SEQ ID NO: 1.

[0131] In the examples, the deamidase is selected from polypeptides having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with the mature polypeptide of SEQ ID NO: 3.

[0132] In the examples, the deamidase is selected from polypeptides having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with the mature polypeptide of SEQ ID NO: 5.

[0133] In the examples, the deamidase is selected from polypeptides having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with the mature polypeptide of SEQ ID NO: 7.

[0134] In the examples, the deamidase is selected from polypeptides having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with the mature polypeptide of SEQ ID NO: 9.

[0135] For the purposes of this invention, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. [Journal of Molecular Biology] 48: 443-453) is used to determine the sequence identity between two amino acid sequences as the output of "longest identity". This algorithm is implemented in the Niedel program using the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. [Trends in Genetics] 16: 276-277) (preferably version 6.6.0 or later). The parameters used are a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. For the Niedel program to report the longest identity, the non-brief (-nobrief) option must be specified on the command line. The Niedel-marked "longest identity" output is calculated as follows: (identical residues × 100) / (alignment length - total number of vacancies in the alignment) In the context of this invention, the term "variant" means a polypeptide having enzymatic activity that contains alterations (i.e., substitutions, insertions, and / or deletions) at one or more (e.g., several) positions. Substitution means replacing an amino acid occupying a position with a different amino acid; deletion means removing an amino acid occupying a position; and insertion means adding one or more (e.g., several) amino acids (e.g., 1-5 amino acids) adjacent to and immediately following the amino acid occupying a position.

[0136] Amino acid alterations can be minor, i.e., conserved amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; typically small deletions of 1–30 amino acids; small N-terminal or C-terminal extensions, such as methionine residues at the N-terminus; small linker peptides of up to 20–25 residues; or small extensions that facilitate purification by altering net charge or another function (such as polyhistidine fragments, antigenic epitopes, or binding domains).

[0137] Examples of conserved substitutions are found in the following group: basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine, and valine), aromatic amino acids (phenylalanine, tryptophan, and tyrosine), and small amino acids (glycine, alanine, serine, threonine, and methionine). Amino acid substitutions that do not typically alter specific activity are known in the art and are described, for example, by H. Neurath and RL Hill, 1979, in *The Proteins*, Academic Press, New York. Common substitutions are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0138] Alternatively, these amino acid alterations have the property of changing the physicochemical properties of the peptide. For example, these amino acid alterations can affect the peptide's thermal stability, change its substrate specificity, and alter its optimal pH.

[0139] Essential amino acids in peptides can be identified using procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, a single alanine mutation is introduced at each residue in the molecule, and the enzyme activity of the resulting mutant molecule is tested to identify amino acid residues essential to the molecule's activity. See also Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The active site of the enzyme or other biological interactions can also be determined by combining mutations of amino acids at hypothetical contact sites with physical analysis of the structure, such as by techniques like NMR, crystallography, electron diffraction, or photoaffinity labeling. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acids can also be inferred from comparisons with related peptides.

[0140] Using known mutagenesis, recombination, and / or tampering methods, followed by relevant screening procedures, one or more amino acid substitutions, deletions, and / or insertions can be made and tested, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95 / 17413; or WO 95 / 22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30: 10832-10837; US Patent No. 5,223,409; WO 92 / 06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7:127).

[0141] Mutagenesis / reorganization methods can be combined with high-throughput, automated screening methods to detect the activity of cloned, mutagenesis-encoded peptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17: 893-896). Mutagenesis-encoded DNA molecules encoding active peptides can be recovered from host cells and rapidly sequenced using standard methods in the art. These methods allow for the rapid determination of the importance of individual amino acid residues within the peptide.

[0142] The protein deamidase used in the method of the present invention can be added at a concentration of 0.01-20 IPA (U) / g substrate protein (e.g., 0.1-12 IPA (U) / g substrate protein, 0.5-7 IPA (U) / g substrate protein). In some embodiments, the protein deamidase used in the method of the present invention is added at a concentration in the range of 0.3-4 IPA (U) / g substrate protein (e.g., 0.4-2.5 IPA (U) / g substrate protein).

[0143] The activity of deamidase (protein glutaminase) can be measured using the assay described in Example 1. The activity assay consists of two separate parts: (1) an enzymatic step in which ammonia is formed by the catalysis of the protein deamidase; and (2) a non-enzymatic detection step in which the ammonia formed in step (1) is derivatized into an indophenol blue compound with maximum absorption at 630 nm. The amount of enzyme that produces 1 μmol of ammonia per minute at 37 °C is defined as 1 unit (expressed in indophenol assay units: IPA (U)). The activity can be determined relative to known strength standards.

[0144] The enzyme dosage will depend on parameters such as temperature, incubation time, and the intended use of the modified legume protein. Those skilled in the art will know how to determine the optimal enzyme dosage.

[0145] Without wishing to be bound by any particular theory, the inventors of this invention believe that using protein deamidases to produce modified legume proteins (e.g., aqueous dispersions of modified legume proteins) contributes to the excellent benefits reported herein, including but not limited to improved colloidal stability.

[0146] The invention is further defined by the following numbered embodiments: Example 1. A method for obtaining an aqueous dispersion of modified legume protein, the method comprising the following steps: (a) Provide an aqueous solution of legume protein isolate with a protein content in the range of 1%-20% (w / w); (b) Treating the aqueous solution from step (a) with a protein deamidase to obtain enzymatically deamidated legume protein; and (c) The enzymatically deamidated legume protein from step (b) is subjected to heat treatment to obtain an aqueous dispersion of the modified legume protein. In steps (b) and (c), the pH is in the range of pH 6-8, and The legume protein isolates described herein are derived from or obtained from peas, soybeans, broad beans, lentils, or any combination thereof.

[0147] Example 2. The method as described in Example 1, wherein after storage at a temperature of at least 4°C for 15 minutes, the modified legume protein aqueous dispersion has improved colloidal stability compared to an aqueous dispersion of the modified legume protein prepared using a similar method in which a protein deamidase is not added to an aqueous solution of the legume protein isolate.

[0148] Example 3. The method as described in Example 1, wherein after storage at a temperature of at least 4°C for 15 minutes, the modified legume protein aqueous dispersion has improved colloidal stability compared to an aqueous dispersion of modified legume protein prepared using a similar method but without the use of protein deamidase and a heat treatment step following treatment with said protein deamidase.

[0149] Example 4. The method as described in any of the preceding examples, wherein the protein deamidase treatment in step (b) is carried out at a temperature in the range of 50°C-70°C for 15-90 minutes.

[0150] Example 5. The method as described in any of the preceding examples, wherein the protein deamidase treatment in step (b) is carried out at a temperature in the range of 55°C-70°C for 15-90 minutes.

[0151] Example 6. The method as described in any of the preceding examples, wherein the protein deamidase treatment in step (b) is carried out at a temperature in the range of 60°C-70°C for 15-90 minutes.

[0152] Example 7. The method as described in any of the preceding examples, wherein the protein deamidase treatment in step (b) is carried out at a temperature in the range of 60°C-65°C for 15-90 minutes.

[0153] Example 8. The method as described in any of the preceding examples, wherein the protein deamidase treatment in step (b) is performed for 30-60 minutes.

[0154] Example 9. The method as described in any of the preceding examples, wherein no stabilizer and / or emulsifier is present in any of steps (a), (b) and / or (c).

[0155] Example 10. The method as described in any of the preceding examples, wherein no stabilizer and / or emulsifier is present in any of steps (a), (b), and (c).

[0156] Example 11. The method as described in any of the preceding examples, wherein the protein content of the aqueous solution in step (a) is in the range of 3%-20% (w / w), 1%-15% (w / w), 3%-15% (w / w), or 3%-10% (w / w).

[0157] Example 12. The method as described in any of the preceding examples, wherein the protein content of the aqueous solution in step (a) is in the range of 5%-15% (w / w).

[0158] Example 13. The method as described in any of the preceding examples, wherein the lipid content of the aqueous solution in step (a) is in the range of 0%-2% (w / w), such as 0%-1% (w / w), such as in the range of 0.4%-0.8% (w / w).

[0159] Example 14. The method as described in any of the preceding examples, wherein the heat treatment in step (c) is performed for 5-30 minutes at a temperature in the range of 80°C-125°C, such as for 10-20 minutes at a temperature in the range of 85°C-121°C.

[0160] Example 15. The method as described in any of the preceding examples, wherein the heat treatment in step (c) is performed at a temperature in the range of 85°C to 95°C for 10 to 20 minutes, such as 10 minutes at 90°C or 15 minutes at 85°C.

[0161] Example 16. The method as described in any one of Examples 1-14, wherein the heat treatment in step (c) is performed for 10-20 minutes at a temperature in the range of 110°C-121°C, such as for 15 minutes at about 115°C.

[0162] Example 17. The method as described in any of the preceding examples, wherein the heat treatment in step (c) further comprises ultra-high temperature (UHT) treatment lasting for 1-120 seconds at a temperature in the range of 126°C-150°C, such as lasting for 2-90 seconds at a temperature in the range of 130°C-145°C.

[0163] Example 18. The method as described in any one of Examples 1-13, wherein the heat treatment in step (c) is performed for 1-120 seconds at a temperature in the range of 121°C-150°C, for 1-90 seconds at a temperature in the range of 130°C-145°C, for 30 seconds at 130°C, for 3-4 seconds at 140°C, or for 1-2 seconds at 145°C.

[0164] Example 19. The method as described in any one of Examples 1-13, wherein the heat treatment comprises a first step of heat treatment for 5-30 minutes at a temperature in the range of 80°C-125°C, such as for 10-20 minutes at a temperature in the range of 85°C-121°C, followed by a second step of heat treatment for 1-120 seconds at a temperature in the range of 126°C-150°C, such as for 1-90 seconds at a temperature in the range of 130°C-145°C.

[0165] Example 20. The method as described in any of the preceding examples, wherein the legume protein isolate is derived from or obtained from peas, soybeans, or combinations thereof.

[0166] Example 21. The method as described in any of the preceding examples, wherein the legume protein isolate is derived from or obtained from peas.

[0167] Example 22. The method as described in any of the preceding examples, wherein, when mixed with an acidic beverage, the modified legume protein isolate aqueous dispersion has improved stability compared to an aqueous dispersion of the modified legume protein prepared by a similar method in which a protein deamidase is not added to an aqueous solution of the legume protein isolate.

[0168] Example 23. The method as described in Example 22, wherein the acidic beverage is a coffee beverage or a tea beverage, optionally a geothermal coffee beverage or a hot tea beverage.

[0169] Example 24. The method as described in any of the preceding examples, wherein, when mixed with an acidic beverage, the modified legume protein aqueous dispersion has improved stability, such as reduced flocculation levels, compared to an aqueous dispersion of the modified legume protein prepared by a similar method in which a protein deamidase is not added to an aqueous solution of the legume protein isolate.

[0170] Example 25. The method as described in any of the preceding examples, wherein, when mixed with an acidic beverage, the modified legume aqueous dispersion has improved stability, such as reduced flocculation levels, compared to an aqueous dispersion of the modified legume prepared using a similar method in which a protein deamidase is not added to an aqueous solution of the legume protein isolate and a heat treatment step following treatment with a protein deamidase is not used.

[0171] Example 26. The method as described in any of the preceding examples, wherein after storage at a temperature of at least 4°C for at least 15 minutes, the modified legume protein aqueous dispersion has a reduced level of protein precipitation compared to an aqueous dispersion of the modified legume protein prepared by a similar method in which a protein deamidase is not added to an aqueous solution of the legume protein isolate.

[0172] Example 27. The method as described in any of the preceding examples, wherein after storage at a temperature of at least 4°C for at least 15 minutes, the modified legume protein aqueous dispersion has a reduced level of protein precipitation compared to an aqueous dispersion of the modified legume protein prepared by a similar method in which a protein deamidase is not added to an aqueous solution of the legume protein isolate and a heat treatment step following treatment with a protein deamidase is not used.

[0173] Example 28. The method as described in any of the preceding examples, wherein the aqueous dispersion of the modified legume protein comprises: lipids, sugars, proteins, vitamins, minerals, amino acids, flavorings, dietary fiber, salts, and any combination thereof.

[0174] Example 29. The method as described in the preceding examples, wherein the additional food ingredient is a lipid, such as an oil, preferably a vegetable oil, more preferably soybean oil, and / or a sugar, such as sucrose.

[0175] Example 30. The method as described in any of the preceding examples, wherein the aqueous dispersion of the modified legume protein is a soy beverage, pea beverage, broad bean beverage, lentil beverage, or a beverage containing any combination thereof.

[0176] Example 31. The method as described in any of the preceding examples, wherein the aqueous dispersion of the modified legume protein is a pea beverage, a soy beverage, or a beverage comprising a combination thereof.

[0177] Example 32. The method as described in any of the preceding examples, wherein the aqueous dispersion of the modified legume protein is a pea beverage.

[0178] Example 33. The method as described in any of the preceding examples, wherein the aqueous dispersion of the modified legume protein is substantially free of additives such as stabilizers and / or emulsifiers.

[0179] Example 34. The method as described in any of the preceding examples, wherein the aqueous dispersion of the modified legume protein is suitable for storage at a temperature of 3°C to 30°C.

[0180] Example 35. The method as described in any of the preceding examples, wherein the aqueous dispersion of the modified legume protein is suitable for storage at a temperature of 3°C-5°C, such as at about 4°C, or at a temperature of 16°C-24°C, such as at about 20°C.

[0181] Example 36. The method as described in any of the preceding examples, wherein the protein deamidase is derived from or obtained from a species of the genus *Chlorella*, more preferably from or obtained from *Chlorella utilis* or *Chlorella viscera*.

[0182] Example 37. The method as described in any of the preceding examples, wherein the protein deamidase comprises a polynucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with SEQ ID NO: 2, 4, 6, 8, or 10.

[0183] Example 38. The method as described in any of the preceding examples, wherein the protein deamidase comprises a polynucleotide sequence of SEQ ID NO: 2, 4, 6, 8 or 10.

[0184] Example 39. An aqueous dispersion of a modified legume protein in the pH range of 6-8, wherein the modification is obtained by enzymatic deamidation with a protein deamidase, and wherein the legume protein is derived from or obtained from a legume protein isolate, such as a legume protein isolate derived from or obtained from peas, soybeans, broad beans, lentils, or any combination thereof.

[0185] Example 40. An aqueous dispersion as described in Example 39, wherein the legume protein is derived from or obtained from peas, soybeans, or a combination thereof.

[0186] Example 41. An aqueous dispersion as described in any one of Examples 39-40, wherein the legume protein is derived from or obtained from peas.

[0187] Example 42. An aqueous dispersion as described in any one of Examples 39-41, wherein the modified legume protein aqueous dispersion is a dairy alternative beverage, such as a pea-based beverage, a soy-based beverage, or a beverage containing a combination thereof.

[0188] Example 43. An aqueous dispersion as described in any one of Examples 39-42, wherein the aqueous dispersion further comprises a protein deamidase.

[0189] Example 44. An aqueous dispersion as described in any one of Examples 39-43, wherein the protein deamidase comprises a polypeptide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with SEQ ID NO: 2, 4, 6, 8, or 10.

[0190] Example 45. The aqueous dispersion as described in any one of Examples 39-44, characterized in that it has undergone a heat treatment step.

[0191] Example 46. An aqueous dispersion as described in the preceding examples, wherein the heat treatment is performed after enzymatic deamidation with the protein deamidase.

[0192] Example 47. An aqueous dispersion as described in any one of Examples 45 or 46, wherein the heat treatment is performed for 5-30 minutes at a temperature in the range of 80°C-125°C, such as for 10-20 minutes at a temperature in the range of 85°C-121°C, and / or for 1-120 seconds at a temperature in the range of 126°C-150°C, such as for 2-90 seconds at a temperature in the range of 130°C-145°C.

[0193] Example 48. An aqueous dispersion as described in any one of Examples 45 or 46, wherein the heat treatment comprises a first step of heat treatment at a temperature in the range of 80°C-125°C for 5-30 minutes, such as at a temperature in the range of 85°C-121°C for 10-20 minutes, followed by a second step of heat treatment at a temperature in the range of 126°C-150°C for 1-120 seconds, such as at a temperature in the range of 130°C-145°C for 1-90 seconds.

[0194] Example 49. An aqueous dispersion as described in any one of Examples 39-48, wherein the aqueous dispersion is substantially free of added stabilizers and / or emulsifiers.

[0195] Example 50. An aqueous dispersion as described in any one of Examples 39-49, characterized in that the aqueous dispersion has improved colloidal stability compared to an aqueous dispersion of modified legume protein prepared without the use of protein deamidase.

[0196] Example 51. An aqueous dispersion as described in any one of Examples 45-50, characterized in that the aqueous dispersion has improved colloidal stability compared to an aqueous dispersion of modified legume protein prepared without the use of protein deamidase and after a heat treatment step following treatment with said protein deamidase.

[0197] Example 52. Use of protein deamidase in improving colloidal stability in the production of aqueous dispersions of modified legume proteins.

[0198] Example 53. Use as described in Example 52, wherein the aqueous dispersion of the modified legume protein has a pH in the range of pH 6-8.

[0199] Example 54. Use as described in any one of Examples 52-53, wherein the modified legume protein is derived from or obtained from legume protein isolates of peas, soybeans, broad beans, lentils or any combination thereof.

[0200] Example 55. Use as described in any one of Examples 52-54, wherein the modified legume protein is derived from or obtained from legume protein isolates of peas, soybeans or any combination thereof.

[0201] Example 56. Use as described in any one of Examples 52-55, wherein the modified legume protein is derived from or obtained from legume protein isolates from peas.

[0202] Example 57. Use as described in any one of Examples 52-56, wherein the protein deamidase comprises a polypeptide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with SEQ ID NO: 2, 4, 6, 8, or 10.

[0203] Example 58. Use as described in any one of Examples 52-57, wherein the aqueous dispersion is a dairy alternative beverage, such as a pea-based dairy alternative beverage, a soy-based dairy alternative beverage, or a dairy alternative beverage comprising a combination of soy and peas.

[0204] Example 59. Use as described in any one of Examples 52-58, wherein the aqueous dispersion of the modified legume protein is used in acidic beverages, such as hot acidic beverages.

[0205] Example 60. Use as described in any one of Examples 52-59, wherein after storage at a temperature of at least 4°C for at least 15 minutes, the modified legume protein aqueous dispersion has improved colloidal stability compared to an aqueous dispersion of the modified legume protein prepared without the use of protein deamidase.

[0206] Example 61. Use as described in any one of Examples 52-60, wherein after storage at a temperature of at least 4°C for at least 15 minutes, the modified legume protein aqueous dispersion has a reduced level of protein precipitation compared to an aqueous dispersion of the modified legume protein prepared without the use of protein deamidase.

[0207] Example 62. Use of protein deamidase and heat treatment in improving colloidal stability in the production of aqueous dispersions of modified legume proteins in the pH range of 6-8.

[0208] Example 63. Use as described in Example 62, wherein the modified legume protein is derived from or obtained from legume protein isolates of peas, soybeans, broad beans, lentils or any combination thereof.

[0209] Example 64. Use as described in any one of Examples 62 or 63, wherein the modified legume protein is derived from or obtained from legume protein isolates of peas, soybeans or combinations thereof, preferably derived from or obtained from peas.

[0210] Example 65. Use as described in any one of Examples 62-64, wherein the heat treatment is performed after the protein deamidase treatment.

[0211] The inventions described and claimed herein are not limited to the specific embodiments disclosed herein, as these embodiments are intended to serve as illustrative examples of several aspects of the invention. Any equivalent embodiments, together with combinations of one or more of these embodiments, are intended to be included within the scope of the invention.

[0212] This document cites several references, the disclosures of which are incorporated herein by reference in their entirety. The invention is further described through the following examples, which should not be construed as limiting the scope of the invention.

[0213] Example Material enzymes The following enzymes are used throughout the example: Protein deamidase The protein glutaminase derived from *Chlorobacterium visceratum* (strain formerly known as *Chlorobacterium* species-62563) has a mature polypeptide sequence as shown in SEQ ID NO: 2. Propeptide cleavage was achieved by treating the deamidase in SEQ ID NO: 1 with a site-specific endopeptidase. The site-specific endopeptidase used was derived from *Bacillus licheniformis* (…). Bacillus licheniformis The glutamyl endopeptidase obtained after maturation is the polypeptide shown in SEQ ID NO: 2.

[0214] Example 1: Assay of protein deamidase activity The activity assay consists of two separate parts: 1) An enzymatic step in which ammonia is formed through the catalytic action of a protein deamidase; and 2) Non-enzymatic detection step, wherein the ammonia formed in step (1) is derivatized into a blue indophenol compound with maximum absorption at 630 nm.

[0215] In step (1), ammonia is produced by deamidation of a protein deamidase. In step (2), the generated ammonia reacts with phenol under alkaline conditions to form dioxane. This reaction is catalyzed by sodium pentacyanonitroferro(III) ferric(III) nitrosyl ferrate (sodium nitroprusside). "Colorimetric reagent solution A" contains phenol and sodium nitroprusside. "Colorimetric reagent solution B" provides the alkaline reaction conditions. The intermediate is then oxidized by adding sodium hypochlorite ("Colorimetric reagent solution C") to form indophenol blue. This compound absorbs visible light at 630 nm. The enzyme activity is then calculated using a standard curve.

[0216] Measurement procedure: The enzymatic step involving ammonia formation (1) Reagents: Diluent for assay: 0.2 M sodium phosphate buffer, 0.01% Triton X-100, pH 6.5.

[0217] Assay buffer: Same as above. Used for preparing stock solutions and diluting samples for protein deamidases (hereinafter referred to as "enzymes").

[0218] Substrate solution: 30 mM Z-Gln-Gly (Merck C6154-1G), in the assay dilution (check pH after dissolution).

[0219] Termination solution: 0.4 M TCA.

[0220] Standard: NH4Cl diluted in the assay dilution solution (ammonium standard for IC, Merck 59755-100ML, 1000 mg / L NH4Cl). + (in water) (see also the "Standard Curve" section).

[0221] The enzyme product is dissolved / diluted in the assay buffer and a suitable dilution is prepared to produce a linear assay response.

[0222] Incubation: 1. Add 10 μL of diluted enzyme sample in triplicate to the wells of a 96-well microtiter plate (MTP).

[0223] 2. Add 100 μL of substrate solution to each well.

[0224] 3. For blank samples, add 100 μL of 0.4 M TCA solution.

[0225] 4. Seal the board with a transparent sealing film.

[0226] 5. Incubate the plate at 37°C and 500 rpm for 10 minutes on a thermostatic mixer equipped with a heated lid.

[0227] 6. To terminate the reaction, carefully add 100 μL of 0.4 M TCA solution (except for the blank sample, which already contains TCA).

[0228] Total reaction volume: 210 μL.

[0229] Step (2) Ammonia Detection Step Reagents: Colorimetric reagent A: 4% (w / v) phenol, 0.015% (w / v) sodium pentacyanonitroferro(III) ferrate dihydrate (sodium nitroprusside) (Na2[Fe(CN)5NO) 2H2O).

[0230] Colorimetric reagent B: 5% (w / v) potassium hydroxide.

[0231] Colorimetric reagent C: 28% (w / v) potassium carbonate, 6% (v / v) sodium hypochlorite (Sigma-Aldrich 239305-25 ml, effective Cl2 <5%).

[0232] Incubation: 1. Transfer 15 μL from each well in step (1) to a new 96-well MTP.

[0233] 2. Transfer 45 μL of Milli-Q water into each well.

[0234] 3. Add 30 μL of colorimetric reagent B to each well (on the lab bench, gently shake to mix).

[0235] 4. Add 60 μL of colorimetric reagent A to each well (shake gently by hand on the lab bench to mix).

[0236] 5. Add 60 μL of colorimetric reagent C to each well (shake gently by hand on the lab bench to mix).

[0237] 6. Color development: Carefully seal the plate and place it on the lab bench for 30 minutes.

[0238] 7. Carefully transfer the MTP to the microplate reader and measure the absorbance at 630 nm.

[0239] Total reaction volume: 210 μL Standard curve: Standard stock solution: 1000 mg NH4 + / L.

[0240] In the ammonia detection procedure, a standard curve is prepared by adding ammonium standards at different dilutions to the determination dilution buffer. Specifically, 15 μL of diluted ammonia standard is mixed with 45 μL of water, and then the colorimetric reagents B, A, and C are added in the order given above.

[0241] The amount of enzyme that produces 1 μmol of ammonia per minute at 37°C is defined as 1 unit (indophenol assay unit; IPA (U)): in It can be abbreviated as in • C NH4+ It is the ammonia concentration in the reaction solution obtained from the ammonium standard curve (i.e., taking into account the dilution of the pre-diluted ammonium standard solution in the ammonia derivatization step).

[0242] • 18.04 is the molecular weight of ammonium used in the standard solution.

[0243] • V 反应 It is the reaction volume in the pore when ammonia is generated (210 μL).

[0244] • V 酶 This is the volume (10 μL) of enzyme solution added to the well when ammonia is generated.

[0245] • V NH3 The detection volume is the reaction volume in the well when detecting ammonia (210 μL).

[0246] Example 2: Colloidal stability of modified legume-based beverages In this example, pea protein isolate was used to test whether the method according to the invention could be used to prepare legume-based beverages with improved colloidal stability.

[0247] Pea beverages were prepared in the laboratory using the following procedure. A 10% solution (m / v) (pH 7) of pea protein isolate (82.2% protein content based on dry solids) was prepared by mixing with deionized H₂O. The 10% pea beverage solution was then treated with protein deamidase (dosages of 0.6 IPA(U) / g protein and 2 IPA(U) / g protein). The reaction was carried out at 60°C for 1 hour, followed by enzyme inactivation at 85°C for 10 minutes, and then cooled to room temperature on ice. A control sample (pH 7) was prepared following the same procedure as above, but without the use of protein deamidase. Pea protein samples (both the protein deamidase-treated sample and the control sample) without additives (e.g., no emulsifiers or stabilizers) were then diluted to a protein content of 3.2% and mixed. Colloidal stability was visually observed after the samples had stood for 15 minutes. The results of the colloidal stability test were presented in [the table / documentation]. Figure 1 As shown in the image. Figure 1The effects of adding a protein deamidase to the method of the present invention are shown, and the suitability of the protein deamidase in the preparation of dispersions from pea protein isolates is clearly demonstrated. In particular, and as illustrated herein, improved pea protein isolate-based dispersions treated with a protein deamidase according to the method disclosed herein are obtained, exhibiting improved colloidal stability, as can be visually observed, with no sedimentation or flocculation in the protein deamidase-treated samples. Figure 1 (The sample on the right with the check mark below the sample).

[0248] In another experiment, different pea protein isolates from Cosucra (Cosucra C9, 83% protein content on a dry basis) were tested by preparing a 10% solution (m / v) (pH 7) by mixing pea protein isolates with deionized H2O. The 10% pea beverage solution was then treated with protein deamidase (dose 2 IPA(U) / g protein). The reaction was carried out at 60°C for 1 hour, followed by enzyme inactivation at 85°C for 10 minutes, and then cooled to room temperature on ice. A control sample (pH 7) was prepared again using the same procedure, but without protein deamidase. The additive-free pea protein sample was then diluted to a protein content of 6.4% and mixed. Colloidal stability was visually observed after the sample was allowed to stand for 15 minutes. The results of this study confirmed the findings described above. Figure 1 The previous findings shown indicate that protein deamidases effectively improve the colloidal stability of dispersions prepared from pea protein isolates using the method claimed herein.

[0249] To confirm that the improved colloidal stability was indeed a result of protein deamidase treatment of legume protein, rather than a heat treatment step alone, a simple experiment was conducted. An aqueous dispersion of legume protein was prepared following the same procedure as described above, except that the protein deamidase treatment step was omitted. Two different samples were prepared: a first sample without heat treatment and a second sample treated at 85°C for 10 minutes. The results were... Figure 2 As shown in the figure, the sample indicated by the check mark below the sample is the sample that has undergone heat treatment. Figure 2 The sample in the left image was prepared using 4% double-layered pea protein isolate, and Figure 2 The sample in the image on the right was prepared using 8% Ingredion pea protein isolate. All tested samples were at pH 7. As seen for both sample groups, heat treatment with only the PPI-based solution resulted in some reduction in sedimentation in the samples; however, no improvement in dispersibility or colloidal stability was observed, thus confirming that improved colloidal stability is only observed when protein deamidase treatment is performed in conjunction with the heat treatment step. Figure 1(The image on the right with the checkmark below the sample).

[0250] In summary, this article reports and Figure 1 and 2 The results illustrated clearly demonstrate the excellent results obtained using the method of the present invention in obtaining modified legume proteins, particularly aqueous dispersions of legume proteins, with improved dispersibility and colloidal stability. Furthermore, data not shown here further confirm that the modified legume protein aqueous dispersions exhibit improved stability both immediately and after storage when mixed with hot, acidic beverages, particularly coffee beverages.

[0251] Example 3: Effects of protein deamidases and heat treatment on the colloidal stability of legume-based beverages In this example, two different commercial pea protein isolates were used to test whether the method according to the invention (particularly the combination of protein deamidase and heat treatment) could produce a legume-based beverage with improved colloidal stability.

[0252] Pea-based beverage samples were prepared using the following procedure. The samples included in the study are shown in Table 1.

[0253] Double-layered pea protein isolate (82.2% protein content based on dry solids) was mixed with deionized H₂O to prepare a 10% solution (m / v). The 10% pea beverage solution was then treated with protein deamidase (dose of 2 IPA(U) / g protein). The enzymatic reaction with protein deamidase was carried out at 65°C or 70°C for 1 hour. Control samples were included that were not heat-treated and / or treated with protein deamidase. For samples heat-treated after enzyme treatment, this was carried out at 85°C for 10 minutes, followed by cooling to room temperature on ice. After enzyme treatment, unheat-treated samples were directly cooled to room temperature on ice. The additive-free (e.g., no emulsifiers or stabilizers) samples were then diluted to a protein content of 3.2% (w / w) and mixed.

[0254] After allowing the samples to stand at room temperature for 15 minutes, the colloidal stability was visually inspected. Results from the colloidal stability test are shown below. Figure 3 The study clearly demonstrated the effectiveness of using a combination of protein deamidase and heat treatment in preparing aqueous dispersions of pea protein isolates. This was visually apparent as no particle aggregation, sedimentation, or flocculation occurred in samples subjected to both protein deamidase and heat treatment. Specifically, and as shown in [the study / exhibition]... Figure 3Visual inspection of samples 5 and 9 (prepared by treatment with protein deamidase followed by heat inactivation) showed optimal colloidal stability, with no particle aggregation or sedimentation observed in these samples. Not wishing to be bound by any particular theory, the inventors of this invention believe that heat treatment contributes to improving the colloidal stability of aqueous dispersions of legumes by increasing the solubility of legume proteins.

[0255] It is also confirmed that the protein deamidases for which protection and disclosure are claimed herein can operate at higher incubation temperatures, such as those exemplified herein as 65°C and 70°C.

[0256] Table 1. Pea-based beverage samples prepared from pea protein isolate (4% dry matter) using protein deamidase and / or heat treatment. CTRL, Control; PD, Protein deamidase; RT, Room temperature; temp, Temperature In another experiment, pea protein isolate from Cosucra (Cosucra C9, 83% protein content on a dry basis) was tested by preparing a 10% solution (m / v) by mixing the pea protein isolate with deionized H2O. The samples included in the study are provided in Table 2. The 10% pea beverage solution was then treated with protein deamidase (dose 2 IPA(U) / g protein). The enzymatic reaction was carried out at 65°C or 70°C for 1 hour. Control samples untreated and / or untreated with protein deamidase were included. For samples heat-treated after enzyme treatment, this was carried out at 85°C for 10 minutes, followed by cooling to room temperature on ice. Untreated samples were cooled directly to room temperature on ice after enzyme treatment. Samples without additives (e.g., no emulsifiers or stabilizers) were then diluted to a protein content of 3.2% (w / w) and mixed.

[0257] After allowing the sample to stand at room temperature for 15 minutes, visually inspect the colloidal stability (see...). Figure 4 The results of this study confirm the findings mentioned above. Figure 3 The previous finding shown is that a combination of protein deamidase treatment followed by heat treatment of legume proteins can improve colloidal stability. This is exemplified by... Figure 4 As seen in samples 5 and 9, these samples were prepared by incubating with protein deamidase at 65°C or 70°C, respectively, followed by heat inactivation at 85°C for 10 minutes, demonstrating the best colloidal stability among all tested samples.

[0258] Table 2. Pea-based beverage samples prepared from Cosucra C9 pea protein isolate (8% dry matter) using protein deamidase and / or heat treatment. CTRL, Control; PD, Protein deamidase; RT, Room temperature; temp, Temperature

Claims

1. A method for obtaining an aqueous dispersion of modified legume protein, the method comprising the following steps: (a) Provide an aqueous solution of legume protein isolate with a protein content in the range of 1%-20% (w / w); (b) Treating the aqueous solution from step (a) with a protein deamidase to obtain enzymatically deamidated legume protein; and (c) The enzymatically deamidated legume protein from step (b) is subjected to heat treatment to obtain an aqueous dispersion of the modified legume protein. In steps (b) and (c), the pH is in the range of pH 6-8, and The legume protein isolates described herein are derived from or obtained from peas, soybeans, broad beans, lentils, or any combination thereof, and The modified legume protein aqueous dispersion, after being stored at a temperature of at least 4°C for 15 minutes, exhibits improved colloidal stability compared to an aqueous dispersion of modified legume protein prepared using a similar method but without the use of protein deamidase and a heat treatment step following treatment with the protein deamidase.

2. The method of claim 1, wherein the protein deamidase treatment in step (b) is performed at a temperature in the range of 50°C-70°C, such as at a temperature in the range of 60°C-70°C for 15-90 minutes, or such as for 30-60 minutes.

3. The method of claim 1 or 2, wherein no stabilizer and / or emulsifier is present in any one of steps (a), (b) and / or (c).

4. The method as described in any of the preceding claims, wherein the protein content of the aqueous solution in step (a) is in the range of 3%-20% (w / w), such as 5%-15% (w / w).

5. The method as described in any of the preceding claims, wherein the lipid content of the aqueous solution in step (a) is in the range of 0%-2% (w / w), such as 0%-1% (w / w).

6. The method as claimed in any of the preceding claims, wherein the heat treatment in step (c) is performed for 5-30 minutes at a temperature in the range of 80°C-125°C, such as for 10-20 minutes at a temperature in the range of 85°C-121°C.

7. The method of any of the preceding claims, wherein the legume protein isolate is derived from or obtained from peas, soybeans, or combinations thereof.

8. The method of any of the preceding claims, wherein the protein deamidase is derived from or obtained from a species of the genus *Chlorella*, such as *Chlorella utilis* or *Chlorella viviparus*.

9. The method of any of the preceding claims, wherein the aqueous dispersion of the modified legume protein further comprises one or more additional food ingredients selected from the group consisting of lipids, sugars, proteins, vitamins, minerals, amino acids, flavorings, dietary fiber, salt, and any combination thereof.

10. An aqueous dispersion of a modified legume protein in the pH range of 6-8, wherein the modification is obtained by enzymatic deamidation with a protein deamidase followed by heat treatment, and wherein the legume protein is derived from or obtained from legume protein isolates of peas, soybeans, broad beans, lentils, or any combination thereof.

11. The aqueous dispersion of claim 10, wherein the legume protein is derived from or obtained from legume protein isolates of peas, soybeans, or combinations thereof.

12. The aqueous dispersion of any one of claims 10 or 11, wherein the aqueous dispersion is substantially free of added stabilizers and / or emulsifiers.

13. Use of protein deamidase in the production of aqueous dispersions of modified legume proteins in the pH range of 6-8 for improving colloidal stability, wherein the modified legume protein is derived from or obtained from legume protein isolates of peas, soybeans, broad beans, lentils, or any combination thereof.

14. The use as claimed in claim 13, wherein the modified legume protein aqueous dispersion has improved colloidal stability compared to an aqueous dispersion of the modified legume protein prepared without the use of protein deamidase.

15. The use of protein deamidase and heat treatment in the production of aqueous dispersions of modified legume proteins in the pH range of 6-8 for improving colloidal stability, wherein the modified legume protein is derived from or obtained from legume protein isolates of peas, soybeans, broad beans, lentils, or any combination thereof.

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