Use of protein deamidase in process for obtaining seed-based dairy alternative beverage with improved stability

Abstract

The present invention relates to the use of a protein deamidase for obtaining a seed-based dairy alternative beverage with improved stability.

Description

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

[0002] This invention relates to a novel process for obtaining seed-based dairy alternative beverages with improved stability, including the use of protein deamidases. Background Technology

[0003] In recent years, there has been an increase in the number of people pursuing vegan, vegetarian, or non-dairy diets for health reasons. Additionally, food products made from animal milk (such as cow's milk) are receiving increasing attention due to their high environmental costs. These factors are driving growing demand for dairy alternatives to traditionally milk-based foods, including milk, cheese, and yogurt.

[0004] One category of dairy alternatives that has garnered significant attention in recent years is seed-based food products, such as almond-based, pea-based, or soy-based beverages. Almonds, peas, and soybeans are nutrient-rich and low in calories, and some health benefits associated with their consumption include weight loss, lowering blood cholesterol levels, and reducing the risk of heart disease. Furthermore, seed-based beverages have a pleasant flavor and a relatively high fat and protein content compared to their carbohydrate content; therefore, these beverages do not cause spikes in blood sugar levels, making them suitable food product choices for people with diabetes and those following low-carbohydrate diets. These factors contribute to the popularity of seed-based beverages as an alternative to animal-derived milk.

[0005] However, similar to other plant proteins, seed proteins (such as almond protein or pea / soy protein) tend to have poor dispersibility and stability, and exhibit aggregation under heat and / or low pH conditions. This limits the use of seed-based beverages in acidic hot beverages such as coffee and tea.

[0006] Deamidation is known to improve the solubility of plant proteins, thereby improving functional properties such as foaming activity, foaming stability, emulsifying activity, and emulsifying stability. For example, WO 2020 / 176469 A1 describes the use of protein glutaminase in the production of stable protein solutions. To obtain stable almond milk, a stabilizer (gellan gum) is required. Similarly, WO 2022 / 045152 A1 describes treating commercially available almond milk (Rude Health) with protein glutaminase “Amano” 500 (Amano Enzyme Co., Ltd.), followed by heat inactivation and cooling, and then mixing with Nestlé decaffeinated coffee solution (pH 5.2). The pH of the decaffeinated coffee solution after adding almond milk is 5.8. All exemplary milks in WO 2022 / 045152 A1 are prepared by at least a deamidation reaction at 50°C for 5 hours.

[0007] The object of this invention is to identify improved methods for producing dairy alternative beverages, particularly seed-based dairy alternative beverages, with improved technical functional properties (e.g., improved stability to heat and / or low pH). Summary of the Invention

[0008] The inventors of this invention have unexpectedly discovered that by treating a seed material slurry with a protein deamidase, wherein the treatment with the protein deamidase is carried out in the presence of a chloride salt, and / or the protein content of the seed material slurry is at least 3% (w / w), more protein is dissolved, and a seed-based dairy alternative beverage is obtained, which has improved dispersibility, reduced flocculation risk, and excellent stability, particularly when mixed with acidic beverages such as coffee or tea. Furthermore, using the methods disclosed and claimed herein, a lower dose of protein deamidase is required to obtain the improved properties of the seed-based dairy alternative beverage.

[0009] Therefore, the present invention provides a method for obtaining a seed-based dairy product alternative beverage, the method comprising the following steps: (a) Providing a slurry of seed material in water; (b) The seed material in water is treated with a protein deamidase to obtain an enzymatically deamidated seed material slurry; (c) Optionally, the pulp of the enzymatically deamidated seed material is diluted to obtain a diluted pulp of the enzymatically deamidated seed material; and (d) Heat-treating the optionally diluted slurry of the enzymatically deamidinated seed material to obtain the seed-based dairy alternative beverage. The treatment with the protein deamidase is carried out in the presence of chloride salt, and / or the slurry of step (a) has a protein content of at least 3% (w / w).

[0010] The present invention also provides a method for obtaining an almond-based dairy product alternative beverage, the method comprising the following steps: (a) Provide an almond paste in water; (b) The pulp of almond material in water is treated with a protein deamidase to obtain an enzymatically deamidated almond material pulp; (c) Optionally, the pulp of the enzymatically deamidated almond material is diluted to obtain a diluted pulp of the enzymatically deamidated almond material; and (d) Heat-treating the optionally diluted slurry of the enzymatically deamidated almond material to obtain the almond-based dairy alternative beverage, wherein the treatment with the protein deamidase is carried out in the presence of a chloride salt, and / or the slurry of step (a) has a protein content of at least 3% (w / w).

[0011] The method of this invention enables producers of seed-based dairy alternative beverages, such as almond-based or legume-based beverages, to obtain beverages with excellent stability and sensory properties. Examples of such products include almond-based drinks and pea / soybean-based drinks for barista applications. Furthermore, the improved dispersibility and stability of seed-based dairy alternative beverages obtained according to the disclosed method can be achieved with shorter enzyme incubation times and lower enzyme incubation temperatures, thereby further enabling manufacturers to save time and energy.

[0012] Therefore, the present invention also relates to seed-based dairy alternative beverages obtainable by any of the methods disclosed herein. The improved stability of seed-based dairy alternative beverages prepared using the methods of the present invention further avoids or reduces the need to add emulsifiers and / or stabilizers during beverage production, and thus also meets consumer demand for clean-label dairy alternative beverages.

[0013] This invention also relates to the use of protein deamidases in the production of seed-based dairy alternative beverages. In particular, this invention relates to the use of protein deamidases in the production of almond-based or pea / soybean-based dairy alternative beverages to improve stability to heat and / or low pH (e.g., as in acidic beverages). Attached Figure Description

[0014] Figure 1 The stability of almond-based beverages in filtered coffee at room temperature for 2 minutes was demonstrated.

[0015] Figure 2 The stability of almond-based beverages in filtered coffee at room temperature for 50 minutes was demonstrated.

[0016] Figure 3 The stability of almond-based beverages in filtered coffee at room temperature for 2 minutes was demonstrated.

[0017] Figure 4 The stability of almond-based beverages in filtered coffee at room temperature for 3 minutes was demonstrated.

[0018] Figure 5 The stability of almond-based beverages in filtered coffee at room temperature for 5 minutes was demonstrated.

[0019] Figure 6 The stability of almond-based beverages prepared at enzyme incubation temperatures of 30°C and 60°C in filtered coffee at room temperature for 5 minutes was demonstrated.

[0020] Figure 7 The stability of an almond-based beverage prepared at an enzyme incubation temperature of 30°C was demonstrated after 5 minutes in filtered coffee at room temperature.

[0021] sequence

[0022] SEQ ID NO: 1: Derived from Visceral Chlorella ( Chryseobacterium viscerum Protein deamidase (strain formerly known as Chrysobacterium) Chryseobacterium (Species 62563), which has a mature polypeptide sequence as shown in SEQ ID NO: 2.

[0023] SEQ ID NO: 2: Mature polypeptide sequence of protein deamidase derived from Visceral Chrysobacterium.

[0024] SEQ ID NO: 3: Derived from *Cyclophorus pyogenes* ( Chryseobacterium proteolyticum The protein deamidase has a mature polypeptide sequence as shown in SEQ ID NO: 4.

[0025] SEQ ID NO: 4: Mature polypeptide sequence of protein deamidase derived from Chrysobacterium utilis.

[0026] SEQ ID NO: 5: Derived from *Aureobacillus davidii* (Brucella davidii) Chryseobacterium gambrini The protein deamidase has a mature polypeptide sequence as shown in SEQ ID NO: 6.

[0027] SEQ ID NO: 6: Mature polypeptide sequence of protein deamidase derived from *Aureobacterium brevesense*.

[0028] SEQ ID NO: 7: Derived from Culex pipiens lucida ( Chryseobacterium culicisThe protein deamidase has a mature polypeptide sequence as shown in SEQ ID NO: 8.

[0029] SEQ ID NO: 8: Mature polypeptide sequence of protein deamidase derived from Culex pipiens.

[0030] SEQ ID NO: 9: Originating from sewage-borne Chlorella vulgaris ( Chryseobacterium defluvii The protein deamidase has a mature polypeptide sequence as shown in SEQ ID NO: 10.

[0031] SEQ ID NO: 10: Mature polypeptide sequence of protein deamidase derived from *Cryptospirobacter septicemia*. Detailed Implementation

[0032] Based on this detailed description, the following definitions apply. Note that the singular forms “a / an” and “the” include plural indicators unless the context explicitly indicates otherwise.

[0033] As used herein, the terms “drinks,” “milk,” and “beverages” are used interchangeably and have the same meaning unless otherwise defined or explicitly indicated by the context.

[0034] Unless otherwise defined or explicitly indicated by the context, all percentages are weight percentages (percentage w / w or "% (w / w)").

[0035] Unless otherwise defined or explicitly indicated by the context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0036] The term "dairy alternative beverage" refers to a food product that can be used as a substitute for conventional dairy-based food products, such as animal-derived dairy beverages. The dairy alternative beverage according to the invention is a seed-based dairy alternative beverage. In the context of this invention, the terms "seed," "grain," or "legume" refer to the reproductive structure of a plant, and when used, for example, to describe "seed material" and "seed-based dairy alternative beverage," they mean a food source that can be ingested by humans and animals (including domesticated animals, such as companion animals) in its unprocessed and / or processed form.

[0037] In some embodiments, the seed material is obtained from or derived from nuts. Examples of nuts include, but are not limited to, almonds, cashews, coconuts, hazelnuts, macadamia nuts, pistachios, peanuts, pecans, and walnuts.

[0038] In some embodiments, the seed material is derived from or obtained from legumes. Examples of legumes include, but are not limited to, soybeans, peas, chickpeas, mung beans, broad beans, lupins, and lentils.

[0039] Seed-based dairy alternative beverages may or may not be combined with additional food ingredients to produce seed-based dairy alternative beverages. Additional food ingredients that may be added to seed-based dairy alternative beverages can be any food ingredient that a person skilled in the art would find useful. Additional food ingredients can be solid or liquid ingredients. Additional food ingredients may or may not be plant-based. In some embodiments, the additional food ingredient is water.

[0040] Other food ingredients that may be added to seed-based dairy alternative beverages include, but are not limited to, lipids (e.g., oils, especially vegetable oils), sugars (e.g., sucrose), proteins, synthetic amino acids in various forms, dietary fiber, salt, minerals, flavorings, vitamins, and any combination thereof.

[0041] In this embodiment, lipids are added to the seed-based dairy alternative beverage and / or the slurry of seed material. The lipids can be vegetable oils or mixtures of vegetable oils. Lipids can be selected from rapeseed oil, flaxseed oil, safflower oil, soybean oil, olive oil, sunflower oil, palm oil, and combinations thereof. In one embodiment, the lipid is rapeseed oil, sunflower oil, or a combination thereof. The selection of a suitable lipid can be based on the type of seed-based dairy alternative beverage desired.

[0042] In one embodiment, sugar is added to the seed-based dairy alternative beverage and / or the slurry of the seed material. In one embodiment, the sugar is sucrose and / or fructose.

[0043] In embodiments, additional food ingredients that may be added to the seed-based dairy alternative beverage and / or seed material slurry are selected from the following list: sodium chloride, dicalcium carbonate, dicalcium phosphate, tricalcium phosphate, calcium carbonate, and any combination thereof. In a preferred embodiment, sodium chloride is added to the seed material slurry. Those skilled in the art know how to determine the appropriate amount of additional ingredients to be added to the seed material slurry and / or seed-based dairy alternative beverage. In one embodiment, sodium chloride is used, and its concentration ranges from, for example, 0.05% to 0.2% (w / w) based on the dairy alternative beverage.

[0044] In one embodiment, vitamins and / or minerals are added to the seed-based dairy alternative beverage. In another embodiment, vitamins and / or minerals are added to the pulp of the seed material. Vitamins may be vitamin A, vitamin C, vitamin D, vitamin E, vitamin B12, thiamine (vitamin B1), riboflavin (vitamin B2), niacin (vitamin B3), vitamin B6, vitamin K, folic acid (vitamin B9), and mixtures thereof. Minerals may be calcium, phosphorus, magnesium, sodium, potassium, chloride, iron, zinc, iodine, selenium, copper, and mixtures thereof.

[0045] Seed-based dairy alternative beverages can be fortified with plant-based dairy alternative powders (e.g., like soy milk powder) or with concentrated or isolated proteins (e.g., like soy protein or pea protein isolates or soy protein or pea protein concentrates). In embodiments, seed-based dairy alternative beverages are fortified, such as almond-based drinks fortified with pea protein or soy protein. In embodiments, seed-based dairy alternative beverages are fortified with calcium carbonate (CaCO3), such as almond-based drinks fortified with CaCO3. Seed-based dairy alternative beverages fortified with both concentrated or isolated proteins and CaCO3 are also contemplated. Optionally, fortified seed-based dairy alternative beverages can be further formulated with stabilizers (e.g., gellan gum). In one embodiment, the seed-based dairy alternative beverage is an almond drink containing CaCO3 and gellan gum. In the context of this invention, the fortification and formulation of seed-based dairy alternative beverages are carried out after the methods claimed and disclosed herein (i.e., after protein deamidase treatment). Therefore, in embodiments of the invention, the treatment of the seed material slurry in water with protein deamidase, optional dilution and heat treatment are carried out in the absence of added emulsifiers and / or stabilizers, and the resulting seed-based dairy alternative beverage is then fortified, for example, with CaCO3 and / or gellan gum.

[0046] Seed material may be in the form of an aqueous solution or suspension of a seed-based dairy substitute powder. Alternatively, seed material may be any other suitable formulation obtained from seeds, such as an aqueous suspension of a powder obtained from seeds. Seed material may be any combination of the above.

[0047] Preferably, the protein content of the seed-based dairy alternative beverage is at least 0.5% (w / w).

[0048] Preferably, the protein content of the seed-based dairy alternative beverage is up to 8% (w / w).

[0049] In the examples, the seed-based dairy alternative beverages have a protein content of about 0.5% (w / w), 1% (w / w), 1.5% (w / w), 2% (w / w), 2.5% (w / w), 3% (w / w), 3.5% (w / w), or 4% (w / w).

[0050] In a preferred embodiment, the seed-based dairy alternative beverage has a protein content of about 1% (w / w).

[0051] Preferably, the lipid content of the seed-based dairy alternative beverage is at least 1% (w / w).

[0052] Preferably, the lipid content of the seed-based dairy alternative beverage is up to 5% (w / w).

[0053] It can standardize and / or homogenize dairy alternative beverages. It can pasteurize or otherwise heat-treat dairy alternative beverages.

[0054] Dairy alternative beverages obtained according to the methods disclosed herein do not require the addition of emulsifiers and / or stabilizers to achieve their desired properties. Specifically, using the methods disclosed herein, seed-based dairy alternative beverages (e.g., almond-based beverages) can be obtained that are substantially free of added emulsifiers and / or stabilizers and still possess improved stability (particularly improved stability to heat and / or low pH). In the examples, “substantially free” means 0% (w / w). Therefore, seed-based dairy alternative beverages that do not flocculate or precipitate even after prolonged storage or after mixing with hot and / or acidic beverages (e.g., coffee or tea drinks) can be obtained. As used herein, the terms “emulsifier” and “stabilizer” mean added emulsifiers and stabilizers, i.e., ingredients that are not naturally or inherently present in the materials used to prepare the seed-based dairy alternative beverages. Examples of such emulsifiers and stabilizers include, but are not limited to, thickeners (e.g., carboxymethyl cellulose, gellan gum, hydroxypropyl starch, and agar) and emulsifiers (e.g., monoglycerides and diglycerides).

[0055] The seed-based dairy alternative beverages obtained by the method of the present invention have improved stability, including improved storage stability. Therefore, the dairy alternative beverages can be stored before consumption. Storage does not affect the properties of the dairy alternative beverages, and they also substantially do not precipitate or flocculate over time. In the context of the present invention, when used to describe seed-based dairy alternative beverages (e.g., almond-based beverages), the term "storage stability" or "storage-stable" means that the beverage is resistant to flocculation or precipitation both immediately after its production and after prolonged storage under typical storage conditions for consumer beverages and beverage additive products, and after being combined with acidic beverages (e.g., coffee or tea drinks). The stability of the dairy alternative beverages can be determined by any method known in the art for such assessment, including by visual evaluation and taste testing.

[0056] The seed-based dairy alternative beverages disclosed herein are largely unaffected by acidic (i.e., low pH) food matrices (such as coffee or tea beverages). Therefore, in one embodiment, the seed-based dairy alternative beverage is used in acidic beverages (e.g., sports drinks, coffee drinks, or tea drinks). In a preferred embodiment, the seed-based dairy alternative beverage is an almond-based beverage suitable for barista applications, such as for preparing coffee drinks containing coffee and an almond-based beverage. In another preferred embodiment, the seed-based dairy alternative beverage is a pea-based / soy-based beverage for use in barista applications. In the context of this invention, "barista beverage" or "for use in barista applications" means a dairy alternative beverage suitable for mixing with acidic beverages, and this dairy alternative beverage may or may not contain additional ingredients to produce a beverage that produces better foam and has a higher viscosity, thereby enabling consistently stable pouring and latte art creation. Compared to standard seed-based dairy alternative beverages, seed-based barista beverages may contain more fat and / or more protein.

[0057] The use of the seed-based dairy alternative beverage according to the invention in obtaining ready-to-drink beverages is also considered.

[0058] In the context of this invention, "acidic / acidic food matrix" or "acidic beverage" means a sports drink, coffee drink, or tea drink with a pH in the range of 3-6 (e.g., pH in the range of 4-6, such as pH in the range of about 4.5-5.5, such as pH in the range of about 4.8-5.1). For example, an acidic beverage can be coffee with a pH below 5.0. An acidic beverage can also be a tea beverage, such as a tea beverage based on Indian tea blends, spiced tea blends, black tea (e.g., Assam and Darjeeling), green tea, Earl Grey tea, oolong tea, and rooibos tea. Such tea beverages can be used to prepare Indian spiced lattes. In embodiments, seed-based dairy alternative beverages are stable when added to acidic beverages with a pH below 5.1. Such acidic beverages can be, for example, filtered coffee, espresso drinks, etc.

[0059] Dairy alternative beverages include seed-based beverages, creamer, etc. Examples of seed-based beverages include almond drinks, cashew drinks, chickpea drinks, coconut drinks, fava bean drinks, hazelnut drinks, lentil drinks, lupin drinks, macadamia nut drinks, mung bean drinks, pistachio drinks, pea drinks, peanut drinks, pecan drinks, soy drinks, walnut drinks, and beverages containing any combination thereof. In an embodiment, the dairy alternative beverage is an almond drink, a pea drink, a soy drink, or any combination thereof. In a preferred embodiment, the dairy alternative beverage is an almond-based beverage. In another preferred embodiment, the dairy alternative beverage is a pea-based beverage or a soy-based beverage.

[0060] The dairy alternative beverage of the present invention is derived from or obtained from seed material, which is or is derived from the edible part of a plant. In some embodiments, the seed material is derived from or obtained from almonds, cashews, chickpeas, coconuts, broad beans, hazelnuts, macadamia nuts, mung beans, lentils, lupins, pistachios, peas, peanuts, pecans, soybeans, walnuts, or any combination thereof. In a preferred embodiment, the seed material is obtained from or derived from almonds, peas, soybeans, or any combination thereof.

[0061] In some embodiments, the seed material is heat-treated. In some embodiments, the seed material is dehydrated. In some embodiments, the seed material is dehulled, ground, wet-milled, and / or dry-milled. In some embodiments, the seed material is a powder (e.g., almond flour) or a paste (e.g., almond paste) or any combination thereof. In some embodiments, the seed material is pulverized or ground to produce a paste, such as almond paste.

[0062] In the method of this invention, seed material is suspended in water to provide a seed material slurry, and the resulting seed material slurry in water is enzymatically treated with a protein deamidase to obtain an enzymatically deamidated seed material slurry. In the context of this invention, the term "enzymatically deamidated seed material" means seed material that has undergone protein deamidase treatment to achieve deamidation. Those skilled in the art will know suitable analytical methods for determining the enzymatic deamidation of seed material. One such method is illustrated in Example 1 by measuring the free ammonium content (NH4).

[0063] In one aspect of the invention, during protein deamidase treatment, the protein content of the seed material pulp is at least 3% (w / w), for example at least 3.5% (w / w), at least 4% (w / w), at least 4.5% (w / w), at least 5% (w / w), at least 5.5% (w / w), at least 6.5% (w / w), at least 7% (w / w), at least 7.5% (w / w), at least 8% (w / w), at least 8.5% (w / w), at least 9% (w / w), at least 9.5% (w / w), or at least 10% (w / w).

[0064] In a preferred embodiment, the protein content of the seed material slurry is in the range of 4%-20% (e.g., about 5%-10% (w / w)), such as about 5% (w / w), about 8% (w / w), or about 10% (w / w). In one embodiment, the protein content of the seed material slurry is at least 5% (w / w) during protein deamidase treatment.

[0065] In some embodiments, a slurry of enzymatically deamidated seed material is diluted to obtain a diluted slurry of the enzymatically deamidated seed material, which is then heat-treated to obtain a seed-based dairy alternative beverage. As used herein, "diluted slurry of enzymatically deamidated seed material" means a suspension of enzymatically deamidated seed material with a protein content of less than 3% (w / w). Dilution can be obtained by adding a diluent that is deemed useful by those skilled in the art. Examples of diluents used in the methods of the present invention include, but are not limited to, aqueous solutions, such as water, plant-based dairy alternative beverages, or combinations thereof. In one embodiment, the slurry of the enzymatically deamidated seed material is diluted with water.

[0066] In one embodiment, the protein content of the diluted slurry of the enzymatically deamidated seed material is at most 2.5% (w / w). In a preferred embodiment, the protein content of the diluted slurry of the enzymatically deamidated seed material is in the range of 0.1%-2.5% (w / w), preferably in the range of 0.5%-2% (w / w), and more preferably in the range of 1%-2% (w / w). In a preferred embodiment, the protein content of the diluted slurry of the enzymatically deamidated seed material is about 1% (w / w). In some embodiments, the protein content of the diluted slurry of the enzymatically deamidated seed material is about 2.5% (w / w), wherein the seed material is derived from or obtained from peas, soybeans, or combinations thereof. In other embodiments, the protein content of the diluted slurry of the enzymatically deamidated seed material is about 1.5% (w / w), wherein the seed material is derived from or obtained from almonds.

[0067] In one embodiment, a method for obtaining an almond-based dairy product alternative beverage is provided, the method comprising the following steps: (a) Provide a pulp of almond material in water with a protein content of at least 3% (w / w); (b) The pulp of almond material in water is treated with a protein deamidase to obtain an enzymatically deamidated almond material pulp; (c) Diluting the slurry of the enzymatically deamidated almond material to obtain a diluted slurry of the enzymatically deamidated almond material; and (d) Heat-treating the optionally diluted slurry of the enzymatically deamidated almond material to obtain the almond-based dairy alternative beverage.

[0068] The inventors unexpectedly discovered that by treating a slurry of seed material with a protein content of at least 3% (w / w) (e.g., about 5%, 8%, or 10% (w / w)) with a protein deamidase, followed by dilution to, for example, a protein content of 1%, and then heat-treating it, for example, to inactivate the protein deamidase, a seed-based dairy alternative beverage was obtained, which, when used as a dairy alternative beverage, exhibits improved or increased stability to heat and / or low pH. This has been experimentally demonstrated (see Examples 2-5), for example, the almond-based dairy alternative beverage prepared according to the method disclosed herein exhibits improved stability when mixed with warm filtered coffee beverages (whether immediately upon mixing or after 1 hour).

[0069] In a preferred embodiment, a method for obtaining a seed-based dairy alternative beverage is provided, the method comprising the following steps: (a) Provide a slurry of seed material in water with a protein content of 5%-20% (w / w); (b) The seed material in water is treated with a protein deamidase to obtain an enzymatically deamidated seed material slurry; (c) Diluting the pulp of the enzymatically deamidated seed material to obtain a diluted pulp of the enzymatically deamidated seed material with a protein content of 1%-2.5% (w / w), for example 1%-2% (w / w); and (d) The diluted slurry of the enzymatically deamidated seed material is heat-treated to obtain the seed-based dairy alternative beverage.

[0070] In some embodiments, the method of the present invention further includes the following steps: (e) Separating the seed-based dairy alternative beverage into a solid stream and a liquid stream; (f) Harvesting the liquid stream as a liquid seed-based dairy alternative beverage; and (g) Optionally, the protein deamidase is inactivated.

[0071] The inventors have discovered that for legume substrates (such as pea or soybean materials), the step of diluting an enzymatically deamidated legume material into a slurry can be optionally performed to obtain a diluted slurry of enzymatically deamidated legume material.

[0072] Therefore, the present invention provides a method for obtaining a soy-based dairy product alternative beverage, the method comprising the following steps: (a) Provide a slurry of legume material in water with a protein content of at least 3% (w / w); (b) The slurry of the legume material in water is treated with a protein deamidase to obtain an enzymatically deamidated legume material slurry; (c) Optionally, the slurry of the enzymatically deamidated legume material is diluted to obtain a diluted slurry of the enzymatically deamidated legume material; and (d) Heat-treating the diluted slurry of the enzymatically deamidinated legume material to obtain the legume-based dairy alternative beverage, wherein the legume material is derived from or obtained from soybeans, peas, chickpeas, mung beans, broad beans, lupins, lentils or any combination thereof, preferably soybeans and / or peas.

[0073] In a preferred embodiment, the protein content of the slurry of the bean material in step (a) in water is at least 6% (w / w), or even more preferably at least 8% (w / w), such as at least 9% (w / w).

[0074] In another aspect of the invention, the enzymatic treatment with protein deamidase is carried out in the presence of a chloride salt. In the context of this invention, the term "chloride salt" has its conventional meaning in the art and includes chloride salts relevant to food applications. In a preferred embodiment, the chloride salt is selected from potassium chloride and sodium chloride. In a preferred embodiment, the chloride salt is sodium chloride.

[0075] Without being bound by any particular theory, the inventors believe that the presence of chloride salts promotes the enzymatic deamidation of proteins in seed materials during treatment with protein deamidases, while simultaneously stabilizing the enzymes and proteins in the pulp. Therefore, embodiments of the present invention relate to the use of protein deamidases and chloride salts in the production of seed-based dairy alternative beverages to improve stability and deamidation rate. In the context of this invention, "deamidation rate" refers to the speed or frequency at which proteins in seed materials undergo deamidation when incubated with a protein deamidase. The deamidation rate can be assessed based on the amount of ammonium and / or soluble proteins present in the pulp of the deamidated seed material.

[0076] Preferably, the amount of chloride salt is based on at least 0.05% (w / w) of the seed material in the water slurry.

[0077] Preferably, the amount of chloride salt is at most 2% (w / w) of the seed material slurry in water.

[0078] In a preferred embodiment, the amount of chloride salt is 0.10% (w / w) or 0.15% (w / w) of the seed material slurry in water. In a preferred embodiment, the chloride salt is sodium chloride, and the amount of sodium chloride is about 0.10-0.15% (w / w) of the seed material slurry in water.

[0079] The inventors of this invention have discovered that by treating a seed material slurry in water with a protein deamidase in the presence of a chloride salt (e.g., sodium chloride and / or potassium chloride), seed-based dairy alternative beverages, particularly nut-based beverages, can be obtained. Such beverages not only contain more dissolved protein but also exhibit improved dispersibility and stability when mixed with acidic beverages (e.g., coffee or tea drinks). This has been experimentally demonstrated (see Example 5), for example, an almond-based dairy alternative beverage prepared according to the method disclosed herein exhibits improved stability when mixed with a warm, filtered coffee drink.

[0080] In the context of this invention, stability to heat and / or low pH means that the seed-based dairy alternative beverage can maintain its stability and integrity when exposed to elevated temperatures and pH levels within an acidic range, as may occur when mixed with warm, acidic beverages (e.g., coffee drinks). In the context of this invention, "low pH" typically refers to a pH value less than 7, preferably less than 5, which indicates acidic conditions. Preferably, the stability of the seed-based dairy alternative beverage is at a pH less than 5. For example, the stability and integrity of the beverage can be seen from the absence of any precipitation or flocculation when the seed-based dairy alternative beverage is mixed with warm, acidic beverages. Therefore, the seed-based dairy alternative beverage of this invention is resistant to flocculation and precipitation in warm, acidic beverages.

[0081] When enzymatic deamidation of seed material is carried out in the presence of chloride salts, substrates with low protein content (e.g., 1% (w / w)) can be enzymatically treated. Furthermore, compared to processes that do not use chloride salts during enzyme incubation, lower doses of protein deamidase can be used, thus significantly reducing manufacturing costs. Adding chloride salts during enzymatic treatment (particularly protein deamidase treatment) further eliminates the need for additional steps in formulating seed-based dairy alternative beverages, thereby further optimizing the overall process for obtaining the final beverage intended for consumers.

[0082] Therefore, in some embodiments, the present invention provides a method for obtaining a seed-based dairy product alternative beverage, the method comprising the following steps: (a) Providing a slurry of seed material in water; and (b) The seed material in water is treated with a protein deamidase to obtain the seed-based dairy alternative beverage, wherein the treatment with the protein deamidase is carried out in the presence of a chloride salt.

[0083] In a preferred embodiment, the seed material treated with protein deamidase in the presence of chloride salt is derived from or obtained from nuts, preferably from or derived from almonds.

[0084] The temperature of the protein deamidase used to treat the seed material slurry in water is maintained in the range of 10°C to 80°C, for example, in the range of 20°C to 65°C, so that the seed material is enzymatically deamidated by the protein deamidase to produce enzymatically deamidated seed material. In some embodiments, the slurry temperature is maintained between 15°C to 40°C, 25°C to 40°C, 30°C to 45°C, 35°C to 50°C, 40°C to 55°C, 50°C to 60°C, or 50°C to 65°C. In other embodiments, the slurry temperature is maintained at about 20°C, about 25°C, about 30°C, about 35°C, about 40°C, about 45°C, about 50°C, about 55°C, or about 60°C.

[0085] In some embodiments, the slurry containing the added protein deamidase is maintained at 10°C–80°C (e.g., 20°C–65°C) for at least 10 minutes to allow enzymatic deamidation of the seed material. In some embodiments, the slurry is maintained for about 10, about 15, about 20, about 25, about 30, about 60, about 120, about 180, or about 240 minutes to allow enzymatic deamidation of the seed material. In some embodiments, the slurry is maintained for at least about 10, 30, 60, or 90 minutes. In some embodiments, the slurry is maintained for 30 minutes. In some embodiments, the slurry is maintained for 60 minutes. In some embodiments, the slurry is maintained at 50°C–60°C for about 30–60 minutes.

[0086] In some embodiments, lipids are added to the slurry. Lipids may be added before, during, or after treatment with a protein deamidase. In one embodiment, lipids are added before treatment with a protein deamidase. In another embodiment, lipids are added after treatment with a protein deamidase. The lipids may be selected from rapeseed oil, linseed oil, safflower oil, soybean oil, olive oil, sunflower oil, palm oil, and combinations thereof. In one embodiment, the lipid is soybean oil.

[0087] The methods used (including temperature range, pH, and enzymatic treatment duration) will vary depending on the seed material and the enzymes added to the slurry. Technicians will know how to determine the optimal process parameters based on the seed material and enzymes used. Preferably, during slurry treatment of the seed material with protein deamidase, the pH is in the range of pH 5-8.

[0088] Enzymatic deamidation of seed material can inactivate protein deamidases. Enzyme inactivation can occur at any step after hydrolysis. In some embodiments, enzyme inactivation is achieved by heat treatment. In some embodiments, the heat treatment is performed at 85°C-95°C for 5-30 minutes. In another embodiment, the heat treatment is performed at 85°C-95°C for 10 minutes. In some embodiments, the heat treatment is performed at 90°C for 5, 10, 15, 20, 25, or 30 minutes.

[0089] In some embodiments, the enzyme is inactivated by ultra-high temperature (UHT) treatment. UHT treatment can be direct or indirect. In some embodiments, UHT treatment is performed at 135°C-154°C for 1-10 seconds. In other embodiments, UHT treatment is performed at 140°C-150°C for 3, 4, 5, 6, 7, 8, 9, or 10 seconds. In still other embodiments, UHT treatment is performed at 140°C-145°C for 3, 4, 5, 6, 7, 8, 9, or 10 seconds. In some embodiments, UHT treatment is performed at 143°C for 4, 5, 6, 7, or 8 seconds.

[0090] After optional enzyme inactivation, the slurry of enzymatically deamidated seed material can be cooled. The enzymatically deamidated seed material can be used directly to obtain seed-based dairy alternative beverages, or it can be separated into solid and liquid streams, for example, by centrifugation. Centrifugation can occur in a sedimentation centrifuge. After centrifugation, the liquid stream can be harvested or collected and used as an aqueous solution containing the enzymatically deamidated seed material. The liquid stream may still contain some solid matter. In some embodiments, the liquid stream contains 1%-80% solids. In other embodiments, the liquid stream contains 1%-10%, 5%-20%, 10%-25%, 20%-35%, 25%-40%, 30%-45%, 35%-50%, 40%-55%, 45%-60%, 50%-65%, 55%-70%, 60%-75%, or 65%-80% solids. In some embodiments, the liquid stream contains 10%-15% solids.

[0091] In some embodiments, the liquid stream is further processed to remove water, also known as concentration. Concentration also increases the relative amount of solids in the concentrated liquid stream. In some embodiments, water removal concentrates the product of enzymatic treatment. Concentration can be achieved by evaporating water from the liquid stream. In some embodiments, the concentrated liquid stream contains 10%-100% solids. In other embodiments, the concentrated liquid stream contains 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%, or 90%-100% solids. In some embodiments, water removal increases the viscosity of the dairy alternative beverage.

[0092] In some embodiments, the liquid stream is used directly as a liquid seed-based dairy alternative beverage. Additional food ingredients may be added to the liquid stream to produce the dairy alternative beverage. In some embodiments, the liquid stream is derived from almond, pea, or soy materials. For example, almond-derived, pea-derived, or soy-derived liquid streams may be formulated using, for example, sodium chloride (NaCl), sugar, and flavoring agents. Furthermore, liquid streams may be formulated using, for example, calcium carbonate, protein, sugar, and flavoring agents. It may be homogenized. It may be UHT or ESL treated and aseptically packaged. The final product may be sold as a seed-based dairy alternative beverage, such as an almond-based beverage, a pea-based beverage, or a soy-based beverage.

[0093] The seed material slurry can be further processed, for example, by treatment with additional enzymes, including, for example, additional modifying enzymes or hydrolases. Thus, in some embodiments, the seed material slurry is further treated with one or more hydrolases selected from the group consisting of pectinase, hemicellulase, xylanase, β-glucanase, mannanase, glucanase, glucosylamylase, isoamylase, α-amylase, β-amylase, and mixtures thereof. In some embodiments, the additional modifying enzyme or hydrolases are added together with a protein deamidase, and the enzymatic treatment is performed simultaneously. In other embodiments, the modifying enzyme or hydrolases are added before or after the protein deamidase, and the enzymatic treatment is performed in a separate step of the method. The enzymes used in the methods of the present invention can be added to the slurry containing the seed material in any suitable form (e.g., in liquid form, particularly a stabilizing liquid), or they can be added as a substantially dry powder or granules. For example, granules can be produced as disclosed in U.S. Patent Nos. 4,106,991 and 4,661,452. Liquid enzyme preparations can be stabilized, for example, by adding sugars, sugar alcohols, or lactic acid according to established procedures. Other enzyme stabilizers are well known in the art. Additionally, enzymes can be added to a slurry containing seed material in any suitable manner, such as as individual components (added separately or sequentially), or as a single step or in a composition.

[0094] Protein deamidase In the method of the present invention, seed material is treated with a protein deamidase to obtain enzymatically deamidated seed material. In a preferred embodiment of the method of the present invention, seed material derived from or obtained from almonds is treated with a protein deamidase to obtain enzymatically deamidated almond material. In one aspect of the present invention, enzymatic deamidation is carried out using a protein deamidase in the presence of a chloride salt, preferably in the presence of sodium chloride.

[0095] In this invention, protein deamidase refers to an enzyme that acts directly on the amide groups of the amino acid side chains that make up a protein to cause deamidation and release ammonia without cleaving the peptide bonds of the protein or causing the protein to crosslink.

[0096] The term "deamidase" refers to the activity of protein-glutamine glutaminase (also known as glutamylpeptide glutaminase), as described in EC 3.5.1.44, which catalyzes the hydrolysis of γ-amides of glutamine substituted at the carboxyl position or both the α-amino and carboxyl positions, such as L-glutamylglycine and L-phenylalanyl-L-glutamylglycine. Therefore, deamidases can deamidate glutamine residues in proteins to glutamate residues, and deamidases are also called protein glutamine deamidases. Deamidases include Cys-His-Asp catalytic triads (e.g., Cys-156, His-197, and Asp-217, as described in Hashizume et al., "Crystal structures of protein glutaminase and its proforms converted into enzyme-substrate complex"). Journal of Biological Chemistry (See [Journal of Biochemistry], Vol. 286, No. 44, pp. 38691–38702) and belongs to InterPro entry IPR041325.

[0097] Deamidases may also include protein asparaginases, which act directly on the amide group of the side chain of asparagine residues in a protein to release ammonia and thus convert the asparagine residues to aspartic acid residues. In this invention, either protein glutaminase or protein asparaginase, or a combination of both, can be used as the protein deamidase. An example of a protein deamidase used in this invention is protein glutaminase.

[0098] The protein deamidases used in the methods of this invention can be obtained from microorganisms of any genus. For the purposes of this invention, the term "obtained from," as used herein in conjunction with a given source, should mean that the polypeptide encoded by the polynucleotide is produced by that source or by a strain that has inserted a polynucleotide from that source. In one embodiment, the polypeptide obtained from a given source is secreted extracellularly.

[0099] Protein deamidases can be obtained from microorganisms using any suitable technique. For example, an enzyme preparation can be obtained by fermenting a suitable microorganism and subsequently isolating the protein deamidase preparation from the resulting fermentation broth or microorganism using methods known in the art. Protein deamidases can also be obtained using recombinant DNA technology. Such methods typically involve culturing host cells transformed with a recombinant DNA vector containing a DNA sequence encoding the protein deamidase, and operatively linking the DNA sequence to a suitable expression signal such that the enzyme can be expressed in a culture medium under conditions allowing enzyme expression, and recovering the enzyme from the culture. The DNA sequence can also be incorporated into the genome of a host cell. The DNA sequence can be genomic, cDNA, or synthetically derived, or any combination thereof, and can be isolated or synthesized according to methods known in the art.

[0100] Protein deamidases can be purified. As used herein, the term "purified" includes protein deamidase proteins that are substantially free of insoluble components from the producing organism. The term "purified" also includes protein deamidase proteins that are substantially free of insoluble components from the native organism from which the enzyme was obtained. Preferably, the enzyme can also be separated from some soluble components of the source organism and the culture medium. More preferably, separation can be performed by one or more unit operations: filtration, precipitation, or chromatography.

[0101] There are no particular limitations on the type or source of the protein deamidase used in this invention. Examples of protein deamidases include those derived from the genera *Chlorella* and *Flavobacterium*. Flavobacterium genus), Stenotrophomonas ( Empedobacter genus), Sphingosine Bacillus ( Sphingobacterium genus), Chlorella ( Aureobacterium genus) or genus of aromatic fungi ( Myroides Protein deamidases (genus).

[0102] Protein deamidases can originate from any of the sources mentioned herein. In the context of this invention, the term "originating from" means that the enzyme can be isolated from the organism in which it naturally exists, i.e., the amino acid sequence of the protein deamidase is identical to that of the natural polypeptide. The term "originating from" also means that the enzyme can be recombinantly produced in a host organism, and the recombinant enzyme has an amino acid sequence identical to that of the natural enzyme, or has a modified amino acid sequence (e.g., with one or more amino acids that are deleted, inserted, and / or substituted), i.e., the recombinant enzyme is a mutant of the natural amino acid sequence. The meaning of "natural enzyme" includes natural variants. Furthermore, the term "originating from" includes enzymes synthesized, for example, through peptide synthesis. The term "originating from" also includes enzymes that have been modified in vivo or in vitro, for example, through glycosylation, phosphorylation, etc. For recombinant enzymes, the term "originating from" refers to the identity of the enzyme, not the identity of the host organism from which the recombinant enzyme is produced.

[0103] In some embodiments, the protein deamidase may be derived from the genus *Chlorella*, such as *Chlorella vulgaris*, *Chlorella brevis*, *Chlorella culminatus*, *Chlorella septicemia*, or *Chlorella pyrenoidosa*. In some embodiments, the deamidase in the method of the present invention is derived from or obtained from *Chlorella vulgaris*.

[0104] EP1839491 discloses the presence of Corynebacterium glutamicum ( Corynebacterium glutamicum The clone of a protein glutaminase from *Chlorella utilis* expressed in the sample. Deamidases are also commercially available, for example, protein glutaminases derived from *Chlorella*, such as "Amano PG500" (manufactured by Amano Enzyme Products Co., Ltd.).

[0105] For example, protein deamidases can be obtained from the culture medium of the aforementioned microorganisms.

[0106] Protein deamidases are produced by microbial cells in an inactive proform containing a propeptide domain tightly bound to the deamidase domain. This proform is expressed as a fusion protein with reduced deamidase activity to protect the viability of the host cell. Essentially, the fusion protein is post-processed to remove the propeptide and release the active deamidase outside the host cell. However, in recombinant expression systems, the fusion protein is secreted outside the host cell as an inactive proform containing the propeptide. The propeptide can then be enzymatically cleaved to separate it from the mature deamidase. The protein deamidase of the methods and compositions of the present invention is a mature deamidase in which the propeptide has been removed. In some embodiments, the propeptide is enzymatically cleaved by an endopeptidase. In some embodiments, the propeptide may still be present in the composition containing the mature deamidase.

[0107] The recombinant mature protein deamidases used in the method of this invention comprise polypeptides of SEQ ID NO: 2, 4, 6, 8, and 10. Each mature protein deamidase is derived from a proto-deamidase polypeptide comprising polypeptides of SEQ ID NO: 1, 3, 5, 7, and 9, respectively. The proto-deamidase polypeptide contains a propeptide fused to the N-terminus of a deamidase identical to that of the polypeptides of SEQ ID NO: 2, 4, 6, 8, or 10. The propeptide can be enzymatically cleaved from the proto-deamidase polypeptide to release the mature deamidase. A naturally occurring propeptide sequence is provided in the proto-deamidase polypeptide.

[0108] The methods and compositions of the present invention comprise a mature deamidase and, optionally, a second polypeptide derived from the propeptide of the deamidase. The second polypeptides described herein are mutant variants of naturally occurring propeptides. These variant propeptide sequences have been found to bind weakly to their corresponding deamidases, making them more readily cleaved enzymatically after recombinant expression and secretion from host cells. The polypeptides of SEQ ID NO: 1 to 10 are derived from species of the genus *Chlorella* and described in PCT application PCT / EP2023 / 055936; filed on March 8, 2023 (which is incorporated herein by reference).

[0109] After expressing the original polypeptide in a recombinant expression system, the propeptide is cleaved using a site-specific endopeptidase, leaving an active mature deamidase. In some embodiments, the cleaved propeptide is not purified from the mature deamidase. Therefore, the propeptide can be present in a composition containing a mature deamidase.

[0110] According to a preferred embodiment, the protein deamidase used in the process of the present invention is derived from or obtained from species of the genus *Chlorella*, such as *Chlorella utilis* or *Chlorella viviparus*.

[0111] In the context of this invention, the term "mature polypeptide" means a polypeptide in its mature form after N-terminal processing (e.g., removal of a signal peptide). A "signal peptide" is an amino acid sequence attached to the N-terminal portion of a protein that promotes its secretion outside the cell. The mature form of an extracellular protein lacks a signal peptide, which is cleaved during the secretion process.

[0112] In one embodiment, the deamidase is selected from polypeptides having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with SEQ ID NO: 2.

[0113] In one embodiment, the deamidase is selected from polypeptides having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with SEQ ID NO: 4.

[0114] In one embodiment, the deamidase is selected from polypeptides having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with SEQ ID NO: 6.

[0115] In one embodiment, the deamidase is selected from polypeptides having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with SEQ ID NO: 8.

[0116] In one embodiment, the deamidase is selected from polypeptides having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with SEQ ID NO: 10.

[0117] In one embodiment, the deamidase is selected from polypeptides having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with the mature polypeptide of SEQ ID NO: 1.

[0118] In one embodiment, the deamidase is selected from polypeptides having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with the mature polypeptide of SEQ ID NO: 3.

[0119] In one embodiment, the deamidase is selected from polypeptides having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with the mature polypeptide of SEQ ID NO: 5.

[0120] In one embodiment, the deamidase is selected from polypeptides having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with the mature polypeptide of SEQ ID NO: 7.

[0121] In one embodiment, the deamidase is selected from polypeptides having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with the mature polypeptide of SEQ ID NO: 9.

[0122] For the purposes of this invention, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. [Journal of Molecular Biology] 48: 443-453) is used to determine the sequence identity between two amino acid sequences as the output of "longest identity". This algorithm is implemented in the Niedel program using the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. [Trends in Genetics] 16: 276-277) (preferably version 6.6.0 or later). The parameters used are a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. For the Niedel program to report the longest identity, the non-brief (-nobrief) option must be specified in the command line. The Niedel-marked "longest identity" output is calculated as follows: (Identical residues × 100) / (Alignment length - Total number of vacancies in the alignment) In the context of this invention, the term "variant" means a polypeptide having enzymatic activity that contains alterations (i.e., substitutions, insertions, and / or deletions) at one or more (e.g., several) positions. Substitution means replacing an amino acid occupying a position with a different amino acid; deletion means removing an amino acid occupying a position; and insertion means adding one or more (e.g., several) amino acids (e.g., 1-5 amino acids) adjacent to and immediately following an amino acid occupying a position.

[0123] Amino acid alterations can be minor, i.e., conserved amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; typically small deletions of 1–30 amino acids; small N-terminal or C-terminal extensions, such as methionine residues at the N-terminus; small linker peptides of up to 20–25 residues; or small extensions that facilitate purification by altering net charge or another function, such as polyhistidine fragments, antigenic epitopes, or binding domains.

[0124] Examples of conserved substitutions are found in the following group: basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine, and valine), aromatic amino acids (phenylalanine, tryptophan, and tyrosine), and small amino acids (glycine, alanine, serine, threonine, and methionine). Amino acid substitutions that do not typically alter specific activity are known in the art and are described, for example, by H. Neurath and RL Hill, 1979, in *The Proteins*, Academic Press, New York. Common substitutes are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0125] Alternatively, these amino acid alterations have the property of changing the physicochemical properties of the peptide. For example, these amino acid alterations can affect the peptide's thermal stability, change its substrate specificity, and alter its optimal pH.

[0126] Essential amino acids in peptides can be identified using procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, a single alanine mutation is introduced at each residue in the molecule, and the enzyme activity of the resulting mutant molecule is tested to identify amino acid residues essential to the molecule's activity. See also Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The active site of the enzyme or other biological interactions can also be determined by combining mutations of amino acids at hypothetical contact sites with physical analysis of the structure, such as by techniques like NMR, crystallography, electron diffraction, or photoaffinity labeling. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acids can also be inferred from comparisons with related peptides.

[0127] Using known mutagenesis, recombination, and / or tampering methods, followed by relevant screening procedures, one or more amino acid substitutions, deletions, and / or insertions can be made and tested, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95 / 17413; or WO 95 / 22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30: 10832-10837; US Patent No. 5,223,409; WO 92 / 06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7:127).

[0128] Mutagenesis / reorganization methods can be combined with high-throughput, automated screening methods to detect the activity of cloned, mutagenesis-encoded peptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17: 893-896). Mutagenesis-encoded DNA molecules encoding active peptides can be recovered from host cells and rapidly sequenced using standard methods in the art. These methods allow for the rapid determination of the importance of individual amino acid residues within the peptide.

[0129] The protein deamidase used in the method of the present invention can be added at a concentration of 0.01-20 IPA (U) / g substrate protein (e.g., 0.1-15 IPA (U) / g substrate protein, 0.5-10 IPA (U) / g substrate protein). In some embodiments, the protein deamidase used in the method of the present invention is added at a concentration in the range of 2.0-6.5 IPA (U) / g substrate protein (e.g., 2.5-5 IPA (U) / g substrate protein).

[0130] In a preferred embodiment, during the enzymatic treatment step with a protein deamidase, the dosage of the protein deamidase used in the method of the present invention is lower than the dosage of the protein deamidase used in the same method but without the addition of a chloride salt (preferably sodium chloride). In another preferred embodiment, the dosage of the protein deamidase used in the method of the present invention is lower than the dosage of the protein deamidase used in the same method but wherein the protein content of the seed material in the water slurry is at least 3% (w / w).

[0131] Without being bound by any particular theory, the inventors believe that the addition of chloride salts (especially sodium chloride) and / or the use of high-protein slurries (>3% (w / w)) during protein deamidase treatment not only improves the deamidation of the seed material slurry but also enables the production of seed-based dairy alternative beverages with improved stability to heat and low pH, particularly in warm, acidic beverages such as coffee drinks.

[0132] The activity of deamidase (protein glutaminase) was measured using the assay described in Example 1. The activity assay consisted of two separate parts: (1) an enzymatic step in which ammonia was formed by the catalysis of the protein deamidase; and (2) a non-enzymatic assay step in which the ammonia formed in step (1) was derivatized into an indophenol blue compound with maximum absorption at 630 nm. The amount of enzyme that produces 1 μmol of ammonia per minute at 37 °C was defined as 1 unit (expressed in indophenol assay units: IPA (U)). The activity was determined relative to known intensity standards.

[0133] The enzyme dosage will depend on parameters such as temperature, incubation time, and dairy alternative formulation. Those skilled in the art will know how to determine the optimal enzyme dosage.

[0134] Without being bound by any particular theory, the inventors believe that using protein deamidases to produce enzymatically deamidated seed materials for use in the production of seed-based dairy alternative beverages contributes to the superior benefits reported herein, including but not limited to improved stability of seed-based dairy alternative beverages to heat and / or low pH.

[0135] The invention is further defined by the following numbered embodiments:

[0136] Example 1. A method for obtaining a seed-based dairy product alternative beverage, the method comprising the following steps: (a) Providing a slurry of seed material in water; (b) The seed material in water is treated with a protein deamidase to obtain an enzymatically deamidated seed material slurry; (c) Optionally, the pulp of the enzymatically deamidated seed material is diluted to obtain a diluted pulp of the enzymatically deamidated seed material; and (d) Heat-treating the optionally diluted slurry of the enzymatically deamidinated seed material to obtain the seed-based dairy alternative beverage. The treatment with the protein deamidase is carried out in the presence of chloride salt, and / or the slurry of step (a) has a protein content of at least 3% (w / w).

[0137] Example 2. The method as described in Example 1, wherein the treatment in step (b) is carried out in the range of 10°C to 80°C, for example, in the range of 10°C to 65°C, 25°C to 40°C, 30°C to 45°C, 35°C to 50°C, 40°C to 55°C, 50°C to 65°C or 50°C to 60°C.

[0138] Example 3. The method as described in any of the preceding examples, wherein the treatment in step (b) is performed at a temperature in the range of 20°C to 60°C, for example, in the range of 50°C to 60°C.

[0139] Example 4. The method as described in any of the preceding examples, wherein the process in step (b) lasts for at least 10 minutes, at least 30 minutes, or at least 60 minutes.

[0140] Example 5. The method as described in any of the preceding examples, wherein the processing in step (b) is performed for 15-90 minutes, for example, 30-60 minutes.

[0141] Example 6. The method as described in any of the preceding examples, wherein the treatment in step (b) is performed at 50°C-60°C for 30-60 minutes.

[0142] Example 7. The method as described in any of the preceding examples, wherein the chloride salt is selected from potassium chloride and sodium chloride.

[0143] Example 8. The method as described in any of the preceding examples, wherein the chloride salt is sodium chloride.

[0144] Example 9. The method as described in any of the preceding examples, wherein the amount of chloride salt is 0.05%-2% chloride salt (w / w) based on the slurry, preferably 0.07%-0.2% (w / w) based on the slurry, for example, about 0.10% (w / w) or about 0.15% (w / w) based on the slurry.

[0145] Example 10. The method as described in any of the preceding examples, wherein the amount of the chloride salt is 0.1%-0.15% chloride salt (w / w) based on the slurry.

[0146] Example 11. The method as described in any of the preceding examples, wherein the protein content of the slurry of seed material in water is in the range of 0.1%-3% (w / w), for example in the range of 0.1%-2.5% (w / w), 0.5%-1.5% (w / w), or 1%-2% (w / w), for example about 1% (w / w).

[0147] Example 12. The method as described in any one of Examples 1-10, wherein the protein content of the slurry of the seed material in water is in the range of 4%-20% (w / w), preferably in the range of 5%-15% (w / w), more preferably in the range of 5%-10% (w / w), and the protein content of the diluted slurry of the enzymatically deamidated seed material is at most 2.5% (w / w), preferably wherein the protein content of the diluted slurry of the enzymatically deamidated seed material is in the range of 0.1%-2.5% (w / w), for example in the range of 0.5%-2% (w / w), for example in the range of 1%-2% (w / w), for example about 1% (w / w).

[0148] Example 13. The method as described in any of the preceding examples, the method further comprising adding lipids during or after step (a) or (b), wherein the lipid content of the pulp or dairy alternative beverage is 1%-5% (w / w).

[0149] Example 14. The method as described in Example 13, wherein the lipid content is 3% (w / w).

[0150] Example 15. The method of any one of Examples 13 or 14, wherein the lipid is an oil, such as a vegetable oil selected from the group consisting of rapeseed oil, sunflower oil, or mixtures thereof.

[0151] Example 16. The method as described in any of the preceding examples, wherein no stabilizer and / or emulsifier is present during steps (a), (b), (c) and / or (d).

[0152] Example 17. The method as described in any of the preceding examples, wherein no stabilizer and / or emulsifier is present during any of steps (a), (b), (c), and (d).

[0153] Example 18. The method as described in any of the preceding examples, wherein the seed-based dairy alternative beverage is substantially free of added emulsifiers and / or stabilizers.

[0154] Example 19. The method as described in any of the preceding examples, wherein the diluted slurry of the enzymatically deamidated seed material is obtained by diluting the slurry of the enzymatically deamidated seed material with an aqueous suspension such as water, a plant-based dairy alternative beverage, or a combination thereof.

[0155] Example 20. The method as described in any of the preceding examples, wherein the seed material is obtained from or derived from almonds, cashews, chickpeas, coconuts, broad beans, hazelnuts, lentils, lupins, macadamia nuts, mung beans, pistachios, peas, peanuts, pecans, soybeans, walnuts, or any combination thereof.

[0156] Example 21. The method as described in any of the preceding examples, wherein the seed material is obtained from or derived from almonds, peas, soybeans, or any combination thereof.

[0157] Example 22. The method as described in any of the preceding examples, wherein the seed material is obtained from or derived from almonds.

[0158] Example 23. The method of any one of Examples 1-21, wherein the seed material is obtained from or derived from peas, soybeans or a combination thereof.

[0159] Example 24. The method as described in any of the preceding examples, wherein the seed-based dairy alternative beverage is an almond beverage, cashew beverage, chickpea beverage, coconut beverage, fava bean beverage, hazelnut beverage, lentil beverage, lupin beverage, macadamia nut beverage, mung bean beverage, pistachio beverage, pea beverage, peanut beverage, pecan beverage, soy beverage, walnut beverage, or any combination thereof.

[0160] Example 25. The method as described in any of the preceding examples, wherein the seed-based dairy alternative beverage is an almond beverage, a pea beverage, a soy beverage, or any combination thereof.

[0161] Example 26. The method as described in any of the preceding examples, wherein the seed-based dairy alternative beverage is an almond drink.

[0162] Example 27. The method of any one of Examples 1-25, wherein the seed-based dairy alternative beverage is a pea-based beverage or a soy-based beverage.

[0163] Example 28. The method as described in any of the preceding examples, wherein the seed-based dairy alternative beverage is used in an acidic beverage such as a sports drink, coffee drink, or tea drink, preferably a coffee drink.

[0164] Example 29. The method as described in any of the preceding examples, wherein the seed-based dairy alternative beverage is used in barista applications and / or as a ready-to-drink beverage.

[0165] Example 30. The method as described in any of the preceding examples, wherein the seed-based dairy alternative beverage comprises one or more additional food ingredients selected from the group consisting of lipids, sugars, proteins, vitamins, minerals, amino acids, flavorings, dietary fiber, salt, water, and any combination thereof.

[0166] Example 31. The method as described in Example 30, wherein the additional food ingredient is lipid, such as oil, preferably vegetable oil, and / or sugar, such as sucrose, and / or calcium carbonate.

[0167] Example 32. The method as described in any of the foregoing embodiments, further comprising the following steps: (e) Separating the seed-based dairy alternative beverage into a solid stream and a liquid stream; (f) Harvesting the liquid stream as a liquid seed-based dairy alternative beverage; and (g) Optionally, the protein deamidase is inactivated.

[0168] Example 33. The method as described in any of the preceding examples, wherein the protein deamidase is inactivated by heat treatment such as ultra-high temperature (UHT) treatment.

[0169] Example 34. The method as described in any of the preceding examples, wherein the protein deamidase is derived from or obtained from a species of the genus *Chlorella*, for example, derived from or obtained from *Chlorella utilis* or *Chlorella viviparus*.

[0170] Example 35. The method as described in any of the preceding examples, wherein the protein deamidase comprises a polynucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with SEQ ID NO: 2, 4, 6, 8, or 10.

[0171] Example 36. The method as described in any of the preceding examples, wherein the protein deamidase comprises a polynucleotide sequence of SEQ ID NO: 2, 4, 6, 8 or 10.

[0172] Example 37. The method as described in any of the preceding examples, wherein the seed material is further modified by treatment with additional modifying enzymes and / or hydrolytic enzymes.

[0173] Example 38. The method as described in any of the preceding examples, wherein the seed-based dairy alternative beverage has increased stability to heat and / or low pH compared to a seed-based dairy alternative beverage obtained using the same method but without protein deamidase, wherein the treatment with the protein deamidase is carried out in the presence of chloride salt, and / or the slurry of step (a) has a protein content of at least 3% (w / w).

[0174] Example 39. The method as described in any of the preceding examples, wherein a lower dose of protein deamidase is used compared to the use of the same method but where the protein deamidase treatment is carried out in the absence of chloride salts and / or the slurry of step (a) does not have a protein content of at least 3% (w / w).

[0175] Example 40. A seed-based dairy alternative beverage, which can be obtained by any of the methods described in any of the preceding examples.

[0176] Example 41. A seed-based dairy alternative beverage as described in Example 40, characterized in that, compared with a seed-based dairy alternative beverage obtained using the same method but without protein deamidase, the seed-based dairy alternative beverage has improved stability to heat and / or low pH.

[0177] Example 42. A seed-based dairy alternative beverage as described in Example 40, characterized in that, compared with a seed-based dairy alternative beverage obtained using the same method but without protein deamidase and chloride salt, the seed-based dairy alternative beverage has improved stability to heat and / or low pH.

[0178] Example 43. A seed-based dairy alternative beverage as described in any one of Examples 40-42, said seed-based dairy alternative beverage for use in acidic beverages such as sports drinks, coffee drinks or tea drinks, preferably coffee drinks.

[0179] Example 44. A seed-based dairy alternative beverage as described in any one of Examples 40-43, said seed-based dairy alternative beverage for use in barista applications or ready-to-drink beverages.

[0180] Example 45. A seed-based dairy alternative beverage as described in any one of Examples 40-44, wherein the seed-based dairy alternative beverage further comprises a protein deamidase.

[0181] Example 46. A seed-based dairy alternative beverage as described in any one of Examples 40-45, wherein the protein deamidase comprises a polypeptide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with SEQ ID NO: 2, 4, 6, 8, or 10.

[0182] Example 47. A seed-based dairy alternative beverage as described in any one of Examples 40-46, wherein the seed-based dairy alternative beverage further comprises a chloride salt, preferably wherein the chloride salt is sodium chloride.

[0183] Example 48. Use of protein deamidase in improving stability in the production of seed-based dairy alternative beverages.

[0184] Example 49. Use as described in Example 48, wherein the improved stability is improved stability under heat and / or low pH.

[0185] Example 50. Use as described in any one of Examples 48-49, wherein the seed-based dairy alternative beverage is obtained using the method described in any one of Examples 1-39.

[0186] Example 51. Use as described in any one of Examples 48-50, wherein the protein deamidase comprises a polypeptide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with SEQ ID NO: 2, 4, 6, 8, or 10.

[0187] Example 52. Use as described in any one of Examples 48-51, wherein the seed-based dairy alternative beverage is an almond-based beverage, a pea-based beverage, a soy-based beverage, or a beverage containing any combination thereof, preferably an almond-based beverage, a pea-based beverage, or a soy-based beverage for barista applications.

[0188] Example 53. Use as described in any one of Examples 48-52, wherein the seed-based dairy alternative beverage has improved stability against heat and / or low pH compared to seed-based dairy alternative beverages obtained without the use of protein deamidases.

[0189] Example 54. Use of protein deamidase and chloride salt in improving stability in the production of seed-based dairy alternative beverages.

[0190] Example 55. Use as described in Example 54, wherein the improved stability is improved stability under heat and / or low pH.

[0191] Example 56. Use of protein deamidase and chloride salt in the production of seed-based dairy alternative beverages to improve stability and / or degree of deamidation.

[0192] Example 57. Use as described in any one of Examples 54-56, wherein the chloride salt is selected from potassium chloride and sodium chloride, preferably wherein the chloride salt is sodium chloride.

[0193] Example 58. Use as described in any one of Examples 54-57, wherein the amount of chloride salt in the seed-based dairy alternative beverage is 0.05%-2% chloride salt (w / w), preferably 0.07%-0.2% (w / w).

[0194] Example 59. Use as described in any one of Examples 54-58, wherein the amount of chloride salt in the seed-based dairy alternative beverage is about 0.10% (w / w) or about 0.15% (w / w).

[0195] Example 60. Use as described in any one of Examples 54-59, wherein the seed-based dairy alternative beverage is obtained using the method described in any one of Examples 1-39.

[0196] Example 61. Use as described in any one of Examples 54-60, wherein the seed-based dairy alternative beverage has improved stability against heat and / or low pH compared to seed-based dairy alternative beverages obtained without the use of protein deamidases and chloride salts.

[0197] Example 62. Use as described in any one of Examples 54-61, wherein the seed-based dairy alternative beverage is an almond beverage, a pea beverage, a soy beverage, or any combination thereof.

[0198] Example 63. Use as described in any one of Examples 54-62, wherein the seed-based dairy alternative beverage is an almond drink.

[0199] Example 64. A method for obtaining a seed-based dairy product alternative beverage, the method comprising the following steps: (a) Provide a slurry of seed material in water with a protein content of at least 3% (w / w); (b) The seed material in water is treated with a protein deamidase to obtain an enzymatically deamidated seed material slurry; (c) Diluting the pulp of the enzymatically deamidated seed material to obtain a diluted pulp of the enzymatically deamidated seed material; and (d) The diluted slurry of the enzymatically deamidated seed material is heat-treated to obtain the seed-based dairy alternative beverage.

[0200] Example 65. The method as described in the foregoing examples, the method further comprising adding a chloride salt during steps (a) and / or (b).

[0201] Example 66. The method described in the preceding examples, wherein the chloride salt is selected from potassium chloride and sodium chloride, preferably wherein the chloride salt is sodium chloride.

[0202] Example 67. The method as described in any one of Examples 65-66, wherein the concentration of chloride salt based on the seed-based dairy alternative beverage is in the range of 0.05%-0.2% (w / w), for example about 0.10% (w / w) or about 0.15% (w / w).

[0203] Example 68. The method of any one of Examples 64-67, wherein the protein content of the diluted slurry of the enzymatically deamidated seed material is at most 2.5% (w / w).

[0204] Example 69. The method as described in any one of Examples 64-68, wherein the protein content of the diluted slurry of the enzymatically deamidated seed material is in the range of 0.1%-2.5% (w / w).

[0205] Example 70. The method as described in any one of Examples 64-69, wherein the protein content of the diluted slurry of the enzymatically deamidated seed material is in the range of 0.5%-2% (w / w).

[0206] Example 71. The method as described in any one of Examples 64-79, wherein the protein content of the diluted slurry of the enzymatically deamidated seed material is in the range of 1%-2%, for example, about 1% (w / w).

[0207] Example 72. The method as described in any one of Examples 64-71, wherein the protein content of the pulp of the enzymatically deamidinated seed material is in the range of 4%-20% (w / w).

[0208] Example 73. The method as described in any one of Examples 64-72, wherein the protein content of the pulp of the enzymatically deamidated seed material is in the range of 5%-15% (w / w).

[0209] Example 74. The method of any one of Examples 64-73, wherein the protein content of the pulp of the enzymatically deamidated seed material is in the range of 5%-10% (w / w), for example about 5%, 8% or 10% (w / w).

[0210] Example 75. The method of any one of Examples 64-74, wherein the seed material is obtained from or derived from almonds, cashews, chickpeas, coconuts, broad beans, hazelnuts, lentils, lupins, macadamia nuts, mung beans, pistachios, peas, peanuts, pecans, soybeans, walnuts, or any combination thereof.

[0211] Example 76. The method of any one of Examples 64-75, wherein the seed material is obtained from or derived from almonds, peas, soybeans or any combination thereof.

[0212] Example 77. A method for obtaining an almond-based dairy product alternative beverage, the method comprising the following steps: (a) Provide a pulp of almond material in water with a protein content of at least 3% (w / w); (b) The pulp of almond material in water is treated with a protein deamidase to obtain an enzymatically deamidated almond material pulp; (c) Diluting the slurry of the enzymatically deamidated almond material to obtain a diluted slurry of the enzymatically deamidated almond material; and (d) The diluted slurry of the enzymatically deamidated almond material is heat-treated to obtain the almond-based dairy alternative beverage.

[0213] Example 78. The method as described in Example 77, wherein the protein content of the diluted slurry of the enzymatically deamidated almond material is in the range of 0.1%-2.5% (w / w), preferably in the range of 0.5%-2% (w / w), more preferably in the range of 1%-2% (w / w), and most preferably about 1% (w / w).

[0214] Example 79. The method as described in any one of Examples 77-78, wherein the protein content of the diluted pulp of the enzymatically deamidated almond material is in the range of 1%-2% (w / w), for example about 1% (w / w).

[0215] Example 80. The method as described in any one of Examples 77-79, wherein the protein content of the pulp of the enzymatically deamidinated almond material is 4%-20% (w / w), preferably in the range of 5%-15% (w / w), and more preferably in the range of 5%-10% (w / w).

[0216] Example 81. The method of any one of Examples 77-80, wherein the protein content of the pulp of the enzymatically deamidated almond material is in the range of 5%-10% (w / w), for example about 5%, 8% or 10% (w / w).

[0217] Example 82. A method for obtaining a soy-based dairy product alternative beverage, the method comprising the following steps: (a) Provide a slurry of legume material in water with a protein content of at least 3% (w / w); (b) The slurry of the legume material in water is treated with a protein deamidase to obtain an enzymatically deamidated legume material slurry; (c) Optionally, the slurry of the enzymatically deamidated legume material is diluted to obtain a diluted slurry of the enzymatically deamidated legume material; and (d) Heat-treating the optionally diluted slurry of the enzymatically deamidated legume material to obtain the legume-based dairy alternative beverage.

[0218] Example 83. A method for obtaining a soy-based dairy product alternative beverage, the method comprising the following steps: (a) Provide a slurry of legume material in water with a protein content of at least 3% (w / w); (b) The slurry of the legume material in water is treated with a protein deamidase to obtain an enzymatically deamidated legume material slurry; (c) Diluting the slurry of the enzymatically deamidated legume material to obtain a diluted slurry of the enzymatically deamidated legume material; and (d) Heat-treating the diluted slurry of the enzymatically deamidated legume material to obtain the legume-based dairy alternative beverage.

[0219] Example 84. The method as described in Example 82 or 83, wherein the protein content of the slurry of legume material in water is in the range of 5%-10% (w / w), for example about 5%, 8% or 10% (w / w).

[0220] Example 85. The method as described in any one of Examples 82-84, wherein the protein content of the diluted pulp of the enzymatically deamidated almond material is in the range of 0.1%-2.5% (w / w).

[0221] Example 86. The method of any one of Examples 82-85, wherein the legume material is derived from or obtained from peas, soybeans or a combination thereof.

[0222] Example 87. A method for obtaining a seed-based dairy product alternative beverage, the method comprising the following steps: (a) Providing a slurry of seed material in water; (b) The seed material in water is treated with a protein deamidase to obtain an enzymatically deamidated seed material slurry, wherein the treatment with the protein deamidase is carried out in the presence of a chloride salt.

[0223] Example 88. The method as described in the preceding examples, wherein the seed-based dairy alternative beverage has improved stability against heat and / or low pH compared to seed-based dairy alternative beverages obtained using similar methods but without protein deamidase and chloride salt.

[0224] Example 89. The method as described in any one of Examples 87-88, wherein the treatment in step (b) is performed using a lower dose of protein deamidase compared to using the same method but without adding chloride salt in step (b).

[0225] Example 90. The method of any one of Examples 87-89, wherein the seed-based dairy alternative beverage is an almond drink.

[0226] Example 91. The method of any one of Examples 87-90, wherein the chloride salt is selected from potassium chloride and sodium chloride, preferably sodium chloride.

[0227] Example 92. The method as described in any one of Examples 87-91, wherein the amount of chloride salt is 0.05%-2% chloride salt (w / w) based on the slurry, preferably 0.07%-0.2% (w / w) based on the slurry, for example, 0.10% (w / w) or 0.15% (w / w) based on the slurry.

[0228] Example 93. The method as described in any one of Examples 87-92, wherein the protein content of the slurry of seed material in water is in the range of 0.1%-3% (w / w), for example in the range of 0.5%-2% (w / w), for example about 1% (w / w) or 1.5% (w / w).

[0229] Example 94. The method as described in any one of Examples 87-93, wherein the treatment in step (b) is performed at a temperature in the range of 10°C-65°C, for example in the range of 15°C-40°C, for example in the range of 20°C-30°C.

[0230] Example 95. The method as described in any one of Examples 87-94, wherein the treatment in step (b) is performed at a temperature in the range of 50°C to 65°C.

[0231] Example 96. The method as described in any one of Examples 87-95, wherein the treatment in step (b) is performed for at least 10 minutes, preferably 15-90 minutes, for example 30-60 minutes.

[0232] Example 97. A method for obtaining an almond-based dairy product alternative beverage, the method comprising the following steps: (a) Providing a paste of almond material in water; and (b) The slurry of almond material in water is treated with a protein deamidase to obtain the almond-based dairy alternative beverage, wherein the treatment with the protein deamidase is carried out in the presence of a chloride salt.

[0233] Example 98. The method as described in Example 97, wherein the chloride salt is selected from potassium chloride and sodium chloride, preferably sodium chloride.

[0234] Example 99. The method as described in any one of Examples 97-98, wherein the amount of chloride salt is 0.05%-2% chloride salt (w / w) based on the slurry, preferably 0.07%-0.2% (w / w) based on the slurry, for example, 0.10% (w / w) or 0.15% (w / w) based on the slurry.

[0235] Example 100. The method as described in any one of Examples 97-99, wherein the protein content of the slurry of almond material in water is in the range of 0.1%-3% (w / w), for example in the range of 0.5%-2% (w / w), for example about 1% (w / w) or 1.5% (w / w).

[0236] The inventions described and claimed herein are not limited to the specific embodiments disclosed herein, as these embodiments are intended to serve as illustrative examples of several aspects of the invention. Any equivalent embodiments, together with combinations of one or more of these embodiments, are intended to be included within the scope of the invention.

[0237] This document cites several references, the disclosures of which are incorporated herein by reference in their entirety. The invention is further described through the following examples, which should not be construed as limiting the scope of the invention.

[0238] Example Material enzymes The following enzymes are used throughout the example: Protein deamidaseThe protein glutaminase, derived from *Cyclocarya* species 62563, has the mature polypeptide sequence shown in SEQ ID NO: 2. Propeptide cleavage was achieved by treating the deamidase in SEQ ID NO: 1 with a site-specific endopeptidase. The site-specific endopeptidase used was derived from *Bacillus licheniformis* (…). Bacillus licheniformis The glutamyl endopeptidase of [organism name missing]. The active deamidase obtained after maturation is the polypeptide shown in SEQ ID NO: 2. *Citrus* species 62563 was isolated from soil samples collected in Sibhult, Sweden in September 2013.

[0239] Example 1: Assay of protein deamidase activity The protein deamidase activity assay consists of two separate parts: 1) An enzymatic step in which ammonia is formed through the catalytic action of a protein deamidase; and 2) Non-enzymatic detection step, wherein the ammonia formed in step (1) is derivatized into a blue indophenol compound with maximum absorption at 630 nm.

[0240] In step (1), ammonia is produced by deamidation of a protein deamidase. In step (2), the generated ammonia reacts with phenol under alkaline conditions to form dioxane. This reaction is catalyzed by sodium pentacyanonitroferro(III) ferric(III) nitrosyl ferrate (sodium nitroprusside). "Colorimetric reagent solution A" contains phenol and sodium nitroprusside. "Colorimetric reagent solution B" provides the alkaline reaction conditions. The intermediate is then oxidized by adding sodium hypochlorite ("Colorimetric reagent solution C") to form indophenol blue. This compound absorbs visible light at 630 nm. The enzyme activity is then calculated using a standard curve.

[0241] Measurement procedure: The enzymatic step involving ammonia formation (1) Reagents: Diluent for assay: 0.2 M sodium phosphate buffer, 0.01% Triton X-100, pH 6.5.

[0242] Assay buffer: Same as above. Used for preparing stock solutions and diluting samples for protein deamidases (hereinafter referred to as "enzymes").

[0243] Substrate solution: 30 mM Z-Gln-Gly (Merck C6154-1G), in the assay dilution (check pH after dissolution).

[0244] Termination solution: 0.4 M TCA.

[0245] Standard: NH4Cl diluted in the assay dilution solution (ammonium standard for IC, Merck 59755-100ML, 1000 mg / L NH4Cl). + (in water) (see also the "Standard Curve" section).

[0246] The enzyme product is dissolved / diluted in the assay buffer and a suitable dilution is prepared to produce a linear assay response.

[0247] Incubation: 1. Add 10 μL of diluted enzyme sample in triplicate to the wells of a 96-well microtiter plate (MTP).

[0248] 2. Add 100 μL of substrate solution to each well.

[0249] 3. For blank samples, add 100 μL of 0.4 M TCA solution.

[0250] 4. Seal the board with a transparent sealing film.

[0251] 5. Incubate the plate at 37°C and 500 rpm for 10 minutes on a thermostatic mixer equipped with a heated lid.

[0252] 6. To terminate the reaction, carefully add 100 μL of 0.4 M TCA solution (except for the blank sample, which already contains TCA).

[0253] Total reaction volume: 210 μL Step (2) Ammonia Detection Step Reagents: Colorimetric reagent A: 4% (w / v) phenol, 0.015% (w / v) sodium pentacyanonitrosylferric(III) dihydrate (sodium nitroprusside) (Na2[Fe(CN)5NO]∙2H2O).

[0254] Colorimetric reagent B: 5% (w / v) potassium hydroxide.

[0255] Colorimetric reagent C: 28% (w / v) potassium carbonate, 6% (v / v) sodium hypochlorite (Sigma-Aldrich 239305-25ml, <5% can be replaced with Cl2).

[0256] Incubation: 1. Transfer 15 μL from each well in step (1) to a new 96-well MTP.

[0257] 2. Transfer 45 μL of Milli-Q water into each well.

[0258] 3. Add 30 μL of colorimetric reagent B to each well (shake gently by hand on the lab bench to mix).

[0259] 4. Add 60 μL of colorimetric reagent A to each well (shake gently by hand on the lab bench to mix).

[0260] 5. Add 60 μL of colorimetric reagent C to each well (shake gently by hand on the lab bench to mix).

[0261] 6. Color development: Carefully seal the plate and place it on the lab bench for 30 minutes.

[0262] 7. Carefully transfer the MTP to the microplate reader and measure the absorbance at 630 nm.

[0263] Total reaction volume: 210 μL Standard curve: Standard stock solution: 1000 mg NH4 + / L.

[0264] In the ammonia detection procedure, a standard curve is prepared by adding ammonium standards at different dilutions to the determination dilution buffer. Specifically, 15 μL of diluted ammonia standard is mixed with 45 μL of water, and then the colorimetric reagent is added in the order given above (B, A, and C).

[0265] The amount of enzyme that produces 1 μmol of ammonia per minute at 37°C is defined as 1 unit (indophenol assay unit; IPA (U)): in It can be abbreviated as in

[0266] • C NH4+ It is the ammonia concentration in the reaction solution obtained from the ammonium standard curve (i.e., taking into account the dilution of the pre-diluted ammonium standard solution in the ammonia derivatization step).

[0267] • 18.04 is the molecular weight of ammonium used in the standard solution.

[0268] • V 反应 It is the reaction volume in the pore when ammonia is generated (210 μL).

[0269] • V 酶 This is the volume (10 μL) of enzyme solution added to the well when ammonia is generated.

[0270] • V NH3 The detection volume is the reaction volume in the well when detecting ammonia (210 μL).

[0271] Example 2: Testing of protein deamidases in the production of almond beverages with protein contents of 1%, 2.5%, 5%, and 10%. Almond paste (from KoRo, 21%-25% protein) was suspended in deionized water to achieve a final protein concentration of 1%, 2.5%, 5%, or 10%, and mixed in a hot mixer. Protein deamidase was added at concentrations of 0, 2.7, or 4 IPA (U) / g protein, and the mixture was kept at 60°C for 1 hour. After incubation, samples with 1% protein were directly heat-treated at 90°C for 15 min to inactivate the enzyme, while samples with 2.5%, 5%, or 10% protein were separated and either directly heat-treated or diluted to 1% protein and then heat-treated (90°C / 15 min). The samples were then homogenized using an Ultra-turrax at 14,000 rpm for 1 min to produce the final beverage sample.

[0272] Then, the beverage stability, soluble protein, viscosity, and stability in coffee were measured.

[0273] After centrifuging the beverage sample at 21,000 g for 10 min, the soluble protein in the supernatant was measured. Protein determination was performed using a LECO analyzer (the nitrogen content after combustion and reduction was determined by the Dumas method, and N2 was detected using a conductivity detector). The protein factor was 5.18.

[0274] Viscosity was measured using an Anton Paar Modular Compact rheometer MCR 302. For this purpose, a 21 g beverage sample was weighed into a cup and placed in the measuring cell (C-ETD160 / ST-SN81248612, paddle: ST24-2D / 2V / 2V-30-SN29679). Analysis was performed at 20°C using the "Low Viscosity" program.

[0275] The stability of the beverage was visually assessed. The stability of the beverage sample in coffee was assessed by mixing 4 parts coffee with 1 part cold beverage sample using warm filtered coffee (pH: approximately 4.95-5.01, temperature: 58℃-63℃), allowing the mixture to stand for 5-10 minutes, and visually assessing for coagulation / precipitation.

[0276] result: All blank beverage samples (enzyme-free; see [link]). Figures 1-4Samples marked "0 U" (indicating the absence of protein deamidase) are unstable, showing an emulsion layer on top, precipitation at the bottom, and a relatively clear, non-turbid middle layer. All samples containing the enzyme are more stable, showing an emulsion layer on top, no or only a small amount of precipitation, and a milky white colloidal layer in the middle to stabilize the protein. Samples heat-treated at protein concentrations exceeding 1% are very viscous or solid.

[0277] The stability of soluble proteins, viscosity, and beverage samples in coffee is given in Table 1 below. The stability in coffee is also... Figure 1 and Figure 2 The photos show that undiluted samples after deamidase treatment were labeled "1%", while diluted samples after deamidase treatment were labeled "2.5% to 1%", "5% to 1%" or "10% to 1%".

[0278] Table 1: Viscosity, soluble protein, and stability in coffee of beverage samples. Samples incubated at 5% or 10% protein and then directly heat-treated were too viscous to be analyzed ("na" in the table).

[0279] The results show that incubating almond paste with 5%-10% protein content with protein deamidase (2.7 or 4 IPA (U) / g protein) at 60°C for 60 min, followed by dilution to 1% protein content and heat treatment at 90°C for 15 min, yielded a final beverage with low viscosity and good stability (no coagulation) in coffee (samples 8.2 and 9.2, 11.2 and 12.2). Samples prepared without protein deamidase coagulated (samples 7.2 and 10.2).

[0280] If the initial dilution to 1% protein and incubation with protein deamidase at 60°C for 60 min, followed by heat treatment at 90°C for 15 min, resulted in a beverage with low viscosity but unstable in coffee, i.e., sample coagulation (samples 2 and 3). The blank sample (prepared without protein deamidase) also coagulated (sample 1).

[0281] Almond paste with 2.5% protein was incubated with protein deamidase (2.7 or 4 IPA (U) / g protein) at 60°C for 60 min, then diluted to 1% protein, and then heat-treated at 90°C for 15 min to obtain a final beverage with low viscosity but poor stability in coffee (samples 5.2 and 6.2). Samples prepared without protein deamidase also coagulated (sample 4.2).

[0282] If almond paste with 5%-10% protein is incubated with protein deamidase at 60°C for 60 min, followed by heat treatment at 90°C for 15 min, the final beverage will be very viscous or even solid (sample numbers 7-12).

[0283] When comparing the concentrations of soluble protein in different samples, they were very similar across all samples with a protein content of 1%, indicating that protein deamidases functioned at all tested protein concentrations.

[0284] Viscosities also exhibited the same trend, with results being comparable across all enzyme-treated samples at a final protein content of 1%. Blank samples (prepared without protein deamidase) tended to have higher viscosity when incubated at high protein levels and diluted to 1% before heat treatment.

[0285] Example 3: Testing of protein deamidases in the production of almond beverages with protein contents of 1%, 2.5%, 5%, and 8%. Almond paste (from Kro Corporation, 21%-25% protein) was suspended in deionized water to achieve a final protein concentration of 1%, 2.5%, 5%, or 8%, and mixed in a hot mixer. Protein deamidase was added at concentrations of 0, 2.7, or 4 IPA (U) / g protein, and the mixture was kept at 60°C for 1 hour. After incubation, samples with 1% protein were directly heat-treated at 90°C for 15 min to inactivate the enzyme, while samples with 2.5%, 5%, or 8% protein were diluted to 1% protein and then heat-treated (90°C / 15 min). The samples were then homogenized using an Ultra-turrax at 14,000 rpm for 1 min. Beverage stability, total and soluble protein, viscosity, and stability in coffee were measured in the final beverage as described in Example 2.

[0286] result: All blank beverage samples (enzyme-free; see [link]). Figures 1-4 Samples marked "0 U" (indicating no protein deamidase was added) are unstable, showing an emulsified layer on top, a precipitate at the bottom, and a relatively clear, non-turbid middle layer. All samples treated with protein deamidase are more stable, showing an emulsified layer on top, no or only a small amount of precipitate, and a milky white colloidal layer in the middle to stabilize the protein.

[0287] Soluble protein, viscosity, and stability in coffee are given in Table 2. Stability in coffee is also... Figure 3 and Figure 4 The photograph shows that the undiluted sample after deamidase treatment is labeled "1%" ( Figure 3Samples diluted after deamidase treatment were labeled as "2.5% to 1%", "5% to 1%", "8% to 1%" or "10% to 1%". Figure 4 ).

[0288] Table 2: Viscosity, soluble protein, and stability of the final almond beverage in coffee.

[0289] The overall results confirmed what was shown in Example 2, in which the tested protein content was 8% instead of 10%. Incubation with protein deamidase at higher protein contents (5% or 8%) and dilution to 1% before heat inactivation improved stability in coffee while maintaining other parameters (such as soluble protein and viscosity) comparable to the sample incubated at 1% protein.

[0290] Example 4: Testing low doses of protein deamidase and measuring NH4 production in the production of almond beverages with protein contents of 1%, 5%, and 8%. Almond paste (from Korro, 21%–25% protein) was suspended in deionized water to achieve a final protein concentration of 1%, 5%, or 8%, and mixed in a hot mixer. Protein deamidase was added at concentrations of 0, 0.7, 1.3, 2.7, or 4 IPA (U) / g protein, and the mixture was kept at 60°C for 1 hour. After incubation, samples with 1% protein were directly heat-treated at 90°C for 15 min to inactivate the enzyme, while samples with 5% or 8% protein were diluted to 1% protein and then heat-treated (90°C / 15 min). The samples were then homogenized using an Ultra-turrax at 14,000 rpm for 1 min. Beverage stability, soluble protein, and stability in coffee were measured in the final beverage as described in Example 2.

[0291] The generation of NH4 is measured using the ammonia detection procedure (step 2 of the determination procedure) as described in Example 1.

[0292] result: All blank beverage samples (without added protein deamidase) were unstable, showing an emulsion layer on top, sediment at the bottom, and a relatively clear, non-turbid middle layer. All samples containing the enzyme were more stable, showing an emulsion layer on top, no or only a small amount of sediment, and a milky white colloidal layer in the middle to stabilize the protein.

[0293] Soluble proteins, NH4 produced, and stability in coffee are given in Table 3.

[0294] Table 3: Soluble protein content and stability in coffee of the final almond beverage

[0295] Similar to Examples 2 and 3 above, incubation at 1% protein and direct heat treatment produced almond beverages that were unstable in coffee. However, for samples obtained using higher enzyme doses (samples 10, 14, and 15), incubation at 5% or 8% protein, followed by dilution to 1% protein and then heat treatment, produced beverages stable in coffee. In samples prepared with the same enzyme dose but incubated at higher protein concentrations, soluble protein was slightly higher, and NH4 production was significantly higher in these samples (e.g., sample 15 compared to 10 and 5), indicating that the enzyme performed better at higher protein concentrations.

[0296] Example 5: Testing protein deamidase in the production of almond beverages with and without NaCl, containing 1% protein. Almond paste (from Kro Corporation, 21%–25% protein) was suspended in deionized water to achieve a final protein concentration of 1% and mixed in a hot mixer. Protein deamidase was added at an increased dose as shown in Table 4A, and the mixture was incubated at 60°C for 1 hour. After incubation, the sample was heat-treated at 90°C for 15 min to inactivate the enzyme. Beverage stability, soluble protein content, and stability in coffee were measured in the final beverage as described in Example 2.

[0297] A similar set of samples was prepared, except that 0.15% NaCl was added to the almond suspension before incubation, as shown in Table 4B. The protein content of the almond paste used was 24.5%.

[0298] result: The blank beverage sample (without added protein deamidase) was unstable, showing an emulsion layer on top, a precipitate at the bottom, and a relatively clear, non-turbid middle layer. All samples treated with protein deamidase were more stable, showing an emulsion layer on top, no or only a small amount of precipitate, and a milky white colloidal layer in the middle to stabilize the protein. Compared to the blank sample without NaCl, the blank sample containing NaCl (without added protein deamidase) had a larger precipitate and a smaller emulsion layer.

[0299] Soluble proteins and their stability in coffee are shown in Tables 4A and 4B.

[0300] Table 4A: Soluble protein and stability in coffee of final almond beverage without NaCl

[0301] Table 4B: Soluble protein and stability in coffee of the final almond beverage containing 0.15% NaCl.

[0302] Based on the results shown in Tables 4A and 4B, it can be concluded that adding NaCl to the process according to the present invention improves the stability of the final beverage in coffee. Meanwhile, NaCl tends to slightly reduce the level of soluble proteins.

[0303] To test the correlation between the timing of chloride salt addition and the results, a second experiment was conducted. For this purpose, a 1% almond protein slurry was prepared by mixing one (1) part almond matrix (25% protein) with 24 parts distilled water. The slurry was then divided into 13 equal samples, each 80 g. The slurries were thoroughly mixed, and protein deamidase and sodium chloride (NaCl) were added according to the dosages in Table 5.

[0304] Samples were incubated at 60°C for 60 minutes in the presence of protein deamidase alone (samples 2 to 7 in Table 5) or in the presence of protein deamidase and NaCl (samples 8 to 13 in Table 5). Sample 1 in Table 5 was included as a blank control, containing neither protein deamidase nor NaCl. After incubation, samples were heat-treated at 90°C for 15 minutes to inactivate the enzymes, followed by cooling with ice water. Samples were homogenized for 1 minute using an Ultra-turrax set to 14,000 rpm. Samples were then refrigerated until further analysis.

[0305] Table 5: Samples treated with different concentrations of protein deamidase and with or without the addition of NaCl during enzyme incubation.

[0306] After rotating at 14,000 rpm for 10 minutes, the soluble protein in the supernatant was determined using a LECO analyzer (the nitrogen content after combustion and reduction was determined using the Dumas method, and N2 was detected using a conductivity detector). The protein factor was 5.18. The ammonium content (NH4) in the supernatant was also determined after rotating at 14,000 rpm for 10 minutes using the ammonia detection procedure described in Example 1 (step 2 of the assay procedure). The sample was diluted 3-fold.

[0307] The stability of the beverage samples was visually assessed. The stability of the beverage samples in coffee was assessed by mixing four portions of coffee with one portion of cold beverage sample using warm filtered coffee (medium roast, pH: approx. 4.95-5.01, temperature: 58°C-63°C), allowing the mixture to stand for 5 minutes, and visually assessing for coagulation / precipitation.

[0308] Table 6: Soluble protein, ammonium content, and stability in coffee of the beverage samples in Table 5.

[0309] The total protein content in the analyzed samples ranged from 1.05% to 1.09%.

[0310] Stability in coffee also Figure 5 As shown in the photo.

[0311] As clearly supported by the data presented in this paper, it has been found that adding a small amount of chloride salt (e.g., 0.15% sodium chloride (NaCl)) to 1% protein almond pulp during protein deamidase treatment (samples 2–7) improved enzyme efficiency, resulting in the production of more NH4 and a higher content of soluble protein compared to samples where NaCl was added after enzyme incubation (samples 8–13). Furthermore, samples with enzyme doses of 4 IPA (U) / g protein or higher and NaCl was added before incubation showed improved stability in coffee, indicating that the effect of NaCl is related to enzyme performance, rather than some stabilizing effect of almond protein in coffee.

[0312] Example 6: Testing protein deamidase and phosphate / citrate levels in the production of almond beverages with 1% protein. Almond paste (from Koro Company, 21%-25% protein) was suspended in deionized water to a final protein concentration of 1% and mixed in a hot mixer. The paste was then divided into smaller samples of 50g each. K₂HPO₄ or NaCl was added to the samples according to Table 7 below. Protein deamidase was added at the dosage shown in Table 7, and the mixture was incubated at 60°C for 1 hour. After incubation, the samples were heat-treated at 90°C for 15 minutes to inactivate the enzyme. The NH₄ production and stability in coffee of the final beverage were measured as described in Examples 1 and 3, respectively. The NH₄ production and stability in coffee are shown in Table 7.

[0313] Table 7: Stability of the generated NH4 and final almond beverage in coffee.

[0314] When comparing NH4 generation in samples treated with protein deamidase, it was clear that the addition of NaCl again improved overall enzyme performance, resulting in higher NH4 generation in samples containing NaCl (samples 19-24) than in samples with only protein deamidase (samples 2-4) or samples with K2HPO4 (samples 8-16). When comparing the stability of different samples in coffee, deamidation achieved with protein deamidase and NaCl (samples 19-24) was sufficient at the highest enzyme dosage. Samples with 0.5% or more of added K2HPO4 were stable at low to medium enzyme dosages; at the highest enzyme dosage, stability in coffee required only 0.15% K2HPO4.

[0315] Overall, the results indicate that adding 0.1%–0.15% (w / w) NaCl in combination with protein deamidase improves enzyme performance, as evidenced by a higher degree of deamidation. This same effect was not observed when using K₂HPO₄. The effect on coffee stability may be due to different mechanisms: the addition of NaCl improves enzyme performance, leading to a higher degree of deamidation and thus improving protein stability, while the addition of K₂HPO₄ does not directly affect the enzyme but acts as a buffer, increasing pH and thus improving protein stability. Since sodium chloride is commonly used for the fortification of final dairy alternative beverages, its use during enzymatic treatment with protein deamidases has long been beneficial. Furthermore, the role of sodium chloride allows manufacturers to use alternative salts besides, for example, K₂HPO₄, enabling a simplified method for producing clean-label seed-based dairy alternative beverages, preferably for barista applications.

[0316] Example 7: Testing of protein deamidase in the production of almond beverages with 1% and 8% protein content at 30°C and 60°C. Almond paste (from Kro Corporation, 21%–25% protein) was suspended in deionized water to a final protein concentration of 1% or 8% and mixed in a hot mixer. The mixture was divided into smaller batches of 50 g / bottle and protein deamidase was added at a concentration of 0–1.3–2.7–5.4–10.8 IPA (U) / g protein. Incubation was completed at 30°C or 60°C for 1 hour. After incubation, the 1% protein sample was directly heat-treated at 90°C for 15 minutes to inactivate the enzyme, while the 8% protein sample was diluted to 1% protein and then heat-treated at 90°C for 15 minutes. The samples were then homogenized using an Ultra-turrax at 14,000 rpm for 1 minute. The soluble protein, NH4 production, and stability in coffee were measured in the final beverage.

[0317] After centrifuging the sample at 21,000 g for 10 min, the soluble protein in the supernatant was measured. Protein determination was performed using a LECO analyzer (the nitrogen content after combustion and reduction was determined by the Dumas method, and N2 was detected using a conductivity detector). The protein factor was 5.18.

[0318] The ammonium content (NH4) was determined using the ammonia detection procedure (step 2 of the determination procedure) as described in Example 1.

[0319] Stability in coffee was measured using warm coffee (pH: approximately 4.95–5.01, temperature: 58°C–63°C), in which four (4) portions of coffee were mixed with one (1) portion of cold almond beverage, the mixture was left to stand for 5–10 minutes, and then coagulation / precipitation was visually assessed.

[0320] Soluble proteins, NH4 production, and stability in coffee are given in Table 8 below. Stability in coffee is also... Figure 6 As shown in the photo.

[0321] Table 8: Total and soluble protein, NH4 concentration, and stability of almond beverages in coffee

[0322] As seen in the results in Table 8, a positive effect was also observed when enzyme incubation was performed at lower temperatures (e.g., 30°C) compared to higher protein concentrations (8% protein vs. 1% protein) at 60°C. That is, the enzyme was more efficient at higher protein concentrations, resulting in higher soluble protein, more NH4 production, and better stability in coffee. For samples incubated at 8% protein, enzyme incubation at 60°C yielded stable beverages in coffee at enzyme doses of 2.7 IPA (U) / g protein or higher. Stability was observed at 5.4 IPA (U) / g protein or higher when enzyme incubated at 30°C. All samples incubated at 1% protein were unstable in coffee.

[0323] At the same enzyme dose, samples incubated with 8% protein had higher soluble protein (10%-50%) compared to those incubated with 1% protein, especially in the lower enzyme dose range, while at higher protein concentrations, NH4 production was significantly higher in all samples (40%-100%).

[0324] As expected, the enzyme performance at 30°C was lower than that at 60°C, as observed from the NH produced.

[0325] Example 8: Testing protein deamidase in the production of almond beverages with 1% protein content, with NaCl added before or after enzyme incubation at 30°C. Almond paste (from Koro Company, 21%–25% protein) was suspended in deionized water to a final protein concentration of 1% and mixed in a hot mixer. The paste was then divided into 13 equal portions, 80 g each. 0.15% NaCl was added to samples 2–7 (NaCl added before enzyme incubation). Protein deamidase was added at an increased dose as shown in Table 9, and the mixture was kept at 30°C for 1 hour. After incubation, samples 1–7 were heat-treated at 90°C for 15 minutes to inactivate the enzyme. Just before heat inactivation, 0.15% NaCl was added to samples 8–13 (NaCl added after incubation). Soluble protein, NH4 production, and stability in coffee were measured in the final beverage as described in Examples 1 and 2.

[0326] Soluble proteins, NH4 produced, and stability in coffee are shown in Table 9. Stability in coffee is also... Figure 7 As shown in the photo.

[0327] Table 9: Soluble protein and stability in coffee of almond beverage samples.

[0328] The results showed that NaCl significantly improved overall enzyme performance. Samples with NaCl added during incubation (samples 2-7) exhibited much higher levels of soluble protein and NH4 formation compared to samples with NaCl added after incubation (samples 8-13). Furthermore, at the highest test enzyme dose of 6.7 IPA(U) / g protein, samples with NaCl added before incubation showed improved stability in coffee, indicating that the effect of NaCl is related to enzyme performance, rather than protein interaction or stabilization in coffee.

[0329] Compared to Example 5, an improvement in overall enzyme performance was also observed with NaCl at 30°C. However, as can be expected, a slightly higher enzyme dose was required to achieve the same performance at 30°C compared to 60°C.

[0330] Example 9: Testing of protein deamidases in dairy alternative beverages with pea protein isolates containing 2.45%, 4.9%, and 9.8% protein. Enzyme reaction Pea protein isolate (Roquette Nutralys S85F 2.0, protein concentration: 82%) was suspended in tap water to achieve final protein concentrations of 2.45%, 4.9%, or 9.8%, and mixed using a magnetic stirrer. Protein deamidase was added at concentrations of 0, 0.67, 2, or 6 IPA (U) / g protein, and the mixture was incubated at 60°C for 1 hour in a FINEPCR combi-D24 spinner (spin speed set to 7). After incubation, the sample was heat-treated at 85°C for 10 minutes to inactivate the enzyme. The protein solubility, degree of deamidation, and coffee stability of the samples were then analyzed.

[0331] Protein solubility measurement Each sample was priced at 15,000. g After centrifugation for 10 minutes, the protein solubility in the supernatant was measured. The protein content was then determined using a LECO analyzer (the Dumas method was used to determine the nitrogen content after combustion and reduction, and a conductivity detector was used to detect N2). The protein factor was 6.25.

[0332] Protein deamidation measurement The degree of deamidation was measured using an ammonia test kit from HACH (HACH, High Range Ammonia Test N Tube Reagent (0-50 mg / LN)). Using the HACH kit and the procedure described below, the amount of ammonia released from deamidated pea protein isolates (PPIs) with and without protein deamidase treatment (2.45%, 4.9%, or 9.8% protein concentration), as well as the total amount of ammonia released from the PPIs via acid hydrolysis, was measured.

[0333] The procedure is as follows: 1. For the deamidation degree test, dilute the 9.8% PPI pasteurized solution to a 6% protein concentration using DI water. Test the 2.45% PPI and 4.9% PPI pasteurized solutions directly without dilution.

[0334] 2. The degree of deamidation of the stock solution (2.45%), (4.9%), and (6%) diluted solution was tested using a Hach reagent kit.

[0335] 3. The total ammonia released was analyzed by drawing 1000 µL of the control sample solution, adding 500 µL of 6 M HCl, and treating at 100 °C for 3 h. After acid hydrolysis, 28 mL of pH 7.0 buffer (0.1 M citrate-0.2 M Na₂HPO₄ buffer) and 550 µL of 6 M NaOH were added to adjust the pH of the hydrolysis mixture to 7.0, resulting in a total dilution of 30-fold. The released ammonia was determined using a Hach kit.

[0336] 4. The release of free ammonia was measured by aspirating 1000 µL of the deamidase-treated sample solution and centrifuging at 12,000 rpm for 10 minutes at ambient temperature. Then, pH 7.0 buffer (0.1 M citrate-0.2 M Na₂HPO₄ buffer) was added to the supernatant, followed by a total dilution of 2–10-fold depending on the ammonia release level. 100 µL was then aspirated for free ammonia analysis.

[0337] 5. The degree of deamidation is defined as the ratio between the amount of free ammonia released by the deamidase reaction (measured in step 4) and the total amount of ammonia released by hydrochloric acid protein hydrolysis (from glutamine and asparagine, measured in step 3).

[0338] Hach Company, AmVer Salicylate Test'N Tube Method: 1. Start program 343 N, ammonia HR TNT.

[0339] 2. Prepare a blank: Add 0.1 mL of ammonia-free water to an AmVer™ diluent reagent Test 'N Tube for high-range ammonia nitrogen.

[0340] 3. Sample preparation: Add 0.1 mL of sample to an AmVer™ diluent reagent Test 'N Tube for high-range ammonia nitrogen.

[0341] 4. Add the contents of a salicylic acid ammonium reagent powder pillow for 5 mL samples to each vial.

[0342] 5. Add the contents of one cyanuric acid ammonia reagent powder pillow to each vial.

[0343] 6. Place the caps on both vials. Shake well to dissolve the powder.

[0344] 7. Start the instrument timer. Begin the 20-minute reaction time.

[0345] 8. Clean the empty vials.

[0346] 9. Insert the blank vial into the 16 mm cell scaffold.

[0347] 10. Zero the display. The display shows 0.0 mg / L NH3–N.

[0348] 11. Clean the sample vials.

[0349] 12. Insert the sample vial into the 16 mm cell scaffold.

[0350] 13. Readings. Results are expressed in mg / L NH3–N.

[0351] Coffee stability assessment The stability of the PPI in coffee was measured using warm instant Nestlé Gold coffee. Instant coffee was prepared according to the instructions, which recommended mixing 2 grams of instant coffee powder with 150 mL of tap water boiled at 85°C (tap water 11 dH, pH 6.5-6.8). The PPI solution was adjusted to a protein concentration of 2.45% and then mixed with the prepared coffee. The PPI solution was then mixed with the hot (85°C) coffee according to the recipe. The mixture was allowed to stand for 5-10 minutes, and then visually assessed for coagulation / precipitation.

[0352] result As shown in Tables 10-12 below, at the same enzyme dosage but with different initial pea protein concentrations, the degree of deamidation improved with increasing pea protein concentration, especially for a pea protein concentration of 9.8% (Table 11). At an enzyme dosage of 2 IPA (U) / g protein, the degree of deamidation improved from 11.7% to 15.1%, and at an enzyme dosage of 6 IPA (U) / g protein, the degree of deamidation improved from 15.9% to 19.5% (Table 11).

[0353] Compared with low dry matter, deamidation under high dry matter conditions resulted in a more significant improvement in solubility (Table 10), and the degree of deamidation was also significantly improved at 9.8% dry matter.

[0354] Similarly, when deamidation was performed at high protein concentrations, coffee stability was significantly improved compared to low protein concentrations (Table 12). At 0.67 IPA (U) / g protein, a deamidated pea protein solution with a protein concentration of 9.8% was stable at an isolate:coffee ratio of 1:3, while pea protein concentrations of 2.45% and 4.9% were unstable. Furthermore, at 6 IPA (U) / g protein, only a deamidated pea protein solution with a protein concentration of 9.8% was stable at an isolate:coffee ratio of 1:7. Similarly, at an enzyme dosage of 2 IPA (U) / g protein, deamidated pea protein with a protein concentration of 4.9% or higher was stable at an isolate:coffee ratio of 1:4, while a deamidated pea protein solution with a protein concentration of 2.45% was unstable.

[0355] Table 10: Solubility Improvement of Different PPI Samples

[0356] Table 11: Deamidation degree of samples with different PPI

[0357] Table 12: Coffee stability of samples with different PPI .

Claims

1. A method for obtaining a seed-based dairy product alternative beverage, the method comprising the following steps: (a) Providing a slurry of seed material in water; (b) The seed material in water is treated with a protein deamidase to obtain an enzymatically deamidated seed material slurry; (c) Optionally, the pulp of the enzymatically deamidated seed material is diluted to obtain a diluted pulp of the enzymatically deamidated seed material; and (d) Heat-treating the optionally diluted slurry of the enzymatically deamidinated seed material to obtain the seed-based dairy alternative beverage. The treatment with the protein deamidase is carried out in the presence of chloride salt, and / or the slurry of step (a) has a protein content of at least 3% (w / w).

2. The method of claim 1, wherein the treatment in step (b) is performed in the range of 10°C to 80°C, for example, in the range of 20°C to 60°C, 15°C to 40°C, 20°C to 30°C or 50°C to 60°C, preferably wherein the treatment in step (b) is performed for 15 to 90 minutes, for example, 30 to 60 minutes.

3. The method as described in any of the preceding claims, wherein the chloride salt is selected from potassium chloride and sodium chloride, preferably sodium chloride.

4. The method as described in any of the preceding claims, wherein the amount of the chloride salt is 0.05%-2% (w / w) of the chloride salt based on the slurry, preferably 0.07%-0.2% (w / w) of the chloride salt based on the slurry, for example, about 0.10% (w / w) or about 0.15% (w / w) of the slurry.

5. The method as described in any of the preceding claims, wherein the protein content of the slurry of seed material in water is in the range of 0.1%-3% (w / w), for example in the range of 0.1%-2.5% (w / w), 0.5%-1.5% (w / w), or 1%-2% (w / w), for example about 1% (w / w).

6. The method according to any one of claims 1-4, wherein the protein content of the slurry of the seed material in water is in the range of 4%-20% (w / w), preferably in the range of 5%-15% (w / w), more preferably in the range of 5%-10% (w / w), and the protein content of the diluted slurry of the enzymatically deamidated seed material is at most 2.5% (w / w), preferably wherein the protein content of the diluted slurry of the enzymatically deamidated seed material is in the range of 0.1%-2.5% (w / w), for example in the range of 0.5%-2% (w / w), for example in the range of 1%-2% (w / w), for example about 1% (w / w).

7. The method as described in any of the preceding claims, wherein no stabilizer and / or emulsifier is present during steps (a), (b), (c) and / or (d).

8. The method of any of the preceding claims, wherein the seed material is obtained from or derived from almonds, cashews, chickpeas, coconuts, broad beans, hazelnuts, lentils, lupins, macadamia nuts, mung beans, pistachios, peas, peanuts, pecans, soybeans, walnuts, or any combination thereof, preferably obtained from or derived from almonds, peas, soybeans, or any combination thereof.

9. The method as claimed in any of the preceding claims, wherein the seed-based dairy alternative beverage is an almond beverage, cashew beverage, chickpea beverage, coconut beverage, broad bean beverage, hazelnut beverage, lentil beverage, lupin beverage, macadamia nut beverage, mung bean beverage, pistachio beverage, pea beverage, peanut beverage, pecan beverage, soy beverage, walnut beverage, or any combination thereof, preferably an almond beverage, pea beverage, soy beverage, or any combination thereof.

10. The method as claimed in any of the preceding claims, wherein the seed-based dairy alternative beverage is used in an acidic beverage such as a sports drink, a coffee drink, or a tea drink, preferably a coffee drink.

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

12. The method of any of the preceding claims, wherein the seed-based dairy alternative beverage has increased stability to heat and / or low pH compared to a seed-based dairy alternative beverage prepared using the same method but without protein deamidase.

13. The method as claimed in any of the preceding claims, wherein a lower dose of protein deamidase is used compared to the use of the same method but where the protein deamidase treatment is carried out in the absence of chloride salts and / or the slurry of step (a) does not have a protein content of at least 3% (w / w).

14. A method for obtaining an almond-based dairy product alternative beverage, the method comprising the following steps: (a) Provide an almond paste in water; (b) The pulp of almond material in water is treated with a protein deamidase to obtain an enzymatically deamidated almond material pulp; (c) Optionally, the pulp of the enzymatically deamidated almond material is diluted to obtain a diluted pulp of the enzymatically deamidated almond material; and (d) Heat-treating the optionally diluted slurry of the enzymatically deamidated almond material to obtain the almond-based dairy alternative beverage. The treatment with the protein deamidase is carried out in the presence of chloride salt, and / or the slurry of step (a) has a protein content of at least 3% (w / w).

15. A seed-based dairy alternative beverage, which can be obtained by the method described in any one of claims 1-14.

16. Use of protein deamidase in the production of seed-based dairy alternative beverages to improve stability, preferably wherein the seed-based dairy alternative beverage is an almond beverage, a pea beverage, a soy beverage, or any combination thereof.

Images