Method for obtaining a plant-based ready-to-drink coffee or tea beverage.

Abstract

The present invention relates to the use of protein deamidase in obtaining plant-based ready-to-drink coffee or tea beverages having improved stability, particularly improved storage stability.

Description

Technical Field

[0001] Reference to Sequence Listing This application contains a sequence listing in a computer-readable format. The computer-readable format is incorporated herein by reference.

[0002] The present invention relates to the use of protein deamidase in a method for obtaining a plant-based ready-to-drink coffee or tea beverage having improved stability, particularly improved storage stability.

Background Art

[0003] In recent years, due to health reasons, the number of people seeking vegan, vegetarian, or non-dairy diets has been increasing. Furthermore, animal milk, particularly food made from cows, is increasingly recognized for its high environmental cost. These factors have led to a greater demand for dairy alternative plant-based foods for traditional dairy-derived foods including milk, creamers, cheeses, yogurts, and ice creams.

[0004] For example, ready-to-drink (RTD) acidic beverages such as sports drinks, RTD coffee beverages, and RTD tea beverages are sold worldwide as immediately consumable, pre-packaged, portable beverages. RTD acidic beverages can be stored and consumed at room temperature and low temperatures. Some of the more common commercially available RTD beverages include, but are not limited to, iced coffee, coffee latte, cold brew coffee, and iced milk tea. Many of the RTD beverages sold today are based on animal milk, although RTD beverages based on dairy alternative beverages, such as coconut milk-based iced coffee sold by ALOHA, have also been recently introduced to the market.

[0005] Dairy alternative plant-based beverages should meet the same requirements as conventional dairy beverages in terms of storage stability, free from phase separation, creaming, gelling, taste, and / or sedimentation. In particular, plant-based beverages must meet consumer expectations and demands regarding taste and texture, including a smooth mouthfeel, creaminess, and rich flavor. In this regard, certain challenges that remain unresolved include the insufficient solubility of plant proteins, off-flavors caused by plant proteins, and the tendency of plant proteins to precipitate in acidic products such as coffee or tea beverages.

[0006] U.S. Patent Application Publication No. 2022 / 0079187A1 discloses non-dairy analogues, such as pea milk, containing deamidated purified protein components, which reduce immediate feathering when mixed with heated coffee beverages. All exemplified non-dairy analogues suitable for mixing with heated coffee are prepared using at least gellan gum and phosphates. This disclosure does not relate to ready-to-drink coffee or tea beverages and does not demonstrate any improvement in the long-term stability of coffee and plant-based milk mixtures.

[0007] U.S. Patent Application Publication No. 2022 / 0151255A1 discloses nut milk treated with protein deamidase to reduce immediate coagulation that occurs when nut milk is mixed with a weakly acidic to weakly alkaline liquid such as coffee. The exemplified nut milk has only been tested for immediate coagulation in heated (90°C) coffee, and this disclosure does not describe any storage stability of the mixed coffee and nut milk, nor is it related to ready-to-drink coffee or tea beverages.

[0008] U.S. Patent Application Publication No. 2022 / 0142210A1 discloses a protein solution and a method for preparing it, including the use of a protein deamidase for processing a solution containing a protein and a stabilizer. Long-term stability is tested only with respect to pasteurized formulations containing deamidated pea protein, pectin gum, gellan gum, and juice concentrate. This disclosure does not describe the use of protein deamidase to improve the stability, in particular storage stability, of plant-based ready-to-drink coffee or tea beverages.

[0009] U.S. Patent Application Publication No. 2023 / 0240312A1 discloses protein deamidase-treated vegetable milks, such as oat milk or pea milk, that exhibit improved dispersibility, as measured by coagulation tendency, when added to heated coffee. This disclosure does not refer to the use of protein deamidase to obtain ready-to-drink coffee or tea beverages with storage stability. [Overview of the Initiative] [Problems that the invention aims to solve]

[0010] Therefore, an object of the present invention is to identify an improved process for obtaining a plant-based ready-to-drink coffee or tea beverage with improved stability, and in particular a plant-based ready-to-drink coffee or tea beverage with improved storage stability. [Means for solving the problem]

[0011] The inventors have surprisingly discovered that by treating plant material with protein deamidase, it is possible to obtain plant material with improved dispersibility and reduced risk of aggregation, both during mixing and long-term storage, when mixed with acidic beverages, particularly coffee or tea beverages. As a result, it is possible to obtain a plant-based ready-to-drink acidic beverage that is storage-stable and sensory-satisfying, including a blend of enzymatically deamidated plant material and an acidic beverage, which can be stored for several weeks before consumption under low temperature (refrigerated) conditions or at room temperature without suffering product degradation. Therefore, the plant-based ready-to-drink coffee or tea beverage claimed herein not only meets consumer demands for satisfactory sensory characteristics but also ensures storage stability.

[0012] Therefore, the present invention relates to a method for obtaining a plant-based ready-to-drink coffee or tea beverage, (a) A step of providing an aqueous solution containing an enzymatically deamidated plant material, (b) A step of mixing the aqueous solution from step (a) with a coffee or tea beverage to obtain a plant-based ready-to-drink coffee or tea beverage, (c) A step of storing a plant-based ready-to-drink coffee or tea beverage for at least 4 hours, preferably at least 8 hours, and more preferably at least 12 hours before consumption. This provides a method that includes [something].

[0013] The method claimed herein includes the further advantage that the mixing of an aqueous solution containing enzymatically deamidated plant material with a coffee or tea beverage can be carried out under both low-temperature conditions and at room temperature, for example, at temperatures in the range of 3°C to 30°C, for example, at temperatures in the range of 4°C to 8°C, or alternatively at temperatures in the range of 18°C ​​to 22°C. This provides manufacturers of plant-based RTD coffee or tea beverages with an improved and cost-effective production method.

[0014] Accordingly, the present invention further relates to plant-based ready-to-drink coffee or tea beverages obtained according to any of the methods claimed herein. Plant-based RTD coffee or tea beverages obtained according to the methods of the present invention have improved immediate and long-term stability. In particular, the inventors have found that the plant-based RTD coffee or tea beverages claimed herein are resistant to flocculation both immediately after preparation and for several weeks of storage, under both refrigerated and room temperature conditions. Thus, plant-based RTD coffee or tea beverages can be obtained that have functional properties, including creaminess, taste, and stability, which are required and expected by consumers. The improved stability of plant-based RTD coffee or tea beverages prepared according to the methods of the present invention further avoids the need to add emulsifiers and stabilizers to the final product, and thus also satisfies consumer requirements for clean-label plant-based foods. Thus, plant-based RTD coffee or tea beverages claimed herein and prepared using the methods claimed herein contain satisfying sensory properties without compromising health, thereby providing a healthier, more portable food that is high in protein and low in fat and sugar, and free of additives.

[0015] The present invention also provides the use of protein deamidase in the production of plant-based ready-to-drink coffee or tea beverages. In particular, the present invention provides the use of protein deamidase in the production of plant-based ready-to-drink coffee or tea beverages with storage stability. [Brief explanation of the drawing]

[0016] [Figure 1] This shows the stability of a soy beverage in instant coffee at 4°C.

[0017] [Figure 2] This demonstrates the stability of pea beverage in coffee extract at 4°C.

[0018] [Figure 3]Shows the stability of a soybean beverage in Earl Grey tea at 4°C.

[0019] [Figure 4] Shows the stability of a soybean beverage in Lipton black tea at 4°C.

[0020] [Figure 5] Shows the stability of a broad bean beverage in Earl Grey tea at 4°C.

[0021] [Figure 6] Shows the stability of a broad bean beverage in Lipton black tea at 4°C.

[0022] [Figure 7] Shows the stability of a soybean beverage in Lipton green tea at 4°C.

[0023] [Figure 8] Shows the stability of a broad bean beverage in Lipton green tea at 4°C.

[0024] [Figure 9] Shows the stability of a broad bean beverage in Lipton jasmine tea at 4°C.

[0025] [Figure 10] Shows the stability of an RTD coffee beverage prepared using a broad bean-based beverage, showing both immediate stability (day 0) and storage stability (day 7).

[0026] [Figure 11] Shows the stability of an RTD coffee beverage prepared using a broad bean-based beverage blended with phosphate, showing both immediate stability (day 0) and storage stability (day 7).

Mode for Carrying Out the Invention

[0027] Array A protein deamidase derived from Chryseobacterium viscerum (formerly known as Chryseobacterium sp-62563), possessing the mature polypeptide sequence shown in Sequence ID No. 1: Sequence ID No. 2. Sequence ID 2: Mature polypeptide sequence of protein deamidase derived from Chryseobacterium viscerum. A protein deamidase derived from Chryseobacterium proteolyticum, possessing the mature polypeptide sequence shown in Sequence ID No. 3: Sequence ID No. 4. Sequence ID 4: Mature polypeptide sequence of protein deamidase from Chryseobacterium proteolyticum. A protein deamidase derived from Chryseobacterium gambrini, possessing the mature polypeptide sequence shown in Sequence ID No. 5:Sequence ID No. 6. Sequence ID 6: Mature polypeptide sequence of protein deamidase derived from Chryseobacterium gambrini. A protein deamidase derived from Chryseobacterium culicis, possessing the mature polypeptide sequence shown in Sequence IDs 7 and 8. Sequence ID 8: Mature polypeptide sequence of protein deamidase derived from Chryseobacterium culicis. A protein deamidase derived from Chryseobacterium defluvii, possessing the mature polypeptide sequences shown in SEQ ID NOs: 9 and 10. Sequence ID 10: Mature polypeptide sequence of protein deamidase derived from Chryseobacterium defluvii.

[0028] Depending on the mode of carrying out the invention, the following definitions apply. Note that the singular forms "a," "an," and "the" refer to multiple objects unless otherwise clearly indicated by the context.

[0029] As used herein, the terms “drink” and “beverage” are interchangeable and have the same meaning.

[0030] Unless otherwise defined or clearly indicated by context, all percentages are weight percentages (percent w / w or "%(w / w)").

[0031] Unless otherwise specifically defined or indicated by context, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention pertains.

[0032] In this specification, the terms “RTD acidic beverage,” “RTD coffee or tea beverage,” “RTD beverage,” and “ready-to-drink” beverage, also called “RTD beverage,” refer to liquid food products that can be consumed directly at the time of purchase without requiring any additional preparation steps, such as the addition of water, heating, cooling, or cooking. The ready-to-drink coffee or tea beverage claimed herein comprises at least a mixture of enzymatically deamidated plant material and a coffee or tea beverage, or may be combined with or not combined with additional food ingredients to produce a ready-to-drink coffee or tea beverage. The ready-to-drink coffee or tea beverage may be ingested by humans or animals, preferably by humans. The ready-to-drink coffee or tea beverage claimed herein is plant-based.

[0033] The plant material may be in the form of an aqueous solution or suspension of a plant-based dairy substitute powder. Alternatively, the plant material may be any other suitable preparation obtained from a plant, such as an aqueous suspension of flour obtained from a plant, for example, from a part of a plant. The plant material may be any combination of the above.

[0034] The plant material used to obtain plant-based ready-to-drink beverages is subjected to enzymatic treatment with protein deamidase to obtain an aqueous solution containing the enzymatically deamidated plant material. Therefore, in relation to the present invention, the term “enzymatically deamidated plant material” means plant material treated with protein deamidase for deamidation. Those skilled in the art will know of appropriate analytical methods for determining the enzymatic deamidation of plant material. One such method is by measuring the free ammonium content (NH4) (step 2 of the assay procedure), as exemplified in Example 1 disclosed herein.

[0035] In some embodiments, additional food components are added to the plant-based ready-to-drink beverage. The additional food component may be any food component that would be useful to those skilled in the art. The additional food component may be a solid or a liquid component. The additional food component may be plant-based or non-plant-based. In some embodiments, the additional food component is water.

[0036] Plant-based ready-to-drink coffee or tea beverages may be fortified with, for example, plant-based dairy substitute powders such as soy milk powder, or concentrated or isolated proteins such as soy protein isolate, soy protein concentrate, pea protein isolate, or pea protein concentrate. In one embodiment, the plant-based ready-to-drink coffee or tea beverage is fortified, for example, an oat-based beverage fortified with pea protein, or a soy-based beverage fortified with soy protein. In one embodiment, the plant-based ready-to-drink coffee or tea beverage is an oat-based beverage fortified with pea or soy protein to a protein level of 2-3% (w / w). In another embodiment, the plant-based ready-to-drink coffee or tea beverage is a soy-based beverage fortified with soy or pea protein to a protein level of 6-8% (w / w).

[0037] Soybeans and peas belong to the legume family (Fabaceae). Based on their protein content, soybean and pea protein products can be classified into three main categories: soybean / pea flour, soybean / pea protein concentrate (SPC or PPC), and soybean / pea protein isolate (SPI or PPI), with the highest protein content found in isolates, followed by concentrates, and finally flour.

[0038] Preferably, the plant-based ready-to-drink coffee or tea beverage has a protein content of at least 0.03% (w / w).

[0039] Preferably, the plant-based ready-to-drink coffee or tea beverage has a protein content of up to 2% (w / w).

[0040] In a preferred embodiment, the plant-based ready-to-drink coffee or tea beverage has a protein content of approximately 1.5% (w / w).

[0041] Preferably, the plant-based ready-to-drink coffee or tea beverage has a lipid content of at least 0.5% (w / w).

[0042] Preferably, the plant-based ready-to-drink coffee or tea beverage has a maximum lipid content of 3% (w / w).

[0043] In a preferred embodiment, the plant-based ready-to-drink coffee or tea beverage has a lipid content of about 1.5% (w / w).

[0044] Additional food ingredients that may be added to plant-based ready-to-drink coffee or tea beverages include, but are not limited to, lipids, e.g., oils, especially vegetable oils; sugars, e.g., sucrose; proteins, various forms of synthetic amino acids; dietary fiber; salts; minerals; flavorings; vitamins; and any combination thereof.

[0045] In one embodiment, lipids are added to a plant-based ready-to-drink coffee or tea beverage and / or to an aqueous solution containing enzymatically deamidated plant material. The lipids may be vegetable oils or mixtures of vegetable oils. 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. The selection of an appropriate lipid may depend on the type of plant-based ready-to-drink coffee or tea beverage desired.

[0046] In further embodiments, sugars are optionally added together with lipids to plant-based ready-to-drink coffee or tea beverages and / or to aqueous solutions containing enzymatically deamidated plant materials. In one embodiment, the sugar is sucrose. In preferred embodiments, sucrose and soybean oil are added to aqueous solutions containing enzymatically deamidated plant materials.

[0047] In one embodiment, the salt is added to a plant-based ready-to-drink coffee or tea beverage and / or to an aqueous solution containing enzymatically deamidated plant material. The salt may be sodium chloride, dicalcium carbonate, dicalcium phosphate, tricalcium phosphate, calcium carbonate, or any combination thereof.

[0048] In one embodiment, vitamins and / or minerals are added to a plant-based ready-to-drink coffee or tea beverage and / or to an aqueous solution containing enzymatically deamidated plant material. Vitamins may include 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 include calcium, phosphorus, magnesium, sodium, potassium, chloride, iron, zinc, iodine, selenium, copper, and mixtures thereof.

[0049] A plant-based ready-to-drink coffee or tea beverage obtained according to the method claimed herein does not require the addition of emulsifiers and / or stabilizers to achieve the properties claimed herein. In particular, using the method claimed herein yields a plant-based ready-to-drink coffee or tea beverage with improved stability, especially improved storage stability. This means that the plant-based ready-to-drink coffee or tea beverage does not aggregate or precipitate even after long-term storage, such as several weeks or months. Thus, in one embodiment, the plant-based ready-to-drink coffee or tea beverage is substantially free of added emulsifiers and / or stabilizers. In another embodiment, an aqueous solution containing enzymatically deamidated plant material is substantially free of added emulsifiers and / or stabilizers. For example, an aqueous solution containing enzymatically deamidated plant material may be a plant-based dairy alternative beverage, such as a pea, soybean, or oat-based beverage, which is essentially free of added emulsifiers and / or stabilizers.

[0050] As used herein, the terms “emulsifier” and “stabilizer” mean added emulsifiers and stabilizers, i.e., components not found naturally in the materials used to prepare plant-based ready-to-drink coffee or tea beverages. Examples of such emulsifiers and stabilizers include, but are not limited to, thickeners such as carboxymethylcellulose, gellan gum, hydroxypropyl starch, and agar, as well as emulsifiers such as monoglycerides and diglycerides.

[0051] In connection with the present invention, the terms “substantially absent” and “essentially absent” are used interchangeably to describe compositions, products, or processes that contain only trace or negligible amounts of a particular substance, component, or process characteristic. This indicates that the presence of the specified substance or process characteristic is minimal and does not affect the overall characteristics or function of the present invention. In one embodiment, “essentially absent” or “substantially absent” means 0% (w / w) or 0% (w / v).

[0052] Using the method of the present invention, a plant-based ready-to-drink coffee or tea beverage with stability, particularly storage stability, can be obtained without the need to add buffer salts such as citrates or phosphates during the production of the plant-based ready-to-drink coffee or tea beverage, or when blending the resulting plant-based ready-to-drink coffee or tea beverage. This has been confirmed by the data shown in Example 6 of this specification.

[0053] In one embodiment, an aqueous solution containing enzymatically deamidated plant material is substantially free of added buffer salts.

[0054] In further embodiments, aqueous solutions containing enzymatically deamidated plant material are obtained using a method that is substantially free of added buffer salts.

[0055] In another embodiment, a plant-based ready-to-drink coffee or tea beverage is substantially free of added buffer salts.

[0056] In another embodiment, a method for obtaining a plant-based ready-to-drink coffee or tea beverage is carried out in the absence of added buffer salts.

[0057] Examples of buffer salts include chloride salts such as sodium chloride and potassium chloride, citrates, and phosphates such as tricalcium phosphate, potassium phosphate, dipotassium phosphate, sodium phosphate, and disodium phosphate.

[0058] In some embodiments, the buffer salt added is a phosphate.

[0059] In further embodiments, the aqueous solution containing the enzymatically deamidated plant material is substantially free of added phosphates.

[0060] The plant-based ready-to-drink coffee or tea beverages obtained by the method of the present invention have improved stability, particularly improved storage stability. Therefore, the plant-based ready-to-drink coffee or tea beverages are stored for at least 4 hours, preferably at least 8 hours, and more preferably at least 12 hours before consumption. This long-term storage does not affect the properties of the plant-based RTD beverage, and the plant-based RTD beverage remains free from precipitation and aggregation over time. In relation to the present invention, when the terms “stability” or “stable” are used to describe a plant-based ready-to-drink beverage, it means that the beverage is resistant to aggregation or precipitation both immediately after its manufacture and after long-term storage under typical storage conditions for consumer beverages and beverage additive products. In relation to the present invention, the stability and storage stability of plant-based ready-to-drink coffee or tea beverages should be understood not only as resistance to feathering when an aqueous solution containing enzymatically deamidated plant material is added to the coffee or tea beverage, but also as resistance to precipitation, aggregation, and sedimentation that may occur over time when the RTD coffee or tea beverage is stored. Stability can be determined by any method known in the art for evaluation, including visual evaluation. The plant-based ready-to-drink coffee or tea beverages claimed herein are largely unaffected by acidic food matrices, such as coffee or tea beverages, used to prepare the plant-based ready-to-drink beverages. Furthermore, the plant-based RTD coffee or tea beverages are stable over time, i.e., they are stable against precipitation of proteins and other components contained therein under refrigerated and room temperature conditions for extended periods such as 7 days, 14 days, 21 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, and 12 months, for example, 4 months or 8 months.

[0061] The plant-based ready-to-drink coffee or tea beverages claimed herein are storage-stable and can be stored under refrigerated or room temperature conditions. In relation to the present invention, room temperature conditions mean a temperature of about 15 to 25°C, for example, about 18 to 22°C, and low temperature and / or refrigerated conditions mean a temperature of about 2 to 5°C, for example, about 4°C. In one embodiment, the plant-based ready-to-drink coffee or tea beverage is intended for storage at 3 to 30°C, for example, 3 to 5°C and / or 15 to 25°C. In one embodiment, the plant-based ready-to-drink coffee or tea beverage is intended for storage at about 4°C and / or about 18 to 22°C.

[0062] In one embodiment, the plant-based ready-to-drink coffee or tea beverage is a canned or packaged beverage. The plant-based RTD coffee or tea beverage may be contained in and stored in any type of can or packaging material deemed suitable by those skilled in the art.

[0063] In some embodiments, the aqueous solution containing the enzymatically deamidated plant material and the coffee or tea beverage have a temperature in the range of 3°C to 30°C before and / or during mixing. In some embodiments, the aqueous solution containing the enzymatically deamidated plant material and the coffee or tea beverage have a temperature in the range of 3°C to 30°C before mixing. In some embodiments, the aqueous solution containing the enzymatically deamidated plant material and the coffee or tea beverage have essentially the same temperature at the time of mixing. The ability to mix the coffee or tea beverage with the enzymatically deamidated plant material at low and / or room temperature provides manufacturers of plant-based ready-to-drink coffee or tea beverages with an improved manufacturing method that is cost-effective, energy-efficient, and more reliable, i.e., avoids the risk of aggregation during and / or after the preparation of plant-based RTD coffee or tea beverages.

[0064] In some embodiments, the ratio of an aqueous solution containing enzymatically deamidated plant material to a coffee or tea beverage is 1:10 to 10:1 based on weight / weight, for example, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 and 1:10, or 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1 and 9:1. In one embodiment, the ratio of an aqueous solution containing enzymatically deamidated plant material to a coffee or tea beverage is 1:1. In another embodiment, the ratio of an aqueous solution containing enzymatically deamidated plant material to a coffee or tea beverage is 1:4. In yet another embodiment, the ratio of an aqueous solution containing enzymatically deamidated plant material to a coffee or tea beverage is 3:1.

[0065] Preferably, the aqueous solution containing the enzymatically deamidated plant material has a protein content of at least 0.3% (w / w).

[0066] Preferably, the aqueous solution containing the enzymatically deamidated plant material has a maximum protein content of 5% (w / w).

[0067] In a preferred embodiment, the aqueous solution containing the enzymatically deamidated plant material has a protein content of about 1% (w / w).

[0068] In another preferred embodiment, an aqueous solution containing enzymatically deamidated plant material has a protein content of about 2% (w / w).

[0069] Preferably, the aqueous solution containing the enzymatically deamidated plant material has a lipid content of at least 0.5% (w / w).

[0070] Preferably, the aqueous solution containing the enzymatically deamidated plant material has a maximum lipid content of 4% (w / w).

[0071] In preferred embodiments, the aqueous solution containing the enzymatically deamidated plant material has a lipid content of about 1.5%, about 2%, or about 3.5% (w / w).

[0072] In relation to the present invention, the terms “acid / acidic food matrix,” “acidic beverage,” and “acidic drink” mean a coffee or tea beverage having a low pH, i.e., a maximum pH of 7. Therefore, in one embodiment, the coffee or tea beverage has a pH of 3 to 7, for example, 4.5 to 6.5. In a preferred embodiment, the coffee or tea beverage has a pH of 5.0 to 5.5. In relation to the present invention, the coffee or tea beverage may have a pH in the range of 3 to 7, for example, a pH in the range of about 4 to 6, for example, a pH in the range of about 4.5 to 5.5. For example, the coffee beverage may be a highly acidic coffee with a pH of less than 5.0. Examples of highly acidic coffees include, but are not limited to, espresso and ristretto. The coffee beverage may also be a coffee beverage prepared from instant coffee, for example, Nescafé Gold instant coffee or coffee extract. The acidic beverage may also be a tea beverage, for example, a tea beverage based on a chai tea blend, a spice tea blend, for example, black tea such as Assam and Darjeeling, green tea, Earl Grey, oolong tea, and rooibos tea. Such tea beverages may be used to prepare chai lattes or milk tea beverages.

[0073] In one embodiment, a plant-based ready-to-drink beverage prepared by mixing a coffee or tea beverage with an aqueous solution containing enzymatically deamidated plant material has a pH of 5 to 7, for example, 5.2 to 6.5.

[0074] In some embodiments, an aqueous solution containing enzymatically deamidated plant material is i. A process to obtain a slurry of plant material in water, Ii. A step of treating the slurry from step (i) with protein deamidase to obtain an aqueous solution containing deamidated plant material in water, iii. A step to selectively inactivate protein deamidase and It is obtained using a method that includes [a specific method].

[0075] The protein deamidase used to process the slurry of plant material in water is maintained at a temperature in the range of 20–80°C, and as a result, the plant material is enzymatically deamidated by the protein deamidase, resulting in enzymatically deamidated plant material. In some embodiments, the slurry is maintained at a temperature of 25–40°C, 30–45°C, 35–50°C, 40–55°C, 50–60°C, or 50–65°C. In further embodiments, the slurry is maintained at a temperature of about 20°C, 25°C, about 30°C, about 35°C, about 40°C, about 45°C, about 50°C, about 55°C, or about 60°C.

[0076] In some embodiments, the added protein deamidase is held at a temperature of 20–80°C for at least 10 minutes to enable enzymatic deamidation of the plant material. In some embodiments, the slurry is held for about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 60 minutes, about 120 minutes, about 180 minutes, or about 240 minutes to enable enzymatic deamidation of the plant material. In some embodiments, the slurry is held for at least about 10 minutes, 30 minutes, or 60 minutes. In some embodiments, the slurry is held for 30 minutes. In some embodiments, the slurry is held for 60 minutes.

[0077] In a preferred embodiment, the slurry to which protein deamidase has been added is held at a temperature in the range of 50-60°C for about 30-60 minutes.

[0078] In some embodiments, a method for obtaining an aqueous solution containing enzymatically deamidated plant material substantially does not involve added emulsifiers and / or stabilizers and / or added buffer salts. In some embodiments, a slurry of plant material in water is treated with protein deamidase in the absence of added buffer salts.

[0079] In some embodiments, lipids are added to the slurry. Lipids may be added before, during, or after the slurry is treated with protein deamidase. In one embodiment, lipids are added before the slurry is treated with protein deamidase. In another embodiment, lipids are added after the slurry is treated with protein deamidase. 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.

[0080] The process used, including the temperature range, pH, and length of enzyme treatment, will vary depending on the plant material and the enzyme added to the slurry. Those skilled in the art will know how to determine the best process parameters based on the plant material and enzyme used.

[0081] After enzymatic deamidation of the plant material, the protein deamidase may be inactivated. The enzyme may be inactivated in any step after hydrolysis. In some embodiments, the enzyme is inactivated by heat treatment. In some embodiments, the heat treatment is at 85-95°C for 5-30 minutes. In further embodiments, the heat treatment is at 85-95°C for 10 minutes. In some embodiments, the heat treatment is at 95°C for 5, 10, 15, 20, 25, or 30 minutes.

[0082] In some embodiments, the enzyme is inactivated by ultra-high temperature (UHT) treatment. The UHT treatment may be direct or indirect. In some embodiments, the UHT treatment is at 135-154°C for 1-10 seconds. In further embodiments, the UHT treatment is at a temperature of 140-150°C for 3, 4, 5, 6, 7, 8, 9, or 10 seconds. In further embodiments, the UHT treatment is at a temperature of 140-145°C for 3, 4, 5, 6, 7, 8, 9, or 10 seconds. In some embodiments, the UHT treatment is at 143°C for 4, 5, 6, 7, or 8 seconds. In some embodiments, the UHT treatment is at 110-125°C, for example, 121°C, for 10-600 seconds, for example, about 60 seconds. In some embodiments, the UHT treatment is an in-container sterilization treatment, the plant-based ready-to-drink coffee or tea beverage is canned or packaged, and the UHT treatment is performed at 110-125°C for 10-40 minutes.

[0083] After optional enzymatic inactivation, the aqueous solution containing the enzymatically deamidated plant material may be cooled. The hydrolyzed plant material can be separated into a solid stream and a liquid stream, for example, by centrifugation. Centrifugation may be performed in a decanter centrifuge. Following centrifugation, the liquid stream can be recovered or collected and used as an aqueous solution containing the enzymatically deamidated plant material. The liquid stream may still contain some solid matter. In some embodiments, the liquid stream contains 1-80% solids. In further 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.

[0084] In some embodiments, the liquid stream is further processed to remove water. This is also called concentration. Concentration increases the relative amount of solids in the concentrated liquid stream. In some embodiments, water removal concentrates hydrolysis products, i.e., free sugars. Concentration can occur by evaporation of water in the liquid stream. In some embodiments, the concentrated liquid stream contains 10–100% solids. In further 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 will increase the viscosity of the aqueous solution containing the enzymatically deamidated plant material.

[0085] In some embodiments, the liquid stream is used directly as an aqueous solution containing enzymatically deamidated plant material. Additional food components can be added to the liquid stream to produce an aqueous solution containing enzymatically deamidated plant material. In some embodiments, the liquid stream is derived from soybean or pea material. For example, this soybean or pea-derived liquid stream may be formulated with, for example, sodium chloride (NaCl), oil, sugar, and flavoring agents. It may be homogenized. It can be subjected to UHT or ESL treatment and aseptically filled.

[0086] In some embodiments, aqueous solutions containing enzymatically deamidated plant materials constitute plant-based dairy alternative beverages. Examples of plant-based dairy alternative beverages include soy beverages, cashew beverages, macadamia beverages, coconut beverages, pea beverages, rice beverages, quinoa beverages, flax beverages, hemp beverages, oat beverages, and any combination thereof. In one embodiment, the plant-based beverage is a soy beverage, a pea beverage, or any combination thereof. In some embodiments, the plant-based beverage is prepared from a soy protein isolate or a pea protein isolate.

[0087] The plant material is the edible part of a plant or derived from the edible part of a plant. In some embodiments, the plant material is derived from the edible part of a starchy plant. In some embodiments, the edible part of a plant may be a tuber, root, stem, cob, legume, fruit, or seed. In some embodiments, the plant is a cereal, and the plant material is a cereal, also called a whole grain, or derived from a cereal. In further embodiments, the cereal may be derived from maize, rice, millet, milo, quinoa, oats, or rye. In further embodiments, the cereal may be derived from barley, wheat, buckwheat, or rye. In some embodiments, the plant material is the tuber or root (including rhizome) of a potato, sweet potato, cassava, tiger nut (chuffa nut), canna, or tapioca, or derived therefrom. In some embodiments, the plant material is the fruit of a banana, jackfruit, or breadfruit, or derived therefrom. In some embodiments, the plant material is or is derived from hemp, faba, sago, pea, or soybean plants.

[0088] In some embodiments, the plant material is heat-treated. In some embodiments, the plant material is dehydrated. In some embodiments, the plant material is hulled, powdered, wet-ground, and / or dry-ground. In some embodiments, the plant material is corn flour, rice flour, barley flour, wheat flour, buckwheat flour, millet flour, quinoa flour, oat flour, rye flour, potato flour, sweet potato flour, cassava flour, tiger nut flour, tapioca flour, hemp flour, nut flour, pea flour, soybean flour, bean flour, hulled oats, hulled barley, hulled wheat, hulled peas, hulled beans, or any combination thereof. In preferred embodiments, the plant material is oat material, pea material, soybean material, or any combination thereof.

[0089] In some embodiments, plant material is suspended in water to produce an aqueous solution containing plant material, where the ratio of plant material to water is 1:3 to 1:10 (w / w), for example, 1:3 to 1:8 (w / w) or 1:6 to 1:10 (w / w).

[0090] Aqueous solutions containing enzymatically deamidated plant material can be subjected to further processing, such as treatment with further enzymes, including further hydrolytic enzymes. In some embodiments, aqueous solutions containing enzymatically deamidated plant material can be standardized and / or homogenized.

[0091] An aqueous solution containing enzymatically deamidated plant material may be further treated with one or more hydrolytic enzymes selected from the group consisting of pectinase, hemicellulase, xylanase, beta-glucanase, mannanase, glucanase, glucoamylase, isoamylase, alpha-amylase, beta-amylase, and mixtures thereof. The enzymes used in the method of the present invention may be added to a slurry containing plant material in liquid form, in any suitable form such as a stabilized liquid, or it may be added substantially as a dry powder or granules. Granules may be produced, for example, as disclosed in U.S. Patent No. 4,106,991 and U.S. Patent No. 4,661,452. The liquid enzyme preparation may be stabilized, for example, by adding sugar or sugar alcohol or lactic acid according to established methods. Other enzyme stabilizers are well known in the art.

[0092] The inventors have found that protein deamidase makes it possible to obtain a sterile, plant-based ready-to-drink coffee or tea beverage that is stable against elevated temperature and acidic conditions. Therefore, one embodiment provides a method for obtaining a sterile, plant-based ready-to-drink coffee or tea beverage, - A process to obtain a slurry of plant material in water, - A step of treating a slurry of plant material in water with protein deamidase to obtain an aqueous solution containing deamidated plant material in water, - A step of optionally inactivating protein deamidase by subjecting an aqueous solution containing deamidated plant material in water to heat treatment, - A step of mixing an aqueous solution containing enzymatically deamidated plant material with a coffee or tea beverage to obtain a plant-based ready-to-drink coffee or tea beverage, - A plant-based ready-to-drink coffee or tea beverage is heat-treated, preferably UHT-treated, to obtain a sterilized plant-based ready-to-drink coffee or tea beverage, optionally, the plant-based ready-to-drink coffee or tea beverage is a canned or packaged beverage. - A process of storing sterilized plant-based ready-to-drink coffee or tea beverages for at least 4 hours before consumption. With regard to a method including the above, preferably, this method is substantially free of added emulsifiers, stabilizers and / or added buffer salts.

[0093] Protein deamidase In the method of the present invention, the plant material is treated with a protein deamidase to obtain an enzymatically deamidated plant material.

[0094] In this invention, protein deamidase refers to an enzyme that directly acts on the amide group of the side chain of amino acids that make up a protein, causing deamidation without cleaving the peptide bonds of the protein and cross-linked proteins, thereby releasing ammonia.

[0095] The term "deamidase" refers to the protein-glutaminase (also known as glutaminylpeptide glutaminase) activity described in EC 3.5.1.44 that catalyzes the hydrolysis of glutamine gamma-amides (e.g., L-glutaminylglycine and L-phenylalanyl-L-glutaminylglycine) substituted at the carboxyl position or at both the alpha-amino and carboxyl positions. Thus, deamidase can deamidate glutamine residues in proteins to glutamic acid residues and is also called protein glutamine deamidase. Deamidase contains three catalytic residues (Cys-156, His-197, and Asp-217, as shown in, for example, Hashizume et al. "Crystal structures of protein glutaminase and its pro forms converted into enzyme-substrate complex," Journal of Biological Chemistry, vol.286, no.44, pp.38691-38702) and belongs to InterPro entry IPR041325.

[0096] Other examples of deamidases include protein asparaginases, which directly act on the amide group of the side chain of an asparagine residue contained in a protein to release ammonia, thereby converting the asparagine residue to an aspartic acid residue. In the present invention, either one of protein glutaminase and protein asparaginase can be used as the protein deamidase, or both can be used in combination. An example of a protein deamidase used in the present invention is protein glutaminase.

[0097] The protein deamidase used in the method of the present invention may be obtained from a microorganism of any genus. For the purposes of the present invention, the term "obtained from" as used herein in relation to a given source means that a polypeptide encoded by a polynucleotide is produced by the source or a strain into which a polynucleotide derived from the source has been inserted. In one embodiment, the polypeptide obtained from a given source is secreted extracellularly.

[0098] Protein deamidases can be obtained from microorganisms by the use of any suitable technique. For example, enzyme preparations can be obtained by fermentation of a suitable microorganism by a method known in the art, and isolation of the resulting fermented broth or subsequent protein deamidase preparation from the microorganism. Protein deamidases can also be obtained by the use of recombinant DNA techniques. Such methods typically involve culturing host cells transformed with a recombinant DNA vector containing a DNA sequence encoding a protein deamidase and a DNA sequence operably linked to an appropriate expression signal so as to enable the expression of the enzyme in a culture medium under conditions that allow for the recovery of the enzyme from the medium. The DNA sequence can also be incorporated into the genome of the host cell. The DNA sequence may be of genomic, cDNA, or synthetic origin, or any combination thereof, and can be isolated or synthesized according to methods known in the art.

[0099] The protein deamidase may be purified. As used herein, the term “purified” includes protein deamidase enzyme proteins that are essentially free from insoluble components derived from the producing organism. The term “purified” also includes protein deamidase enzyme proteins that are essentially free from the insoluble components of the native organism from which it is obtained. Preferably, it is also separated from some of the soluble components of the organism and the culture medium from which it is derived. More preferably, it is separated by one or more unit operations: filtration, precipitation, or chromatography.

[0100] The type or origin of the protein deamidase used in the present invention is not particularly limited. Examples of protein deamidases include those derived from the genera Chryseobacterium, Flavobacterium, Empedobacter, Sphingobacterium, Aureobacterium, or Myroides.

[0101] Protein deamidases may originate from any source mentioned herein. In this context, “originate” means that the enzyme may be isolated from an organism in which it naturally occurs, i.e., the amino acid sequence of the protein deamidase is identical to that of the natural polypeptide. “Originate” also means that the enzyme can be produced by recombination in a host organism, having an amino acid sequence identical to or modified to that of the natural enzyme, for example, having one or more amino acids deleted, inserted, and / or substituted; i.e., a recombinant enzyme that is a variant of the natural amino acid sequence. “Natural enzyme” includes natural variants. Furthermore, “originate” includes enzymes produced by synthesis, for example, by peptide synthesis. “Originate” also encompasses enzymes that have been modified (whether in vivo or in vitro), for example, by glycosylation, phosphorylation, etc. With respect to recombinant enzymes, “originate” refers to the identity of the enzyme, not the identity of the host organism in which the enzyme is recombinantly produced.

[0102] In some embodiments, the protein deamidase may be derived from a species of the genus Chryseobacterium, such as Chryseobacterium viscerum (this strain was previously referred to as Chryseobacterium sp-62563), C. gambrini, C. culicis, C. defluvii, or C. proteolyticum. In some embodiments, the deamidase in the method of the present invention is derived from or obtained from Chryseobacterium viscerum.

[0103] European Patent No. 1839491 discloses the cloning of protein glutaminase from Chryseobacterium proteolyticum expressed in Corynebacterium glutamicum. Furthermore, deamidases, such as protein glutaminases derived from the genus Chryseobacterium, such as "Amano PG500" (manufactured by Amano Enzyme Inc.), are commercially available.

[0104] For example, protein deamidase can be obtained from the culture broth of the above-mentioned microorganisms.

[0105] Protein deamidase is produced by microbial cells as an inactive proform containing a propeptide domain firmly bound to the deamidase domain. The proform is expressed as a fusion protein and has reduced deamidase activity that protects the viability of the host cell. By nature, the fusion protein is post-processed to remove the propeptide and release 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 and separated from the mature deamidase. The protein deamidase of the methods and compositions of the present invention is mature deamidase from 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.

[0106] The recombinant mature protein deamidases used in the method of the present invention comprise the polypeptides of SEQ ID NOs: 2, 4, 6, 8, and 10. Each mature protein deamidase is derived from a deamidase proform polypeptide comprising the polypeptides of SEQ ID NOs: 1, 3, 5, 7, and 9, respectively. The proform polypeptide contains a propeptide at its N-terminus that is fused to the same deamidase as that of the polypeptides of SEQ ID NOs: 2, 4, 6, 8, or 10. The propeptide can be enzymatically cleaved from the proform polypeptide to release the mature deamidase. Naturally occurring propeptide sequences are provided in the proform polypeptide.

[0107] 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 mutants of naturally occurring propeptides. These mutant propeptide sequences have been found to bind less strongly to their corresponding deamidases, resulting in more readily enzymatic cleavage after recombinant expression and secretion from host cells. The polypeptides of Sequence IDs 1-10 are derived from species of the genus Chryseobacterium and are described in PCT application PCT / EP2023 / 055936, filed on 8 March 2023, which is incorporated herein by reference.

[0108] Following expression of the proform polypeptide in a recombinant expression system, the propeptide was cleaved using site-directed endopeptidase, leaving behind active mature deamidase. In some embodiments, the cleaved propeptide was not purified from the mature deamidase. Therefore, the propeptide may be present in the composition together with the mature deamidase.

[0109] According to a preferred embodiment, the protein deamidase used in the method of the present invention is derived from or obtained from a Chryseobacterium species, such as Chryseobacterium proteolyticus or Chryseobacterium viscerum.

[0110] In relation to the present invention, the term "mature polypeptide" refers to the polypeptide in its mature form after N-terminal processing (e.g., removal of the signal peptide). The "signal peptide" is a sequence of amino acids attached to the N-terminal portion of a protein that promotes the secretion of the protein outside the cell. The mature form of the extracellular protein lacks the signal peptide, which is cleaved during the secretion process.

[0111] In one embodiment, the deamidase is selected from a polypeptide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with SEQ ID NO: 2.

[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: 4.

[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: 6.

[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: 8.

[0115] In one embodiment, the deamidase is selected from a polypeptide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with SEQ ID NO: 10.

[0116] In one embodiment, the deamidase is selected from a polypeptide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with the mature polypeptide of SEQ ID NO: 1.

[0117] In one embodiment, the deamidase is selected from a polypeptide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with the mature polypeptide of SEQ ID NO: 3.

[0118] In one embodiment, the deamidase is selected from a polypeptide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with the mature polypeptide of SEQ ID NO: 5.

[0119] In one embodiment, the deamidase is selected from a polypeptide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with the mature polypeptide of SEQ ID NO: 7.

[0120] In one embodiment, the deamidase is selected from a polypeptide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with the mature polypeptide of SEQ ID NO: 9.

[0121] For the purposes of this invention, sequence identity between two amino acid sequences is determined as output data labeled "Longest Identity" using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol.48:443-453), which is preferably implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277), version 6.6.0 or later. The parameters used are a gap-open penalty of 10, a gap-extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The -nobrief option must be specified on the command line for the Needle program to report the longest identity. The Needle output labeled "Longest Identity" is calculated as follows: (Number of identical residues × 100) / (Length of alignment - Total number of gaps in the alignment)

[0122] In relation to the present invention, the term “mutant” means a polypeptide having enzymatic activity that includes modifications, i.e., substitutions, insertions, and / or deletions at one or more (e.g., several) positions. Substitution means exchanging 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 to 5 amino acids, adjacent to or immediately following an amino acid occupying a position.

[0123] Amino acid changes may be minor, i.e., conservative amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; small deletions, typically 1 to 30 amino acids; small amino or carboxyl terminal extensions, such as amino-terminal methionine residues; small linker peptides of up to 20 to 25 residues; or small extensions that facilitate purification by altering the net charge or another function, such as a polyhistidine tract, antigen epitope, or binding domain.

[0124] Examples of conservative substitutions are found in the groups of basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine, and valine), aromatic amino acids (phenylalanine, tryptophan, and tyrosine), and low molecular weight amino acids (glycine, alanine, serine, threonine, and methionine). Amino acid substitutions that generally do not alter specific activity are well known in the art and are described, for example, in H. Neurath and RLHill, 1979, *The Proteins*, Academic Press, New York. Common substitutions include 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, amino acid changes are those that alter the physicochemical properties of a polypeptide. For example, amino acid changes can affect the thermal stability of a polypeptide, alter substrate specificity, and change the optimal pH.

[0126] Essential amino acids in polypeptides 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 into any residue within the molecule to identify amino acid residues critical to the molecule's activity, and the resulting mutant molecules are tested for enzyme activity. See also Hilton et al., 1996, J. Biol. Chem. 271:4699-4708. The active site of an enzyme or other biological interactions can also be determined by physical analysis of the structure, measured by techniques such as nuclear magnetic conjugate, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutations of the hypothetical contact site amino acids. For example, see 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 alignment with related polypeptides.

[0127] Substitutions, deletions, and / or insertions of single or multiple amino acids can be performed and tested using known mutagenesis, recombination, and / or shuffling methods, followed by relevant screening procedures such as those disclosed in Reidhaar-Olson and Sauer, 1988, Science 241:53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86:2152-2156; International Publication No. 95 / 17413; or International Publication No. 95 / 22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30:10832-10837; U.S. Patent No. 5,223,409; International Publication No. 92 / 06204), and region-specific mutagenesis (Derbyshire et al., 1986, Gene 46:145; Ner et al., 1988, DNA 7:127).

[0128] Mutagenesis / shuffling methods can be combined with high-throughput automated screening methods to detect the activity of cloned and mutation-induced polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17; 893-896). Mutageneised DNA molecules encoding the active polypeptide can be recovered from host cells and rapidly sequenced using standard methods in this technique. This method allows for rapid determination of the importance of individual amino acid residues within the polypeptide.

[0129] The protein deamidase used in the method of the present invention may be added at a concentration of 0.01 to 20 IPA(U) / g substrate protein, for example, 0.1 to 12 IPA(U) / g substrate protein or 0.5 to 7 IPA(U) / g substrate protein. In some embodiments, the protein deamidase used in the method of the present invention is added at a concentration in the range of 0.3 to 2 IPA(U) / g substrate protein, for example, 0.5 to 1.5 IPA(U) / g substrate protein.

[0130] Deamidase (protein glutaminase) activity was measured using the assay described in Example 1. The activity assay consists of two separate parts: (1) an enzymatic step in which ammonia is formed by the catalytic action of protein deamidase, and (2) a non-enzymatic detection step in which the ammonia formed in step (1) is derivatized into a blue indophenol compound having an absorption maximum at 630 nm. The amount of enzyme that produces 1 μmol of ammonia per minute at 37°C is defined as 1 unit (indophenol assay unit: given in IPA(U)). Activity can be determined against a declared strength standard.

[0131] The enzyme dosage will depend on parameters such as temperature, incubation time, and dairy product substitution recipes. Those skilled in the art will know how to determine the optimal enzyme dosage.

[0132] While we do not wish to be bound by any particular theory, we believe that the use of protein deamidase to obtain enzymatically deamidated plant material for use in the production of plant-based ready-to-drink coffee or tea beverages contributes to the excellent benefits reported herein, including, but not limited to, improved stability, including storage stability, of plant-based ready-to-drink acidic or tea beverages.

[0133] The present invention is further defined by the following numbered embodiments.

[0134] Embodiment 1. A method for obtaining a plant-based ready-to-drink acidic beverage, (a) A step of providing an aqueous solution containing an enzymatically deamidated plant material, (b) A step of mixing the aqueous solution from step (a) with an acidic beverage to obtain a plant-based ready-to-drink acidic beverage. A method for which a plant-based ready-to-drink acidic beverage, including [ingredient], is stored for at least 4 hours before consumption.

[0135] Embodiment 2. A method for obtaining a plant-based ready-to-drink coffee or tea beverage, (a) A step of providing an aqueous solution containing an enzymatically deamidated plant material, (b) A step of mixing the aqueous solution from step (a) with a coffee or tea beverage to obtain a plant-based ready-to-drink coffee or tea beverage, (c) A plant-based ready-to-drink coffee or tea beverage is stored for at least 4 hours before consumption. Methods that include...

[0136] Embodiment 3. The method according to any one of Embodiments 1 to 2, wherein a plant-based ready-to-drink coffee or tea beverage is stored for at least 8 hours before consumption.

[0137] Embodiment 4. The method according to any one of Embodiments 1 to 3, wherein a plant-based ready-to-drink coffee or tea beverage is stored for at least 12 hours before consumption.

[0138] Embodiment 5. The method according to any one of Embodiments 1 to 4, wherein the aqueous solution in step (a) and the coffee or tea beverage in step (b) have temperatures in the range of 3°C to 30°C before and / or during mixing in step (b).

[0139] Embodiment 6. The method according to any one of Embodiments 1 to 5, wherein the aqueous solution in step (a) and the coffee or tea beverage in step (b) have a temperature in the range of 3°C to 30°C before mixing in step (b).

[0140] Embodiment 7. The method according to any one of Embodiments 1 to 6, wherein the aqueous solution of step (a) and the coffee or tea beverage of step (b) have temperatures in the range of 3°C to 12°C before and / or during mixing in step (b), for example, 3°C to 10°C, 4°C to 9°C, or 5°C to 8°C.

[0141] Embodiment 8. The method according to any one of Embodiments 1 to 7, wherein the aqueous solution in step (a) and the coffee or tea beverage in step (b) have a temperature in the range of 3°C to 12°C, for example, 3°C to 10°C, 4°C to 9°C, or 5°C to 8°C, before mixing in step (b).

[0142] Embodiment 9. The method according to any one of Embodiments 1 to 6, wherein the aqueous solution of step (a) and the coffee or tea beverage of step (b) have temperatures in the range of 15°C to 25°C, for example, 16°C to 24°C, 17°C to 22°C, or 18°C ​​to 21°C, before and / or during mixing in step (b).

[0143] Embodiment 10. The method according to any one of Embodiments 1 to 6, wherein the aqueous solution in step (a) and the coffee or tea beverage in step (b) have temperatures in the range of 15°C to 25°C, for example, 16°C to 24°C, 17°C to 22°C, or 18°C ​​to 21°C, before mixing in step (b).

[0144] Embodiment 11. The method according to any one of Embodiments 1 to 10, wherein the aqueous solution containing an enzymatically deamidated plant material and the coffee or tea beverage are essentially the same temperature before mixing.

[0145] Embodiment 12. The method according to any one of Embodiments 1 to 11, wherein the aqueous solution of step (a) has a protein content in the range of 0.3 to 5% (w / w), for example, 1 to 3% (w / w).

[0146] Embodiment 13. Any one of Embodiments 1 to 12, wherein the aqueous solution of step (a) has a lipid content in the range of 0.5 to 4% (w / w), for example, 1 to 2% (w / w), for example, about 1.5% (w / w).

[0147] Embodiment 14. The method according to any one of Embodiments 1 to 13, wherein the aqueous solution is a plant-based dairy alternative beverage.

[0148] Embodiment 15. The method according to any one of Embodiments 1 to 14, wherein the aqueous solution is a soybean beverage, a cashew beverage, a macadamia nut beverage, a coconut beverage, a pea beverage, a rice beverage, a quinoa beverage, a flaxseed beverage, a hemp beverage, an oat beverage, or any combination thereof.

[0149] Embodiment 16. The method according to any one of Embodiments 1 to 15, wherein the aqueous solution is a pea beverage, a soybean beverage, or a combination thereof.

[0150] Embodiment 17. The method according to Embodiment 16, wherein the soy beverage is prepared from soy flour, soy protein isolate, soy protein concentrate, or any combination thereof, preferably from soy protein isolate.

[0151] Embodiment 18. The method according to Embodiment 16, wherein the pea beverage is prepared from pea flour, pea protein isolate, pea protein concentrate, or any combination thereof, preferably from pea protein isolate.

[0152] Embodiment 19. The method according to Embodiment 15, wherein the aqueous solution is an oat-based beverage.

[0153] Embodiment 20. The method according to any one of Embodiments 1 to 19, wherein an aqueous solution containing a plant-based ready-to-drink coffee or tea beverage and / or enzymatically deamidated plant material substantially contains no added emulsifiers and / or stabilizers.

[0154] Embodiment 21. The method according to any one of Embodiments 1 to 20, wherein a plant-based ready-to-drink coffee or tea beverage is substantially free of added emulsifiers and / or stabilizers.

[0155] Embodiment 22. The method according to any one of Embodiments 1 to 21, wherein the aqueous solution containing a plant-based ready-to-drink coffee or tea beverage and / or enzymatically deamidated plant material is substantially free of gum such as gellan gum and pectin gum.

[0156] Embodiment 23. The method according to any one of Embodiments 1 to 22, wherein an aqueous solution containing a plant-based ready-to-drink coffee or tea beverage and / or enzymatically deamidated plant material substantially contains no added buffer salts.

[0157] Embodiment 24. The method according to any one of Embodiments 1 to 23, wherein the plant-based aqueous solution containing the enzymatically deamidated plant material is substantially free of added buffer salts.

[0158] Embodiment 25. Any one of Embodiments 23 to 24, wherein the buffer salt is a chloride salt, a citrate, a phosphate, or any combination thereof.

[0159] Embodiment 26. Any one of Embodiments 23 to 25, wherein the buffer salt is a phosphate.

[0160] Embodiment 27. The method according to any one of Embodiments 1 to 26, wherein the plant-based ready-to-drink coffee or tea beverage has a pH of 3 to 7, for example, 3.5 to 6.5, 4.5 to 6.5, or 5.0 to 5.5.

[0161] Embodiment 28. The method according to any one of Embodiments 1 to 27, wherein the plant-based ready-to-drink coffee or tea beverage is a canned or packaged beverage.

[0162] Embodiment 29. The method according to any one of Embodiments 1 to 28, wherein a plant-based ready-to-drink coffee or tea beverage is intended for storage at a temperature of 3°C to 30°C.

[0163] Embodiment 30. The method according to any one of Embodiments 1 to 29, wherein a plant-based ready-to-drink coffee or tea beverage is for storage at temperatures of 3°C to 5°C, e.g., about 4°C, or 16°C to 24°C, e.g., about 20°C.

[0164] Embodiment 31. The method according to any one of Embodiments 1 to 30, wherein the plant-based ready-to-drink coffee or tea beverage further comprises one or more additional food ingredients selected from the group consisting of lipids, sugars, proteins, vitamins, minerals, amino acids, flavorings, dietary fiber, salt, and any combination thereof.

[0165] Embodiment 32. The method according to Embodiment 31, wherein the additional food components are lipids, for example, oils, preferably vegetable oils, more preferably soybean oils, and / or sugars, for example, sucrose.

[0166] Embodiment 33. The method according to any one of Embodiments 1 to 32, wherein a plant-based ready-to-drink coffee or tea beverage is heat-treated.

[0167] Embodiment 34. The method according to any one of Embodiments 1 to 33, wherein a plant-based ready-to-drink coffee or tea beverage is heat-treated, and the heat treatment is ultra-high temperature (UHT) treatment, extended shelf life (ESL) treatment, or pasteurization, preferably UHT treatment.

[0168] Embodiment 35. The method according to any one of Embodiments 33 to 34, wherein the heat treatment is performed after step (b) and before step (c).

[0169] Embodiment 36. The method according to any one of Embodiments 1 to 35, wherein the coffee or tea beverage is a coffee beverage.

[0170] Embodiment 37. The method according to any one of Embodiments 1 to 35, wherein the coffee or tea beverage is a tea beverage.

[0171] Embodiment 38. The method according to any one of Embodiments 1 to 37, wherein the plant material is soybean, cashew, macadamia, coconut, pea, bean, rice, quinoa, flax, hemp, or oat, preferably derived from oat, pea, or soybean.

[0172] Embodiment 39. The method according to any one of Embodiments 1 to 38, wherein the plant material is derived from peas or soybeans.

[0173] Embodiment 40. The method according to any one of Embodiments 1 to 39, wherein the ratio of the aqueous solution to the coffee or tea beverage is 1:10 to 10:1 based on w / w.

[0174] Embodiment 41. The method according to any one of Embodiments 1 to 40, wherein the ratio of the aqueous solution to the coffee or tea beverage is 1:1, 1:4, or 3:1 on a w / w basis.

[0175] Embodiment 42. An aqueous solution containing an enzymatically deamidated plant material, i. A process to obtain a slurry of plant material in water, ii. A step of treating the slurry from step (i) with protein deamidase to obtain an aqueous solution containing deamidated plant material in water, iii. A step to selectively inactivate protein deamidase and The method according to any one of Embodiments 1 to 41, obtained by using a method including the method.

[0176] Embodiment 43. The method of Embodiment 42, wherein the treatment with protein deamidase is carried out at 50-60°C for 30-60 minutes.

[0177] Embodiment 44. The method according to any one of Embodiments 42 to 43, further comprising processing in step ii with one or more hydrolytic enzymes selected from the group consisting of pectinase, hemicellulase, xylanase, beta-glucanase, mannanase, glucanase, glucoamylase, isoamylase, alpha-amylase, beta-amylase, and mixtures thereof.

[0178] Embodiment 45. The method according to any one of Embodiments 42 to 44, wherein a protein deamidase, and, in accordance with Embodiment 44, one or more hydrolases, are inactivated by a heat treatment such as ultra-high temperature (UHT) treatment.

[0179] Embodiment 46. The method according to any one of Embodiments 42 to 45, wherein step (ii) is substantially free of added emulsifiers, stabilizers and / or buffer salts, preferably free of added buffer salts.

[0180] Embodiment 47. The method according to any one of Embodiments 1 to 46, wherein the aqueous solution containing enzymatically deamidated plant material is derived from or obtained from the genus Chryseobacterium, for example, Chryseobacterium proteolyticus or Chryseobacterium viscerum.

[0181] Embodiment 48. The method according to any one of Embodiments 1 to 47, wherein an aqueous solution containing enzymatically deamidated plant material is obtained using a protein deamidase containing 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 NOs: 2, 4, 6, 8, or 10.

[0182] Embodiment 49. The method according to any one of Embodiments 1 to 48, wherein an aqueous solution containing enzymatically deamidated plant material is obtained using a protein deamidase containing the polynucleotide sequence of SEQ ID NO: 2, 4, 6, 8, or 10.

[0183] Embodiment 50. A plant-based ready-to-drink acidic beverage that can be obtained by the method described in any one of Embodiments 1 to 49.

[0184] Embodiment 51. The plant-based ready-to-drink acidic beverage according to Embodiment 50, further comprising protein deamidase.

[0185] Embodiment 52. A plant-based ready-to-drink acidic beverage according to any one of Embodiments 50 to 51, 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 NOs: 2, 4, 6, 8, or 10.

[0186] Embodiment 53. A plant-based ready-to-drink acidic beverage according to any one of Embodiments 50 to 52, which is a plant-based ready-to-drink coffee or tea beverage.

[0187] Embodiment 54. A plant-based ready-to-drink acidic beverage according to any one of Embodiments 50 to 53, characterized in that it is substantially free of added emulsifiers, stabilizers and / or buffer salts.

[0188] Embodiment 55. A plant-based ready-to-drink acidic beverage according to any one of Embodiments 50 to 54, characterized in that it is substantially free of added buffer salts.

[0189] Embodiment 56. A plant-based ready-to-drink acidic beverage according to any one of Embodiments 50 to 55, characterized in that it is substantially free of added phosphates.

[0190] Embodiment 57. A plant-based ready-to-drink acidic beverage according to any one of Embodiments 50 to 56, characterized by being heat-treated.

[0191] Embodiment 58. A plant-based ready-to-drink acidic beverage according to any one of Embodiments 50 to 57, characterized by being subjected to UHT treatment, ESL treatment, or pasteurization.

[0192] Embodiment 59. A plant-based ready-to-drink coffee or tea beverage comprising an enzymatically deamidated plant material and a coffee or tea beverage.

[0193] Embodiment 60. A plant-based ready-to-drink coffee or tea beverage according to Embodiment 59, characterized in that it is essentially free of added buffer salts, emulsifiers, and / or stabilizers.

[0194] Embodiment 61. A plant-based ready-to-drink coffee or tea beverage according to any one of Embodiments 59 to 60, characterized by being storage stable.

[0195] Embodiment 62. A pea-based and / or soy-based ready-to-drink coffee or tea beverage that can be obtained by a method embodied in any one of Embodiments 1 to 49.

[0196] Embodiment 63. A pea and / or soybean-based ready-to-drink coffee or tea beverage according to Embodiment 62, characterized in that it is essentially free of added buffer salts, emulsifiers and / or stabilizers.

[0197] Embodiment 64. Use of protein deamidase in the production of plant-based ready-to-drink acidic beverages to improve storage stability.

[0198] Embodiment 65. The use according to Embodiment 64, wherein the plant-based ready-to-drink acidic beverage is a plant-based ready-to-drink coffee or tea beverage.

[0199] Embodiment 66. The use according to any one of Embodiments 64 to 65, 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 NOs: 2, 4, 6, 8, or 10.

[0200] Embodiment 67. The use according to any one of Embodiments 64 to 66, wherein the protein deamidase is used in the absence of added buffer salts, emulsifiers and / or stabilizers.

[0201] Embodiment 68. The use according to any one of Embodiments 64 to 67, wherein the plant-based ready-to-drink acidic beverage is a pea and / or soy-based ready-to-drink acidic beverage.

[0202] The invention described herein and claimed is not to be limited to the scope of the specific embodiments disclosed herein, for these embodiments are intended to illustrate several aspects of the invention. Any equivalent embodiments are intended to be within the scope of the invention, and to be one or more combinations of the embodiments.

[0203] Various references are cited herein, and their disclosures are incorporated by reference in their entirety. The present invention will be further illustrated by the following embodiments, which should not be construed as limiting the scope of the invention. [Examples]

[0204] material enzyme Throughout the examples, the following enzymes are used: Protein deamidase: A protein deamidase derived from Chryseobacterium viscerum (this strain was previously called Chryseobacterium sp-62563) possessing the mature polypeptide sequence shown in Sequence ID No. 2. Propeptide cleavage was achieved by treating the deamidase of Sequence ID No. 1 with a site-specific endopeptidase. The site-specific endopeptidase used was glutamyl endopeptidase derived from Bacillus licheniformis. The active deamidase obtained after maturation was the polypeptide shown in Sequence ID No. 2.

[0205] Example 1: Protein deamidase activity assay The activity assay consists of two distinct, isolated parts: 1) An enzymatic process in which ammonia is formed by the catalytic action of protein deamidase, 2) A non-enzymatic detection step in which the ammonia formed in step (1) is derivatized into a blue indophenol compound having an absorption maximum at 630 nm. It consists of.

[0206] In step (1), ammonia is generated by the deamidation of protein deamidase. In step (2), the generated ammonia reacts with phenol under alkaline conditions to form dioxyphenylamine. The reaction is catalyzed by sodium pentacyanonitrosylferrate(III) (sodium nitroprusside). "Coloring reagent solution A" contains phenol and sodium nitroprusside. "Color development reagent solution B" provides alkaline reaction conditions. The intermediate is then oxidized by adding sodium hypochlorite ("color development 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.

[0207] Assay procedure Step (1) Enzymatic step involving ammonia formation reagent: Assay dilution: 0.2M sodium phosphate buffer, 0.01% Triton® X-100, pH 6.5. Assay buffer: Same as above. Used to prepare stock and diluted samples of protein deamidase (hereinafter referred to as "enzyme"). Substrate solution: 30 mM Z-Gln-Gly (Merck C6154-1G) in the assay dilution solution (check the pH after dissolution). Stop solution: 0.4M TCA. Standard: NH4Cl (Ammonium standard for IC, Merck 59755-100ML, 1000mg / L NH4) + The solution (in water) was diluted with the assay dilution solution (see also the section on "Standard Curve").

[0208] The enzyme product is dissolved / diluted in the assay buffer to prepare a suitable dilution that yields a linear assay response.

[0209] Warmth: Add 1.10 μL of diluted enzyme sample to each well of a 96-well microtiter plate (MTP), three times each. Add 2,100 μL of substrate solution to each well. 3. For blank samples, add 100 μL of 0.4 M TCA solution. 4. Use a transparent plate sealer to seal the plate. 5. Keep the plate warm for 10 minutes at 37°C and 500 rpm on a thermomix with a lid heating function. 6. To stop the reaction, carefully add 100 μL of 0.4 M TCA solution (except for blank samples that already contain TCA). Total reaction volume: 210 μL.

[0210] Step 2) Ammonia detection step reagent: Chromogenic reagent A: 4% (w / v) phenol, 0.015% (w / v) sodium pentacyanonitrosylferrate (III) dihydrate (sodium nitroprusside) (Na2[Fe(CN)5NO]·2H2O). Colorimetric reagent B: 5% (w / v) potassium hydroxide. Colorimetric reagent C: 28% (w / v) potassium carbonate, 6% (v / v) sodium hypochlorite (Sigma-Aldrich 239305-25ml, less than 5% usable Cl2).

[0211] Warmth: 1. Transfer 15 μL from each well in step (1) to a new 96-well MTP. Transfer 2.45 μL of Milli-Q water to each well. 3. Add 30 μL of chromogenic reagent B to each well (mix gently by hand on a laboratory table). 4. Add 60 μL of chromogenic reagent A to each well (mix gently by hand on a laboratory table). 5. Add 60 μL of chromogenic reagent C to each well (mix gently by hand on a laboratory table). 6. Color development: Carefully seal the plate and leave it on the laboratory table for 30 minutes. 7. Carefully transfer the MTP to a plate reader and measure its absorbance at 630 nm. Total reaction volume: 210 μL Standard curve: Standard stock solution: 1000 mg NH4 + / L.

[0212] The standard curve is prepared by adding a diluted ammonium standard to the assay dilution buffer during the ammonia detection step. Specifically, 15 μL of diluted ammonia standard is mixed with 45 μL of water, and then the chromogenic reagents are added in the order described above: B, A, and C.

[0213] 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)):

number

number

number

[0214] Example 2: Preparation of a leguminous plant-based beverage Preparation of soy beverages 100 mL of commercially available soy beverage (DouBenDou WeiZhen soy beverage manufactured by DaLi group; protein content: 2.5 g, fat: 1.7 g, carbohydrates: 7.2 g (per 100 mL of product); ingredients: water, soybeans (non-GMO), sucrose) was treated with protein deamidase at an amount of 2 IPA(U) / g protein in the soy beverage. The enzymatic treatment was carried out at 60°C for 1 hour. Next, the enzyme was inactivated at 85°C for 10 minutes. The resulting soy beverage was cooled to room temperature (20-25°C). A control sample of the soy beverage was prepared using the same procedure, but without the use of protein deamidase.

[0215] Preparation of pea beverage The pea beverage was prepared in the laboratory using the following procedure. A 10% solution (m / v) was prepared by mixing ShuangTa pea protein isolate (82.2% protein content based on dry solid) with deionized H2O. The 10% pea beverage solution was then treated with protein deamidase at doses of 0.6 IPA(U) / g protein and 2 IPA(U) / g protein. The reaction was carried out at 60°C for 1 hour, after which the pea beverage solution containing soybean oil (Nissin), sucrose, and H2O was homogenized according to the recipe (at 8000 rpm for 10 minutes); protein: 3.4 g; soybean oil: 1.9 g; and sucrose: 3.4 g (per 100 g). After homogenization, the pea beverage was pasteurized at 85°C for 10 minutes with stirring. The sample was then cooled to 5°C. A control sample of pea beverage was prepared using the same procedure without the use of protein deamidase. The pH of the pea beverage was approximately 7.0 in both the enzyme-treated sample and the control sample.

[0216] Example 3: Improvement of the stability of pea-based and soy-based beverages mixed with coffee and treated with protein deamidase. Stability of soy beverages in instant coffee drinks An instant coffee beverage was prepared by dissolving 2 g of Nescafe Gold instant coffee (Nestlé) in 150 g of 85°C warm water. Next, 2 g of soy beverage prepared according to Example 2 was added to 20 g of the instant coffee beverage. In the enzymatically deamidated sample (treated with 2 IPA(U) / g protein), no coagulation was observed during mixing, but in the non-enzymatically treated sample, clear aggregation was observed and it immediately settled to the bottom of the test tube. Next, the samples were stored in a refrigerator (approximately 4°C) for storage stability testing. The results of the storage stability test are shown in Table 1 (Y: present, N: absent) and Figure 1 below. The pH of the soy-based coffee beverage was approximately 5.2.

[0217] [Table 1]

[0218] As can be seen from Table 1 and Figure 1, the use of protein deamidase to prepare enzymatically deamidated plant-based beverages yielded soy-based ready-to-drink coffee beverages that did not exhibit visible aggregation after storage for 2, 9, and 16 days.

[0219] Stability of pea beverages in coffee Coffee was prepared using commercially available Milan Gold coffee beans (Grand Espresso No. 2). These beans were ground and brewed using a De'Longhi® coffee machine (Type: ECAM23.420.SW) under the Extra Strong and Extra Long coffee taste settings.

[0220] Two g and eight g of pea beverage prepared according to Example 2 were mixed with eight g of extracted coffee at room temperature. The results are shown in Table 2 and Figure 2. When 2 g of pea beverage was mixed with eight g of coffee extract, the pH changed from 5.0 to approximately 5.5, but when eight g of pea beverage was mixed with eight g of coffee extract, the final pH was approximately 6.0.

[0221] [Table 2]

[0222] As can be seen from Table 2 and Figure 2, the use of protein deamidase to prepare enzymatically deamidated plant-based beverages yielded pea-based ready-to-drink coffee beverages that did not exhibit visible aggregation immediately after mixing (t=0) or after storage for 28 and 35 days.

[0223] Example 4: Improvement of the stability of pea-based and soy-based beverages mixed with tea and treated with protein deamidase. Stability of soy beverages in black tea In relation to this invention, two types of commercially available black tea were tested. In the first test, a black tea extract was prepared using commercially available Earl Grey tea bags (2 grams of tea per tea bag). Two tea bags were mixed with 100 mL of 85°C warm water and kept warm for 30 minutes. Then, the tea bags were removed from the liquid and discarded. Next, 2 g of soy beverage prepared according to Example 2 was mixed with 20 g of Earl Grey black tea beverage. For storage stability testing, the samples were stored in a refrigerator (4°C). The results of the stability test are shown in Table 3 and Figure 3. The pH of the Earl Grey tea and soy beverage mixture was approximately 5.3.

[0224] [Table 3]

[0225] As can be seen from Table 3 and Figure 3, the use of protein deamidase to prepare enzymatically deamidated soy-based beverages yielded soy-based ready-to-drink tea beverages that did not show visible aggregation after storage for 2, 9, and 16 days.

[0226] In the second test, a tea extract was prepared using commercially available Lipton tea. Four tea bags were mixed with 100 mL of 85°C warm water and kept warm for 30 minutes. The tea bags were then removed from the liquid and discarded. Next, 3 g of soy beverage prepared according to Example 2 was mixed with 9 g of Lipton tea extract. For storage stability testing, the samples were stored in a refrigerator (4°C). The results of the stability test are shown in Table 4 and Figure 4. The pH of the Lipton tea and soy beverage mixture was approximately 5.4.

[0227] [Table 4]

[0228] As can be seen from Table 4 and Figure 4, the use of protein deamidase to prepare enzymatically deamidated plant-based beverages yielded soy-based ready-to-drink tea beverages that did not show visible aggregation after storage for 2, 9, and 16 days.

[0229] Stability of pea beverages in black tea In relation to this invention, two types of commercially available black tea were tested. In the first test, a black tea extract was prepared using commercially available Earl Grey tea bags. Two tea bags were mixed with 100 mL of 85°C warm water and kept warm for 30 minutes. Then, the tea bags were removed from the liquid and discarded. Next, 3 g of pea beverage prepared according to Example 2 was mixed with 9 g of Earl Grey tea extract. For storage stability testing, the samples were stored in a refrigerator (4°C). The results of the stability test are shown in Table 5 and Figure 5. The pH of the Earl Grey tea and pea beverage mixture was approximately 5.7.

[0230] [Table 5]

[0231] As can be seen from Table 5 and Figure 5, the use of protein deamidase to prepare enzymatically deamidated plant-based beverages yielded pea-based ready-to-drink acidic beverages without visible aggregation after storage for 2, 9, and 16 days.

[0232] In the second test, a tea extract was prepared using commercially available Lipton tea. Two tea bags were mixed with 100 mL of 85°C warm water and kept warm for 30 minutes. The tea bags were then removed from the liquid and discarded. Next, 3 g of pea beverage prepared according to Example 2 was mixed with 9 g of Lipton tea extract. For storage stability testing, the samples were stored in a refrigerator (4°C). The results of the stability test are shown in Table 6 and Figure 6. The pH of the Lipton tea and pea beverage mixture was approximately 5.7.

[0233] [Table 6]

[0234] As can be seen from Table 6 and Figure 6, the use of protein deamidase to prepare enzymatically deamidated plant-based beverages yielded pea-based ready-to-drink tea beverages without visible aggregation after storage for 2, 9, and 16 days. The slight aggregation observed in samples treated with lower amounts of protein deamidase, i.e., 0.6 IPA(U) / g protein, confirms the optimal range of protein deamidase.

[0235] Stability of soy beverages in green tea Green tea extract was prepared using commercially available Lipton green tea bags. Four tea bags were mixed with 100 mL of 85°C warm water and kept warm for 30 minutes. The tea bags were then removed from the liquid and discarded. Next, 3 g of soy beverage prepared according to Example 2 was mixed with 9 g of green tea extract. For storage stability testing, the samples were stored in a refrigerator (4°C). The results of the stability test are shown in Table 7 and Figure 7. The pH of the Lipton green tea and soy beverage mixture was approximately 5.9.

[0236] [Table 7]

[0237] As can be seen from Table 7 and Figure 7, the use of protein deamidase to prepare enzymatically deamidated plant-based beverages yielded soy-based ready-to-drink tea beverages that did not show visible aggregation after storage for 2, 9, and 16 days.

[0238] Stability of pea beverage in green tea Green tea was prepared using commercially available Lipton green tea bags. Two tea bags were mixed with 100 mL of 85°C warm water and incubated for 30 minutes. Then, the tea bags were removed from the liquid and discarded. Next, 3 g of the pea beverage prepared according to Example 2 was mixed with 9 g of the green tea extract. For the storage stability test, the samples were stored in a refrigerator (4°C). The results of the stability test are shown in Table 8 and Figure 8. The pH of the Lipton green tea and pea beverage mixture was approximately 6.0.

[0239]

Table 8

[0240] As can be seen from Table 8 and Figure 8, in order to prepare an enzymatically deamidated plant-based beverage, by using protein deamidase at an enzyme dosage of 2 IPA (U) / g protein, ready-to-drink tea beverages without visible aggregation were obtained after storage for 2, 9, and 16 days. The optimal range of protein deamidase was confirmed by the slight aggregation observed for samples treated with a lower amount of protein deamidase, i.e., 0.6 IPA (U) / g protein.

[0241] Stability of Pea Beverage in Jasmine Herb Tea A jasmine herb tea extract was prepared using commercially available Lipton jasmine tea bags. Two tea bags were mixed with 100 mL of 85°C warm water and incubated for 30 minutes. Then, the tea bags were removed from the liquid and discarded. Next, 3 g of the pea beverage prepared according to Example 2 was mixed with 9 g of the jasmine herb tea extract. For the storage stability test, the samples were stored in a refrigerator (4°C). The results of the stability test are shown in Table 9 and Figure 9. The pH of the Lipton jasmine tea and pea beverage mixture was approximately 6.1.

[0242]

Table 9

[0243] As can be seen from Table 9 and Figure 9, using protein deamidase at a PD dose of 2 IPA(U) / g protein to prepare enzymatically deamidated plant-based beverages yielded ready-to-drink tea beverages without visible aggregation after storage for 2, 9, and 16 days. The slight aggregation observed for samples treated with lower amounts of protein deamidase, i.e., 0.6 IPA(U) / g protein, confirms the optimal range for protein deamidase.

[0244] Example 5: Role of pH and tannic acid in the stability of pea and soy beverages treated with protein deamidase. Tannic acid (tannin) is a type of polyphenol compound found in tea and coffee, and tends to have a bitter and astringent taste. The role of tannic acid in the curing of plant-based beverages was investigated. Tannic acid (CAS 1401-55-4) was purchased from Sigma Aldrich.

[0245] Soybean and pea beverages prepared according to Example 2, as well as a control sample (without the addition of protein deamidase), were included in the test. The soybean and pea beverages were each mixed with a 1% (m / v) tannic acid solution, and the pH of the beverage was recorded when agglutination was observed. Next, the pH of another portion of the soybean or pea beverage was adjusted using 0.02 mol / L HCl to match the recorded pH of the tannic acid-treated plant-based beverage. The HCl-treated soybean and pea beverages were then visually inspected to determine if agglutination could be observed. The results from the visual inspection are shown in Table 10. Based on these observations, it was concluded that not only pH but also tannic acid affects the agglutination of plant-based beverages. Therefore, it was concluded that treatment of plant-based beverages with protein deamidase not only makes the plant-based beverages more stable to acidic pH but also improves their resistance to polyphenol-induced coagulation effects, such as those caused by tannic acid found in coffee and tea.

[0246] [Table 10]

[0247] Example 6: Testing of phosphate-containing and phosphate-free protein deamidases in pea-based ready-to-drink coffee beverages. Enzyme reaction Pea protein samples: Pea protein isolate (Roquette Nutralys S85F 2.0, protein content: 82%) was suspended in tap water until the final protein content was 10%, and then mixed with a magnetic stirrer. Protein deamidase was tested at concentrations of 0 (control sample), 6, and 12 IPA(U) / g protein, and these three samples were incubated at 60°C for 1 hour in a FINEPCR combi-D24 rotisserie set to a rotation speed of 7.

[0248] Coffee sample: The coffee used was Milan Gold coffee beans (Arabica and Robusta blend, medium roast), brewed in a Juracoffee machine with a water-to-bean ratio of 8:1.

[0249] Pea milk preparation The pea protein sample was incubated with protein deamidase, and then the pea milk sample was prepared by compounding it according to the recipe in Table 11 below. The materials were thoroughly mixed in a VORWERK Thermomix set to a rotation speed of 2.5 and a temperature of 60°C. The pea milk sample was then homogenized at a rotation speed of 8 for 3 minutes at 60°C.

[0250] [Table 11]

[0251] After mixing, the pea milk sample was pasteurized at 90°C for 10 minutes under stirring. Then, the sample was transferred to ice water and cooled to room temperature.

[0252] For some soybean milk samples, dipotassium phosphate (K2HPO4·3H2O) at 500 mg / 100 g of soybean milk was added to the soybean milk before mixing with coffee.

[0253] RTD coffee beverage Soybean milk samples were mixed with coffee samples at different ratios and placed in an autoclave at 121 °C for 5 minutes. Then, the RTD coffee samples were transferred to ice water and cooled to room temperature.

[0254] Next, for an accelerated experiment to mimic stability during shelf life, the RTD coffee samples were placed in sealed bottles and put into an oven at 60 °C.

[0255] Coagulation of the RTD coffee samples was evaluated by visual inspection. The sealed bottles were inverted, and the observed precipitation was evaluated by visually inspecting the content of the precipitate formed at the bottom of the flask.

[0256] From the results shown in Tables 12 and 13 (and corresponding Figures 10 and 11), it was confirmed that coagulation of the RTD coffee samples can be avoided with protein deamidase alone, both when phosphate is added and when it is not added.

[0257] [Table 12]

[0258] [Table 13]

Claims

1. A method for obtaining a plant-based ready-to-drink coffee or tea beverage, (a) A step of providing an aqueous solution containing an enzymatically deamidated plant material, (b) A step of mixing the aqueous solution from step (a) with a coffee or tea beverage to obtain the plant-based ready-to-drink coffee or tea beverage, (c) A step of storing the plant-based ready-to-drink coffee or tea beverage for at least 4 hours, preferably at least 8 hours, and more preferably at least 12 hours before consumption. Methods that include...

2. The method according to claim 1, wherein the aqueous solution and the coffee or tea beverage in step (a) have a temperature in the range of 3 to 30°C before and / or during the mixing in step (b).

3. The method according to claim 1 or 2, wherein the aqueous solution in step (a) has a protein content in the range of 0.3 to 5% (w / w), such as 1 to 3% (w / w).

4. The method according to any one of claims 1 to 3, wherein the aqueous solution in step (a) has a lipid content in the range of 0.5 to 4% (w / w), such as 1 to 2% (w / w).

5. The method according to any one of claims 1 to 4, wherein the aqueous solution in step (a) is a plant-based dairy alternative beverage.

6. The method according to any one of claims 1 to 5, wherein the aqueous solution containing the enzymatically deamidated plant material and / or the plant-based ready-to-drink coffee or tea is substantially free of added emulsifiers and / or stabilizers.

7. The method according to any one of claims 1 to 6, wherein the plant-based ready-to-drink coffee or tea beverage is a canned or packaged beverage.

8. The method according to any one of claims 1 to 7, wherein the plant-based ready-to-drink coffee or tea beverage is intended for storage at a temperature of 3 to 30°C.

9. The method according to any one of claims 1 to 8, wherein the plant-based ready-to-drink coffee or tea beverage comprises one or more additives selected from the group consisting of lipids, sugars, proteins, vitamins, minerals, amino acids, flavorings, dietary fiber, salt, and any combination thereof.

10. The method according to any one of claims 1 to 9, wherein the plant material is oats, peas, or soybeans, preferably derived from peas or soybeans.

11. The method according to any one of claims 1 to 10, wherein the ratio of aqueous solution to coffee or tea beverage is 1:10 to 10:1 based on w / w.

12. The aqueous solution containing the enzymatically deamidated plant material, i. A process to obtain a slurry of plant material in water, ii. A step of treating the slurry from step (i) with protein deamidase to obtain an aqueous solution containing deamidated plant material in water, iii. A step of optionally inactivating the protein deamidase. The method according to any one of claims 1 to 11, obtained by using a method including the method.

13. The method according to any one of claims 1 to 12, wherein the plant-based ready-to-drink coffee or tea beverage is heat-treated, optionally the heat treatment being ultra-high temperature (UHT), extended shelf life (ESL) treatment, or pasteurization.

14. The method according to any one of claims 1 to 13, wherein the aqueous solution containing the enzymatically deamidated plant material and / or the plant-based ready-to-drink coffee or tea beverage is substantially free of added buffer salts, preferably substantially free of added phosphates.

15. The method according to any one of claims 1 to 14, wherein the aqueous solution containing the enzymatically deamidated plant material is obtained using a protein deamidase derived from or obtained from the species Chryseobacterium.

16. A plant-based ready-to-drink coffee or tea beverage obtained by the method according to any one of claims 1 to 15.

17. Use of protein deamidase in the production of plant-based ready-to-drink coffee or tea beverages to improve stability, preferably to improve storage stability.

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