Modified leguminous protein with improved colloidal stability

Abstract

The present invention relates to novel methods of using protein deamidase in obtaining an aqueous dispersion of modified leguminous protein with improved colloidal stability.

Description

[0001] MODIFIED LEGUMINOUS PROTEIN WITH IMPROVED COLLOIDAL STABILITY

[0002] Reference to sequence listing

[0003] This application contains a Sequence Listing in computer readable form. The computer readable form is incorporated herein by reference.

[0004] FIELD OF THE INVENTION

[0005] The present invention relates to the use of a protein deamidase and heat treatment in methods for obtaining an aqueous dispersion of modified leguminous protein with improved colloidal stability.

[0006] BACKGROUND OF THE INVENTION

[0007] The number of people pursuing vegan, vegetarian or non-dairy diets for health and other reasons has increased in recent years. Further, food products made from animals, particularly cows, are increasingly recognized for their high environmental costs. These factors are leading to a greater demand for dairy alternative plant-based food products to replace foods traditionally derived from milk, including milk, creamer, cheese, yogurt, and ice cream.

[0008] Legumes are recognized as a valuable and low-cost source of high-quality protein products, such as, e.g., concentrates and isolates. Industrial scale production typically has applied soybean as the protein source, yet, pea protein, such as, e.g., pea protein isolate, offers a promising alternative to soy-derived protein products.

[0009] Plant proteins, including leguminous plant protein, often have low solubility and poor functional properties and may require extensive modification to obtain an industrially viable product which meets the consumers’ demands in terms of taste, texture, and appearance. Such extensive modifications may pose significant challenges to manufacturers of plant proteins and plant protein derived food products, in particular when it comes to obtaining plant-based food products which fulfil the consumer’s requirements for clean label while, at the same time, keeping the cost of manufacturing low.

[0010] Deamidation is known to improve the solubility of plant proteins and thereby improve functional properties of the plant proteins, including foaming activity, foaming stability, emulsification activity and emulsification stability. Deamidation can be chemical or enzymatic, and in the latter case, in particular protein deamidases offer an effective means for modifying plant proteins without compromising the organoleptic or techno-functional properties of the modified plant protein.

[0011] EP 4201 216 A1 describes plant milk treated with a protein deamidase to thereby prevent the coagulation of the plant milk in the case of adding to a hot liquid beverage, a hot liquid food, etc. EP 4 201 216 A1 is thus concerned with preventing aggregation when plant milk is mixed with a heated liquid food or beverage, such as coffee. EP 4201 216 A1 does not describe use of protein deamidase and heat treatment for improving colloidal stability of aqueous dispersions of modified leguminous proteins.

[0012] US 2023 / 0345970 A1 describes deamidated pea protein isolates, processes for making a deamidated pea protein isolate, and use of the deamidated pea protein isolate in food and beverage compositions. US 2023 / 0345970 A1 is not concerned with the use of protein deamidase and heat treatment for improving colloidal stability of aqueous dispersions of modified leguminous proteins.

[0013] In view of the above, it is an object of the present invention to identify improved methods for obtaining aqueous dispersions of modified leguminous protein with improved colloidal stability.

[0014] SUMMARY OF THE INVENTION

[0015] The present inventor has surprisingly found that by treating an aqueous solution of leguminous protein isolate with a protein deamidase followed by a step of heat treatment, an aqueous dispersion of modified leguminous protein is obtained which has improved colloidal stability, dispersibility and reduced risk of flocculation, even during storage. Thereby, a stable, organoleptically satisfactory aqueous dispersion of modified leguminous protein can be obtained which has the added benefit of being particularly suitable for mixing with other beverages, such as acidic beverages. Thus, an aqueous dispersion of modified leguminous protein can be obtained which has both improved solubility and taste and improved beverage stability.

[0016] The modified leguminous protein as claimed herein thus not only meets consumers’ demands in terms of satisfactory organoleptic properties, but also achieves storage and beverage stability.

[0017] The invention therefore provides a method of obtaining an aqueous dispersion of a modified leguminous protein, comprising the steps of:

[0018] (a) providing an aqueous solution of leguminous protein isolate having a protein content in the range of 1-20% (w / w);

[0019] (b) treating the aqueous solution of step (a) with a protein deamidase to obtain an enzymatically deamidated leguminous protein; and

[0020] (c) heat treating the enzymatically deamidated leguminous protein of step (b) to obtain the aqueous dispersion of modified leguminous protein, wherein in steps (b) and (c) the pH is in the range of pH 6-8, and wherein the leguminous protein isolate is derived or obtained from pea, soy, fava bean, lentil, or any combination thereof.

[0021] By using the herein claimed and disclosed method, an aqueous dispersion of modified leguminous protein is obtained, which after storage for 15 minutes at a temperature of at least 4°C, has an improved colloidal stability compared to an aqueous dispersion of modified leguminous protein prepared using a similar method but without the use of a protein deamidase and heat treatment to obtain the aqueous dispersion of modified leguminous protein.

[0022] In the context of this invention, it is essential to differentiate between colloidal stability and coffee stability, as both terms refer to distinct 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 without the separation of its constituent phases over time. The method of the present invention results in an aqueous dispersion of modified leguminous protein with improved colloidal stability. This means that the aqueous dispersion remains homogeneously mixed without settling or aggregation of the protein particles even after prolonged storage. The improvement in colloidal stability is achieved through enzymatic deamidation and subsequent heat treatment, which ensures that the protein particles stay well- dispersed in the solution. Coffee stability, on the other hand, pertains specifically to the ability of an aqueous dispersion of leguminous 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 the homogeneity and preventing separation or curdling when the aqueous dispersion of leguminous protein is combined with the acidic beverages.

[0023] Preferably, the enzymatic treatment with the protein deamidase is carried out at a temperature in the range of 50-70°C for 15-90 minutes. In an embodiment, the treatment with the protein deamidase is at carried out at a temperature in the range of 60-70°C for 30-60 minutes. As shown in Example 3 and Figure 3 and 4, the herein disclosed methods for obtaining aqueous dispersions of leguminous proteins with improved colloidal stability can be carried out at short enzyme incubation times and at high enzyme incubation temperatures. These improvements mean less downtime, reduced energy consumption, and the ability to produce a high-quality product more efficiently.

[0024] The modified leguminous protein obtained according to the methods of the invention has an improved colloidal stability which is achieved without the need for added emulsifiers and / or stabilizers and without the need for adjusting pH outside the range of pH 6-8. Thus, a modified leguminous protein is obtained which requires minimum processing and can be labelled as a clean-label product. Thus, in some preferred embodiments, an emulsifier and / or stabilizer is not present in any of steps (a), (b) and / or (c).

[0025] Using the methods of the invention, an aqueous dispersion of modified leguminous protein can be obtained which has improved immediate and long-term colloidal stability and improved stability when mixed with acidic beverages. Thus, the invention further relates to an aqueous dispersion of modified leguminous protein having a pH in the range of 6-8, wherein the modified leguminous protein is obtained by treatment with a protein deamidase, followed by a heating step, and wherein the leguminous protein is derived or obtained from a leguminous protein isolate of pea, soy, fava bean, lentil, or any combination thereof. In particular, the inventor has found that the aqueous dispersion of modified leguminous protein according to the invention is resistant to flocculation, both immediately after its preparation and after days, or weeks, of storage, both when stored at refrigerated conditions and at room temperature. Thus, an aqueous dispersion of modified leguminous protein can be obtained which has the functional properties, including creaminess, taste, and stability, which consumers demand and expect.

[0026] The improved stability of the aqueous dispersion of modified leguminous protein obtained according to the methods as claimed herein further avoids the need for adding emulsifiers and / or stabilizers, both during the process of obtaining the final product and to the final product. Thus, the aqueous dispersion of modified leguminous protein disclosed herein and obtained using the methods of the invention, not only comprises satisfactory organoleptic properties, but may be used in obtaining healthier, clean-label dairy alternative food products which are high in protein and low in added fat and sugar.

[0027] The invention also provides for the use of a protein deamidase in the production of an aqueous dispersion of modified leguminous protein to improve colloidal stability.

[0028] The invention also provides for the use of a protein deamidase and heat treatment in the production of an aqueous dispersion of modified leguminous protein to improve colloidal stability.

[0029] BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 illustrates colloidal stability of legume-based suspensions obtained using the methods disclosed herein with or without the addition of a protein deamidase.

[0031] Figure 2 illustrates colloidal stability of legume-based suspensions prepared with or without a step of heat treatment.

[0032] Figure 3 illustrates colloidal stability of legume-based suspensions obtained with or without protein deamidase and / or heat treatment.

[0033] Figure 4 illustrates colloidal stability of legume-based suspensions obtained with or without protein deamidase and / or heat treatment.

[0034] SEQUENCES

[0035] SEQ ID NO: 1: Protein deamidase derived from Chryseobacterium viscerum (the strain has formerly been referred to as Chryseobacterium sp-62563) having the mature polypeptide sequence shown as SEQ ID NO: 2.

[0036] SEQ ID NO: 2: Mature polypeptide sequence of protein deamidase derived from Chryseobacterium sp-62563.

[0037] SEQ ID NO: 3: Protein deamidase derived from Chryseobacterium proteolyticum having the mature polypeptide sequence shown as SEQ ID NO: 4.

[0038] SEQ ID NO: 4: Mature polypeptide sequence of protein deamidase derived from Chryseobacterium proteolyticum. SEQ ID NO: 5: Protein deamidase derived from Chryseobacterium gambrini having the mature polypeptide sequence shown as SEQ ID NO: 6.

[0039] SEQ ID NO: 6: Mature polypeptide sequence of protein deamidase derived from Chryseobacterium gambrini.

[0040] SEQ ID NO: 7: Protein deamidase derived from Chryseobacterium culicis having the mature polypeptide sequence shown as SEQ ID NO: 8.

[0041] SEQ ID NO: 8: Mature polypeptide sequence of protein deamidase derived from Chryseobacterium culicis.

[0042] SEQ ID NO: 9: Protein deamidase derived from Chryseobacterium defluvii having the mature polypeptide sequence shown as SEQ ID NO: 10.

[0043] SEQ ID NO: 10: Mature polypeptide sequence of protein deamidase derived from Chryseobacterium defluvii.

[0044] DETAILED DESCRIPTION OF THE INVENTION

[0045] In accordance with this detailed description, the following definitions apply. Note that the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0046] Reference to "about" a value or parameter herein includes aspects that are directed to that value or parameter per se. For example, description referring to "about X" includes the aspect "X". When used in combination with measured values, "about" includes a range that encompasses at least the uncertainty associated with the method of measuring the particular value and can include a range of plus or minus two standard deviations around the stated value.

[0047] Unless defined otherwise or clearly indicated by context, all percentages are percentage by weight (percent w / w or “% (w / w)”).

[0048] The drawings featured in the figures are for the purpose of illustrating certain convenient embodiments of the invention and are not to be considered as limitation thereto.

[0049] Furthermore, it will be appreciated that embodiments described in connection with one of the aspects described herein may equally be applied to the other aspects, unless otherwise stated.

[0050] Unless defined otherwise or clearly indicated by 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 belongs.

[0051] The present invention is based on the surprising and inventive finding that an aqueous dispersion of a modified leguminous protein, which has improved colloidal stability, dispersibility and reduced risk of flocculation, even after storage or mixing with acidic beverages, can be obtained by treating a leguminous protein isolate with a protein deamidase followed by a step of heat treatment. As a result, a stable, such as, e.g., a shelf-stable, organoleptically satisfactory modified leguminous protein is obtained which when provided as an aqueous dispersion of the modified leguminous protein has the added benefit of being particularly suitable for mixing with other beverages, such as acidic beverages.

[0052] The surprising findings reported herein are exemplified in Examples 2 and 3 and illustrated in at least Figures 1-4, and based on these general observations, the inventor expects that the same benefits can be obtained using the methods of the invention on any suitable leguminous protein isolate derived or obtained from pea, soy, fava bean, lentil, or any combination thereof.

[0053] In a first aspect, the present invention relates to a method of obtaining an aqueous dispersion of modified leguminous protein, comprising the steps of:

[0054] (a) providing an aqueous solution of leguminous protein isolate having a protein content in the range of 1-20% (w / w);

[0055] (b) treating the aqueous solution of step (a) with a protein deamidase to obtain an enzymatically deamidated leguminous protein; and

[0056] (c) heat treating the enzymatically deamidated leguminous protein of step (b) to obtain the aqueous dispersion of modified leguminous protein, wherein in steps (b) and (c) pH is in the range of pH 6-8, and wherein the leguminous protein isolate is derived or obtained from pea, soy, fava bean, lentil, or any combination thereof.

[0057] The term “modified leguminous protein” is meant as a leguminous protein having been treated with a protein deamidase and subjected to heat treatment. The term “enzymatically deamidated leguminous protein” thus means a leguminous protein treated with a protein deamidase for deamidation. A person skilled in the art will know of suitable analytical methods to determine that enzymatic deamidation of a leguminous protein has occurred. One such method is exemplified in Example 1 by the measurement of free ammonium content (NH4). The modified leguminous protein can be ingested by humans or animals, preferably by humans. The modified leguminous protein claimed herein is plant-based.

[0058] The modified leguminous protein may be in an aqueous dispersion and may be referred to as an aqueous dispersion of a modified leguminous protein. The term “aqueous dispersion” has its normal meaning which is well known and understood by those of skill in the art. It means a liquid system in which very small solid particles are uniformly dispersed in an aqueous solution, such as, e.g., water.

[0059] The aqueous dispersion of modified leguminous protein as claimed herein comprises at least an enzymatically deamidated leguminous protein and may or may not be combined with additional food ingredients to produce the aqueous dispersion of modified leguminous protein. In some embodiments the aqueous dispersion of modified leguminous protein is intended for use as a beverage, e.g. a dairy alternative milk or drink. As used herein, the terms “drink", “milk” and "beverage" are used interchangeably and have the same meaning unless otherwise stated. The modified leguminous protein obtained according to the present invention exhibits an improved colloidal stability and improved acidic and / or heat stability and can be incorporated in a wide variety of food products. For example, the modified leguminous protein can be used directly as a food and has certain commercial value.

[0060] In the context of the present invention, the term “colloidal stability” means the ability of the particles in a liquid, such as an aqueous solution, to remain stable in dispersion. The ability of the modified leguminous protein to remain stable in dispersion without, e.g., formation of aggregates or agglomerates leading to change in particle size in dispersion, is meant as colloidal stability. Furthermore, the modified leguminous protein of the invention has improved resistance towards sedimentation, i.e., resistance towards particles settling. The colloidal stability and sedimentation of the modified leguminous protein may be determined by visual examination of an aqueous dispersion of the modified leguminous protein. The visual inspection may be carried out immediately and after longer storage, e.g., by placing the aqueous dispersion of modified leguminous protein in a transparent beaker allowing for the visual examination both immediately after pouring the liquid and following storage. Alternatively or in addition to the visual inspection, the colloidal stability and sedimentation may also be evaluated based on particle size analyses, such as, e.g., particle size distribution in the aqueous suspension. For example, the visual examination may be performed after 5-60 minutes incubation of the aqueous suspension of modified protein at room temperature or under refrigerated conditions.

[0061] Without wishing to be bound by any particular theory, it is believed that the colloidal stability of the modified leguminous protein obtained according to the methods disclosed herein is achieved by treating the leguminous protein isolate with a protein deamidase. Thus, in some embodiments the modified leguminous protein has an improved colloidal stability compared to a modified leguminous protein prepared using a similar method where a protein deamidase is not added to the aqueous solution of leguminous protein isolate.

[0062] In particular, after storage for 15 minutes at a temperature of at least 4°C, the aqueous dispersion of modified leguminous protein has an improved colloidal stability compared to an aqueous dispersion of modified leguminous protein prepared using a similar method where a protein deamidase is not added to the aqueous solution of leguminous protein isolate.

[0063] In particular, after storage for 15 minutes at a temperature of at least 4°C, the aqueous dispersion of modified leguminous protein has an improved colloidal stability compared to an aqueous dispersion of modified leguminous protein prepared using a similar method but without a step of heat treatment following the treatment with the protein deamidase. As confirmed at least by the results shown in Example 3 and Figures 3 and 4, the use of protein deamidase, followed by a step of heat treatment, results in aqueous dispersions of leguminous proteins having superior colloidal stability. The modified leguminous protein, such as the aqueous dispersion of modified leguminous protein, of the present invention is shelf-stable and can be stored under refrigerated conditions or at room temperature conditions.

[0064] In the context of the invention, room temperature conditions comprise temperatures at about 15-25°C, such as about 18-22°C, and cold and / or refrigerated conditions means temperatures at about 2-5°C, such as about 4°C.

[0065] The modified leguminous protein, and in particular the aqueous dispersion of the modified leguminous protein, has improved beverage stability. Without the wish of being bound by any particular theory, the improved colloidal stability, in particular the improved beverage stability, reported herein, is not just a result of improved solubility of the protein in the dispersion. I.e. the modified leguminous protein and aqueous dispersion of modified leguminous protein both have improved protein solubility in liquid dispersion and further an improved stability in liquid. This is seen, e.g., when mixing the aqueous dispersion of modified leguminous protein with an acidic, optionally hot, beverage, such as a coffee drink or a tea drink, wherein no feathering or sedimentation is visible upon mixing.

[0066] Thus, in some embodiments, the aqueous dispersion of modified leguminous protein has an improved stability when mixed with an acidic beverage. Also, in some embodiments, the aqueous dispersion of modified leguminous protein has an improved stability when mixed with a hot acidic beverage.

[0067] In some embodiments, the aqueous dispersion of modified leguminous protein is suitable for mixing with a hot acidic beverage, such as a coffee drink or a tea drink. In the context of the invention, a hot acidic beverage means a beverage having a temperature of at least 85°C when mixed with the aqueous dispersion of modified leguminous protein. 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 aqueous dispersion of modified leguminous protein. In some embodiments, the aqueous dispersion of modified leguminous protein has an improved stability, such as a reduced level of feathering, when mixed with a hot acidic beverage compared to an aqueous dispersion of modified leguminous protein prepared using a similar method where a protein deamidase is not added to the aqueous solution of leguminous protein isolate.

[0068] The aqueous dispersion of modified leguminous protein is stable over time, i.e., the aqueous dispersion is stable against precipitation of the proteins and other components contained therein. The stability may be observed immediately upon obtaining the aqueous dispersion of modified protein and may be observed visually 5 minutes, 10 minutes, 15 minutes or even 30 minutes after obtainment of the aqueous dispersion of modified protein.

[0069] The aqueous dispersion of modified leguminous protein is stable over an extended period of time, i.e., the aqueous dispersion is stable against precipitation of the proteins and other components contained therein under refrigerated and room temperature conditions for a period of time of 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, such as 4 months or 8 months.

[0070] Preferably, the leguminous protein isolate has a protein content of at least 80% (w / w dry matter basis).

[0071] Preferably, the leguminous protein isolate has a protein content of at most 95% (w / w dry matter basis).

[0072] In preferred embodiments, the leguminous protein isolate has a protein content of about 82% (w / w dry matter basis).

[0073] Preferably, the modified leguminous protein has a lipid content of at least 0% (w / w dry matter basis).

[0074] Preferably, the modified leguminous protein has a lipid content of at most 10% (w / w dry matter basis).

[0075] In a preferred embodiment, the modified leguminous protein has a lipid content of about 5% (w / w dry matter basis).

[0076] In some embodiments, additional food ingredients are added to the modified leguminous protein or to the aqueous dispersion of modified leguminous protein. The additional food ingredients may be any food ingredient deemed useful by a practitioner of skill in the art. The additional food ingredient may be a solid or liquid ingredient. The additional food ingredient may or may not be plant-based. In some embodiments, the additional food ingredient is water.

[0077] The modified leguminous protein or the aqueous dispersion of modified leguminous protein may be fortified with a plant-based dairy alternative powder, such as, e.g., a soymilk powder, or concentrated or isolated protein, such as soy protein isolate, soy protein concentrate, pea protein isolate or pea protein concentrate. In some embodiments, the aqueous dispersion of modified leguminous protein is a pea-based drink fortified with a soy protein or a pea protein to a protein level of 1-20% (w / w).

[0078] The additional food ingredients which may be added to the modified leguminous protein or to the aqueous dispersion of modified leguminous protein, include, but are not limited to, e.g., lipids, such as oils, in particular plant oils, sugars, such as sucrose, proteins, various forms of synthetic amino acids, dietary fibres, salts, minerals, flavouring agents, vitamins, and any combinations thereof.

[0079] In some embodiments, lipid is added to the aqueous solution of leguminous protein isolate and / or to the modified leguminous protein and / or to the aqueous dispersion of modified leguminous protein. The lipid may be a plant oil or a mixture of plant oils. The lipid may be selected from rapeseed oil, flaxseed oil, safflower oil, flaxseed oil, soybean oil, olive oil, sunflower oil, palm oil and combinations thereof. In an embodiment the lipid is a soybean oil. The selection of suitable lipid may be based on the type of food product desired.

[0080] In some embodiments, sugar is added, optionally together with a lipid, to the aqueous solution of leguminous protein isolate and / or to the modified leguminous protein and / or to the aqueous dispersion of modified leguminous protein. In an embodiment the sugar is sucrose. In preferred embodiments, sucrose and soybean oil is added to the aqueous solution of leguminous protein isolate.

[0081] In some embodiments, salt is added to the aqueous solution of leguminous protein isolate and / or to the modified leguminous protein and / or to the aqueous dispersion of modified leguminous protein. The salt may be sodium chloride, dicalcium carbonate, dicalcium phosphate, tricalcium phosphate, calcium carbonate and any combinations thereof.

[0082] In some embodiments, vitamins and / or minerals are added to the aqueous solution of leguminous protein isolate and / or to the modified leguminous protein and / or to the aqueous dispersion of modified leguminous protein. The 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. The mineral may be calcium, phosphorous, magnesium, sodium, potassium, chloride, iron, zinc, iodine, selenium, copper, and mixtures thereof.

[0083] The modified leguminous protein and the aqueous dispersion of modified leguminous protein obtained according to the methods claimed herein does not require the addition of emulsifiers and / or stabilizers to achieve the properties claimed herein. In particular, using the methods of the invention, a modified leguminous protein is obtained which has improved stability, in particular improved colloidal stability, without the need for adding emulsifiers and / or stabilizer during any of the steps of producing the final product or to the final product.

[0084] The aqueous dispersion of modified leguminous protein does not flocculate or precipitate even after longer storage, such as after several weeks or months of storage. Thus, in preferred embodiments, the modified leguminous protein and / or the aqueous dispersion of modified leguminous protein is essentially free of added emulsifiers and / or stabilizers. Also, in preferred embodiments, a stabilizer and / or emulsifier is not present in any of the steps of the methods claimed herein.

[0085] As used herein, the term “essentially free of is used to describe a composition or a product that contains only trace amounts or negligible quantities of a particular substance or component. It indicates that the presence of the specified substance or component is minimal and does not impact the overall characteristics or functionality of the invention. In an embodiment, "essentially free of means 0% (w / w) or 0% (w / v).

[0086] As used herein, the terms “emulsifier” and “stabilizer” are meant as added emulsifiers and stabilizers, i.e., ingredients not naturally found in the material used for preparing the modified leguminous protein. Examples of such emulsifiers and stabilizers include, but are not limited to, thickening agents, such as, e.g., carboxymethylcellulose, gellan gum, hydroxypropyl starch and agar, and emulsifiers, such as, e.g., monoglyceride and diglyceride.

[0087] In the context of the present invention, the leguminous protein is derived or obtained from pea, soy, fava bean, lentil, or any combinations thereof. Soy, pea, fava bean and lentil all belong to the family of legumes or Fabaceae. Based on protein contents, leguminous protein products may be classified into three main categories: flours, concentrates, and isolates, with the highest protein contents being in the isolates followed by concentrates and lastly flours.

[0088] In the context of the invention, the leguminous protein is derived or obtained from a leguminous protein isolate. The term “leguminous protein isolate” refers to a composition in which the protein content is increased as compared to that of pre-processed state ( / .e., plant protein raw materials) due to extraction or concentration of the proteins. Typically, such isolate refers to a composition in which the protein content is of 80% (w / w) or more, for example, a range of 80-99% (w / w). For some commercial soybean protein isolates or pea protein isolates, protein content is about 82% (w / w) or above. Some purified soybean protein isolates or pea protein isolates can reach a protein content of 90% (w / w) or above.

[0089] The leguminous protein isolate used in the methods of the present invention may be commercially available isolates or may be prepared using methods known to the person skilled in the art. The leguminous protein isolate may be a spray-dried or freeze-dried leguminous protein isolate.

[0090] In the methods of the invention, the leguminous protein is derived or obtained from a leguminous protein isolate such as of a pea protein isolate, a soy protein isolate, a fava bean protein isolate, a lentil protein isolate, or any combinations thereof. In preferred embodiments, the leguminous protein is derived or obtained from a pea protein isolate, a soy protein isolate, or a combination thereof.

[0091] In the methods of the invention, the leguminous protein isolate is provided as an aqueous solution of leguminous protein isolate having a protein content in the range of 1-20% (w / w). The term “aqueous solution of leguminous protein isolate” means an aqueous liquid, such as water, in which a leguminous protein isolate, e g. in the form of powder, is dissolved. The aqueous solution of protein isolate may or may not have a uniform distribution of the leguminous protein isolate in the aqueous solution.

[0092] In preferred embodiments, the aqueous solution of leguminous protein isolate has a protein content in the range of 3-20% (w / w). In further embodiments, the aqueous solution of leguminous 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 an embodiment, the aqueous solution of leguminous protein isolate has a protein content in the range of 5-15% (w / w).

[0093] In some embodiments, the aqueous solution of leguminous 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 leguminous protein isolate has a lipid content of about 0.5% (w / w) or about 1% (w / w).

[0094] The protein deamidase used to treat the aqueous solution of leguminous protein isolate is held at a temperature in the range of 20-80°C, so that the leguminous protein isolate is enzymatically deamidated by the protein deamidase to produce an enzymatically deamidated leguminous protein. In some embodiments, the aqueous solution of leguminous protein isolate is held ata temperature between 25-40°C, 30-45°C, 35-50°C, 40-55°C, 50-60°C, 50-65°C, 55-70°C, or 55-65°C. In further embodiments, the aqueous solution of leguminous protein isolate is held at a temperature of 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.

[0095] In an embodiment, the treatment with the protein deamidase is carried out at a temperature in the range of 50-70°C, such as at a temperature in the range of 55-70°C. In an embodiment, the treatment with the protein deamidase is at a temperature in the range of 60- 70°C, such as at a temperature in the range of 60-65°C.

[0096] In some embodiments, the aqueous solution of leguminous protein isolate with the added protein deamidase is held at 20-80°C for at least 10 minutes to allow for enzymatic deamidation of the leguminous protein. In some embodiments, the aqueous solution of leguminous 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 for enzymatic deamidation of the leguminous protein isolate. In some embodiments, the aqueous solution of leguminous protein isolate is held for at least about 10, 30, 60, or 90 minutes. In some embodiments, the aqueous solution of leguminous protein isolate is held for 30 minutes. In some embodiments, the aqueous solution of leguminous protein isolate is held for 60 minutes.

[0097] Preferably, the treatment with the protein deamidase is carried out at a temperature in the range of 50-70°C for 15-90 minutes. In an embodiment, the treatment with the protein deamidase is at carried out at a temperature in the range of 60-70°C for 30-60 minutes.

[0098] In the methods of the invention, pH is in the range of pH 6-8. For example, in the methods disclosed herein, the pH may be about 6.2, 6.5, 6.7, 7, 7.2, 7.5, or 7.7. In the context of the invention, the pH may be measured on any of the aqueous solution of leguminous protein isolate, the aqueous solution of enzymatically deamidated leguminous protein, and the aqueous dispersion of modified leguminous protein.

[0099] According to the methods disclosed herein, the pH needs not be adjusted to pH lower than 6 during any of the steps of obtaining the modified leguminous protein or the aqueous dispersion of the modified leguminous protein. Thus, a simpler production method is achieved, which does not influence the final properties of the modified leguminous protein or the aqueous dispersion of the modified leguminous protein, in particular which does not influence the colloidal stability of the modified leguminous protein or the aqueous dispersion of modified leguminous protein.

[0100] Additional parameters, such as temperature ranges and the length of enzymatic treatment, will vary depending on the leguminous protein, further enzymes used, and the desired end product. The skilled person will know how to determine suitable further process parameters based on, e.g., the leguminous protein, end product, and enzymes used. After the enzymatic deamidation of the leguminous protein, heat treatment is performed. The heat treatment also inactivates the protein deamidase. In some embodiments, the heat treatment is 80-125°C for 5-30 minutes. For example, the heat treatment is 85-121°C for 10-20 minutes. In some embodiments, the heat treatment is 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 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 115°C for 2, 5, 10, 15, or 20 minutes. In some embodiments, the heat treatment is 120°C for 2, 5, 10, 15, or 20 minutes.

[0101] In some embodiments, the heat treatment further comprises an Ultra High Temperature (UHT) treatment at a temperature in the range of 126-150°C for 1-120 seconds, such as at a temperature in the range of 130-145°C for 1-90 seconds.

[0102] In some embodiments, the heat treatment, and also inactivation of the protein deamidase, is an Ultra High Temperature (UHT) treatment. The UHT treatment may be direct or indirect. In some embodiments, the UHT treatment is carried out at a temperature in the range of 121-150°C for 1-120 seconds. In some embodiments, the UHT treatment is at a temperature in the range of 130-145°C for 1-90 seconds. In further embodiments, the UHT treatment is 135-154°C for 1-10 seconds. In further embodiments, the UHT treatment is 140-150°C for 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 seconds. In further embodiments, the UHT treatment is 140-145°C for 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 seconds. In some embodiments, the UHT treatment is 143°C for 1, 2, 3, 4, 5, 6, 7, or 8 seconds.

[0103] In some embodiment, the heat treatment is performed in two steps, comprising a first step of heat treatment at a temperature in the range of 80-125°C for 5-30 minutes, such as at a temperature in the range of 85-121°C for 10-20 minutes, followed by a UHT treatment at a temperature in the range of 126-150°C for 1-120 seconds, such as at a temperature in the range of 130-145°C for 1-90 seconds.

[0104] After the heat treatment, the aqueous dispersion of modified leguminous protein may be cooled. The aqueous dispersion may be separated into a solid and a liquid stream, for example by centrifugation. Centrifugation may occur in a decanter centrifuge. Following centrifugation, the liquid stream may be harvested or collected and used as the aqueous dispersion of modified leguminous protein. The liquid stream may still comprise some solid matter. In some embodiments, the liquid stream comprises 1-80% solids. In further embodiments, the liquid stream comprises 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 comprises 10-15% solids.

[0105] In some embodiments, the aqueous dispersion of modified leguminous protein is not separated into a solid and a liquid stream, i.e. the aqueous dispersion of modified leguminous protein is not subjected to a step of centrifugation. In such embodiments, the aqueous dispersion of modified leguminous protein is used directly, e.g., to obtain a dairy alternative food product, such as, e.g., a dairy alternative drink. The aqueous dispersion of modified leguminous protein obtained according to the methods of the invention has improved dispersibility and colloidal stability irrespective of whether a step of centrifugation is performed or not.

[0106] In some embodiments, the liquid stream is used directly as the aqueous dispersion of modified leguminous protein. Additional food ingredients may be added to the liquid stream to produce the aqueous dispersion of modified leguminous protein. For example, the leguminous protein derived liquid stream may be formulated using for instance sodium chloride (NaCI), oil, sugar, and flavouring agents. It may be homogenized. It may be UHT or ESL treated and aseptically packed.

[0107] In some embodiments, the aqueous dispersion of modified leguminous protein is a dairy alternative drink. Examples of dairy alternative drinks include soy drinks, pea drinks, fava bean drinks, lentil drinks and drinks comprising any combination thereof. In an embodiment, the plantbased beverage is a soy drink, a pea drink, or a drink comprising a combination thereof. In some embodiments, the dairy alternative drink is prepared from soy protein isolate or pea protein isolate.

[0108] The aqueous dispersion of modified protein finds applicability in a wide range of applications, including, but not particularly limited to, use as a dairy alternative beverage, such as a dairy alternative “milk”, use for mixing with other types of beverages, such as for mixing with acidic beverages, such as coffee or tea, to obtain ready-to-drink (RTD) acidic beverages, and use as a protein drink.

[0109] In some embodiments, the aqueous dispersion of modified leguminous protein is a canned beverage or a packaged beverage. The aqueous dispersion of modified leguminous protein may be contained and stored in any type of can or packaging material deemed suitable by the person skilled in the art. In some embodiments, the canned beverage or a packaged beverage of the aqueous dispersion of modified leguminous protein is subjected to canned sterilization. For example, the canned sterilization may be at 80-125°C for 5-30 minutes, preferably at 110-121°C for 10-20 minutes.

[0110] The aqueous solution of leguminous protein isolate may be subjected to further processing, such as, e.g., treatment with further enzymes, including, e.g., further hydrolyzing enzymes. Such further enzymes include, but are not limited to, one or more of the hydrolyzing enzymes selected from the group of pectinases, hemicellulases, xylanases, beta-glucanases, mannanases, glucanases, glucoamylases, iso-amylases, alpha-amylases, beta-amylases, and mixtures thereof.

[0111] The enzymes used in the methods of the invention may be added to the aqueous dispersion comprising leguminous protein isolate in any suitable form, such as in the form of a liquid, in particular a stabilized liquid, or it may be added as a substantially dry powder or granulate. Granulates may be produced, e.g., as disclosed in US Patent No. 4,106,991 and US Patent No. 4,661,452. Liquid enzyme preparations may, for instance, be stabilized by adding a sugar or sugar alcohol or lactic acid according to established procedures. Other enzyme stabilizers are well-known in the art.

[0112] In a further aspect, the present invention relates to an aqueous dispersion of modified leguminous protein having a pH in the range of 6-8, wherein the modification is obtained by enzymatic deamidation with a protein deamidase followed by a step of heat treatment, and wherein the leguminous protein is derived or obtained from a leguminous protein isolate of pea, soy, fava bean, lentil, or any combination thereof, preferably a leguminous protein isolate of pea, soy, or a combination thereof.

[0113] In a further aspect, the present invention relates to the use of a protein deamidase in the production of an aqueous dispersion of modified leguminous protein having a pH in the range of pH 6-8 to improve colloidal stability, wherein the modified leguminous protein is derived or obtained from a leguminous protein isolate of pea, soy, fava bean, lentil, or any combination thereof.

[0114] In a further aspect, the present invention relates to the use of a protein deamidase and heat treatment in the production of an aqueous dispersion of modified leguminous protein having a pH in the range of pH 6-8 to improve colloidal stability, wherein the modified leguminous protein is derived or obtained from a leguminous protein isolate of pea, soy, fava bean, lentil, or any combination thereof.

[0115] Protein deamidase

[0116] In the methods of the invention, a leguminous protein isolate is treated with a protein deamidase to obtain an aqueous dispersion of modified leguminous protein.

[0117] In the present invention, a protein deamidase refers to an enzyme having an effect of directly acting on an amide group of a side chain of an amino acid that constitutes a protein to cause deamidation and release ammonia without cleaving a peptide bond of the protein and crosslinking proteins.

[0118] The term “deamidase” means a protein-glutamine glutaminase (also known as glutaminylpeptide glutaminase) activity, as described in EC 3.5.1.44, which catalyses the hydrolysis of the gamma-amide of glutamine substituted at the carboxyl position or both the alphaamino and carboxyl positions, e.g., L-glutaminylglycine and L-phenylalanyl-L-glutaminylglycine. Thus, deamidases can deamidate glutamine residues in proteins to glutamate residues and are also referred to as protein glutamine deamidase. Deamidases comprise a Cys-His-Asp catalytic triad (e.g., Cys-156, His-197, and Asp-217, as shown in Hashizume et al. “Crystal structures of protein glutaminase and its pro forms converted into enzyme-substrate complex”, Journal of Biological Chemistry, vol. 286, no. 44, pp. 38691-38702) and belong to the InterPro entry IPR041325.

[0119] Deamidase may also include a protein asparaginase that directly acts on an amide group of a side chain of an asparagine residue contained in a protein to release ammonia and thus converts the asparagine residue into an aspartate residue. In the present invention, as a protein deamidase, any one of the protein glutaminase and the protein asparaginase can be used, or both can be used in combination. One example of the protein deamidase used in the present invention is a protein glutaminase.

[0120] A protein deamidase to be used in a method of the present invention may be obtained from microorganisms of any genus. For purposes of the present invention, the term “obtained from” as used herein in connection with a given source shall mean that the polypeptide encoded by a polynucleotide is produced by the source or by a strain in which the polynucleotide from the source has been inserted. In an embodiment, the polypeptide obtained from a given source is secreted extracellularly.

[0121] The protein deamidase may be obtained from a microorganism by use of any suitable technique. For instance, an enzyme preparation may be obtained by fermentation of a suitable microorganism and subsequent isolation of a protein deamidase preparation from the resulting fermented broth or microorganism by methods known in the art. The protein deamidase may also be obtained by use of recombinant DNA techniques. Such method normally comprises cultivation of a host cell transformed with a recombinant DNA vector comprising a DNA sequence encoding the protein deamidase and the DNA sequence being operationally linked with an appropriate expression signal such that it is capable of expressing the enzyme in a culture medium under conditions permitting the expression of the enzyme and recovering the enzyme from the culture. The DNA sequence may also be incorporated into the genome of the host cell. The DNA sequence may be of genomic, cDNA or synthetic origin or any combinations of these, and may be isolated or synthesized in accordance with methods known in the art.

[0122] The protein deamidase may be purified. The term "purified" as used herein covers protein deamidase enzyme protein essentially free from insoluble components from the production organism. The term "purified" also covers protein deamidase enzyme protein essentially free from insoluble com-ponents from the native organism from which it is obtained. Preferably, it is also separated from some of the soluble components of the organism and culture medium from which it is derived. More preferably, it is separated by one or more of the unit operations: filtration, precipitation, or chromatography.

[0123] The types or origins of the protein deamidase used in the present invention are not particularly limited. Examples of the protein deamidase includes protein deamidases derived from Chryseobacterium genus, Flavobacterium genus, Empedobacter genus, Sphingobacterium genus, Aureobacterium genus, or Myroides genus. The protein deamidase may be derived from any of the sources mentioned herein. The term “derived” means in this context that the enzyme may have been isolated from an organism where it is present natively, i.e., the amino acid sequence of the protein deamidase is identical to a native polypeptide. The term “derived” also means that the enzyme may have been produced recombinantly in a host organism, the recombinantly produced enzyme having either an amino acid sequence which is identical to a native enzyme or having a modified amino acid sequence, e.g. having one or more amino acids which are deleted, inserted and / or substituted, i.e. a recombinantly produced enzyme which is a mutant of a native amino acid sequence. Within the meaning of a native enzyme are included natural variants. Furthermore, the term “derived” includes enzymes produced synthetically by, e.g., peptide synthesis. The term “derived” also encompasses enzymes which have been modified, e.g., by glycosylation, phosphorylation, etc., whether in vivo or in vitro. With respect to recombinantly produced enzymes the term “derived from” refers to the identity of the enzyme and not the identity of the host organism in which it is produced recombinantly.

[0124] In some embodiments, the protein deamidase may be derived from Chryseobacterium genus, such as Chryseobacterium sp-62563, C. gambrini, C. culicis, C. defluvii, or C. proteolyticum. In some embodiments, the deamidase in the methods of the invention is derived from or obtained from Chryseobacterium sp-62563.

[0125] EP1839491 discloses cloning of a protein glutaminase from Chryseobacterium proteolyticum expressed in Corynebacterium glutamicum. Deamidases are also commercially available, e.g., protein glutaminases derived from Chryseobacterium genus, for example, "Amano PG500” (manufactured by Amano Enzyme Inc.).

[0126] For example, protein deamidases can be obtained from a culture broth of the abovedescribed microorganisms.

[0127] Protein deamidases are produced by microbial cells in an inactive proform, which comprises a propeptide domain tightly bound to a deamidase domain. The proform is expressed as a fusion protein, which has reduced deamidase activity to protect the viability of the host cell. In nature, the fusion protein is post-processed to remove the propeptide and release the active deamidase outside of the host cell. However, in recombinant expression systems, the fusion protein is secreted outside of the host cell as an inactive proform comprising the propeptide. The propeptide may then be enzymatically cleaved off to separate it from the mature deamidase. The protein deamidases of the methods and compositions of the present invention are mature deamidases where the propeptide has been removed. In some embodiments, the propeptide was cleaved enzymatically by an endopeptidase. In some embodiments, the propeptide may still be present in the composition comprising the mature deamidase.

[0128] The recombinant, mature protein deamidases used in the methods of the invention comprises the polypeptide of SEQ ID NOs: 2, 4, 6, 8, and 10. Each mature protein deamidase is derived from a deamidase proform polypeptide, which comprises the polypeptide of SEQ ID NO: 1, 3, 5, 7, and 9, respectively. The preform polypeptide comprises a propeptide at the N-terminal end, fused to a deamidase which is the same as that of the polypeptide of SEQ ID NOs: 2, 4, 6, 8, or 10. The propeptide may be enzymatically cleaved from the proform polypeptide to release the mature deamidase. Naturally occurring propeptide sequences are provided in the proform polypeptide.

[0129] The methods and compositions of the invention include a mature deamidase and optionally a second polypeptide which is derived from the propeptide of a deamidase. The second polypeptides described herein are mutated variants of the naturally occurring propeptides. These variant propeptide sequences have been found to bind less strongly to their corresponding deamidase, so that they are more easily enzymatically cleaved off after recombinant expression and secretion from of the host cell. The polypeptides of SEQ ID NOs: 1 to 10 are derived from Chryseobacterium spp. and are described in the Applicant’s PCT application with publication number WO 2023 / 170177 A1 , herein incorporated by reference.

[0130] After expression of the proform polypeptide in a recombinant expression system, a sitespecific endopeptidase is used to cleave off the propeptide, leaving an active, mature deamidase. In some embodiments, the cleaved propeptide is not purified away from the mature deamidase. Therefore, the propeptide may be present in the composition with the mature deamidase.

[0131] According to a preferred embodiment the protein deamidase applied in the process of the invention is derived from or obtained from a Chryseobacterium species, e.g., Chryseobacterium proteolyticum or Chryseobacterium viscerum.

[0132] In the context of the present invention, the term “mature polypeptide” means a polypeptide in its mature form following N terminal processing (e.g., removal of signal peptide). A "signal peptide" is a sequence of amino acids attached to the N-terminal portion of a protein, which facilitates the secretion of the protein outside the cell. The mature form of an extracellular protein lacks the signal peptide, which is cleaved off during the secretion process.

[0133] In an embodiment the deamidase is selected from a polypeptide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to SEQ ID NO: 2.

[0134] In an embodiment the deamidase is selected from a polypeptide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to SEQ ID NO: 4.

[0135] In an embodiment the deamidase is selected from a polypeptide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to SEQ ID NO: 6.

[0136] In an embodiment the deamidase is selected from a polypeptide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to SEQ ID NO: 8. In an embodiment the deamidase is selected from a polypeptide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to SEQ ID NO: 10.

[0137] In an embodiment the deamidase is selected from a polypeptide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to a mature polypeptide of SEQ ID NO: 1.

[0138] In an embodiment the deamidase is selected from a polypeptide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to a mature polypeptide of SEQ ID NO: 3.

[0139] In an embodiment the deamidase is selected from a polypeptide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to a mature polypeptide of SEQ ID NO: 5.

[0140] In an embodiment the deamidase is selected from a polypeptide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to a mature polypeptide of SEQ ID NO: 7.

[0141] In an embodiment the deamidase is selected from a polypeptide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to a mature polypeptide of SEQ ID NO: 9.

[0142] For purposes of the present invention, the sequence identity between two amino acid sequences is determined as the output of “longest identity” using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle pro-gram of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 6.6.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. In order for the Needle program to report the longest identity, the -nobrief option must be specified in the command line. The output of Needle labelled “longest identity” is calculated as follows:

[0143] (Identical Residues x 100) / (Length of Alignment - Total Number of Gaps in Alignment)

[0144] In the context of the present invention, the term “variant” means a polypeptide having enzymatic activity comprising an alteration, i.e., a substitution, insertion, and / or deletion, at one or more (e.g., several) positions. A substitution means replacement of the amino acid occupying a position with a different amino acid; a deletion means removal of the amino acid occupying a position; and an 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.

[0145] The amino acid changes may be of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; small deletions, typically of 1-30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a polyhistidine tract, an antigenic epitope or a binding domain.

[0146] Examples of conservative substitutions are within the groups of 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 generally alter specific activity are known in the art and are described, for example, by H. Neurath and R. L. Hill, 1979, In, The Proteins, Academic Press, New York. Common substitutions are Ala / Ser, Val / lle, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / lle, Leu / Val, Ala / Glu, and Asp / Gly.

[0147] Alternatively, the amino acid changes are of such a nature that the physico-chemical properties of the polypeptides are altered. For example, amino acid changes may affect the thermal stability of the polypeptide, alter the substrate specificity, change the pH optimum, and the like.

[0148] Essential amino acids in a polypeptide can be identified according to 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, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for enzymatic activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271 : 4699-4708. The active site of the enzyme or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labelling, in conjunction with mutation of putative contact site amino acids. See, for ex-ample, 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 an alignment with a related polypeptide.

[0149] Single or multiple amino acid substitutions, deletions, and / or insertions can be made and tested using known methods of mutagenesis, recombination, and / or shuffling, followed by a relevant screening procedure, 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; U.S. 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).

[0150] Mutagenesis / shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide.

[0151] A protein deamidase to be used in the methods of the invention may be added at a concentration of 0.01-20 IPA(U) / g substrate protein, such as 0.1-12 IPA(U) / g substrate protein, 0.5-7 IPA(U) / g substrate protein. In some embodiments, the protein deamidase to be used in the methods of the invention is added at a concentration in the range of 0.3-4 IPA(U) / g substrate protein, such as 0.4-2.5 IPA(U) / g substrate protein.

[0152] Deamidase (protein glutaminase) activity may be measured using the assay described in Example 1. The activity assay consists of two separate de-coupled parts: (1) an enzymatic step wherein ammonia is formed by the catalytic action of the protein deamidase; and (2) a non- enzymatic detection step, wherein the ammonia formed in step (1) is derivatized to a blue indophenol compound with an absorption maximum at 630 nm. The amount of enzyme producing 1 pmol ammonia per minute at 37°C is defined as 1 unit (given in Indophenol Assay Unit: IPA(U)). The activity may be determined relative to a standard of declared strength.

[0153] The enzymes dosage will depend on parameters such as the temperature, the incubation time, and the intended use of the modified leguminous protein. The skilled person will know how to determine the optimal enzyme dosage.

[0154] Without wishing to be bound by any particular theory, the inventor believes that the use of protein deamidase to yield a modified leguminous protein, such as, e.g., an aqueous dispersion of modified leguminous protein, contribute to the superior benefits reported herein, including, but not limited to, the improved colloidal stability.

[0155] The invention is further defined by the following numbered embodiments:

[0156] Embodiment 1. A method of obtaining an aqueous dispersion of a modified leguminous protein, comprising the steps of:

[0157] (a) providing an aqueous solution of leguminous protein isolate having a protein content in the range of 1-20% (w / w);

[0158] (b) treating the aqueous solution of step (a) with a protein deamidase to obtain an enzymatically deamidated leguminous protein; and

[0159] (c) heat treating the enzymatically deamidated leguminous protein of step (b) to obtain the aqueous dispersion of modified leguminous protein, wherein in steps (b) and (c) pH is in the range of pH 6-8, and wherein the leguminous protein isolate is derived or obtained from pea, soy, fava bean, lentil, or any combination thereof.

[0160] Embodiment 2. Method of embodiment 1 , wherein after storage for 15 minutes at a temperature of at least 4°C the aqueous dispersion of modified leguminous protein has an improved colloidal stability compared to aqueous dispersion of modified leguminous protein prepared using a similar method where a protein deamidase is not added to the aqueous solution of leguminous protein isolate.

[0161] Embodiment 3. Method of embodiment 1 , wherein after storage for 15 minutes at a temperature of at least 4°C the aqueous dispersion of modified leguminous protein has an improved colloidal stability compared to an aqueous dispersion of modified leguminous protein prepared using a using a similar method but without the use of a protein deamidase and a step of heat treatment following the treatment with the protein deamidase.

[0162] Embodiment 4. Method of any of the preceding embodiments, wherein the treatment with the protein deamidase in step (b) is carried out at a temperature in the range of 50-70°C for 15- 90 minutes.

[0163] Embodiment 5. Method of any of the preceding embodiments, wherein the treatment with the protein deamidase in step (b) is carried out at a temperature in the range of 55-70°C for 15- 90 minutes.

[0164] Embodiment 6. Method of any of the preceding embodiments, wherein the treatment with the protein deamidase in step (b) is carried out at a temperature in the range of 60-70°C for 15- 90 minutes.

[0165] Embodiment 7. Method of any of the preceding embodiments, wherein the treatment with the protein deamidase in step (b) is carried out at a temperature in the range of 60-65°C for 15- 90 minutes.

[0166] Embodiment 8. Method of any of the preceding embodiments, wherein the treatment with the protein deamidase in step (b) is carried out for 30-60 minutes.

[0167] Embodiment 9. Method of any of the preceding embodiments, wherein a stabilizer and / or emulsifier is not present in any of steps (a), (b) and / or (c).

[0168] Embodiment 10. Method of any of the preceding embodiments, wherein a stabilizer and / or emulsifier is not present in any of steps (a), (b) and (c).

[0169] Embodiment 11. Method of any of the preceding embodiments, wherein the aqueous solution of step (a) has a protein content in the range of 3-20% (w / w), in the range of 1-15% (w / w), in the range of 3-15% (w / w), or in the range of 3-10% (w / w).

[0170] Embodiment 12. Method of any of the preceding embodiments, wherein the aqueous solution of step (a) has a protein content in the range of 5-15% (w / w).

[0171] Embodiment 13. Method of any of the preceding embodiments, wherein the aqueous solution of step (a) has a lipid content 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).

[0172] Embodiment 14. Method of any of the preceding embodiments, wherein the heat treatment in step (c) is carried out at a temperature in the range of 80-125°C for 5-30 minutes, such as at a temperature in the range of 85-121°C for 10-20 minutes. Embodiment 15. Method of any of the preceding embodiments, wherein the heat treatment in step (c) is carried out at a temperature in the range of 85-95°C for 10-20 minutes, such as at 90°C for 10 minutes or at 85°C for 15 minutes.

[0173] Embodiment 16. Method of any of embodiments 1-14, wherein the heat treatment in step (c) is carried out at a temperature in the range of 110-121°C for 10-20 minutes, such as at about 115°C for 15 minutes.

[0174] Embodiment 17. Method of any of the preceding embodiments, wherein the heat treatment in step (c) further comprises an Ultra High Temperature (UHT) treatment at a temperature in the range of 126-150°C for 1-120 seconds, such as at a temperature in the range of 130-145°C for 2-90 seconds.

[0175] Embodiment 18. Method of any of embodiments 1-13, wherein the heat treatment in step (c) is at a temperature in the range of 121-150°C for 1-120 seconds, such as at a temperature in the range of 130-145°C for 1-90 seconds, such as at 130°C for 30 seconds, at 140°C for 3-4 seconds, or at 145°C for 1-2 seconds.

[0176] Embodiment 19. Method of any of embodiments 1-13, wherein the heat treatment comprises a first step of heat treatment at a temperature in the range of 80-125°C for 5-30 minutes, such as at a temperature in the range of 85-121°C for 10-20 minutes, followed by a second step of heat treatment at a temperature in the range of 126-150°C for 1-120 seconds, such as at a temperature in the range of 130-145°C for 1-90 seconds.

[0177] Embodiment 20. Method of any of the preceding embodiments, wherein the leguminous protein isolate is derived or obtained from pea, soy, or a combination thereof.

[0178] Embodiment 21. Method of any of the preceding embodiments, wherein the leguminous protein isolate is derived or obtained from pea.

[0179] Embodiment 22. Method of any of the preceding embodiments, wherein, when mixed with an acidic beverage, the aqueous dispersion of modified leguminous protein isolate has an improved stability compared to an aqueous dispersion of modified leguminous protein prepared using a similar method where a protein deamidase is not added to the aqueous solution of leguminous protein isolate.

[0180] Embodiment 23. Method of embodiment 22, wherein the acidic beverage is a coffee drink or a tea drink, optionally a hot coffee drink or a hot tea drink.

[0181] Embodiment 24. Method of any of the preceding embodiments, wherein, when mixed with an acidic beverage, the aqueous dispersion of modified leguminous protein has an improved stability, such as a reduced level of feathering, compared to an aqueous dispersion of modified leguminous protein prepared using a similar method where a protein deamidase is not added to the aqueous solution of leguminous protein isolate.

[0182] Embodiment 25. Method of any of the preceding embodiments, wherein, when mixed with an acidic beverage, the aqueous dispersion of modified leguminous protein has an improved stability, such as a reduced level of feathering, compared to an aqueous dispersion of modified leguminous protein prepared using a similar method where a protein deamidase is not added to the aqueous solution of leguminous protein isolate and wherein a step of heat treatment following the treatment with the protein deamidase is not used.

[0183] Embodiment 26. Method of any of the preceding embodiments, wherein the aqueous dispersion of modified leguminous protein after storage for at least 15 minutes at a temperature of at least 4°C has a reduced level of precipitation of the protein compared to an aqueous dispersion of modified leguminous protein prepared using a similar method where a protein deamidase is not added to the aqueous solution of leguminous protein isolate.

[0184] Embodiment 27. Method of any of the preceding embodiments, wherein the aqueous dispersion of modified leguminous protein after storage for at least 15 minutes at a temperature of at least 4°C has a reduced level of precipitation of the protein compared to an aqueous dispersion of modified leguminous protein prepared using a similar method where a protein deamidase is not added to the aqueous solution of leguminous protein isolate and wherein a step of heat treatment following the treatment with the protein deamidase is not used.

[0185] Embodiment 28. Method of any of the preceding embodiments, wherein the aqueous dispersion of modified leguminous protein further comprises one or more additional food ingredients selected from the group of lipids, sugars, proteins, vitamins, minerals, amino acids, flavouring agents, dietary fibres, salts, and any combination thereof.

[0186] Embodiment 29. Method of the preceding embodiment, wherein the additional food ingredient is a lipid, such as an oil, preferably a plant oil, more preferably a soybean oil, and / or a sugar, such as a sucrose.

[0187] Embodiment 30. Method of any of the preceding embodiments, wherein the aqueous dispersion of modified leguminous protein is a soy drink, a pea drink, a fava bean drink, a lentil drink, or a drink comprising any combinations thereof.

[0188] Embodiment 31. Method of any of the preceding embodiments, wherein the aqueous dispersion of modified leguminous protein is a pea drink, a soy drink or a drink comprising a combination thereof.

[0189] Embodiment 32. Method of any of the preceding embodiments, wherein the aqueous dispersion of modified leguminous protein is a pea drink.

[0190] Embodiment 33. Method of any of the preceding embodiments, wherein the aqueous dispersion of modified leguminous protein is essentially free of additives, such as stabilizers and / or emulsifiers.

[0191] Embodiment 34. Method of any of the preceding embodiments, wherein the aqueous dispersion of modified leguminous protein is meant for storage at a temperature of 3-30°C.

[0192] Embodiment 35. Method of any of the preceding embodiments, wherein the aqueous dispersion of modified leguminous protein is meant for storage at a temperature of 3-5°C, such as at about 4°C, or at a temperature of 16-24°C, such as about 20°C. Embodiment 36. Method of any of the preceding embodiments, wherein the protein deamidase is derived from or obtained from a Chryseobacterium species, more preferably from Chryseobacterium proteolyticum or Chryseobacterium viscerum.

[0193] Embodiment 37. Method of any of the preceding embodiments, wherein the protein deamidase comprises a polynucleotide sequence with at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to SEQ ID NOs: 2, 4, 6, 8, or 10.

[0194] Embodiment 38. Method of any of the preceding embodiments, wherein the protein deamidase comprises the polynucleotide sequence of SEQ ID NOs: 2, 4, 6, 8, or 10.

[0195] Embodiment 39. An aqueous dispersion of modified leguminous protein having a pH in the range of 6-8, wherein the modification is obtained by enzymatic deamidation with a protein deamidase and wherein the leguminous protein is derived or obtained from a leguminous protein isolate, such as from a leguminous protein isolate of pea, soy, fava bean, lentil, or any combinations thereof.

[0196] Embodiment 40. Aqueous dispersion of embodiment 39, wherein the leguminous protein is derived or obtained from pea, soy, or a combination thereof.

[0197] Embodiment 41. Aqueous dispersion of any of embodiments 39-40, wherein the leguminous protein is derived or obtained from pea.

[0198] Embodiment 42. Aqueous dispersion of any of embodiments 39-41 , wherein the aqueous dispersion of modified leguminous protein is a dairy alternative drink, such as a pea-based drink, a soy-based drink or a drink comprising a combination thereof.

[0199] Embodiment 43. Aqueous dispersion of any of embodiments 39-42, further comprising a protein deamidase.

[0200] Embodiment 44. Aqueous dispersion of any of embodiments 39-43, wherein the protein deamidase comprises a polypeptide sequence with at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to SEQ ID NOs: 2, 4, 6, 8, or 10.

[0201] Embodiment 45. Aqueous dispersion of any of embodiments 39-44, characterized by having been subjected to a step of heat treatment.

[0202] Embodiment 46. Aqueous dispersion of the preceding embodiment, wherein the heat treatment is carried out after the enzymatic deamidation with the protein deamidase.

[0203] Embodiment 47. Aqueous dispersion of any of embodiments 45 or 46, wherein the heat treatment is at a temperature in the range of 80-125°C for 5-30 minutes, such as at a temperature in the range of 85-121°C for 10-20 minutes, and / or at a temperature in the range of 126-150°C for 1-120 seconds, such as at a temperature in the range of 130-145°C for 2-90 seconds.

[0204] Embodiment 48. Aqueous dispersion of any of embodiments 45 or 46, wherein the heat treatment comprises a first step of heat treatment at a temperature in the range of 80-125°C for 5-30 minutes, such as at a temperature in the range of 85-121°C for 10-20 minutes, followed by a second step of heat treatment at a temperature in the range of 126-150°C for 1-120 seconds, such as at a temperature in the range of 130-145°C for 1-90 seconds.

[0205] Embodiment 49. Aqueous dispersion of any of embodiments 39-48 which is essentially free of added stabilizer and / or emulsifier.

[0206] Embodiment 50. Aqueous dispersion of any of embodiments 39-49 characterized by having an improved colloidal stability compared to an aqueous dispersion of modified leguminous protein prepared without use of a protein deamidase.

[0207] Embodiment 51. Aqueous dispersion of any of embodiments 45-50 characterized by having an improved colloidal stability compared to an aqueous dispersion of modified leguminous protein prepared without use of a protein deamidase and a step of heat treatment after the treatment with the protein deamidase.

[0208] Embodiment 52. Use of a protein deamidase in the production of an aqueous dispersion of modified leguminous protein to improve colloidal stability.

[0209] Embodiment 53. Use according to embodiment 52, wherein the aqueous dispersion of modified leguminous protein has a pH in the range of pH 6-8.

[0210] Embodiment 54. Use according to any of embodiments 52-53, wherein the modified leguminous protein is derived or obtained from a leguminous protein isolate of pea, soy, fava bean, lentil, or any combinations thereof.

[0211] Embodiment 55. Use according to any of embodiments 52-54, wherein the modified leguminous protein is derived or obtained from a leguminous protein isolate of pea, soy, or any combination thereof.

[0212] Embodiment 56. Use according to any of embodiments 52-55, wherein the modified leguminous protein is derived or obtained from a leguminous protein isolate of pea.

[0213] Embodiment 57. Use according to any of embodiments 52-56, wherein the protein deamidase comprises a polypeptide sequence with at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to SEQ ID NOs: 2, 4, 6, 8, or 10.

[0214] Embodiment 58. Use according to any of embodiments 52-57, wherein the aqueous dispersion is a dairy alternative drink, such as a pea-based dairy alternative drink, a soy-based dairy alternative drink or a dairy alternative drink comprising a combination of soy and pea.

[0215] Embodiment 59. Use according to any of embodiments 52-58, wherein the aqueous dispersion of modified leguminous protein is for use in acidic beverages, such as hot acidic beverages.

[0216] Embodiment 60. Use according to any of embodiments 52-59, wherein the aqueous dispersion of modified leguminous protein after storage for at least 15 minutes at a temperature of at least 4°C has an improved colloidal stability compared to an aqueous dispersion of modified leguminous protein prepared without the use of a protein deamidase. Embodiment 61. Use according to any of embodiments 52-60, wherein the aqueous dispersion of modified leguminous protein after storage for at least 15 minutes at a temperature of at least 4°C has a reduced level of precipitation of the protein compared to an aqueous dispersion of modified leguminous protein prepared without the use of a protein deamidase.

[0217] Embodiment 62. Use of a protein deamidase and heat treatment in the production of an aqueous dispersion of modified leguminous protein having a pH in the range of pH 6-8 to improve colloidal stability.

[0218] Embodiment 63. Use according to embodiment 62, wherein the modified leguminous protein is derived or obtained from a leguminous protein isolate of pea, soy, fava bean, lentil, or any combinations thereof.

[0219] Embodiment 64. Use according to any of embodiments 62 or 63, wherein the modified leguminous protein is derived or obtained from a leguminous protein isolate of pea, soy, or a combination thereof, preferably from pea.

[0220] Embodiment 65. Use according to any of embodiments 62-64, wherein the heat treatment is after the protein deamidase treatment.

[0221] The invention described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed, since these embodiments are intended as illustrations of several aspects of the invention. Any equivalent embodiments are intended to be within the scope of this invention as well as combinations of one or more of the embodiments.

[0222] Various references are cited herein, the disclosures of which are incorporated by reference in their entireties. The present invention is further described by the following examples which should not be construed as limiting the scope of the invention.

[0223] EXAMPLES

[0224] Materials

[0225] Enzymes

[0226] The following enzymes are used throughout the examples:

[0227] Protein deamidase: Protein glutaminase derived from Chryseobacterium viscerum (the strain has formerly been referred to as Chryseobacterium sp-62563) having the mature polypeptide sequence shown as SEQ ID NO: 2. Cleavage of the pro-peptide was achieved by treating the deamidase of SEQ ID NO: 1 with a site-specific endopeptidase. The site-specific endopeptidase used is a glutamyl endopeptidase from Bacillus licheniformis. The resulting active deamidase after maturation was the polypeptide shown in SEQ ID NO: 2. Example 1: Protein deamidase activity assay

[0228] The activity assay consists of two separate de-coupled parts:

[0229] 1) An enzymatic step wherein ammonia is formed by the catalytic action of the protein deamidase; and

[0230] 2) A non-enzymatic detection step wherein the ammonia formed in step (1) is derivatized to a blue indophenol compound with an absorption maximum at 630 nm.

[0231] In step (1), the ammonia is developed by the deamidating action of the protein deamidase. In step (2), the generated ammonia reacts with phenol to form dioxyphenylamine under alkaline conditions. The reaction is catalysed by sodium pentacyanonitrosylferrate(lll) (sodium nitroprusside). “Color Reagent solution A” contains phenol and sodium nitroprusside. “Color Reagent Solution B” provides alkaline reaction conditions. The intermediate is then oxidized by addition of sodium hypochlorite (“Color 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.

[0232] Assay Procedure:

[0233] Step (1) Enzymatic step with ammonia formation

[0234] Reagents:

[0235] Assay dilution solution: 0.2 M Na-phosphate buffer, 0.01% Triton X-100, pH 6.5.

[0236] Assay buffer: Same as above. Used to prepare stock solution and diluted sample of protein deamidase (referred to in the following as “enzyme”).

[0237] Substrate solution: 30 mM Z-GIn-Gly (Merck C6154-1G) in assay dilution solution (check pH after dissolution).

[0238] Stop solution: 0.4M TCA.

[0239] Standard: NH4CI (Ammonium Standard for IC, Merck 59755-100ML, 1000 mg / L NH4+in water) diluted in assay dilution solution (see also “Standard curve” section).

[0240] Dissolve / dilute enzyme product in assay buffer and prepare suitable dilution resulting in a linear assay response.

[0241] Incubation:

[0242] 1. Add 10 pL of diluted enzyme samples in triplicates to the wells of a 96-well microtiter plate (MTP).

[0243] 2. Add 100 pL of substrate solution to each well.

[0244] 3. For blank samples add 100 pL 0.4M TCA solution.

[0245] 4. Seal the plate using transparent plate sealer. 5. Incubate the plate for 10 minutes at 37°C, 500 rpm, on a thermomixer equipped with a lid heating function.

[0246] 6. To stop the reaction, carefully add 100 pL 0.4M TCA solution (except for the blank samples, which already contain TCA).

[0247] Total reaction volume: 210 pL.

[0248] Step (2) Ammonia detection step

[0249] Reagents:

[0250] Color reagent A: 4% (w / v) Phenol, 0.015% (w / v) sodium pentacyanonitrosylferrate(lll) dihydrate (sodium nitroprusside) (Na2[Fe(CN)5NO]-2H2O).

[0251] Color reagent B: 5% (w / v) Potassium hydroxide.

[0252] Color reagent C: 28% (w / v) Potassium carbonate, 6% (v / v) sodium hypochlorite (Sigma- Aldrich 239305-25ml, < 5% available Cl2).

[0253] Incubation:

[0254] 1. Transfer 15 pL from each well from step (1) into a new 96-well MTP.

[0255] 2. Transfer 45 pL Milli-Q water to each well.

[0256] 3. To each well, add 30pL of color reagent B (on lab table, shake gently by hand to mix).

[0257] 4. To each well, add 60pL of color reagent A (on lab table, shake gently by hand to mix).

[0258] 5. To each well, add 60pL of color reagent C (on lab table, shake gently by hand to mix).

[0259] 6. Color development: Carefully seal the plate and leave it on lab table for 30 minutes.

[0260] 7. Carefully transfer the MTP to a plate reader and measure absorbance at 630 nm.

[0261] Total reaction volume: 210 pL

[0262] Standard curve:

[0263] Standard stock solution: 1000 mg NFV / L.

[0264] The standard curve is prepared by adding dilutions of the ammonium standard in the assay dilution buffer in the ammonia detection step. That is, mixing 15 pL diluted ammonia standard with 45 pL water and then add the color reagents in the order given above; B, A, and C.

[0265] The amount of enzyme producing 1 pmol ammonia per minute at 37°C is defined as 1 unit (Indophenol Assay Unit; IPA(U)): which can be shortened to where

[0266] • CNH4+ is the ammonia concentration in the reaction solution derived from the ammonium standard curve (i.e., taking into account the dilution of the prediluted ammonium standard solution in the ammonia derivatization step).

[0267] • 18.04 is the molecular mass of ammonium used for the standard solution. is the reaction volume in the well when ammonia is generated (210 pL). is the volume of enzyme solution added to the well when ammonia is generated (10 pL).

[0268] • VNH3 detection is the reaction volume in the well when ammonia is detected (210 pL).

[0269] Example 2: Colloidal stability of modified legume-based beverage

[0270] In this example, pea protein isolate was used to test if the method according to the invention could be used to prepare a legume-based beverage with improved colloidal stability.

[0271] Pea beverage was laboratory prepared using the following procedure. The ShuangTa pea protein isolate (protein content 82.2% based on dry solid) was mixed with deionized H2O to prepare a 10% solution (m / v) (pH 7). The 10% pea beverage solution was then treated with protein deamidase at the dosage 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 enzyme deactivation at 85°C for 10 minutes then cooling down on ice to room temperature. A control sample (pH 7) was included prepared following the same procedure described above but without the use of a protein deamidase. The pea protein samples (a protein deamidase treated sample and a control sample) containing no additives (e.g. not emulsifiers or stabilizers) were then diluted to 3.2% protein content and mixed well. The colloidal stability was observed visually after the samples had been left to stand still for 15 minutes. The results from the colloidal stability testing are shown in Figure 1. Figure 1 shows the effect of adding the protein deamidase in the method of the invention and clearly demonstrates the applicability of protein deamidase in preparing dispersions from pea protein isolates. In particular, and as exemplified herein, an improved pea protein isolate-based dispersion, treated with a protein deamidase according to the methods disclosed herein, was obtained having an improved colloidal stability of, as seen visually as no sedimentation or flocculation in the protein deamidase treated sample (Figure 1 , sample to the right marked with check mark underneath sample). In another experiment, a different pea protein isolate from Cosucra (Cosucra C9, protein content 83% based on dry basis) was tested by mixing the pea protein isolate with deionized H2O to prepare a 10% solution (m / v) (pH 7). The 10% pea beverage solution was then treated with protein deamidase at the dosage of 2 I PA(U) / g protein. The reaction was carried out at 60°C for 1 hour followed by enzyme deactivation at 85°C for 10 minutes then cooling down on ice to room temperature. Control sample (pH 7) was again included and prepared following the same procedure but without protein deamidase. The pea protein samples containing no additives were then diluted to 6.4% protein content and mixed well. The colloidal stability was observed visually after the samples had been left to stand still for 15 minutes. The results from this study confirmed the previous findings as described above and shown in Figure 1, i.e. that protein deamidase is effective in improving colloidal stability of dispersions prepared from pea protein isolates using the methods claimed herein.

[0272] To confirm that the improvement in colloidal stability was indeed a result of the protein deamidase treatment of the leguminous protein and not the result of the heat treatment step alone, a simple experiment was performed in which an aqueous dispersion of leguminous protein was prepared following the same procedure as described above, except the step of treatment with protein deamidase was omitted. I.e. two different samples were prepared: a first sample which was not subjected to a step of heat treatment and a second sample which was subjected to heat treatment at 85°C for 10 minutes. The results are shown in Figure 2 wherein the samples shown with a check mark underneath the samples are the samples which have been subjected to the heat treatment. The samples in the picture to the left in Figure 2 were prepared using 4% ShuangTa pea protein isolate, and the samples in the picture to the right in Figure 2 were prepared using 8% Ingredion pea protein isolate. The pH was pH 7 for all tested samples. As seen for both sample groups, the use of only heat treatment of the PPI based solutions leads to some reduction in sedimentation in the samples, however, no improvement in dispersibility and colloidal stability is observed, thus confirming that improved colloidal stability is only observed when protein deamidase treatment, together with the heat treatment step, is performed (Figure 1, right picture marked with check mark underneath sample).

[0273] Taken together, the results reported herein and exemplified in Figures 1 and 2 clearly demonstrate the superior results obtained using the methods of the invention in obtaining modified leguminous protein, in particular aqueous dispersions of leguminous protein, which have improved dispersibility and colloidal stability. Furthermore, from data not shown here, it was further confirmed that the aqueous dispersion of modified leguminous protein had improved stability, both immediate and after storage, when mixed with a hot acidic beverage, in particular when mixed with a coffee drink. Example 3: Effect of protein deamidase and heat treatment on colloidal stability of legumebased beverages

[0274] In this example, two different commercial pea protein isolates were used to test if the method according to the invention, in particular the combined use of protein deamidase and heat treatment, could result in legume-based beverages with improved colloidal stability.

[0275] Pea-based beverage samples were prepared using the following procedure. The samples included in the study are shown in Table 1 .

[0276] ShuangTa pea protein isolate (protein content 82.2% based on dry solid) was mixed with deionized H2O to prepare a 10% solution (m / v). The 10% pea beverage solution was then treated with protein deamidase at the dosage 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 which were either not subjected to heat treatment and / or protein deamidase treatment. For the samples subjected to heat treatment after the enzyme treatment, this was done at 85°C for 10 minutes, followed by cooling down on ice to room temperature. Samples not subjected to heat treatment were directly cooled down on ice to room temperature after the enzyme treatment. The samples, containing no additives (e.g. no emulsifiers or stabilizers), were then diluted to protein content of 3.2% (w / w) and mixed well.

[0277] The colloidal stability was examined visually after the samples had been left to stand still for 15 minutes at room temperature. The results from the colloidal stability testing are shown in Figure 3 and clearly demonstrates the effect of using protein deamidase in combination with heat treatment in preparing aqueous dispersions of pea protein isolates. This is seen visually as no particle aggregation, sedimentation or flocculation occurred in the samples subjected to both protein deamidase and heat treatment. In particular, and as seen from visual inspection of samples 5 and 9 in Figure 3 (samples prepared by treatment with protein deamidase, followed by heat inactivation), gave rise to the best colloidal stability with no particle aggregation or settlement appearing in these samples. Without the wish of being bound by any particular theory, the present inventor believes that heat treatment aids in improving colloidal stability of aqueous dispersions of leguminous proteins by increasing the solubility of the leguminous protein.

[0278] It was also confirmed that the protein deamidase claimed and disclosed herein could work at higher incubation temperatures, as exemplified herein with 65°C and 70°C.

[0279] Table 1 Pea-based beverage samples prepared from Shuangta pea protein isolate (4% dry matter), using protein deamidase and / or heat treatment. CTRL, control; PD, protein deamidase; RT, room temperature; temp, temperature.

[0280] In another experiment, pea protein isolate from Cosucra (Cosucra C9, protein content 83% based on dry basis) was tested by mixing the pea protein isolate with deionized H2O to prepare a 10% solution (m / v). The samples included in the study are provided in Table 2. The 10% pea beverage solution was then treated with protein deamidase at the dosage of 2 IPA(U) / g protein. The enzymatic reaction was carried out at 65°C or 70°C for 1 hour. Control samples were included which were either not subjected to heat treatment and / or protein deamidase treatment. For the samples subjected to heat treatment after the enzyme treatment, this was done at 85°C for 10 minutes, followed by cooling down on ice to room temperature. Samples not subjected to heat treatment were directly cooled down on ice to room temperature after the enzyme treatment. The samples, containing no additives (e.g. no emulsifiers or stabilizers) were then diluted to protein content of 3.2% (w / w) and mixed well.

[0281] The colloidal stability was examined visually after the samples had been left to stand still for 15 minutes at room temperature (see Figure 4). The results from this study confirmed the previous findings as described above and shown in Figure 3, i.e., that colloidal stability could be improved by the combination of protein deamidase treatment, followed by heat treatment of the leguminous protein. This is seen, e.g., from samples 5 and 9 in Figure 4, which samples were prepared by incubation with protein deamidase at 65°C or 70°C, respectively, followed by heat inactivation at 85°C for 10 minutes, showing the best colloidal stability of all samples tested.

[0282] 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

CLAIMS1. A method of obtaining an aqueous dispersion of a modified leguminous protein, comprising the steps of:(a) providing an aqueous solution of leguminous protein isolate having a protein content in the range of 1-20% (w / w);(b) treating the aqueous solution of step (a) with a protein deamidase to obtain an enzymatically deamidated leguminous protein; and(c) heat treating the enzymatically deamidated leguminous protein of step (b) to obtain the aqueous dispersion of modified leguminous protein, wherein in steps (b) and (c) pH is in the range of pH 6-8, and wherein the leguminous protein isolate is derived or obtained from pea, soy, fava bean, lentil, or any combination thereof, and wherein after storage for 15 minutes at a temperature of at least 4°C the aqueous dispersion of modified leguminous protein has an improved colloidal stability compared to an aqueous dispersion of modified leguminous protein prepared using a using a similar method but without the use of a protein deamidase and a step of heat treatment following the treatment with the protein deamidase.

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

3. Method of claim 1 or 2, wherein a stabilizer and / or emulsifier is not present in any of steps (a), (b) and / or (c).

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

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

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

7. Method of any of the preceding claims, wherein the leguminous protein isolate is derived or obtained from pea, soy, or a combination thereof.

8. Method of any of the preceding claims, wherein the protein deamidase is derived or obtained from a Chryseobacterium species, such as from Chryseobacterium proteolyticum or Chryseobacterium viscerum.

9. Method of any of the preceding claims, wherein the aqueous dispersion of modified leguminous protein further comprises one or more additional food ingredients selected from the group of lipids, sugars, proteins, vitamins, minerals, amino acids, flavouring agents, dietary fibres, salts, and any combinations thereof.

10. An aqueous dispersion of modified leguminous protein having a pH in the range of 6-8, wherein the modification is obtained by enzymatic deamidation with a protein deamidase followed by heat treatment, and wherein the leguminous protein is derived or obtained from a leguminous protein isolate of pea, soy, fava bean, lentil, or any combinations thereof.

11. The aqueous dispersion of claim 10, wherein the leguminous protein is derived or obtained from a leguminous protein isolate of pea, soy, or a combination thereof.

12. The aqueous dispersion of any of claims 10 or 11 which is essentially free of added stabilizer and / or emulsifier.

13. Use of a protein deamidase in the production of an aqueous dispersion of modified leguminous protein having a pH in the range of pH 6-8 to improve colloidal stability, wherein the modified leguminous protein is derived or obtained from a leguminous protein isolate of pea, soy, fava bean, lentil, or any combinations thereof.

14. Use of claim 13, wherein the aqueous dispersion of modified leguminous protein has an improved colloidal stability compared to aqueous dispersion of modified leguminous protein prepared without use of a protein deamidase.

15. Use of a protein deamidase and heat treatment in the production of an aqueous dispersion of modified leguminous protein having a pH in the range of pH 6-8 to improve colloidal stability, wherein the modified leguminous protein is derived or obtained from a leguminous protein isolate of pea, soy, fava bean, lentil, or any combinations thereof.

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